FIFTH EDITION FOOD CHEMICALS CODEX Effective January 1, 2004 COMMITTEE ON FOOD CHEMICALS CODEX Food and Nutrition Board INSTITUTE OF MEDICINE OF THE NATIONAL ACADEMIES
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FIFTH EDITION
FOODCHEMICALS
CODEXEffective January 1, 2004
COMMITTEE ON FOOD CHEMICALS CODEX
Food and Nutrition Board
INSTITUTE OF MEDICINEOF THE NATIONAL ACADEMIES
THE NATIONAL ACADEMIES PRESS • 500 Fifth Street, N.W. • Washington, DC 20001
NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whosemembers are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Instituteof Medicine. The members of the committee responsible for the report were chosen for their special competences and with regardfor appropriate balance.
Support for this project was provided by U.S. Food and Drug Administration Contract No. 223-99-2321. The views presented in thisreport are those of the Institute of Medicine Committee on Food Chemicals Codex and are not necessarily those of the funding agency.
COMPLIANCE WITH FEDERAL STATUTES The fact that an article appears in the Food Chemicals Codex or its supplements does notexempt it from compliance with requirements of acts of Congress, with regulations and rulings issued by agencies of the UnitedStates Government under authority of these acts, or with requirements and regulations of governments in other countries that haveadopted the Food Chemicals Codex. Revisions of the federal requirements that affect the Codex specifications will be included inCodex supplements as promptly as practicable.
Library of Congress Cataloging-in-Publication Data
Food chemicals codex / Committee on Food Chemicals Codex, Food andNutrition Board, Institute of Medicine.-- 5th ed.
p. cm.‘‘Effective January 1, 2004.’’Includes index.ISBN 0-309-08866-6 (hardback)
1. Food additives--Standards--United States. 2. Food additives--Analysis. I. Institute of Medicine (U.S.). Committee on Food ChemicalsCodex.
TP455.F66 2003664′.06′021873--dc21 2003010423
Additional copies of this report are available from the National Academies Press, 500 Fifth Street, N.W., Lockbox 285, Washington,DC 20055; (800) 624-6242 or (202) 334-3313 (in the Washington metropolitan area); Internet, http://www.nap.edu.
For more information about the Institute of Medicine, visit the IOM home page at: www.iom.edu.
Copyright 2003 by the National Academy of Sciences. All rights reserved.
Printed in the United States of America.
No part of this publication may be reproduced by any mechanical, photographic, or electronic process, or in the form of a phonographicrecording, nor may it be stored in a retrieval system, transmitted, or otherwise copied for public or private use, without writtenpermission from the publisher, except for official use by the United States Government or by governments in other countries thathave adopted the Food Chemicals Codex.
The serpent has been a symbol of long life, healing, and knowledge among almost all cultures and religions since the beginning ofrecorded history. The serpent adopted as a logotype by the Institute of Medicine is a relief carving from ancient Greece, now heldby the Staatliche Museen in Berlin.
THE NATIONAL ACADEMIESAdvisers to the Nation on Science, Engineering, and Medicine
The National Academy of Sciences is a private, nonprofit, self-perpetuating society ofdistinguished scholars engaged in scientific and engineering research, dedicated to thefurtherance of science and technology and to their use for the general welfare. Upon theauthority of the charter granted to it by the Congress in 1863, the Academy has a mandatethat requires it to advise the federal government on scientific and technical matters. Dr.Bruce M. Alberts is president of the National Academy of Sciences.
The National Academy of Engineering was established in 1964, under the charter ofthe National Academy of Sciences, as a parallel organization of outstanding engineers.It is autonomous in its administration and in the selection of its members, sharing withthe National Academy of Sciences the responsibility for advising the federal government.The National Academy of Engineering also sponsors engineering programs aimed atmeeting national needs, encourages education and research, and recognizes the superiorachievements of engineers. Dr. Wm. A. Wulf is president of the National Academy ofEngineering.
The Institute of Medicine was established in 1970 by the National Academy of Sciencesto secure the services of eminent members of appropriate professions in the examinationof policy matters pertaining to the health of the public. The Institute acts under theresponsibility given to the National Academy of Sciences by its congressional charter tobe an adviser to the federal government and, upon its own initiative, to identify issues ofmedical care, research, and education. Dr. Harvey V. Fineberg is president of the Instituteof Medicine.
The National Research Council was organized by the National Academy of Sciences in1916 to associate the broad community of science and technology with the Academy’spurposes of furthering knowledge and advising the federal government. Functioning inaccordance with general policies determined by the Academy, the Council has becomethe principal operating agency of both the National Academy of Sciences and the NationalAcademy of Engineering in providing services to the government, the public, and thescientific and engineering communities. The Council is administered jointly by bothAcademies and the Institute of Medicine. Dr. Bruce M. Alberts and Dr. Wm. A. Wulfare chair and vice chair, respectively, of the National Research Council.
www.national-academies.org.
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COMMITTEE ON FOOD CHEMICALS CODEX
S. Suzanne Nielsen (Chair 2001− ); Vice-Chair (1998−2000), Department of Food Sci-ence, Purdue University, West Lafayette, IN
Grady W. Chism III (Vice-Chair 2001− ) Department of Food Science and Technology,Ohio State University, Columbus
Michael H. Auerbach, Danisco USA, Inc., Ardsley, NYJonathan DeVries, General Mills, Inc., Minneapolis, MNMark Dreher, McNeil Nutritionals, New Brunswick, NJCarl Frey, Pepsi Cola North America, Valhalla, NYDavid S. Frick, Sensient Colors, Inc., St. Louis, MOGlen Ishikawa, NutraSweet Company, Evanston, ILRichard W. Lane, Unilever Bestfoods NA, Englewood Cliffs, NJJohn W. Salminen, Health Canada, Ottawa, Ontario, CanadaShelly J. Schmidt, Department of Food Science and Human Nutrition, University of
Illinois at Champaign-UrbanaPamela J. White, Food Science and Human Nutrition Department, Iowa State Univer-
sity, Ames
ConsultantAndrew G. Ebert, The Kellen Company, Atlanta, GA
StaffRicardo A. Molins, Study Director (1999− )Marcia S. Lewis, Research Assistant
FORMER MEMBERS AND STAFF OF THE COMMITTEE ON FOODCHEMICALS CODEX, 1998–2003
Andrew Ebert, 1988−1999E. Allen Foegeding, 2000−2001Merle Pierson, 1998−2001Steve Taylor, 1989−2000George Zografi, 1995−1999
Fatima N. Johnson, Study Director (1992−1999)Maria Oria, Program Officer (2002−2003)
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FOOD AND NUTRITION BOARD MEMBERS
Robert M. Russell (Vice-Chair), Jean Mayer USDA Human Nutrition, Research Centeron Aging, Tufts University, Boston, MA
Larry R. Beuchat, Center for Food Safety, University of Georgia, GriffinBenjamin Caballero, Center for Human Nutrition, Johns Hopkins Bloomberg School of
Public Health, Baltimore, MDShiriki Kumanyika, Center for Clinical Epidemiology and Biostatistics, University of
Pennsylvania School of Medicine, PhiladelphiaLynn Parker, Food Research and Action Center, Washington, DCA. Catharine Ross, Nutrition Department, Pennsylvania State University, University ParkBarbara O. Schneeman, Department of Nutrition, University of California, DavisSteve L. Taylor, Department of Food Science and Technology, University of Nebraska-
LincolnCatherine E. Woteki, Iowa State University, AmesBarry L. Zoumas, Alan R. Warehime Professor of Agribusiness, Department of
Agricultural Economics and Rural Sociology, Pennsylvania State University,University Park
StaffAllison A. Yates, DirectorLinda Meyers, Deputy DirectorGail Spears, Administrative AssistantGeraldine Kennedo, Administrative AssistantGary Walker, Financial Associate
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Participants in Committee Activities and Other Programs
Workshop on Criteria for Establishing MicrobiologicalSpecifications for Food Additives(April 25, 2000)
Gary Acuff Rodney GrayDane Bernard Ricardo MolinsRobert Buchanan Merle PiersonRichard Ellis Steve TaylorRussell Flowers Bruce Tompkin
Others Who Provided Assistance, 1998–2003
Nancy Alexander Robert G. BurseyGideon Andemicael Stephen J. Byrd Roger DabbahBen Andreson Christine DaleyJit F. Ang Edward Campbell Alberto DavidovichStephen Ashmead Michael F. Campbell A. de GrootMohamed Bakri Assoumani Richard Cantrill John DeanMichael H. Auerbach Janet Catanach Michael E. DeikerJ. Ayala Claudia Ceniceros G. Christopher C. DeMerlis
Marilyn R. Chambers Laura DepintoK. V. Balakrishnan Noeline Champion Mario Diaz-Cruz, IIIRichard L. Barndt Sharon Chang Sidney A. DoodeCharles H. Barnstein Minn-Chang Cheng Jim DoucetJulie N. Barrows Zak Chowhan James E. DownesKlaus Bauer Eunice Ciurlie Paul DribnenkiA. Allen Bednarczyk James P. Clark Douglas A. DrogoshAlison R. Behling Ross Clark G. DuchateauGint Behreus Ryan M. Clark John DuttonRaffaele Bernetti Warren S. Clark, Jr.Bruce M. Bertram Margaret Clarke Andrew EbertJ. Bertram David B. Clissold Denise L. EdgrenSanford W. Bigelow Melissa Cockayne Karen ElamRobert Biles Kipp A. Coddington James T. ElfstrumAnthony T. Bimbo David Cook Richard EllisDon Blaine Jerry Cook Sheryl E. EllisMel Blum Steven J. Cooke Mark EmpyCheryl Borders C. Arleen Courtney Roy EngelsDon P. Boudreaux Stuart Craig Elizabeth ErmanMarion M. Bradford Richard E. CristolKyd Brenner Raymond V. Croes H. FallahiPhillip R. Bross Eunice Cuirle John FernstromPaul Browning Lance Culbert Peg Fitzkee
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Thomas Fletcher Steven E. Hill Laurent LeducPatrick T. Flynn, Jr. Annet Hoek Ingrid LeutritzBarbara Ford Pat Hoffman Greg LimaGeorge T. Ford, Jr. Ronald D. Hogue Gerard E. LinkowskiJohn V. Fratus Thomas Holland Clara LubarskyJohn Fry James How Po Yam Lui
James HydeRaymond J. MaggioJames A. Gall
Vicente Ibañez Robert MaloneyDebbie GarczynskiKenji Ishii Steven ManheimerJerry D. GargulakMark L. Itzkoff Clodualdo ManingatPaula Gaynor
Ody ManingatJoe GensicK. James Colin D. MayGerstner GerhardJay-lin Jane James P. McCarthyTammy GierkeAllen J. Johnson Diane B. McCollKevin GilliesJerry Jost Gary McDermottJohn P. GleasonDavid R. Joy Larry McGirrVictor C. GoHamish Joyce Bill McKeownMary A. Godshall
Mike McLeanAmy R. Goodfellow Isabelle KamishlianIrving MelcerScott J. Grare Ken KasengrandeDoug J. MeltonRodney J. H. Gray Robert KasikPhilip H. MerrellRichard Green G. Kere KempRich MilitoJames Griffiths Gregory KeselPhilip MingleLisa Gruener Ken KeyteMel Mirliss
Janeen KincaidDewey MitchumMartin Hahn Charles L. KingM. MiyasakaMikyoung Hahn P. P. KirschJohn ModdermanFerid Haji Lori L. KlopfBill MonagleJohn Hallagan Christian KlugThomas J. MuldoonEarl G. Hammond W. F. KohlG. MüllerSue Harris Monique KosseRaja K. MurtiM. Hashimoto Karen KowalewskiAmy MutreChristopher D. Hassall Paul Kuznesof
Ken Hassan Carol Kyck Lyn NaborsTheodore D. Head
Denise NahonJerome H. Heckman Ron Lacock
Donald D. NaragonAllan R. Hedges Frank Lambert
June M. NeadesBarbara B. Heidolph William Lambert
Lisa NevarroRichard Hendricks Lucina LampilaMichael Henkel Kathleen E. Lanshe
Melanie O’DonnellBruce Henkin Richard Larock
Atsuhi OkiyamaJohn L. Herrman Susan Lawlor
Owen J. OlesenJoseph Hickenbottom Brian Lawrence
Stanley T. OmayeDavid H. Hickman Neil Lawson
Aydin ÖrstanJ. J. Higgins Patricia L. LawsonNancy Higley A. Philip Leber
FCC V Participants in Committee Activities / ix
Juhani Paakkanen Sheldon Silbiger Lorraine TwerdokAndy Paterson John M. Simmons R. T. TylerKen Paydon Patrick Smith Tom TynerJohn Pearce Timothy R. SmithBruce Peterson Elisabeth A. Snipes Youichiro UmekiBruce E. Phillips Jennifer SnyderGlyn O. Phillips Charles Sokol Q. Edith Valle C.Jay Piester Kyle A. Spencer Mel Vandenberg
Laura M. Spiegelhoff Cheryl Van DyneGlenn A. Rasmussen Jennifer Spokes Julio C. VegaGregory Redko Erik Sprenne James VergheseDorothee Reuscher Edward A. Steele Marc VermaeulenGreg Reynhout Daniel G. Steffen Uwe VoeklerAlan B. Richards Lewis Stegink Wolfgang A. VoglMichelle Rieckhoff Fred Stone Frank VollaroWilliam Riha David StrausChet A. Roberts Eric Strauss Robert Waller, Jr.Mark Robertson Hiroshi Sugano Glenn WardSusan Rodgers Yoshi-hisa Sugita Cayce WarfBryan Rodriguez Rusell Sydes Jerry WeigelQuinton Rogers Bernard F. Szuhaj Cathy WendlerGlenn Ruskin Richard H. WendtRobin A. Russell Tetsua Taguchi Thomas P. West, Jr.
Todd Talashek Jennifer WhiteGeorge Sanderson David A. Tarizzo Stephanie WinderAntonio Sardella Sarah Taylor Karen WingartzEiji Sato Steve Taylor John T. WoodardDavid A. Saunders Taguchi Tetsuya Hennie A. WorkelRudolph F. Scarpelli Jette Thestrup Karel WrightKevin Schaffler Rani M. Thomas William W. WrightRainer Schnee William R. ThorntonEd Schoenberg Geoff Tomlinson Kohei YamamotoMichael Schrage Josephine M. Torrente Gary L. YinglingGloria T. Seaborn Kathleen Trahanovsky Matt YokotaCatherine Sheehan Keith TriebwasserPaul E. Shelton Bryan Tungland Priscilla S. ZawislakTadahisa Shimoda Samuel Tuthill Randall E. Zigmont
Michael Zviely
Preface
The Fifth Edition of the Food Chemicals Codex (FCC) is a result of the collectiveefforts of the many members, past and present, of the Committee on Food ChemicalsCodex over the past 42 years. The current committee, whose members have broughtall these efforts to fruition with this edition, was appointed following a request fromthe U.S. Food and Drug Administration (FDA) to continue this activity. The chargeto the committee states that ‘‘the committee shall (1) provide information on mattersrelated to the purity of food ingredients used in the United States and shall beknowledgeable of the purity of food ingredients used in other countries; (2) provideinformation on food-grade specifications for food additives, GRAS [generally recog-nized as safe] substances, and any other food substances used as ingredients; and (3)publish specification monographs in a Fifth Edition of the Food Chemicals Codex.To provide such information, the committee shall review proposals from industry,government, and any other source.’’
The FCC project, currently under the Food and Nutrition Board of the Institute ofMedicine of the National Academies, began in 1961, soon after the passage of the1958 Food Additives Amendment to the federal Food, Drug, and Cosmetic Act.Although the FDA had, by regulations and informal statements, defined in generalterms the quality requirements for GRAS and other food chemicals, these requirementswere not sufficiently specific to serve as release, procurement, and acceptance specifi-cations for manufacturers and users of food chemicals. Therefore, regulators andother interested parties believed that the publication of a book of standards designedespecially for food chemicals would promote uniformity of quality and added assur-ance of safety for such chemicals. For these reasons, the Food Protection Committeeof the National Academy of Sciences/National Research Council received requestsin 1958 from its Industry Liaison Panel and other sources to undertake a project toproduce a Food Chemicals Codex comparable in many respects to the United StatesPharmacopeia and the National Formulary for drugs. As a result of these requests,representatives of industry and government agencies agreed that there was a definiteneed for such a Codex and that the Food Protection Committee was a suitable bodyto undertake the project.
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The first edition, published in 1966, was supported by a Public Health Servicegrant and more than 100 supplementary grants from industry, associations, and founda-tions. Its role, which is still that of the Food Chemicals Codex, was to define thequality of food-grade chemicals in terms of identity, strength, and purity based onthe elements of safety and good manufacturing practice. Later editions were supportedby direct contracts with the FDA. Such sponsorship has been sufficient to supportthe publication of 4 earlier editions and 14 supplements in a 42-year span.
SCOPE
The scope of the Food Chemicals Codex has expanded with each new edition.Substances included in the first edition were limited to chemicals added directly tofoods to achieve a desired function. Succeeding editions included these substancesas well as such processing aids as enzymes, extraction solvents, filter media, andboiler water additives; those that are regarded as foods, such as fructose and dextrose,rather than as additives; and those that exhibit a functional effect, not on the foodsto which they are added, but to the human body when the food is consumed. ThisFifth Edition includes 961 monographs from the Fourth Edition; 49 monographs,along with those for 15 flavor chemicals, added in the three supplements to the FourthEdition; and 19 new monographs, along with 33 for flavor chemicals, new to thisFifth Edition, bringing the total to 1077. Because of its regulatory status in countriesother than the United States, and its worldwide use, the Food Chemicals Codexcontains some monographs for chemicals not currently allowed in foods in the UnitedStates. This circumstance is clearly indicated in such monographs.
UPDATING AND DEVELOPING SPECIFICATIONS
The committee has invariably sought to define, using physicochemical and microbio-logical parameters, ingredients prepared under good manufacturing practices as safefor human consumption. Special emphasis has been placed on reducing contaminants,including trace elements, particularly lead. The committee removed Arsenic andMicrobiological Criteria specifications from monographs that were unnecessarilyburdened with them. More importantly, the committee revised the Lead and HeavyMetals Limits Policy by removing the Heavy Metals (as Pb) specifications andreplacing them with specifications for relevant heavy metals. The committee alsodecided, based on research of the Standing Committee on the Scientific Evaluationof Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine, theNational Academies, that the intake of fluoride as a constituent of substances describedin FCC monographs is not expected to significantly add to the human daily fluorideintake. However, because high levels of fluoride have been amply demonstrated tocause toxicological problems, as described in the report, the maintenance of fluoridelimits in selected food additives appears consistent with sound public health policy.Because of the difficulties in analyzing for fluoride in food chemicals, the committeehas adopted a new analytical method for fluoride and will continue to add more whenadequate validation of new methods is submitted.
FCC V Preface / xv
Limits on contaminants, specifically lead and other heavy metals, have been reducedin most monographs in this edition. This trend is expected to continue. Manufacturersand suppliers of food ingredients are encouraged to inform the committee of theirability to supply food ingredients with lead and other heavy metals limits lower thanthose specified in this edition. The arsenic specification remains in relatively fewmonographs in this edition where (1) the ingredient or additive is a high-volumeconsumption item (greater than 25 million pounds a year), (2) the ingredient oradditive is derived from a natural (mineral) source where arsenic may be an intrinsiccontaminant, or (3) there is reason to believe that arsenic constitutes a significantpart of the total heavy metals content.
The committee is cognizant of the need for international harmonization of specifica-tions in today’s world. Efforts were made, where feasible, to harmonize the specifica-tions in this edition with those of other standards-setting organizations, in particularwith those in the Compendium of Food Additive Specifications, prepared by the Foodand Agricultural Organization of the United Nations (FAO)/World Health Organiza-tion (WHO) Joint Expert Committee on Food Additives (JECFA) and published bythe FAO.
FORMAT
Generally the presentation follows that of the Fourth Edition, but a number of signifi-cant changes and additions have been made. As expected, the passage of 7 yearssince the appearance of the Fourth Edition has been accompanied by changes:
T Additional information in terms of FEMA (Flavor and Extract Manufacturers Asso-ciation) numbers has been added to essential oil and other flavor monographs not inthe Flavors Table, Chapter 3.T Infrared Spectra for most substances requiring them for identification purposeshave been rerun and thus are more accurate.T New headers on each page of this book tell readers where they are by chapter,monograph, appendix, or test.T The language in the monograph section has been revised to be more clear, consistent,and concise.T All tests that occurred identically in three or more monographs were moved to theappendices.
FUTURE REVISIONS
The introduction of new food additives as well as constant changes and advances inmanufacturing processes and analytical sciences lead to a need for continued revisionof this compendium.
The committee recognizes the need to initiate an extensive update of the analyticalmethods described in this edition, in such a way that advanced new technologiesare incorporated in the Sixth Edition, while maintaining a balance with other, lesstechnology-intensive methods for use by laboratories and firms that may not have
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access to such advanced technology. The committee specially recognizes the urgencyof updating current chromatographic methods throughout the present edition andintends to complete this goal during the next 5 years. Users of this edition are requestedand encouraged to submit suggestions for updating the specifications as well as thegeneral analytical methods. Constructive criticism and notification of errors shouldalso be brought to the attention of the Food Chemicals Codex, Institute of Medicine,500 Fifth Street, N.W., Washington, D.C. 20001 or <[email protected]>.
LEGAL STATUS
The Food Chemicals Codex has earned international recognition by manufacturers,vendors, and users of food chemicals. The specifications herein serve as the basisfor many buyer and seller contractual agreements.
In the United States, the first edition was given quasi-legal recognition in July1966 by means of a letter of endorsement from FDA Commissioner James L. Goddard,which was reprinted in the book. The letter stated that ‘‘the FDA will regard thespecifications in the Food Chemicals Codex as defining an ‘appropriate food grade’within the meaning of Sec. 121.101(b)(3) and Sec. 121.1000(a)(2) of the food additiveregulations, subject to the following qualification: this endorsement is not construedto exempt any food chemical appearing in the Food Chemicals Codex from compliancewith requirements of Acts of Congress or with regulations and rulings issued by theFood and Drug Administration under authority of such Acts.’’
Subsequently, the specifications in the Second Edition, followed by those in theThird Edition, were cited, by reference, in the U.S. Code of Federal Regulations todefine specific safe ingredients under title 21, in various parts of sections 172, 173,and 184.
In Canada, the current edition of the Food Chemicals Codex, including its supple-ments, is officially recognized in the Canadian Food and Drug Regulations underSection B.01.045(b) as the reference for specifications for food additives. The newAustralia New Zealand Food Authority recognizes the Food Chemicals Codex as aprimary source of identity and purity specifications in its Food Standards Code,Chapter 1 General Food Standards, Part 1.3 Substances Added to Food, Standard1.3.4 Identity and Purity.
REVIEWERS
This report has been reviewed in draft form by individuals chosen for their diverseperspectives and technical expertise, in accordance with procedures approved bythe National Research Council’s Report Review Committee. The purpose of thisindependent review is to provide candid and critical comments that will assist theinstitution in making its published report as sound as possible and to ensure that thereport meets institutional standards for objectivity, evidence, and responsiveness tothe study charge. The review comments and draft manuscript remain confidential toprotect the integrity of the deliberative process. We wish to thank the followingindividuals for their review of this report:
FCC V Preface / xvii
William E. Artz, University of IllinoisJames N. Bemiller, Purdue UniversityRengaswami Chandrasekaran, Purdue UniversitySam Chang, North Dakota State UniversitySusan L. Cuppett, University of NebraskaStephanie Doores, University of PennsylvaniaWilliam Eigel, Virginia Polytechnic and State UniversityRonald Eitenmiller, University of GeorgiaJeffrey M. Farber, Health CanadaHarold R. Faust, PenrecoKenneth Fowkes, Praxair Distribution Inc.Earl Hammond, Iowa State UniversityDonald L. Johnson, ConsultantPaul Lachance, Rutgers UniversityJohn Lichtfield, The Ohio State UniversityHarold M. McNair, Virginia Polytechnic and State UniversityDennis D. Miller, Cornell UniversityDavid B. Min, The Ohio State UniversitySean O’Keefe, Virginia Polytechnic and State UniversityAndrew Proctor, University of ArkansasJenny Scott, National Food Processors AssociationRandy Wehling, University of NebraskaRonald Wrolstad, Oregon State University
Although the reviewers listed above have provided many constructive commentsand suggestions, they were not asked to endorse the conclusions or recommendationsnor did they see the final draft of the report before its release. The review of thisreport was overseen by Barbara P. Klein, University of Illinois. Appointed by theNational Research Council and Institute of Medicine, she was responsible for makingcertain that an independent examination of this report was carried out in accordancewith institutional procedures and that all review comments were carefully considered.Responsibility for the final content of this report rests entirely with the authoringcommittee and the institution.
ACKNOWLEDGMENTS
A compendium of this breadth can only result from the cooperation of many individualsand organizations. Underlying this, the support provided by FDA contract number223-99-2321, monitored by project officers Paul M. Kuznesof and Daniel Folmer, isgratefully acknowledged.
Several monographs and various sections in this edition have portions based onother publications, and are used with permission granted by the parent organizations:the American Chemical Society; the American Oil Chemists Society; the AmericanSociety for Testing and Materials; AOAC International; and the United States Pharma-copeial Convention, Inc. This edition of the Food Chemicals Codex directly referencesthe procedures in the Eighth Edition of the FDA Bacteriological Analytical Manual
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(BAM) for its microbial limit tests. Where the sample size is not defined in the limit,the results are based on the sampling procedures described in BAM.
While participating individuals have been listed on pages vii–ix, the followingorganizations have also been active participants:
American Dairy Products InstituteCorn Refiners AssociationEnzyme Technical AssociationFlavor and Extract Manufacturers AssociationGelatin Manufacturers of EuropeGelatin Manufacturers Institute of AmericaInternational Association of Color ManufacturersInternational Dairy FederationInternational Food Additives CouncilInternational Pectin Producers AssociationInternational Pharmaceutical Excipients CouncilInternational Technical Caramel AssociationNational Association of Chewing Gum ManufacturersSalt InstituteSoy Protein Council
Members of the National Academies Press—Sally S. Stanfield, James M. Gormley,Estelle H. Miller, Dan Parham, and William B. Mason—and staff of the Institute ofMedicine Office of Reports and Communication—Jennifer Bitticks, Jennifer Otten,Bronwyn Schrecker, and Leah Covington—provided valuable support to the FCCstaff toward the publication of this edition.
Success in the complex task of completing the Fifth Edition is due to the dedicationand determination of the members of the Committee on Food Chemicals Codex underthe focused leadership of its successive chairs, Steve L. Taylor and S. Suzanne Nielsen,during the past 58 months, and to those of the Food Chemicals Codex staff, MariaOria and Marcia Lewis.
Washington, D.C. Ricardo A. MolinsSeptember 2003 Study Director
General Information
OPERATING PROCEDURES OF THE FOOD CHEMICALS CODEX
Organization
The Food Chemicals Codex (FCC) project is an activity of the Food and NutritionBoard, a unit of the Institute of Medicine of the National Academies. The immediateresponsibility for developing the Food Chemicals Codex lies with the Board’s Commit-tee on Food Chemicals Codex. The committee consists of 12 to 15 members, chosenfor their expertise in the various aspects of the committee’s work, who are appointed,upon recommendation of the Food and Nutrition Board and the President of theInstitute of Medicine, by the Chairman of the National Research Council. Committeemembers are paid no consulting fees or honoraria and are reimbursed only for expensesincurred while attending meetings and other activities of the committee.1
Functions of the Committee on Food Chemicals Codex
The committee’s principal functions are as follows:
T To establish the general policies and guidelines by which FCC specifications areprepared.
T To evaluate comments submitted by interested parties on any aspect of the specifica-tions and test procedures.
T To propose means by which the specifications may be kept current in reflectingfood-grade quality on the basis of product safety and good manufacturing practices.
T To provide information on issues dealing with specifications for particular sub-stances and analytical test procedures.
1The project scope, a committee roster, and meeting information are accessible on the National Academies’web site. Access <www.nationalacademies.org/cp.nsf> and search by name for ‘‘Food Chemicals Codex.’’
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T To seek the advice of specialists when additional expert opinion is needed in makingdecisions regarding the appropriateness of specifications.
T To establish working groups consisting of committee members and other expertsto address specific issues relevant to monograph development and to report theirfindings and recommendations to the full committee.
T To consider and act on any other issues concerning the development and publicationof specifications and test procedures for food-grade ingredients.
T To approve the final manuscript for review before the publication of any editionof the FCC or its supplements.
Committee business is conducted through a central office at the National Academiesin Washington, D.C. The appointed responsible study director at the Food and NutritionBoard, Institute of Medicine, coordinates all committee activities. The committeemeets in regular session, usually once a year, to discuss the project’s progress,including technical and policy issues relevant to the FCC. One or more members ofthe committee as well as the study director conduct ad hoc meetings on short-termprojects as needed. The committee and study director also organize workshops andsymposia as appropriate to exchange information with interested parties on key issues,whether of broad or limited scope.
Requirements for Listing Substances in the Food Chemicals Codex
The requirements are as follows: (1) the substance is permitted for use in food or infood processing in the United States (or, in certain cases, in other countries in whichFCC specifications are recognized), (2) it is commercially available, and (3) suitablespecifications and analytical test procedures are available to determine its identityand purity.
Criteria for Food Chemicals Codex Grade
The specifications published in the FCC are based primarily on the criteria of safetyand good manufacturing practices (GMP). An FCC-grade substance is one that isprepared under GMP (discussed in detail later in this section) and that is of suchpurity as to ensure that potentially harmful or objectionable contaminants are notpresent at levels that would represent a hazard to the consumer of the foods in whichthe substance is intended to be used. Thus, FCC specifications define substances ofsufficiently high quality to represent a reasonable certainty of safety when they areused under customary conditions of intentional use in food or in food processing.The specifications generally represent acceptable levels of quality and purity of food-grade substances available in the United States and in other countries in which FCCspecifications are recognized. Because the different types of ingredients are diverseand complex, few general criteria can be established that will apply to all substancesfor which FCC specifications are prepared. The committee recognizes that limits andtests cannot be provided to cover all possible unusual or unexpected impurities, the
FCC V General Information / xxi
presence of which would be inconsistent with GMP. This matter is discussed furtherunder Trace Impurities, in the General Provisions, and under General Good Manufac-turing Practices Guidelines for Food Chemicals.
In addition to impurity limits, specifications, where applicable, must include thefollowing: empirical formula, structural formula, and formula weight; description ofthe substance, including physical form, odor (flavoring agents only), and solubility(see the descriptive terms for solubility in the General Provisions); identification;assay (or a quantitative test to serve as an assay); physicochemical characteristicssuch as specific rotation, melting range or solidification point, viscosity, specificgravity, refractive index, and pH; loss on drying or water content; residual solvents;limits for mycotoxins and microbiological contaminants; and limits for byproducts andother adventitious constituents usually occurring in, or arising from the manufacture of,the substance. For safety, the committee deleted taste, as a characteristic of anysubstance, from all monographs, and odor from all but flavor monographs. The dataprovided, taken together, represent a complete compositional understanding of thesubstance. Additional information items include how the substance is to be packagedand stored to maintain its integrity and its functional use(s) in foods. If the substancecontains an ‘‘added substance,’’ mentioning this fact enables the committee to judgewhether the specifications should include it (see Added Substances under GeneralProvisions).
Important Changes That May Affect How Information Is Submitted
Before submitting information to the Committee on Food Chemicals Codex, pleaseread carefully the following paragraphs about the Federal Advisory Committee Act(FACA) Amendments of 1997, section 15, public law number 105-153. This actcreates certain new requirements regarding studies performed for federal governmentagencies by the National Academies: the National Academy of Sciences (NAS), theNational Academy of Engineering (NAE), the Institute of Medicine (IOM), andthe National Research Council (NRC) (collectively referred to as ‘‘the NationalAcademies’’).
The National Academies’ policy applies to any committee (board, panel, etc.)appointed by the National Academies to develop a report (study reports, letter reports,workshop proceedings, summaries of symposia, and other manuscripts derived frominstitutional activities) that is intended for distribution outside the National Academies.
Documents Available to the Public at Committee Meetings Any meeting of acommittee at which anyone other than committee members or officials, agents, oremployees of the institution is present, whether in person or by telephone or audioor video teleconferences, is a ‘‘data-gathering committee meeting.’’ Except as pro-vided by exemptions, all data-gathering committee meetings are open to the public.
Within the capacity of the meeting room, attendance at data-gathering committeemeetings that are open to the public would not be limited. Any person, includingmembers of the news media, may attend as observers (not participants), whetherexplicitly invited or not, provided that the individual is not disruptive. The chair ofthe meeting, assisted by officials and staff of the National Academies, is responsiblefor the conduct of the meeting.
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Documents Unavailable to the Public at Committee Meetings Any committeemeeting at which only committee members and officials, agents, and employees of theNational Academies are present is a ‘‘closed committee meeting.’’ Closed committeemeetings are not open to the public or to any person who is not a committee memberor an official, agent, or employee of the National Academies. Deliberations by acommittee in discussing, preparing, and finalizing a draft written report, includingdeliberations relating to review comments received in connection with review of thedraft report under the National Academies’ report review process, must be conductedin closed meetings.
After each closed committee meeting, the study director for the committee shallprepare a brief summary of the closed committee meeting and post the summaryimmediately on the National Academies’ web site <www.nas.edu>. Except as providedby the exemptions, the brief summary of a closed committee meeting will identifythe committee members present, the topics discussed, materials made available to thecommittee, and such other matters as the study director determines should be included,except that the brief summary will not disclose the substantive content or conclusionsor recommendations of any draft report or discussions thereof or disclose any reportreview comments.
Public Access File A public access file for a committee project is established assoon as the study director creates a project record in the National Academies’ currentprojects system.2
Materials provided at a data-gathering meeting or received by mail or fax from anorganization or lay persons who are not officials, agents, or employees of the NationalAcademies are placed in the public access file. Video tapes, audio tapes, or otheralternative media such as diskettes or slides or viewgraphs presented to the committeeby an organization or by individuals who are not officials, agents, or employees ofthe National Academies are considered by the National Academies to be subject topublic disclosure as well.
Materials Exempt from the Public Access File The study director must requestand receive advance written approval from the National Academies’ Office of GeneralCounsel (OGC) for withholding from the public any material presented to a committeeby an organization or by a person other than an official, agent, or employee of theNational Academies. This request must include adequate documentation to supportsuch withholding. For example, in the case of classified or statutorily protectedinformation, the National Academies must receive a written statement addressed tothe National Academies setting forth sufficient information to enable the NationalAcademies’ Office of General Counsel and the National Research Council’s ExecutiveOffice to confirm that the information in question would be exempt from publicdisclosure under one or more of the Freedom of Information Act (FOIA)3 exemptions.The study director may not distribute to committee members any such materials(containing restrictive legends or markings limiting disclosure) without first consulting
2Access <www.nationalacademies.org/cp.nsf> and search by name for ‘‘Food Chemicals Codex.’’3Access the Freedom of Information Act at <http://www.oalj.dol.gov/public/apa/refrnc/FOIA.HTM>.
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the OGC. Only written materials that the OGC, in consultation with the NationalResearch Council Executive Officer, determines to be exempt from disclosure underthe exemptions to the disclosure requirements of the FOIA will be withheld from thePublic Access File.
Procedures for Submission and Development of Specifications
The committee will consider suggested specifications, such as previously elaborated,submitted with supporting data by any interested party, including food ingredientmanufacturers and suppliers, food processors, and industry associations. Suggestedspecifications should be submitted, in duplicate, to Food Chemicals Codex, Food andNutrition Board, Institute of Medicine, 500 Fifth Street, N.W., Washington, D.C.20001. The committee and/or the project staff examine suggested specifications andoften expand them to meet the general criteria the committee requires. Becausecommittee discussions involving quality characteristics of substances used in foodor food processing might result in sharing privileged or proprietary information,contributors may request that such discussions be held in closed sessions. The finaloutcome of such discussions must be openly shared with all manufacturers, users,and parties interested in the substance discussed; therefore, open discussions arethe norm, except during unusual circumstances. Where privileged or proprietaryinformation is concerned, the project staff can put such information in a format sothat the end results are not associated with particular manufacturers or users. Thecommittee and/or the staff draft a new monograph and send it to the originator (andto any other manufacturers of that substance that can be identified) for comment.After the draft has gone through this process and all necessary revisions have beenmade, the committee votes by mail ballot whether to propose these specifications forpublic comment. If the committee finds deficiencies, or if any questions are raised,the draft is returned to the originator and other interested parties with the committee’scomments and recommendations for improvement. Once a draft has gained committeeacceptance, availability of the proposed specification for comment is announced inthe Federal Register or online at <www.cfsan.fda.gov> or through notices in tradejournals. This notification allows the public and other interested parties as well asmanufacturers and users that may be inadvertently overlooked to provide their com-ments to the committee. Once the public comments are considered and any necessarychanges made, the committee votes to determine whether the monograph is suitablefor publication. Monographs as well as supporting materials such as general tests andinfrared spectra are then reviewed through the National Research Council’s reportreview process and, if approved, are published in the next edition of the FCC or asupplement.
Procedure for Revising Specifications
FCC specifications are subject to revision at any time, and suggested revisions maybe initiated by regulatory bodies, manufacturers, suppliers, or users of the ingredients;by the committee itself; or by any other interested parties. All suggestions for revisionmust be accompanied by supporting data. In the case of revisions of test procedures
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and analytical methods, comparative data for both the existing and suggested proce-dures must be submitted. Where changes in limits or other tolerances are suggested,supporting data should be presented on representative production batches. Suggestionsfor changing the limits of certain impurities (e.g., arsenic, cadmium, lead, fluoride,and mercury) may require the submission of safety data and information concerningthe daily intake of the substance. All suggestions for revision, together with thesupporting data, are reviewed by the Committee on Food Chemicals Codex and/orby the FCC staff. If other manufacturers are involved (and can be identified), theyare also asked to comment. If the committee finds deficiencies, or if any questionsarise, the suggested revised specifications are returned to the originator (and othermanufacturers, where appropriate) with the committee’s comments or questions. Ifagreement cannot be reached at this point between the committee and the originator,or among manufacturers and other interested parties, a special meeting may be heldto discuss the matter, or the parties involved may be invited to one of the committee’sregular meetings to examine the question in depth. Approved revisions are publishedeither in the next edition of the FCC or in a supplement by the same proceduredescribed under Procedures for Submission and Development of Specifications.
Additional Information
FCC users should become thoroughly familiar with the General Provisions pertainingto this edition. Inquiries regarding any aspect of the operation of the FCC projectmay be directed to Food Chemicals Codex, Food and Nutrition Board, Institute ofMedicine, 500 Fifth Street, N.W., Washington, D.C. 20001 (telephone 202-334-2580;facsimile 202-334-2316; email [email protected]). Additionally, all interested parties mayview new and revised materials as presented by the Committee on Food ChemicalsCodex at <www.iom.edu/fcc>.
VALIDATION OF FOOD CHEMICALS CODEX METHODS
Submissions to the Food Chemicals Codex
Submissions for new or revised specifications and analytical methods must containsufficient information to enable committee members to evaluate the proposals. Inmost cases, evaluations involve assessing the clarity and completeness of the analyticalmethods description, determining the need for the methods, and reviewing documenta-tion that the methods have been appropriately validated. Information may vary de-pending on the type of test method involved. However, in most cases a submissionwill consist of the following sections:
Rationale Use this section to identify the need for the analytical method and describethe capability of the specific method proposed and why it is preferred over othertypes of determinations. For revised analytical methods, provide a comparison oflimitations of the existing FCC analytical method and advantages offered by thesuggested method.
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Suggested Analytical Method Use this section to present a complete descriptionof the analytical method sufficiently detailed to enable persons ‘‘skilled in the art’’to replicate it. Include all important operational parameters and specific instructionssuch as reagent preparation, systems suitability tests performance, description ofblanks used, precautions, and explicit formulas for calculating test results.
Data Elements Use this section to provide thorough and complete documentationof the validation of the analytical method. Include summaries of experimental data andcalculations substantiating each of the applicable analytical performance parameters.These parameters are described in the following section.
Validation
Validation of an analytical method is the process of establishing, by laboratory studies,that the performance characteristics of the method meet the requirements for theintended analytical applications. Express performance characteristics in terms of ana-lytical parameters. Each of the recommended parameters is defined in the next sectionof this chapter, along with a delineation of a typical method by which it may bemeasured.
Typical analytical parameters used in assay validation are accuracy, precision,specificity, limit of detection, limit of quantitation, linearity, range, and ruggedness.
AccuracyDefinition The accuracy of an analytical method is the closeness of test results
obtained by that method to the true value. Accuracy may often be expressed as percentrecovery by the assay of known, added amounts of analyte.
Determination Determine the accuracy of an analytical method by applying thatmethod to samples to which known amounts of analyte have been added both aboveand below the normal levels expected in the samples. Calculate the accuracy fromthe test results as the percentage of analyte recovered by the assay.
PrecisionDefinition The precision of an analytical method is the degree of agreement
among individual test results when the procedure is applied repeatedly to multiplesamplings of a homogeneous sample. The precision of an analytical method is usuallyexpressed as the standard deviation or relative standard deviation (coefficient ofvariation). Precision may be a measure of the degree of either reproducibility orrepeatability of the analytical method under normal operating conditions. In thiscontext, reproducibility refers to the use of the analytical procedure in differentlaboratories. Intermediate precision expresses within-laboratory variation, as on differ-ent days, or with different analysts or equipment within the same laboratory. Repeat-ability refers to the use of the analytical procedure within a laboratory over a shorttime, using the same analyst with the same equipment.
Determination Determine the precision of an analytical method by assaying asufficient number of aliquots of a homogeneous sample to be able to calculate statisti-cally valid estimates of standard deviation or relative standard deviation (coefficientof variation). Assays in this context are independent analyses of samples that have
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been carried through the complete analytical procedure from sample preparation tofinal test result.
SpecificityDefinition The specificity of an analytical method is its ability to measure, both
accurately and specifically, the analyte in the presence of components that may beexpected to be present in the sample matrix. Specificity may often be expressed asthe degree of bias of test results obtained by analysis of samples containing addedimpurities, degradation products, or related chemical compounds when comparedwith test results from samples without added substances. The bias may be expressedas the difference in assay results between the two groups of samples. Specificity isa measure of the degree of interference (or absence thereof) in the analysis of complexsample mixtures.
Determination Determine the specificity of an analytical method by comparingtest results obtained from the analysis of samples containing impurities, degradationproducts, or related chemical compounds with those obtained from the analysis ofsamples without these elements. The bias of the assay, if any, is the difference intest results between the two groups of samples.
When impurities or degradation products are unidentified, demonstrate specificityby analyzing samples (with the method in question) containing impurities or degrada-tion products and by comparing the results to those from additional purity assays(e.g., chromatographic assay). The degree of agreement of test results is a measureof the specificity.
Limit of DetectionDefinition The limit of detection is a parameter of limit tests. It is the lowest
concentration of analyte in a sample that can be detected, but not necessarily quanti-tated, under the stated experimental conditions. Thus, limit tests merely substantiatethat the analyte concentration is above or below a certain level. The limit of detectionis usually expressed as the concentration of analyte (e.g., percentage, milligrams pergram, parts per billion) in the sample.
Determination Determining the limit of detection of an analytical method willvary depending on whether it is an instrumental or noninstrumental procedure. Forinstrumental procedures, different techniques may be used. Some investigators deter-mine the signal-to-noise ratio by comparing test results from samples containing knownconcentrations of analyte with those of blank samples and establish the minimum levelat which the analyte can be reliably detected. A signal-to-noise ratio of 2:1 or 3:1 isgenerally accepted. Other investigators measure the magnitude of analytical back-ground response by analyzing a number of blank samples and calculating the standarddeviation of this response. The standard deviation, multiplied by a factor, usually 2or 3, provides an estimate of the limit of detection. This limit is subsequently validatedby the analysis of a suitable number of samples known to be close to or at the limitof detection.
For noninstrumental methods, determine the limit of detection by analyzing sampleswith known concentrations of analyte and by establishing the minimum level at whichthe analyte can reliably be detected.
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Limit of QuantitationDefinition Limit of quantitation is a parameter of quantitative assays for low
levels of compounds in sample matrices, such as impurities and degradation productsin food additives and processing aids. It is the lowest concentration of analyte in asample that can be determined with acceptable precision and accuracy under the statedexperimental conditions. The limit of quantitation is expressed as the concentration ofanalyte (e.g., percentage, milligram per kilogram, parts per billion) in the sample.
Determination Determining the limit of quantitation of an analytical method mayvary depending on whether it is an instrumental or a noninstrumental procedure. Forinstrumental procedures, a common approach is to measure the magnitude of analyticalbackground response by analyzing a number of blank samples and calculating thestandard deviation of this response Multiplying the standard deviation by a factor,usually 10, provides an estimate of the limit of quantitation. This limit is subsequentlyvalidated by the analysis of a suitable number of samples known to be close to or atthe limit of quantitation.
For noninstrumental methods, determine the limit of quantitation by analyzingsamples having known concentrations of analyte and by establishing the minimumlevel at which the analyte can be detected with acceptable accuracy and precision.
Linearity and RangeDefinition of Linearity The linearity of an analytical method is its ability (within
a given range) to elicit test results that are directly, or by a well-defined mathematicaltransformation, proportional to the concentration of analyte in samples within a givenrange. Linearity is usually expressed in terms of the variance around the slope ofthe regression line (correlation coefficient), calculated according to an establishedmathematical relationship from test results obtained by the analysis of samples withvarying concentrations of analyte.
Definition of Range The range of an analytical method is the interval betweenand including the upper and lower levels of analyte that have been demonstrated tobe determined with precision, accuracy, and linearity using the method as written.The range is normally expressed in the same units as test results (e.g., percent,milligrams per kilogram, parts per million) obtained by the analytical method.
Determination of Linearity and Range Determine the linearity of an analyticalmethod by mathematically treating test results obtained from analysis of samples withanalyte concentrations across the claimed range of the method. The treatment isnormally a calculation of a regression line by the method of least squares of testresults versus analyte concentrations. In some cases, to obtain proportionality betweenassays and sample concentrations, the test data may have to be subjected to a mathemat-ical transformation before the regression analysis. The slope of the regression lineand its variance (correlation coefficient) provide a mathematical measure of linearity;the y-intercept is a measure of the potential assay bias.
Validate the range of the method by verifying that the analytical method providesacceptable precision, accuracy, and linearity when applied to samples containinganalyte at the extremes of the range as well as within the range.
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RuggednessDefinition The ruggedness of an analytical method is the degree of reproducibility
of test results obtained by the analysis of the same samples under a variety of normaltest conditions, such as different laboratories, analysts, instruments, lots of reagents,elapsed assay times, assay temperatures, and days. Ruggedness is normally expressedas the lack of influence on test results of operational and environmental variables ofthe analytical method. Ruggedness is a measure of reproducibility of test results undernormal, expected operational conditions from laboratory to laboratory and from analystto analyst.
Determination Determine the ruggedness of an analytical method by analyzingaliquots from homogeneous lots in different laboratories, by different analysts, usingoperational and environmental conditions that may differ but still are within thespecified parameters of the assay. Determine the degree of reproducibility of testresults as a function of the assay variables. This reproducibility may be compared to theprecision of the assay under normal conditions to obtain a measure of the ruggedness ofthe analytical method.
RobustnessThe robustness of an analytical method is a measure of the method’s capacity toremain unaffected by small, but deliberate, variations in method parameters, and itprovides an indication of the method’s reliability during normal use.
Data Elements Required for Assay Validation
FCC assay procedures vary from highly exacting analytical determinations to subjec-tive evaluation of attributes. Considering this variety of assays, it is only logical thatdifferent test methods require different validation schemes. This section covers onlythe most common categories of assays for which validation data should be required.These categories are as follows:
Category I Analytical methods for quantitation of major components of foodadditives or processing aids (including preservatives).
Category II Analytical methods for determination of impurities in food additivesor processing aids. These methods include quantitative assays and limit tests.
Category III Analytical methods for determination of performance characteristics(e.g., solubility, melting point).
For each assay category, different analytical information is needed. In the followingtable, data elements that are normally required for each assay category are listed.
Already-established general assays and tests (e.g., titrimetric method of waterdetermination, identification test) should also be validated to verify their accuracy(and absence of possible interference) when used for a new product or raw material.
The validity of an analytical method can be verified only by laboratory studies.Therefore, documentation of the successful completion of such studies is a basicrequirement for determining whether a method is suitable for its intended applications.Appropriate documentation should accompany any proposal for new or revised com-pendial analytical procedures.
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Data Elements Required for Assay Validation
Assay Category IIAnalyticalPerformance Assay Limit AssayParameter Category I Quantitative Tests Category III
Accuracy Yes Yes * *Precision Yes Yes No YesSpecificity No Yes No *Limit of Detection Yes Yes Yes *Limit of Quantitation No No Yes *Linearity Yes Yes No *Range Yes Yes * *Ruggedness Yes Yes Yes Yes
*May be required, depending on the nature of the specific test.
GENERAL GOOD MANUFACTURING PRACTICES GUIDELINES FOR FOODCHEMICALS4
Food chemicals and other substances employed as adjuncts in foods and as aids in foodprocessing must meet recognized standards of performance and quality for their intendeduses and applications. The requirements contained in the monographs of the FCC pertainto the characteristics of food chemicals at the time of their use.
It is not sufficient, however, for an end product merely to meet the FCC requirements.Production of food-quality chemicals is best achieved by implementing procedures thatplace primary emphasis on preventing defects and deficiencies. Thus, a product must bemade and handled in a sanitary manner, in a way designed either to preclude the formationof undesirable by-products, or to ensure their adequate removal, as well as to preventcontamination, deterioration, mix-up and mislabeling, and the introduction of unusual orunexpected impurities.
Food chemicals are subject to applicable regulations promulgated by the responsiblegovernment agencies in countries in which FCC specifications are recognized. In theUnited States, for example, the pertinent regulations that deal primarily with sanitationare the ‘‘Current Good Manufacturing Practices in Manufacturing, Packing, or HoldingHuman Food.’’5
Beyond requirements related to sanitation, however, manufacturers, processors, packers,and distributors should establish and exercise other appropriate systems of controlsthroughout their operations, including food safety assurance systems such as HazardAnalysis and Critical Control Points (HACCP), where applicable, to ensure that FCCsubstances are safe and otherwise suitable for their intended use. These controls, togetherwith the regulations cited above, constitute ‘‘good manufacturing practices.’’ While the
4These guidelines are presented for information only and are not intended to be mandatory in any sense asregards compliance with FCC specifications.
5Code of Federal Regulations, Title 21, Part 110, which may be obtained from the Superintendent of Documents,U.S. Government Printing Office, Washington, D.C. 20402. Also, Parts 113 and 114 are of interest, particularlywith regard to record keeping.
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details of the application of the principles of good manufacturing practices to the manufac-turing, processing, packing, and distribution of food chemical substances will vary, thefundamental relevance of such principles at all stages of an operation should be recognized.
The principles of good manufacturing practices encompass such considerations as
T Systems of quality control and assurance, including self-auditing procedures.T Clearly defined responsibilities of supervisory and other personnel, all of whom mustbe qualified and adequately trained.T Design, operation and maintenance of buildings and equipment, with attention tohousekeeping, sanitation, pest control, prevention of contamination of product, cleaningof equipment, a calibration program for all instruments and gauges, and environmentallysatisfactory methods of waste disposal.T Documentation of validation studies pertaining to the manufacturing process, laboratorytest methods, and equipment and computer applications, when any such studies are appro-priate.T Written operational instructions that should include such items as
—General instructions and hazards.—Master manufacturing instructions.—Master packaging instructions.—Master specifications for raw materials, in-process materials, packaging materials,
labels, and finished products.—Laboratory test methods.—Control instrumentation and computer applications.—Labeling, holding, and distribution instructions.
T Handling and control, including the testing and approval, of raw materials, process aids,intermediates, and finished products.T Product containers, closures, and labeling (including the control of labels and labeling).T Laboratory and inspection controls and records (including the effect of process changes).T Reserve samples of raw materials and products.T Written records that contain essential operational data for each individual lot of foodchemical and that permit tracing the lot history from the raw materials through manufactur-ing, packaging, holding, and distributing the product.T Product stability and lifespan.T Systems for holding, evaluating, and disposing of rejected products and returned mate-rials.T Procedures for investigating complaints and taking appropriate corrective action.
Some food chemicals have uses other than as food chemicals—in fact, the food-gradematerial may be only a small part of production for industrial or other uses. In suchsituations, the principles of good manufacturing practices must apply, and particularattention must be paid to the suitability of the raw materials used; the prevention of cross-contamination; and the segregation of food chemicals from nonfood chemicals, includingmaterial in process, final product, and product in storage. The necessary controls to ensurethe above must be developed and implemented.
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Note: Depending on the processes used, it frequently is possible to divert the food-grade product from the main product stream as the final steps in producing a food-grade product are approached, and to complete the processing under conditionssuitable for food-grade substances. In such cases, if the diverted material can beadequately characterized by a knowledge of its history, and/or by appropriate analyti-cal testing, it may be considered to be the raw material for the food-grade product.
Biotechnology (processes involving the use of biological systems) is an importantsource of chemicals, enzymes, and other substances used in foods and in food processing.Some food ingredients have long been made by fermentation and by enzymatic processes,but now processes involving genetically modified organisms have become a prominentemerging source of such substances.
The manufacture of food chemicals, whether it involves chemical or biological synthesisand purification, or recovery from natural materials, has a number of characteristics thatmust be taken into account in establishing a system of good manufacturing practice. Forexample, in the production of many chemicals, recycling of process liquors and recoveryfrom waste streams are necessary for reasons of quality, economics, and environmentalprotection. In addition, the production of some food chemicals involves processes in whichchemical and biochemical mechanisms have not been fully elucidated, and thus the methodsand procedures for materials accountability usually will differ from those applicable tothe manufacture of other classes of materials.
Another aspect of good manufacturing practices for food chemicals relates to thepossible presence of objectionable impurities. While the limits and tests provided in theFCC are consistent with the information available to the committee regarding currentmethods of manufacture and common impurities that may be present, it obviously isimpossible to provide limits and tests in each FCC monograph for the detection of allpossible impurities because these may vary with the raw materials and the method ofprocessing used in making the chemical. Thus, to evaluate whether other undesirableimpurities may be present, the manufacturer should understand, to the best degree possiblefor the process at hand, the factors that contribute to the presence of impurities. Solventsas well as impurities in the raw materials and processing aids, all of which might carryinto the final product, must be considered. In synthetic processes, it is necessary similarlyto consider intermediates and the products of side reactions, as well as the possibleformation of isomeric compounds, including epimers and enantiomorphs.
The same general considerations apply to biological processes, be they traditional orbased on newer biotechnology. For products of biotechnology, a review that includesadequate characterization and documentation of the genetic origins of the starting materialsand the characteristics of the process provides the necessary guidance for identifying andsetting levels of undesirable impurities, which need to be controlled to suitable levels orto be absent altogether. Therefore, the active genetic components in the process (e.g.,culture, recombinant DNA) should be known, well characterized, and free from anypotential for introducing biologically significant levels of undesirable constituents (e.g.,toxins, antibiotics, antinutrients, allergens) that cannot be kept out of the final product bypreliminary processing. As in the production of all food chemicals, testing is appropriatewhen possible and particularly to demonstrate the absence of certain toxins and certainspecific DNA sequences.
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Because of the necessity to maintain the purity and integrity of the genetic materialsassociated with the biotechnological process, containment6 is a particularly importantconsideration in preventing cross-contamination as well as the inadvertent release ofbiologically active materials.
Exposure of all products used in foods and food processing to foreign material contami-nation must be prevented. If objectionable impurities from any source, other than thosecovered by FCC requirements, are suspected to be present, good manufacturing practicerequires the manufacturer to ensure that the substance is suitable for its intended applica-tions as a food chemical by applying additional tests and limits. Current analytical technol-ogy should be applied wherever possible.
6See NIH Guidelines for Research Involving Recombinant DNA Molecules, Federal Register, Vol. 51, No.88, pages 16957–16985, May 7, 1986. Copies, which include later revisions of the Guidelines, may be obtainedfrom the Office of Recombinant DNA Activities, National Institutes of Health, Building 31, Room 4B11,Bethesda, MD 20892.
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MONOGRAPHS ADDED TO THE FOOD CHEMICALS CODEX, FIFTH EDITION,FROM SUPPLEMENTS 1 THROUGH 3
Supplement 1 1,3,5-Undecatriene Pork CollagenCalcium Lignosulfonate Veratraldehyde Salatrimbeta-Cyclodextrin Solin OilDimethyl Dicarbonate Supplement 2 Soy Protein ConcentrateGlyceryl Palmitostearate L-Carnitine Sucrose Acetate Isobutyrate4-Hexylresorcinol ErythritolMagnesium Phosphate, Ferric Citrate Flavors
Dibasic, Mixed Hydrates Ferrous Citrate Acetaldehyde Diethyl AcetalManganese Citrate Maltitol 2-Acetyl ThiazoleOlestra Menhaden Oil, Hydrogenated Allyl Phenoxy AcetateSodium Lignosulfonate Menhaden Oil, Refined Allyl PropionateSucrose Fatty Acid Esters Sheanut Oil, Refined BorneolSugar Beet Fiber 2-sec-Butyl CyclohexanoneVitamin K Supplement 3 Butyl 2-Methyl ButyrateWhey Protein Concentrate Acidified Sodium Chlorite Diphenyl EtherWhey, Reduced Lactose Solutions d-FenchoneWhey, Reduced Minerals Aspartame-Acesulfame Salt Fenchyl AlcoholYeast, Autolyzed Curdlan Furfuryl Alcohol
gamma-Cyclodextrin 2-Furyl Methyl KetoneFlavors Polyglycerol Polyricinoleic�-Pentadecalactone Acid
NEW MONOGRAPHS IN THE FOOD CHEMICALS CODEX, FIFTH EDITION
Allura Red Whey Protein Isolate Maltol IsobutyrateArabinogalactan 2-Methoxy 3-(or 5- or 6-)Bohenin Flavors Isopropyl PyrazineBrilliant Blue 2,6-Dimethoxy Phenol 5H-5-Methyl-6,7-Butadiene-Styrene Rubber 3,4-Dimethyl 1,2- dihydrocyclopenta[b]pyrazineCurdlan Cyclopentandione 5-Methyl FurfuralErythrosine 5-Ethyl 3-Hydroxy 4-Methyl Methyl FuroateFast Green 2(5H)-Furanone Methyl HexanoateFerrous Glycinate 3-Ethyl Pyridine Methyl IsovalerateHydrogenated Starch Furfuryl Mercaptan 5-Methyl 2-Phenyl 2-Hexenal
Hydrolysates Geranyl Isovalerate Methyl ThiobutyrateIndigotine 2,3-Heptandione Methyl ValerateSunset Yellow (Z)-3-Hexenyl Butyrate �-Naphthyl Ethyl EtherTartrazine (Z)-3-Hexenyl Formate Phenyl Ethyl CinnamateTransglutaminase Hexyl Butyrate Phenyl Ethyl PropionateTrehalose Hexyl Hexanoate Propyl FormateVegetable Oil Phytosterol Isoamyl Isobutyrate Propyl Mercaptan
Esters Isobutyl Formate SalicylaldehydeWheat Protein Isolate Isobutyl Hexanoate �-TetradecalactoneWhey Mineral Concentrate Linalool Oxide 2-Tridecanone
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FORMER AND CURRENT TITLES OF FOOD CHEMICALS CODEXMONOGRAPHS
Fourth Edition Title Fifth Edition Title
Acacia Gum ArabicAmmonium Hydroxide Ammonia SolutionDL-�-Tocopherol All-rac-�-TocopherolD-�-Tocopherol Concentrate RRR-�-TocopherolTocopherols Concentrate, Mixed RRR-Tocopherols Concentrate, MixedD-�-Tocopheryl Acetate RRR-�-Tocopheryl AcetateDL-�-Tocopheryl Acetate All-rac-�-Tocopheryl AcetateD-�-Tocopheryl Acetate Concentrate RRR-�-Tocopheryl Acetate ConcentrateD-�-Tocopheryl Acid Succinate RRR-�-Tocopheryl Acid Succinate
MONOGRAPHS COMBINED
Fourth Edition Title Fifth Edition Title
Butadiene-Styrene 50/50 Rubber Butadiene-Styrene RubberButadiene-Styrene 75/25 Rubber
Sodium Acetate Sodium AcetateSodium Acetate, Anhydrous
Contents
PREFACE . . . xiii
GENERAL INFORMATION . . . xixOperating Procedures of the Food Chemicals Codex . . . xixValidation of Food Chemicals Codex Methods . . . xxivGeneral Good Manufacturing Practices Guidelines for Food Chemicals. . . xxixLists of New Monographs and Former and Current Titles . . . xxxiii
Section
1 GENERAL PROVISIONS AND REQUIREMENTS APPLYING TOSPECIFICATIONS, TESTS, AND ASSAYS OF THE FOODCHEMICALS CODEX . . . 1
2 MONOGRAPH SPECIFICATIONS . . . 9
3 FLAVOR CHEMICALS . . . 515Specifications for Flavor Chemicals (table) . . . 517Test Methods for Flavor Chemicals . . . 630Gas Chromatographic (GC) Assay of Flavor Chemicals . . . 635
4 INFRARED SPECTRA . . . 637
xi
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5 GENERAL TESTS AND ASSAYS . . . 827Appendix I: Apparatus for Tests and Assays . . . 831Appendix II: Physical Tests and Determinations . . . 834
A. Chromatography . . . 834B. Physicochemical Properties . . . 841C. Others . . . 854
Appendix III: Chemical Tests and Determinations . . . 859A. Identification Tests . . . 859B. Limit Tests . . . 861C. Others . . . 876
Appendix IV: Chewing Gum Base . . . 892Appendix V: Enzyme Assays . . . 896Appendix VI: Essential Oils and Flavors . . . 929Appendix VII: Fats and Related Substances . . . 934Appendix VIII: Oleoresins . . . 944Appendix IX: Rosins and Related Substances . . . 947Appendix X: Carbohydrates (Starches, Sugars, and RelatedSubstances) . . . 951
Solutions and Indicators . . . 962
INDEX . . . 979
1/General Provisions andRequirements Applying toSpecifications, Tests, and Assaysof the Food Chemicals Codex
The General Provisions provide, in summary form, the basicpolicies and guidelines for the interpretation and applicationof the standards, tests, assays, and other specifications of theFood Chemicals Codex and make it unnecessary to repeatthroughout the book those requirements that are pertinent innumerous instances.
Where exceptions to the General Provisions are made, thewording in the individual monograph or general test chaptertakes precedence and specifically indicates the directions orthe intent.
TITLE OF BOOK
The title of this book, including supplements thereto issuedseparately, is the Food Chemicals Codex, Fifth Edition. Itmay be abbreviated to FCC V.
Where the term ‘‘Codex’’ is used without further qualifica-tion in the text of this book, it applies to the Food ChemicalsCodex, Fifth Edition.
INQUIRIES
Inquiries regarding any aspect of the operation of the FoodChemicals Codex may be directed to the Food ChemicalsCodex, Institute of Medicine, 500 Fifth Street, N.W., Wash-ington, D.C. 20001.
CODEX SPECIFICATIONS
Food Chemicals Codex specifications, comprising the De-scription, Requirements, and Tests, are presented in mono-graph form (Section 2) or tabular form (Section 3) for eachsubstance or group of related substances. They are designedto ensure that food additives have a sufficiently high level of
1
quality to be safe under usual conditions of intentional usein foods, both directly or indirectly, or in food processing.Thus, FCC specifications generally represent acceptable levelsof quality and purity of food-grade ingredients available inthe United States (or in other countries in which FCC specifi-cations are recognized).
The titles of FCC monographs are in most instances thecommon or usual names. The FCC specifications applyequally to substances bearing the main titles, synonyms listedunder the main titles, and names derived by transpositionof definitive words in main titles. The Committee on FoodChemicals Codex recognizes that the nomenclature used forflavor chemicals may not be consistent with other authoritativesources.
Although the assays and tests described constitute methodsupon which the specifications of the Food Chemicals Codexdepend, analysts are not prevented from applying alternativemethods if they are satisfied that the procedures used willproduce results of equal or greater accuracy. In the event ofdoubt or disagreement concerning a substance purported tocomply with the requirements of this Codex, only the methodsdescribed herein are applicable and authoritative.
POLICIES AND GUIDELINES
General Policy It is the policy of the Codex to set maximumlimits for trace impurities wherever they are deemed to beimportant for a particular food chemical, and they shall be setat levels consistent with food safety and good manufacturingpractice.
Maximum limits for inorganic trace impurities (e.g., arse-nic, cadmium, fluoride, lead, mercury, selenium) will be in-cluded in any monographs for which consumer safety or manu-facturing experience indicates their desirability. No limits for
2 / General Provisions and Requirements FCC V
arsenic, lead, and other heavy metals are required for flavorchemicals because of the very low levels at which these sub-stances are added to foods.
All requests to increase limits shall be considered on thebasis of the toxicological risk involved, the principles of goodmanufacturing practice, and the availability of the same sub-stances from other sources that meet the FCC limits inquestion.
Added Substances (Policy) FCC specifications are in-tended for application to individual substances (single entities)and not to proprietary blends or other mixtures. Some specifi-cations, however, allow ‘‘added substances’’ (i.e., functionalsecondary ingredients such as anticaking agents, antioxidants,diluents, emulsifiers, and preservatives) intentionally addedwhen necessary to ensure the integrity, stability, utility, orfunctionality of the primary substance in commercial use.
If an FCC monograph allows such additions, each addedsubstance must meet the following requirements: (1) it isapproved for use in foods by the U.S. Food and Drug Adminis-tration or by the responsible government agency in othercountries in which FCC specifications are recognized; (2) itis of appropriate food-grade quality and meets the require-ments of the Food Chemicals Codex, if listed therein; (3) itis used in an amount not to exceed the minimum required toimpart its intended technical effect or function in the primarysubstance; (4) its use will not result in concentrations ofcontaminants exceeding permitted levels in any food as aconsequence of the affected FCC primary substance’s beingused in food; and (5) it does not interfere with the assay andtests prescribed for determining compliance with the FCCrequirements for the primary substance, unless the monographfor the primary substance has provided for such interferences.
Where added substances are specifically permitted in anFCC substance, the label shall state the name(s) and amount(s)of any added substance(s).
Adding substances not specifically provided for and men-tioned by name of function in the monograph of an FCCsubstance will cause the substance to no longer be designatedas an FCC substance. Such a combination is a mixture to bedescribed by disclosure of its ingredients, including any thatare not FCC substances.
Allergens The Committee on Food Chemicals Codex recog-nizes the issue of food allergens, but current limitations regard-ing (1) the threshold levels and (2) the analytical methods todetect allergens at very low levels have thus far preventedthe inclusion in FCC monographs of specifications related toallergens.
Arsenic Specifications (Policy) Arsenic specifications willbe included in monographs only when there is specific reasonfor the committee to believe that arsenic constitutes a likelycontaminant in the substance in question.
Fluoride Limits (Guideline) The Committee on FoodChemicals Codex has established limits for fluoride in numer-ous monographs. For phosphates, this reflects the natural oc-currence of fluoride in the inorganic phosphate starting mate-rial. Fluoride limits in other monographs may reflect thenatural occurrence of fluoride in the article or in reagents
used in food additive manufacture.Following issuance of the Fluoride Limits (Guideline) in
earlier editions of the Food Chemicals Codex, considerableresearch has been completed demonstrating the cariostatic—caries preventing—properties of fluoride. Fluoride is nowadded to many municipal water supplies to provide a levelof 1 mg/L, and many dental products are formulated with it.Fluoride is in the formulation of many over-the-counter dietarysupplements as well.
During and after the time the Food Chemicals Codex,Fourth Edition, was in preparation, the Standing Committeeon the Scientific Evaluation of Dietary Reference Intakes,Food and Nutrition Board, Institute of Medicine, The NationalAcademies, completed a comprehensive review of fluoride(IOM, 1997).1 The committee reviewed the literature, notedthe toxicological manifestations of fluoride in animals andhumans, and established a general No Observed Adverse Ef-fect Level (NOAEL) of 10 mg/day. As fluoride has the abilityto induce fluorosis—mottling of the primary teeth of youngchildren—an upper limit (UL) of 0.7 mg/day was establishedfor infants, with increasing ULs assigned to older children.In addition, an Adequate Intake (AI) of 3 mg/day was assignedfor adult females and of 4 mg/day for adult males, with theUL for both being 10 mg/day.
The intake of fluoride as a constituent of substances de-scribed in FCC monographs, even at the maximum limitsestablished for fluoride, is not expected to significantly addto the human daily fluoride intake from other sources and iswell within the various limits described in the Institute ofMedicine’s committee report. Nonetheless, given that toxico-logical manifestations have been amply demonstrated for fluo-ride, as described in the report, the maintenance of fluoridelimits in drinking water and food, and thus food additives,appears consistent with sound public health policy. Therefore,the Committee on Food Chemicals Codex considers that main-taining fluoride limits for relevant food additives and ingredi-ents is justified.
Because of the difficulties in analyzing for fluoride in foodchemicals, the committee intends to adopt new analyticalmethods for fluoride as soon as adequate validation is submit-ted. Furthermore, in view of the considerable variation influoride limits for additives and ingredients in various nationaland international compendia, the committee deems harmoni-zation of fluoride limits between the FCC and other compendiato be desirable.
FCC Substances Containing Sulfiting Agents (Policy) Ifan FCC substance contains 10 mg/kg or more of any sulfitingagent, the presence of such sulfiting agent shall be indicatedon the labeling.
Labeling (Policy) For purposes of compliance with FoodChemicals Codex monographs, ‘‘labeling’’ means all labelsand other written, printed, or graphic matter (1) on any articleor any of its containers or wrappers or (2) accompanying sucharticle, or otherwise provided by vendors to purchasers for
1IOM (Institute of Medicine). 1997. Dietary Reference Intakes forCalcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington,DC: National Academy Press.
FCC V General Provisions and Requirements / 3
purposes of product identification.In addition to FCC labeling requirements, substances in-
cluded in the Food Chemicals Codex are subject to compliancewith such labeling requirements as may be promulgated bygovernment bodies. Such substances are intended for use infoods, either directly or indirectly, and in food processing.
The name of the substance on a container label, plus thedesignation ‘‘Food Chemicals Codex Grade,’’ ‘‘FCC Grade,’’or simply ‘‘FCC,’’ is a representation by the manufacturer,vendor, or user of the substance that at the time of shipment,it conforms to the specifications in FCC V, including itssupplements that are current at that time.
When an FCC substance is available commercially in solu-tion form or as a component of a mixture, and there is noprovision in the Food Chemicals Codex for such solution ormixture, the manufacturer, vendor, or user may indicate onthe label that the product contains substances meeting FCCspecifications by use of the initials ‘‘FCC’’ after the nameof those components that meet the FCC requirements.
For the labeling of FCC substances in which added sub-stances are permitted, see Added Substances (Policy), above.
For the labeling of FCC substances that contain 10 mg/kgor more of a sulfiting agent, see FCC Substances ContainingSulfiting Agents (Policy), above.
Heavy Metals Limits (Policy) The Committee on FoodChemicals Codex notes the importance of providing limitsfor individual heavy metals as required by the source andcomposition of individual food additives. Thus, it has decidedto remove from most monographs the general heavy metals(as lead) limits and tests and, based on the current level andavailability of scientific information and on the policy statedbelow, to replace them with limits and tests for specific heavymetals such as lead, cadmium, and mercury as may be relevantto each substance.
The committee recognizes the desirability of lowering ex-posure to lead and other heavy metals, especially in the caseof infants and children. Overall exposure to heavy metals ingeneral, and to lead in particular, is a public health concern.Although diet is not the largest source of lead exposure, it isa significant one. While ingestion of FCC substances doesnot represent the major source of dietary lead, it is desirableto lower the lead limits for all FCC substances, particularlyfor those substances consumed in high amounts. Therefore,the committee’s policy is to reduce lead and other heavymetals limits to the lowest extent feasible, especially giventhat more recent evidence shows deleterious neurobehavioraleffects occurring in children exposed to lead at levels belowthose previously considered acceptable.
In setting limits for lead and other heavy metals, the com-mittee considers the amount of a food chemical consumed,the feasibility of manufacturing a product within these limits,and the availability of analytical methods to ensure compli-ance. The constraints of good manufacturing practice and theavailability of reliable analytical methods are often limitingfactors in setting lower limits for lead and other heavy metals.
The committee regards as one of its goals the assurance ofthe safety of properly used food chemicals. This means that
FCC specifications will respond to advances in knowledgeabout new manufacturing methods, analytical techniques, ortoxicology and safety issues.
Microbiological Attributes (Policy) Manufacturers, ven-dors, and users of FCC substances are expected to exercisegood manufacturing practices (GMPs) and to establish foodsafety assurance systems such as Hazard Analysis and CriticalControl Points (HACCP) to ensure that FCC substances aresafe and otherwise suitable for their intended use. FCC sub-stances are expected to meet applicable regulatory require-ments, including microbiological criteria, for safety and qual-ity. According to Codex Alimentarius recommendations forestablishing and applying microbiological criteria for foods,‘‘Mandatory (regulatory) microbiological criteria shall applyto those products and/or points of the food chain where noother more effective tools are available, and where they areexpected to improve the degree of protection to the consumer.Where these are appropriate they shall be product-type specificand only applied at the point of the food chain specified inthe regulation.’’ In addition, businesses may develop microbi-ological criteria for specific food additives or ingredients,processes, and products. The General Policy for microbiologi-cal safety and quality of FCC substances is that such sub-stances be produced, handled, and used in food processingfollowing GMPs and applicable food safety systems. There-fore, the FCC does not list specific microbiological criteriafor FCC substances other than those for which scientificallyvalid data are available to the committee that support theneed for such criteria. In such cases, the Codex Alimentariusprinciples for establishing and applying microbiological crite-ria have been followed.2 These principles include the fol-lowing:
1. Microbiological criteria should be established and appliedonly where there is a definite need and their applicationis practical.
2. Consideration is given toi. the evidence of actual or potential hazards to health;ii. the microbiological status of raw material(s);iii. the effect of processing on the microbiological status
of the ingredient or food additive;iv. the likelihood and consequences of microbial contami-
nation and/or growth during subsequent handling, stor-age, and use;
v. the category(ies) of consumers concerned; andvi. the intended use of the ingredient or food additive.
3. The sampling plan, method, and handling are stated.4. The microorganism(s) included in the criteria is (are)
widely accepted as relevant to the particular ingredient orfood additive—as pathogen(s), as indicator organism(s),or as spoilage organism(s).
5. Limits used in the criteria are based on microbiologicaldata appropriate to the ingredient or food additive andother similar substances.
2Codex Alimentarius Commission. 1997. Joint FAO/WHO Food Stan-dards Programme, Codex Committee on Food Hygiene, Supplement toVolume 1B-1997. Principles for the Establishment and Application ofMicrobiological Criteria for Foods. CAC/GL 21-997. Pp. 47–54.
4 / General Provisions and Requirements FCC V
Mg/Kg and Percent (Policy) Beginning with the SecondSupplement to FCC III, to bring the Codex into concordancewith current nomenclature as used in analytical chemistry,the term ‘‘ppm’’ [parts per million (by weight)] was replacedby ‘‘mg/kg’’ (milligrams per kilogram).
The term ‘‘mg/kg’’ is used for expressing the concentrationsof trace amounts of substances, such as impurities, up to 10mg/kg. Above 10 mg/kg, percent (by weight) is used. Forexample, a monograph requirement equivalent to 20 mg/kgis expressed as 0.002%, or 0.0020%, depending on the numberof significant figures justified by the test specified for use inconjunction with the requirement.
ATOMIC WEIGHTS AND CHEMICAL FORMULAS
The atomic weights used in computing formula weights andvolumetric and gravimetric factors stated in tests and assaysare those recommended in 1991 by the IUPAC Commissionon Isotopic Abundances and Atomic Weights.
Molecular and structural formulas and formula weightsimmediately following titles are included for the purpose ofinformation and are not to be considered an indication of thepurity of the substance. Molecular formulas given in specifica-tions, tests, and assays, however, denote the pure chemicalentity.
ASSAYS AND TESTS
Every FCC substance in commerce, when tested in accordwith these assays and tests, meets all of the requirements inthe monograph defining it.
Many materials in concentrated forms, whether to be usedin food or as test reagents, are a skin or respiratory irritantor are otherwise toxic. Use caution when handling these mate-rials.
Analytical Samples In the description of assays and tests,the approximate quantity of the analytical sample to be usedis usually indicated. The quantity actually used, however,should not deviate by more than 10% from that stated.
Some substances must be dried before a sample is takenfor an assay or test. When a Loss on Drying or Water test isspecified, the undried substance may be used and the resultscalculated on the dried basis, provided that any moisture orother volatile matter in the undried sample does not interferewith the specified assay and test procedures.
The word ‘‘accurately,’’ used in connection with gravimet-ric or volumetric measurements, means that the operationshould be carried out within the limits of error prescribedunder Volumetric Apparatus or Weights and Balances, Appen-dix I. The same significance also applies to the term ‘‘exactly’’or quantitative expressions such as ‘‘100.0 mL’’ or ‘‘50.0mg.’’
The word ‘‘transfer,’’ when used in describing assays andtests, means that the procedure should be carried out quantita-tively.
Apparatus With the exception of volumetric flasks andother exact measuring or weighing devices, directions to usea definite size or type of container or other laboratory appara-
tus are intended only as recommendations, unless otherwisespecified.
Where an instrument for physical measurement, such as athermometer, spectrophotometer, or gas chromatograph, isdesignated by its distinctive name or tradename in a test orassay, a similar instrument of equivalent or greater sensitivityor accuracy may be employed.
Where low-actinic or light-resistant containers are speci-fied, clear glass containers that have been rendered opaqueby application of a suitable coating or wrapping may be used.
Blank Tests Where a blank determination is specified in atest or assay, it is to be conducted using the same quantitiesof the same reagents and by the same procedure repeated inevery detail except that the substance being tested is omitted.
A residual blank titration may be stipulated in assays andtests involving a back titration in which a volume of a volumet-ric solution larger than is required to react with the sampleis added, and the excess of this solution is then titrated witha second volumetric solution. Where a residual blank titrationis specified or where the procedure involves such a titration,a blank is run as directed in the preceding paragraph. Thevolume of the titrant consumed in the back titration is thensubtracted from the volume required for the blank. The differ-ence between the two, equivalent to the actual volume con-sumed by the sample, is the corrected volume of the volumetricsolution to be used in calculating the quantity of the substancebeing determined.
Centrifuge Where the use of a centrifuge is indicated, unlessotherwise specified, the directions are predicated upon theuse of apparatus having an effective radius of about 20 cm(8 in.) and driven at a speed sufficient to clarify the supernatantlayer within 15 min. If necessary, determine the gravity byusing the equation
g = {[(rpm × 2 × �)/60] × rm}/980,
in which rpm is the rotor speed and rm is the mean radius,in centimeters, of the tube holding the sample in the rotor.
Desiccators and Desiccants The expression ‘‘in a desicca-tor’’ means using a tightly closed container of appropriatedesign in which a low moisture content can be maintainedby means of a suitable desiccant. Preferred desiccants includeanhydrous calcium chloride, magnesium perchlorate, phos-phorus pentoxide, and silica gel.
Filtration Where it is directed to ‘‘filter,’’ without furtherqualification, the intent is that the liquid be filtered throughsuitable filter paper or an equivalent device until the filtrateis clear.
Identification The tests described under this heading inmonographs are designed for application to substances takenfrom labeled containers and are provided only as an aid tosubstantiate identification. These tests, regardless of theirspecificity, are not necessarily sufficient to establish proof ofidentity, but failure of a substance taken from a labeled con-tainer to meet the requirements of a prescribed identificationtest means that it does not conform to the requirements ofthe monograph.
FCC V General Provisions and Requirements / 5
Indicators The quantity of an indicator solution used shouldbe 0.2 mL (approximately 3 drops) unless otherwise directedin an assay or test.
Negligible The term ‘‘negligible,’’ as used in some Residueon Ignition specifications, indicates a quantity not exceeding0.5 mg.
Odorless This term, when used in describing a flavoringmaterial, applies to the examination, after exposure to air for15 min, of about 25 g of the material that has been transferredquickly from the original container to an open evaporatingdish of about 100-mL capacity. If the package contains 25 gor less, the entire contents should be examined.
Pressure Measurements The term ‘‘mm Hg’’ used withrespect to pressure within an apparatus, or atmospheric pres-sure, refers to the use of a suitable manometer or barometercalibrated in terms of the pressure exerted by a column ofmercury of the stated height.
Reagents Specifications for reagents are not included in theFood Chemicals Codex. Unless otherwise specified, reagentsrequired in tests and assays should conform to the specifica-tions of the current editions of Reagent Chemicals—AmericanChemical Society Specifications or in the section on ReagentSpecifications in the United States Pharmacopeia. Reagentsnot covered by any of these specifications should be of agrade suitable to the proper performance of the method ofassay or test involved.
Acids and Ammonium Hydroxide When ammonium hy-droxide, glacial acetic acid, hydrochloric acid, hydrofluoricacid, nitric acid, phosphoric acid, or sulfuric acid is calledfor in tests and assays, reagents of ACS grade and strengthsare to be used. (These reagents sometimes are called ‘‘concen-trated,’’ but this term is not used in the Food ChemicalsCodex.)
Alcohol, Ethyl Alcohol, Ethanol When one of these sub-stances is called for in tests and assays, use ACS-grade EthylAlcohol (95%).
Alcohol Absolute, Anhydrous Alcohol, Dehydrated Alco-hol When one of these substances is called for in tests andassays, use ACS-grade Ethyl Alcohol, Absolute.
Water When water is called for in tests and assays andin the preparation of solutions, it shall have been preparedby distillation, ion-exchange treatment, or reverse osmosis.
Water, Carbon Dioxide-Free When this type of water iscalled for, it shall have been boiled vigorously for 5 min ormore, and allowed to cool while protected from absorptionof carbon dioxide from the atmosphere. ‘‘Deaerated water’’is water that has been treated to reduce the content of dissolvedair by suitable means, such as by boiling vigorously for 5min and cooling or by the application of ultrasonic vibration.
Note: Certain chemical reagents specified in FCC testprocedures may be considered to be hazardous or toxicby the Occupational Safety and Health Administration,by the Environmental Protection Agency (under provi-sions of the Toxic Substances Control Act), or by healthauthorities in other countries in which the Food Chemi-cals Codex is recognized. In preparing this edition, the
Committee on Food Chemicals Codex has attemptedto specify use of different reagents where suitable sub-stitutes are known. For some procedures, however, theoriginal chemicals have been retained due to the lackof information on suitable substitutes. In such cases, theanalyst is encouraged to investigate the use of suitablesubstitute reagents, as appropriate, and to inform thecommittee of the results so obtained.
The methods and analytical procedures described in theCodex are designed for use by properly trained personnelin a suitably equipped laboratory. In common with manylaboratory procedures, the methods quoted frequently involvehazardous materials.
In performing the assay or test procedures in the Codex,safe laboratory practices must be followed. This includes theuse of precautionary measures, protective equipment, andwork practices consistent with the chemicals and proceduresused. Before undertaking any assay or procedures describedin this compendium, the individual should be aware of thehazards associated with the chemicals and of the proceduresand means of protecting against them. Material Safety DataSheets, which contain precautionary information related tosafety and health concerns, are available from manufacturersand distributors of many chemicals and should provide helpfulinformation about the safe use of such chemicals.
Reference Standards Some instrumental and chromato-graphic tests and assays specify the use of a reference standard.Where a reference standard is designated as USP, it may beobtained from the United States Pharmacopeia, 12601 Twin-brook Parkway, Rockville, MD 20852 <http://www.usp.org>.Where a reference standard is designated as a NIST (NationalInstitute of Standards and Technology) Standard ReferenceMaterial, it may be obtained from the Standard Reference Mate-rials Program, NIST, 100 Bureau Drive, Stop 2322, Gaithers-burg, MD 20899-2322 <http://ts.nist.gov/ts/htdocs/230/232/232.htm>.
To serve its intended purpose, each reference standard mustbe properly stored, handled, and used. Generally, referencestandards should be stored in their original containers awayfrom heat and protected from light. Follow any special instruc-tions accompanying the containers.
Assay and test results are determined on the basis of com-parison of the test sample with the reference standard thathas been freed from or corrected for volatile residues or watercontent as instructed on the reference standard label. If areference standard is required to be dried before use, transfera sufficient amount to a clean, dry vessel. Do not use theoriginal container as the drying vessel, and do not dry areference standard repeatedly at temperatures above 25°.Where the titrimetric determination of water is required atthe time a reference standard is to be used, proceed as directedin the Karl Fischer Titrimetric Method under Water Determi-nation, Appendix IIB.
Unless a reference standard label bears a specific potencyor content, assume the reference standard is 100.0% pure.
Significant Figures When tolerance limits are expressednumerically, the values are significant to the number of digits
6 / General Provisions and Requirements FCC V
indicated. Record the observed or calculated analytical resultwith only one digit included in the decimal place to the rightof the last place in the limit expression. If this digit is smallerthan 5, eliminate it and leave the preceding digit unchanged.If this digit is greater than 5, eliminate it and increase thepreceding digit by one. If this digit equals 5, eliminate it andincrease the preceding digit by one. For example, a require-ment of not less than 96.0% would not be met by a result of95.94%, but would be met by results of 95.96% or 95.95%,both of which would be rounded to 96.0%. When a range isstated, the upper and lower limits are inclusive so that therange consists of the two values themselves, properly rounded,and all intermediate values between them.
Solutions Prepare all solutions, unless otherwise specified,with water prepared by distillation, ion-exchange treatment,reverse osmosis, or as otherwise indicated in the monograph.
Such expressions as ‘‘1:10’’ or ‘‘10%’’ mean that 1 partby volume of a liquid or 1 part by weight of a solid is to bedissolved in a volume of the diluent or solvent sufficient tomake the finished solution 10 parts by volume. Directions forthe preparation of colorimetric solutions (CS), test solutions(TS), and volumetric solutions (VS), are provided in the sec-tion on Solutions and Indicators under General Tests andAssays, following Appendix X.
Prepare a volumetric solution to have a normality (molarity)within 10% of the stated value and to be standardized to foursignificant figures. When volumetric equivalence factors areprovided in tests and assays, the term ‘‘0.X N (M)’’ is under-stood to mean a VS having a normality (molarity) of exactly0.X000 N (M). If the normality (molarity) of the VS employedin a particular procedure differs from 0.X000, apply an appro-priate correction factor.
Specific Gravity Numerical values for specific gravity, un-less otherwise noted, refer to the ratio of the weight of asubstance in air at 25° to that of an equal volume of waterat the same temperature. Determine specific gravity by anyreliable method, unless otherwise specified.
Temperatures Unless otherwise specified, temperatures areexpressed in centigrade (Celsius) degrees, and all measure-ments are to be made at 25°, unless otherwise directed.
Test Solutions See Solutions and Indicators under GeneralTests and Assays, following Appendix X.
Time Limits Unless otherwise specified, allow 5 min fora reaction to take place when conducting limit tests for traceimpurities such as chloride or iron.
Expressions such as ‘‘exactly 5 min’’ mean that the statedperiod should be accurately timed.
Tolerances The minimum purity tolerances specified forFCC items have been established with the expectation thatthe substances to which they apply will be used as direct orindirect food additives, ingredients, or food-processing aids.These tolerance limits should neither bar the use of lots ofarticles that more nearly approach 100% purity nor constitutea basis for a claim that such lots exceed the quality prescribedby the Food Chemicals Codex.
When a maximum assay tolerance is not given, the assayshould show the equivalent of not more than 100.5%.
Trace Impurities Tests for inherent trace impurities areprovided to limit such substances to levels that are consistentwith good manufacturing practice and that are safe and other-wise unobjectionable under conditions in which the food addi-tive or ingredient is customarily employed.
It obviously is impossible to provide limits and tests ineach monograph for the detection of all possible unusualor unexpected impurities, the presence of which would beinconsistent with good manufacturing practice. The limits andtests provided are those considered to be necessary accordingto currently recognized methods of manufacture and are basedon information available to or provided to the Committee onFood Chemicals Codex. If other methods of manufacture orother than the usual raw materials are used, or if other possibleimpurities may be present, additional tests may be requiredand should be applied, as necessary, by the manufacturer,vendor, or user to demonstrate that the substance is suitablefor its intended application.
Vacuum The unqualified use of the term ‘‘in vacuum’’means a pressure at least as low as that obtainable by anefficient aspirating water pump (not higher than 20 mm Hg).
Water and Loss on Drying In general, for compoundscontaining water of crystallization or adsorbed water, a limittest, to be determined by the Karl Fischer Titrimetric Method,is provided under the heading Water. For compounds in whichthe loss on drying may not necessarily be attributable to water,a limit test, to be determined by other methods, is providedunder the heading Loss on Drying.
Weighing PracticesConstant Weight A direction that a substance is to be
‘‘dried to constant weight’’ means that the drying shouldcontinue until two consecutive weighings differ by not morethan 0.5 mg/g of sample taken, the second weighing to followan additional hour of drying.
The direction ‘‘ignite to constant weight’’ means that theignition should be continued at 800° � 25°, unless otherwisespecified, until two consecutive weighings do not differ bymore than 0.5 mg/g of sample taken, the second weighing tofollow an additional 15 min of ignition.
Tared Container When a tared container, such as a glassfiltering crucible, a porcelain crucible, or a platinum dish, iscalled for in an analytical procedure, it shall be treated as isspecified in the procedure. For example, dried or ignited fora specified time, or to constant weight, cooled in a desiccatoras necessary, and weighed accurately.
Weights and Measures, Symbols and Abbreviations TheInternational System of Units (SI), to the extent possible, isused in most specifications, assays, and tests in this FoodChemicals Codex. The SI metric units, and other units andabbreviations commonly employed, are as follows:
° = degrees centigradekg = kilogramg = gram
FCC V General Provisions and Requirements / 7
mg = milligram�g = microgramng = nanogrampg = picogramL = litermL = milliliter�L = microliterm = metercm = centimeterdm = decimetermm = millimeter�m = micrometer (0.001 mm)nm = nanometerC = coulombA = ampereV = voltmV = millivoltW = wattdc = direct currentft = footin. = inchin.3 = cubic inchgal = gallonlb = poundoz = ounceppm = parts per million (106) partsmg/kg = parts per million (by weight)ppb = parts per billion (109) partsng/kg = parts per billion (by weight)psi = pounds per square inchsp. gr. = specific gravityb.p. = boiling pointm.p. = melting pointid = inside diameterod = outside diameterh = hourmin = minutes = second% = percentN = normalityM = molarity�M = micromolar�mol = micromoleACS = American Chemical SocietyAOAC = AOAC InternationalAOCS = American Oil Chemists SocietyASTM = American Society for Testing and MaterialsCAS = Chemical Abstracts ServiceCFU = colony-forming unit(s)FDA = United States Food and Drug AdministrationFEMA = Flavor and Extract Manufacturers AssociationINS = International Numbering SystemNIST = National Institute of Standards and TechnologyUSP = United States Pharmacopeia
GENERAL SPECIFICATIONS AND STATEMENTS
Certain specifications and statements in the monographs of theFood Chemicals Codex are not amenable to precise descriptionand accurate determination within narrow limiting ranges.Because of the subjective or general nature of these specifica-tions, good judgment, based on experience, must be used ininterpreting and attaching significance to them. Specificationsor statements that are most likely to cause doubt are discussedin the subsequent paragraphs.
Description The material given under this heading in mono-graphs is provided for general information. It includes a de-scription of physical characteristics such as color and formand information on stability under certain conditions of expo-sure to air and light. Statements in this section may also coverapproximate indications of properties such as solubility (seebelow) in various solvents, pH, melting point, and boilingpoint, with numerical values modified by ‘‘about,’’ ‘‘approxi-mately,’’ ‘‘usually,’’ and other comparable nonspecific terms.These characteristics and statements are not requirements, butare provided as information that may assist with the overallevaluation of a food chemical. As the committee revises ex-isting monographs and develops new ones, it will, as appro-priate, include information about why certain specificationsand limits are given.
Solubility Statements included in the Requirements sectionof a monograph under a heading such as Solubility in Alcoholexpress exact requirements and constitute quality specifica-tions.
Statements relating to solubility given in the Description,however, are intended as information regarding approximatesolubilities only and are not to be considered as Codex-qualityrequirements. Such statements are considered to be of minorsignificance as a means of identification or determination ofpurity. For those purposes, dependence must be placed uponother specifications.
Approximate solubilities are indicated by the followingdescriptive terms:
Parts of SolventRequired for 1 Part
Descriptive Term of Solute
Very Soluble less than 1Freely Soluble from 1 to 10Soluble from 10 to 30Sparingly Soluble from 30 to 100Slightly Soluble from 100 to 1000Very Slightly Soluble from 1000 to 10,000Practically Insoluble or Insoluble more than 10,000
Soluble substances, when brought into solution, may showslight physical impurities, such as fragments of filter paper,fibers, and dust particles. Unless excluded by definite testsor other requirements; however, significant amounts of blackspecks, metallic chips, glass fragments, or other insolublematter are not permitted.
8 / General Provisions and Requirements FCC V
Function A statement of function is provided in each mono-graph to indicate the principal applications or technical effectsof the substance in foods or in food processing. The statementis not intended to limit in any way the choice or use of thesubstance or to indicate that it has no other utility.
In preparing the Fifth Edition, the committee considered anumber of new monographs describing substances that may,following ingestion, provide certain health benefits. The com-mittee noted that these substances are intended to exhibit afunctional effect, not on the foods to which they are added,but on the human body, in a manner similar to that of somenutrients, when that food is consumed. They also noted thatthese substances are products of an emerging science and thata comprehensive understanding of their beneficial effects hadyet to be developed at the time their respective monographswere reviewed. The committee, nevertheless, believes thatincluding monographs for these substances in the Food Chemi-cals Codex is consistent with sound public health policy tostandardize the material and minimize possible contaminants.Accordingly, the committee selected the term, ‘‘Sourceof . . . ,’’ in describing the function of these materials. Includ-ing these monographs was approved, taking into account theemerging science, but extensive monograph revisions in futureeditions might be necessary. By including these monographsin this Fifth Edition, the committee neither judges nor endorsesany potential health benefits.
Additionally, because of the current usage of the term ‘‘di-etary supplement,’’ the committee replaced this term with‘‘nutrient.’’
Packaging and Storage Statements in monographs relatingto packaging and storage are advisory in character and areintended only as general information to emphasize instanceswhere deterioration may be accelerated under adverse packag-ing and storage conditions, such as exposure to air, light, ortemperature extremes, or where safety hazards are involved.Additionally, to reduce the risk of intentional or accidentalintroduction of undesirable materials into food substances,containers should be equipped with tamper-resistant closures.
Cool Place A cool place is one where the temperature isbetween 8° and 15° (46° and 59°F). Alternatively, it may bea refrigerator, unless otherwise specified in the monograph.
Excessive Heat Any temperature above 40° (104°F).
Storage Under Nonspecific Conditions Where no specificstorage directions or limitations are provided in the individualmonograph, the conditions of storage and distribution includeprotection from moisture, freezing, and excessive heat. Con-tainers should be stored in secure areas when not in use toreduce the possibility of tampering.
Containers The container is the device that holds the sub-stance and that is or may be in direct contact with it. Theimmediate container is in direct contact with the substanceat all times. The closure is a part of the container. Closuresshould be tamper resistant and tamper evident.
The container should not interact physically or chemicallywith the material that it holds so as to alter its strength, quality,or purity, and the food additive contact surface of the containershould comply with the food additive regulations promulgatedunder the Food, Drug, and Cosmetic Act (or with applicablelaws and regulations in other countries in which FCC specifi-cations are recognized).
Polyunsaturated fats and oils are susceptible to oxidationwhen stored in metal containers, at elevated temperatures,and/or in open containers. Oxidation can be minimized bystoring them in closed, nonmetal containers with minimalheadspace or flushed with nitrogen gas.
Light-Resistant Container A light-resistant container isdesigned to prevent deterioration of the contents beyond theprescribed limits of strength, quality, or purity under the ordi-nary or customary conditions of handling, shipment, storage,and sale. A colorless container may be made light resistantby enclosing it in an opaque carton or wrapper (see alsoApparatus, above).
Well-Closed Container A well-closed container protectsthe contents from extraneous solids and from loss of thechemical under the ordinary or customary conditions of han-dling, shipment, storage, and sale.
Tight Container A tight container protects the contentsfrom contamination of extraneous liquids, solids, or vapors;from loss of the chemical; and from efflorescence, deliques-cence, or evaporation under the ordinary or customary condi-tions of handling, shipment, storage, and sale, and is capableof tight reclosure.
Product Security Tamper-evident packaging closures andsecurity tags should be used. Containers that appear to havebeen opened or otherwise altered by unauthorized personsshould not be used until the purity of the substance has beenconfirmed.
2/Monographs
Acesulfame PotassiumAcesulfame K; 6-Methyl-1,2,3-oxathiazine-4(3H)-one-2,2Dioxide Potassium Salt
NK
SO2OH3C
O
C4H4KNO4S Formula wt 201.24
INS: 950 CAS: [55589-62-3]
DESCRIPTION
Acesulfame Potassium occurs as a white, free-flowing crystal-line powder. It is freely soluble in water and very slightlysoluble in ethanol.
Function Nonnutritive sweetener; flavor enhancer.
REQUIREMENTS
IdentificationA. Add a few drops of sodium cobaltinitrite TS to a solution
of 0.3 g of sample in 1 mL of glacial acetic acid and 5 mLof water. A yellow precipitate forms.
B. Dissolve 10 mg of sample in 1000 mL of water. Thesolution shows an absorption maximum at 227 � 2 nm.Assay Not less than 99.0% and not more than 101.0% ofC4H4KNO4S after drying.
9
Fluoride Not more than 3 mg/kg.Lead Not more than 1 mg/kg.Loss on Drying Not more than 1.0%.Organic Impurities Not more than 20 �g/g of UV-activecompounds.pH of a 1:100 Solution Between 5.5 and 7.5.
TESTS
Assay Dissolve between 200 and 300 mg of sample, pre-viously dried at 105° for 2 h and accurately weighed, in 50mL of glacial acetic acid contained in a 250-mL flask.
Note: Dissolution may be slow.
Add 2 or 3 drops of crystal violet TS to the flask, and titratewith 0.1 N perchloric acid to a blue-green endpoint that persistsfor at least 30 s.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 20.12 mg of C4H4KNO4S.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB, using a 4-g sample.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a solution of 2 g of sample in 20 mL of water,and 2 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Organic Impurities
Reference Standard 4-Hydroxybenzoic acid ethyl ester.Sample Solution A solution of 10 g of sample per liter
of deionized water.
10 / Acetic Acid, Glacial / Monographs FCC V
Procedure (See Chromatography, Appendix IIA.) Use ahigh-performance liquid chromatograph capable of separatingacesulfame potassium and 4-hydroxybenzoic acid ethyl esterwith a resolution of 2. Use a chromatograph equipped witha UV or diode array (227 nm) detector and a 25-cm × 4.6-mm (id) stainless steel column, or equivalent, packed with 3to 5 �m of reversed phase C18 silica gel, or equivalent. Theelution is isocratic. Use a 40:60 (v/v) solution of acetoni-trile:0.01 M/L tetrabutyl ammonium hydrogen sulfate(TBAHS) in water as the mobile phase, with a flow rate ofabout 1 mL/min.
Inject 20 �L of Sample Solution into the chromatograph,and obtain the chromatogram. If peaks other than that causedby acesulfame potassium appear within three times the elutiontime of acesulfame potassium, carry out a second run using20 �L of Sample Solution diluted to 0.2 mg/L.
The sum of the areas of all peaks eluted in the first runwithin three times the elution time of acesulfame potassium,except for the acesulfame potassium peak, does not exceedthe peak area of acesulfame potassium in the second run.pH of a 1:100 Solution Determine as directed under pHDetermination, Appendix IIB.
Packaging and Storage Store in well-closed containers ina cool, dry place.
Acetic Acid, Glacial
H3C OH
O
C2H4O2 Formula wt 60.05
INS: 260 CAS: [64-19-7]
FEMA: 2006
DESCRIPTION
Acetic Acid, Glacial, occurs as a clear, colorless liquid. Itboils at about 118°. When well diluted with water (e.g., 1:100),it has a vinegar odor and taste. It is miscible with water, withalcohol, and with glycerin.
Function Acidifier; flavoring agent.
REQUIREMENTS
Identification A 1:3 aqueous solution gives positive testsfor Acetate, Appendix IIIA.Assay Not less than 99.5% and not more than 100.5%, byweight, of C2H4O2.Lead Not more than 0.5 mg/kg.Nonvolatile Residue Not more than 0.005%.
Readily Oxidizable Substances Passes test.Solidification Point Not cooler than 15.6°.
TESTS
Assay Transfer about 2 mL of sample into a tared, glass-stoppered flask, and accurately weigh. Add 40 mL of water,then add phenolphthalein TS, and titrate with 1 N sodiumhydroxide. Each milliliter of 1 N sodium hydroxide is equiva-lent to 60.05 mg of C2H4O2.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Nonvolatile Residue Evaporate 19 mL (20 g) of sample,accurately measured, in a tared dish on a steam bath, and dryat 105° for 1 h.Readily Oxidizable Substances Dilute 2 mL of sample ina glass-stoppered container with 10 mL of water, and add 0.1mL of 0.1 N potassium permanganate. The pink color doesnot change to brown within 2 h.Solidification Point Determine as directed under Solidifica-tion Point, Appendix IIB.
Packaging and Storage Store in tightly closed containers.
Acetone2-Propanone; Dimethyl Ketone
CH3COCH3
C3H6O Formula wt 58.08
CAS: [67-64-1]
DESCRIPTION
Acetone occurs as a clear, colorless, volatile liquid. It ismiscible with water, with alcohol, with ether, with chloroform,and with most volatile oils.
Caution: Acetone is highly flammable.
Function Extraction solvent.
REQUIREMENTS
Identification Mix 0.1 mL of sample with 10 mL of water,add 5 mL of 1 N sodium hydroxide, warm, and add 5 mL ofiodine TS. A yellow precipitate of iodoform forms.Assay Not less than 99.5% and not more than 100.5% ofC3H6O, by weight.Acidity (as acetic acid) Not more than 0.002%.Aldehydes (as formaldehyde) Not more than 0.002%.Alkalinity (as ammonia) Not more than 10 mg/kg.Distillation Range Within a range of 1°, including 56.1°.
FCC V Monographs / Acetone Peroxides / 11
Lead Not more than 1 mg/kg.Methanol Not more than 0.05%.Nonvolatile Residue Not more than 10 mg/kg.Phenols Passes test.Refractive Index Between 1.358 and 1.360.Solubility in Water Passes test.Specific Gravity Not greater than 0.7880 at 25°/25° (equiv-alent to 0.7930 at 20°/20°).Substances Reducing Permanganate Passes test.Water Not more than 0.5%.
TESTS
Assay Transfer about 1 g of sample, accurately weighed,into a 1000-mL flask containing 20 mL of water, and diluteto volume with water. Place 10 mL of this solution into aglass-stoppered flask, add 25 mL of sodium hydroxide TS,and allow the mixture to stand for 5 min. Add 25 mL of 0.1N iodine, stopper the flask, allow the contents to stand in acold, dark place for 10 min, and add 30 mL of 1 N sulfuricacid. Titrate the excess iodine with 0.1 N sodium thiosulfate,using starch TS as the indicator. Perform a blank determination(see General Provisions), and make any necessary correction.Each milliliter of 0.1 N iodine is equivalent to 0.9675 mg ofC3H6O.Acidity (as acetic acid) Mix 38 mL of sample with an equalvolume of carbon dioxide-free water, add 0.1 mL of phenol-phthalein TS, and titrate with 0.1 N sodium hydroxide. Notmore than 0.1 mL is required to produce a pink color.Aldehydes (as formaldehyde) Prepare a Sample Solution bydiluting 2.5 mL of sample with 7.5 mL of water. Prepare aStandard Solution containing 40 �g of formaldehyde in 10mL of water. Add 0.15 mL of a 5% solution of 5,5-dimethyl-1,3-cyclohexanedione in alcohol to each solution, and evapo-rate on a steam bath until the Acetone is volatilized. Diluteto 10 mL with water, and cool quickly in an ice bath whilestirring vigorously. Any turbidity produced in the SampleSolution does not exceed that produced in the Standard So-lution.Alkalinity (as ammonia) Add 1 drop of methyl red TS to25 mL of water, add 0.1 N sulfuric acid until a red color justappears, then add 23 mL of sample, and mix. Not more than0.1 mL of 0.1 N sulfuric acid is required to restore the red color.Distillation Range Determine as directed under DistillationRange, Appendix IIB.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Methanol Prepare a Sample Solution by diluting 10 mL ofsample to 100 mL with water. Prepare a Standard Solutionin water containing 40 �g of methanol in each milliliter. Add0.2 mL of 10% phosphoric acid and 0.25 mL of 1:20 potassiumpermanganate solution to 1 mL of each solution. Allow themixtures to stand for 15 min, then add 0.3 mL of 1:10 sodiumbisulfite solution to each, and shake until colorless. Slowlyadd 5 mL of ice-cold 80% sulfuric acid, keeping the mixturescold during the addition. Add 0.1 mL of 1:100 chromotropicacid solution, mix, and digest on a steam bath for 20 min.
Any violet color produced in the Sample Solution does notexceed that produced in the Standard Solution.Nonvolatile Residue Evaporate 125 mL (about 100 g) ofsample to dryness in a tared dish on a steam bath, dry theresidue at 105° for 30 min, cool, and weigh.Phenols Evaporate 3 mL of sample to dryness at 60°. Add3 drops of a solution of 100 mg of sodium nitrite in 5 mL ofsulfuric acid to the residue, allow the mixture to stand forabout 3 min, and then carefully add 3 mL of 2 N sodiumhydroxide. No color appears.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Water Mix 38 mL of sample with an equalvolume of carbon dioxide-free water. The solution remainsclear for at least 30 min.Specific Gravity Determine by any reliable method (seeGeneral Provisions).Substances Reducing Permanganate Transfer 10 mL ofsample into a glass-stoppered cylinder, add 0.05 mL of 0.1N potassium permanganate, mix, and allow to stand for 15min. The pink color does not entirely disappear.Water Determine as directed under Water Determination,Appendix IIB, using freshly distilled pyridine instead of meth-anol as the solvent.
Packaging and Storage Store in tight containers remotefrom fire.
Acetone Peroxides
INS: 929 CAS: [1336-17-0]
DESCRIPTION
Acetone Peroxides, usually mixed with an edible carrier suchas cornstarch, occur as a fine, white, free-flowing powder.They are a mixture of monomeric and linear dimeric acetoneperoxides (mainly 2,2-hydroperoxypropane), with minor pro-portions of higher polymers.
Caution: Acetone Peroxides are strong oxidizingagents. Avoid exposure to the skin and eyes.
Function Bleaching agent; maturing agent; dough condi-tioner.
REQUIREMENTS
Identification Dissolve about 20 mg of sample in 5 mL of1:10 sulfuric acid, allow to stand for a few minutes, andadd a drop of potassium permanganate TS. The pink colordisappears.Assay A sample yields an amount of hydrogen peroxideequivalent to not less than 16.0% of Acetone Peroxides.Lead Not more than 4 mg/kg.
12 / Acetylated Monoglycerides / Monographs FCC V
TESTS
Assay Transfer about 200 mg of sample, accuratelyweighed, into a 250-mL beaker, add 50 mL of 1:10 sulfuricacid, allow to stand for at least 3 min, stirring occasionally,and titrate with 0.1 N potassium permanganate to a light pinkcolor that persists for at least 20 s. Calculate the total perox-ides, P, as grams of hydrogen peroxide equivalents per 100g of the sample, by the equation
P = V × N × 0.017 × 100/W,
in which V and N are the volume and exact normality, respec-tively, of the potassium permanganate; 0.017 is the milliequi-valent weight of hydrogen peroxide; and W is the weight, ingrams, of the sample taken. Multiply the value P so obtainedby 1.6 to convert to percent Acetone Peroxides.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 4 �g of lead (Pb) ion in the control.
Packaging and Storage Store in tightly closed containersin a cool, dry place, preferably below 24°.
Acetylated MonoglyceridesAcetylated Mono- and Diglycerides; Acetic and Fatty AcidEsters of Glycerol; Acetoglycerides
CH2
CH
CH
OR1
OR2
OR3
in which R1, R2, and R3 each may be a fatty acid moiety, COCH3, or H. At least one R must be a fatty acid, and at least one must be COCH3.
INS: 472a
DESCRIPTION
Acetylated Monoglycerides occur as clear, thin liquids orsolids, ranging in color from white to pale yellow. They consistof partial or complete esters of glycerin with a mixture ofacetic acid and edible fat-forming fatty acids. They may bemanufactured by the interesterification of edible fats withtriacetin and glycerin in the presence of catalytic agents, fol-lowed by molecular distillation, or by the direct acetylationof edible monoglycerides with acetic anhydride and withoutthe use of a catalyst or molecular distillation. They are insolu-ble in water, but are soluble in alcohol, in acetone, and inother organic solvents, the extent of solubility depending onthe degree of esterification and the melting range.
Function Emulsifier; coating agent; texture-modifyingagent; solvent; lubricant.
REQUIREMENTS
Acid Value Not more than 6.Lead Not more than 2 mg/kg.Reichert-Meissl Value Between 75 and 200.
The following specifications should conform to the representa-tions of the vendor: Free Glycerin, Iodine Value, and Saponifi-cation Value.
TESTS
Acid Value Determine as directed in Method II under AcidValue, Appendix VII.Free Glycerin Determine as directed under Free Glycerinor Propylene Glycol, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Reichert-Meissl Value Determine as directed underReichert-Meissl Value, Appendix VII.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.
Packaging and Storage Store in well-closed containers.
N-Acetyl-L-MethionineN-Acetyl-L-2-amino-4-(methylthio)butyric Acid
CH3SCH2CH2CHCOOH
HNCOCH3
C7H13NO3S Formula wt 191.25
CAS: [65-82-7]
DESCRIPTION
N-Acetyl-L-Methionine occurs as a colorless or lustrous,white, crystalline solid or a white powder. It is soluble inwater, in alcohol, in alkali solutions, and in dilute mineralacids, but practically insoluble in ether.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as those
View IR
FCC V Monographs / Acid Hydrolysates of Proteins / 13
of a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC7H13NO3S, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.5%.Optical (Specific) Rotation [�]D
20°: Between –18.0° and–22.0° after drying.Residue on Ignition Not more than 0.1%.
TESTS
Assay Transfer about 250 mg of the sample, accuratelyweighed, into a glass-stoppered flask, and add 100 mL ofwater, 5 g of dibasic potassium phosphate, 2 g of monobasicpotassium phosphate, and 2 g of potassium iodide. Mix wellto dissolve, add 50.0 mL of 0.1 N iodine, stopper the flask,and mix. Allow to stand for 30 min, add starch TS indicator,and then titrate the excess iodine with 0.1 N sodium thiosulfate.Perform a residual blank titration. Each milliliter of 0.1 Niodine is equivalent to 9.563 mg of C7H13NO3S.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Dry at 105° for 2 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 2 g of the previously dried sample in sufficientwater to make 100 mL.Residue on Ignition Ignite 1 g as directed in Method Iunder Residue on Ignition (Sulfated Ash), Appendix IIC.
Packaging and Storage Store in tightly closed, light-resis-tant containers.
Acid Hydrolysates of Proteins
Acid-Hydrolyzed Proteins; Hydrolyzed Vegetable Protein(HVP); Hydrolyzed Plant Protein (HPP); Hydrolyzed(Source) Protein Extract; Acid-Hydrolyzed Milk Protein
DESCRIPTION
Acid Hydrolysates of Proteins occur as liquids, pastes, pow-ders, or granules. They are composed primarily of aminoacids, small peptides (peptide chains of five or fewer aminoacids), and salts resulting from the essentially complete hydro-lysis of peptide bonds in edible proteinaceous materials, cata-lyzed by food-grade acids and/or heat. Cleavage of peptidebonds typically ranges from a low of 85% to essentially 100%.In processing, the protein hydrolysates may be treated withsafe and suitable alkaline materials. The edible proteinaceousmaterials used as raw materials are derived from corn, soy,
wheat, yeast, peanuts, rice, or other safe and suitable vegetableor plant sources, or from milk.
Function Flavoring agent; flavor enhancer.
REQUIREMENTS
Note: Perform all calculations on the dried basis. Evap-orate liquid and paste samples to dryness in a suitabletared container; then, as for the powdered and granularforms, dry to constant weight at 105° (see GeneralProvisions).
Assay (Total Nitrogen; TN) Not less than 4.0%.�-Amino Nitrogen (AN) Not less than 3.0%.�-Amino Nitrogen/Total Nitrogen (AN/TN) Percent RatioNot less than 62.0% and not more than 85.0% when calculatedon an ammonia nitrogen-free basis.Ammonia Nitrogen (NH3-N) Not more than 1.5%.3-Chloropropane-1,2-diol (3-CPD) Not more than 1 mg/kg.1,3-Dichloro-2-propanol (DCP) Not more than 0.05 mg/kg.Glutamic Acid Not more than 20.0% as glutamic acid(C5H9NO4) and not more than 35.0% of the total amino acids.Insoluble Matter Not more than 0.5%.Lead Not more than 3 mg/kg.Potassium Not more than 30.0%.Sodium Not more than 20.0%.
TESTS
Assay (Total Nitrogen; TN) Determine as directed underNitrogen Determination, Appendix IIIC.�-Amino Nitrogen (AN) Determine as directed under �-Amino Nitrogen Determination, Appendix IIIC.�-Amino Nitrogen/Total Nitrogen (AN/TN) Percent RatioCalculate by dividing the percent of �-amino nitrogen (AN)by the percent of total nitrogen (TN) as corrected for ammonianitrogen (NH3-N) according to the formula
100[(AN − NH3-N)/(TN − NH3-N)].
Ammonia Nitrogen (NH3-N) Determine as directed underAmmonia Nitrogen, Appendix IIIC.3-Chloropropane-1,2-diol (3-CPD)
3-CPD Stock Solution Transfer 12.5 mg of reagent-grade3-chloropropane-1,2-diol (3-CPD), accurately weighed, intoa 100-mL volumetric flask, dilute to volume with ethyl acetate,and mix.
Dilute 3-CPD Solution Dilute 5 mL of 3-CPD Stock Solu-tion to 100 mL with ethyl acetate to yield a solution containing6.25 �g/mL of 3-CPD.
Internal Standard Solution Transfer 50 mg of 1-chlorote-tradecane into a 50-mL volumetric flask, and dilute to volumewith ethyl acetate. Dilute 1 mL of this solution to 100 mLwith ethyl acetate to yield a solution containing 10 �g/mLof 1-chlorotetradecane.
Standard SolutionsA. Pipet 2 mL of Dilute 3-CPD Solution and 2.5 mL of
Internal Standard Solution into a 25-mL volumetric flask,
14 / Acid Hydrolysates of Proteins / Monographs FCC V
dilute to volume with ethyl acetate, and mix. The resultingStandard Solution A contains 0.5 �g/mL of 3-CPD.
B. Pipet 8 mL of Dilute 3-CPD Solution and 2.5 mL ofInternal Standard Solution into a 25-mL volumetric flask,dilute to volume with ethyl acetate, and mix. The resultingStandard Solution B contains 2.0 �g/mL of 3-CPD.
C. Pipet 16 mL of Dilute 3-CPD Solution and 2.5 mL ofInternal Standard Solution into a 25-mL volumetric flask,dilute to volume with ethyl acetate, and mix. The resultingStandard Solution C contains 4.0 �g/mL of 3-CPD.
Procedure (See Chromatography, Appendix IIA) Use agas chromatograph equipped with an electrolytic conductivitydetector operated in the halogen mode and fitted either witha capillary injector operated in the splitless mode or with apurged, packed injector with a glass insert. Use a 30-m ×0.53-mm (id), fused-silica column, or equivalent, coated with1-�m Supelcowax 10 or an equivalent bonded carbowax col-umn fitted with a 50-cm retention gap of 0.53-mm, deacti-vated, fused silica, or equivalent. Set the column temperatureto 170° for 5 min, raise the temperature at a rate of 5°/minto 250°, and hold it at that temperature for 10 min. Maintainthe injector temperature at 225°. Use helium as the carriergas at a flow rate of 8 mL/min.
Use hydrogen as the reactant gas at a flow rate of 30 mL/min, and use 1-propanol as the solvent at a flow rate throughthe cell of 0.5 mL/min or at the manufacturer’s specified flowrate for the optimum operation of the electrolytic conductivitydetector. The reactor temperature should be 900°, with a basetemperature of 275°. Minimize contamination of the reactiontube by venting flow from the column at all times, except forthe time during which compounds of interest elute.
Inject 1 �L of each Standard Solution A, B, and C, intothe gas chromatograph. Calculate the response area ratios of3-CPD to the Internal Standard Solution for each StandardSolution. Plot the response area ratios versus the microgramsof 3-CPD in each Standard Solution to obtain the standardcurve.
Adjust an accurately weighed sample, as needed, with 20%aqueous sodium chloride to obtain a Sample Solution with asolids content of 36%. Transfer a 20-g aliquot of the SampleSolution into a 20-mL Extrelut NT column (EM Science,Gibbstown, NJ), or equivalent, and allow it to equilibrate for15 min. Elute the column with 150 mL of ethyl acetate,collecting the eluent in a 250-mL short-neck, round-bottomflask with a 24/40 joint. Using a rotary evaporator at 50°,concentrate the eluent to a volume of approximately 3 mL.Add 0.5 mL of Internal Standard Solution to the eluent,transfer this mixture to a 4-dram screw-cap vial, and diluteto a volume of 5.0 mL. Inject 1 �L into the gas chromatograph,measure its response area ratio of 3-CPD to the InternalStandard Solution, and determine from the standard curve themicrograms of 3-CPD in the 20-g aliquot taken.1,3-Dichloro-2-propanol (DCP)
Eluent Transfer 850 mL of chromatographic-grade pen-tane and 150 mL of chromatographic-grade diethyl ether intoa suitable container, and mix well.
DCP Stock Solution Transfer 50 mg of reagent-grade 1,3-dichloro-2-propanol (DCP), accurately weighed, into a 50-mL volumetric flask, dilute to volume with Eluent, and mix.
Dilute DCP Solution Stepwise and quantitatively dilutethe DCP Stock Solution with Eluent to obtain a final solutioncontaining 1 �g/mL of DCP.
Internal Standard Solution Transfer 50 mg of trichloro-benzene into a 50-mL volumetric flask, dilute to volume withEluent, and mix. Use a 1:1000 dilution of this solution as theInternal Standard Solution.
Standard Solutions Pipet 1-, 2-, 3-, and 4-mL portions ofDilute DCP Solution into separate 50-mL volumetric flasks.Add 1.0 mL of Internal Standard Solution to each, dilute tovolume with Eluent, and mix.
Sample Preparation Dissolve 5.0 g of sample, accuratelyweighed, in a minimal volume of 20% aqueous sodium chlo-ride solution. Quantitatively transfer this solution to an Ex-trelut NT column (EM Science, Gibbstown, NJ), or equivalent.After 15 min, elute the column with three 20-mL portions ofEluent, and collect all the eluate. Carefully evaporate theeluate to less than 4 mL. Add 1.0 mL of Internal StandardSolution, and dilute with Eluent, as necessary, to bring thefinal volume to 5.0 mL.
Procedure (See Chromatography, Appendix IIA) Use agas chromatograph equipped with a split injector and an elec-tron-capture detector and fitted with a 50-m × 0.2-mm (id),fused-silica column (Carbowax 20M, or equivalent) coatedwith dimethylpolysiloxane, or equivalent. Use nitrogen as thecarrier gas at a flow rate of 8 mL/min. Before use, preconditionthe column by heating it at 200° and the detector at 300° for24 h. Set the injector temperature at 250° and the electron-capture detector at 300°, and program the column temperatureas follows: Maintain for 10 min at 115°, raise rapidly at 30°/min to 200°, and maintain at 200° for 12 min.
Inject 1.0 �L of each of the four Standard Solutions intothe gas chromatograph. For each Standard Solution, calculatethe response area ratios of DCP to the Internal StandardSolution. Plot the response area ratios versus the microgramsof DCP in each Standard Solution to obtain the standard curve.
Similarly, inject 1.0 �L of Sample Preparation. Measureits response ratio, and determine from the standard curve themicrograms of DCP in the sample taken.Glutamic Acid Determine as directed under Glutamic Acid,Appendix IIIC.Insoluble Matter Transfer about 5 g of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, add 75 mL ofwater, cover the flask with a watch glass, and boil gently for2 min. Filter the solution through a tared filtering crucible,dry at 105° for 1 h, cool, and weigh.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB using a Sample Solution prepared as directed fororganic compounds, and 3 �g of lead (Pb) ion in the control.Potassium
Standard Solution Transfer 38.20 mg of reagent-gradepotassium chloride, accurately weighed, into a 100-mL volu-metric flask, dissolve in and dilute to volume with deionizedwater, and mix. Transfer 5.0 mL of this solution into a 1000-
FCC V Monographs / Acidified Sodium Chlorite Solutions / 15
mL volumetric flask, dilute to volume with deionized water,and mix. Each milliliter contains 1.0 �g of potassium (K).
Sample Solution Transfer 1.00 � 0.05 g of previouslydried sample, accurately weighed, into a silica or porcelaindish. Ash in a muffle furnace at 550° for 2 to 4 h. Allow theash to cool, and dissolve in 5 mL of 20% hydrochloric acid,warming the solution if necessary to complete solution of theresidue. Filter the solution through acid-washed filter paperinto a 1000-mL volumetric flask. Wash the filter paper withhot water, dilute to volume, and mix. Use a 1:300 aqueousdilution as the Sample Solution.
Spectrophotometer Use any suitable atomic absorptionspectrophotometer.
Procedure Determine the absorbance of each solution at766.5 nm, following the manufacturer’s instructions for opti-mum operation of the spectrophotometer. The absorbance ofthe Sample Solution does not exceed that of the StandardSolution.Sodium
Standard Solution Transfer 25.42 mg of reagent-gradesodium chloride, accurately weighed, into a 100-mL volumet-ric flask, dissolve in and dilute to volume with deionizedwater, and mix. Transfer 5.0 mL of this solution into a 1000-mL volumetric flask, dilute to volume with deionized water,and mix. Each milliliter of the final Standard Solution contains0.5 �g of sodium (Na). Prepare a 1:100 aqueous dilution ofthis solution to obtain the final working Standard Solution.
Sample Solution Transfer 1.00 � 0.05 g of previouslydried sample, accurately weighed, into a silica or porcelaindish. Ash in a muffle furnace at 550° for 2 to 4 h. Allow theash to cool, and dissolve in 5 mL of 20% hydrochloric acid,warming the solution if necessary to complete solution of theresidue. Filter the solution through acid-washed filter paperinto a 100-mL volumetric flask. Wash the filter paper withhot water, dilute to volume, and mix. Prepare a 1:4000 aqueousdilution of this solution to obtain the final Sample Solution.
Spectrophotometer Use any suitable atomic absorptionspectrophotometer.
Procedure Determine the absorbance of each solution at589.0 nm, following the manufacturer’s instructions for opti-mum operation of the spectrophotometer. The absorbance ofthe Sample Solution does not exceed that of the StandardSolution.
Packaging and Storage Store in well-closed containers.
Acidified Sodium Chlorite Solutions
DESCRIPTION
Acidified Sodium Chlorite (ASC) Solutions occur as clear,colorless to pale-yellow liquids. The ASC Solutions are equi-librium mixtures of sodium chlorite (NaClO2) and chlorousacid (HClO2). ASC Solutions are produced by lowering the
pH of a sodium chlorite solution with a safe and suitable acidto achieve a pH within the range 2.3 to 3.9 depending on theintended use.
Function Antimicrobial agent in processing water used tospray, dip, rinse, or store food before processing, to be fol-lowed by rinsing in potable water or by blanching, cooking,or canning; sanitizer for hard surfaces; broad-spectrum bacte-ricide, virucide, fungicide, and sporicide.
REQUIREMENTS
Lead Not more than 1 mg/kg.Mercury Not more than 1 mg/kg.pH Between 2.3 and 3.9.
Note: The pH is chosen depending on the application;it controls the concentration of metastable chlorous acid,which rapidly breaks down into chlorine dioxide, chlo-ride, and in some applications, chlorate.
Caution: To minimize the evolution of hazardous chlo-rine dioxide gas, do not adjust the pH below 2.3.
Sodium Chlorite Between 40 and 1200 ppm, depending onthe application.
TESTS
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a 1.0-mL portion of sample mixed with 5 mLof water and 11 mL of 2.7 N hydrochloric acid, and 10 �gof lead (Pb) ion in the control.Mercury Determine as directed under Mercury Limit Test,Appendix IIIB, using the following as the Sample Prepara-tion: Transfer 2.0 mL of sample into a 50-mL beaker; add10 mL of water, 1 mL of 1:5 sulfuric acid, and 1 mL of a1:25 potassium permanganate solution; cover with a watchglass; boil for a few seconds; and cool.pH Determine as directed under pH Determination, Appen-dix IIB.Sodium Chlorite (21 CFR 173.325; ‘‘Determination of So-dium Chlorite: 50 ppm to 1500 ppm,’’ Alcide Corporation)
Sample For solutions containing 40 to 250 ppm, use a100-g sample; for those containing 250 to 500 ppm, use a 50-gsample; for those containing 500 to 1100 ppm, use a 20-g sam-ple; for those containing 1100 to 1500 ppm, use a 15-g sample.
Procedure Transfer the sample into a tared 250-mL Erlen-meyer flask, and record the weight to the nearest 0.1 mg. Adda magnetic stirring bar. Add approximately 2 g of potassiumiodide, place the flask over a magnetic stirrer, and stir untilthe potassium iodide crystals dissolve (about 1 min). Add 1 mLof 6 N hydrochloric acid, and stir for 30 s. While continuouslystirring, titrate the liberated iodine with standardized 0.025 Nsodium thiosulfate (Na2S2O3). When most of the brown iodinecolor has faded, add 2 mL of starch indicator solution, andtitrate to a clear endpoint, allowing adequate mixing timebetween additions of titrant near the endpoint. Record thevolume of titrant, V, in milliliters.
Calculation Calculate the amount of Sodium Chlorite, inparts per million, by the equation
16 / Aconitic Acid / Monographs FCC V
ppm of Sodium Chlorite = (V × N × 90.44 × 1000)/(W × 4),
in which V is the volume, in milliliters, of titrant; N is thenormality of the sodium thiosulfate titrant; 90.44 is the molec-ular weight of Sodium Chlorite; 1000 is a conversion factorfrom milligrams per gram to parts per million; W is the weight,in grams, of the sample; and 4 is the milliequivalents ofsodium thiosulfate per milliequivalent of Sodium Chlorite.
Alternative Methods The concentration of SodiumChlorite also can be determined using ion chromatogra-phy by following U.S. Environmental ProtectionAgency Method 300.11 or amperometrically by follow-ing American Public Health Association Method 4500-ClO2.
2
Packaging and Storage Store in closed, opaque containers.Avoid exposure to sun or ultraviolet light because chlorinedioxide gas will generate in the solution.
Aconitic AcidEquisetic Acid; Citridic Acid; Achilleic Acid; 1-Propene-1,2,3-tricarboxylic Acid
C
C
H COOH
HOOC CH2 COOH
C6H6O6 Formula wt 174.11
CAS: [499-12-7]
FEMA: 2010
DESCRIPTION
Aconitic Acid occurs in the leaves and tubers of Aconitumnapellus L. (Fam. Ranunculaceae) and various species ofAchillea and Equisetum, in beet root, and in sugar cane. Itmay be synthesized by the dehydration of citric acid by sulfuricor methanesulfonic acid. Aconitic Acid from the abovesources has the ‘‘trans’’ configuration. It has a melting pointof 195° to 200° with decomposition. It is practically odorlessand has a winy taste. It is soluble in water and in alcohol andis slightly soluble in ether.
Function Flavoring substance and adjuvant.
1Hautman, Daniel P. and Munch, David J. ‘‘Method 300.1: Determina-tion of inorganic anions in drinking water by ion chromatography, Revi-sion 1.0.’’ U.S. Environmental Protection Agency, Office of GroundWater and Drinking Water. 1997. Online. Available: <http://www.epa.-gov/OGWDW/methods/sourcalt.html> [accessed November 18, 2002].
2Franson, MA, ed. 1998. Standard methods 4500-ClO2, amperometricmethod II. In: Standard Methods for the Examination of Water andWastewater, 20th Ed. Baltimore, MD: APHA/AWWA/WEF. Pp. 4-73–4-79.
REQUIREMENTS
Identification The infrared spectrum of the sample, deter-mined neat as a potassium bromide dispersion, exhibits infra-red absorption bands at 3030, 2630, and 1720 cm–1. An aque-ous solution of the substance exhibits major absorption peaksat 411 and 432 nm, with little or no absorption at 389 nm.Assay Not less than 98.0% and not more than 100.5% ofC6H6O6, calculated on the anhydrous basis.Lead Not more than 0.5 mg/kg.Oxalate Passes test.Readily Carbonizable Substances Passes test.Residue on Ignition Not more than 0.1%.Water Not more than 0.5%.
TESTS
Assay Dissolve about 3 g of sample, accurately weighed,in 40 mL of water, add phenolphthalein TS, and titrate with1 N sodium hydroxide. Each milliliter of 1 N sodium hydroxideis equivalent to 58.04 mg of C6H6O6.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 10-g sample.Oxalate Neutralize 10 mL of a 1:10 aqueous solution with6 N ammonium hydroxide, add 5 drops of 2.7 N hydrochloricacid, cool, and add 2 mL of calcium chloride TS. No turbiditydevelops.Readily Carbonizable Substances Transfer 1.0 g of sam-ple, finely powdered, into a 22- × 175-mm test tube previouslyrinsed with 10 mL of 95% sulfuric acid and allowed to drainfor 10 min. Add 10 mL of 95% sulfuric acid, agitate the tubeuntil solution is complete, and immerse the tube in a waterbath at 90° � 1° for 60 � 0.5 min, keeping the level of theacid below the level of the water during the heating period.Cool the tube in a stream of water, and transfer the acidsolution into a color comparison tube. The color of the acidsolution is not darker than that of the same volume of MatchingFluid K (see Readily Carbonizable Substances, Appendix IIB)in a similar matching tube, viewing the tubes vertically againsta white background.Residue on Ignition Determine as directed in Method Iunder Residue on Ignition, Appendix IIC, igniting a 4-gsample.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tightly closed containers.
FCC V Monographs / Agar / 17
Adipic AcidHexanedioic Acid; 1,4-Butanedicarboxylic Acid
HOOC(CH2)4COOH
C6H10O4 Formula wt 146.14
INS: 355 CAS: [124-04-9]
DESCRIPTION
Adipic Acid occurs as white crystals or a crystalline powder.It is not hygroscopic. It is freely soluble in alcohol, solublein acetone, and slightly soluble in water.
Function Buffer; neutralizing agent.
REQUIREMENTS
Assay Not less than 99.6% and not more than 101.0% ofC6H10O4, calculated on the anhydrous basis.Lead Not more than 2 mg/kg.Melting Range Between 151.5° and 154°.Residue on Ignition Not more than 0.002%.Water Not more than 0.2%.
TESTS
Assay Mix about 1.5 g of sample, accurately weighed, with75 mL of recently boiled and cooled water contained in a250-mL glass-stoppered Erlenmeyer flask, add phenolphtha-lein TS, and titrate with 0.5 N sodium hydroxide to the firstappearance of a faint pink endpoint that persists for at least30 s, shaking the flask as the endpoint is approached. Eachmilliliter of 0.5 N sodium hydroxide is equivalent to 36.54mg of C6H10O4.Lead Determine as directed in Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Residue on Ignition Transfer 100.0 g of sample into a tared125-mL platinum dish that has been previously cleaned byfusing with 5 g of potassium pyrosulfate or bisulfate, followedby boiling in 2 N sulfuric acid and rinsing with water. Meltthe sample completely over a gas burner, then ignite the meltwith the burner. After ignition starts, lower or remove theflame to prevent the sample from boiling and to keep it burningslowly until it is completely carbonized. Ignite at 850° in amuffle furnace for 30 min or until the carbon is completelyremoved, then cool and weigh.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Agar
INS: 406 CAS: [9002-18-0]
DESCRIPTION
Agar is commercially available as white to pale yellow bundlesconsisting of thin, membranous agglutinated strips, or in cut,flaked, granulated, or powdered forms. Agar is a generic namegiven to a group of related molecules with a repeating unitof agarobiose formed basically by D- and L-galactose unitsinterlinked with �-1,3 and �-1,4 linkages. Approximately ev-ery tenth D-galactopyranose unit contains a sulfate ester group.It is extracted from the cellular walls of agarophyte seaweed,considering as such the red seaweed from phylum Rodophyta,which belong to the Gelidiceae, Gracilariaceae, and Ahnphel-tiaceae families. It is insoluble in cold water, but it is solublein boiling water.
Function Stabilizer; emulsifier; thickener.
REQUIREMENTS
IdentificationA. Place a few fragments of unground sample or a small
amount of the powder on a slide, add a few drops of water,and examine microscopically. The sample appears granularand somewhat filamentous. A few fragments of the spiculesof sponges and a few frustules of diatoms may be present.
B. While stirring continuously, boil 1 g of sample with 65mL of water for 10 min, and adjust to a concentration of1.5%, by weight, with hot water. A clear liquid results thatcongeals between 32° and 39° to form a firm, resilient gelthat does not liquefy below 85°.Arsenic Not more than 3 mg/kg.Ash (Acid-Insoluble) Not more than 0.5%, calculated onthe dried basis.Ash (Total) Not more than 6.5%, calculated on the driedbasis.Gelatin Passes test.Insoluble Matter Not more than 1.0%.Lead Not more than 5 mg/kg.Loss on Drying Not more than 20.0%.Starch Passes test.Water Absorption Passes test.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Ash (Acid-Insoluble) Determine as directed under Ash(Acid-Insoluble), Appendix IIC.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Gelatin Dissolve about 1 g of sample in 100 mL of boilingwater, and allow the solution to cool to about 50°. Add 5 mL
18 / DL-Alanine / Monographs FCC V
of trinitrophenol TS to 5 mL of the solution. No turbidityforms within 10 min.Insoluble Matter Add sufficient water to 7.5 g of sampleto make 500 g, boil for 15 min, and readjust to the originalweight. Add hot water to 100 g of the mixture to make 200mL, heat almost to boiling, filter while hot through a taredfiltering crucible, rinse the container with several portions ofhot water, and pass the rinsings through the crucible. Dry thecrucible and its contents at 105° to constant weight, cool, andweigh. The weight of the residue does not exceed 15 mg.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 5 h. Cutunground sample into 2- to 5-mm square pieces before drying.Starch Boil 100 mg of sample in 100 mL of water, cool,and add a few drops of iodine TS. No blue color appears.Water Absorption Place 5 g of sample into a 100-mLgraduated cylinder, fill to volume with water, mix, and allowto stand at about 25° for 24 h. Pour the contents of the cylinderthrough moistened glass wool, allowing the water to draininto another 100-mL graduated cylinder. Not more than 75mL of water is obtained.
Packaging and Storage Store in well-closed containers.
DL-AlanineDL-2-Aminopropanoic Acid
CH3CH(NH2)COOH
C3H7NO2 Formula wt 89.09
CAS: [302-72-7]
DESCRIPTION
DL-Alanine occurs as a white crystalline powder. It is freelysoluble in water, but sparingly soluble in alcohol. The pH ofa 1:20 aqueous solution is between 5.5 and 7.0. It melts withdecomposition at about 198°. It is optically inactive.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC3H7NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.
Loss on Drying Not more than 0.3%.Residue on Ignition Not more than 0.2%.
TESTS
Assay Dissolve about 200 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to a blue-green endpoint.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 8.909 mg of C3H7NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
L-AlanineL-2-Aminopropanoic Acid
CH3CCOOH
H NH2
C3H7NO2 Formula wt 89.09
CAS: [56-41-7]
DESCRIPTION
L-Alanine occurs as a white crystalline powder. It is freelysoluble in water, sparingly soluble in alcohol, and insolublein ether. The pH of a 1:20 aqueous solution is between 5.5and 7.0.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC3H7NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.
View IRView IR
FCC V Monographs / Alginic Acid / 19
Loss on Drying Not more than 0.3%.Optical (Specific) Rotation [�]D
20°: Between +13.5° and+15.5°, calculated on the dried basis; or [�]D
25°: between +13.2°and +15.2°, calculated on the dried basis.Residue on Ignition Not more than 0.2%.
TESTS
Assay Dissolve about 200 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to a blue-green endpoint.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 8.909 mg of C3H7NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 10 g of a previously dried sample in sufficient 6N hydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
Alginic Acid
(C6H8O6)n Formula wt, calculated 176.13Formula wt, actual (avg.) 200.00
INS: 400 CAS: [9005-32-7]
DESCRIPTION
Alginic Acid occurs as a white to yellow-white, fibrous pow-der. It is a hydrophilic colloidal carbohydrate extracted fromvarious species of brown seaweeds (phaeophyceae) with dilutealkali. It may be described chemically as a linear glycurono-glycan consisting mainly of �-1,4 linked D-mannuronic andL-guluronic acid units in the pyranose ring form. Alginic Acidis insoluble in water, readily soluble in alkaline solutions, andinsoluble in organic solvents. The pH of a 3:100 suspensionin water is between 2.0 and 3.4.
Function Stabilizer; thickener; emulsifier.
REQUIREMENTS
IdentificationA. Add 1 mL of calcium chloride TS to 5 mL of a 1:150
solution of sample in 0.1 N sodium hydroxide. A voluminousgelatinous precipitate forms.
B. Add 1 mL of 2 N sulfuric acid to 5 mL of the solutionprepared for Identification Test A. A heavy gelatinous precipi-tate forms.
C. Place about 5 mg of sample into a test tube, add 5 mLof water, 1 mL of a freshly prepared 1:100 naphtholresorcin-ol:ethanol solution, and 5 mL of hydrochloric acid. Heat themixture to boiling, boil gently for about 3 min, and then coolto about 15°. Transfer the contents of the test tube to a 30-mL separator with the aid of 5 mL of water, and extract with15 mL of isopropyl ether. Perform a blank determination (seeGeneral Provisions), and make any necessary correction. Theisopropyl ether extract from the sample exhibits a deeperpurple hue than that from the blank.Assay A sample yields not less than 20% and not morethan 23% of carbon dioxide (CO2), corresponding to between91.0% and 104.5% of (C6H8O6)n (equiv wt 200.00), calculatedon the dried basis.Arsenic Not more than 3 mg/kg.Lead Not more than 5 mg/kg.Loss on Drying Not more than 15.0%.Residue on Ignition (Sulfated Ash) Not more than 8.0%,calculated on the dried basis.
TESTS
Assay Determine as directed under Alginates Assay, Appen-dix IIIC. Each milliliter of 0.25 N sodium hydroxide consumedin the assay is equivalent to 25 mg of (C6H8O6)n (equiv wt200.00).Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead ion (Pb) in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Residue on Ignition (Sulfated Ash) Determine as directedin Method I under Residue on Ignition, Appendix IIC, ignitinga 3-g sample.
Packaging and Storage Store in well-closed containers.
20 / Allura Red / Monographs FCC V
Allura Red1
Allura Red AC; CI 16035; Class: Monoazo
OCH3
NaO3S
H3C
N N
HO
SO3Na
C18H14N2O8S2Na2 Formula wt 496.43
INS: 129 CAS: [25956-17-6]
DESCRIPTION
Allura Red occurs as a red-brown powder or granule. It isprincipally the disodium salt of 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid. It dis-solves in water to give a solution red at neutrality and in acidand dark red in base. It is insoluble in ethanol.
Function Color.
REQUIREMENTS
Identification An aqueous solution containing 16.4 mg/Lexhibits absorbance intensities (A) and wavelength maximaas follows: at pH 7, A = 0.87 at 500 nm; at pH 1, A = 0.83at 490 nm (both neutral and acid solutions exhibit a shoulderat about 410 nm); and at pH 13, A = 0.37 at 500 nm, and A =0.41 at 450 nm.Assay Not less than 85.0% total coloring matters.Arsenic Not more than 3 mg/kg.Ether Extracts Not more than 0.2%.Lead Not more than 10 mg/kg.Loss on Drying (Volatile Matter) at 135°, Chlorides, andSulfates (as sodium salts) Not more than 15.0% in combi-nation.Subsidiary Colors
Disodium 6-Hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-8-(2-methoxy-5-methyl-4-sulfophenoxy)-2-naphthalene-sulfonic Acid Not more than 1.0%.
Higher and Lower Sulfonated Subsidiary Colors (as sodiumsalts) Not more than 1.0% each.
1To be used or sold for use to color food that is marketed in the UnitedStates, this color additive must be from a batch that has been certifiedby the U.S. Food and Drug Administration (FDA). If it is not from anFDA-certified batch, it is not a permitted color additive for food use inthe United States, even if it is compositionally equivalent. The nameFD&C Red No. 40 can be applied only to FDA-certified batches of thiscolor additive. Allura Red is a common name given to the uncertifiedcolorant. See the monograph entitled FD&C Red No. 40 for directionsfor producing an FDA-certified batch.
Uncombined Intermediates and Products of Side Reac-tions
4-Amino-5-methoxy-o-toluenesulfonic Acid Not morethan 0.2%.
Disodium 6,6′-Oxybis(2-naphthalenesulfonic Acid) Notmore than 1.0%.
Sodium 6-Hydroxy-2-naphthalenesulfonic Acid Not morethan 0.3%.Water-Insoluble Matter Not more than 0.2%.
TESTS
Assay Determine the total color strength as the weight per-cent of the sample taken using Methods I and II in TotalColor under Colors, Appendix IIIC. Express the Total Coloras the average of the two results.
Method I (Sample Preparation) Transfer 175 to 225 mgof sample, accurately weighed, into a 1-L volumetric flask,dissolve in and dilute to volume with water. The absorptivity(a) for Allura Red is 0.052 mg/L/cm at 502 nm.
Method II (Sample Preparation) Transfer approximately0.2 g of sample, accurately weighed, into the titration flask.The stoichiometric factor (Fs) for Allura Red is 8.06.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Chloride Determine as directed in Sodium Chloride underColors, Appendix IIIC.Ether Extracts Determine as directed in Ether Extractsunder Colors, Appendix IIIC.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Loss on Drying (Volatile Matter) at 135° Determine asdirected in Loss on Drying (Volatile Matter) under Colors,Appendix IIIC.Subsidiary Colors
Solvent System Use a solvent system composed of 20 mLeach of acetonitrile, dioxane, ethyl acetate, isoamyl alcohol,and water and 4 mL of ammonium hydroxide.
Standard Solution Transfer approximately 1 g of purifiedAllura Red (free of subsidiary colors), accurately weighed,into a 50-mL volumetric flask. Add 50 mg each of lowerand higher sulfonated subsidiary colors, accurately weighed,dissolve in and dilute to volume with water. Store in the dark.
Sample Solution Transfer approximately 2 g of sample,accurately weighed, into a 100-mL volumetric flask, dissolvein and dilute to volume with water.
Procedure Spot 3-�L aliquots of Sample Solution andStandard Solution side by side 3 cm from the bottom of a20- × 20-cm glass plate coated with a 0.25-mm layer ofSilica Gel G. Up to seven samples and standards may be runsimultaneously.
When the plate has air dried for 15 min, develop it in anunlined tank equilibrated with the Solvent System for at least20 min. Allow the solvent front to reach to within 3 cm fromthe top of the plate.
Allow the plate to dry in a fume hood, and by visualinspection, compare the intensities of the lower and higher
FCC V Monographs / Aluminum Ammonium Sulfate / 21
sulfonated subsidiary colors with those in the Standard Solu-tion. If the subsidiary colors in the Sample Solution appearmore concentrated than those in the Standard Solution, deter-mine the quantity of each, using a densitometer set to monitorthe absorbance maximum of each. Calculate the concentra-tions of the subsidiary colors in percent (P), if present above0.1%, by the equation
P = (A × p)/AS,
in which A is the area of the densitometer curve, p is thepercent of subsidiary color in the Standard Solution, and AS
is the area of the densitometer curve for the subsidiary colorin the Standard Solution.Sulfate Determine as directed in Sodium Sulfate under Col-ors, Appendix IIIC.Uncombined Intermediates and Products of Side Reac-tions Determine as directed for Method II in UncombinedIntermediates and Products of Side Reactions under Colors,Appendix IIIC, injecting 20 �L of the following Sample Prep-aration: Transfer 0.25 g of sample, accurately weighed, intoa 100-mL volumetric flask. Dissolve in and dilute to volumewith 0.1 M disodium borate (Na2B4O7).Water-Insoluble Matter Determine as directed in Water-Insoluble Matter under Colors, Appendix IIIC.
Packaging and Storage Store in well-closed containers.
Almond Oil, Bitter, FFPA
Bitter Almond Oil Free from Prussic Acid
CAS: [8013-76-1]
DESCRIPTION
Almond Oil, Bitter, FFPA, occurs as a colorless to slightlyyellow liquid with a strong almond aroma and a slightlyastringent, mild taste. It is a volatile oil obtained from thenuts of the bitter almond tree, Prunus amygdalus Batsch var.amara (De Candolle) Focke (Fam. Rosaceae), apricot kernel(Prunus armeniaca L.), and other fruit kernels containingamygdalin. It is prepared by steam distillation of a water-macerated, powdered, and pressed cake that has been speciallytreated and redistilled to remove hydrocyanic acid. It is solublein most fixed oils and in propylene glycol, slightly solublein mineral oil, and insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.
Assay Not less than 95.0% of aldehydes, calculated as benz-aldehyde (C7H6O).Acid Value Not more than 8.0.Chlorinated Compounds Passes test.Hydrocyanic Acid Passes test (about 0.015%).Optical (Specific) Rotation Optically inactive, or not morethan �0.15°.Refractive Index Between 1.541 and 1.546 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 1.040 and 1.050.
TESTS
Assay Determine as directed under Aldehydes, AppendixVI, using about 1 mL of sample, accurately weighed, and53.05 as the equivalence factor (e) in the calculation.Acid Value Determine as directed under Acid Value, Ap-pendix VI.Chlorinated Compounds Determine as directed underChlorinated Compounds, Appendix VI.Hydrocyanic Acid Transfer 1 mL of sample into a test tube.Add 1 mL of water, 5 drops of a 1:10 solution of sodiumhydroxide, and 5 drops of a 1:10 solution of ferrous sulfate.Shake thoroughly, and acidify with 0.5 N hydrochloric acid.No blue precipitate or color appears.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a 100-mmtube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesto form a clear solution in 2 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Aluminum Ammonium Sulfate
Ammonium Alum
AlNH4(SO4)2·12H2O Formula wt 453.32
INS: 523 CAS: [7784-25-0]
DESCRIPTION
Aluminum Ammonium Sulfate occurs as large, colorless crys-tals, white granules, or a powder. One gram dissolves in 7mL of water at 25° and in about 0.3 mL of boiling water. Its
View IR
22 / Aluminum Potassium Sulfate / Monographs FCC V
solutions are acid to litmus. It is insoluble in alcohol, and isfreely, but slowly, soluble in glycerin.
Function Buffer; neutralizing agent.
REQUIREMENTS
Identification A 1:20 aqueous solution gives positive testsfor Aluminum, for Ammonium, and for Sulfate, Appendix IIIA.Assay Not less than 99.5% and not more than 100.5% ofAlNH4(SO4)2·12H2O.Alkalies and Alkaline Earths Passes test.Fluoride Not more than 0.003%.Lead Not more than 3 mg/kg.Selenium Not more than 0.003%.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in 50 mL of water, add 50.0 mL of 0.05 M disodium EDTAand 20 mL of pH 4.5 buffer solution (77.1 g of ammoniumacetate and 57 mL of glacial acetic acid in 1000 mL ofsolution), and boil gently for 5 min. Cool, and add 50 mL ofalcohol and 2 mL of dithizone TS. Back titrate with 0.05M zinc sulfate to a bright rose-pink color. Perform a blankdetermination (see General Provisions), and make any neces-sary correction. The milliliters of 0.05 M disodium EDTAconsumed is equivalent to 50 minus the milliliters of 0.05 Mzinc sulfate used. Each milliliter of 0.05 M disodium EDTAconsumed is equivalent to 22.67 mg of AlNH4(SO4)2·12H2O.Alkalies and Alkaline Earths Completely precipitate thealuminum from a boiling solution of 1 g of sample in 100mL of water by adding enough 6 N ammonium hydroxide torender the solution distinctly alkaline to methyl red TS, andfilter. Evaporate the filtrate to dryness, and ignite. The weightof the residue does not exceed 5 mg.Fluoride Determine as directed in Method V under FluorideLimit Test, Appendix IIIB.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Selenium Determine as directed in Method II under Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.
Packaging and Storage Store in well-closed containers.
Aluminum Potassium Sulfate
Potassium Alum
AlK(SO4)2·12H2O Formula wt 474.38
INS: 522 CAS: [7784-24-9]
DESCRIPTION
Aluminum Potassium Sulfate occurs as large, transparent crys-tals or crystalline fragments, or as a white crystalline powder.
One gram dissolves in 7.5 mL of water at 25° and in about0.3 mL of boiling water. Its solutions are acid to litmus. It isinsoluble in alcohol, but is freely soluble in glycerin.
Function Buffer; neutralizing agent; firming agent.
REQUIREMENTS
Identification A 1:20 aqueous solution gives positive testsfor Aluminum, for Potassium, and for Sulfate, Appendix IIIA.Assay Not less than 99.5% and not more than 100.5% ofAlK(SO4)2·12H2O.Ammonium Salts Passes test.Fluoride Not more than 0.003%.Lead Not more than 3 mg/kg.Selenium Not more than 0.003%.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in 50 mL of water, add 50.0 mL of 0.05 M disodium EDTAand 20 mL of pH 4.5 buffer solution (77.1 g of ammoniumacetate and 57 mL of glacial acetic acid in 1000 mL of aqueoussolution), and boil gently for 5 min. Cool, and add 50 mL ofalcohol and 2 mL of dithizone TS. Back titrate with 0.05M zinc sulfate to a bright rose-pink color. Perform a blankdetermination (see General Provisions), and make any neces-sary correction. The milliliters of 0.05 M disodium EDTAconsumed is equivalent to 50 minus the milliliters of 0.05 Mzinc sulfate used. Each milliliter of 0.05 M disodium EDTAis equivalent to 23.72 mg of AlK(SO4)2·12H2O.Ammonium Salts Add 1 g of sample to 10 mL of 1 Nsodium hydroxide in a small beaker, and heat on a steam bathfor 1 min. The odor of ammonia is not perceptible.Fluoride Determine as directed in Method V under FluorideLimit Test, Appendix IIIB.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Selenium Determine as directed in Method II under Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.
Packaging and Storage Store in well-closed containers.
Aluminum Sodium Sulfate
Soda Alum; Sodium Alum
AlNa(SO4)2 Formula wt, anhydrous 242.09AlNa(SO4)2·12H2O Formula wt, dodecahydrate 458.29
INS: 521 CAS: anhydrous [10102-71-3]CAS: dodecahydrate [7784-28-3]
DESCRIPTION
Aluminum Sodium Sulfate occurs as colorless crystals, whitegranules, or a powder. It is anhydrous or may contain up to
FCC V Monographs / Aluminum Sulfate / 23
12 molecules of water of hydration. The anhydrous form isslowly soluble in water. The dodecahydrate is freely solublein water, and it effloresces in air. Both forms are insolublein alcohol.
Function Buffer; neutralizing agent; firming agent.
REQUIREMENTS
Identification A sample responds to the flame test for So-dium, Appendix IIIA, and gives positive tests for Aluminumand for Sulfate, Appendix IIIA.Assay Anhydrous: Not less than 99.0% and not more than104.0% of AlNa(SO4)2 after drying; Dodecahydrate: Not lessthan 99.5% of AlNa(SO4)2 after drying.Ammonium Salts Passes test.Fluoride Not more than 0.003%.Lead Not more than 3 mg/kg.Loss on Drying Anhydrous: Not more than 10%; Dodecahy-drate: Not more than 47.2%.Neutralizing Value Anhydrous: Between 104 and 108.Selenium Not more than 0.003%.
TESTS
Assay Accurately weigh about 500 mg of sample, pre-viously dried as directed in the test for Loss on Drying (below),moisten with 1 mL of glacial acetic acid, and dissolve in 50mL of water, warming gently on a steam bath until solutionis complete. Cool, neutralize with 6 N ammonium hydroxide,add 50.0 mL of 0.05 M disodium EDTA and 20 mL of pH4.5 buffer solution (77.1 g of ammonium acetate and 57 mLof glacial acetic acid in 1000 mL of aqueous solution), andboil gently for 5 min. Cool, and add 50 mL of alcohol and2 mL of dithizone TS. Back titrate with 0.05 M zinc sulfateto a bright pink color. Perform a blank determination (seeGeneral Provisions), and make any necessary correction. Themilliliters of 0.05 M disodium EDTA consumed is equivalentto 50 minus the milliliters of 0.05 M zinc sulfate used. Eachmilliliter of 0.05 M disodium EDTA consumed is equivalentto 12.10 mg of AlNa(SO4)2.Ammonium Salts Heat 1 g of sample with 10 mL of 1 Nsodium hydroxide on a steam bath for 1 min. The odor ofammonia is not perceptible.Fluoride Determine as directed in Method V under the Fluo-ride Limit Test, Appendix IIIB, using a 1.76-g sample.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Anhydrous: Determine as directed underLoss on Drying, Appendix IIC, drying a sample at 200° for16 h; Dodecahydrate: Determine as directed for the anhydroussample, but dry the sample first at 50° to 55° for 1 h, thenat 200° for 16 h.Neutralizing Value Transfer 500 mg of anhydrous sample,accurately weighed, into a 200-mL Erlenmeyer flask, add 30mL of water and 4 drops of phenolphthalein TS, and boiluntil the sample dissolves. Add 13.0 mL of 0.5 N sodiumhydroxide, boil for a few seconds, and titrate with 0.5 Nhydrochloric acid to the disappearance of the pink color, add-
ing the acid dropwise and agitating vigorously after eachaddition. Calculate the neutralizing value, as parts of NaHCO3
equivalent to 100 parts of the sample, by the formula
8.4V,
in which V is the volume, in milliliters, of 0.5 N sodiumhydroxide consumed by the sample.Selenium Determine as directed in Method II under Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.
Packaging and Storage Store in tight containers.
Aluminum Sulfate
Al2(SO4)3 Formula wt, anhydrous 342.14Al2(SO4)3·18H2O Formula wt, octadecahydrate 666.41
INS: 520 CAS: anhydrous [10043-01-3]CAS: octadecahydrate [7784-31-8]
DESCRIPTION
Aluminum Sulfate occurs as a white powder, as shining plates,or as crystalline fragments. It is anhydrous or contains 18molecules of water of crystallization. Because of efflores-cence, the hydrate may have a composition approximatingthe formula Al2(SO4)3·14H2O. One gram of the hydrate dis-solves in about 2 mL of water. The anhydrous approachesthe same solubility, but the rate of solution is so slow that itinitially appears to be relatively insoluble. The pH of a 1:20aqueous solution is 2.9 or above.
Function Firming agent.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Aluminum and for Sulfate, Appendix IIIA.Assay Anhydrous: Not less than 99.5% of Al2(SO4)3, calcu-lated on the ignited basis; Octadecahydrate: Not less than99.5% and not more than 114.0% of Al2(SO4)3·18H2O, corres-ponding to not more than approximately 101.7% ofAl2(SO4)3·14H2O.Alkalies and Alkaline Earths Passes test (about 0.4%).Ammonium Salts Passes test.Fluoride Not more than 0.003%.Lead Not more than 3 mg/kg.Loss on Ignition Anhydrous: Not more than 5%.
Note: This Requirement does not apply toAl2(SO4)3·18H2O.
Selenium Not more than 0.003%.
TESTS
Assay Accurately weigh an amount of sample equivalentto about 4 g of Al2(SO4)3, transfer into a 250-mL volumetric
24 / Ambrette Seed Oil / Monographs FCC V
flask, dissolve in and dilute to volume with water, and mix.Pipet 10 mL of this solution into a 250-mL beaker, add 25.0mL of 0.05 M disodium EDTA and 20 mL of pH 4.5 buffersolution (77.1 g of ammonium acetate and 57 mL of glacialacetic acid in 1000 mL of aqueous solution), and boil gentlyfor 5 min. Cool, and add 50 mL of alcohol and 2 mL ofdithizone TS. Back titrate with 0.05 M zinc sulfate to a brightpink color. Perform a blank determination (see General Provi-sions), and make any necessary correction. The milliliters of0.05 M disodium EDTA consumed is equivalent to 50 minusthe milliliters of 0.05 M zinc sulfate used. Each milliliter of0.05 M disodium EDTA consumed is equivalent to 8.554 mgof Al2(SO4)3 or to 16.66 mg of Al2(SO4)3·18H2O.Alkalies and Alkaline Earths Add a few drops of methylred TS to a boiling solution of 2 g of sample in 150 mL ofwater, and then add 6 N ammonium hydroxide until the colorof the solution just changes to a distinct yellow. Add hotwater to restore the original volume, and filter while hot.Evaporate 75 mL of the filtrate to dryness, and ignite toconstant weight. Not more than 4 mg of residue remains.Ammonium Salts Heat 1 g of sample with 10 mL of 1 Nsodium hydroxide on a steam bath for 1 min. The odor ofammonia is not perceptible.Fluoride Determine as directed in Method V under the Fluo-ride Test, Appendix IIIB, using 1.67 g of sample.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Ignition Anhydrous: Accurately weigh about 2 gof sample, and ignite, preferably in a muffle furnace, at about500° for 3 h.
Note: This Test does not apply to Al2(SO4)3·18H2O.
Selenium Determine as directed in Method II under Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.
Packaging and Storage Store in well-closed containers.
Ambrette Seed Oil
Ambrette Seed Liquid CAS: [8015-62-1]
DESCRIPTION
Ambrette Seed Oil occurs as a clear yellow to amber liquidwith the strong, musky odor of ambrettolide. It is a volatileoil obtained by steam distillation from the partially dried andcrushed seeds of the plant Abelmoschus moschatus Moench,syn. Hibiscus abelmoschus L. (Fam. Malvaceae). It is refinedby solvent extraction to remove fatty acids or by precipitationof the fatty acid salts. It is soluble in most fixed oils and inmineral oil, often with cloudiness, but relatively insoluble inglycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 3.0.Optical (Specific) Rotation Between –2.5° and +3°.Refractive Index Between 1.468 and 1.485 at 20°.Saponification Value Between 140 and 200.Specific Gravity Between 0.898 and 0.920.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a 100-mmtube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed in Saponifica-tion Value under Esters, Appendix VI, using about 1 g ofsample, accurately weighed.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Ammonia Solution
Ammonium Hydroxide; Stronger Ammonia Water
NH3 Formula wt 17.03
INS: 527 CAS: [7664-41-7]
DESCRIPTION
Ammonia Solution occurs as a clear, colorless liquid. Uponexposure to air it loses ammonia rapidly. Its specific gravityis about 0.90.
Caution: Ammonia Solution is irritating to the oralmucosa and respiratory tract. Perform tests in a well-ventilated fume hood.
Function pH control agent; surface finishing agent; boilerwater additive.
REQUIREMENTS
Identification Hold a glass rod wet with hydrochloric acidnear the surface of the sample liquid. Dense, white fumesevolve.
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FCC V Monographs / Ammoniated Glycyrrhizin / 25
Assay Not less than 27.0% and not more than 30.0%, byweight, of NH3.Lead Not more than 0.5 mg/kg.Nonvolatile Residue Not more than 0.02%.Readily Oxidizable Substances Passes test.
TESTS
Assay Accurately tare a 125-mL glass-stoppered Erlen-meyer flask containing 35.0 mL of 1 N sulfuric acid. Cool asample, contained in its original bottle, to 10° or cooler.Partially fill a 10-mL graduated pipet from near the bottomof this sample. (Do not use vacuum to draw up the sample.)Wipe off any liquid adhering to the outside of the pipet, anddiscard the first milliliter of sample. Hold the pipet just abovethe surface of the acid, and transfer 2 mL into the flask,leaving at least 1 mL in the pipet. Stopper the flask, mix, andweigh again to obtain the weight of the sample. Add methylred TS, and titrate the excess acid with 1 N sodium hydroxide.Each milliliter of 1 N sulfuric acid is equivalent to 17.03 mgof NH3.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Nonvolatile Residue Evaporate 11 mL (10 g) of sample ina tared platinum or porcelain dish to dryness, dry at 105° for1 h, cool, and weigh.Readily Oxidizable Substances Dilute 4 mL of samplewith 6 mL of water, and add a slight excess of 2 N sulfuricacid and 0.1 mL of 0.1 N potassium permanganate. The pinkcolor does not completely disappear within 10 min.
Packaging and Storage Store in tight containers at a tem-perature not exceeding 25°.
Ammoniated Glycyrrhizin
CAS: [1407-03-0]
DESCRIPTION
Ammoniated Glycyrrhizin occurs as a brown powder. It isprecipitated by acid from the water extract of dried and groundrhizomes and roots of Glycyrrhiza glabra or related Glycyrr-hiza (licorice root) (Fam. Leguminosae) and neutralized withdilute ammonia. Suitable diluents may be added.
Function Flavoring agent; flavor enhancer.
REQUIREMENTS
Identification A sample gives positive tests for Ammonium,Appendix IIIA.Assay Not less than 22.0% and not more than 32.0% ofmonoammonium glycyrrhizinate (C42H65NO16), calculated onthe dried basis.
Ash (Total) Not more than 2.5%.Loss on Drying Not more than 6.0%.
TESTS
Assay (Based on AOAC method 982.19.)Apparatus (See Chromatography, Appendix IIA.) Use a
high-performance liquid chromatograph operated at roomtemperature with a 10-�m particle size, 30-cm × 4-mm (id),C18 reverse-phase column (�Bondapak C18 column, WatersCorp., 34-T Maple Street, Milford, MA 01757, or equivalent).Maintain the Mobile Phase at a pressure and flow rate (typi-cally 2.0 mL/min) capable of giving the required elution time(see System Suitability in High-Performance Liquid Chroma-tography). Use an ultraviolet detector that monitors absorptionat 254 nm (0.2 to 0.1 AUFS range).
Mobile Phase Add 380 mL of acetonitrile and 10 mL ofglacial acetic acid to 610 mL of glass-distilled water that hasbeen filtered through a 0.45-�m filter (Millipore, or equiva-lent). Mix, and de-gas thoroughly.
Standard Solution Dissolve about 10 mg of Monoammon-ium Glycyrrhizinate Standard for analytical use (Sigma, orequivalent), accurately weighed, in 20 mL of a 1:1 (v/v)solution of acetonitrile:water. Filter the solution through a0.45-�m filter (Millipore, or equivalent). Prepare fresh daily.
Note: Correct the weight of the Monoammonium Gly-cyrrhizinate Standard taken for the percent loss on dry-ing shown on its label.
Assay Solution Dissolve about 40 g of sample, accuratelyweighed, in 20 mL of water. Filter the solution through a0.45-�m filter (Millipore, or equivalent).
System Suitability Inject duplicate 10-�L portions of theStandard Solution into the chromatograph. The retention timeof monoammonium glycyrrhizinate is approximately 6 min.Adjust the operating conditions if necessary. The mean stan-dard deviation for replicate injections is not more than 2.0%.
Procedure Separately inject, in duplicate, 10-�L volumesof the Standard Solution and the Assay Solution into thechromatograph, and determine the mean peak area for eachsolution. Calculate the percent monoammonium glycyrrhizi-nate, equivalent to C42H65NO16, in the portion of sample takenby the formula
100 × (20CS/WU) × (AU/AS),
in which CS is the concentration, in milligrams per milliliter,of the Standard Solution; WU is the weight, in milligrams, ofthe sample taken; and AU and AS are the peak areas of theAssay Solution and the Standard Solution, respectively.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 1-g sample at 105° for 1 h.
Packaging and Storage Store in a tightly closed containerin a cool, dry place.
26 / Ammonium Alginate / Monographs FCC V
Ammonium Alginate
Algin
(C6H7O6NH4)n Formula wt, calculated 193.16Formula wt, actual (avg.) 217.00
INS: 403 CAS: [9005-34-9]
DESCRIPTION
Ammonium Alginate occurs as a white to yellow, fibrous orgranular powder. It is the ammonium salt of alginic acid (seethe monograph for Alginic Acid). It dissolves in water to forma viscous, colloidal solution. It is insoluble in alcohol and inhydroalcoholic solutions in which the alcohol content isgreater than about 30% by weight. It is insoluble in chloro-form, in ether, and in acids having a pH lower than about 3.
Function Stabilizer; thickener; emulsifier.
REQUIREMENTS
IdentificationA. Add 1 mL of calcium chloride TS to 5 mL of a 1:100
aqueous solution. A voluminous, gelatinous precipitate forms.B. Add 1 mL of 2.7 N sulfuric acid to 10 mL of a 1:100
aqueous solution. A heavy, gelatinous precipitate forms.C. Place about 5 mg of sample into a test tube, add 5
mL of water, 1 mL of a freshly prepared 1:100 solutionnaphtholresorcinol:ethanol, and 5 mL of hydrochloric acid.Heat the mixture to boiling, boil gently for about 3 min, andthen cool to about 15°. Transfer the contents of the test tubeto a 30-mL separator with the aid of 5 mL of water, andextract with 15 mL of isopropyl ether. Perform a blank deter-mination (see General Provisions), and make any necessarycorrection. The isopropyl ether extract from the sample exhib-its a deeper purple hue than that from the blank.
D. Add 5 mL of 1 N sodium hydroxide to about 1 g ofsample contained in a test tube, and shake the mixture briefly.The odor of ammonia is evident.Assay A sample yields not less than 18% and not morethan 21% of carbon dioxide (CO2), corresponding to between88.7% and 103.6% of Ammonium Alginate (equiv wt 217.00),calculated on the dried basis.Arsenic Not more than 3 mg/kg.Lead Not more than 5 mg/kg.Loss on Drying Not more than 15.0%.Residue on Ignition (Sulfated Ash) Not more than 7.0%,calculated on the dried basis.
TESTS
Assay Determine as directed under Alginates Assay, Appen-dix IIIC. Each milliliter of 0.25 N sodium hydroxide consumedin the assay is equivalent to 27.12 mg of Ammonium Alginate(equiv wt 217.00).Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead ion (Pb) in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Residue on Ignition (Sulfated Ash) Determine as directedin Method I under Residue on Ignition, Appendix IIC, usinga 3-g sample.
Packaging and Storage Store in well-closed containers.
Ammonium Bicarbonate
NH4HCO3 Formula wt 79.06
INS: 503(ii) CAS: [1066-33-7]
DESCRIPTION
Ammonium Bicarbonate occurs as white crystals or as a crys-talline powder. It volatilizes rapidly at 60°, dissociating intoammonia, carbon dioxide, and water, but it is quite stable atroom temperature. One gram dissolves in about 6 mL of water.It is insoluble in alcohol.
Function Alkali; leavening agent.
REQUIREMENTS
Identification A sample gives positive tests for Ammoniumand for Bicarbonate, Appendix IIIA.Assay Not less than 99.0% and not more than 100.5% ofNH4HCO3.Chloride Not more than 0.003%.Lead Not more than 3 mg/kg.Nonvolatile Residue Not more than 0.05% (0.55% for prod-ucts containing a suitable anticaking agent).Sulfur Compounds (as SO4) Not more than 0.007%.
TESTS
Assay Dissolve about 3 g of sample, accurately weighed,in 40 mL of water. Add 2 drops of methyl red TS, and whileconstantly stirring, titrate with 1 N hydrochloric acid, addingthe acid slowly, until the solution becomes faintly pink. Heatthe solution to boiling, cool, and continue the titration until thefaint pink color no longer fades after boiling. Each milliliter of1 N hydrochloric acid is equivalent to 79.06 mg of NH4HCO3.Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB, usinga 500-mg sample. Any turbidity produced by the sample doesnot exceed that produced by a control containing 15 �g ofchloride ion (Cl).Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.
FCC V Monographs / Ammonium Chloride / 27
Nonvolatile Residue Transfer 4 g of sample into a tareddish, add 10 mL of water, and evaporate to dryness on asteam bath. Heat the dish at 105° for 1 h, cool in a desiccator,and weigh.Sulfur Compounds (as SO4) Dissolve 4 g of sample in 40mL of water, add about 10 mg of sodium carbonate and 1mL of 30% hydrogen peroxide, and evaporate the solution todryness on a steam bath. Treat the residue as directed in theSulfate Limit Test under Chloride and Sulfate Limit Tests,Appendix IIIB. Any turbidity produced by the sample doesnot exceed that produced by a control containing 280 �g ofsulfate ion (SO4).
Packaging and Storage Store in well-closed containers.
Ammonium Carbonate
INS: 503(i) CAS: [10361-29-2]
DESCRIPTION
Ammonium Carbonate occurs as a white powder or as hard,white or translucent masses. It consists of ammonium bicarbon-ate (NH4HCO3) and ammonium carbamate (NH2·COONH4)in varying proportions. On exposure to air it becomes opaqueand is finally converted into porous lumps or a white powderof ammonium bicarbonate because of the loss of ammonia andcarbon dioxide. One gram dissolves slowly in about 4 mL ofwater. Its solutions are alkaline to litmus.
Function Buffer; leavening agent; neutralizing agent.
REQUIREMENTS
Identification When heated, a sample volatilizes withoutcharring, and the vapor is alkaline to moistened litmus paper.A 1:20 aqueous solution effervesces when an acid is added.Assay Not less than 30.0% and not more than 34.0% of NH3.Chloride Not more than 0.003%.Lead Not more than 3 mg/kg.Nonvolatile Residue Not more than 0.05%.Sulfur Compounds (as SO4) Not more than 0.005%.
TESTS
Assay Place about 10 mL of water in a weighing bottle,tare the bottle and its contents, add about 2 g of sample, andaccurately weigh. Transfer the contents of the bottle to a 250-mL flask, and while mixing, slowly add 50.0 mL of 1 Nsulfuric acid, allowing for the release of carbon dioxide. Whensolution has been effected, wash down the sides of the flaskwith a few milliliters of water, add a few drops of methylorange TS, and titrate the excess acid with 1 N sodium hydrox-ide. Each milliliter of 1 N sulfuric acid is equivalent to 17.03mg of NH3.
Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB, usingthe residue of the following: Dissolve 500 mg of sample in10 mL of hot water, add about 5 mg of sodium carbonate,and evaporate to dryness on a steam bath. Any turbidityproduced does not exceed that shown in a control containing15 �g of chloride ion (Cl).Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Nonvolatile Residue Transfer 4 g of sample into a tareddish, add 10 mL of water, and evaporate on a steam bath.Heat the dish at 105° for 1 h, cool in a desiccator, and weigh.Sulfur Compounds Determine as directed in the SulfateLimit Test under Chloride and Sulfate Limit Tests, AppendixIIIB, using the residue of the following: Dissolve 4 g of samplein 40 mL of water, add about 10 mg of sodium carbonateand 1 mL of 30% hydrogen peroxide, and evaporate thesolution to dryness on a steam bath. Any turbidity produceddoes not exceed that shown in a control containing 200 �gof sulfate (SO4) ion.
Packaging and Storage Store in tight, light-resistant con-tainers, preferably at a temperature not exceeding 30°.
Ammonium Chloride
NH4Cl Formula wt 53.49
INS: 510 CAS: [12125-02-9]
DESCRIPTION
Ammonium Chloride occurs as colorless crystals or as a white,fine or coarse, crystalline powder. It is somewhat hygroscopic.One gram dissolves in 2.6 mL of water at 25°, in 1.4 mL ofboiling water, in about 100 mL of alcohol, and in about 8mL of glycerin. The pH of a 1:20 solution is between 4.5and 6.0.
Function Yeast food; dough conditioner.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Ammonium and for Chloride, Appendix IIIA.Assay Not less than 99.0% of NH4Cl after drying.Lead Not more than 4 mg/kg.Loss on Drying Not more than 0.5%.
TESTS
Assay Dissolve about 200 mg of sample, previously driedover silica gel for 4 h and accurately weighed, in about 40mL of water contained in a glass-stoppered flask. While agitat-ing the mixture, add 3 mL of nitric acid, 5 mL of nitrobenzene,
28 / Ammonium Phosphate, Dibasic / Monographs FCC V
and 50.0 mL of 0.1 N silver nitrate; shake vigorously; add 2mL of ferric ammonium sulfate TS; and titrate the excess silvernitrate with 0.1 N ammonium thiocyanate. Each milliliter of0.1 N silver nitrate is equivalent to 5.349 mg of NH4Cl.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using 4 �g of lead (Pb) ion in the controlLoss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample over silica gel for 4 h.
Packaging and Storage Store in tight containers.
Ammonium Phosphate, Dibasic
Diammonium Hydrogen Phosphate; DiammoniumPhosphate
(NH4)2HPO4 Formula wt 132.06
INS: 342(ii) CAS: [7783-28-0]
DESCRIPTION
Ammonium Phosphate, Dibasic, occurs as white crystals, acrystalline powder, or granules. It is freely soluble in water.The pH of a 1:100 aqueous solution is between 7.6 and 8.2.
Function Buffer; dough conditioner; leavening agent;yeast food.
REQUIREMENTS
Identification A 1:20 aqueous solution gives positive testsfor Ammonium and for Phosphate, Appendix IIIA.Assay Not less than 96.0% and not more than 102.0% of(NH4)2HPO4.Arsenic Not more than 3 mg/kg.Fluoride Not more than 10 mg/kg.Lead Not more than 4 mg/kg.
TESTS
Assay Dissolve about 600 mg of sample, accuratelyweighed, in 40 mL of water, and titrate to a pH of 4.6 with0.1 N sulfuric acid. Each milliliter of 0.1 N sulfuric acid isequivalent to 13.21 mg of (NH4)2HPO4.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 35 mLof water.Fluoride Determine as directed in Method IV under Fluo-ride Limit Test, Appendix IIIB, using a 2-g sample, BufferSolution A, and 0.1 mL of Fluoride Standard Solution.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.
Packaging and Storage Store in tightly closed containers.
Ammonium Phosphate, Monobasic
Ammonium Dihydrogen Phosphate; MonoammoniumPhosphate
NH4H2PO4 Formula wt 115.03
INS: 342(i) CAS: [7722-76-1]
DESCRIPTION
Ammonium Phosphate, Monobasic, occurs as white crystals,a crystalline powder, or granules. It is freely soluble in water.The pH of a 1:100 aqueous solution is between 4.3 and 5.0.
Function Buffer; dough conditioner; leavening agent;yeast food.
REQUIREMENTS
Identification A 1:20 aqueous solution gives positive testsfor Ammonium and for Phosphate, Appendix IIIA.Assay Not less than 96.0% and not more than 102.0% ofNH4H2PO4.Arsenic Not more than 3 mg/kg.Fluoride Not more than 10 mg/kg.Lead Not more than 4 mg/kg.
TESTS
Assay Dissolve about 500 mg of sample, accuratelyweighed, in 50 mL of water, and titrate to a pH of 8.0 with0.1 N sodium hydroxide. Each milliliter of 0.1 N sodiumhydroxide is equivalent to 11.50 mg of NH4H2PO4.Arsenic Determine as directed under Arsenic Limit Test, Ap-pendix IIIB, using a solution of 1 g of sample in 35 mL of water.Fluoride Determine as directed in Method IV under the Fluo-ride Limit Test, Appendix IIIB, using a 2-g sample, Buffer Solu-tion B, and 0.1 mL of Fluoride Standard Solution.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.
Packaging and Storage Store in tightly closed containers.
Ammonium Saccharin1,2-Benzisothiazolin-3-one 1,1-Dioxide Ammonium Salt
N
SO2
NH4
C7H8N2O3S Formula wt 200.21
DESCRIPTION
Ammonium Saccharin occurs as white crystals or as white,
FCC V Monographs / Ammonium Saccharin / 29
crystalline powder. It is freely soluble in water. The pH of a1:3 aqueous solution is between 5 and 6.
Function Nonnutritive sweetener.
REQUIREMENTS
IdentificationA. Dissolve about 100 mg of sample in 5 mL of a 1:20
solution of sodium hydroxide, evaporate to dryness, and gentlyfuse the residue over a small flame until ammonia no longerevolves. After the residue has cooled, dissolve it in 20 mLof water, neutralize the solution with 2.7 N hydrochloric acid,and filter. Add 1 drop of ferric chloride TS to the filtrate. Aviolet color appears.
B. Mix 20 mg of sample with 40 mg of resorcinol, cau-tiously add 10 drops of sulfuric acid, and heat the mixture ina liquid bath at 200° for 3 min. After cooling, add 10 mL ofwater and an excess of 1 N sodium hydroxide. A fluorescentgreen liquid results.
C. A 1:10 aqueous solution gives positive tests for Ammo-nium, Appendix IIIA.
D. Add 1 mL of hydrochloric acid to 10 mL of a 1:10aqueous solution. A crystalline precipitate of saccharin forms.Wash the precipitate well with cold water and dry at 105°for 2 h. The saccharin thus obtained melts between 226° and230° (see Melting Range or Temperature, Appendix IIB).Assay Not less than 98.0% and not more than 101.0% ofC7H8N2O3S, calculated on the anhydrous basis.Benzoate and Salicylate Passes test.Lead Not more than 2 mg/kg.Readily Carbonizable Substances Passes test.Selenium Not more than 0.003%.Toluenesulfonamides Not more than 0.0025%.Water Not more than 0.3%.
TESTS
Assay With the aid of 10 mL of water, quantitatively transferabout 500 mg of sample, accurately weighed, into a separatorAdd 2 mL of 2.7 N hydrochloric acid, and extract the precipi-tated saccharin, first with 30 mL, then with five 20-mL por-tions of a solvent comprising 9:1 (v/v) chloroform:alcohol.Filter each extract through a small filter paper moistened withthe solvent mixture, and evaporate the combined filtrates todryness on a steam bath with the aid of a current of air.Dissolve the residue in 75 mL of hot water, cool, add phenol-phthalein TS, and titrate with 0.1 N sodium hydroxide. Per-form a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N sodiumhydroxide is equivalent to 20.02 mg of C7H8N2O3S.Benzoate and Salicylate Add 3 drops of ferric chloride TSto 10 mL of a 1:20 aqueous solution previously acidified with5 drops of glacial acetic acid. No precipitate or violet colorappears.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.
Readily Carbonizable Substances Determine as directedunder Readily Carbonizable Substances, Appendix IIB, using200 mg of sample dissolved in 5 mL of 95% sulfuric acidand kept at 48° to 50° for 10 min. The color is no darkerthan that of Matching Fluid A.Selenium Determine as directed in Method I under the Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.Toluenesulfonamides
Methylene Chloride Use a suitable grade (such as thatobtainable from Burdick & Jackson Laboratories, Inc.), equiv-alent to the product obtained by distillation in an all-glassapparatus.
Internal Standard Stock Solution Transfer 100.0 mg of95% n-tricosane (obtainable from Chemical Samples Co.) intoa 10-mL volumetric flask, dissolve in and dilute to volumewith n-heptane, and mix.
Stock Standard Preparation Transfer 20.0 mg each ofreagent-grade o-toluenesulfonamide and p-toluenesulfon-amide into a 10-mL volumetric flask, dissolve in and diluteto volume with methylene chloride, and mix.
Diluted Standard Preparations Pipet 0.1, 0.25, 1.0, 2.5,and 5.0 mL, respectively, of the Stock Standard Preparationinto five 10-mL volumetric flasks. Pipet 0.25 mL of the Inter-nal Standard Stock Solution into each flask, dilute each tovolume with methylene chloride, and mix. These solutionscontain, respectively, 20, 50, 200, 500, and 1000 �g/mL ofeach toluenesulfonamide, plus 250 �g of n-tricosane.
Test Preparation (See Chromatography, Appendix IIA.)Dissolve 2.00 g of sample in 8.0 mL of 5% sodium bicarbonatesolution, and mix the solution thoroughly with 10.0 g ofchromatographic siliceous earth (Celite 545, Johns-Manville,or equivalent). Transfer the mixture into a 250- × 25-mmchromatographic tube having a fritted-glass disk and a Teflonstopcock at the bottom and a reservoir at the top. Pack thecontents of the tube by tapping the column on a paddedsurface, and then by tamping firmly from the top. Place 100mL of methylene chloride in the reservoir, and adjust thestopcock so that 50 mL of eluate is collected in 20 to 30 min.Add 25 �L of Internal Standard Stock Solution to the eluate,mix, and then concentrate the solution to a volume of 1.0 mLin a suitable concentrator tube fitted with a modified Snydercolumn, using a Kontes tube heater maintained at 90°.
Procedure Inject 2.5 �L of the Test Preparation into asuitable gas chromatograph equipped with a flame-ionizationdetector and a 3-m × 2-mm (id) glass column, or equivalent,packed with 3% phenyl methyl silicone (OV-17, AppliedScience Laboratories, Inc., or equivalent) on 100- to 120-mesh, silanized, calcined, diatomaceous silica (Gas-ChromQ, Applied Science, or equivalent).
Caution: The glass column should extend into the injec-tor for on-column injection and into the detector baseto avoid contact with metal.
Maintain the column at 108°. Set the injection port temperatureto 225° and the detector to 250°. Use helium as the carrier gas,with a flow rate of 30 mL/min. Set the instrument attenuationsetting so that 2.5 �L of the Diluted Standard Preparationcontaining 200 �g/mL of each toluenesulfonamide gives aresponse of 40% to 80% of full-scale deflection. Record the
30 / Ammonium Sulfate / Monographs FCC V
chromatogram, note the peaks for o-toluenesulfonamide, p-toluenesulfonamide, and the n-tricosane internal standard, andcalculate the areas for each peak by suitable means. Theretention times for o-toluenesulfonamide, p-toluenesulfon-amide, and n-tricosane are about 5, 6, and 15 min, respectively.
In a similar manner, obtain the chromatograms for 2.5-�Lportions of each of the five Diluted Standard Preparations,and for each solution, determine the areas of the o-toluenesul-fonamide, p-toluenesulfonamide, and n-tricosane peaks. Fromthe values thus obtained, prepare standard curves by plottingconcentration of each toluenesulfonamide, in micrograms permilliliter, versus the ratio of the respective toluenesulfonamidepeak area to that of n-tricosane. From the standard curve,determine the concentration, in micrograms per milliliter, ofeach toluenesulfonamide in the Test Preparation. Divide eachvalue by 2 to convert the result to milligrams per kilogramof the toluenesulfonamide in the 2-g sample taken for analysis.
Note: If the toluenesulfonamide content of the sampleis greater than about 500 mg/kg, the impurity maycrystallize out of the methylene chloride concentrate(see Test Preparation). Although this level of impurityexceeds that permitted by the specification, the analysismay be completed by diluting the concentrate withmethylene chloride containing 250 �g of n-tricosane permilliliter, and by applying appropriate dilution factorsin the calculation. Care must be taken to redissolvecompletely any crystalline toluenesulfonamide to givea homogeneous solution.
Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Ammonium Sulfate
(NH4)2SO4 Formula wt 132.14
INS: 517 CAS: [7783-20-2]
DESCRIPTION
Ammonium Sulfate occurs as colorless or white crystals orgranules that decompose at temperatures above 280°. Onegram is soluble in about 1.5 mL of water. It is insoluble inalcohol. The pH of a 0.1 M solution is between 4.5 and 6.0.
Function Dough conditioner; yeast nutrient.
REQUIREMENTS
Identification A sample gives positive tests for Ammoniumand for Sulfate, Appendix IIIA.Assay Not less than 99.0% and not more than 100.5% of(NH4)2SO4.
Lead Not more than 3 mg/kg.Residue on Ignition Not more than 0.25%.Selenium Not more than 0.003%.
TESTS
Assay Transfer about 2 g of sample, accurately weighed,into a 250-mL flask, and dissolve it in 100 mL of water. Add40 mL of a mixture of equal volumes of formaldehyde andwater, previously neutralized to phenolphthalein TS with 1N sodium hydroxide. Mix, allow to stand for 30 min, andtitrate the mixture with 1 N sodium hydroxide to a pinkendpoint that persists for 5 min. Each milliliter of 1 N sodiumhydroxide is equivalent to 66.06 mg of (NH4)2SO4.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Selenium Determine as directed in Method II under Sele-nium Limit Test, Appendix IIIB, using 200 mg of sample.
Packaging and Storage Store in well-closed containers.
Amyris Oil, West Indian Type
Sandalwood Oil, West Indian Type
DESCRIPTION
Amyris Oil, West Indian Type, occurs as a clear, pale yellow,viscous liquid having a distinct odor suggestive of sandal-wood. It is the volatile oil obtained by steam distillation fromthe wood of Amyris balsamifera L. (Fam. Rutaceae). It issoluble in most fixed oils and usually in mineral oil. It issoluble in an equal volume of propylene glycol, the solutionoften becoming opalescent on further dilution. It is practicallyinsoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseshown in the respective spectrum in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 3.0.Angular Rotation Between +10° and +53°.Ester Value Not more than 7.Ester Value after Acetylation Between 115 and 165.Refractive Index Between 1.503 and 1.512 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.943 and 0.976.
View IR
FCC V Monographs / Angelica Seed Oil / 31
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed in Ester Value underEsters, Appendix VI, using about 5 g of sample, accuratelyweighed.Ester Value after Acetylation Determine as directed underTotal Alcohols, Appendix VI, using about 2 g of the driedacetylated oil, accurately weighed. Reflux for a period of 2h. Calculate the Ester Value after Acetylation by the formula
A × 28.05/B,
in which A is the number of milliliters of 0.5 N alcoholicpotassium hydroxide consumed in the saponification, and Bis the weight, in grams, of the acetylated oil used in the test.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 80% alcohol, often with opalescence.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made of aluminumor glass or that are lined with tin.
Angelica Root Oil
CAS: [8015-64-3]
DESCRIPTION
Angelica Root Oil occurs as a pale yellow to deep amberliquid with a warm, pungent odor and bittersweet taste. It isobtained by steam distillation of the dried slender rootlets ofAngelica archangelica L. (Fam. Umbelliferae). It is solublein most fixed oils, slightly soluble in mineral oil, but relativelyinsoluble in glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 7.0.Angular Rotation Between 0° and +46°.Ester Value Between 10 and 65.
Refractive Index Between 1.473 and 1.487 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.850 and 0.880.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed under Ester Value, Ap-pendix VI, using about 5 g of sample, accurately weighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 1 mL of 90% alcohol, often with turbidity, and remains insolution on further addition of alcohol to a total of 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined. The oil increases inspecific gravity and viscosity during storage.
Angelica Seed Oil
DESCRIPTION
Angelica Seed Oil occurs as a light yellow liquid having asweeter and more delicate aroma than the root oil. It is obtainedby steam distillation of the fresh seeds of Angelica archan-gelica L. (Fam. Umbelliferae). It is soluble in most fixedoils, slightly soluble in mineral oil, but relatively insolublein glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseshown in the respective spectrum in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 3.0.Angular Rotation Between +4° and +16°.Ester Value Between 14.0 and 32.0.Refractive Index Between 1.480 and 1.488 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.853 and 0.876.
View IR
View IR
32 / Anise Oil / Monographs FCC V
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed under Ester Determina-tion, Appendix VI, using about 5 g of sample, accuratelyweighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 4 mL of 90% alcohol, often with considerable turbidity,and it remains in solution on further addition of alcohol to atotal of 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Anise Oil
CAS: [8007-70-3]
DESCRIPTION
Anise Oil occurs as a colorless to pale yellow, strongly refrac-tive liquid with the characteristic odor and taste of anise. Itis obtained by steam distillation of the dried ripe fruit ofPimpinella anisum L. (Fam. Umbelliferae) or Illicium verumHooker filius (Fam. Magnoliaceae).
Note: If solid material has separated, carefully warmthe sample until it is completely liquefied, and mixbefore using it.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between +1° and –1°.Phenols Passes test.Refractive Index Between 1.553 and 1.560 at 20°.Solidification Point Not lower than 15°.Solubility in Alcohol Passes test.Specific Gravity Between 0.978 and 0.988.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Phenols Prepare a 1:3 solution of recently distilled samplein 90% alcohol. It is neutral to moistened litmus paper, andadding 1 drop of ferric chloride TS to 5 mL of the solutionproduces no blue or brown color.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solidification Point Determine as directed under Solidifica-tion Point, Appendix IIB.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI, using 3 mL of 90% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Annatto Extracts
INS: 160b CAS: [1393-63-1]
DESCRIPTION
Annatto Extracts occur as dark red solutions, emulsions, orsuspensions in water or oil or as dark red powders. The extractis prepared from annatto seed, Bixa orellana L. (Fam. Bixa-ceae), using a food-grade extraction solvent. Bixin is theprincipal pigment of oil-soluble Annatto Extracts. Norbixinis the principal pigment of alkaline water-soluble AnnattoExtracts. Commercial preparations are usually mixtures ofbixin, norbixin, and other carotenoids.
Function Color.
REQUIREMENTS
IdentificationA. Oil- and Water-Soluble Annatto Extracts Oil-soluble
Annatto Extracts diluted with acetone exhibit absorbance max-ima at 439, 470, and 501 nm. Water-soluble Annatto Extractsdiluted with water exhibit absorbance maxima at 451 to 455nm and 480 to 484 nm.
B. Carr-Price Reaction (See Chromatography, Appen-dix IIA.) Prepare a small chromatography column by fillinga 200- × 7-mm glass tube, stoppered with glass wool, withalumina (80- to 200-mesh) slurried in toluene so that thesettled alumina fills about 2⁄3 of the tube. Using a rubber outlettube and clamp, adjust the flow rate to about 30 drops/min.
Oil-Soluble Annatto Extracts Add to the top of the alu-mina column 3 mL of a solution containing sufficient sample,
View IR
FCC V Monographs / �-Apo-8′-Carotenal / 33
in toluene, to impart a color equivalent to a solution of 0.1%potassium dichromate. Elute with toluene until a pale yellowfraction is washed from the column. Wash the column withthree 10-mL volumes of dry acetone, add 5 mL of Carr-PriceReagent (see Solutions and Indicators), and allow it to runonto the top of the column. The orange-red zone (bixin) atthe top of the column immediately turns blue-green.
Water-Soluble Annatto Extracts Transfer 2 mL or 2 g ofsample into a 50-mL separatory funnel, and add sufficient 2N sulfuric acid to make the solution acidic to pH test paper(pH 1 to 2). Dissolve the red precipitate of norbixin by mixingthe solution with 50 mL of toluene. Discard the water layer,and wash the toluene phase with water until it no longergives an acid reaction. Remove any undissolved norbixin bycentrifugation or filtration, and dry the solution over anhy-drous sodium sulfate. Transfer 3 to 5 mL of the dry solutionto the top of an alumina column prepared as described above.Elute the column with toluene, three 10-mL volumes of dryacetone, and 5 mL of Carr-Price Reagent (see Solutions andIndicators) added to the top of the column. The orange-redband of norbixin immediately turns blue-green.Arsenic Not more than 3 mg/kg.Color Intensity A sample meets the representations of thevendor.Lead Not more than 10 mg/kg.Residual Solvents Acetone: Not more than 0.003%; Hex-anes: Not more than 0.0025%; Isopropyl Alcohol: Not morethan 0.005%; Methyl Alcohol: Not more than 0.005%; Trichlo-roethylene and Dichloromethane: Not more than 0.003%, indi-vidually or in combination.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Color Intensity
Oil-Soluble Annatto Extracts Transfer a sample, accu-rately weighed, into a solution of 1% glacial acetic acid inacetone, and dilute to a suitable volume (absorbance of 0.5to 1.0). Filter the sample to clarify if necessary. Measure theabsorbance at 454 nm, and calculate the color intensity bythe formula
A/(b × c),
in which A is the absorbance of the Sample Solution; b is thelength, in centimeters, of the cell; and c is the concentration,in grams per liter, of the Sample Solution.
Water-Soluble Annatto Extracts Determine as directedunder Oil-Soluble Annatto Extracts (above), but dissolve thesample in 0.1 M sodium hydroxide, and measure the ab-sorbance at 453 nm.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Residual Solvents Determine as directed under ResidualSolvent, Appendix VIII.
Packaging and Storage Store under refrigeration in full,well-closed containers that are made from steel or aluminumand that are suitably lined.
�-Apo-8′-CarotenalApocarotenal; APO
CH3H3C
CH3
CHO
CH3CH3
CH3 CH3
C30H40O Formula wt 416.65
INS: 160e CAS: [1107-26-2]
DESCRIPTION
�-Apo-8′-Carotenal occurs as a fine, crystalline powder witha dark, metallic sheen. It is freely soluble in chloroform andsparingly soluble in acetone, but it is insoluble in water. Itmelts at 136° to 140° with decomposition.
Function Color.
REQUIREMENTS
IdentificationA. Determine the absorbance of Sample Solution B, pre-
pared as directed in the Assay (below), at 488 nm and at 460nm. The ratio A488/A460 is between 0.77 and 0.85.
B. Determine the absorbance of Sample Solution B at 460nm, and that of Sample Solution A, prepared as directed inthe Assay (below), at 332 nm. The ratio A332/A460 is between0.63 and 0.75.Assay Not less than 96.0% and not more than 101.0% ofC30H40O.Arsenic Not more than 1 mg/kg.Lead Not more than 10 mg/kg.Residue on Ignition Not more than 0.2%.
TESTS
Assay (Note: Carry out all work in low-actinic glasswareand in subdued light.)
Sample Solution A Transfer about 40 mg of sample, accu-rately weighed, into a 100-mL volumetric flask, dissolve in10 mL of acid-free chloroform, dilute to volume with cyclo-hexane, and mix. Pipet 2 mL of this solution into a 50-mLvolumetric flask, dilute to volume with cyclohexane, and mix.
Sample Solution B Pipet 5 mL of Sample Solution A intoa 50-mL volumetric flask, dilute to volume with cyclohexane,and mix.
Procedure Determine the absorbance of Sample SolutionB in a 1-cm cell at the wavelength of maximum absorption
34 / Arabinogalactan / Monographs FCC V
at about 460 nm, with a suitable spectrophotometer, usingcyclohexane as the blank. Calculate the quantity, in milli-grams, of C30H40O in the sample taken by the formula
25,000A/264,
in which A is the absorbance of the solution and 264 is theabsorptivity of pure �-Apo-8′-Carotenal.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in tight, light-resistant con-tainers under inert gas.
Arabinogalactan
Larch Fiber; Larch Gum
INS: 409 CAS: [9036-66-2]
DESCRIPTION
Arabinogalactan occurs as a white to yellow-white, coarse orfine powder. It is the dried water extract from the wood ofthe larch trees Larix occidentalis and Larix laricina (Fam.Pinaceae). It is a highly branched polysaccharide that has amolecular weight of 15,000 to 60,000 daltons and is composedof galactose units and arabinose units in the approximate ratioof 6:1. It is freely dispersible in hot or cold water. It is insolublein alcohol.
Function Dietary fiber; humectant; stabilizer.
REQUIREMENTS
Assay (Total Carbohydrates) Not less than 80% of Arabino-galactan.Identification Add 20 g of sample to 20 mL of water, andstir until completely dissolved. Pour the solution into a 500-mL beaker, and add 100 mL of water. Transfer 7 mL of theresulting solution into 250-mL beaker and add 0.2 mL ofdiluted lead subacetate TS. No precipitate forms. Add 280mL of 95% ethyl alcohol to the remainder of the solution. Aprecipitate forms.Ash (Total) Not more than 10.0%.Lead Not more than 0.1 mg/kg.Insoluble Matter Not more than 0.1%.Loss on Drying Not more than 8.0%.Protein Not more than 1.0%.Starch Passes test.Carbohydrates (Total) Not less than 80.0%.
TESTS
Assay (Total Carbohydrates) The remainder, after sub-tracting from 100% the sum of the percentages of Ash, Losson Drying, and Protein, represents the percent of total carbo-hydrates (as arabinogalactan) in the sample.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Insoluble Matter Dissolve 5 g of sample in about 100 mLof water contained in a 250-mL Erlenmeyer flask, add 10 mLof 2.7 N hydrochloric acid, and boil gently for 15 min. Usesuction to filter the hot solution through a tared, filtered cruci-ble; wash thoroughly with hot water; dry at 105° for 2 h; andweigh.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 5 h.Protein Determine as directed under Nitrogen Determina-tion, Appendix IIIC, transferring about 3.5 g of sample, accu-rately weighed, into a 500-mL Kjeldahl flask. The percent ofprotein equals the percent of N × 6.25.Starch Add a few drops of iodine TS to a 1:10 aqueoussolution. No blue or red color appears.
Packaging and Storage Store in well-closed containers.
L-ArginineL-2-Amino-5-guanidinovaleric Acid
NHCH2CH2CH2CCOOH
C
NH2
NH H NH2
C6H14N4O2 Formula wt 174.20
CAS: [74-79-3]
DESCRIPTION
L-Arginine occurs as white crystals or as a white crystallinepowder. It is soluble in water, insoluble in ether, and sparinglysoluble in alcohol. It is strongly alkaline, and its water solu-tions absorb carbon dioxide from the air.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.
View IR
FCC V Monographs / L-Arginine Monohydrochloride / 35
Assay Not less than 98.5% and not more than 101.5% ofC6H14N4O2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 1.0%.Optical (Specific) Rotation [�]D
20°: Between +26.0° and+27.9°, calculated on the dried basis; or [�]D
25°: Between+25.8° and +27.7°, calculated on the dried basis.Residue on Ignition Not more than 0.2%.
TESTS
Assay Dissolve about 200 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to a green endpoint or until the blue colordisappears completely. Each milliliter of 0.1 N perchloricacid consumed in the assay is equivalent to 8.710 mg ofC6H14N4O2.
Caution: Handle perchloric acid in an appropriatefume hood.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying the sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 8 g of a previously dried sample in sufficient 6 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
L-Arginine MonohydrochlorideL-2-Amino-5-guanidinovaleric Acid Monohydrochloride
NHCH2CH2CH2CCOOH
C NH2HNH
NH2·HCl
C6H14N4O2·HCl Formula wt 210.66
CAS: [1119-34-2]
DESCRIPTION
L-Arginine Monohydrochloride occurs as a white or nearlywhite crystalline powder. It is soluble in water, slightly soluble
in hot alcohol, and insoluble in ether. It is acidic and meltswith decomposition at about 235°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H14N4O2·HCl, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Optical (Specific) Rotation [�]D
20°: Between +21.3° and23.5°, calculated on the dried basis; or [�]D
25°: Between 21.3°and +23.4°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 100 mg of sample, previously driedat 105° for 3 h and accurately weighed, in 2 mL of formicacid, add exactly 15.0 mL of 0.1 N perchloric acid, and heaton a water bath for 30 min.
Caution: Handle perchloric acid in an appropriatefume hood.
After cooling, add 45 mL of glacial acetic acid, and titratethe excess perchloric acid with 0.1 N sodium acetate, determin-ing the endpoint potentiometrically. Perform a blank determi-nation (see General Provisions), and make any necessarycorrection. Each milliliter of 0.1 N perchloric acid is equivalentto 10.53 mg of C6H14N4O2·HCl.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying the sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 8 g of a previously dried sample in sufficient 6 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
View IR
36 / Ascorbic Acid / Monographs FCC V
Ascorbic AcidVitamin C; L-Ascorbic Acid
O
OH OH
OHOCH2C
OH
H
C6H8O6 Formula wt 176.13
INS: 300 CAS: [50-81-7]
DESCRIPTION
Ascorbic Acid occurs as white or slightly yellow crystals oras powder. It melts at about 190°. It gradually darkens onexposure to light, is reasonably stable in air when dry, butrapidly deteriorates in solution in the presence of air. Onegram is soluble in about 3 mL of water and in about 30 mLof alcohol. It is insoluble in chloroform and in ether.
Function Antioxidant; meat-curing aid; nutrient.
REQUIREMENTS
IdentificationA. A 1:50 aqueous solution slowly reduces alkaline cupric
tartrate TS at 25°, but more readily upon heating.B. The infrared absorption spectrum of a potassium bro-
mide dispersion of the sample exhibits maxima at the samewavelengths as those of a similar preparation of USP AscorbicAcid Reference Standard.Assay Not less than 99.0% and not more than 100.5% ofC6H8O6.Lead Not more than 2 mg/kg.Optical (Specific) Rotation [�]D
25°: Between +20.5° and+21.5°.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 400 mg of sample, accuratelyweighed, in a mixture of 100 mL of water, recently boiledand cooled, and 25 mL of 2 N sulfuric acid. Titrate the solutionimmediately with 0.1 N iodine, adding starch TS near theendpoint. Each milliliter of 0.1 N iodine is equivalent to 8.806mg of C6H8O6.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 1 g of sample in 10 mL of carbon dioxide-freewater.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in tight, light-resistant con-tainers.
Ascorbyl PalmitatePalmitoyl L-Ascorbic Acid
O
OH OH
OC
OH
H
H2COOC(H2C)14H3C
C22H38O7 Formula wt 414.54
INS: 304 CAS: [137-66-6]
DESCRIPTION
Ascorbyl Palmitate occurs as a white or yellow-white powder.It is very slightly soluble in water and in vegetable oils. Onegram dissolves in about 4.5 mL of alcohol.
Function Antioxidant.
REQUIREMENTS
Identification A 1:10 solution in alcohol decolorizes dichlo-rophenol–indophenol TS.Assay Not less than 95.0% of C22H38O7, calculated on thedried basis.Lead Not more than 2 mg/kg.Loss on Drying Not more than 2%.Melting Range Between 107° and 117°.Optical (Specific) Rotation [�]D
25°: Between +21° and +24°,calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 300 mg of sample, accuratelyweighed, in 50 mL of alcohol contained in a 250-mL Erlen-meyer flask, add 30 mL of water, and immediately titratewith 0.1 N iodine to a yellow color that persists for at least30 s. Each milliliter of 0.1 N iodine is equivalent to 20.73mg of C22H38O7.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in a vacuum oven at 560°to 600° for 1 h.Melting Range Determine as directed in Procedure forClass Ia, under Melting Range or Temperature, Appendix IIB.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 1 g of sample in 10 mL of methanol.
FCC V Monographs / Aspartame / 37
Residue on Ignition Determine as directed in Method Iunder Residue on Ignition, Appendix IIC, igniting a 2-gsample.
Packaging and Storage Store in tightly closed containers,preferably in a cool, dry place.
L-AsparagineL-�-Aminosuccinamic Acid
H2NCOCH2CCOOH
H NH2
C4H8N2O3 Formula wt, anhydrous 132.12C4H8N2O3·H2O Formula wt, monohydrate 150.13
CAS: anhydrous [70-47-3]CAS: monohydrate [5794-13-8]
DESCRIPTION
L-Asparagine occurs as white crystals or as a crystalline pow-der. It is soluble in water and practically insoluble in alcoholand in ether. Its solutions are acid to litmus. It melts atabout 234°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.0% and not more than 101.5% ofC4H8N2O3, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Between 11.5% and 12.5%.Optical (Specific) Rotation [�]D
20°: Between +33.0° and+36.5°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 130 mg of sample, previously driedat 130° for 3 h and accurately weighed, in 3 mL of formicacid and 50 mL of glacial acetic acid, and titrate with 0.1 Nperchloric acid, determining the endpoint potentiometrically.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.21 mg of C4H8N2O3.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed for
organic compounds, and using 5 �g of lead (Pb) ion in thecontrol.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 130° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 10 g of a previously dried sample in sufficient 6N hydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
AspartameN-L-�-Aspartyl-L-phenylalanine 1-Methyl Ester; APM
OCH3
O
HONH
OH
H NH2O
C14H18N2O5 Formula wt 294.31
INS: 951 CAS: [22839-47-0]
DESCRIPTION
Aspartame occurs as a white, crystalline powder. It is sparinglysoluble in water and slightly soluble in alcohol. The pH of a0.8% solution is between about 4.5 and 6.0.
Function Sweetener; sugar substitute; flavor enhancer.
REQUIREMENTS
Identification The infrared absorption spectrum of a potas-sium bromide dispersion of sample exhibits maxima only atthe same wavelengths as those of a similar preparation ofUSP Aspartame Reference Standard.Assay Not less than 98.0% and not more than 102.0% ofC14H18N2O5, calculated on the dried basis.5-Benzyl-3,6-dioxo-2-piperazineacetic Acid Not morethan 1.5%.Lead Not more than 1 mg/kg.Loss on Drying Not more than 4.5%.Optical (Specific) Rotation [�]D
20°: Between +14.5° and+16.5°, calculated on the dried basis.Other Related Substances Not more than 2.0%.Residue on Ignition Not more than 0.2%.
TESTS
Assay Transfer about 300 mg of sample, accuratelyweighed, to a 150-mL beaker, dissolve in 1.5 mL of 96%
View IR
38 / Aspartame / Monographs FCC V
formic acid, and add 60 mL of glacial acetic acid. Add crystalviolet TS, and titrate immediately with 0.1 N perchloric acidto a green endpoint.
Caution: Handle perchloric acid in an appropriatefume hood.
Note: Use 0.1 N perchloric acid previously standardizedto a green endpoint. A blank titration exceeding 0.1mL may be due to excessive water content and maycause loss of visual endpoint sensitivity.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 29.43 mg of C14H18N2O5.5-Benzyl-3,6-dioxo-2-piperazineacetic Acid
Mobile Phase Weigh and transfer 5.6 g of potassiumphosphate monobasic into a 1-L flask, add 820 mL of water,and dissolve. Adjust the pH to 4.3 using phosphoric acid, add180 mL of methanol, and mix. Filter through a 0.45-�m disk,and de-gas.
Diluting Solvent Add 200 mL of methanol to 1800 mLof water, and mix.
Impurity Standard Preparation Transfer about 25 mg ofUSP 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid ReferenceStandard, accurately weighed, into a 100-mL volumetric flask.Add 10 mL of methanol, and dissolve. Dilute to volume withwater, and mix. Pipet 15 mL of this solution into a 50-mLvolumetric flask, dilute to volume with Diluting Solvent, andmix. Use a freshly prepared solution.
Sample Preparation Transfer about 50 mg of sample,accurately weighed, to a 10-mL volumetric flask. Dilute tovolume with Diluting Solvent, and mix. Use a freshly preparedsolution.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a suitable high-performance liquid chromato-graph equipped with a detector measuring at 210 nm and a250- × 4.6-mm (id) column packed with octadecyl silanizedsilica (10-�m Partisil ODS-3, or equivalent), and operatedunder isocratic conditions at 40°. The flow rate of the MobilePhase is about 2 mL/min.
System Suitability The area responses of three replicateinjections of Impurity Standard Preparation show a relativestandard deviation of not more than 2.0%.
Procedure Separately inject equal 20-�L portions of Im-purity Standard Preparation and Sample Preparation into thechromatograph, and record the chromatograms (the approxi-mate retention time of 5-benzyl-3,6-dioxo-2-piperazineaceticacid is 4 min, and the approximate retention time of Aspartameis 11 min). Measure the peak area response of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in each chromatogram. Calcu-late the percentage of 5-benzyl-3,6-dioxo-2-piperazineaceticacid in the sample by the formula
1000(AUCS)/(ASWU),
in which AU and AS are the peak area responses of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the Sample Preparationand in the Impurity Standard Preparation, respectively; CS
is the concentration, in milligrams per milliliter, of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the Impurity Standard
Preparation; and WU is the weight, in milligrams, of Aspar-tame taken for the Sample Preparation.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 1-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 4 g of sample in sufficient 15 N formic acid tomake 100 mL. Make the determination within 30 min ofpreparing the Sample Solution.Other Related Impurities Proceed as directed in the testfor 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid (above), ex-cept use the following in place of the Standard Preparation:
Other Related Substances Standard Preparation Pipet 2mL of the Sample Preparation from the test for 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid into a 100-mL volumetricflask, dilute to volume with the Diluting Solvent, and mix.
Procedure Inject about 20-�L portions of the Other Re-lated Substances Standard Preparation and the Sample Prepa-ration into the chromatograph, and record the chromatogramfor a time equal to twice the retention time of Aspartame. Inthe chromatogram obtained from the Sample Preparation, thesum of the responses of all secondary peaks, other than thatfor 5-benzyl-3,6-dioxo-2-piperazineacetic acid, is not morethan the response of the Aspartame peak obtained in thechromatogram from the Other Related Substances StandardPreparation.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers ina cool, dry place.
FCC V Monographs / Aspartame-Acesulfame Salt / 39
Aspartame-Acesulfame SaltAPM-Ace; [2-carboxy-�-(N-(b-methoxycarbonyl-2-phenyl)ethylcarbamoyl)]ethanaminium 6-methyl-4-oxo-1,2,3-oxathiazin-3-ide-2,2-dioxide; L-Phenylalanine, L-�-aspartyl-2-methyl ester compound with 6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (1:1)
CH2
HOOCCH2CHCONHCHCOOCH3
NH3
OSO2N
O
CH3
C18H23O9N3S Formula wt 457.45
CAS: [106372-55-8]
DESCRIPTION
Aspartame-Acesulfame Salt occurs as a white, crystallinepowder. It is sparingly soluble in water and slightly solublein alcohol.
Function Sweetener.
REQUIREMENTS
Identification An infrared absorption spectrum of a potas-sium bromide dispersion of Aspartame-Acesulfame Salt ex-hibits maxima only at the same wavelengths as those of atypical spectrum as shown in the section on Infrared Spectra,using the same test conditions as specified therein.Assay Not less than 63.0% and not more than 66.0% ofaspartame, calculated on the dried basis. Not less than 34.0%and not more than 37.0% of acesulfame, calculated as acidformed on the dried basis.5-Benzyl-3,6-dioxo-2-piperazineacetic Acid (DKP) Notmore than 0.5%.Lead Not more than 1 mg/kg.Loss on Drying Not more than 0.5%.Optical (Specific) Rotation [�]D
20°: Between +14.5° and+16.5°, calculated on the dried basis.Other Related Impurities Not more than 1.0%.Potassium Not more than 0.5%.
TESTS
Assay (Note: Use a combination pH-electrode for all titra-tions.) Dissolve 0.100 to 0.150 g of sample, accurately
weighed, in 50 mL of ethanol. Under a flow of nitrogen,titrate the solution with standardized 0.1 N tetrabutylammon-ium hydroxide in methanol or 2-propanol. Determine the vol-ume of titrant needed to reach the first equivalence point (V1
mL) and the second equivalence point (V2 mL).Perform a blank titration with 50 mL of ethanol. Calculate
the percent Acesulfame and Aspartame, respectively, takenby the formulas
[(V1 – VB) × N × 163]/(10 × W),
[(V2 – V1) × N × 294]/(10 × W),
in which V1, V2, and VB are the number of milliliters of 0.1N tetrabutylammonium hydroxide in methanol or 2-propanolused for the sample (first and second equivalence points) andthe blank, respectively; N is the normalityof the tetrabutylam-monium hydroxide; W is the weight, in grams, of sampletaken; and 163 and 294 are the formula weights of acesulfameand aspartame, respectively.5-Benzyl-3,6-dioxo-2-piperazineacetic Acid
Mobile Phase Dissolve 5.6 g of potassium phosphate,monobasic, accurately weighed, in 820 mL of water containedin a 1-L flask. Use phosphoric acid to adjust the pH to 4.3,add 180 mL of methanol, and mix. Filter through a 0.45-�mdisk, and de-gas.
Diluting Solvent Add 200 mL of methanol to 1800 mLof water, and mix.
Standard Preparation Transfer about 25 mg of USP Ref-erence Standard 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid,accurately weighed, into a 100-mL volumetric flask. Add 10mL of methanol, and dissolve. Dilute to volume with water,and mix. Pipet 15 mL of this solution into a 50-mL volumetricflask, dilute to volume with the Diluting Solvent, and mix.Use a freshly prepared solution on the day of use.
Sample Preparation Transfer about 50 mg of sample,accurately weighed, into a 10-mL volumetric flask. Dilute tovolume with Diluting Solvent, and mix. Use a freshly preparedsolution on the day of use.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a suitable high-performance liquid chromato-graph equipped with a detector measuring at 210 nm and a250- × 4.6-mm (id) column packed with octadecyl silanizedsilica (10-�m Partisil ODS-3, or equivalent), and operatedunder isocratic conditions at 40°. The flow rate of the MobilePhase is about 2 mL/min.
System Suitability The area responses of three replicateinjections of the Standard Preparation show a relative stan-dard deviation of not more than 2.0%.
Procedure Separately inject equal 20-�L portions of theStandard Preparation and the Sample Preparation into thechromatograph, and record the chromatograms (the approxi-mate retention time of 5-benzyl-3,6-dioxo-2-piperazineaceticacid is 4 min, and the approximate retention time of aspartameis 11 min). Measure the peak area response of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in each chromatogram.
Calculation Calculate the percent 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the sample taken by the formula
1000 × (AUCS)/(ASWU),
View IR
40 / DL-Aspartic Acid / Monographs FCC V
in which AU and AS are the peak area responses of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the Sample Preparationand in the Standard Preparation, respectively; CS is the con-centration, in milligrams per milliliter, of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the Standard Preparation; and WU
is the weight, in milligrams, of sample taken for the SamplePreparation.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 6.2 g of sample in sufficient 15 N formic acid tomake 100 mL. Make the determination within 30 min ofpreparation of the Sample Solution. Divide the calculatedspecific rotation by 0.646 to correct for the Aspartame contentin Aspartame-Acesulfame Salt.Other Related Impurities Determine as directed in the testfor 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid (above), butuse the following Standard Preparation and Procedure:
Standard Preparation Pipet 1.5 mL of the Sample Prepa-ration from the test for 5-Benzyl-3,6-dioxo-2-piperazineaceticAcid into a 100-mL volumetric flask, dilute to volume withthe Diluting Solvent, and mix.
Procedure Separately inject equal 20-�L portions of theStandard Preparation and the Sample Preparation into thechromatograph, and record the chromatograms for a timeequal to twice the retention time of Aspartame. In the chroma-togram obtained from the Sample Preparation, the sum ofthe responses of all secondary peaks, other than those for 5-benzyl-3,6-dioxo-2-piperazineacetic acid and Acesulfame, isnot more than the response of the Aspartame peak obtainedin the chromatogram from the Standard Preparation.Potassium
Standard Solutions Transfer 190.7 mg of potassium chlo-ride, previously dried at 105° for 2 h, into a 1000-mL volumet-ric flask, dilute to volume with water, and mix. Transfer 100.0mL of this solution to a second 1000-mL volumetric flask,dilute to volume with water, and mix to obtain a Stock Solutioncontaining 10 �g of potassium per milliliter (equivalent to19.07 �g of potassium chloride). Pipet 10.0-, 15.0-, and 20.0-mL aliquots of the Stock Solution into separate 100-mL volu-metric flasks; add 2.0 mL of a 1:5 solution of sodium chlorideand 1.0 mL of hydrochloric acid to each; dilute with waterto volume; and mix. The Standard Solutions obtained contain,respectively, 1.0, 1.5, and 2.0 �g of potassium per milliliter.
Test Solution Transfer about 3.0 g of sample, accuratelyweighed, into a 500-mL volumetric flask, dilute to volumewith water, and mix. Transfer 10 mL of this solution into a100-mL volumetric flask and add 2.0 mL of a 1:5 sodiumchloride solution and 1.0 mL of hydrochloric acid, dilute tovolume with water, and mix. Filter the solution.
Procedure Concomitantly determine the absorbances ofthe Standard Solutions and the Test Solution at the potassiumemission line of 766.5 nm, using a suitable atomic absorptionspectrophotometer equipped with a potassium hollow-cathode
lamp and an air–acetylene flame, using water as the blank.Plot the absorbance of the Standard Solutions versus concen-tration, in micrograms per milliliter, of potassium, and drawthe straight line best fitting the plotted points. From the graphso obtained, determine the concentration, C, in microgramsper milliliter, of potassium in the Test Solution. Calculate thepercent potassium in the sample taken by the formula
500C/W,
in which W is the quantity, in milligrams, of sample taken toprepare the Test Solution.
Packaging and Storage Store in well-closed containers ina cool, dry place.
DL-Aspartic AcidDL-Aminosuccinic Acid
HOOCCH2CH(NH2)COOH
C4H7NO4 Formula wt 133.10
CAS: [617-45-8]
DESCRIPTION
DL-Aspartic Acid occurs as colorless or white crystals. It isslightly soluble in water, but insoluble in alcohol and in ether.It is optically inactive and melts with decomposition atabout 280°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC4H7NO4, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 200 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to a green endpoint or until the blue colordisappears completely.
Caution: Handle perchloric acid in an appropriatefume hood.
View IR
FCC V Monographs / Azodicarbonamide / 41
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.31 mg of C4H7NO4.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
L-Aspartic AcidL-Aminosuccinic Acid
HOOCCH2CCOOH
H NH2
C4H7NO4 Formula wt 133.10
CAS: [56-84-8]
DESCRIPTION
L-Aspartic Acid occurs as white crystals or as a crystallinepowder. It is slightly soluble in water, but insoluble in alcoholand in ether. It melts at about 270°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC4H7NO4, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.25%.Optical (Specific) Rotation [�]D
20°: Between +24.5° and+26.0°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 200 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to a green endpoint or until the blue colordisappears completely.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.31 mg of C4H7NO4.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 8 g of a previously dried sample in sufficient 6 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
AzodicarbonamideAzodicarboxylic Acid Diamide
H2N C
O
N N C
O
NH2
C2H4N4O2 Formula wt 116.08
INS: 927a CAS: [123-77-3]
DESCRIPTION
Azodicarbonamide occurs as a yellow to orange-red, crystal-line powder. It is practically insoluble in water and in mostorganic solvents. It is slightly soluble in dimethyl sulfoxide.It melts above 180° with decomposition.
Function Maturing agent for flour.
REQUIREMENTS
Identification A solution of 35 mg of sample in 1000 mLof water exhibits an ultraviolet absorption maximum at about245 nm.Assay Not less than 98.6% and not more than 100.5% ofC2H4N4O2 after drying.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.5%.Nitrogen Between 47.2% and 48.7%.pH of a 2% Suspension Not less than 5.0.Residue on Ignition Not more than 0.15%.
TESTS
Assay Transfer about 225 mg of sample, previously driedin a vacuum oven at 50° for 2 h and accurately weighed, into
View IR
42 / Balsam Peru Oil / Monographs FCC V
a 250-mL glass-stoppered iodine flask. Add about 23 mL ofdimethyl sulfoxide to the flask, washing any adhered sampledown with the solvent, then stopper the flask, and place about2 mL of the solvent in the cup or lip of the flask. Swirloccasionally, until complete solution of the sample is effected,and then loosen the stopper to drain the remainder of solventinto the flask and to rinse down any dissolved sample intothe solution. Add 5.0 g of potassium iodide followed by15 mL of water, then immediately pipet 10 mL of 0.5 Nhydrochloric acid into the flask, and rapidly stopper. Swirluntil the potassium iodide dissolves, and allow to stand for20 to 25 min protected from light. Titrate the liberated iodinewith 0.1 N sodium thiosulfate to the disappearance of theyellow color. Titrate with additional thiosulfate if any yellowcolor appears within 15 min. Perform a blank determination(see General Provisions) on a solution consisting of 25 mLof dimethyl sulfoxide, 5.0 g of potassium iodide, 15 mL ofwater, and 5 mL of 0.5 N hydrochloric acid, and make anynecessary correction. Each milliliter of 0.1 N sodium thiosul-fate is equivalent to 5.804 mg of C2H4N4O2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead ion (Pb) in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in a vacuum oven at 50°for 2 h.Nitrogen Transfer about 50 mg of sample into a 100-mLKjeldahl flask, add 3 mL of concentrated hydriodic acid solu-tion (57% freshly assayed), and digest the mixture with gentleheating for 1.25 h, adding sufficient water, when necessary,to maintain the original volume. Increase the heat at the endof the digestion period, and continue heating until the volumeis reduced by about one-half. Cool to room temperature, add1.5 g of potassium sulfate, 3 mL of water, and 4.5 mL ofsulfuric acid, and heat until iodine fumes no longer evolve.Allow the mixture to cool, wash down the sides of the flaskwith water, heat until charring occurs, and again cool to roomtemperature. Add 40 mg of mercuric oxide to the charredmaterial, heat until the color of the solution is pale yellow, thencool, wash down the sides of the flask with a few milliliters ofwater, and digest the mixture for an additional 3 h. Cool thedigest, add 20 mL of ammonia-free water, 16 mL of a 50%sodium hydroxide solution, and 5 mL of a 44% sodium thiosul-fate solution. Immediately connect the flask to a distillationapparatus as directed under Nitrogen Determination, Appen-dix IIIC, and distill, collecting the distillate in 10 mL of a4% boric acid solution. Add a few drops of methyl red–methylene blue TS to the distillate, and titrate with 0.05 Nsulfuric acid. Perform a blank determination (see GeneralProvisions), and make any necessary correction. Each millili-ter of 0.05 N sulfuric acid is equivalent to 0.7004 mg ofnitrogen.pH of a 2% Suspension Determine as directed under pHDetermination, Appendix IIB, using the following solution:Add 2 g of sample to 100 mL of water, and agitate the mixturewith a power stirrer for 5 min.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1.5-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
Balsam Peru Oil
CAS: [8007-00-9]
DESCRIPTION
Balsam Peru Oil occurs as a yellow to pale brown, slightlyviscous liquid having a sweet, balsamic odor. It is obtainedby extraction or distillation of Peruvian Balsam obtained fromMyroxylon pereirae Royle Klotzsche (Fam. Leguminosae).Occasionally, crystals may occur within the liquid. It is solublein most fixed oils, and is soluble, with turbidity, in mineraloil. It is partly soluble in propylene glycol, but it is practicallyinsoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Between 30 and 60.Angular Rotation Between –1° and +2°.Ester Value Between 200 and 225.Refractive Index Between 1.567 and 1.579 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 1.095 and 1.110.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed in Ester Value underEsters, Appendix VI, using about 1 g of sample, accuratelyweighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 90% alcohol and remains in solution upondilution to 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
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FCC V Monographs / Basil Oil, European Type / 43
Basil Oil, Comoros Type
Basil Oil Exotic; Basil Oil, Réunion Type
DESCRIPTION
Basil Oil, Comoros Type, occurs as a light yellow liquidwith a spicy odor. It is obtained by steam distillation of theflowering tops or the entire plant of Ocimum basilicum L.(Fam. Lamiaceae). It may be distinguished from other types,such as basil oil, European type, by its camphoraceous odorand physicochemical constants. It is soluble in most fixed oilsand, with turbidity, in mineral oil. One milliliter is soluble in20 mL of propylene glycol with slight haziness, but it isinsoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 1.0.Angular Rotation Between –2° and +2°.Ester Value after Acetylation Between 25 and 45.Refractive Index Between 1.512 and 1.520 at 20°.Saponification Value Between 4 and 10.Solubility in Alcohol Passes test.Specific Gravity Between 0.952 and 0.973.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value after Acetylation Determine as directed underLinalool Determination, Appendix VI, using about 2.5 g of thedry acetylated oil, accurately weighed, for the saponification.Calculate the Ester Value after Acetylation by the formula
a × 28.05/b,
in which a is the volume, in milliliters, of 0.5 N alcoholicpotassium hydroxide consumed in the saponification, and bis the weight of the dry acetylated oil, in grams, used in the test.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed in Saponifica-tion Value under Esters, Appendix VI, using about 5 g ofsample, accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 4 mL of 80% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Basil Oil, European Type
Basil Oil, Italian Type; Sweet Basil OilCAS: [8015-73-4]
DESCRIPTION
Basil Oil, European Type, occurs as a pale yellow to yellowliquid with a floral-spicy odor. It is obtained by the steamdistillation of the flowering tops or the entire plant of Ocimumbasilicum L. It may be distinguished from other types, suchas basil oil, Comoros type, or basil oil, Réunion type, by itsmore floral odor and its physicochemical constants. It is solu-ble in most fixed oils and, with turbidity, in mineral oil. Onemilliliter is soluble in 20 mL of propylene glycol with slighthaziness, but it is insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Acid Value Not more than 2.5.Angular Rotation Between –5° and –15°.Ester Value after Acetylation Between 140 and 180.Refractive Index Between 1.483 and 1.493 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.900 and 0.920.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value after Acetylation Determine as directed underLinalool Determination, Appendix VI, using 2.5 g of thedry acetylated oil, accurately weighed, for the saponification.Calculate the Ester Value after Acetylation by the formula
a × 28.05/b,
in which a is the number of milliliters of 0.5 N alcoholicpotassium hydroxide consumed in the saponification, and bis the weight, in grams, of the acetylated oil used in the test.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.
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44 / Bay Oil / Monographs FCC V
Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI, using 4 mL of 80% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Bay Oil
Myrcia Oil
DESCRIPTION
Bay Oil occurs as a yellow or brown-yellow liquid with apleasant, aromatic odor and a pungent, spicy taste. It is thevolatile oil distilled from the leaves of Pimenta acris Kostel(Fam. Myrtaceae). It is soluble in alcohol and in glacial aceticacid. Its solutions in alcohol are acid to litmus.
Function Flavoring agent.
REQUIREMENTS
IdentificationA. The infrared absorption spectrum of the sample exhibits
relative maxima at the same wavelengths as those shown inthe respective spectrum in the section on Infrared Spectra,using the same test conditions as specified therein.
B. Shake 1 mL of sample with 20 mL of hot water, andfilter. The filtrate gives not more than a slight acid reactionwith litmus, and on the addition of 1 drop of ferric chloride TSyields only a transient gray-green, not a blue or purple, color.Assay Not less than 50% and not more than 65%, by volume,of phenols.Angular Rotation Levorotatory, but not more than –3°.Refractive Index Between 1.507 and 1.516 at 20°.Specific Gravity Between 0.950 and 0.990.
TESTS
Assay Determine as directed under Phenols, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using in a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers in acool place protected from light.
Beeswax, White
White Wax
INS: 901
DESCRIPTION
Beeswax, White, occurs as a yellow-white solid, somewhattranslucent in thin layers, with a faint, characteristic odor,free from rancidity. It is the bleached, purified wax from thehoneycomb of the bee Apis mellifera L. (Fam. Apidae), andit consists primarily of myricyl palmitate (myricin), ceroticacid and ester, and some high-carbon paraffins. Its specificgravity is about 0.95. Beeswax, White, is insoluble in waterand sparingly soluble in cold alcohol. Boiling alcohol dis-solves cerotic acid and part of the myricin. It is completelysoluble in chloroform, in ether, and in fixed and volatile oils.It is partly soluble in cold carbon disulfide and is completelysoluble in it at temperatures of 30° or above.
Function Surface-finishing (glazing) agent; release agent;raw material for flavoring agent.
REQUIREMENTS
Acid Value Between 17 and 24.Carnauba Wax Passes test.Ester Value Between 72 and 79.Fats, Japan Wax, Rosin, and Soap Passes test.Lead Not more than 5 mg/kg.Melting Range Between 62° and 65°.Saponification Cloud Test Passes test.
TESTS
Acid Value Warm about 3 g of sample, accurately weighed,in a 200-mL flask with 25 mL of absolute alcohol, previouslyneutralized to phenolphthalein with potassium hydroxide, un-til the sample is melted. Shake the mixture, add 1 mL ofphenolphthalein TS, and titrate the warm solution with 0.5 Nalcoholic potassium hydroxide to a permanent, faint pinkcolor. Save this solution for the Ester Value test.Carnauba Wax Place 100 mg of sample in a test tube, andadd 20 mL of n-butanol. Immerse the test tube in boilingwater, and shake the mixture gently until solution is complete.Transfer the test tube into a beaker of water at 60°, and allowit to cool to room temperature. A loose mass of fine, needle-like crystals separate from a clear mother liquor. Under themicroscope, the crystals appear as loose needles or stellateclusters, and no amorphous masses are observed, indicatingthe absence of carnauba wax.Ester Value Add 25.0 mL of 0.5 N alcoholic potassiumhydroxide and 50 mL of alcohol to the solution resulting fromthe determination of Acid Value, heat the mixture under areflux condenser for 4 h, and titrate the excess alkali with 0.5N hydrochloric acid. Perform a residual blank titration, andcalculate the Ester Value as the number of milligrams of
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FCC V Monographs / Bentonite / 45
potassium hydroxide required for each gram of the sampletaken for the test.Fats, Japan Wax, Rosin, and Soap Boil 1 g of sample for30 min with 35 mL of a 1:7 sodium hydroxide solution,maintaining the volume by the occasional addition of water,and cool the mixture. The wax separates and the liquid remainsclear. Filter the cold mixture, and acidify the filtrate withhydrochloric acid. No precipitate forms.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Melting Range Determine as directed in Procedure forClass II under Melting Range or Temperature, Appendix IIB.Saponification Cloud Test
Saponifying Solution Dissolve 40 g of potassium hydrox-ide in about 900 mL of aldehyde-free alcohol maintained ata temperature of 15° until solution is complete. Warm to roomtemperature, and add sufficient aldehyde-free alcohol to make1000 mL.
Procedure Transfer 3.00 g of sample into a round-bottom,100-mL boiling flask provided with a ground-glass joint, add30 mL of the Saponifying Solution, attach a reflux condenserto the flask, and heat the mixture gently on a steam bath for2 h. At the end of this period, remove the reflux condenser,insert a thermometer into the solution, and place the flask inan 80° water bath. Rotate the flask while both the bath andthe solution cool to 65°. The solution shows no cloudinessor globule formation before this temperature is reached.
Packaging and Storage Store in well-closed containers.
Beeswax, Yellow
Yellow Wax
INS: 901 CAS: [8012-89-3]
DESCRIPTION
Beeswax, Yellow, occurs as a yellow to gray-brown solidwith an agreeable, honey odor. It is the purified wax fromthe honeycomb of the bee Apis mellifera L. (Fam. Apidae)and consists primarily of myricyl palmitate (myricin), ceroticacid and ester, and some high-carbon paraffins. It is somewhatbrittle when cold, and presents a dull, granular, noncrystallinefracture when broken. It becomes pliable at a temperature ofabout 35°. Its specific gravity is about 0.95. Beeswax, Yellow,is insoluble in water and sparingly soluble in cold alcohol.Boiling alcohol dissolves cerotic acid and part of the myricin.It is completely soluble in chloroform, in ether, and in fixedand volatile oils. It is partly soluble in cold carbon disulfideand completely soluble in it at temperatures of 30° or above.
Function Candy glaze and polish; raw material for flavor-ing agent.
REQUIREMENTS
Acid Value Between 18 and 24.Carnauba Wax Passes test.Ester Value Between 72 and 77.Fats, Japan Wax, Rosin, and Soap Passes test.Lead Not more than 5 mg/kg.Melting Range Between 62° and 65°.Saponification Cloud Test Passes test.
TESTS
Proceed as directed in the monograph for Beeswax, White.
Packaging and Storage Store in well-closed containers.
Bentonite
Smectite; Aluminum Silicate
INS: 558 CAS: [1302-78-9]
DESCRIPTION
Bentonite occurs commercially as powders ranging in colorsand tints from off white to pale brown to gray depending onthe cations present in natural deposits. It comprises naturalsmectite clays consisting primarily of colloidal hydrated alu-minum silicates of the montmorillonite or hectorite type ofminerals with varying quantities of alkalies, alkaline earths,and iron. It is insoluble in water, in alcohol, in dilute acids,and in alkalies.
Function Clarifying, filter agent.
REQUIREMENTS
IdentificationA. With intense agitation, add 2 g of sample, in small
portions, to 100 mL of water. Allow the mixture to stand for12 h to ensure complete hydration. Place 2 mL of the mixtureso obtained on a suitable glass slide, and allow it to air dryat room temperature to produce an oriented film. Place theslide in a vacuum desiccator over a free surface of ethyleneglycol. Evacuate the desiccator, and close the stopcock sothat ethylene glycol saturates the desiccator chamber. Allowthe slide to stand for 12 h. Record the X-ray diffraction patternusing a copper source, and calculate the d values. The largestpeak corresponds to a d value between 15.0 and 17.2 Å.Prepare a random powder specimen of sample, and determinethe d values in the region between 1.48 and 1.54 Å. The peakis between 1.492 and 1.504 Å or between 1.510 and 1.540 Å.
B. Add 1 g of potassium nitrate and 3 g of anhydroussodium carbonate to 0.5 g of sample contained in a metalcrucible, heat until the mixture has melted, and allow it tocool. Add 20 mL of boiling water to the residue, mix, filter,
46 / Benzoic Acid / Monographs FCC V
and wash the residue with 50 mL of water. Add 1 mL ofhydrochloric acid and 5 mL of water to the residue, and filter.Add 1 mL of 10 N sodium hydroxide to the filtrate, filter,and add 3 mL of 2 M ammonium chloride. A gelatinous,white precipitate forms.Arsenic Not more than 5 mg/kg.Coarse Particles Not more than 0.5% of sample is retainedon a 75-�m sieve.Gel Formation Passes test.Lead Not more than 0.004%.Loss on Drying Not more than 8.0%.Microbial Limits:
Aerobic Plate Count Not more than 1000 CFU per gram.E. coli Negative in 25 g.
pH of a 1:50 Dispersion Between 8.5 and 10.5.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using 5.0 mL of the Standard Arsenic Solutionand a 25-mL aliquot of the following Sample Solution: Trans-fer 8.0 g of dried sample into a 250-mL beaker containing100 mL of 1:25 hydrochloric acid, mix, cover with a watchglass, and boil gently, stirring occasionally, for 15 min withoutallowing excessive foaming. Filter the hot supernatant liquidthrough a rapid-flow filter paper into a 200-mL volumetricflask, and wash the filter with four 25-mL portions of hot,1:25 hydrochloric acid, collecting the washings in the volu-metric flask. Cool the combined filtrates to room temperature,add 1:25 hydrochloric acid to volume, and mix.Coarse Particles Add 100 mL of water to 20 g of sample,and mix for 15 min at not less than 5000 rpm. Transfer themixture to a wet sieve of nominal mesh aperture (75 �m),previously dried at 100° to 105° and weighed, and wash withthree 500-mL volumes of water, ensuring that any agglomer-ates are dispersed. Dry at 100° to 105°, and weigh. Thedifference in weight corresponds to the measure of coarseparticles.Gel Formation Mix 6 g of sample with 300 mg of magne-sium oxide. Add the mixture, in several divided portions, to200 mL of water contained in a blender jar with an approxi-mately 500-mL capacity. Blend thoroughly for 5 min at highspeed, transfer 100 mL of the mixture into a 100-mL graduatedcylinder, and leave undisturbed for 24 h. Not more than 2mL of supernatant liquid appears on the surface.Lead (Note: The Standard Preparation and the Test Prepa-ration may be modified, if necessary, to obtain solutions ofsuitable concentrations, adaptable to the linear or workingrange of the spectrophotometer used.)
Standard Preparation On the day of use, dilute 3.0 mL ofLead Nitrate Stock Solution (see the Flame Atomic AbsorptionMethod under Lead Limit Test, Appendix IIIB) to 100 mL withwater. Each milliliter of the Standard Preparation contains theequivalent of 3 �g of lead.
Test Preparation Transfer 3.75 g of dried sample into a250-mL beaker containing 100 mL of 1:25 hydrochloric acid,stir, cover with a watch glass, and boil for 15 min. Cool toroom temperature, and allow the insoluble matter to settle.Decant the supernatant liquid through a rapid-flow filter paper
into a 400-mL beaker. Wash the filter with four 25-mL por-tions of hot water, collecting the filtrate in the 400-mL beaker.Concentrate the combined extracts by gentle boiling to ap-proximately 20 mL. If a precipitate forms, add 2 to 3 dropsof nitric acid, heat to boiling, and cool to room temperature.Filter the concentrated extracts through a rapid-flow filterpaper into a 50-mL volumetric flask. Transfer the remainingcontents of the 400-mL beaker through the filter paper andinto the flask with water. Dilute to volume with water, and mix.
Procedure Determine the absorbances of the Test Prepa-ration and the Standard Preparation at 284 nm in a suitableatomic absorption spectrophotometer equipped with a leadhollow-cathode lamp, deuterium arc background correction,and a single-slot burner, using an oxidizing air–acetyleneflame. The absorbance of the Test Preparation is not greaterthan that of the Standard Preparation.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Microbial Limits (Note: Current methods for the followingtests may be found online at <www.cfsan.fda.gov/~ebam/bam-toc.html>):
Aerobic Plate CountE. coli
pH of a 1:50 Dispersion Disperse 4.0 g of sample in 200mL of water, mixing vigorously to facilitate wetting, anddetermine as directed under pH Determination, Appendix IIB.
Packaging and Storage Store in tight containers.
Benzoic Acid
COOH
C7H6O2 Formula wt 122.12
INS: 210 CAS: [65-85-0]
DESCRIPTION
Benzoic Acid occurs as white crystals, scales, or needles. Itbegins to sublime at about 100° and is volatile with steam.One gram is soluble in 275 mL of water at 25°, in 20 mL ofboiling water, in 3 mL of alcohol, in 5 mL of chloroform,and in 3 mL of ether. It is soluble in fixed and in volatileoils and is sparingly soluble in hexane.
Function Preservative; antimicrobial agent.
REQUIREMENTS
Identification Dissolve 1 g of sample in a 20:1 (v/v) mixtureof water and 1 N sodium hydroxide, filter the solution, and
FCC V Monographs / Benzoyl Peroxide / 47
add about 1 mL of ferric chloride TS. A buff-colored precipi-tate forms.Assay Not less than 99.5% and not more than 100.5% ofC7H6O2, calculated on the anhydrous basis.Lead Not more than 2.0 mg/kg.Readily Carbonizable Substances Passes test.Readily Oxidizable Substances Passes test.Residue on Ignition Not more than 0.05%.Solidification Point Between 121° and 123°.Water Not more than 0.7%.
TESTS
Assay Dissolve about 500 mg of sample, accuratelyweighed, in 25 mL of 50% alcohol previously neutralizedwith 0.1 N sodium hydroxide, add phenolphthalein TS, andtitrate with 0.1 N sodium hydroxide. Each milliliter of 0.1 Nsodium hydroxide is equivalent to 12.21 mg of C7H6O2.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Readily Carbonizable Substances Dissolve 500 mg ofsample in 5 mL of 95% sulfuric acid, and proceed as directedunder Readily Carbonizable Substances, Appendix IIB. Theresulting color is no darker than Matching Fluid Q.Readily Oxidizable Substances Add 0.1 N potassium per-manganate, dropwise, to a mixture of 100 mL of water and1.5 mL of sulfuric acid heated to 100°, until a pink colorpersists for 30 s. Dissolve 1.0 g of sample in the hot solution,and titrate with 0.1 N potassium permanganate to a pinkcolor that persists for 15 s. The volume of 0.1 N potassiumpermanganate consumed does not exceed 0.5 mL.Residue on Ignition Ignite 2 g as directed in Method Iunder Residue on Ignition (Sulfated Ash), Appendix IIC.Solidification Point Determine as directed under Solidifica-tion Point, Appendix IIB.Water Determine as directed in the Karl Fischer TitrimetricMethod under Water Determination, Appendix IIB, usingmethanol in pyridine (1:2) as the solvent.
Packaging and Storage Store in well-closed containers.
Benzoyl Peroxide
C O O C
O O
C14H10O4 Formula wt 242.23
INS: 928 CAS: [94-36-0]
DESCRIPTION
Benzoyl Peroxide occurs as a colorless, crystalline solid. Itis insoluble in water, slightly soluble in alcohol, and solublein chloroform and in ether. It melts between 103° and 106°with decomposition.
Caution: Benzoyl Peroxide, especially in the dry form,is a dangerous, highly reactive oxidizing material andhas been known to explode spontaneously. Observesafety precautions printed on the label of the container.
Function Bleaching agent.
REQUIREMENTS
Identification Add 50 mL of 0.5 N alcoholic potassiumhydroxide to 500 mg of sample, heat gradually to boiling,and continue boiling for 15 min. Cool, dilute to 200 mLwith water, and make the solution strongly acid with 0.5 Nhydrochloric acid. Extract with ether, dry the extract withanhydrous sodium sulfate, and then evaporate to dryness ona steam bath. The residue of benzoic acid so obtained meltsbetween 121° and 123° (see Melting Range or Temperature,Appendix IIB).Assay Not less than 96.0% of C14H10O4.Lead Not more than 4 mg/kg.
TESTS
Assay Dissolve about 250 mg of sample, accuratelyweighed, in 15 mL of acetone contained in a 100-mL glass-stoppered bottle, and add 3 mL of a 1:2 solution of potassiumiodide. Swirl for 1 min, then immediately titrate with 0.1 Nsodium thiosulfate (without the addition of starch TS). Eachmilliliter of 0.1 N sodium thiosulfate is equivalent to 12.11mg of C14H10O4.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds from the residue of the following mixture,and 4 �g of lead (Pb) ion in the control: Mix 1 g of samplewith 10 mL of 1 N sodium hydroxide, slowly evaporate todryness on a steam bath, and cool.
Packaging and Storage Store in the original container, andobserve the safety precautions printed on the label.
48 / Bergamot Oil, Coldpressed / Monographs FCC V
Bergamot Oil, Coldpressed
CAS: [8007-75-8]FEMA: 2153
DESCRIPTION
Bergamot Oil, Coldpressed, occurs as a green to yellow-greenor yellow-brown liquid with a fragrant, sweet-fruity odor. Itis a volatile oil obtained by pressing, without the aid of heat,the fresh peel of the fruit of Citrus bergamia Risso et Poiteau(Fam. Rutaceae). It is miscible with alcohol and with glacialacetic acid. It is soluble in most fixed oils, but is insolublein glycerin and in propylene glycol. It may contain a suitableantioxidant.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 36.0% of esters, calculated as linalylacetate (C12H20O2).Angular Rotation Between +12° and +30°.Refractive Index Between 1.465 and 1.468 at 20°.Residue on Evaporation Not more than 6.0%.Solubility in Alcohol Passes test.Specific Gravity Between 0.875 and 0.880.Ultraviolet Absorbance Not less than 0.32.
TESTS
Assay Determine as directed in Ester Determination underEsters, Appendix VI, using a 2-g sample, accurately weighed,but heat the mixture for 30 min on the steam bath. Use 98.15as the equivalence factor (e) in the calculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, using an Abbé or other refractometer of equal or greateraccuracy.Residue on Evaporation Determine as directed under Resi-due on Evaporation, Appendix VI, heating a sample for 5 h.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 90% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).Ultraviolet Absorbance Determine as directed under Ultra-violet Absorbance of Citrus Oils, Appendix VI, using about 50mg of sample, accurately weighed. The absorbance maximumoccurs at 315 � 3 nm.
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
BHAButylated Hydroxyanisole
OH
OCH3
C(CH3)3
C11H16O2 Formula wt 180.25
INS: 320 CAS: [25013-16-5]
DESCRIPTION
BHA occurs as a white or slightly yellow, waxy solid. It ispredominantly 3-tert-butyl-4-hydroxyanisole (3-BHA), withvarying amounts of 2-tert-butyl-4-hydroxyanisole (2-BHA).It melts between 48° and 63°. It is freely soluble in alcoholand in propylene glycol, and insoluble in water.
Function Antioxidant.
REQUIREMENTS
Identification Add 2 mL of sodium borate TS and 1 mLof a 1:10,000 solution of 2,6-dichloroquinonechlorimide:ab-solute alcohol to 5 mL of a 1:10,000 solution of sample in72% alcohol, and mix. A blue color appears.Assay Not less than 98.5% of C11H16O2.Residue on Ignition Not more than 0.05%.
TESTS
AssayInternal Standard Use 4-tert-butylphenol.Internal Standard Solution Dissolve about 500 mg of In-
ternal Standard, accurately weighed, in acetone contained ina 100-mL volumetric flask, add acetone to volume, and mix.
Standard Preparation Dissolve together accuratelyweighed quantities of USP Reference Standards 3-tert-Butyl-4-hydroxyanisole and 2-tert-Butyl-4-hydroxyanisole to finalconcentrations of 9 mg/mL and 1 mg/mL, respectively, insufficient Internal Standard Solution to make 10 mL.
Assay Preparation Dissolve about 100 mg of sample,accurately weighed, in the Internal Standard Solution con-tained in a 10-mL volumetric flask, dilute to volume with theInternal Standard Solution, and mix.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flame-ionization detector and containing a 1.8-m × 2-mm (id) stain-less-steel column, or equivalent, packed with 10% siliconeGE XE-60, or equivalent. Maintain the column isothermallyat a temperature between 175° and 185°, and use helium asthe carrier gas at a flow rate of 30 mL/min. Chromatographa sufficient number of injections of the Standard Preparation,
View IR
FCC V Monographs / Biotin / 49
and record the areas as directed under Procedure to ensurethat the relative standard deviation does not exceed 2.0% forthe 3-tert-butyl-4-hydroxyanisole isomer and 6.0% for the 2-tert-butyl-4-hydroxyanisole isomer; the resolution betweenthe isomers is not less than 1.3; and the tailing factor doesnot exceed 2.0.
Procedure Separately inject suitable portions (about 5�L) of the Standard Preparation and the Assay Preparationinto the gas chromatograph, and record the chromatograms.Measure the areas under the peaks for each isomer and theInternal Standard in each chromatogram, and calculate thequantity, (I), in milligrams, of each isomer in the sample takenby the equation
I = 10 × CS × (RU/RS),
in which CS is the concentration, in milligrams per milliliter,of the isomer in the Standard Preparation; RU is the ratio ofthe area of the isomer to that of the Internal Standard in thechromatogram from the Assay Preparation; and RS is the ratioof the area of the isomer to that of the Internal Standard inthe chromatogram from the Standard Preparation. Calculatethe weight, in milligrams, of C11H16O2 in the sample takenby adding the quantities of the two isomers.Residue on Ignition Determine as directed in Method Iunder Residue on Ignition, Appendix IIC, igniting a 10-gsample.
Packaging and Storage Store in well-closed containers.
BHTButylated Hydroxytoluene; 2,6-Di-tert-butyl-p-cresol
OH
CH3
C(CH3)3(CH3)3C
C15H24O Formula wt 220.35
INS: 321 CAS: [128-37-0]
DESCRIPTION
BHT occurs as a white, crystalline solid. It is freely solublein alcohol, and insoluble in water and in propylene glycol.
Function Antioxidant.
REQUIREMENTS
Identification Dissolve 200 mg of 3,3′-dimethoxybenzidinedihydrochloride in a mixture of 40 mL of methanol and 60mL of 1 N hydrochloric acid. Add 5 mL of the resultingdianisidine solution to 10 mL of water and 2 mL of a 3:100solution of sodium nitrite. Add this solution to 10 mL of a1:100,000 solution of sample:methanol. An orange-red colorappears within 3 min. Add 5 mL of chloroform, and shake.The chloroform layer exhibits a purple or magenta color thatfades when exposed to light.Assay Not less than 99.0% of C15H24O.Residue on Ignition Not more than 0.002%.
TESTS
Assay The solidification point (see Solidification Point, Ap-pendix IIB) of a sample is not lower than 69.2°, indicating apurity of not less than 99.0% of C15H24O.Residue on Ignition Transfer about 50 g of sample, accu-rately weighed, into a tared crucible, ignite until thoroughlycharred, and cool. Moisten the ash with 1 mL of sulfuric acid,and complete the ignition by heating for 15-min periods at800° � 25° to constant weight.
Packaging and Storage Store in well-closed containers.
Biotincis-Hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-valericAcid; d-Biotin
O
CHN NH
HC CH
H2C CH(CH2)4COOHS
C10H16N2O3S Formula wt 244.31
CAS: [58-85-5]
DESCRIPTION
Biotin occurs as a practically white, crystalline powder. It isstable to air and heat. One gram dissolves in about 5000 mLof water at 25° and in about 1300 mL of alcohol; it is moresoluble in hot water and in dilute alkali, and it is insolublein other common organic solvents.
Function Nutrient.
REQUIREMENTS
IdentificationA. The infrared absorption spectrum of a potassium bro-
mide dispersion of the sample exhibits relative maxima at the
50 / Birch Tar Oil, Rectified / Monographs FCC V
same wavelengths as those of a similar preparation of USPBiotin Reference Standard.
B. A saturated solution of sample in warm water decolor-izes bromine TS, added dropwise.Assay Not less than 97.5% and not more than 100.5% ofC10H16N2O3S.Lead Not more than 2 mg/kg.Melting Range Between 229° and 232°, with decompo-sition.Optical (Specific) Rotation [�]D
20°: Between +89° and +93°.
TESTS
Assay Mix about 500 mg of sample, accurately weighed,with 100 mL of water, add phenolphthalein TS, and whileheating and stirring continuously, slowly titrate the suspensionwith 0.1 N sodium hydroxide to a pink color. Each milliliterof 0.1 N sodium hydroxide is equivalent to 24.43 mg ofC10H16N2O3S.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutionin 0.1 N sodium hydroxide containing 500 mg of sample ineach 25 mL.
Packaging and Storage Store in tight containers.
Birch Tar Oil, Rectified
DESCRIPTION
Birch Tar Oil, Rectified, occurs as a clear, dark brown liquidwith a strong leather odor. It is the pyroligneous oil obtainedby dry distillation of the bark and the wood of Betula pendulaRoth and related species of Betula (Fam. Betulaceae) andrectified by steam distillation. It is soluble in most fixed oils,but it is insoluble in glycerin, in mineral oil, and in propyleneglycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Solubility in Alcohol Passes test.Specific Gravity Between 0.886 and 0.950.
TESTS
Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of absolute alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Black Pepper Oil
CAS: [8006-82-4]
DESCRIPTION
Black Pepper Oil occurs as an almost colorless to slightlygreen liquid with the characteristic odor of pepper and arelatively mild taste. It is the volatile oil obtained by steamdistillation from the dried, unripened fruit of the plant Pipernigrum L. (Fam. Piperaceae). It is soluble in most fixed oils,in mineral oil, and in propylene glycol. It is sparingly solublein glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Angular Rotation Between –1° and –23°.Refractive Index Between 1.479 and 1.488 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.864 and 0.884.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 95% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
View IR
View IR
FCC V Monographs / Bois de Rose Oil / 51
Bohenin1,3-Behenic-2-oleic Glyceride
CH2OC(CH2)20CH3
O
CHOC(CH2)7CH
CH2OC(CH2)20CH3
OCH(CH2)7CH3
O
DESCRIPTION
Bohenin occurs as a white to light tan, waxy solid. It is atriglyceride containing behenic acid at the 1- and 3-positionsand oleic acid at the 2-position. Behenic acid is a saturatedfatty acid that occurs naturally in peanuts, most seed fats,animal milk fat, and marine oils. It is produced by the interes-terification of triolein and ethyl behenate in the presence ofa suitable lipase enzyme preparation. It melts at approximately52°. It is insoluble in water; soluble in hexane, in chloroform,and in acetone; and slightly soluble in hot ethanol.
Function Tempering aid and antibloom agent in the manu-facture of chocolate and chocolate coatings.
REQUIREMENTS
Identification Bohenin exhibits the following compositionprofile of fatty acids determined as directed under Fatty AcidComposition, Appendix VII:
Fatty Acid: 16:0 18:0 18:1 20:0 22:0 24:0Weight % (Range): <1.5 <3.0 >25.0 <7.0 >58.0 <3.0
Acid Value Not more than 0.3Diglycerides Not more than 5.0%.Iodine Value Between 24 and 30.Lead Not more than 0.5 mg/kg.Peroxide Value Not more than 0.3 meq/kgSaponification Value Between 162 and 172.Triglycerides Not less than 95.0%.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VII.Diglycerides and Triglycerides
Sample Preparation Transfer 5 g of sample, accuratelyweighed, into a 50-mL beaker, and warm at 80° to melt.Transfer 300 �L of melted sample into a 10-mL volumetricflask, add 9 mL of acetone, and swirl to dissolve. If crystalsof Bohenin form, warm the flask to 40° in a water bath. Aftercomplete dissolution, fill the flask to 10 mL with acetone.
Standard Preparation Proceed as directed for the SamplePreparation, substituting a Bohenin standard (available fromLaroda Fine Chemicals AB, Limhamnsgardens allé 9, 216 16LIMHAMN Växel, Sweden) for the sample.
Procedure (See Chromatography, Appendix IIA.) Use asuitable high-performance liquid chromatograph equippedwith differential refractometer, autosampler injection unit,mobile-phase degasser, column heating block or oven, and acomputing integrator. The column is Lichrosorb RP-18 250-mm × 4.5 mm (id) (GL Science, Inc., or equivalent) andYMC-Pack ODA-A A-303 250-mm × 4.5 mm (id) (YMCCompany, Ltd., or equivalent) connected in a series, or equiva-lent, maintained at 50°. Use 80:20 acetone:acetonitrile as theeluent, at a flow rate of 2 mL/min.
Equilibrate the column at 50° by pumping eluent at 0.9 mL/min, until a stable baseline is obtained. Using the autosampler,inject duplicate 30-�L measures of Sample Preparation andStandard Preparation.
Calculation Calculate the percent of diglycerides in theSample Preparation by the following formula:
100(DG/SA)
in which DG is the total sum of the peak areas at retentiontimes between 11 and 14 min and SA is the total sum of allpeak areas.
Calculate the percent of triglycerides in the Sample Prepa-ration by the following formula:
%T = 100[(SA − DG)/SA].
Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Peroxide Value Determine as directed under PeroxideValue, Appendix VII.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.
Packaging and Storage Store in closed containers awayfrom excessive heat.
Bois de Rose Oil
DESCRIPTION
Bois de Rose Oil occurs as a colorless to pale yellow liquidwith a slightly camphoraceous, pleasant, floral odor. It is thevolatile oil obtained by steam distillation from the chippedwood of Aniba rosaeodora var. amazonica Ducke (Fam. Lau-raceae). The oils from the coastal region of Brazil and theAmazon valley tend to differ in odor and in linalool contentfrom that produced in the Loreto province of Peru. It is solublein most fixed oils and in propylene glycol. It is soluble in
View IR
52 / Brilliant Blue / Monographs FCC V
mineral oil, occasionally with turbidity, but it is only slightlysoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 82.0% and not more than 92.0% oftotal alcohols, calculated as linalool (C10H18O).Angular Rotation Between –4° and +6°.Distillation Range Not less than 70% distills between 195°and 205°.Refractive Index Between 1.462 and 1.470 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.868 and 0.889.
TESTS
Assay Determine as directed under Linalool Determination,Appendix VI, using about 1.2 g of acetylated sample, accu-rately weighed.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Distillation Range Determine as directed under DistillationRange, Appendix IIB, using 50 mL of the sample, previouslydried over anhydrous sodium sulfate, and a 125-mL distilla-tion flask.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 6 mL of 60% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Brilliant Blue1
Brilliant Blue FCF; CI 42090; Class: Triphenylmethane
C
N(C2H5)CH2
N(C2H5)CH2SO3
SO3Na
SO3Na
C37H34N2O9S3Na2 Formula wt 792.86
INS: 133 CAS: [3844-45-9]
DESCRIPTION
Brilliant Blue occurs as a dark purple to bronze powder orgranules. It is principally the disodium salt of ethyl[4-[p-[ethyl(m-sulfobenzyl)amino]-�-(o-sulfophenyl)benzylidene]-2,5-cyclohexadien-1-ylidene](m-sulfobenzyl) ammonium hy-droxide inner salt. It dissolves in water to give a solutiongreen-blue at neutrality, green in weak acid, and yellow instronger acid. Addition of base to its neutral solution producesa violet color only on boiling. When dissolved in concentratedsulfuric acid, it yields a yellow solution that turns green whendiluted with water. It is slightly soluble in ethanol.
Function Color.
REQUIREMENTS
Identification A freshly prepared aqueous solution con-taining 10 mg/L exhibits absorbance intensities (A) and wave-length maxima as follows: at pH 7, A = 1.11 at 630 nm; atpH 1, A = 0.95 at 629 nm, and A = 0.2 at 410 nm; and at pH13, A = 1.29 at 630 nm, and A = 0.15 at 408 nm.Assay Not less than 85.0% coloring matters.Arsenic Not more than 3 mg/kg.Chromium Not more than 0.0005%.Ether Extracts (combined) Not more than 0.2%.Lead Not more than 10 mg/kg.Leuco Base Not more than 5.0%.
1 To be used or sold for use to color food that is marketed in the UnitedStates, this color additive must be from a batch that has been certifiedby the U.S. Food and Drug Administration (FDA). If it is not from anFDA-certified batch, it is not a permitted color additive for food use inthe United States, even if it is compositionally equivalent. The nameFD&C Blue No. 1 can be applied only to FDA-certified batches of thiscolor additive. Brilliant Blue is a common name given to the uncertifiedcolorant. See the monograph entitled FD&C Blue No. 1 for directionsfor producing an FDA-certified batch.
FCC V Monographs / Brominated Vegetable Oil / 53
Loss on Drying (Volatile Matter) at 135°, Chloride, andSulfate (as sodium salts) Not more than 15.0% in combi-nation.Manganese Not more than 0.001%.Subsidiary Colors Not more than 6.0%, combined, of iso-meric disodium salts of ethyl[4-[p-[ethyl(p-sulfobenzyl)amino]-I-(o-sulfophenyl)benzylidene]-2,5-cyclohexadien-1-ylidene](p-sulfobenzyl) ammonium hydroxide inner salt, andethyl[4-[p-[ethyl(o-sulfobenzyl)amino]-�-(o-sulfophenyl)benzylidene] -2,5-cyclohexadien-1-ylidene] (o-sulfobenzyl)ammonium hydroxide inner salt.Uncombined Intermediates and Products of Side Reac-tions
o-, m-, and p-Sulfobenzaldehydes Not more than 1.5%,combined.
N-ethyl,N-(m-Sulfobenzyl)sulfanilic Acid Not more than0.3%.Water-Insoluble Matter Not more than 0.2%.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Chloride Determine as directed in Sodium Chloride underColors, Appendix IIIC.Chromium Determine as directed in Chromium under Col-ors, Appendix IIIC.Ether Extracts (combined) Determine as directed in EtherExtracts under Colors, Appendix IIIC.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Leuco Base Determine as directed in Leuco Base underColors, Appendix IIIC, using the following Sample Solution:Transfer approximately 120 mg of sample, accuratelyweighed, into a 1-L volumetric flask, and dissolve in anddilute to volume with water.Loss on Drying (Volatile Matter) at 135° Determine asdirected in Loss on Drying (Volatile Matter) under Colors,Appendix IIIC.Manganese A 1:20 aqueous solution gives positive tests forManganese, Appendix IIIA.Subsidiary Colors
Solvent System Use a solvent system composed of 50 mLof acetonitrile, 50 mL of isoamyl alcohol, 15 mL of 2-buta-none, 5 mL of water, and 5 mL of ammonium hydroxide.
Sample Solution Transfer approximately 1 g of sample,accurately weighed, into a 100-mL volumetric flask. Fill theflask about 3⁄4 full with water, place it in the dark for 1 h,dilute to volume with water, and mix well.
Procedure Spot 0.1 mL of the Sample Solution in a lineacross a 20- × 20-cm glass plate coated with a 0.25-mm layerof Silica Gel G, approximately 3 cm from the bottom edge.Allow the plate to dry for about 20 min in the dark, thendevelop with the Solvent System in an unlined tank equilibratedfor at least 20 min before the plate is inserted. Allow thesolvent front to reach within about 3 cm of the top of theplate. Dry the developed plate in the dark.
When the plate has dried, scrape off all the colored bandsabove the Brilliant Blue, which remains close to the origin,into a 30-mL beaker. Extract the subsidiary colors with three6-mL portions of 95% ethanol, or until no color remains onthe gel by visual inspection. Record the volume of ethanolused and the spectrum of the solution between 400 and 700nm. Calculate the percent of subsidiary colors by the formula
(A × V × 100)/(a × W × b),
in which A is the absorbance at the wavelength maximum; V isthe volume, in milliliters, of the solution; a is the absorptivity(0.126 mg/L/cm); W is the weight, in milligrams, of the sam-ple; and b is the pathlength of the cell.Sulfate Determine as directed in Sodium Sulfate under Col-ors, Appendix IIIC.Total Color Determine the total color strength as the weightpercent of the sample taken using Methods I and II in TotalColor under Colors, Appendix IIIC. Express the Total Coloras the average of the two results.
Method I (Sample Preparation) Transfer 50 to 75 mg ofsample, accurately weighed, into a 1-L volumetric flask, anddissolve in and dilute to volume with water. The absorptivity(a) for Brilliant Blue is 0.164 mg/L/cm at 630 nm.
Method II (Sample Preparation) Transfer approximately0.5 g of sample, accurately weighed, into the titration flask.The stoichiometric factor (Fs) for Brilliant Blue is 2.52.Uncombined Intermediates and Products of Side Reac-tions Determine as directed for Method I in UncombinedIntermediates and Products of Side Reactions under Colors,Appendix IIIC. Calculate the concentrations of m-sulfobenzal-dehyde and N-ethyl-N-(3-sulfobenzyl)-sulfanilic acid usingthe following absorptivities:
m-Sulfobenzaldehyde, a = 0.0495 mg/L/cm at 246 nm(acid solution).
N-Ethyl-N-(3-sulfobenzyl)-sulfanilic acid, a = 0.078 mg/L/cm at 277 nm (alkaline solution).Water-Insoluble Matter Determine as directed in Water-Insoluble Matter under Colors, Appendix IIIC.
Packaging and Storage Store in well-closed containers.
Brominated Vegetable Oil
DESCRIPTION
Brominated Vegetable Oil occurs as a pale yellow to darkbrown, viscous, oily liquid. It is a bromine addition productof vegetable oil or oils. It is soluble in chloroform, in ether,in hexane, and in fixed oils, and is insoluble in water.
Function Flavoring agent; beverage stabilizer.
REQUIREMENTS
Identification Mix about 0.2 mL of sample with 1 g ofanhydrous sodium carbonate in a suitable crucible, cover the
54 / Butadiene-Styrene Rubber / Monographs FCC V
mixture with an additional 1 g of sodium carbonate, compactthe mixture by gentle tapping, and heat the crucible over anopen flame until the crucible turns red. Cool the crucible andits contents, dissolve the residue in 20 mL of hot water, andfilter. Add 1.7 N nitric acid to the filtrate until effervescenceceases, then add 1 mL of silver nitrate TS. A curdy, yellowprecipitate forms that is insoluble in nitric acid but solublein an excess of stronger ammonia water.Free Bromine Passes test.Free Fatty Acids (as oleic acid) Not more than 2.5%.Iodine Value Not more than 16.Specific Gravity Within the range specified by the vendor.
TESTS
Free Bromine Dissolve 1 g of sample in 20 mL of acetone,add 1 g of sodium iodide, and allow the mixture to stand ina stoppered flask in the dark for 30 min, with occasionalshaking. Add 25 mL of water and 1 mL of starch TS. Noblue color appears.Free Fatty Acids (as oleic acid) Determine as directed underFree Fatty Acids, Appendix VII, using 28.2 as the equivalencefactor (e) in the calculation for oleic acid. Titrate with theappropriate normality of sodium hydroxide solution, shakingvigorously, to the first permanent pink color of the sameintensity as that of the neutralized alcohol (if the sample colorinterferes, titrate to a pH of 8.5, determined with a suitableinstrument).Iodine Value Determine as directed under Iodine Value,Appendix VII.Specific Gravity Determine by any reliable method (seeGeneral Provisions) at the temperature specified by thevendor.
Packaging and Storage Store in well-closed containers.
Butadiene-Styrene Rubber
CH CHCH2 CH2
C C
H H
CH2 CH2n n n
DESCRIPTION
Butadiene-Styrene Rubber occurs as a synthetic liquid latexor solid rubber produced by the emulsion polymerization ofbutadiene and styrene, using fatty acid soaps as emulsifiers,and a suitable catalyst, molecular weight regulator (if re-quired), and shortstop. It also occurs as a solid rubber producedby the solution copolymerization of butadiene and styrene ina hexane solution, using butyl lithium as a catalyst. Solventsand volatiles are removed by processing with hot water or bydrum drying.
The latex, which has a pH between 9.5 and 11.0 and asolids content between 26% and 63%, is coagulated with orwithout other food-grade ingredients in a heated kettle. Thecoagulated mass is squeezed to drain off sera, and the coagu-lum is washed with hot water (with or without alkali), and itis rinsed with water until the batch is neutral. Finally, thecoagulum is dried to remove residual volatiles. When butadi-ene-styrene rubber is purchased in the latex form, it must bewashed by the preceding or an equivalent procedure. In thecase of the solvent-polymerized product, solvent and volatilesare removed by processing in hot water or by drum drying.
Both of the solid forms are supplied by the manufacturereither as a slab or as a uniform, free-flowing crumb and maycontain a suitable food-grade antioxidant. The crumb form, inaddition, may contain a suitable food-grade partitioning agent.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Note: The following Requirements apply to the solidrubber as supplied by the manufacturer or to the washedand dried coagulum obtained from the latex as describedabove.
Identification Identify emulsion-polymerized Butadiene-Styrene Rubber latex and solid by comparing their infraredabsorption spectra with the respective four typical spectra asshown in the section on Infrared Spectra. Prepare latex sam-ples by first drying them at 105° for 4 h, then by dissolvingthem in hot toluene and evaporating on a potassium bromideplate. Prepare solid samples by dissolving them in hot tolueneand evaporating on a potassium bromide plate.Bound Styrene Between 1.0% and 50.0%.Cadmium Not more than 1 mg/kg.Lead Not more than 3 mg/kg.Lithium Not more than 0.0075%.Mercury Not more than 3 mg/kg.Quinones Not more than 0.002%.Residual Hexane Not more than 0.01%.Residual Styrene Not more than 0.003%.
TESTS
Bound Styrene Determine as directed under Bound Styrene,Appendix IV.Cadmium Determine as directed under Cadmium LimitTest, Appendix IIIB, or by the following procedure:
Note: For this assay, use reagent-grade chemicals withthe lowest practicable Sb, As, Bi, Cd, Cu, Pb, Hg, Ag,and Sn levels, and use only high-purity water and gases.Rinse all glass- and plasticware twice with 10% nitricacid and twice with 10% hydrochloric acid, and thenrinse thoroughly with High-Purity Water.
High-Purity Water Obtain High-Purity Water from amixed-bed strong-acid, strong-base ion exchange apparatuscapable of producing water of more than 15-megohm re-sistivity.
FCC V Monographs / Butadiene-Styrene Rubber / 55
10% Nitric Acid Solution Slowly add 100 mL of nitricacid to 500 mL of High-Purity Water contained in a 1000-mLvolumetric flask. Dilute to volume with High-Purity Water.
10% Hydrochloric Acid Solution Slowly add 100 mL ofhydrochloric acid to 500 mL of High-Purity Water containedin a 1000-mL volumetric flask. Dilute to volume with High-Purity Water.
Cadmium Stock Solution Use any commercially availableNIST traceable 1000 ppm plasma-grade standard stock solu-tion of cadmium.
Cadmium Calibration Standards Tare three clean, dry 4-oz polyethylene bottles (or equivalent). Add approximately50 g of High-Purity Water to each. Slowly add 28 � 1 g ofconcentrated nitric acid, mix thoroughly, slowly add 12 � 1g of concentrated hydrochloric acid, and mix thoroughly again.Using a precision micropipet, add 10, 50, and 500 �L, respec-tively, of Cadmium Stock Solution to one of each of the bottles.Dilute each solution to 100.0 � 0.1 g with High-Purity Water,and mix thoroughly to obtain calibration standards with 0.1,0.5, and 5.0 mg/kg, respectively.
Calibration Blank Solution Tare a clean, dry 4-oz polyeth-ylene bottle (or equivalent). Add approximately 50 g of High-Purity Water. Slowly add 28 � 1 g of concentrated nitricacid, mix thoroughly, slowly add 12 � 1 g of concentratedhydrochloric acid, and mix thoroughly again. Dilute the solu-tion to 100.0 � 0.1 g with High-Purity Water, and mix thor-oughly.
Note: For the solutions listed below, use a Parr ClosedDigestion Vessel (catalog number 4748) with polyethyl-ene vessel liners. Any equivalent apparatus may be usedif the predigestion fortification recoveries are withinthe specifications noted below.
Fortification Solution Use any commercially availableNIST traceable 1000 ppm plasma-grade standard stock solu-tion of cadmium.
Sample Digestion Fortification Preparation Weigh a rep-resentative sample on a balance with 0.1-mg precision (seeWeights and Balances under Apparatus for Tests and Assays,Appendix I). Transfer the sample to a digestion vessel that hasbeen cleaned according to the manufacturer’s specifications.Slowly add 5.0 mL of concentrated nitric acid to the digestionvessel, seal, and heat the vessel for 8 to 16 h at 210° � 5°.Allow the vessel to cool to room temperature, and quantita-tively transfer its contents into a clean, dry, tared 1-oz polyeth-ylene bottle. Slowly add concentrated hydrochloric acid toachieve a final concentration of 10% (w/w), and dilute to anappropriate final mass with High-Purity Water.
Digestion Blank Preparation Transfer 5.0 mL of concen-trated nitric acid into a digestion vessel that has been cleanedaccording to the manufacturer’s specifications, seal, and heatthe vessel for 8 to 16 h at 210° � 5°. Allow the vesselto cool to room temperature, and quantitatively transfer itscontents into a clean, dry, tared 1-oz polyethylene bottle.Slowly add concentrated hydrochloric acid to achieve a finalconcentration of 10% (w/w), and dilute to an appropriate finalmass with High-Purity Water.
Digestion Fortification Preparation Prepare as directedunder Sample Preparation (above), except immediately before
heating the sample, add 25 �L of the Fortification Solution.Determine the recovery of the Digestion Fortification Prepa-ration by analyzing the Sample Digestion Fortification Prepa-ration for cadmium. Calculate the percent recovery for cad-mium by subtracting the unfortified assay result from thefortified assay result and multiplying the difference, in milli-grams per kilogram, by 100. The fortification level for cad-mium is 1 mg/kg. Acceptable recoveries are in the 85% to110% range.
Procedure Use an Inductively Coupled Plasma AtomicEmission Spectrometer (ICP-AES), or equivalent instrumen-tation with similar capabilities. Follow the instrument manu-facturer’s instructions for setting instrument parameters forassay of cadmium. Select appropriate background correctionpoints for the cadmium analyte according to the recommenda-tions of the instrument manufacturer. Select analytical wave-lengths to yield adequate sensitivity and freedom from inter-ference.
Analyze the Calibration Blank Solution. Results for cad-mium should indicate a concentration of less than 0.01 mg/kg. If the results are not less than 0.01 mg/kg, repeat theanalysis. In the event that reanalysis is unsuccessful, takesteps consistent with the manufacturer’s recommendationsto identify and remediate the sources of contamination orinterference. Do not proceed with the analysis until the sourcesof contamination or interference have been identified andcorrected.
Subsequently analyze all three Cadmium Calibration Stan-dards, from lowest concentration to highest. Results for eachof the calibration standards should indicate concentrations of100 � 5 mg/kg, 500 � 25 mg/kg, and 5000 � 250 mg/kg,respectively. If the results are not as indicated, repeat theanalysis. In the event that reanalysis is unsuccessful, takesteps consistent with the manufacturer’s recommendationsto identify and remediate the sources of contamination orinterference. Do not proceed with the analysis until the sourcesof contamination or interference have been identified andaddressed. After successful calibration of the instrument forcadmium, reanalyze the Calibration Blank Solution to demon-strate that there is no carryover of the cadmium.
Next, analyze the prepared samples, digestion blanks, anddigestion fortification samples in groups of no more than ten.At a minimum, each group should contain a digestion blank,a prepared sample, a second replicate of the prepared sample,and that same sample prepared as a fortification sample. Ana-lyze the Calibration Blank Solution followed by any of theCadmium Calibration Standards between each group of tensamples.
To determine the calibration curve, aspirate the CadmiumCalibration Standards and the Calibration Blank Solution. Ifpossible, use the calibration function incorporated in the ICP-AES instrument’s soft- or firmware. If necessary, plot instru-ment response versus concentration of cadmium. Fit this linewith a linear equation of the form y = mx + b, in which y isinstrument response, m is the slope of the best-fit line, x isconcentration, and b is the y intercept of the best-fit line. Thecorrelation coefficient for the best-fit line should be ≥0.99.Concentrations of cadmium in the calibration blanks, calibra-
56 / Butadiene-Styrene Rubber / Monographs FCC V
tion standards, digestion blanks, samples, and fortified sam-ples can be directly read from the ICP-AES when using itssoft- or firmware, or it can be calculated from the best-fitequation.Lead Determine as directed under the Cadmium Test(above), except substitute ‘‘lead’’ for ‘‘cadmium,’’ and use thefollowing for the Stock Solution and the Standard Solutions:
Lead Stock Solution Use any commercially availableNIST traceable 1000 ppm plasma-grade standard stock solu-tion of lead.
Lead Calibration Standards Tare three clean, dry 4-ozpolyethylene bottles (or equivalent). Add approximately 50 gof High-Purity Water to each. Slowly add 28 � 1 g of concen-trated nitric acid, mix thoroughly, slowly add 12 � 1 g ofconcentrated hydrochloric acid, and mix thoroughly again.Using a precision micropipet, add 10, 50, and 500 �L, respec-tively, of Lead Stock Solution to one of each of the bottles.Dilute each solution to 100.0 � 0.1 g with High-Purity Water,and mix thoroughly to obtain calibration standards with 0.1,0.5, and 5.0 mg/kg, respectively.Lithium
Atomic Absorption Spectrophotometer Use a suitable in-strument, equipped with a lithium hollow-cathode lamp, capa-ble of measuring the radiation absorbed by lithium in the670-nm spectral band.
Standard Solution Transfer 399.3 mg of reagent-gradelithium carbonate to a 1000-mL volumetric flask, dissolve ina minimal amount of 1:1 hydrochloric acid:water, dilute tovolume with water, and mix. Transfer 10.0 mL of this solutionto a 100-mL volumetric flask, dilute to volume with water,and mix. Finally, transfer 10.0 mL of this solution to a second100-mL volumetric flask, add 1.0 mL of hydrochloric acid,dilute to volume with water, and mix. This solution contains75 �g of lithium per 100 mL.
Sample Solution Accurately weigh 1 g of a solid-rubbersample, wrap it tightly in ashless filter paper, and place it ina tared platinum crucible. Heat in an oven at 100° for 15 min,and then transfer to a muffle furnace programmed to reach500° within 1 to 3 h after introduction of the sample. Removethe crucible from the furnace 15 to 20 min after 500° hasbeen reached, and cool in a desiccator. Quantitatively transferthe contents of the crucible to a 100-mL volumetric flask,using 1 mL of hydrochloric acid and water, dilute to volumewith water, and mix.
Procedure Following the manufacturer’s instructions foroperating the atomic absorption spectrophotometer, aspiratea suitable portion of the Standard Solution through the flame.In a similar manner, aspirate a suitable portion of the SampleSolution. Any absorbance produced by the Sample Solutionshould not exceed that produced by the Standard Solution.Mercury Determine as directed under the Cadmium Test(above), except substitute ‘‘mercury’’ for ‘‘cadmium,’’ anduse the following for the Stock Solution and the StandardSolutions:
Mercury Stock Solution Use any commercially availableNIST traceable 1000 ppm plasma-grade standard stock solu-tion of mercury.
Mercury Calibration Standards Tare three clean, dry 4-oz polyethylene bottles (or equivalent). Add approximately50 g of High-Purity Water to each. Slowly add 28 � 1 g ofconcentrated nitric acid, mix thoroughly, slowly add 12 �1 g of concentrated hydrochloric acid, and mix thoroughlyagain. Using a precision micropipet, add 10, 50, and 500 �L,respectively, of Mercury Stock Solution to one of each of thebottles. Dilute each solution to 100.0 � 0.1 g with High-PurityWater, and mix thoroughly to obtain calibration standards with0.1, 0.5, and 5.0 mg/kg, respectively.Quinones Determine as directed under Quinones, Appen-dix IV.Residual Hexane (Note: The isooctane, 2,2,4-trimethylpen-tane, used in this test should be of chromatographic-gradequality.)
Internal Standard Stock Solution Transfer 150 mg of n-nonane, accurately weighed, to a 50-mL volumetric flask,dilute to volume with isooctane, and mix.
Dilute Internal Standard Solution Pipet 10.0 mL of Inter-nal Standard Stock Solution into a 100-mL volumetric flask,dilute to volume with isooctane, and mix. Pipet 5.0 mL ofthis solution into a 250-mL volumetric flask, dilute to volumewith isooctane, and mix. Each milliliter of the final solutioncontains 6 �g of n-nonane.
Hexane Standard Solution Transfer 150 mg of n-hexane,accurately weighed, to a 50-mL volumetric flask, dilute tovolume with isooctane, and mix. Pipet 1.0 mL of this solutioninto a 100-mL volumetric flask, dilute to volume with isooc-tane, and mix. Finally, pipet 10.0 mL of this solution and10.0 mL of Internal Standard Stock Solution into a 50-mLvolumetric flask, dilute to volume with isooctane, and mix.
Sample Preparation Accurately weigh 1.5 g of a solid-rubber sample, transfer it into a 4-oz bottle, and pipet 25.0mL of the Dilute Internal Standard Solution into the bottle.Stopper the bottle, and shake mechanically overnight to dis-solve the rubber. Add 50 mL of methanol to precipitate thepolymer, and shake vigorously for 15 min. Allow the mixtureto settle, and decant the liquid phase into a 250-mL separator.Wash the polymer with 25 mL of methanol, and add the washto the separator. Add 50 to 75 mL of cold water to theseparator, and shake vigorously for 1 min, venting periodicallyto release any pressure. Allow the phases to separate, drainoff the bottom (aqueous) phase, and rewash the isooctanephase with a second 50-mL portion of cold water. Shake again,allow to separate, and drain off the bottom layer. Transfer 10mL of the isooctane phase to a 20-mL vial for the analysis.
Procedure Use a gas chromatograph equipped with aflame-ionization detector and a 3-m × 3-mm stainless steelcolumn, or equivalent, packed with 60- to 80-mesh Chro-mosorb P containing 15% didecyl phthalate and capable ofseparating hexane, isooctane, and n-nonane, or equivalent.Maintain the column isothermally at 120°. Set the injectionport temperature to 240° and the detector to 250°. Use heliumas the carrier gas, flowing at a rate of 30 mL/min. Use adigital integrator or computer for data acquisition, althoughany mode (other than triangulation and planimetry) that givesaccurate and reliable measurement of the peak areas is satis-factory.
FCC V Monographs / Butane / 57
Obtain chromatograms of duplicate 5-�L portions of theHexane Standard Solution, and measure the areas under thehexane and n-nonane peaks. In a similar manner, obtain chro-matograms of duplicate 5-�L portions of the Sample Prepara-tion, and measure the areas under the hexane and n-nonanepeaks. The peak area ratio of hexane to n-nonane (i.e., sumof hexane areas divided by sum of n-nonane areas) producedby the Sample Preparation does not exceed that produced bythe Hexane Standard Solution.Residual Styrene Determine as directed under Residual Sty-rene, Appendix IV.
Packaging and Storage Store in well-closed containers.
Butanen-Butane
CH3CH2CH2CH3
C4H10 Formula wt 58.12
CAS: [106-97-8]
DESCRIPTION
Butane occurs as a colorless, flammable gas. One volume ofwater dissolves 0.15 volume; 1 volume of alcohol dissolves18 volumes; 1 volume of ether dissolves 25 or 30 volumes,respectively, at 17° and 770 mm Hg. Its boiling temperatureis –0.5°.
Function Propellant; aerating agent.
REQUIREMENTS
Caution: Butane is highly flammable and explosive.Observe precautions and perform sampling and analyti-cal operations in a well-ventilated fume hood.
IdentificationA. The infrared absorption spectrum of sample exhibits
maxima, among others, at about the following wavelengths,in �m: 3.4 (vs), 6.8 (s), 7.2 (m), and 10.4 (m).
B. The vapor pressure of a test sample, obtained as directedin the Sampling Procedure (below) and determined at 21° bymeans of a suitable pressure gauge, is between 205 and 235kPa absolute (30 and 34 psia, respectively).Assay Not less than 97.0% of C4H10.Acidity of Residue Passes test.High-Boiling Residue Not more than 5 mg/kg.Sulfur Compounds Passes test.Water Not more than 10 mg/kg.
TESTS
Sampling Procedure Use a stainless steel sampling cylin-der equipped with a stainless steel valve and having a capacity
of not less than 200 mL and a pressure rating of 240 psi ormore. Dry the cylinder with the valve open at 110° for 2 h,and evacuate the hot cylinder to less than 1 mm of mercury.Close the valve, and cool and weigh the cylinder. Tightlyconnect one end of a charging line to the sample container,and loosely connect the other end to the sampling cylinder.Carefully open the sample container, and allow the sampleto flush out the charging line through the loose connection.Avoid excessive flushing that causes moisture to freeze inthe charging line and connections. Tighten the fitting on thesampling cylinder, and open its valve, allowing the sampleto flow into the evacuated cylinder. Continue sampling untilthe desired amount of sample is obtained, then close thesample container valve, and finally, close the sampling cylin-der valve.
Caution: Do not overload the sampling cylinder.
Weigh the charged sampling cylinder again, and calculate thesample weight.Assay
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a thermal-conductivity detector and containing a 6-m × 3-mm aluminumcolumn, or equivalent, packed with 10 weight percent tetra-ethylene glycol dimethyl ether liquid phase on a support ofcrushed firebrick (GasChrom R, or equivalent), which hasbeen calcined or burned with a clay binder above 900° andsilanized, or equivalent. Use helium as the carrier gas at aflow rate of 50 mL/min, and maintain the temperature of thecolumn at 33°.
System Suitability The peak responses obtained for thesample in the chromatograms from duplicate determinationsagree within 1%.
Procedure Connect one sample cylinder to the chromato-graph through a suitable sampling valve and a flow controlvalve downstream from the sampling valve. Flush the liquidsample through the sampling valve, taking care to avoid trap-ping gas or air in the valve. Inject a suitable volume, typically2 �L, of sample into the chromatograph, and record the chro-matogram.
Calculation Calculate the purity of the sample using thefollowing formula:
(100 × B)/(x + y + z . . . )
in which B is the sample response and x, y, z . . . representthe sum of all the responses in the chromatogram.Acidity of Residue Add 10 mL of water to the residueobtained in High-Boiling Residue (below), mix by swirlingfor about 30 s, add 2 drops of methyl orange TS, insert thestopper in the tube, and shake vigorously. No pink or redcolor appears in the aqueous layer.High-Boiling Residue Prepare a cooling coil from coppertubing [about 6.1 m × 6 mm (od)] to fit into a suitable vacuum-jacketed flask. Immerse the cooling coil in a mixture of dryice and acetone in a vacuum-jacketed flask, and connect oneend of the tubing to a sample cylinder (see Sampling Proce-dure, above). Carefully open the sample cylinder valve, flushthe cooling coil with about 50 mL of the liquified sample,and discard this portion of liquid. Continue delivering liquid
58 / Butylated Hydroxymethylphenol / Monographs FCC V
from the cooling coil, and collect it in a previously chilled1000-mL sedimentation cone until the cone is filled to the1000-mL mark (approximately 600 g). Allow the liquid toevaporate, using a warm water bath maintained at about 40°to reduce evaporating time. When all of the liquid has evapo-rated, rinse the sedimentation cone with two 50-mL portionsof pentane, and combine the rinsings in a tared 150-mL evapo-rating dish. Transfer 100 mL of the pentane solvent to asecond, tared 150-mL evaporating dish, place both evaporat-ing dishes on a water bath, evaporate to dryness, and heat thedishes in an oven at 100° for 60 min. Cool the dishes in adesiccator, and weigh. Repeat the heating for 15-min periodsuntil successive weighings are within 0.1 mg. The weight ofthe residue obtained from the sample is the difference betweenthe weights of the residues in the two evaporating dishes.Calculate the milligrams per kilogram of high-boiling residuebased on a sample weight of 600 g.Sulfur Compounds Carefully open the container valve toproduce a moderate flow of gas. Do not direct the gas streamtoward the face, but deflect a portion of the stream towardthe nose. The gas is free from the characteristic odor of sulfurcompounds.Water Determine as directed under Water Determination,Appendix IIB, but use the following modifications: (a) Providethe closed-system titrating vessel with an opening and passthrough it a coarse-porosity gas dispersion tube connected toa sampling cylinder. (b) Dilute the reagent with anhydrousmethanol to give a water equivalence factor of between 0.2and 1.0 mg/mL; age this diluted solution for not less than 16h before standardization. (c) Obtain a 100-g sample as directedin the Sampling Procedure (above), and introduce the sampleinto the titration vessel through the gas dispersion tube at arate of about 100 mL of gas per minute; if necessary, heatthe sampling cylinder gently to maintain this flow rate.
Packaging and Storage Store in tight cylinders protectedfrom heat.
Butylated Hydroxymethylphenol
OH
(CH3)3C C(CH3)3
CH2OH
C15H24O2 Formula wt 236.35
DESCRIPTION
Butylated Hydroxymethylphenol occurs as a nearly white,crystalline solid. It is freely soluble in alcohol, and insolublein water and in propylene glycol.
Function Antioxidant.
REQUIREMENTS
Identification Butylated Hydroxymethylphenol may beidentified by its solidification point, as determined in theAssay.Assay Not less than 98.0% of C15H24O2.
TESTS
Assay Determine as directed under Solidification Point, Ap-pendix IIIB. The sample’s solidification point is not lowerthan 140°, indicating a purity of not less than 98.0%, byweight, of C15H24O2.
Packaging and Storage Store in well-closed containers.
1,3-Butylene GlycolButane-1,3-diol
CH2OHCH2CHOHCH3
C4H10O2 Formula wt 90.12
CAS: [107-88-0]
DESCRIPTION
1,3-Butylene Glycol occurs as a clear, colorless, hygroscopic,viscous liquid. It is miscible with water, with acetone, andwith ether in all proportions, but is immiscible with fixedoils. It dissolves most essential oils and synthetic flavoringsubstances.
Function Solvent for flavoring agents.
REQUIREMENTS
Assay Not less than 99.0% of C4H10O2.Distillation Range Between 200° and 215°.Lead Not more than 2 mg/kg.Specific Gravity Between 1.004 and 1.006 at 20°.
TESTS
Assay Prepare an acetylating reagent, within one week ofuse, by mixing 3.4 mL of water and 130 mL of acetic anhydridewith 1000 mL of anhydrous pyridine. For the Assay, pipet 20mL of this reagent into a 250-mL iodine flask, and add about1 g of sample, accurately weighed. Attach a dry reflux con-denser to the flask, and reflux for 1 h. Allow the flask to coolto room temperature, then rinse the condenser with 50 mL ofchilled (10°) carbon dioxide-free water, allowing the waterto drain into the flask. Stopper the flask, cool to below 20°, add
FCC V Monographs / Calcium Acetate / 59
phenolphthalein TS, and titrate with 0.5 N sodium hydroxide,swirling the contents of the flask continuously during thetitration. Perform a blank determination (see General Provi-sions), and make any necessary correction. Each milliliter of0.5 N sodium hydroxide is equivalent to 2.253 mg of C4H10O2.Distillation Range Determine as directed under DistillationRange, Appendix IIB.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in well-closed containers.
Caffeine1,3,7-Trimethylxanthine
N
NN
N
O CH3
CH3
O
H3C
C8H10N4O2 Formula wt, anhydrous 194.19C8H10N4O2·H2O Formula wt, monohydrate 212.21
CAS: anhydrous [58-08-2]
DESCRIPTION
Caffeine occurs as a white powder or as white, glisteningneedles, usually matted together. It may be compacted orcompressed into free-flowing granules or pellets. It is odorlessand has a bitter taste. Caffeine is anhydrous or contains onemolecule of water of hydration. Its solutions are neutral tolitmus. The hydrate is efflorescent in air, and 1 g is solublein about 50 mL of water, in 75 mL of alcohol, in about 6 mLof chloroform, and in 600 mL of ether.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate whether it is anhydrous or hydrous.Identification
A. Dissolve about 5 mg of sample in 1 mL of hydrochloricacid contained in a porcelain dish, add 50 mg of potassiumchlorate, and evaporate on a steam bath to dryness. Invert thedish over a vessel containing a few drops of 6 N ammoniumhydroxide. The residue acquires a purple color, which disap-pears on the addition of a solution of a fixed alkali.
B. The infrared absorption spectrum of a mineral oil disper-sion of the sample, previously dried at 80° for 4 h, exhibits
relative maxima at the same wavelengths as those of a similarpreparation of USP Caffeine Reference Standard.Assay Not less than 98.5% and not more than 101.0% ofC8H10N4O2, calculated on the anhydrous basis.Lead Not more than 1 mg/kg.Melting Range Anhydrous: Between 235° and 237.5°.Other Alkaloids Passes test.Readily Carbonizable Substances Passes test.Residue on Ignition Not more than 0.1%.Water Anhydrous: Not more than 0.5%; Hydrous: Not morethan 8.5%.
TESTS
Assay Dissolve about 170 mg of finely powdered sample,accurately weighed, in 5 mL of glacial acetic acid with warm-ing. Cool, add 10 mL of acetic anhydride and 20 mL oftoluene, and titrate with 0.1 N perchloric acid, determining theendpoint potentiometrically. Each milliliter of 0.1 N perchloricacid is equivalent to 19.42 mg of C8H10N4O2.
Caution: Handle perchloric acid in an appropriatefume hood.
Lead Determine as directed for Method I in the FlameAtomic Absorption Spectrophotometric Method under LeadLimit Test, Appendix IIIB, using a 3-g sample.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB, using a sample previouslydried at 80° for 4 h.Other Alkaloids Add a few drops of mercuric–potassiumiodide TS to 5 mL of a 1:50 aqueous solution. No precipitateforms.Readily Carbonizable Substances Dissolve 500 mg ofsample in 5 mL of 95% sulfuric acid. The resulting coloris no darker than that of Matching Fluid D under ReadilyCarbonizable Substances, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store hydrous caffeine in tight con-tainers and anhydrous caffeine in well-closed containers.
Calcium Acetate
Ca(C2H3O2)2 Formula wt 158.17
INS: 263 CAS: [62-54-4]
DESCRIPTION
Calcium Acetate occurs as a fine, white, bulky powder. It isfreely soluble in water and slightly soluble in alcohol.
View IR
60 / Calcium Acid Pyrophosphate / Monographs FCC V
Function Buffer; stabilizer; firming agent.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Calcium and for Acetate, Appendix IIIA.Assay Not less than 99.0% and not more than 100.5% ofCa(C2H3O2)2, calculated on the anhydrous basis.Chloride Not more than 0.05%.Fluoride Not more than 0.005%.Lead Not more than 2 mg/kg.Sulfate Not more than 0.1%.Water Not more than 7.0%.
TESTS
Assay Dissolve about 300 mg of sample, accuratelyweighed, in 150 mL of water containing 2 mL of 2.7 Nhydrochloric acid. While stirring, preferably with a magneticstirrer, add about 30 mL of 0.05 M disodium EDTA from a50-mL buret, then add 15 mL of 1 N sodium hydroxide and300 mg of hydroxy naphthol blue indicator, and continue thetitration to a blue endpoint. Each milliliter of 0.05 M disodiumEDTA is equivalent to 7.909 mg of Ca(C2H3O2)2.Chloride Determine as directed in Chloride Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 40-mg sample does not exceed that producedby a control containing 20 �g of chloride (Cl) ion.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB, except in the Procedure, use10 mL of 1 N hydrochloric acid to dissolve the sample.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Sulfate Determine as directed in Sulfate Test under Chlorideand Sulfate Limit Tests, Appendix IIIB. Any turbidity pro-duced by a 200-mg sample does not exceed that produced bya control containing 200 �g of sulfate (SO4) ion.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Calcium Acid Pyrophosphate
CaH2P2O7 Formula wt 216.04
CAS: [35405-51-7]
DESCRIPTION
Calcium Acid Pyrophosphate occurs as a fine, white, acidicpowder. It is insoluble in water, but it is soluble in dilutehydrochloric and nitric acids.
Function Leavening agent; nutrient.
REQUIREMENTS
IdentificationA. Dissolve about 100 mg of sample by warming it with
a mixture of 5 mL of 2.7 N hydrochloric acid and 5 mL ofwater; add dropwise, while shaking, 2.5 mL of 6 N ammoniumhydroxide; and then add 5 mL of ammonium oxalate TS. Awhite precipitate forms.
B. Dissolve 100 mg of sample in 100 mL of 1.7 N nitricacid. Add 0.5 mL of this solution to 30 mL of quimociac TS.A yellow precipitate does not form. Heat the remaining portionof the sample solution for 10 min at 95°, and then add 0.5mL of the solution to 30 mL of quimociac TS. A yellowprecipitate forms immediately.Assay Not less than 95.0% and not more than 100.5% ofCaH2P2O7.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 2 mg/kg.Loss on Ignition Not more than 10.0%.
TESTS
Assay Dissolve about 300 mg of sample, accuratelyweighed, in 10 mL of 2.7 N hydrochloric acid. Add about120 mL of water and a few drops of methyl orange TS, andboil for 30 min. Keep the volume and pH of the solutionconstant during the boiling period by adding hydrochloricacid or water if necessary. Add 2 drops of methyl red TS and30 mL of ammonium oxalate TS, then add, dropwise, withconstant stirring, a mixture of equal volumes of 6 N ammo-nium hydroxide and water until the pink color of the indicatorjust disappears. Digest on a steam bath for 30 min, cool toroom temperature, allow the precipitate to settle, and filterthe supernatant liquid through a sintered-glass filter crucibleusing gentle suction. Wash the precipitate in the beaker withabout 30 mL of cold (below 20°) wash solution, prepared bydiluting 10 mL of ammonium oxalate TS to 1000 mL withwater. Allow the precipitate to settle, and pour the supernatantliquid through the filter. Repeat this washing by decantationthree more times. Using the wash solution, transfer the precipi-tate as completely as possible to the filter. Finally, wash thebeaker and the filter with two 10-mL portions of cold (below20°) water. Place the sintered-glass filter crucible in the bea-ker, and add 10 mL of water and 50 mL of cold, 1:6 sulfuricacid. Add 35 mL of 0.1 N potassium permanganate from aburet, and stir until the color disappears. Heat to about 70°,and complete the titration with 0.1 N potassium permanganate.Each milliliter of 0.1 N potassium permanganate is equivalentto 5.40 mg of CaH2P2O7.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 5 mL of2.7 N hydrochloric acid.Fluoride Determine as directed under Fluoride Limit Test,Appendix IIIB, using 1.0 g of sample, accurately weighed.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.
FCC V Monographs / Calcium Ascorbate / 61
Loss on Ignition Transfer about 1 g of sample, accuratelyweighed, into a suitable tared crucible, ignite at 800° � 25°for 30 min, cool in a desiccator, and weigh.
Packaging and Storage Store in well-closed containers.
Calcium Alginate
Algin
[(C6H7O6)2Ca]n Formula wt, calculated 195.16Formula wt, actual (avg.) 219.00
INS: 404 CAS: [9005-35-0]
DESCRIPTION
Calcium Alginate occurs as a white to yellow, fibrous orgranular powder. It is the calcium salt of alginic acid (see themonograph for Alginic Acid). It is insoluble in water, but itis soluble in alkaline solutions or in solutions of substancesthat combine with the calcium. It is insoluble in organicsolvents.
Function Stabilizer; thickener; emulsifier.
REQUIREMENTS
Identification Place about 5 mg of sample into a test tube,add 5 mL of water, 1 mL of a freshly prepared 1:100 solutionof naphtholresorcinol:ethanol, and 5 mL of hydrochloric acid.Heat the mixture to boiling, boil gently for about 3 min, andthen cool to about 15°. Transfer the contents of the test tubeinto a 30-mL separator with the aid of 5 mL of water, andextract with 15 mL of isopropyl ether. Perform a blank deter-mination (see General Provisions), and make any necessarycorrection. The isopropyl ether extract from the sample exhib-its a deeper purple hue than that from the blank.Assay A sample yields not less than 18% and not morethan 21% of carbon dioxide (CO2), corresponding to between89.6% and 104.5% of Calcium Alginate (equiv wt 219.00),calculated on the dried basis.Arsenic Not more than 3 mg/kg.Lead Not more than 5 mg/kg.Loss on Drying Not more than 15.0%.
TESTS
Assay Determine as directed under Alginates Assay, Appen-dix IIIC. Each milliliter of 0.25 N sodium hydroxide consumedin the assay is equivalent to 27.38 mg of Calcium Alginate(equiv wt 219.00).Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.
Packaging and Storage Store in well-closed containers.
Calcium Ascorbate
O OHOCH2C
O OHOH
H 2
Ca·2H2O
C12H14CaO12·2H2O Formula wt 426.34
INS: 302 CAS: [5743-27-1]
DESCRIPTION
Calcium Ascorbate occurs as a white to slightly yellow, crys-talline powder. It is soluble in water, slightly soluble in alco-hol, and insoluble in ether. The pH of a 1:10 aqueous solutionis between 6.8 and 7.4.
Function Antioxidant.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Calcium, Appendix IIIA, and it decolorizes dichlorophe-nol–indophenol TS.Assay Not less than 98.0% and not more than 100.5% ofC12H14CaO12·2H2O.Lead Not more than 2 mg/kg.Optical (Specific) Rotation [�]D
25°: Between +95° and +97°.Oxalate Passes test.
TESTS
Assay Dissolve about 300 mg of sample, accuratelyweighed, in 50 mL of water in a 250-mL Erlenmeyer flask,and immediately titrate with 0.1 N iodine to a pale yellowcolor that persists for at least 30 s. Each milliliter of 0.1 Niodine is equivalent to 10.66 mg of C12H14CaO12·2H2O.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 1 g of sample in each 20 mL.
62 / Calcium Bromate / Monographs FCC V
Oxalate Add 2 drops of glacial acetic acid and 5 mL of a1:10 calcium acetate solution to a solution of 1 g of samplein 10 mL of water. The solution remains clear after standingfor 5 min.
Packaging and Storage Store in tight containers, preferablyin a cool, dry place.
Calcium Bromate
Ca(BrO3)2·H2O Formula wt 313.90
INS: 924b CAS: [10102-75-7]
DESCRIPTION
Calcium Bromate occurs as a white, crystalline powder. It isvery soluble in water.
Function Maturing agent; oxidizing agent.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution in 2.7 N hydrochloric acid
imparts a transient yellow-red color to a nonluminous flame.B. Add sulfurous acid dropwise to a 1:20 aqueous solution.
A yellow color develops that disappears upon the addition ofan excess of sulfurous acid.Assay Not less than 99.8% and not more than 100.5% ofCa(BrO3)2·H2O.Lead Not more than 4 mg/kg.
TESTS
Assay Dissolve about 900 mg of sample, accuratelyweighed, in 50 mL of water in a 250-mL glass-stopperedErlenmeyer flask. Add 3 g of potassium iodide, followed by3 mL of hydrochloric acid. Allow the mixture to stand for 5min, add 100 mL of cold water, and titrate the liberated iodinewith 0.1 N sodium thiosulfate, adding starch TS near theendpoint. Perform a blank determination (see General Provi-sions), and make any necessary correction. Each milliliterof 0.1 N sodium thiosulfate is equivalent to 26.16 mg ofCa(BrO3)2·H2O.Lead
Sample Solution Dissolve 2 g of sample in 10 mL ofwater, add 10 mL of hydrochloric acid, and evaporate todryness on a steam bath. Dissolve the residue in 5 mL ofhydrochloric acid, again evaporate to dryness, and then dis-solve the residue in 40 mL of water.
Procedure Determine as directed under Lead Limit Test,Appendix IIIB, using a 20-mL portion of the Sample Solution,and 4 �g of lead (Pb) ion in the control.
Packaging and Storage Store in well-closed containers.
Calcium Carbonate
CaCO3 Formula wt 100.09
INS: 170(i) CAS: [471-34-1]
DESCRIPTION
Calcium Carbonate occurs as a fine, white or colorless, micro-crystalline powder. It is stable in air, and it is practicallyinsoluble in water and in alcohol. The presence of any ammo-nium salt or carbon dioxide increases its solubility in water,but the presence of any alkali hydroxide reduces the solubility.
Function pH control agent; nutrient; dough conditioner;firming agent; yeast nutrient.
REQUIREMENTS
Identification A sample dissolves, with effervescence, in 1N acetic acid, in 2.7 N hydrochloric acid, and in 1.7 N nitricacid, and the resulting solutions, after boiling, give positivetests for Calcium, Appendix IIIA.Assay Not less than 98.0% and not more than 100.5% ofCaCO3 after drying.Acid-Insoluble Substances Not more than 0.2%.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 3 mg/kg.Loss on Drying Not more than 2%.Magnesium and Alkali Salts Not more than 1%.
TESTS
Assay Transfer about 200 mg of sample, previously driedat 200° for 4 h and accurately weighed, into a 400-mL beaker,add 10 mL of water, and swirl to form a slurry. Cover thebeaker with a watch glass, and introduce 2 mL of 2 N hydro-chloric acid from a pipet inserted between the lip of the beakerand the edge of the watch glass. Swirl the contents of thebeaker to dissolve the sample. Wash down the sides of thebeaker, the outer surface of the pipet, and the watch glass,and dilute the contents to about 100 mL with water. Whilestirring, preferably with a magnetic stirrer, add about 30 mLof 0.05 M disodium EDTA from a 50-mL buret, then add 15mL of 1 N sodium hydroxide and 300 mg of hydroxy naphtholblue indicator, and continue the titration to a blue endpoint.Each milliliter of 0.05 M disodium EDTA is equivalent to5.004 mg of CaCO3.Acid-Insoluble Substances Suspend 5 g of sample in 25mL of water, agitate while cautiously adding 25 mL of 1:2hydrochloric acid, and add water to make a volume of about200 mL. Heat the solution to boiling, cover, digest on a steambath for 1 h, cool, and filter. Wash the precipitate with wateruntil the last washing shows no chloride with silver nitrateTS, and then ignite it. The weight of the residue does notexceed 10 mg.
FCC V Monographs / Calcium Chloride / 63
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 10 mLof 2.7 N hydrochloric acid.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB.Lead
Sample Solution Cautiously dissolve 5 g of sample in 25mL of 1:2 hydrochloric acid, and evaporate to dryness on asteam bath. Dissolve the residue in about 15 mL of water,and dilute to 25 mL (1 mL = 200 mg).
Procedure Determine as directed under Lead Limit Test,Appendix IIIB, using a 20-mL portion of the Sample Solution,and 12 �g of lead (Pb) ion in the control.
Alternatively, determine as directed in the APDC ExtractionMethod under Lead Limit Test.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 200° for 4 h.Magnesium and Alkali Salts Mix 1 g of sample with 40mL of water, carefully add 5 mL of hydrochloric acid, mix,and boil for 1 min. Rapidly add 40 mL of oxalic acid TS,and stir vigorously until precipitation is well established. Im-mediately add 2 drops of methyl red TS, then add 6 N ammo-nium hydroxide, dropwise, until the mixture is just alkaline,and cool. Transfer the mixture to a 100-mL cylinder, diluteto 100 mL with water, and let it stand for 4 h or overnight.Decant the clear, supernatant liquid through a dry filter paper,and place 50 mL of the clear filtrate in a platinum dish. Add0.5 mL of sulfuric acid, and evaporate the mixture on a steambath to a small volume. Carefully evaporate the remainingliquid to dryness over a free flame, and continue heating untilthe ammonium salts have been completely decomposed andvolatilized. Finally, ignite the residue to constant weight. Theweight of the residue does not exceed 5 mg.
Packaging and Storage Store in well-closed containers.
Calcium Chloride
CaCl2 Formula wt, anhydrous 110.98CaCl2·2H2O Formula wt, dihydrate 147.01
CAS: anhydrous [10043-52-4]INS: 509 CAS: dihydrate [10035-04-8]
DESCRIPTION
Calcium Chloride occurs as white, hard fragments, granules,or powder. It is anhydrous or contains two molecules of waterof hydration. It is deliquescent. It is soluble in water andslightly soluble in alcohol. The pH of a 1:20 aqueous solutionis between 4.5 and 11.0.
Function Firming agent.
REQUIREMENTS
Labeling Indicate whether it is anhydrous or the dihydrate.
Identification A 1:10 aqueous solution gives positive testsfor Calcium and for Chloride, Appendix IIIA.Assay Anhydrous: Not less than 93.0% and not more than100.5% of CaCl2; Dihydrate: Not less than 99.0% and notmore than 107.0% of CaCl2·2H2O.Acid-Insoluble Matter Anhydrous: Not more than 0.02%;no particles per kilogram of sample greater than 2 mm in anydimension.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.004%.Lead Not more than 5 mg/kg.Magnesium and Alkali Salts Anhydrous: Not more than5.0%; Dihydrate: Not more than 4.0%.
TESTS
Assay Transfer about 1.5 g of sample, accurately weighed,into a 250-mL volumetric flask, dissolve it in a mixture of100 mL of water and 5 mL of 2.7 N hydrochloric acid, diluteto volume with water, and mix. Transfer 50.0 mL of thissolution into a suitable container, and add 50 mL of water.While stirring, preferably with a magnetic stirrer, add about30 mL of 0.05 M disodium EDTA from a 50-mL buret, thenadd 15 mL of 1 N sodium hydroxide and 300 mg of hydroxynaphthol blue indicator, and continue the titration to a blueendpoint. Each milliliter of 0.05 M disodium EDTA is equiva-lent to 5.55 mg of CaCl2 or 7.35 mg of CaCl2·2H2O.Acid-Insoluble Matter Anhydrous: Place a 32-mm (od) lin-tine disk filter1 in a suitable filter assembly comprising a 2.5-L screw-cap bottle cut in half horizontally and fitted with arubber washer with a 35-mm od and a 25-mm id, followedby the lintine disk, a 20-mesh stainless steel screen with a35-mm od, and a bottle cap with a 25-mm hole in the top.With the filter at the bottom, wash the assembly with 100 mLof 1:300 acetic acid, followed by 100 mL of water. Remove thedisk from the assembly, place it on a watch glass, and drythe combination at 105° for 2 h.
Dissolve 1 kg of sample in 3 L of water containing 10 mLof glacial acetic acid. Allow the solution to cool, and filter itthrough the lintine disk. Rinse the walls of the filter assemblyso that all insoluble matter is transferred to the disk, and washwith 100 mL of water. Place the disk on the same watch glassmentioned above, and dry at 105° for 2 h, being careful atall times not to lose any particles that may be on the disk.The difference in the two weights is the weight of the acid-insoluble matter.
Place the disk under a low-power magnifier (4× to 10×magnification). Using a millimeter rule, measure the largestdimension of each particle (or as many as may be necessary)on the disk. No particles greater than 2 mm in any dimensionare present.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 10 mLof water.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB.
1Available from Filter Fabrics, Inc., 814 E. Jefferson, Goshen, IN46526; 219-533-3114.
64 / Calcium Chloride Solution / Monographs FCC V
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a solution of 1 g of sample in 20 mL of water,and 5 �g of lead (Pb) ion in the control.Magnesium and Alkali Salts Dissolve 1.0 g of sample inabout 50 mL of water, add 500 mg of ammonium chloride,mix, and boil for 1 min. Rapidly add 40 mL of oxalic acidTS, and stir vigorously until precipitation is well established.Immediately add 2 drops of methyl red TS, then add 6 Nammonium hydroxide, dropwise, until the mixture is just alka-line, and cool. Transfer the mixture to a 100-mL cylinder,dilute with water to 100 mL, let it stand for 4 h or overnight,then decant the clear, supernatant liquid through a dry filterpaper. Add 0.5 mL of sulfuric acid to 50 mL of the clearfiltrate in a platinum dish, and evaporate the mixture on asteam bath to a small volume. Carefully evaporate the re-maining liquid to dryness over a free flame, and continueheating until the ammonium salts have been completely de-composed and volatilized. Finally, ignite the residue to con-stant weight. The weight of the residue does not exceed 25mg for the anhydrous or 20 mg for the dihydrate.
Packaging and Storage Store in tight containers.
Calcium Chloride Solution
DESCRIPTION
Calcium Chloride Solution occurs as a clear to slightly turbid,colorless or slightly colored liquid at room temperature. It isnominally available in a concentration range of about 35%to 45% of CaCl2.
Function Sequestrant; firming agent.
REQUIREMENTS
Identification When diluted to a concentration of about1:10 (CaCl2 basis), a sample gives positive tests for Calciumand for Chloride, Appendix IIIA.Assay Not less than 90.0% and not more than 110.0%, byweight, of the labeled amount of calcium chloride, expressedas CaCl2.Alkalinity [as Ca(OH)2] Not more than 0.3%.Fluoride Not more than 0.004%, calculated on the amountof CaCl2 as determined in the Assay.Lead Not more than 4 mg/kg, calculated on the amount ofCaCl2 as determined in the Assay.Magnesium and Alkali Salts Not more than 5.0%, calcu-lated on the amount of CaCl2 as determined in the Assay.
TESTS
Assay Transfer an accurately weighed amount of SampleSolution, equivalent to about 1 g of CaCl2, into a 250-mL
volumetric flask, add 5 mL of 2.7 N hydrochloric acid and100 mL of water to dissolve, dilute to volume with water,and mix. Transfer 50.0 mL of this solution into a suitablecontainer, and add 50 mL of water. While stirring, preferablywith a magnetic stirrer, add about 30 mL of 0.05 M disodiumEDTA from a 50-mL buret, then add 15 mL of 1 N sodiumhydroxide and 300 mg of hydroxy naphthol blue indicator,and continue the titration to a blue endpoint. Each milliliterof 0.05 M disodium EDTA is equivalent to 5.55 mg of CaCl2.Alkalinity [as Ca(OH)2] Dilute an accurately weighedamount of Sample Solution, equivalent to about 5 g of CaCl2,to 50 mL with water, add phenolphthalein TS, and titrate with0.1 N hydrochloric acid. Each milliliter of 0.1 N hydrochloricacid is equivalent to 3.71 mg of Ca(OH)2.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB, using as the sample an accu-rately weighed amount of Sample Solution equivalent to 1 gof CaCl2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using as the sample an accurately weighed amountof Sample Solution equivalent to 1 g of CaCl2 diluted to 10mL with water, and 4 �g of lead (Pb) ion in the control.Magnesium and Alkali Salts Using an accurately weighedamount of Sample Solution, equivalent to 1.0 g of CaCl2, diluteto 50 mL with water, add 500 mg of ammonium chloride, mix,and boil for about 1 min. Rapidly add 40 mL of oxalic acidTS, and stir vigorously until precipitation is well established.Immediately add 2 drops of methyl red TS, then add 6 Nammonium hydroxide, dropwise, until the mixture is just alka-line, and cool. Transfer the mixture into a 100-mL cylinder,dilute to 100 mL with water, let it stand for 4 h or overnight,and then decant the clear, supernatant liquid through a dryfilter paper. Add 0.5 mL of sulfuric acid to 50 mL of theclear filtrate in a platinum dish, and evaporate the mixtureon a steam bath to a small volume. Carefully evaporate theremaining liquid to dryness over a free flame, and continueheating until the ammonium salts are completely decomposedand volatilized. Finally, ignite the residue to constant weight.The weight of the residue does not exceed 25 mg.
Packaging and Storage Store in tight containers.
Calcium CitrateTricalcium Citrate
CH2(COO)C(OH)(COO)CH2COO2
Ca3
Ca3(C6H5O7)2·4H2O Formula wt 570.50
INS: 333 CAS: [5785-44-4]
DESCRIPTION
Calcium Citrate occurs as a fine, white powder. It is veryslightly soluble in water, but it is insoluble in alcohol.
FCC V Monographs / Calcium Disodium EDTA / 65
Function Sequestrant; buffer; firming agent.
REQUIREMENTS
IdentificationA. Dissolve 500 mg of sample in 10 mL of water and 2.5
mL of 1.7 N nitric acid, add 1 mL of mercuric sulfate TS,heat to boiling, and then add potassium permanganate TS. Awhite precipitate forms.
B. Completely ignite 500 mg of sample at as low a tempera-ture as possible, cool, and dissolve the residue in a mixtureof 10 mL of water and 1 mL of glacial acetic acid. Filter,and add 10 mL of ammonium oxalate TS to the filtrate. Avoluminous, white precipitate forms that is soluble in hydro-chloric acid.Assay Not less than 97.5% and not more than 100.5% ofCa3(C6H5O7)2 after drying.Fluoride Not more than 0.003%.Lead Not more than 2 mg/kg.Loss on Drying Between 10.0% and 14.0%.
TESTS
Assay Dissolve about 350 mg of sample, previously driedat 150° for 4 h and accurately weighed, in a mixture of 10mL of water and 2 mL of 2.7 N hydrochloric acid, and diluteto about 100 mL with water. While stirring, preferably witha magnetic stirrer, add about 30 mL of 0.05 M disodium EDTAfrom a 50-mL buret, add 15 mL of 1 N sodium hydroxide and300 mg of hydroxy naphthol blue indicator, and continue thetitration to a blue endpoint. Each milliliter of 0.05 M disodiumEDTA is equivalent to 8.300 mg of Ca3(C6H5O7)2.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB, using a 1-g sample, accuratelyweighed. Use 1.0, 5.0, and 10.0 mL of the Sodium FluorideSolution (equivalent to 5.0, 25.0, and 50.0 mg/kg of fluoride,respectively) to prepare the Calibration Curve, and use 10mL of water and only 10 mL of 1 N hydrochloric acid todissolve the sample as directed under Procedure.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample for 4 h at 150°.
Packaging and Storage Store in well-closed containers.
Calcium Disodium EDTACalcium Disodium Ethylenediaminetetraacetate; CalciumDisodium (Ethylenedinitrilo)tetraacetate; Calcium DisodiumEdetate
H2C
COO
NCH2CH2
N
CH2
OOC
NaOOCCH2
Ca
CH2COONa
2H2O
C10H12CaN2Na2O8·2H2O Formula wt 410.30
INS: 385 CAS: [23411-34-9]
DESCRIPTION
Calcium Disodium EDTA occurs as white, crystalline gran-ules or as a white to off white powder. It is slightly hygroscopicand is stable in air. It is freely soluble in water.
Function Preservative; sequestrant.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution responds to the oxalate test
for Calcium and to the flame test for Sodium, Appendix IIIA.B. The infrared absorption spectrum of a mineral oil disper-
sion of sample exhibits maxima only at the same wavelengthsas those of a similar preparation of USP Edetate CalciumDisodium Reference Standard.
C. Add 2 drops of ammonium thiocyanate TS and 2 dropsof ferric chloride TS to 5 mL of water contained in a testtube. Add about 50 mg of sample to the deep red solution soobtained, and mix. The deep red color disappears.Assay Not less than 97.0% and not more than 102.0% ofC10H12CaN2Na2O8, calculated on the anhydrous basis.Lead Not more than 4 mg/kg.Magnesium-Chelating Substances Passes test.Nitrilotriacetic Acid Not more than 0.1%.pH of a 1:100 Solution Between 6.5 and 7.5.Water Not more than 13.0%.
TESTS
Assay Transfer about 1.2 g of sample, accurately weighed,into a 250-mL beaker, and dissolve in 75 mL of water. Add25 mL of 1 N acetic acid and 1.0 mL of diphenylcarbazoneTS, and titrate slowly with 0.1 M mercuric nitrate to the firstappearance of a purple color. Each milliliter of 0.1 M mercuricnitrate is equivalent to 37.43 mg of C10H12CaN2Na2O8.Lead Determine as directed for the Dithizone Method underLead Limit Test, Appendix IIIB, using a Sample Solutionprepared as directed for organic compounds, but use 70%
66 / Calcium Gluconate / Monographs FCC V
perchloric acid instead of 30% hydrogen peroxide to decom-pose the sample.
Caution: Handle perchloric acid in an appropriatefume hood.
The resulting solution meets the requirements of the LeadLimit Test, Appendix IIIB, using 4 �g of lead (Pb) ion in thecontrol.Magnesium-Chelating Substances Transfer 1 g of sample,accurately weighed, into a small beaker, and dissolve it in 5mL of water. Add 5 mL of a buffer solution prepared bydissolving 67.5 g of ammonium chloride in 200 mL of water,adding 570 mL of ammonium hydroxide, and diluting withwater to 1000 mL. Then add 5 drops of eriochrome black TSto the buffered solution, and titrate with 0.1 M magnesiumacetate to the appearance of a deep wine red color. Not morethan 2.0 mL is required.Nitrilotriacetic Acid
Mobile Phase Add 10 mL of a 1:4 solution of tetrabuty-lammonium hydroxide in methanol to 200 mL of water, andadjust with 1 M phosphoric acid to a pH of 7.5 � 0.1. Transferthe solution into a 1000-mL volumetric flask, add 90 mL ofmethanol, dilute to volume with water, mix, filter through amembrane filter (0.5-�m or finer porosity), and de-gas.
Cupric Nitrate Solution Prepare an aqueous solution con-taining about 10 mg of cupric nitrate per milliliter.
Stock Standard Solution Transfer about 100 mg of nitrilo-triacetic acid, accurately weighed, into a 10-mL volumetricflask, add 0.5 mL of ammonium hydroxide, and mix. Diluteto volume with water, and mix.
Standard Preparation Transfer 1.0 g of sample into a100-mL volumetric flask, add 100 �L of Stock StandardSolution, dilute to volume with Cupric Nitrate Solution, andmix. Sonicate, if necessary, to achieve complete solution.
Test Preparation Transfer 1.0 g of sample into a 100-mL volumetric flask, dilute to volume with Cupric NitrateSolution, and mix. Sonicate, if necessary, to achieve completesolution.
Chromatographic System (See Chromatography, Appen-dix IIIA.) Set up the system with reference to High-Perform-ance Liquid Chromatography. The chromatograph has a 254-nm detector and a 15-cm × 4.6-mm column that contains 5-to 10-mm porous microparticles of silica bonded to octylsilane(Zorbax 8, or equivalent). Set the flow rate to about 2 mL/min. Chromatograph three replicate injections of the StandardPreparation, and record the peak responses as directed underProcedure. The relative standard deviation is not more than2.0%, and the resolution factor between nitrilotriacetic acidand Calcium Disodium EDTA is not less than 4.0.
Procedure Separately inject equal volumes (about 50 �L)of the Standard Preparation and the Test Preparation intothe chromatograph, record the chromatograms, and measurethe responses for the major peaks. The retention times areabout 3.5 min for nitrilotriacetic acid and 9 min for CalciumDisodium EDTA. The response of the nitrilotriacetic acidpeak of the Test Preparation does not exceed the differencebetween the nitrilotriacetic acid peak responses obtained fromthe Standard Preparation and the Test Preparation.
pH of a 1:100 Solution Determine as directed under pHDetermination, Appendix IIB.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Calcium Gluconate
CH2OH(CHOH)4COO2Ca
C12H22CaO14 Formula wt, anhydrous 430.38C12H22CaO14·H2O Formula wt, monohydrate 448.39
CAS: anhydrous [299-28-5]INS: 578
DESCRIPTION
Calcium Gluconate occurs as white, crystalline granules orpowder. It is anhydrous or contains one molecule of water ofhydration. It is stable in air. One gram dissolves slowly inabout 30 mL of water at 25° and in about 5 mL of boilingwater. It is insoluble in alcohol and in many other organicsolvents. Its solutions are neutral to litmus.
Function Firming agent; stabilizer; texturizer.
REQUIREMENTS
Labeling Indicate whether it is anhydrous or the monohy-drate.Identification
A. A 1:50 aqueous solution gives positive tests for Cal-cium, Appendix IIIA.
B. Dissolve a quantity of sample in water, heating in awater bath at 60° if necessary, to obtain a Test Solution con-taining 10 mg/mL. Similarly, prepare a Standard Solutionof USP Potassium Gluconate Reference Standard in water,diluting to 10 mg/mL. To prepare a Spray Reagent, dissolve2.5 g of ammonium molybdate in about 50 mL of 2 N sulfuricacid in a 100-mL volumetric flask, add 1.0 g of ceric sulfate,swirl to dissolve, dilute with 2 N sulfuric acid to volume, andmix. Apply separate 5-�L portions of the Test Solution andthe Standard Solution on a suitable thin-layer chromatographicplate (see Chromatography, Appendix IIA) coated with a0.25-mm layer of chromatographic silica gel, and allow todry. Develop the chromatogram in a solvent system consistingof a 50:30:10:10 mixture of alcohol, water, ammonium hy-droxide, and ethyl acetate until the solvent front has movedabout three-fourths of the length of the plate. Remove theplate from the chamber, and dry at 110° for 20 min. Allowto cool, and spray with Spray Reagent. After spraying, heatthe plate at 110° for about 10 min. The principal spot obtained
FCC V Monographs / Calcium Hydroxide / 67
from the Test Solution corresponds in color, size, and Rf valueto that obtained from the Standard Solution.Assay Anhydrous: Not less than 98.0% and not more than102.0% of C12H22CaO14, calculated on the dried basis; Mono-hydrate: Not less than 98.0% and not more than 102.0% ofC12H22CaO14·H2O, calculated on the as-is basis.Lead Not more than 2 mg/kg.Loss on Drying Anhydrous: Not more than 3.0%; Monohy-drate: Not more than 2.0%.Sucrose and Reducing Sugars Not more than 1.0%.
TESTS
Assay Dissolve about 800 mg of sample, accuratelyweighed, in 100 mL of water containing 2 mL of 2.7 Nhydrochloric acid. While stirring, preferably with a magneticstirrer, add about 30 mL of 0.05 M disodium EDTA from a50-mL buret, then add 15 mL of 1 N sodium hydroxide and300 mg of hydroxy naphthol blue indicator, and continue thetitration to a blue endpoint. Each milliliter of 0.05 M disodiumEDTA is equivalent to 21.52 mg of C12H22CaO14 or 22.42mg of C12H22CaO14·H2O.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 16 h.Sucrose and Reducing Sugars Transfer 1.0 g of sampleinto a 250-mL conical flask, and add 20 mL of hot water todissolve the sample. Cool, add 25 mL of alkaline cupric citrateTS, cover the flask, and boil gently for 5 min, accuratelytimed. Cool rapidly to room temperature, add 25 mL of 0.6N acetic acid, 10.0 mL of 0.1 N iodine, and 10 mL of 2.7N hydrochloric acid. Immediately titrate with 0.1 N sodiumthiosulfate, using starch TS as the indicator. Perform a blankdetermination (see General Provisions), and make any neces-sary correction. Each milliliter of 0.1 N sodium thiosulfateconsumed is equivalent to 2.7 mg of reducing substances (asdextrose).
Packaging and Storage Store in well-closed containers.
Calcium Glycerophosphate
C3H7CaO6P Formula wt 210.14
INS: 383 CAS: [27214-00-2]
DESCRIPTION
Calcium Glycerophosphate occurs as a fine, white powder. Itis somewhat hygroscopic. One gram dissolves in about 50mL of water at 25°. It is more soluble in water at a lower
temperature, and citric acid increases its solubility in water.It is insoluble in alcohol.
Function Nutrient.
REQUIREMENTS
Identification A saturated sample solution gives positivetests for Calcium, Appendix IIIA.Assay Not less than 98.0% and not more than 100.5% ofC3H7CaO6P, after drying.Alkalinity Passes test.Lead Not more than 4 mg/kg.Loss on Drying Not more than 12.0%.
TESTS
Assay Accurately weigh about 2 g of sample, previouslydried at 150° for 4 h, and dissolve it in 100 mL of water and5 mL of 2.7 N hydrochloric acid. Transfer the solution intoa 250-mL volumetric flask, dilute to volume with water, andmix well. Pipet 50.0 mL of this solution into a suitable con-tainer, and add 50 mL of water. While stirring, preferablywith a magnetic stirrer, add about 30 mL of 0.05 M disodiumEDTA from a 50-mL buret, then add 15 mL of 1 N sodiumhydroxide and 300 mg of hydroxy naphthol blue indicator,and continue the titration to a blue endpoint. Each milliliterof 0.05 M disodium EDTA is equivalent to 10.51 mg ofC3H7CaO6P.Alkalinity A solution of 1 g of sample in 60 mL of waterrequires not more than 1.5 mL of 0.1 N sulfuric acid forneutralization, using 3 drops of phenolphthalein TS as indi-cator.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying the sample at 150° for 4 h.
Packaging and Storage Store in tight containers.
Calcium Hydroxide
Slaked Lime
Ca(OH)2 Formula wt 74.10
INS: 526 CAS: [1305-62-0]
DESCRIPTION
Calcium Hydroxide occurs as a white powder. One gramdissolves in 630 mL of water at 25°, and in 1300 mL of
68 / Calcium Iodate / Monographs FCC V
boiling water. It is soluble in glycerin and in a saturatedsolution of sucrose but insoluble in alcohol.
Function Buffer; neutralizing agent; firming agent.
REQUIREMENTS
IdentificationA. Mix a sample with from 3 to 4 times its weight of
water. The sample forms a smooth magma. The clear, superna-tant liquid from the magma is alkaline to litmus.
B. Mix 1 g of sample with 20 mL of water, and addsufficient glacial acetic acid to effect solution. The resultingsolution gives positive tests for Calcium, Appendix IIIA.Assay Not less than 95.0% and not more than 100.5% ofCa(OH)2.Acid-Insoluble Substances Not more than 0.5%.Arsenic Not more than 3 mg/kg.Carbonate Passes test.Fluoride Not more than 0.005%.Lead Not more than 2 mg/kg.Magnesium and Alkali Salts Not more than 4.8%.
TESTS
Assay Transfer about 1.5 g of sample, accurately weighed,into a beaker, and gradually add 30 mL of 2.7 N hydrochloricacid. When solution is complete, transfer it into a 500-mLvolumetric flask, rinse the beaker thoroughly, add the rinsingsto the flask, dilute to volume with water, and mix. Transfer50.0 mL of this solution into a suitable container, and add 50mL of water. While stirring, preferably with a magnetic stirrer,add about 30 mL of 0.05 M disodium EDTA from a 50-mLburet, then add 15 mL of 1 N sodium hydroxide and 300 mgof hydroxy naphthol blue indicator, and continue the titrationto a blue endpoint. Each milliliter of 0.05 M disodium EDTAis equivalent to 3.705 mg of Ca(OH)2.Acid-Insoluble Substances Dissolve 2 g of sample in 30mL of 1:3 hydrochloric acid, and heat to boiling. Filter themixture through a suitable tared, porous-bottom porcelaincrucible, wash the residue with hot water until the last washingis free from chloride, ignite at 800° � 25° for 45 min, cool,and weigh.
Note: Avoid exposing the crucible to sudden tempera-ture changes.
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 15 mLof 2.7 N hydrochloric acid.Carbonate Mix 2 g of sample with 50 mL of water, and addan excess of 2.7 N hydrochloric acid. The solution produces nomore than a slight effervescence.Fluoride Determine as directed under the Fluoride LimitTest, Appendix IIIB, using 1.0 g of sample, accuratelyweighed.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a solution of 1 g of sample in 15 mL of 2.7N hydrochloric acid, and 5 �g of lead (Pb) ion in the control.
Magnesium and Alkali Salts Dissolve 500 mg of samplein a mixture of 30 mL of water and 10 mL of 2.7 N hydrochlo-ric acid, and boil for 1 min. Rapidly add 40 mL of oxalic acidTS, and stir vigorously until precipitation is well established.Immediately add 2 drops of methyl red TS; then add 6 Nammonium hydroxide, dropwise, until the mixture is just alka-line; and cool. Transfer the mixture into a 100-mL cylinder,dilute to 100 mL with water, let it stand for 4 h or overnight,then decant the clear, supernatant liquid through a dry filterpaper. Add 0.5 mL of sulfuric acid to 50 mL of the clearfiltrate contained in a tared platinum dish, and evaporate themixture on a steam bath to a small volume. Carefully evaporatethe remaining liquid to dryness over a free flame, and continueheating until the ammonium salts have been completely de-composed and volatilized. Finally, ignite the residue at 800°� 25° to constant weight.
Packaging and Storage Store in tight containers.
Calcium Iodate
Ca(IO3)2·H2O Formula wt 407.90
INS: 916 CAS: [7789-80-2]
DESCRIPTION
Calcium Iodate occurs as a white powder. It is slightly solublein water, and insoluble in alcohol.
Function Maturing agent; dough conditioner.
REQUIREMENTS
Identification Add 1 drop of starch TS and a few drops of20% hypophosphorous acid to 5 mL of a saturated solutionof sample. A transient blue color appears.Assay Not less than 99.0% and not more than 101.0% ofCa(IO3)2·H2O.Lead Not more than 4 mg/kg.
TESTS
Assay Dissolve about 600 mg of sample, accuratelyweighed, in 10 mL of 70% perchloric acid and 10 mL ofwater, heating gently if necessary, and dilute with water to250.0 mL.
Caution: Handle perchloric acid in an appropriatefume hood.
Transfer 50.0 mL of this solution to a 250-mL glass-stopperedErlenmeyer flask, add 1 mL of 70% perchloric acid and 5 gof potassium iodide, stopper the flask, and swirl briefly. Letthe solution stand for 5 min, then titrate with 0.1 N sodiumthiosulfate, adding starch TS just before the endpoint is
FCC V Monographs / Calcium Lactobionate / 69
reached. Each milliliter of 0.1 N sodium thiosulfate is equiva-lent to 3.398 mg of Ca(IO3)2·H2O.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.
Packaging and Storage Store in well-closed containers.
Calcium Lactate2-Hydroxypropanoic Acid Calcium Salt
CH3CH(OH)COO Ca·xH2O2
C6H10CaO6·xH2O Formula wt, anhydrous 218.22
INS: 327 CAS: [814-80-2]
DESCRIPTION
Calcium Lactate occurs as a white to cream-colored, crystal-line powder or granules. It contains up to five moleculesof water of crystallization. The pentahydrate is somewhatefflorescent and at 120° becomes anhydrous. It is soluble inwater and practically insoluble in alcohol.
Function Buffer; dough conditioner; yeast nutrient.
REQUIREMENTS
Identification A 1:20 aqueous solution gives positive testsfor Calcium and for Lactate, Appendix IIIA.Assay Not less than 98.0% and not more than 101.0% ofC6H10CaO6, calculated on the dried basis.Acidity Passes test (about 0.45%, as lactic acid).Fluoride Not more than 0.0015%.Lead Not more than 2 mg/kg.Loss on Drying Pentahydrate: Between 22.0% and 27.0%;Trihydrate: Between 15.0% and 20.0%; Monohydrate: Be-tween 5.0% and 8.0%; Dried Form: Not more than 3.0%.Magnesium and Alkali Salts Not more than 1%.
TESTS
Assay Dissolve an accurately weighed amount of sample,equivalent to about 350 mg of C6H10CaO6, in 150 mL ofwater containing 2 mL of 2.7 N hydrochloric acid. Whilestirring, preferably with a magnetic stirrer, add about 30 mLof 0.05 M disodium EDTA from a 50-mL buret, then add 15mL of 1 N sodium hydroxide and 300 mg of hydroxy naphtholblue indicator, and continue the titration with the disodiumEDTA to a blue endpoint. Each milliliter of 0.05 M disodiumEDTA is equivalent to 10.91 mg of C6H10CaO6.Acidity Dissolve 1 g of sample in 20 mL of water, add 3drops of phenolphthalein TS, and titrate with 0.1 N sodiumhydroxide. Not more than 0.5 mL of titrant is required.
Fluoride Determine as directed in Method I (3.3-g sample)or Method III (1.0-g sample) under Fluoride Limit Test, Ap-pendix IIIB.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 3-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying 1.5 g of sample at 120° for 4 h.Magnesium and Alkali Salts Mix 1 g of sample with 40mL of water, carefully add 1 mL of hydrochloric acid, boilfor 1 min, and rapidly add 40 mL of oxalic acid TS. Immedi-ately add 2 drops of methyl red TS, then add 6 N ammoniumhydroxide, dropwise from a buret, until the mixture is justalkaline, and cool to room temperature. Transfer the mixtureinto a 100-mL graduate, dilute with water to 100 mL, mix,and allow to stand for 4 h or overnight. Decant the clear,supernatant liquid through a dry filter paper, transfer 50 mLof the clear filtrate to a tared platinum dish, and add 0.5 mLof sulfuric acid. Evaporate to a small volume on a steam bath,then carefully heat over a free flame to dryness, and continueheating to complete decomposition and volatilization of theammonium salts. Finally, ignite the residue to constant weight.The weight of the residue does not exceed 5 mg.
Packaging and Storage Store in tight containers.
Calcium LactobionateCalcium 4-(�,D-Galactosido)-D-gluconate
OCa
OOH·2H2OHO
H OH H OH
O OH H H
HO H HO H
H H HO O
O O
OHOOH
OH
HO
O
HOOH
OHOH
C24H42CaO24 Formula wt, anhydrous 754.66
INS: 399 CAS: [5001-51-4]
DESCRIPTION
Calcium Lactobionate occurs as a white to cream-colored,free-flowing powder. It readily forms double salts, such asthe chloride, bromide, and gluconate. It is anhydrous whenobtained by spray-drying, or the dihydrate when obtained bycrystallization. It is freely soluble in water, but insoluble in
70 / Calcium Lignosulfonate / Monographs FCC V
alcohol and in ether. It decomposes at about 120°. The pHof a 1:10 aqueous solution is between 6.5 and 7.5.
Function Firming agent in dry pudding mixes; nutrient.
REQUIREMENTS
Labeling Indicate whether the product has been obtainedthrough spray-drying or from crystallization.Identification
A. The infrared absorption spectrum of a potassium bro-mide dispersion of the sample, previously dried at 105° for8 h, exhibits relative maxima at the same wavelengths asthose of a similar preparation of USP Calcium LactobionateReference Standard.
B. A sample gives positive tests for Calcium, AppendixIIIA.Calcium Content Not less than 5.05% and not more than5.55% of calcium (Ca), calculated on the dried basis.Halides Not more than 0.04%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 8.0%.Optical (Specific) Rotation [�]D
20°: Between +23° and +25°.Reducing Substances Not more than 1.0%.Sulfate Not more than 0.7%.
TESTS
Calcium Content Dissolve about 1.5 g of sample, accu-rately weighed, in 100 mL of water containing 2 mL of 2.7N hydrochloric acid. While stirring, preferably with a mag-netic stirrer, add about 30 mL of 0.05 M disodium EDTAfrom a 50-mL buret, then add 15 mL of 1 N sodium hydroxideand 300 mg of hydroxy naphthol blue indicator, and continuethe titration to a blue endpoint. Each milliliter of 0.05 Mdisodium EDTA is equivalent to 2.004 mg of calcium (Ca).Halides Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB. A1.2-g sample shows no more turbidity than 0.7 mL of 0.020N hydrochloric acid.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 3-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 8 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 500 mg of sample, calculated on the anhydrousbasis, in each 10 mL.Reducing Substances Transfer 1.0 g of sample into a 250-mL conical flask, dissolve it in 20 mL of water, and add 25mL of alkaline cupric citrate TS. Cover the flask, boil thecontents gently for 5 min, accurately timed, and cool rapidlyto room temperature. Add 25 mL of 0.6 N acetic acid, 10.0mL of 0.1 N iodine, and 10 mL of 3 N hydrochloric acid,and titrate with 0.1 N sodium thiosulfate, adding 3 mL ofstarch TS as the endpoint is approached. Perform a blankdetermination (see General Provisions), make any necessarycorrection, and note the difference in volumes of 0.1 N sodium
thiosulfate required. Each milliliter of the difference in volumeof 0.1 N sodium thiosulfate consumed is equivalent to 2.7 mgof reducing substances (as dextrose).Sulfate Transfer about 25 g of sample, accurately weighed,into a 600-mL beaker, dissolve it in 200 mL of water, adjustthe solution to a pH between 4.5 and 6.5 with 2.7 N hydrochlo-ric acid, and filter if necessary. Heat the filtrate or clearsolution to just below the boiling point, then while stirringvigorously, add 10 mL of barium chloride TS, boil gently for5 min, and allow the solution to stand for at least 2 h, or,preferably, overnight. Collect the precipitate of barium sulfateon a suitable, tared crucible, wash until free from chloride,dry, and ignite at 600° to constant weight. The weight ofbarium sulfate so obtained, multiplied by 0.412, representsthe weight of sulfate (SO4) in the sample taken.
Packaging and Storage Store in well-closed containers.
Calcium Lignosulfonate
CAS: [8061-52-7]
DESCRIPTION
Calcium Lignosulfonate occurs as a brown, amorphous poly-mer. It is obtained from the spent sulfite and sulfate pulpingliquor of wood or from the sulfate (kraft) pulping process. Itmay contain up to 30% reducing sugars. It is soluble in water,but not in any of the common organic solvents. The pH of a1:100 aqueous solution is between approximately 3 and 11.
Function Binder; dispersant.
REQUIREMENTS
IdentificationA. A 0.15-g/L aqueous solution gives positive tests for
Calcium, Appendix IIIA.B. Dissolve 100 mg of sample in 50 mL of water. Add 1
mL each of 10% acetic acid and 10% sodium nitrite solutionsto this solution. Mix the solution by swirling, and allow it tostand for 15 min at room temperature. A brown color appears.
C. The ultraviolet absorption spectrum of a 0.1-g/L aque-ous solution at pH 5 exhibits a peak between 275 and 280 nm.Assay Not less than 5.0% sulfonate sulfur.Calcium Not more than 7.0%.Lead Not more than 1 mg/kg.Loss on Drying Not more than 10.0%.Reducing Sugars Not more than 30.0%.Residue on Ignition Not more than 20.0%.Viscosity of a 50% Solution Not more than 3000 centi-poises.
FCC V Monographs / Calcium Lignosulfonate / 71
TESTS
Assay (as Sulfonate Sulfur) Dissolve 1.0 g of sample, accu-rately weighed, in 400 mL of water contained in a beaker.Direct a gentle stream of nitrogen gas over the liquid’s surface.Add 10 mL of nitric acid, and swirl the solution thoroughlyuntil the reaction subsides. Add 10 mL of 70% perchloricacid, and swirl thoroughly again.
Caution: Handle perchloric acid in an appropriatefume hood.
Place the uncovered beaker on a hot plate, and heat the con-tents vigorously until the center of the bottom of the beakerbecomes clear. Remove the beaker, and cool to room tempera-ture. Add 5 mL of hydrochloric acid, and heat again untilwhite fumes evolve. After cooling, dilute the solution to ap-proximately 100 mL with water, adjust to pH 6 � 0.2 with10% sodium hydroxide, and heat the solution to boiling. Add15 mL of 10% barium chloride solution, and leave the solutionovernight in a fresh beaker in a steam bath at 90° to 95°.Filter through ashless filter paper (Whatman No. 42, or equiva-lent), and wash the precipitate with 200 mL of warm water.Transfer the paper and precipitate to a tared crucible. Heatthe crucible slowly on a Bunsen burner to expel moisture.Place the crucible and contents in a muffle furnace at 850°for 1 h. Let the crucible cool in a desiccator, and then weighthe residue to the nearest 0.0001 g. Calculate the percent ofsulfonate sulfur by the formula
(R/S) × 13.7,
in which R is the weight, in grams, of the residue; S is theweight, in grams, of the sample taken; and 13.7 is the conver-sion factor.Calcium
Strontium Chloride Solution While stirring, add 164.7 gof 60% perchloric acid to 500 mL of water contained in a 1-L beaker.
Caution: Handle perchloric acid in an appropriatefume hood.
Then, while stirring, add 15.2 g of strontium chloride hexahy-drate, stirring until solution is complete. Transfer the solutioninto a 1-L volumetric flask, and dilute to volume at roomtemperature with water. Mix the contents by inverting thestoppered flask several times.
Standard Solution Dilute a certified Calcium StandardSolution (NIST, or equivalent), quantitatively and stepwise,with water to obtain a Standard Solution containing 0.7 mgof calcium per milliliter of water. Store the Standard Solutionin polyethylene bottles because of its instability in glass.
Sample Solution Dilute 1 g of sample, previously driedand accurately weighed, to 10.0 mL with water. If the initialSample Solution is not particle free, filter through a 0.45-�mdisposable Millipore filter, discarding the first few millilitersof filtrate. Pipet 5 mL of Strontium Chloride Solution into a50-mL volumetric flask, and add 5.0 mL of the filtrate. Diluteto volume with water, and mix well.
Procedure Using a suitably calibrated atomic absorptionspectrophotometer and following the manufacturer’s instruc-tions for optimum operation of the spectrophotometer, deter-mine the absorbance of the Standard Solution and the SampleSolution at 422.7 nm. The absorbance of the Sample Solutionis not greater than that of the Standard Solution.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on DryingAppendix IIC, drying a sample at 105° for 24 h.Reducing Sugars (Note: The Copper Reagent Solution usedin this test must be prepared several days in advance of use.)
Lead Subacetate Solution Dissolve 80 g of lead subacetatein 220 mL of water. Stir overnight, and filter through WhatmanNo. 42 filter paper, or equivalent. Dilute the supernatant solu-tion to a specific gravity of 1.254 with freshly boiled water.
Copper Reagent Solution Dissolve 28 g of anhydrousdibasic sodium phosphate and 40 g of potassium sodiumtartrate (KNaC4H4O6·4H2O) in 700 mL of water. Add 100mL of 1 N sodium hydroxide and 8 g of copper sulfate pentahy-drate, followed by 180 g of anhydrous sodium sulfate. Add0.7134 g of potassium iodate, and dilute to 1 L. Allow tostand for several days, then filter the clear top part of thesolution through a medium-porosity sintered-glass funnel.
Dextrose Standard Solution Dissolve 140 mg of drieddextrose, accurately weighed, in 500 mL of water.
Dibasic Sodium Phosphate Solution Dissolve 19 g of so-dium phosphate, dibasic, heptahydrate, in 100 mL of water.
Procedure Dissolve 1 g of sample, accurately weighed,in 150 mL of water, and adjust the pH to between 6.9 and7.2 with sodium hydroxide solution or acetic acid. Add LeadSubacetate Solution in increments until no further precipita-tion is observed. Bring the volume to 250.0 mL with water,and mix well. Centrifuge the mixture, pipet 10 mL of thesupernatant into a 50-mL volumetric flask, and dilute to about35 mL with water. Add 2 mL or more of Dibasic SodiumPhosphate Solution until no further precipitation forms. Diluteto 50 mL with water, and mix. Centrifuge at 2100 × gravityfor 10 min. Pipet 5 mL of the supernatant solution into a testtube containing exactly 5 mL of Copper Reagent Solution,and mix. Loosely plug the tube, and place it in a boiling waterbath for 40 min � 10 s. At the end of the heating period,cool the tube immediately in cold water. Add 2 mL of 2.5%potassium iodide solution and 1.5 mL of 2 N sulfuric acid.Mix well, and titrate with 0.005 N sodium thiosulfate, usingstarch as the indicator, and note the volume of 0.005 N sodiumthiosulfate consumed as VS. Run a corresponding blank titra-tion, VR, using 5 mL of water and 5 mL of Copper ReagentSolution.
Repeat the entire procedure with the dextrose standard (5mL of Dextrose Standard Solution and 5 mL of Copper Re-agent Solution), noting the volume of 0.005 N sodium thiosul-fate consumed as VD. Run a corresponding blank titration,VB, using 5 mL of water and 5 mL of Copper Reagent Solution.
Calculate the percent of reducing sugars by the formula
35 × (VB − VS)/(VB − VD),
72 / Calcium Oxide / Monographs FCC V
in which VB − VS is the number of milliliters of 0.005 Nsodium thiosulfate consumed by the 5-mL aliquot of sample,and VB − VD is the number of milliliters of 0.005 N sodiumthiosulfate consumed by 5 mL of Dextrose Standard Solution.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Viscosity of a 50% Solution Dissolve 200 g of sample,calculated on the dried basis and accurately weighed, in 200mL of water contained in a 500-mL beaker. Equilibrate thesolution at 25°, and measure its viscosity with a Brookfieldviscometer A (model LVG, or equivalent), using a number 2spindle at 20 rpm.
Packaging and Storage Store in well-closed containers.
Calcium Oxide
Lime
CaO Formula wt 56.08
INS: 529 CAS: [1305-78-8]
DESCRIPTION
Calcium Oxide occurs as hard, white or gray-white massesor granules, or as a white to gray-white powder. One gramdissolves in about 840 mL of water at 25° and in about 1740mL of boiling water. It is soluble in glycerin but insoluble inalcohol.
Function pH control agent; nutrient; dough conditioner;yeast food.
REQUIREMENTS
Identification Slake 1 g of sample with 20 mL of water,and add glacial acetic acid until the sample is dissolved. Theresulting solution gives positive tests for Calcium, Appen-dix IIIA.Assay Not less than 95.0% and not more than 100.5% ofCaO after ignition.Acid-Insoluble Substances Not more than 1%.Alkalies or Magnesium Not more than 3.6%.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.015%.Lead Not more than 2 mg/kg.Loss on Ignition Not more than 10.0%.
TESTS
Assay Ignite about 1 g of sample to constant weight, anddissolve the ignited sample, accurately weighed, in 20 mL of2.7 N hydrochloric acid. Cool the solution, dilute to 500.0mL with water, and mix. Pipet 50.0 mL of this solution intoa suitable container, and add 50 mL of water. While stirring,
preferably with a magnetic stirrer, add about 30 mL of 0.05M disodium EDTA from a 50-mL buret, then add 15 mL of1 N sodium hydroxide and 300 mg of hydroxy naphthol blueindicator, and continue the titration to a blue endpoint. Eachmilliliter of 0.05 M disodium EDTA is equivalent to 2.804mg of CaO.Acid-Insoluble Substances Slake 5 g of sample, then mixit with 100 mL of water and sufficient hydrochloric acid,added dropwise, to effect solution. Boil the solution, cool,add hydrochloric acid, if necessary, to make the solutiondistinctly acid, and filter through a tared glass filter crucible.Wash the residue with water until free of chlorides, dry at105° for 1 h, cool, and weigh.Alkalies or Magnesium Dissolve 500 mg of sample in 30mL of water and 15 mL of 2.7 N hydrochloric acid. Heat thesolution, and boil for 1 min. Rapidly add 40 mL of oxalicacid TS, and stir vigorously. Add 2 drops of methyl red TS,and neutralize the solution with 6 N ammonium hydroxide toprecipitate the calcium completely. Heat the mixture on asteam bath for 1 h, cool, dilute to 100 mL with water, mixwell, and filter. Add 0.5 mL of sulfuric acid to 50 mL of thefiltrate, then evaporate to dryness, and ignite to constantweight in a tared platinum crucible at 800° � 25°.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 15 mLof 2.7 N hydrochloric acid.Fluoride Determine as directed under Fluoride Limit Test,Appendix IIIB, using a 1.0-g sample, accurately weighed.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a solution of 1 g of sample in 15 mL of 2.7N hydrochloric acid, and 5 �g of lead (Pb) ion in the control.Loss on Ignition Ignite 1 g of sample to constant weightin a tared platinum crucible at 1100° � 50°.
Packaging and Storage Store in tight containers.
Calcium PantothenateD-Calcium Pantothenate; Dextro Calcium Pantothenate
HOCH2C(CH3)2CH(OH)CONH(CH2)2COO Ca2
C18H32CaN2O10 Formula wt 476.54
CAS: [137-08-6]
DESCRIPTION
Calcium Pantothenate occurs as a slightly hygroscopic, whitepowder. It is the calcium salt of the dextrorotatory isomer ofpantothenic acid. It is stable in air. One gram dissolves inabout 3 mL of water. It is soluble in glycerin, but is practicallyinsoluble in alcohol, in chloroform, and in ether.
FCC V Monographs / Calcium Pantothenate, Calcium Chloride Double Salt / 73
Function Nutrient.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution gives positive tests for Cal-
cium, Appendix IIIA.B. The infrared absorption spectrum of a potassium bro-
mide dispersion of the sample, previously dried at 105° for3 h, exhibits relative maxima at the same wavelengths asthose of a similar preparation of USP Calcium PantothenateReference Standard.
C. Boil 50 mg of sample in 5 mL of 1 N sodium hydroxidefor 1 min, cool, and add 5 mL of 1 N hydrochloric acid and2 drops of ferric chloride TS. A strong yellow color appears.Assay Not less than 97.0% and not more than 103.0% ofDextrorotatory Calcium Pantothenate (C18H32CaN2O10) afterdrying.Alkalinity Passes test.Alkaloids Passes test.Calcium Content Not less than 8.2% and not more than8.6% of calcium (Ca) after drying.Lead Not more than 2 mg/kg.Loss on Drying Not more than 5.0%.Optical (Specific) Rotation [�]D
25°: Between +25.0° and+27.5° after drying.
TESTS
Assay (Use low-actinic glassware throughout this pro-cedure.)
Mobile Phase Transfer 2.0 mL of phosphoric acid into a2-L volumetric flask, and dilute to volume with water. Filterthe solution through a 0.45-�m pore-size disk.
Internal Standard Preparation Transfer about 80 mg ofp-hydroxybenzoic acid, accurately weighed, into a 1000-mLvolumetric flask, dissolve in 5 mL of alcohol, dilute to volumewith Mobile Phase, and mix.
Standard Preparation Transfer about 15 mg of USP Cal-cium Pantothenate Reference Standard, previously dried at105° for 3 h and accurately weighed, into a 25-mL volumetricflask. Dilute to volume with Internal Standard Preparation,and mix.
Sample Preparation Proceed as directed for the StandardPreparation, using an accurately weighed amount of sampleequivalent to about 15 mg of Calcium Pantothenate that hasbeen previously dried at 105° for 3 h.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a high-performance liquid chromatographequipped with an ultraviolet detector that measures at 210nm. Under typical conditions, the instrument contains a 15-cm × 3.9-mm (id) column packed with octadecylsilanizedsilica (10-�m �Bondapak C 18, or equivalent.) The flow rateis about 1.5 mL/min.
System Suitability Three replicate injections of the Stan-dard Preparation show a relative standard deviation of notmore than 2.0%.
Procedure Separately inject equal volumes (about 10 �L)of the Standard Preparation and the Sample Preparation into
the chromatograph, record the chromatograms, and measurethe peak responses obtained for the Sample Preparation andthe Standard Preparation. The relative retention times are 0.5for Calcium Pantothenate and 1.0 for p-hydroxybenzoic acid.Calculate the quantity, in milligrams, of C18H32CaN2O10 inthe portion of sample taken by the formula
25C(RU/RS),
in which C is the concentration, in milligrams per milliliter,of USP Calcium Pantothenate Reference Standard in the Stan-dard Preparation, and RU and RS are the ratios of the peakresponses obtained for Calcium Pantothenate and p-hydroxy-benzoic acid from the Sample Preparation and the StandardPreparation, respectively.Alkalinity Dissolve 1 g of sample in 15 mL of recentlyboiled and cooled water in a small flask. As soon as solutionis complete, add 1.0 mL of 0.1 N hydrochloric acid, then add0.05 mL of phenolphthalein TS, and mix. No pink colorappears within 5 s.Alkaloids Dissolve 200 mg of sample in 5 mL of water,and add 1 mL of 2.7 N hydrochloric acid and 2 drops ofmercuric–potassium iodide TS. No turbidity develops within1 min.Calcium Content Dissolve about 950 mg of sample, pre-viously dried at 105° for 3 h and accurately weighed, in 100mL of water containing 2 mL of 2.7 N hydrochloric acid.While stirring, preferably with a magnetic stirrer, add about30 mL of 0.05 M disodium EDTA from a 50-mL buret, thenadd 15 mL of 1 N sodium hydroxide and 300 mg of hydroxynaphthol blue indicator, and continue the titration to a blueendpoint. Each milliliter of 0.05 M disodium EDTA is equiva-lent to 2.004 mg of calcium (Ca).Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 500 mg of sample, previously dried at 105° for 3 h.
Packaging and Storage Store in tight containers.
Calcium Pantothenate, Calcium ChlorideDouble SaltCalcium Chloride Double Salt of DL- or D-CalciumPantothenate
C18H32CaN2O10·CaCl2 Formula wt 587.52
CAS: [6363-38-8]
DESCRIPTION
Calcium Pantothenate, Calcium Chloride Double Salt occursas a white, free-flowing, fine powder. It is a chemical complex
74 / Calcium Pantothenate, Racemic / Monographs FCC V
composed of approximately equimolecular quantities of dex-trorotatory (D) or racemic (DL) calcium pantothenate and cal-cium chloride. It is freely soluble in water, but insoluble inalcohol. Its solutions in water are alkaline to litmus.
Function Nutrient.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution gives positive tests for Cal-
cium, Appendix IIIA.B. Dissolve 50 mg of sample in 5 mL of 1 N sodium
hydroxide, and filter. Add 1 drop of cupric sulfate TS to thefiltrate. A deep blue color appears.
C. Stir 1.0 g of dried sample with 15 mL of dimethylform-amide for 5 min. Centrifuge the mixture, transfer 2.0 mL ofthe clear supernatant liquid to a weighing dish, evaporate itunder vacuum on a steam bath, and dry the residue in an ovenat 105° for 1 h. The weight, in grams, of the residue, composedof uncombined calcium pantothenate and calcium chloride,multiplied by 750 equals the percentage of uncomplexed mate-rial in the sample. It does not exceed 10.0% of the weight ofthe sample.Assay Not less than 45.0% and not more than 55.0% ofCalcium Pantothenate (C18H32CaN2O10) after drying.Arsenic Not more than 3 mg/kg.Calcium Content Between 12.4% and 13.6% of calcium(Ca) after drying.Chloride Content (as Cl) Between 10.5% and 12.1% ofchloride after drying.Lead Not more than 2 mg/kg.Loss on Drying Not more than 5.0%.
TESTS
Assay Determine as directed in the monograph for CalciumPantothenate.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 25 mLof water.Calcium Content Determine as directed in the monographfor Calcium Pantothenate.Chloride Content (as Cl) Transfer about 1 g of sample,previously dried in vacuum for 1 h and accurately weighed,into a 250-mL beaker, and add sufficient water to make 100mL. Equip a pH meter with glass and silver electrodes, andset it on the ‘‘+ millivolt’’ scale. Insert the electrodes and amotor-driven, glass stirring rod into the sample beaker. Add1 to 2 drops of methyl orange TS. Stir, and add, dropwise,10% nitric acid until a pink color appears, then add 10 mLin excess. Titrate the solution with 0.1 N silver nitrate to areading of +1.0 millivolt on the pH meter. Each milliliter of0.1 N silver nitrate is equivalent to 3.545 mg of chloride.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in vacuum at 100° for 1 h.
Packaging and Storage Store in tight containers.
Calcium Pantothenate, Racemic
C18H32CaN2O10 Formula wt 476.54
CAS: [6381-63-1]
DESCRIPTION
Calcium Pantothenate, Racemic, occurs as a white, slightlyhygroscopic powder. It is a mixture of the calcium salts ofthe dextrorotatory (D) and levorotatory (DL) isomers of panto-thenic acid. It is optically inactive. It is stable in air and freelysoluble in water. It is soluble in glycerin, and is practicallyinsoluble in alcohol, in chloroform, and in ether. Its solutionsare neutral or alkaline to litmus.
Note: The physiological activity of Racemic CalciumPantothenate is approximately one-half that of the dex-trorotatory isomer.
Function Nutrient.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution gives positive tests for Cal-
cium, Appendix IIIA.B. The infrared absorption spectrum of a potassium bro-
mide dispersion of the sample, previously dried at 105° for3 h, exhibits relative maxima at the same wavelengths asthose of a similar preparation of USP Calcium PantothenateReference Standard.
C. Boil 50 mg of sample in 5 mL of 1 N sodium hydroxidefor 1 min, cool, and add 5 mL of 1 N hydrochloric acid and2 drops of ferric chloride TS. A strong yellow color appears.Assay Not less than 97.0% and not more than 103.0% ofCalcium Pantothenate (C18H32CaN2O10) after drying.Alkalinity Passes test.Alkaloids Passes test.Calcium Content Not less than 8.2% and not more than8.6% of calcium (Ca) after drying.Lead Not more than 2 mg/kg.Loss on Drying Not more than 5.0%.Optical (Specific) Rotation [�]D
25°: Between –0.05° and+0.05° after drying.
TESTS
Assay Determine as directed under Assay in the monographfor Calcium Pantothenate.Alkalinity Dissolve 1 g of sample in 15 mL of recentlyboiled and cooled water in a small flask. As soon as solutionis complete, add 1.6 mL of 0.1 N hydrochloric acid, then add0.05 mL of phenolphthalein TS, and mix. No pink colorappears within 5 s.Alkaloids Dissolve 200 mg of sample in 5 mL of water,and add 1 mL of 2.7 N hydrochloric acid and 2 drops ofmercuric–potassium iodide TS. No turbidity develops within1 min.
FCC V Monographs / Calcium Phosphate, Dibasic / 75
Calcium Content Dissolve about 950 mg of sample, pre-viously dried at 105° for 3 h and accurately weighed, in 100mL of water containing 2 mL of 2.7 N hydrochloric acid.While stirring, preferably with a magnetic stirrer, add about30 mL of 0.05 M disodium EDTA from a 50-mL buret, thenadd 15 mL of 1 N sodium hydroxide and 300 mg of hydroxynaphthol blue indicator, and continue the titration to a blueendpoint. Each milliliter of 0.05 M disodium EDTA is equiva-lent to 2.004 mg of calcium (Ca).Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 500 mg of sample, previously dried at 105° for 3h, in each 10-mL portion.
Packaging and Storage Store in tight containers.
Calcium Peroxide
CaO2 Formula wt 72.08
INS: 930 CAS: [1305-79-9]
DESCRIPTION
Calcium Peroxide occurs as a white or yellow powder orgranular material. It decomposes in moist air, is practicallyinsoluble in water, and dissolves in acids, forming hydrogenperoxide. A 1:100 aqueous slurry has a pH of about 12.
Function Dough conditioner; oxidizing agent.
REQUIREMENTS
Identification Cautiously dissolve 250 mg of sample in 5mL of glacial acetic acid, and add a few drops of a saturatedsolution of potassium iodide. Iodine is liberated. Add 20 mLof water and sufficient sodium thiosulfate TS to remove theiodine color. The resulting solution gives positive tests forCalcium, Appendix IIIA.Assay Not less than 60.0% of CaO2.Fluoride Not more than 0.005%.Lead Not more than 4 mg/kg.
TESTS
Assay Transfer about 1 g of sample, accurately weighed,into an Erlenmeyer flask, add 30 mL of water and 30 mL of85% phosphoric acid:water (1:1 v/v), and titrate immediatelywith 0.5 N potassium permanganate to the first faint pinkcolor that persists for 1 min. Each milliliter of 0.5 N potassiumpermanganate is equivalent to 18.02 mg of CaO2.
Fluoride Determine as directed under Fluoride Limit Test,Appendix IIIB, using 1.0 g of sample, accurately weighed.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using 10 mL of the following solution and 4 �g oflead (Pb) ion in the control: Transfer 4.0 g of sample, accu-rately weighed, into a 250-mL beaker, cautiously add 50 mLof nitric acid, and evaporate just to dryness on a steam bath.Add 20 mL of nitric acid, repeat the evaporation, cool, anddissolve the residue in sufficient water containing 4 drops ofnitric acid to make 40.0 mL.
Packaging and Storage Store in tight containers, and avoidcontact with readily oxidizable materials. Observe the safetyprecautions printed on the label of the original container.
Calcium Phosphate, Dibasic
Dicalcium Phosphate
CaHPO4 Formula wt, anhydrous 136.06CaHPO4·2H2O Formula wt, dihydrate 172.09
INS: 341(ii) CAS: anhydrous [7757-93-9]CAS: dihydrate [7789-77-7]
DESCRIPTION
Calcium Phosphate, Dibasic, occurs as a white powder. It isanhydrous or contains two molecules of water of hydration.It is stable in air. It is insoluble in alcohol, is practicallyinsoluble in water, but is readily soluble in dilute hydrochloricand nitric acids.
Function Leavening agent; dough conditioner; nutrient;yeast food.
REQUIREMENTS
Labeling Indicate whether it is anhydrous or the dihydrate.Identification
A. Dissolve about 100 mg of sample by warming it witha mixture of 5 mL of 2.7 N hydrochloric acid and 5 mL ofwater. Add 2.5 mL of 6 N ammonium hydroxide, dropwise,with shaking, and then add 5 mL of ammonium oxalate TS.A white precipitate forms.
B. Add 10 mL of ammonium molybdate TS to 10 mL ofa warm 1:100 aqueous solution in a slight excess of nitricacid. A yellow precipitate of ammonium phosphomolybdateforms.Assay Anhydrous or Dihydrate: Not less than 97.0% andnot more than 105.0%.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 2 mg/kg.Loss on Ignition Anhydrous: Between 7.0% and 8.5%; Di-hydrate: Between 24.5% and 26.5%.
76 / Calcium Phosphate, Monobasic / Monographs FCC V
TESTS
Assay Dissolve, with the aid of gentle heat if necessary,about 250 mg of sample, accurately weighed, in a mixture of5 mL of hydrochloric acid and 3 mL of water containedin a 250-mL beaker equipped with a magnetic stirrer, andcautiously add 125 mL of water. With constant stirring, add,in the order named, 0.5 mL of triethanolamine, 300 mg ofhydroxy naphthol blue indicator, and from a 50-mL buret,about 23 mL of 0.05 M disodium ethylenediaminetetraacetate.Add a 45:100 sodium hydroxide solution until the initial redcolor changes to clear blue, continue to add it dropwise untilthe color changes to violet, then add an additional 0.5 mL.The pH is between 12.3 and 12.5. Continue the titration,dropwise, with the 0.05 M disodium ethylenediaminetetraace-tate to the appearance of a clear blue endpoint that persistsfor not less than 60 s. Each milliliter of 0.05 M disodiumethylenediaminetetraacetate is equivalent to 6.803 mg ofCaHPO4 or to 8.604 mg of CaHPO4·2H2O.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 5 mL of2.7 N hydrochloric acid.Fluoride (Note: Prepare and store all solutions in plasticcontainers.)
Buffer Solution Dissolve 73.5 g of sodium citrate in waterto make 250 mL of solution.
Standard Solution Dissolve an accurately weighed quan-tity of USP Sodium Fluoride RS quantitatively in water toobtain a solution containing 1.1052 mg/mL. Transfer 20.0mL of the resulting solution into a 100-mL volumetric flaskcontaining 50 mL of Buffer Solution, dilute to volume withwater, and mix. Each milliliter of this solution contains 100�g of fluoride ion.
Sample Solution Transfer 2.0 g of sample to a beaker con-taining a plastic-coated stirring bar, add 20 mL of water and 2.0mL of hydrochloric acid, and stir until dissolved. Add 50.0 mLof Buffer Solution and sufficient water to make 100 mL.
Electrode System Use a fluoride-specific, ion-indicatingelectrode and a silver–silver chloride reference electrode con-nected to a pH meter capable of measuring potentials with aminimum reproducibility of �0.2 mV.
Standard Response Line Transfer 50.0 mL of Buffer Solu-tion and 2.0 mL of hydrochloric acid into a beaker, and addwater to make 100 mL. Add a plastic-coated stirring bar, insertthe electrodes into the solution, stir for 15 min, and read thepotential, in millivolts.Continuestirring, andat 5-min intervals,add 100 �L, 100 �L, 300 �L, and 500 �L of Standard Solution,reading the potential 5 min after each addition. Plot the loga-rithms of the cumulative fluoride ion concentrations (0.1, 0.2,0.5, and 1.0 �g/mL) versus potential, in millivolts.
Procedure Rinse and dry the electrodes, insert them intothe Sample Solution, stir for 5 min, and read the potential, inmillivolts. From the measured potential and the Standard Re-sponse Line, determine the concentration, C, in micrograms permilliliter, of fluoride ion in the Sample Solution. Calculate thepercentage of fluoride in the sample taken by the formula
C × 0.005.
Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.
Loss on Ignition Ignite about 3 g of sample, accuratelyweighed, preferably in a muffle furnace, at 800° to 825° toconstant weight.
Packaging and Storage Store in tightly closed containers.
Calcium Phosphate, Monobasic
Monocalcium Phosphate; Calcium Biphosphate; AcidCalcium Phosphate
Ca(H2PO4)2 Formula wt, anhydrous 234.05Ca(H2PO4)2·H2O Formula wt, monohydrate 252.07
CAS: anhydrous [7758-23-8]INS: 341(i) CAS: monohydrate [10031-30-8]
DESCRIPTION
Calcium Phosphate, Monobasic, occurs as white crystals orgranules or as a granular powder. It is anhydrous or containsone molecule of water of hydration, but because of its deli-quescent nature, more than the calculated amount of watermay be present. It is sparingly soluble in water and is insolublein alcohol.
Function Buffer; dough conditioner; firming agent; leaven-ing agent; nutrient; yeast food; sequestrant.
REQUIREMENTS
Labeling Indicate the state of hydration.Identification
A. Dissolve 100 mg of sample by warming it in a mixtureof 2 mL of 2.7 N hydrochloric acid and 8 mL of water. Add5 mL of ammonium oxalate TS. A white precipitate forms.
B. Add ammonium molybdate TS to a warm solution ofsample in a slight excess of nitric acid. A yellow precipitateof ammonium phosphomolybdate forms.Assay Anhydrous: Not less than 16.8% and not more than18.3% of Ca; Monohydrate: Not less than 15.9% and notmore than 17.7% of Ca.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 2 mg/kg.Loss on Drying Monohydrate: Not more than 1%.Loss on Ignition Anhydrous: Between 14.0% and 15.5%.
TESTS
Assay Accurately weigh a portion of sample equivalent toabout 475 mg of Calcium Phosphate, Monobasic, Anhydrous[Ca(H2PO4)2], dissolve it in 10 mL of 2.7 N hydrochloricacid, add a few drops of methyl orange TS, and boil for 5min, keeping the volume and pH of the solution constantduring the boiling period by adding hydrochloric acid or water
FCC V Monographs / Calcium Phosphate, Tribasic / 77
as necessary. Add2 drops ofmethyl red TSand 30 mLof ammo-nium oxalate TS, then while constantly stirring, add, dropwise,a mixture of equal volumes of 6 N ammonium hydroxide andwater until the pink color of the indicator just disappears. Digeston a steam bath for 30 min, cool to room temperature, allow theprecipitate to settle, and filter the supernatant liquid through asintered-glass crucible, using gentle suction. Wash the precipi-tate in the beaker with about 30 mL of cold (below 20°) washsolution, prepared by diluting 10 mL of ammonium oxalate TSto 1000 mL with water. Allow the precipitate to settle, and pourthe supernatant liquid through the filter. Repeat this washingby decantation three more times.Using the wash solution, trans-fer the precipitate as completely as possible to the filter. Finally,wash the beaker and the filter with two 10-mL portions of cold(below 20°) water. Place the sintered-glass crucible in the bea-ker, and add 100 mL of water and 50 mL of cold 1:6 sulfuricacid. Add 35 mL of 0.1 N potassium permanganate from a buret,and stir until the color disappears. Heat to about 70°, and com-plete the titration with 0.1 N potassium permanganate. Eachmilliliter of 0.1 N potassium permanganate is equivalent to2.004 mg of Ca.Arsenic Determine as directed under Arsenic Limit Test, Ap-pendix IIIB, using a solution of 1 g of sample in 5 mL of 2.7 Nhydrochloric acid.Fluoride Anhydrous: Determine as directed in Method II un-der the Fluoride Limit Test, Appendix IIIB. Monohydrate: Pro-ceed as directed under Fluoride in the monograph for CalciumPhosphate, Dibasic.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Monohydrate: Determine as directed underLoss on Drying, Appendix IIC, drying a sample at 60° for 3 h.Loss on Ignition Anhydrous: Ignite, preferably in a mufflefurnace, about 3 g of sample, accurately weighed, at 800° for30 min.
Packaging and Storage Store in well-closed containers.
Calcium Phosphate, Tribasic
Tricalcium Phosphate; Precipitated Calcium Phosphate;Calcium Hydroxyapatite
Ca3(PO4)2 Formula wt 310.18Ca5OH(PO4)3 Formula wt 502.31Ca10(OH)2(PO4)6 Formula wt 1004.61
INS: 341(iii) CAS: [7758-87-4]CAS: [1306-06-5]
CAS: [62974-97-4]
DESCRIPTION
Calcium Phosphate, Tribasic, occurs as a white powder thatis stable in air. It consists of a variable mixture of calcium
phosphates. It is insoluble in alcohol and almost insolublein water, but it dissolves readily in dilute hydrochloric andnitric acids.
Function Anticaking agent; buffer; nutrient; clouding agent.
REQUIREMENTS
IdentificationA. Add ammonium molybdate TS to a warm solution of
sample in a slight excess of nitric acid. A yellow precipitateforms.
B. Dissolve about 100 mg of sample by warming it with5 mL of 2.7 N hydrochloric acid and 5 mL of water; whileshaking, add 1 mL of 6 N ammonium hydroxide, dropwise;and then add 5 mL of ammonium oxalate TS. A white precipi-tate forms.Assay Not less than 34.0% and not more than 40.0% ofcalcium (Ca).Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.0075%.Lead Not more than 2 mg/kg.Loss on Ignition Not more than 10.0%.
TESTS
Assay Determine as directed in the monograph for CalciumPhosphate, Dibasic, using a 150-mg sample, accuratelyweighed. Each milliliter of 0.05 M disodium EDTA is equiva-lent to 2.004 mg of Ca.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 5 mL of2.7 N hydrochloric acid.Fluoride (Note: Prepare and store all solutions in plasticcontainers.)
Buffer Solution, Standard Solution, and Electrode Sys-tem Prepare as directed under Fluoride in the monographfor Calcium Phosphate, Dibasic.
Standard Response Line Prepare as directed under Fluo-ride in the monograph for Calcium Phosphate, Dibasic, exceptuse 3.0 mL of hydrochloric acid instead of 2.0 mL.
Procedure Determine as directed under Fluoride in themonograph for Calcium Phosphate, Dibasic, except use 3.0mL of hydrochloric acid instead of 2.0 mL.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Ignition Ignite, preferably in a muffle furnace,about 3 g of sample, accurately weighed, at 800° to 825° toconstant weight.
Packaging and Storage Store in well-closed containers.
78 / Calcium Propionate / Monographs FCC V
Calcium Propionate
(CH3CH2COO)2Ca
C6H10CaO4 Formula wt 186.22
INS: 282 CAS: [4075-81-4]
DESCRIPTION
Calcium Propionate occurs as white crystals or as a crystallinesolid. The pH of a 1:10 aqueous solution is between 7.5 and10.5. One gram dissolves in about 3 mL of water.
Function Preservative; mold inhibitor.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution gives positive tests for Cal-
cium, Appendix IIIA.B. Upon ignition at a relatively low temperature, a sample
yields an alkaline residue that effervesces with acids.Assay Not less than 98.0% and not more than 100.5% ofC6H10CaO4, calculated on the anhydrous basis.Fluoride Not more than 0.003%.Insoluble Substances Not more than 0.2%.Lead Not more than 2 mg/kg.Magnesium (as MgO) Passes test (about 0.4%).Water Not more than 5.0%.
TESTS
Assay Dissolve about 400 mg of sample, accuratelyweighed, in 100 mL of water. While stirring, preferably witha magnetic stirrer, add about 30 mL of 0.05 M disodiumEDTA from a 50-mL buret, then add 15 mL of 1 N sodiumhydroxide and 300 mg of hydroxy naphthol blue indicator,and continue the titration to a blue endpoint. Each milliliterof 0.05 M disodium EDTA is equivalent to 9.311 mg ofC6H10CaO4.Fluoride Determine as directed in Method III under theFluoride Limit Test, Appendix IIIB, using a 1.0-g sample.Insoluble Substances Dissolve 10 g of sample in 100 mLof hot water, filter through a tared filtering crucible, wash theinsoluble residue with hot water, and dry at 105° to constantweight.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Magnesium (as MgO) Place 400.0 mg of sample, 5 mL of2.7 N hydrochloric acid, and about 10 mL of water in a smallbeaker, and dissolve the sample by heating on a hot plate.Evaporate the solution to a volume of about 2 mL, and cool.Transfer the residual liquid into a 100-mL volumetric flask,dilute to volume with water, and mix. Dilute 7.5 mL of thissolution to 20 mL with water, add 2 mL of 1 N sodiumhydroxide and 0.05 mL of a 1:1000 solution of Titan yellow(Clayton yellow), mix, allow to stand for 10 min, and shake.
Any color does not exceed that produced by 1.0 mL of Magne-sium Standard Solution (see Solutions and Indicators) in thesame volume as that of a control containing 2.5 mL of thesample solution (corresponding to 10 mg of sample) and thequantities of the reagents used in the test.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tightly closed containers.
Calcium Pyrophosphate
Ca2P2O7 Formula wt 254.10
INS: 450(vi) CAS: [7790-76-3]
DESCRIPTION
Calcium Pyrophosphate occurs as a fine, white powder. It isinsoluble in water, but is soluble in dilute hydrochloric andnitric acids.
Function Buffer; neutralizing agent; nutrient.
REQUIREMENTS
IdentificationA. Dissolve about 100 mg of sample by warming it with
a mixture of 5 mL of 2.7 N hydrochloric acid and 5 mL ofwater; while shaking, add 2.5 mL of 6 N ammonium hydrox-ide, dropwise, and then add 5 mL of ammonium oxalate TS.A white precipitate forms.
B. Dissolve 100 mg of sample in 100 mL of 1.7 N nitricacid. Add 0.5 mL of this solution to 30 mL of quimociac TS.A yellow precipitate does not form. Heat the remaining portionof the sample solution for 10 min at 95°, and then add 0.5mL of the solution to 30 mL of quimociac TS. A yellowprecipitate forms immediately.Assay Not less than 96.0% of Ca2P2O7.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 2 mg/kg.Loss on Ignition Not more than 1%.
TESTS
Assay Dissolve about 300 mg of sample, accuratelyweighed, in 10 mL of 2.7 N hydrochloric acid, add about 120mL of water and a few drops of methyl orange TS, and boilfor 30 min, keeping the volume and pH of the solution constantduring the boiling period by adding hydrochloric acid or water,if necessary. Add 2 drops of methyl red TS and 30 mL ofammonium oxalate TS, then, while constantly stirring, add,dropwise, a mixture of equal volumes of 6 N ammoniumhydroxide and water until the pink color of the indicator just
FCC V Monographs / Calcium Saccharin / 79
disappears. Digest on a steam bath for 30 min, cool to roomtemperature, allow the precipitate to settle, and filter the super-natant liquid through a sintered-glass crucible, using gentlesuction. Wash the precipitate in the beaker with about 30 mLof cold (below 20°) wash solution, prepared by diluting 10 mLof ammonium oxalate TS to 1000 mL. Allow the precipitate tosettle, and pour the supernatant liquid through the filter. Repeatthis washing by decantation three more times. Using the washsolution, transfer the precipitate as completely as possible tothe filter. Finally, wash the beaker and the filter with two 10-mL portions of cold (below 20°) water. Place the sintered-glass crucible in the beaker, and add 100 mL of water and50 mL of cold 1:6 sulfuric acid. Add 35 mL of 0.1 N potassiumpermanganate from a buret, and stir until the color disappears.Heat to about 70°, and complete the titration with 0.1 Npotassium permanganate. Each milliliter of 0.1 N potassiumpermanganate is equivalent to 6.35 mg of Ca2P2O7.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 5 mL of2.7 N hydrochloric acid.Fluoride Determine as directed under Fluoride Limit Test,Appendix IIIB, using 1.0 g of sample, accurately weighed.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Ignition Ignite about 1 g of sample, accuratelyweighed, preferably in a muffle furnace at 800° to 825° for30 min.
Packaging and Storage Store in well-closed containers.
Calcium Saccharin1,2-Benzisothiazolin-3-one 1,1-Dioxide Calcium Salt
SO2
N
2
Ca·3½H2O
O
C14H8CaN2O6S2·31⁄2H2O Formula wt 467.48
INS: 954 CAS: anhydrous [6485-34-3]
DESCRIPTION
Calcium Saccharin occurs as white crystals or as a white,crystalline powder. One gram is soluble in 1.5 mL of water.
Function Nonnutritive sweetener.
REQUIREMENTS
IdentificationA. Dissolve about 100 mg of sample in 5 mL of a 1:20
solution of sodium hydroxide, evaporate to dryness, and gently
fuse the residue over a small flame until ammonia no longerevolves. After the residue has cooled, dissolve it in 20 mLof water, neutralize the solution with 2.7 N hydrochloric acid,and filter. Add 1 drop of ferric chloride TS to the filtrate. Aviolet color appears.
B. Mix 20 mg of sample with 40 mg of resorcinol, cau-tiously add 10 drops of sulfuric acid, and heat the mixture ina liquid bath at 200° for 3 min. After cooling, add 10 mL ofwater and an excess of 1 N sodium hydroxide. A fluorescentgreen liquid results.
C. A 1:10 aqueous solution gives positive tests for Calcium,Appendix IIIA.
D. Add 1 mL of hydrochloric acid to 10 mL of a 1:10aqueous solution. A crystalline precipitate of saccharin forms.Wash the precipitate well with cold water, and dry at 105°for 2 h. The saccharin thus obtained melts between 226° and230° (see Melting Range or Temperature, Appendix IIB).Assay Not less than 98.0% and not more than 101.0% ofC14H8CaN2O6S2, calculated on the anhydrous basis.Benzoate and Salicylate Passes test.Lead Not more than 2 mg/kg.Readily Carbonizable Substances Passes test.Selenium Not more than 0.003%.Toluenesulfonamides Not more than 0.0025%.Water Not more than 15.0%.
TESTS
Assay With the aid of 10 mL of water, quantitatively transferabout 500 mg of sample, accurately weighed, into a separator.Add 2 mL of 2.7 N hydrochloric acid, and extract the precipi-tated saccharin, first with 30 mL, then with five 20-mL por-tions, of a solvent comprising 9:1 (v/v) chloroform:alcohol.Filter each extract through a small filter paper moistened withthe solvent mixture, and evaporate the combined filtrates todryness on a steam bath with the aid of a current of air.Dissolve the residue in 75 mL of hot water, cool, add phenol-phthalein TS, and titrate with 0.1 N sodium hydroxide. Per-form a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N sodiumhydroxide is equivalent to 20.22 mg of C14H8CaN2O6S2.Benzoate and Salicylate Add 3 drops of ferric chloride TSto 10 mL of a 1:20 aqueous solution previously acidified with5 drops of glacial acetic acid. No precipitate or violet colorappears.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Readily Carbonizable Substances Determine as directedunder Readily Carbonizable Substances, Appendix II, using200 mg of sample dissolved in 5 mL of 95% sulfuric acidand kept at 48° to 50° for 10 min. The color is no darkerthan that of Matching Fluid A.Selenium Determine as directed in Method I under the Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.Toluenesulfonamides
Methylene Chloride Use a suitable grade (such as thatobtainable from Burdick & Jackson Laboratories, Inc.), equiv-
80 / Calcium Silicate / Monographs FCC V
alent to the product obtained by distillation in an all-glassapparatus.
Internal Standard Stock Solution Transfer 100.0 mg of95% n-tricosane (obtainable from Chemical Samples Co.) intoa 10-mL volumetric flask, dissolve in and dilute to volumewith n-heptane, and mix.
Stock Standard Preparation Transfer 20.0 mg each ofreagent-grade o-toluenesulfonamide and p-toluenesulfon-amide into a 10-mL volumetric flask, dissolve in and diluteto volume with methylene chloride, and mix.
Diluted Standard Preparations Pipet 0.1, 0.25, 1.0, 2.5,and 5.0 mL, respectively, of the Stock Standard Preparationinto five 10-mL volumetric flasks. Pipet 0.25 mL of the Inter-nal Standard Stock Solution into each flask, dilute each tovolume with methylene chloride, and mix. These solutionscontain, respectively, 20, 50, 200, 500, and 1000 �g/mL ofeach toluenesulfonamide, plus 250 �g of n-tricosane.
Test Preparation (See Chromatography, Appendix IIA.)Dissolve 2.00 g of sample in 8.0 mL of 5% sodium bicarbonatesolution, and mix the solution thoroughly with 10.0 g ofchromatographic siliceous earth (Celite 545, Johns-Manville,or equivalent). Transfer the mixture into a 250- × 25-mmchromatographic tube, or equivalent, having a fritted-glassdisk and a Teflon stopcock at the bottom and a reservoir atthe top. Pack the contents of the tube by tapping the columnon a padded surface, and then by tamping firmly from thetop. Place 100 mL of methylene chloride in the reservoir, andadjust the stopcock so that 50 mL of eluate is collected in 20to 30 min. Add 25 �L of Internal Standard Stock Solutionto the eluate, mix, and then concentrate the solution to avolume of 1.0 mL in a suitable concentrator tube fitted witha modified Snyder column, using a Kontes tube heater main-tained at 90°.
Procedure Inject 2.5 �L of the Test Preparation into asuitable gas chromatograph equipped with a flame-ionizationdetector and a 3-m × 2-mm (id) glass column, or equivalent,packed with 3% phenyl methyl silicone (OV-17, AppliedScience Laboratories, Inc., or equivalent) on 100- to 120-mesh, silanized, calcined, diatomaceous silica (Gas-ChromQ, Applied Science, or equivalent).
Caution: The glass column should extend into the injec-tor for on-column injection and into the detector baseto avoid contact with metal.
Maintain the column at 180°. Set the injection port tempera-ture to 225° and the detector to 250°. Use helium as thecarrier gas with a flow rate of 30 mL/min. Set the instrumentattenuation so that 2.5 �L of the Diluted Standard Preparationcontaining 200 �g/mL of each toluenesulfonamide gives aresponse of 40% to 80% of full-scale deflection. Record thechromatogram, note the peaks for o-toluenesulfonamide, p-toluenesulfonamide, and the n-tricosane internal standard, andcalculate the areas for each peak by suitable means. Theretention times for o-toluenesulfonamide, p-toluenesulfon-amide, and n-tricosane are about 5, 6, and 15 min, respectively.
In a similar manner, obtain the chromatograms for 2.5-�Lportions of each of the five Diluted Standard Preparations,and for each solution, determine the areas of the o-toluenesul-fonamide, p-toluenesulfonamide, and n-tricosane peaks. From
the values thus obtained, prepare standard curves by plottingthe concentration of each toluenesulfonamide, in microgramsper milliliter, versus the ratio of the respective toluenesulfon-amide peak area to that of n-tricosane. From the standardcurve, determine the concentration, in micrograms per millili-ter, of each toluenesulfonamide in the Test Preparation. Di-vide each value by 2 to convert the result to milligrams perkilogram of the toluenesulfonamide in the 2-g sample takenfor analysis.
Note: If the toluenesulfonamide content of the sampleis greater than about 500 mg/kg, the impurity maycrystallize out of the methylene chloride concentrate(see Test Preparation). Although this level of impurityexceeds that permitted by the specification, the analysismay be completed by diluting the concentrate withmethylene chloride containing 250 �g of n-tricosane permilliliter, and by applying appropriate dilution factorsin the calculation. Care must be taken to redissolvecompletely any crystalline toluenesulfonamide to givea homogeneous solution.
Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Calcium Silicate
INS: 552 CAS: [1344-95-2]
DESCRIPTION
Calcium Silicate occurs as a white to off white, free-flowingpowder that remains so after absorbing relatively largeamounts of water or other liquids. It is a hydrous or anhydroussilicate with varying proportions of CaO and SiO2. It is insolu-ble in water, but it forms a gel with mineral acids. The pHof a 1:20 aqueous slurry is between 8.4 and 12.5.
Function Anticaking agent; filter aid.
REQUIREMENTS
IdentificationA. Mix about 500 mg of sample with 10 mL of 2.7 N
hydrochloric acid, filter, and neutralize the filtrate to litmuspaper with 6 N ammonium hydroxide. The neutralized filtrategives positive tests for Calcium, Appendix IIIA.
B. Prepare a bead by fusing a few crystals of sodiumammonium phosphate on a platinum loop in the flame of aBunsen burner. Place the hot, transparent bead in contact witha sample, and again fuse. Silica floats about in the bead,producing, upon cooling, an opaque bead with a weblikestructure.Fluoride Not more than 10 mg/kg.
FCC V Monographs / Calcium Silicate / 81
Lead Not more than 5 mg/kg.The following additional Requirements should conform to therepresentations of the vendor: Calcium Oxide, Loss on Drying,Loss on Ignition, and Silicon Dioxide.
TESTS
Assay for Silicon Dioxide Transfer about 400 mg of sample,accurately weighed, into a beaker, add 5 mL of water and 10mL of perchloric acid, and heat until dense, white fumes ofperchloric acid evolve.
Caution: Handle perchloric acid in an appropriatefume hood.
Cover the beaker with a watch glass, and continue to heatfor 15 min longer. Allow to cool, add 30 mL of water, filter,and wash the precipitate with 200 mL of hot water. Retainthe combined filtrate and washings for use in the Assay forCalcium Oxide (below). Transfer the filter paper and its con-tents into a platinum crucible, heat slowly to dryness, andthen heat sufficiently to char the filter paper. After cooling,add a few drops of sulfuric acid, and then ignite at about1300° to constant weight. Moisten the residue with 5 dropsof sulfuric acid, add 15 mL of hydrofluoric acid, heat cau-tiously on a hot plate until all of the acid is driven off, andignite to constant weight at a temperature not lower than1000°.
Caution: Handle hydrofluoric acid in an appropriatefume hood.
Cool in a desiccator, and weigh. The loss in weight is equiva-lent to the amount of SiO2 in the sample taken.Assay for Calcium Oxide Using 1 N sodium hydroxide,neutralize to litmus the combined filtrate and washings re-tained in the Assay for Silicon Dioxide (above), and add, whilestirring, about 30 mL of 0.05 M disodium EDTA from a 50-mL buret. Add 15 mL of 1 N sodium hydroxide and 300 mgof hydroxy naphthol blue indicator, and continue the titrationto a blue endpoint. Each milliliter of 0.05 M disodium EDTAis equivalent to 2.804 mg of CaO.Fluoride
0.2 N EDTA/0.2 N TRIS Solution Transfer 18.6 g of diso-dium ethylenediaminetetraacetate (EDTA) and 6.05 g of tris(hydroxymethyl)aminomethane (TRIS), both accuratelyweighed, into a 250-mL beaker. Add 200 mL of hot, deionizedwater, and stir until dissolved. Adjust the pH to 7.5 to 7.6 byadding 5 N sodium hydroxide. Cool the solution, and adjustthe pH to 8.0 with 5 N sodium hydroxide. Transfer the solutioninto a 250-mL volumetric flask, and dilute to volume withdeionized water. Mix well, and store in a plastic container.
Fluoride Stock Solution (1000 mg/kg F) Dissolve 2.210 gof sodium fluoride, accurately weighed, in 50 mL of deionizedwater. Transfer the solution into a 1-L volumetric flask, anddilute to volume. Store this solution and all fluoride solutionsin plastic containers.
100 mg/kg Fluoride Solution Pipet 10 mL of FluorideStock Solution into a 100-mL volumetric flask, and dilute tovolume with deionized water.
On the day of use prepare the following:10 mg/kg Fluoride Solution Pipet 10 mL of 100 mg/kg
Fluoride Solution into a 100-mL volumetric flask, and diluteto volume with deionized water.
1 mg/kg Fluoride Solution Pipet 1 mL of 100 mg/kg Fluo-ride Solution into a 100-mL volumetric flask, and dilute tovolume with deionized water.
Sample Solution Transfer 5 g of sample, accuratelyweighed, into a 150-mL Teflon beaker. Add 40 mL of deion-ized water and 20 mL of 1 N hydrochloric acid. Heat to nearboiling for 1 min while stirring continuously. Cool in an icebath, transfer into a 100-mL volumetric flask, and dilute tovolume with deionized water. The sample does not dissolvecompletely.
Calibration Curve Pipet 20 mL of 10 mg/kg FluorideSolution and 20 mL of 1 mg/kg Fluoride Solution into separate100-mL plastic beakers. Add 10 mL of 0.2 N EDTA/0.2 NTRIS Solution to each beaker. Measure the potential, in milli-volts, of each solution with a suitable fluoride-selective, ion-indicating electrode and a calomel reference electrode con-nected to a pH meter capable of measuring potentials with areproducibility of � 0.2 mV (Orion model 96-09 combinationfluoride electrode, or equivalent). Generate a standard curveby plotting the logarithms of the fluoride ion concentrations,in milligrams per kilogram, of the Fluoride Solutions versusthe potential, in millivolts, or calibrate an Orion ExpandableIon Analyzer EA-940 (or an equivalent instrument) for a directconcentration reading.
Procedure Pipet a 20-mL aliquot of Sample Solution intoa 100-mL plastic beaker, add 10 mL of 0.2 N EDTA/0.2 NTRIS Solution, and measure the solution potential as describedunder Calibration Curve (above). From the measured potentialof the Sample Solution, calculate the concentration, in milli-grams per kilogram, of fluoride ion from the CalibrationCurve.Lead
Lead Nitrate Stock Solution Dissolve 159.8 mg of ACSreagent-grade Lead Nitrate [Pb(NO3)2] in 100 mL of watercontaining 1 mL of nitric acid, dilute to 1000.0 mL withwater, and mix. Each milliliter of this solution contains 100�g of lead (Pb) ion. Prepare and store this solution in glasscontainers that are free from lead salts.
Standard Lead Solution On the day of use, dilute stepwiseand quantitatively an accurately measured volume of LeadNitrate Stock Solution with water to obtain the Standard LeadSolution, which contains 0.25 �g/mL of lead (Pb) ion.
Sample Solution Transfer 5.0 g of sample into a 250-mLbeaker, add 50 mL of 0.5 N hydrochloric acid, cover with awatch glass, and heat slowly to boiling. Boil gently for 15min, cool, and let the undissolved material settle. Decant thesupernatant liquid through Whatman No. 4, or equivalent,filter paper into a 100-mL volumetric flask, retaining as muchas possible of the insoluble material in the beaker. Wash theslurry and beaker with three 10-mL portions of hot water,decanting each washing through the filter paper into the flask.Finally, wash the filter paper with 15 mL of hot water, coolthe filtrate to room temperature, dilute to volume with water,and mix.
82 / Calcium Sorbate / Monographs FCC V
Procedure Set a suitable atomic absorption spectropho-tometer to a wavelength of 217 nm. Adjust the instrument tozero absorbance against water. Read the absorbance of theStandard Lead Solution.
Aspirate the Sample Solution into the spectrophotometer,and measure the absorbance in the same manner. The ab-sorbance obtained from the Sample Solution is not greaterthan that obtained from the Standard Lead Solution.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Loss on Ignition Transfer about 1 g of sample, previouslydried at 105° for 2 h and accurately weighed, into a suitabletared crucible, and ignite at 900° to constant weight.
Packaging and Storage Store in well-closed containers.
Calcium Sorbate2,4-Hexadienoic Acid, Calcium Salt
CH3CH CHCH CHCOO Ca2
C12H14CaO4 Formula wt 262.32
INS: 203 CAS: [7492-55-9]
DESCRIPTION
Calcium Sorbate occurs as a fine, white crystalline powder.It decomposes at about 400°. It is sparingly soluble in waterand practically insoluble in organic solvents, in fats, and inoils.
Function Antimicrobial agent; preservative.
REQUIREMENTS
IdentificationA. Ignite 1 g of sample at 800°. Cool, and slake with 10
mL of water. Add glacial acetic acid until the sample isdissolved, and filter if necessary. The resulting solution givespositive tests for Calcium, Appendix IIIA.
B. Place 200 mg of sample in 5 mL of methanol. Add 0.1mL of 1 N sodium hydroxide, and dissolve in 95 mL of water.After the addition of a few drops of bromine TS, the colordisappears.Assay Not less than 98.0% and not more than 101.0% ofC12H14CaO4, calculated on the dried basis.Acidity (as sorbic acid) Passes test (approximately 1%).Alkalinity [as Ca(OH)2] Passes test (approximately 0.5%).Lead Not more than 2 mg/kg.Loss on Drying Not more than 1.0%.
TESTS
Assay Dissolve about 150 mg of sample, accuratelyweighed, in 50 mL of glacial acetic acid in a 250-mL glass-
stoppered Erlenmeyer flask, warming if necessary to effectsolution. Cool to room temperature, add 2 drops of crystalviolet TS, and titrate with 0.1 N perchloric acid in glacialacetic acid to a blue-green endpoint that persists for at least30 s.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions),and make any necessary correction. Two milliliters of 0.1 Nperchloric acid is equivalent to 26.23 mg of C12H14CaO4.Acidity or Alkalinity Add some drops of methanol, 30 mLof water, and several drops of phenolphthalein TS to 1 g ofsample. If the mixture is colorless, titrate with 0.1 N sodiumhydroxide to a pink color that persists for 15 s. Not morethan 1.0 mL is required. If the mixture is pink, titrate with0.1 N hydrochloric acid. Not more than 1.35 mL is requiredto discharge the pink color.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.
Packaging and Storage Store in tight containers.
Calcium Stearate
CAS: [1592-23-0]
DESCRIPTION
Calcium Stearate occurs as a fine, white to yellow-white,bulky powder. It is a compound of calcium with a mixture ofsolid organic acids obtained from edible sources and consistschiefly of variable proportions of calcium stearate and calciumpalmitate. It is unctuous and free from grittiness. It is insolublein water, in alcohol, and in ether.
Function Anticaking agent; binder; emulsifier.
REQUIREMENTS
IdentificationA. Heat 1 g of sample with a mixture of 25 mL of water
and 5 mL of hydrochloric acid. Fatty acids are liberated,floating as an oily layer on the surface of the liquid. Thewater layer gives positive tests for Calcium, Appendix IIIA.
B. Mix 25 g of sample with 200 mL of hot water, thenadd 60 mL of 2 N sulfuric acid, and heat the mixture, whilestirring frequently, until the fatty acids separate cleanly as atransparent layer. Wash the fatty acids with boiling water untilfree from sulfate, collect them in a small beaker, and warmthem on a steam bath until the water has separated and thefatty acids are clear. Allow the acids to cool, pour off the
FCC V Monographs / Calcium Stearoyl Lactylate / 83
water layer, then melt the acids, filter into a dry beaker, anddry at 105° for 20 min. The solidification point of the fattyacids so obtained is not below 54° (see Solidification Point,Appendix IIB).Assay Not less than 9.0% and not more than 10.5% of CaO,calculated on the dried basis.Free Fatty Acids (as stearic acid) Not more than 3.0%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 4.0%.
TESTS
Assay Boil about 1.2 g of sample, accurately weighed, with50 mL of 0.1 N hydrochloric acid for 10 min, or until thefatty acid layer is clear, adding water if necessary to maintainthe original volume. Cool, filter, and wash the filter and flaskthoroughly with water until the last washing is not acid tolitmus. Neutralize the filtrate to litmus with 1 N sodium hy-droxide. While stirring, preferably with a magnetic stirrer,add about 30 mL of 0.05 M disodium EDTA from a 50-mLburet, then add 15 mL of 1 N sodium hydroxide and 300 mgof hydroxy naphthol blue indicator, and continue the titrationto a blue endpoint. Each milliliter of 0.05 M disodium EDTAis equivalent to 2.804 mg of CaO.Free Fatty Acids (as stearic acid) Transfer 2 g of sample,accurately weighed, into a dry, 125-mL Erlenmeyer flaskcontaining 50 mL of acetone, fit an air-cooled reflux condenseronto the neck of the flask, boil the mixture on a steam bathfor 10 min, and cool. Filter through two layers of WhatmanNo. 42, or equivalent, filter paper, and wash the flask, residue,and filter with 50 mL of acetone. Add phenolphthalein TSand 5 mL of water to the filtrate, and titrate with 0.1 N sodiumhydroxide. Perform a blank determination (see General Provi-sions) using 100 mL of acetone and 5 mL of water, and makeany necessary correction. Each milliliter of 0.1 N sodiumhydroxide is equivalent to 28.45 mg of stearic acid (C18H36O2).Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° to constant weight,using 2-h increments of heating.
Packaging and Storage Store in well-closed containers.
Calcium Stearoyl Lactylate
Calcium Stearoyl-2-Lactylate; Calcium Stearoyl Lactate
INS: 482(i) CAS: [5793-94-2]
DESCRIPTION
Calcium Stearoyl Lactylate occurs as a cream-colored powder.It is a mixture of calcium salts of stearoyl lactic acid, with
minor proportions of other salts of related acids. It is slightlysoluble in hot water.
Function Dough conditioner; stabilizer; whipping agent.
REQUIREMENTS
IdentificationA. Heat 1 g of sample with a mixture of 25 mL of water
and 5 mL of hydrochloric acid. Fatty acids are liberated,floating as an oily layer on the surface of the liquid. Thewater layer gives positive tests for Calcium, Appendix IIIA.
B. Mix 25 g of sample with 200 mL of hot water, thenadd 60 mL of 2 N sulfuric acid, and heat the mixture, whilestirring frequently, until the fatty acids separate cleanly as atransparent layer. Wash the fatty acids with boiling water untilfree from sulfate, collect them in a small beaker, and warmthem on a steam bath until the water has separated and thefatty acids are clear. Allow the acids to cool, pour off thewater layer, then melt the acids, filter into a dry beaker, anddry at 105° for 20 min. The solidification point of the fattyacids so obtained is not below 54° (see Solidification Point,Appendix IIB).Acid Value Between 50 and 86.Calcium Content Between 4.2% and 5.2%.Ester Value Between 125 and 164.Lead Not more than 2 mg/kg.Total Lactic Acid Between 32.0% and 38.0%.
TESTS
Acid Value Transfer about 1 g of sample, accuratelyweighed, to a 125-mL volumetric flask, add 25 mL of alcohol,previously neutralized in phenolphthalein TS, and heat on ahot plate until the sample is dissolved. Cool, add 5 drops ofphenolphthalein TS, and titrate rapidly with 0.1 N sodiumhydroxide to the appearance of the first pink color that persistsfor at least 30 s. Calculate the acid value by the formula
56.1V × N/W,
in which V is the volume, in milliliters, N is the normality ofthe sodium hydroxide solution, and W is the weight, in grams,of the sample taken. Retain the neutralized solution for thedetermination of Ester Value.Calcium Content
Stock Lanthanum Solution Transfer 5.86 g of lanthanumoxide (La2O3) into a 100-mL volumetric flask, wet with afew milliliters of water, slowly add 25 mL of hydrochloricacid, and swirl until the lanthanum oxide is completely dis-solved. Dilute to volume with water, and mix.
Stock Calcium Solution Transfer 124.8 mg of calciumcarbonate (CaCO3) previously dried at 200° for 4 h, into a100-mL volumetric flask, carefully dissolve in 2 mL of 2.7N hydrochloric acid, dilute to volume with water, and mix.This 500-mg/kg calcium solution is commercially available.
Standard Preparations Transfer 10.0 mL of the StockLanthanum Solution into each of three 50-mL volumetricflasks. Using a microliter syringe, transfer 0.20 mL of theStock Calcium Solution into the first flask, 0.40 mL into the
84 / Calcium Sulfate / Monographs FCC V
second flask, and 0.50 mL into the third flask. Dilute eachflask to volume with water, and mix. The flasks contain 2.0,4.0, and 5.0 �g of calcium per milliliter, respectively. Preparethese solutions fresh daily.
Sample Preparation Transfer about 250 mg of sample,accurately weighed, into a 30-mL beaker. While heating, dis-solve the sample in 10 mL of alcohol, and quantitativelytransfer the solution into a 25-mL volumetric flask. Wash thebeaker with two 5-mL portions of alcohol, adding the wash-ings to the flask, dilute to volume with alcohol, and mix.Transfer 5.0 mL of the Stock Lanthanum Solution to a second25-mL volumetric flask. Using a microliter syringe, transfer0.25 mL of the alcoholic solution of the sample to the secondflask, dilute to volume with water, and mix.
Procedure Concomitantly determine the absorbance ofeach Standard Preparation and of the Sample Preparation at422.7 nm, with a suitable atomic absorption spectrophotome-ter, following the operating parameters as recommended bythe manufacturer of the instrument. Plot the absorbance ofthe Standard Preparations versus concentration of calcium,in micrograms per milliliter, and from the curve so obtaineddetermine the concentration, C, in micrograms per milliliter,of calcium in the Sample Preparation. Calculate the quantity,in milligrams, of calcium in the sample taken by the formula
2.5C.
Ester Value Prepare an alcoholic potassium hydroxide solu-tion by dissolving 11.2 g of potassium hydroxide in 250 mLof alcohol and diluting with 25 mL of water. Add 10.0 mLof this solution to the neutralized solution retained in the testfor Acid Value (above). Add 5 drops of phenolphthalein TS,connect a suitable condenser, and reflux for 2 h. Cool, add 5additional drops of phenolphthalein TS, and titrate the excessalkali with 0.1 N sulfuric acid. Perform a blank determination(see General Provisions) using 10.0 mL of the alcoholic potas-sium hydroxide solution, and make any necessary correction.Calculate the ester value by the formula
56.1(B – S) × (N/W),
in which B – S represents the difference between the volumesof 0.1 N sulfuric acid required for the blank and the sample,respectively, N is the normality of the sulfuric acid, and Wis the weight, in grams, of the sample taken.Lead Determine as directed in the Atomic Absorption Spec-trophotometric Method under Lead Limit Test, Appendix IIIB,using a 10-g sample.Total Lactic Acid
Standard Solution Dissolve 1.067 g of lithium lactate(LiC3H5O3) in sufficient water to make 1000.0 mL.
Standard Curve Transfer 10.0 mL of the Standard Solu-tion into a 100-mL volumetric flask, dilute to volume withwater, and mix. Transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of thediluted Standard Solution into separate 100-mL volumetricflasks, dilute each flask to volume with water, and mix. Thesestandards represent 1, 2, 4, 6, and 8 �g of lactic acid permilliliter, respectively. Transfer 1.0 mL of each solution intoseparate test tubes, and continue as directed in the Procedure,beginning with ‘‘Add 1 drop of cupric sulfate TS. . . .’’ Aftercolor development and reading the absorbance values, con-
struct a Standard Curve by plotting absorbance versus micro-grams of lactic acid.
Test Preparation Transfer about 200 mg of sample, accu-rately weighed, into a 125-mL Erlenmeyer flask, add 10 mLof 0.5 N alcoholic potassium hydroxide and 10 mL of water,attach an air condenser, and reflux gently for 45 min. Washthe sides of the flask and the condenser with about 40 mL ofwater, and heat on a steam bath until no odor of alcoholremains. Add 6 mL of 1:2 sulfuric acid, heat until the fattyacids are melted, then cool to about 60°, and add 25 mLof petroleum ether. Swirl the mixture gently, and transferquantitatively to a separator. Collect the water layer in a 100-mL volumetric flask, and wash the petroleum ether layer withtwo 20-mL portions of water, adding the washings to thevolumetric flask. Dilute to volume with water, and mix. Trans-fer 1.0 mL of this solution into a second 100-mL volumetricflask, dilute to volume with water, and mix.
Procedure Transfer 1.0 mL of the Test Preparation intoa test tube, and transfer 1.0 mL of water to a second test tubeto serve as the blank. Treat each tube as follows: Add 1 dropof cupric sulfate TS, swirl gently, and then rapidly add 9.0mL of sulfuric acid from a buret. Loosely stopper the tube,and heat in a water bath at 90° for exactly 5 min. Coolimmediately to below 20° in an ice bath for 5 min, add 3drops of p-phenylphenol TS, shake immediately, and heat ina water bath at 30° for 30 min, shaking the tube twice duringthis time to disperse the reagent. Heat the tube in a waterbath at 90° for exactly 90 s, and then cool immediately to roomtemperature in an ice water bath. Determine the absorbanceof the solution in a 1-cm cell, at 570 nm, with a suitablespectrophotometer, using the blank to set the instrument. Ob-tain the weight, in micrograms, of lactic acid in the portionof the Test Preparation taken for the Procedure by means ofthe Standard Curve.
Packaging and Storage Store in tight containers in a cool,dry place.
Calcium Sulfate
CaSO4 Formula wt, anhydrous 136.14CaSO4·2H2O Formula wt, dihydrate 172.18
INS: 516 CAS: anhydrous [7778-18-9]CAS: dihydrate [10101-41-4]
DESCRIPTION
Calcium Sulfate occurs as a fine, white to slightly yellow-white powder. It is anhydrous or contains two molecules ofwater of hydration.
Function Nutrient; yeast food; dough conditioner; firmingagent; sequestrant.
FCC V Monographs / Candelilla Wax / 85
REQUIREMENTS
Identification Dissolve about 200 mg of sample by warm-ing it with a mixture of 4 mL of 2.7 N hydrochloric acid and16 mL of water. A white precipitate forms when 5 mL ofammonium oxalate TS is added to 10 mL of the solution.Upon the addition of barium chloride TS to the remaining 10mL, a white precipitate forms that is insoluble in hydrochloricand nitric acids.Assay Not less than 98.0% of CaSO4, calculated on thedried basis.Fluoride Not more than 0.003%.Lead Not more than 2 mg/kg.Loss on Drying Anhydrous: Not more than 1.5%; Dihy-drate: Between 19.0% and 23.0%.Selenium Not more than 0.003%.
TESTS
Assay Dissolve 250 mg of sample, accurately weighed, in100 mL of water and 4 mL of 2.7 N hydrochloric acid, boilto effect solution, and cool. While stirring, preferably with amagnetic stirrer, add about 30 mL of 0.05 M disodium EDTAfrom a 50-mL buret, then add 25 mL of 1 N sodium hydroxideand 300 mg of hydroxy naphthol blue indicator, and continuethe titration to a blue endpoint. Each milliliter of 0.05 Mdisodium EDTA is equivalent to 6.807 mg of CaSO4.Fluoride Determine as directed under Fluoride Limit Test,Appendix IIIB, using 1.67 g of sample, accurately weighed.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample to constant weight at 250°.Selenium Determine as directed in Method II under Sele-nium Limit Test, Appendix IIIB, using a 200-mg sample.
Packaging and Storage Store in well-closed containers.
Cananga Oil
CAS: [68606-83-7]
DESCRIPTION
Cananga Oil occurs as a light to deep yellow liquid with aharsh, floral odor suggestive of ylang ylang. It is the oilobtained by distillation from the flowers of the tree Canangaodorata Hook f. et Thoms (Fam. Anonaceae). It is soluble inmost fixed oils and in mineral oil, but it is practically insolublein glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as those
of a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between –15° and –30°.Refractive Index Between 1.495 and 1.505 at 20°.Saponification Value Between 10 and 40.Solubility in Alcohol Passes test.Specific Gravity Between 0.904 and 0.920.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed under Saponi-fication Value, Appendix VI, using about 5 g of sample,accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 95% alcohol, usually becoming cloudy on furtherdilution.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Candelilla Wax
INS: 902 CAS: [8006-44-8]
DESCRIPTION
Candelilla Wax occurs as a hard, yellow-brown, opaque totranslucent wax. It is a purified wax obtained from the leavesof the candelilla plant, Euphorbia antisyphilitica (Fam. Eu-phorbiaceae). Its specific gravity is about 0.983. It is solublein chloroform and in toluene, but insoluble in water.
Function Masticatory substance in chewing gum base; sur-face-finishing agent.
REQUIREMENTS
Identification The infrared absorption spectrum of a meltedsample on a potassium bromide plate exhibits relative maximaat the same wavelengths as those of a typical spectrum asshown in the section on Infrared Spectra, using the same testconditions as specified therein.Acid Value Between 12 and 22.Lead Not more than 3 mg/kg.Melting Range Between 68.5° and 72.5°.Saponification Value Between 43 and 65.
View IR
View IR
86 / Canola Oil / Monographs FCC V
TESTS
Acid Value Determine as directed in Method I under AcidValue, Appendix VII.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV, using 10 �g of lead (Pb) ion in thecontrol.Melting Range Determine as directed in Procedure forClass II under Melting Range or Temperature, Appendix IIB.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.
Packaging and Storage Store in well-closed containers.
Canola Oil
Low Erucic Acid Rapeseed Oil; LEAR Oil
CAS: [120962-03-0]
DESCRIPTION
Canola Oil occurs as a light yellow oil. It is typically obtainedby a combination of mechanical expression followed by n-hexane extraction, from the seed of the plant Brassica juncea,Brassica napus, or Brassica rapa (Fam. Cruciferae). The plantvarieties are those producing oil-bearing seeds with a lowerucic acid (C22:1) content. It is a mixture of triglyceridescomposed of both saturated and unsaturated fatty acids. It isrefined, bleached, and deodorized to substantially remove freefatty acids; phospholipids; color; odor and flavor components;and miscellaneous, other non-oil materials. It can be hydroge-nated to reduce the level of unsaturated fatty acids for func-tional purposes in foods. It is a liquid at 0° and above.
Function Cooking or salad oil; component of margarine orshortening; coating agent; texturizer.
REQUIREMENTS
Labeling Hydrogenated Canola Oil less than fully hydroge-nated must be labeled as Partially Hydrogenated Canola Oil.Identification Unhydrogenated Canola Oil exhibits the fol-lowing composition profile of fatty acids, determined as di-rected under Fatty Acid Composition, Appendix VII.
Fatty Acid: <14 14:0 16:0 16:1 18:0 18:1 18:2Weight % (Range): <0.1 <0.2 <6.0 <1.0 <2.5 >50 <40.0Fatty Acid: 18:3 20:0 20:1 22:0 22:1 24:0 24:1Weight % (Range): <14 <1.0 <2.0 <0.5 <2.0 <0.2 <0.2
Acid Value Not more than 6.Cold Test Passes test.Color (AOCS-Wesson) Not more than 1.5 red/15 yellow.Erucic Acid Not more than 2.0%.
Free Fatty Acids (as oleic acid) Not more than 0.05%.Iodine Value Between 110 and 126.Lead Not more than 0.1 mg/kg.Linolenic Acid Not more than 14.0%.Peroxide Value Not more than 10 meq/kg.Refractive Index Between 1.465 and 1.467 at 40°.Saponifiable Value Between 178 and 193.Stability Not less than 7 h.Sulfur Not more than 10 mg/kg.Unsaponifiable Matter Not more than 1.5%.Water Not more than 0.1%.
TESTS
Acid Value Determine as directed in Method II under AcidValue, Appendix VII.Cold Test Determine as directed under Cold Test, Appen-dix VII.Color (AOCS Wesson) Determine as directed under Color(AOCS-Wesson), Appendix VII, using a 133.4-mm cell.Erucic Acid Determine as part of Fatty Acid Composition,Appendix VII.Free Fatty Acids (as oleic acid) Determine as directed underFree Fatty Acids, Appendix VII, using the following equiva-lence factor (e) in the formula given in the procedure:
Free fatty acids as oleic acid, e = 28.2.
Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Linolenic Acid Determine as directed under Fatty AcidComposition, Appendix VII.Peroxide Value Accurately weigh about 10 g of sample,add 30 mL of a 3:2 mixture of glacial acetic acid:chloroform,and mix. Add 1 mL of a saturated solution of potassiumiodide, and mix for 1 min. Add 100 mL of water, begintitrating with 0.05 N sodium thiosulfate, adding starch TS asthe endpoint is approached, and continue the titration untilthe blue starch color has just disappeared. Perform a blankdetermination (see General Provisions), and make any neces-sary correction. Calculate the peroxide value, as milliequiva-lents of peroxide per kilogram of sample, by the formula
S × N × 1000/W,
in which S is the net volume, in milliliters, of sodium thiosul-fate solution required for the sample; N is the exact normalityof the sodium thiosulfate solution; and W is the weight, ingrams, of the sample taken.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponifiable Value Determine as directed under Saponifi-cation Value, Appendix VII.Stability Determine as directed under Stability, AppendixVII.Sulfur Organosulfur compounds present in the sample reactwith Raney nickel to produce nickel sulfides. Nickel sulfides
FCC V Monographs / Canola Oil / 87
are treated with a strong acid to produce hydrogen sulfide,which is trapped and titrated with mercuric acetate using adithizone indicator.
Caution: This test requires the use of the followinghazardous substances: mercuric acetate, spongy nickel,and dibenzyl disulfide. Conduct the test in a fume hood.
Apparatus Fit a 125-mL, round-bottom boiling flask witha cylindrical filling funnel (20 mL with open top), an STPTFE metering valve stopcock, and a gas inlet tube (see thefigure for Raney Nickel Reduction Apparatus in AppendixIIIC under Sulfur (by Oxidative Microcoulometry). Fit a water-jacketed distillation column with hooks on top of the boilingflask. Fit a piece of glass tubing with ground ST inner jointswith hooks to the distillation column, and connect the distilla-tion column and a gas dispersion tube with ST outer jointswith hooks.
Dibenzyl Disulfide Solution Accurately weigh 0.75 g ofdibenzyl disulfide, and place in a 250-mL volumetric flask.Dilute to volume with methyl isobutyl ketone, and mix.
Sulfur Standard Accurately weigh five 250.0-g samplesof food-grade peanut oil. Transfer 0.0, 1.0, 2.0, 3.0, and 4.0mL of the Dibenzyl Disulfide Solution into the peanut oilsamples; the samples contain 0, 3, 6, 9, and 12 mg/kg ofsulfur, respectively.
Raney Nickel Preparation (Caution: Raney nickel is py-rophoric when dry.) Raney nickel is produced by reactingnickel–aluminum alloy with sodium hydroxide. Each Raneynickel pellet is prepared individually, and each is enoughcatalyst for one determination. To produce one Raney nickelpellet, accurately weigh 1 g of nickel–aluminum alloy powder(50% Ni, 50% Al), place it in a 50-mL centrifuge tube, andchill it in an ice bath. Slowly add 5 mL of water to the tube,and let it stand for 10 min. Then, slowly add 10 mL of 2.5N sodium hydroxide, and allow the mixture to react for 30min. Cap the tube, and place it in a 50° water bath for 2 h.Centrifuge the mixture at 1000 rpm for 10 min, and discardthe supernatant liquid. Wash the pellet twice with 15 mL ofwater and twice with 15 mL of isopropanol, centrifugingbetween each wash. Store the catalyst under isopropanol forno longer than 2 weeks.
Note: Properly dispose of unused Raney Nickel Prepa-ration by transferring it to a 250-mL Erlenmeyer flask,and placing it in a fume hood. Add 20 mL of 60%(w/v) hydrochloric acid, and allow complete digestionof the catalyst.
Caution: Hydrogen gas evolves during the digestionprocess.
Dithizone Indicator Solution Dissolve 10 mg of dithizone(diphenylthiocarbazone) with acetone in a 10-mL volumetricflask, and fill to volume.
Mercuric Acetate Titrant (Caution: Mercuric acetate isa strong irritant when ingested or inhaled or upon dermalexposure.) Transfer 3.82 g of mercuric acetate into a 1000-mL volumetric flask containing 950 mL of water. Add 12.2mL of glacial acetic acid, dilute to volume with water, andmix. Transfer 10.0 mL of this solution into a 100-mL volumet-
ric flask, dilute to volume with water, and mix. The titrantsolution contains 0.0012 M mercuric acetate.
Titration Reagent Blank Add 50.0 mL of 1 N sodiumhydroxide and 50.0 mL of acetone to a 250-mL beaker, andmix. Add 0.5 mL of the Dithizone Indicator Solution, andtitrate with Mercuric Acetate Titrant until the color changesfrom bright amber to red. Record the volume of titrant used.
Procedure Place 15 to 20 g of sample, accuratelyweighed, on the bottom of the boiling flask. Discard theisopropanol from the Raney Nickel Preparation, add 10 mLof 95% isopropanol, mix, and add the mixture to the sample.Attach the water condenser and the nitrogen line to the boilingflask, and adjust the gas flow to 4 psi through the sample.Place a heating mantle under the flask. Immerse the bubblerin a 250-mL beaker containing 50.0 mL of 1 N sodium hydrox-ide, and stir slowly. Boil the sample for 90 min. Add 50 mLof acetone and 0.5 mL of Dithizone Indicator Solution to the250-mL beaker. Add 20 mL of 60% hydrochloric acid intothe filling funnel fitted onto the boiling flask, and adjust thenitrogen flow to 2 to 3 psi. Position the stir bar directly underthe bubbler for maximum dispersion of the hydrogen sulfidebubbles. Slowly add the solution of 60% hydrochloric acidto the boiling flask. Begin the titration with Mercuric AcetateTitrant until the bright amber color changes to red. Addenough hydrochloric acid to turn the solution in the boilingflask green, and then let it boil for 15 min. Continue thetitration throughout the boiling stage, making sure to rinsethe inside of the bubbler with the solution in the beaker byturning off the nitrogen flow until the solution rises to thetop of the vertical tube (the solution usually returns to amberduring the first rinse). Rinse the tube a second time. Continuethe titration, and record the volume of titrant used to thenearest 0.01 mL.
Calculation The concentration of sulfur in the sample,in milligrams per kilogram, is calculated by the followingformula:
(VS – VB) × K/W,
in which VS is the volume, in milliliters, of titrant to theendpoint for the sample; VB is the volume, in milliliters,of titrant to the endpoint for the blank (usually about 0.10mL); K is a constant determined from the calibration ofthe Sulfur Standard (expressed as micrograms of sulfur permilliliter of titrant); and W is the weight, in grams, of thesample taken.
The Sulfur Standards are analyzed, in duplicate, to deter-mine the constant, K, and are calculated by the followingformula:
K = W × C/(VS –VB),
in which W is the weight, in grams, of the Sulfur Standard;C is the concentration, in milligrams per kilogram, of theSulfur Standard; VS is the volume, in milliliters, of titrant forthe Sulfur Standard; and VB is the volume, in milliliters, oftitrant for the Titration Reagent Blank.
88 / Canthaxanthin / Monographs FCC V
Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB. However, in place of 35 to 40 mL of methanol,use 50 mL of a 1:1 chloroform:methanol mixture to dissolvethe sample.
Packaging and Storage Store in tightly closed containers,ensuring no contact with metals, filled to the top or flushedwith nitrogen gas.
Canthaxanthin4,4’-Diketo-�-carotene; Cantha; �-Carotene-4,4′-dione
O
O
C40H52O2 Formula wt 564.85
INS: 161g CAS: [514-78-3]
DESCRIPTION
Canthaxanthin occurs as a dark, crystalline powder. It is solu-ble in chloroform, very slightly soluble in acetone, but insolu-ble in water. It melts at about 207° to 212° with decomposition.
Function Color.
REQUIREMENTS
Identification The absorption spectrum of Sample SolutionB, prepared as directed in the Assay (below), exhibits a maxi-mum near 470 nm.Assay Not less than 96.0% and not more than 101.0% ofC40H52O2.Arsenic Not more than 3 mg/kg.Lead Not more than 10 mg/kg.Mercury Not more than 1 mg/kg.Residue on Ignition Not more than 0.2%.
TESTS
Assay (Note: Carry out all work in low-actinic glasswareand in subdued light.)
Sample Solution A Transfer about 50 mg of sample, accu-rately weighed, into a 100-mL volumetric flask, dissolve in10 mL of acid-free chloroform, immediately dilute to volumewith cyclohexane, and mix. Pipet 5 mL of this solution intoa second 100-mL volumetric flask, dilute to volume withcyclohexane, and mix.
Sample Solution B Pipet 5 mL of Sample Solution A intoa 50-mL volumetric flask, dilute to volume with cyclohexane,and mix.
Procedure Using a suitable spectrophotometer, determinethe absorbance of Sample Solution B in a 1-cm cell at thewavelength of maximum absorption at about 470 nm usingcyclohexane as the blank. Calculate the quantity, in milli-grams, of C40H52O2 in the sample taken by the formula
20,000A/220,
in which A is the absorbance of the solution and 220 is theabsorptivity of pure Canthaxanthin.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds.Mercury Determine as directed under Mercury Limit Test,Appendix IIIB.Residue on Ignition Ignite 1 g of sample as directed underResidue on Ignition, Appendix IIC, using a silica crucible andmoistening the residue with 2 mL of nitric acid and 1 mL ofsulfuric acid.
Packaging and Storage Store in tight, light-resistant con-tainers under inert gas.
Caramel
Caramel Color
INS: 150 CAS: [8028-89-5]
DESCRIPTION
Caramel usually occurs as a dark brown to black liquid orsolid. It is a complex mixture of compounds, some of whichare in the form of colloidal aggregates. Caramel is manufac-tured by heating carbohydrates, either alone or in the presenceof food-grade acids, alkalies, and/or salts. Caramel is producedfrom commercially available, food-grade nutritive sweetenersconsisting of fructose, dextrose (glucose), invert sugar, su-crose, malt syrup, molasses, and/or starch hydrolysates andfractions thereof. The acids that may be used are food-gradesulfuric, sulfurous, phosphoric, acetic, and citric acids; thealkalies are ammonium, sodium, potassium, and calcium hy-droxides; and the salts are ammonium, sodium, and potassiumcarbonate, bicarbonate, phosphate (including mono- and diba-sic), sulfate, and bisulfite. Food-grade antifoaming agents suchas polyglycerol esters of fatty acids may be used as processingaids during its manufacture. Caramel is soluble in water.
Four distinct classes of Caramel can be distinguished bythe reactants used in their manufacture and by specific identifi-cation tests:
FCC V Monographs / Caramel / 89
Class I (Plain Caramel, Caustic Caramel) Prepared byheating carbohydrates with or without acids or alkalis; noammonium or sulfite compounds are used.
Class II (Caustic Sulfite Caramel) Prepared by heatingcarbohydrates with or without acids or alkalis in the presenceof sulfite compounds; no ammonium compounds are used.
Class III (Ammonia Caramel) Prepared by heating carbo-hydrates with or without acids or alkalis in the presence ofammonium compounds; no sulfite compounds are used.
Class IV (Sulfite Ammonia Caramel) Prepared by heatingcarbohydrates with or without acids or alkalis in the presenceof both sulfite and ammonium compounds.
All of these Caramels shall meet the criteria establishedfor Caramel in this monograph.
Function Color.
REQUIREMENTS
Ammoniacal Nitrogen1 Not more than 0.6%, calculated onan equivalent color basis.Arsenic2 Not more than 1 mg/kg.Color Intensity3 Between 0.01 and 0.6 absorbance units(a.u.).Lead2 Not more than 2 mg/kg.Mercury2 Not more than 0.1 mg/kg.4-Methylimidazole1 Not more than 0.025%, calculated onan equivalent color basis.Sulfur Dioxide1 Not more than 0.2%, calculated on anequivalent color basis.Total Nitrogen1 Not more than 3.3%, calculated on anequivalent color basis.Total Sulfur1 Not more than 3.5%, calculated on an equiva-lent color basis.
TESTS
Ammoniacal Nitrogen Transfer 25.0 mL of 0.1 N sulfuricacid into a 500-mL receiving flask, and connect it to a distilla-tion apparatus consisting of a Kjeldahl connecting bulb anda condenser so that the condenser delivery tube is immersedbeneath the surface of the acid solution in the receiving flask.Transfer about 2 g of sample, accurately weighed, into an800-mL long-neck Kjeldahl digestion flask, and add 2 g ofcarbonate-free magnesium oxide, 200 mL of water, and sev-eral boiling chips to the flask. Swirl the digestion flask tomix the contents, and quickly connect it to the distillationapparatus. Heat the digestion flask to boiling, and collectabout 100 mL of distillate in the receiving flask. Wash thetip of the delivery tube with a few milliliters of water, collect-ing the washings in the receiving flask; add 4 or 5 drops of
1These tests are calculated on an equivalent color basis that permitsthe values to be expressed in terms of a Caramel having a color intensitystandardized to 0.1 a.u.
2These tests are calculated on an as-is basis.3Color Intensity is defined as the absorbance of a 0.1% (w/v) solution
of Caramel in water measured in a 1-cm cell at 610 nm and is expressedon a Total Solids basis.
methyl red TS; titrate with 0.1 N sodium hydroxide; andrecord the volume, in milliliters, as S. Conduct a blank deter-mination (see General Provisions), and record the millilitersof 0.1 N sodium hydroxide required as B. Calculate the percentammoniacal nitrogen (equivalent color basis) by the formula
[(B – S) × 0.0014 × 100/W] × 0.1/A610,
in which 0.0014 is the milliequivalent weight of nitrogen for0.1 N sodium hydroxide; W is the weight, in grams, of thesample taken; 0.1 is the basis of color equivalency; and A610
is the color intensity as-is.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds, and 1.0 mL of the Standard ArsenicSolution (1 g As) in the control.Color Intensity (Note: Because Color Intensity is expressedon a solids basis, determine Total Solids first.)
TOTAL SOLIDS
Liquid Samples Use fine quartz sand that passes a No.40, but not a No. 60, sieve and that has been prepared bydigestion with hydrochloric acid, washed acid free, dried, andignited. Mix 30.0 g of the prepared sand, accurately weighed,with 1.5 to 2.0 g of sample, accurately weighed, and dry toconstant weight at 60° under reduced pressure (50 mm Hg).Record the final weight of the sand plus the sample solids.Calculate the percent total solids as follows:
[(WF – WS)/WC] × 100,
in which WF is the final weight, in grams, of the sand plusthe sample solids; WS is the weight, in grams, of the preparedsand taken; and WC is the weight, in grams, of the sampletaken.
Solid Samples, Powdered or Granular Determine as di-rected under Loss on Drying, Appendix IIC, drying a sampleat 60° under reduced pressure (50 mm Hg) to constant weight.Calculate the percent total solids by the formula
[(WD – WB)/(WS – WB)] × 100,
in which WD is the weight, in grams, of the bottle and sampleafter drying; WB is the weight, in grams, of the empty bottle;and WS is the weight, in grams, of the bottle and samplebefore drying.
COLOR INTENSITY
Color Intensity Transfer 100 mg of sample into a 100-mL volumetric flask, dilute to volume with water, and mix.Centrifuge if the solution is cloudy. Determine the absorbance(A610) of the clear solution in a 1-cm cell at 610 nm witha suitable spectrophotometer previously standardized usingwater as the reference. Calculate the color intensity by theformula
(A610 × 100)/S,
in which S is the percent total solids.Lead (Note: For this test, use reagent-grade chemicals withas low a lead content as is practicable as well as high-puritywater and gases. Before use in this analysis, rinse all glasswareand plasticware twice with 10% nitric acid and twice with10% hydrochloric acid, and then rinse them thoroughly with
90 / Caramel / Monographs FCC V
high-purity water, preferably obtained from a mixed-bed,strong-acid, strong-base ion-exchange cartridge capable ofproducing water with an electrical resistivity of 12 to 15megohms.)
Lead Nitrate Stock Solution Dissolve 159.8 mg of ACSReagent-Grade Lead Nitrate [Pb(NO3)2] in 100 mL of watercontaining 1 mL of nitric acid, dilute to 1000.0 mL with water,and mix. Prepare and store this solution in glass containers thatare free from lead salts. Each milliliter of this solution contains100 �g of lead (Pb) ion.
Standard Lead Solution On the day of use, transfer 50.0mL of Lead Nitrate Stock Solution into a 500-mL volumetricflask containing 50 mL of water, add 5 mL of nitric acid,dilute to volume with water, and mix. Each milliliter of Stan-dard Lead Solution contains the equivalent of 10 �g of lead(Pb) ion.
Standard Solutions Prepare a series of lead standard solu-tions serially diluted from the Standard Lead Solution. Pipet2, 5, 10, and 20 mL, respectively, of Standard Lead Solutioninto separate 100-mL volumetric flasks, add 1 mL of nitricacid, dilute to volume, and mix. The Standard Solutions con-tain, respectively, 0.20, 0.50, 1.00 and 2.00 �g of lead permilliliter.
Sample Solution Transfer about 25 g of sample, accuratelyweighed, into an ashing vessel. [Suitable ashing vessels haveapproximately a 100-mL capacity and are flat-bottom plati-num crucibles or dishes, Vycor or quartz tall-form beakers,or Vycor evaporating dishes (Corning Glass Works No. 13180,or equivalent). Discard Vycor vessels when the inner surfacesbecome etched.] Dry the sample overnight at 120° in a forced-draft oven. (The sample must be absolutely dry to preventflowing or spattering in the furnace.) Place the sample in afurnace equipped with a pyrometer to control the temperatureover a range of 260° to 600°, with a variation of less than10°, and set it at 250°. Slowly, in 50° increments, raise thetemperature to 350°, and hold at this temperature until smok-ing ceases. Increase the temperature to 500° in approximately75° increments (the sample must not ignite). Ash for 16 h orovernight at 500°. Remove the sample from the furnace, andallow it to cool. The ash should be white and essentiallycarbon free. If the ash still contains excess carbon particles(i.e., the ash is gray rather than white), proceed as follows:Wet with a minimal amount of water followed by the dropwiseaddition of 0.5 to 3 mL of nitric acid. Dry on a hot plate.Transfer the ash to a furnace set at 250°, slowly increase thetemperature to 500°, and continue heating for 1 to 2 h. Repeatthe nitric acid treatment and ashing, if necessary, to obtain acarbon-free residue.
Note: Local overheating or deflagration may result ifthe sample still contains much intermingled carbon andespecially if much potassium is present in the ash.
Dissolve the residue in 5 mL of 1 N nitric acid, warmingon a steam bath or hot plate for 2 to 3 min to aid solution.Filter, if necessary, and decant through S&S 589 Black Ribbonpaper, or equivalent, into a 50-mL volumetric flask. Repeatwith two 5-mL portions of 1 N nitric acid, filter, and add thewashings to the original filtrate. Dilute to volume with 1 Nnitric acid, and mix to prepare the Sample Solution.
Similarly, prepare duplicate reagent blanks for each Stan-dard Solution and Sample Solution, including any additionalwater and nitric acid if used for sample ashing.
Note: Do not ash nitric acid in a furnace because thelead contaminant will be lost.
Evaporate nitric acid to dryness in an ashing vessel on a steambath or hot plate, and then proceed as above, beginning at‘‘Dissolve the residue in 5 mL of 1 N nitric acid, warmingon a steam bath. . . .’’
Note: Complete the sample preparation and the analysison the same day.
Aqueous Butyl Acetate Use spectral-grade butyl acetate,and saturate it with water.
APDC Solution Transfer 2.00 g of APDC (ammonium 1-pyrrolidinedithiocarbamate) (Aldrich Chemical, or equiva-lent) into a 100-mL volumetric flask, dilute to volume withwater, and mix. Remove insoluble free acid and other impuri-ties normally present by two to three extractions with 10-mLportions of Aqueous Butyl Acetate.
Lead-Free Citric Acid Solution Dissolve 10 g of citricacid in 30 mL of water. While stirring, slowly add ammoniumhydroxide until the pH is between 8.0 and 8.5, using short-range pH paper as an external indicator. Transfer the solutionto a separatory funnel, and extract with 10-mL portions ofDithizone Extraction Solution (under Lead Limit Test, Appen-dix IIIB), until the dithizone solution retains its green coloror remains unchanged. Drain the final dithizone layer, plusabout 1 mL of the aqueous layer, into a beaker, and whilestirring, slowly add 1:1 nitric acid until the pH is between3.5 and 4, again using short-range pH paper as an externalindicator. Transfer this solution into a 100-mL volumetricflask (through a filter, if necessary), dilute to volume withwater, and mix thoroughly.
Pipet 20 mL each of the Standard Solutions, the SampleSolution, and the appropriate reagent blanks into separate 60-mL separatory funnels. Treat each solution as follows: Add4 mL of Citric Acid Solution and 2 to 3 drops of bromocresolgreen TS. The solution should be yellow. Adjust the pHto about 5.4, using ammonium hydroxide initially and thenammonium hydroxide diluted with 4 volumes of water in thevicinity of the color change (the first permanent appearanceof light blue). Add 4 mL of APDC Solution, stopper, andshake for 30 to 60 s. Pipet 5.0 mL of Aqueous Butyl Acetate,stopper the separatory funnel, and shake vigorously for 30 to60 s. Let stand until the layers separate clearly, and drain anddiscard the lower aqueous phase. If an emulsion forms or thesolvent layer is cloudy, drain the solvent layer into a 15-mLcentrifuge tube, cover with aluminum foil or Parafilm (orequivalent), and centrifuge for about 1 min at 2000 rpm.
Procedure Use an atomic absorption spectrophotometerequipped with a 4-in., single-slot burner head. Set the instru-ment to previously determined optimum conditions for organicsolvent aspiration (3 to 5 mL/min) and at a wavelength of283.3 nm. Use an air–acetylene flame adjusted for maximumlead absorption with a fuel-lean flame. Aspirate the blanks,the Standard Solutions, and the Sample Solution, flushingwith water and then with Aqueous Butyl Acetate between
FCC V Monographs / Caramel / 91
measurements. Record the absorbance of the Standard Solu-tions and the Sample Solution (containing butyl acetate), andcorrect for the blanks. Prepare the Standard Curve by plottingthe absorbance of each Standard Solution against its concen-tration, in 1 g of lead per milliliter. (This concentration, inbutyl acetate, is four times that in the aqueous standard.) Fromthe Standard Curve, determine the concentration, C, in 1 g/mL, of the Sample Solution. Calculate the quantity, in milli-grams per kilogram, of lead in the sample by the formula
12.5 × C/W,
in which W is the weight, in grams, of the sample taken.Mercury
Standard Preparation Prepare as directed under MercuryLimit Test, Appendix IIIB, using 1.0 mL of the stock solution,equivalent to 1 �g of mercury, instead of the 2.0 mL specifiedtherein.
Sample Preparation Transfer 5 g of sample into a 250-mL Erlenmeyer flask, and continue as directed in the secondfull paragraph for Sample Solution under Arsenic Limit Test,Appendix IIIB, beginning with ‘‘add 5 mL of sulfuric acidand a few glass beads. . . .’’ After the sample has been digestedand the solution diluted to 35 mL, as directed therein, add 1mL of a 1:25 solution of potassium permanganate, and mix.
Procedure Continue as directed in Procedure under Mer-cury Limit Test, Appendix IIIB. Any absorbance produced bythe Sample Preparation is not more than half that producedby the Standard Preparation, indicating not more than 0.1mg of mercury per kilogram of sample.4-Methylimidazole (4-MeI)
4-Methylimidazole Stock Solution Purify reagent-grade 4-methylimidazole (Aldrich, or equivalent) by redistillation (b.p.92° to 93°, 0.05 mm Hg), and then prepare a stock solutionby transferring 50 mg of the distillate, accurately weighed,into a 50-mL volumetric flask and diluting to volume withtetrahydrofuran (acetone is also an acceptable solvent). Mixthoroughly, and store in a refrigerator.
Standard Solutions Pipet 1.0-, 1.5-, 2.0-, 2.5-, 3.0-, 3.5-,4.0-, and 5.0-mL portions of the 4-Methylimidazole StockSolution into separate 10-mL volumetric flasks, dilute eachto volume with the same solvent used to prepare the stocksolution, and mix. The standards thus prepared represent 4-methylimidazole concentrations (w/v) of 100, 150, 200, 250,300, 350, 400, and 500 mg/L, respectively. Store the StandardSolutions in a refrigerator, and use within 1 month.
Sample Preparation Place a plug of fine glass wool inthe base of a 300- × 22-mm (id) chromatographic tube havinga Teflon stopcock. The column bed, approximately 150 mmtall, should be of uniform consistency, yet open enough toallow elution to occur readily. Transfer 10.0 g of sample,accurately weighed, into a 250-mL polypropylene beaker, andmix thoroughly with 5.0 g of 3 N sodium hydroxide. The pH ofthe mixture should exceed 12. Add 20.0 g of chromatographicsiliceous earth (Johns-Manville Celite 545, or equivalent) tothe beaker, and thoroughly mix with a wide-blade, stainlesssteel spatula until a homogeneous, semidry mixture is ob-tained. Homogeneity is obtained when the color is uniformand no dark clumps are seen. Quantitatively transfer the mix-ture into the column. Place a plug of glass wool on top of
the column, and then allow the column to fall a short distancevertically to help settle the contents.
Rinse the sample beaker with methylene chloride, and pourthe contents into the column with the stopcock open. Allowthe methylene chloride to pass down the column until itreaches the stopcock. Close the stopcock and allow the methyl-ene chloride to remain in contact with the column bed for 5min. Open the stopcock, and pass methylene chloride throughthe column at a rate of 5 mL/min. Collect 200 mL of eluatein a 300-mL round-bottom flask. While maintaining the flaskat a temperature of 35° in a water bath, remove the bulk ofthe solvent from the eluate by rotary vacuum evaporation(350 to 390 mm Hg). Reduce the volume to about 1 mL.During the concentration step, watch the flask carefully toensure that no loss of sample occurs by bumping. Use adisposable Pasteur pipet to quantitatively transfer the extractresidue to a 5-mL volumetric flask, rinsing the flask severaltimes with small (approximately 0.7 mL) portions of the samesolvent used to prepare the original solutions (tetrahydrofuranor acetone) until the dilution mark is reached. Mix thoroughly.
Procedure (See Chromatography, Appendix IIA.) Use asuitable gas chromatograph equipped with a hydrogen flame-ionization detector, and a silanized 1-m × 4-mm (id) glasscolumn, or equivalent, packed with 90- to 100-mesh AnakromABS, or equivalent, containing 7.5% Carbowax 20M and 2%potassium hydroxide, or equivalent. Maintain the column at190° (isothermal). Set the injection point temperature to 200°and the detector temperature to 250°. Use nitrogen as thecarrier gas, with a flow rate of 50 mL/min.
Use an autosampler to inject 5.0 �L of each StandardSolution, and determine their concentrations.
Note: If using manual injections, avoid fractionation inthe syringe needle, and ensure that 5.0 �L is injectedby using the solvent-flush technique with the solventused to prepare the Standard Solutions.
For each Standard Solution chromatogram, obtain the cor-rected peak area. If not using an integrator, calculate thecorrected peak area by multiplying the peak height, in millime-ters, by the peak width at one-half height, in millimeters, bythe proper attenuation and range factors, depending on theparticular apparatus and operating parameters used. Plot eachcorrected peak area thus obtained versus its respective concen-tration of 4-methylimidazole to obtain the standard curve. Inthe same manner, chromatograph a 5.0-�L portion of theSample Preparation, calculate the peak area correspondingto any 4-methylimidazole contained in the sample, and byreference to the standard curve, obtain the content of the 4-methylimidazole in the sample.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X, using 0.5 g of sample, accuratelyweighed.Total Nitrogen Determine as directed in Method II underNitrogen Determination, Appendix IIIC.Total Sulfur Place 1 to 3 g of magnesium oxide or anequivalent quantity of magnesium nitrate, hexahydrate (6.4to 19.2 g); 1 g of powdered sucrose; and 50 mL of nitric acidinto the largest casserole available that fits in an electric mufflefurnace. Transfer about 5 g of sample, accurately weighed,
92 / Caramel / Monographs FCC V
into the casserole when the expected amount of sulfur is 2.5%or less, or 1 g of sample when the expected amount is greaterthan 2.5%. Place the same quantities of reagents in anothercasserole for the blank. Evaporate on a steam bath to theconsistency of paste. Place the casserole in a cold electricmuffle furnace, gradually heat to 525°, and hold at that temper-ature until all nitrogen dioxide fumes are driven off. Cool thecasserole, add 100 mL of water to dissolve the sample, andneutralize to pH 7 with hydrochloric acid, using short-rangepH indicator paper as an external indicator. Add an additional2 mL of hydrochloric acid, filter the solution into a suitablebeaker, heat to boiling, and while stirring, slowly add 20 mLof barium chloride TS to the hot solution. Boil the contentsof the beaker for 5 min, and allow to stand overnight. Filterthe contents of the beaker through a tight, ashless filter paper,and quantitatively transfer the precipitate to the paper. Thor-oughly wash the paper and the precipitate with hot water, andthen transfer the paper to a tared crucible previously ignitedfor 1 h at 800° in a muffle furnace. Dry the paper in thecrucible for 1 h at 105°, and then carefully char it, with freeaccess to air, at low heat over a burner. Gradually increasethe heat to burn away the paper, and ignite the crucible andcontents for 1 h at 800°. Cool and weigh, and calculate thepercent sulfur by the formula
[(WS – WB)/S] × 13.74 × 0.1/A610,
in which WS is the weight, in grams, of the ignited residueof barium sulfate from the sample determination; WB is theweight, in grams, of the ignited residue from the blank determi-nation; and S is the weight, in grams, of the sample taken.
Packaging and Storage Store in well-closed containers andavoid exposure to excessive heating and, for solid products,excessive humidity.
ADDITIONAL INFORMATION
Identification of Classes The four classes of Caramel maybe distinguished from each other by the following methods:
Class I Not more than 50% of the color is bound byDEAE (diethylaminoethyl) cellulose, and not more than 50%of the color is bound by phosphoryl cellulose.
Class II More than 50% of the color is bound by DEAEcellulose, and it exhibits an absorbance ratio (at equal concen-trations) of more than 50.
Class III Not more than 50% of the color is bound byDEAE cellulose, and more than 50% of the color is boundby phosphoryl cellulose.
Class IV More than 50% of the color is bound by DEAEcellulose, and it exhibits an absorbance ratio (at equal concen-trations) of not more than 50.Identification Tests for ClassesAbsorbance Ratio (Note: For the purposes of this test,Absorbance Ratio is defined as the absorbance of Caramel,at equal concentrations, at 280 nm divided by the absorbance at560 nm.) Transfer 100 mg of sample into a 100-mL volumetricflask with the aid of water, dilute to volume, mix, and centri-fuge if the solution is cloudy. Pipet 5.0 mL of the clear solutioninto a 100-mL volumetric flask, dilute to volume with water,
and mix. Using a spectrophotometer equipped with a mono-chromator to provide a band width of 2 nm or less and ofsuch quality that the stray-light characteristic is 0.5% or less,standardized with water as a reference, determine the ab-sorbance of the 0.1% solution in a 1-cm cell at 560 nm andthat of the 1:20 diluted solution at 280 nm. Calculate theabsorbance ratio of the sample by the formula
(AC1 × 20)/AC2,
in which AC1 is the sample absorbance at 280 nm, 20 is thedilution factor, and AC2 is the sample absorbance at 560 nm.Color Bound by DEAE Cellulose (Note: For the purposesof this monograph, Color Bound by DEAE Cellulose is definedas the percent decrease in absorbance of a Caramel solutionat 560 nm after treatment with DEAE cellulose.)
Special Reagent Use DEAE cellulose of 1.0 meq/g capac-ity. DEAE cellulose of higher or lower capacities may beused in proportionately higher or lower quantities.
Procedure Prepare a Sample Solution of approximately0.5 absorbance unit at 560 nm by transferring an appropriateamount of sample into a 100-mL volumetric flask with theaid of 0.025 N hydrochloric acid. Dilute to volume with 0.025N hydrochloric acid, and centrifuge or filter if the solution iscloudy. Add 140 mg of Special Reagent to a 20-mL aliquotof the Sample Solution, mix thoroughly for several minutes,centrifuge or filter, and collect the clear supernatant liquid.Determine the absorbance of the Sample Solution and of thesupernatant liquid in a 1-cm cell at 560 nm, with a suitablespectrophotometer previously standardized using 0.025 N hy-drochloric acid as a reference. Calculate the percent of colorbound by DEAE cellulose by the formula
100[(X1 – X2)/X1],
in which X1 is the absorbance of the Sample Solution at 560nm, and X2 is the absorbance of the supernatant liquid at560 nm.Color Bound by Phosphoryl Cellulose (Note: For the pur-poses of this monograph, Color Bound by Phosphoryl Cellu-lose is defined as the percent decrease in absorbance of aCaramel solution at 560 nm after treatment with phosphorylcellulose.)
Special Reagent Use phosphoryl cellulose (cellulosephosphate) of 1.2 meq/g capacity. Phosphoryl cellulose ofhigher or lower capacities may be used in proportionatelyhigher or lower quantities.
Procedure Transfer 200 to 300 mg of sample into a 100-mL volumetric flask, dilute to volume with 0.025 N hydrochlo-ric acid, and centrifuge or filter if the solution is cloudy. Add1.42 g of Special Reagent to a 40-mL aliquot of the SampleSolution, mix thoroughly for several minutes, centrifuge orfilter, and collect the clear supernatant liquid. Determine theabsorbance of the Sample Solution and of the supernatantliquid in a 1-cm cell at 560 nm with a suitable spectrophotome-ter previously standardized using 0.025 N hydrochloric acid asa reference. Calculate the percent color bound by phosphorylcellulose by the formula
100[(X1 – X2)/X1],
FCC V Monographs / Caraway Oil / 93
in which X1 is the absorbance of the Sample Solution at 560 nm,and X2 is the absorbance of the supernatant liquid at 560 nm.2-Acetyl-4-(5)-tetrahydroxybutylimidazole (THI) [ClassIII (Ammonia Caramel) is the only class of Caramel color foundto contain THI.] Because some countries have a THI limit of25 ppm on an equivalent color basis, the following method fordetermining THI is provided.
2,4-Dinitrophenylhydrazine Hydrochloride (DNPH) Add5 g of reagent-grade 2,4-dinitrophenylhydrazine to 10 mL ofhydrochloric acid contained in a 100-mL Erlenmeyer flask,and gently shake the latter until the free base (red) is convertedto the hydrochloride (yellow). Add 100 mL of ethanol, andheat the mixture on a steam bath until all of the solids havedissolved. Cool to room temperature, and after the solutionhas crystallized, filter off the hydrochloride. Wash the hydro-chloride with ether, dry at room temperature, and store in adesiccator. Upon storage, the hydrochloride slowly convertsto the free base. The latter can be removed by washing withpurified (peroxide-free) dimethoxyethane.
DNPH Hydrochloride Reagent Prepare the reagent bymixing 0.5 g of DNPH Hydrochloride with 15 mL of 5%methanol in dimethoxyethane for 30 min. Store in a refrigera-tor at 4°. When properly prepared and stored, this reagent isstable for at least 3 months.
THI–DNPH Standard Add 0.5 g of DNPH Hydrochlorideto 1 mL of hydrochloric acid, followed by 10 mL of ethylalcohol, and heat on a steam bath until solution. Add 100 mgof THI to the hot solution—crystallization begins in a fewminutes. Filter off the THI–DNPH when the suspensionreaches room temperature. Obtain the THI–DNPH Standardby recrystallizing the THI–DNPH with a hydrochloric acid–ethyl alcohol mixture (1 drop of hydrochloric acid per 5 mLof ethyl alcohol). The yield is 70% to 80% based on theTHI used. When stored in the refrigerator, the THI–DNPHStandard is stable for at least 1 year.
Stock THI–DNPH Solution Dissolve about 10 mg of THI–DNPH Standard, accurately weighed, in a 100-mL volumetricflask, and dilute to volume with absolute carbonyl-free metha-nol. Dilute a portion of this solution tenfold with methanol.The THI concentration, in milligrams per liter, of the StockTHI–DNPH Solution is 0.47 times that of the THI–DNPH.When stored in the refrigerator, the Stock THI–DNPH Solutionis stable for at least 20 weeks.
Cation-Exchange Resin (Strong) Dowex 50 AG × 8, pro-ton form, 100- to 200-mesh.
Cation-Exchange Resin (Weak) Amberlite CG AG 50 I,proton form, 100- to 200-mesh. (Sediment two or three timesbefore use.)
Carbonyl-Free Methanol Add 5 g of Girard’s Reagent P(Aldrich, or equivalent) and 0.2 mL of hydrochloric acid to500 mL of methanol, and reflux for 2 h. Distill the refluxedmethanol through a short Vigreux column, and store in tightlyclosed bottles.
Purified Dimethoxyethane Use distillation to purify di-methoxyethane from 2,4-dinitrophenylhydrazine in the pres-ence of acid, and redistill it from sodium hydroxide. Immedi-ately before use, pass it through a column of neutral aluminato remove peroxides.
Combination Columns Connect a dropping funnel andtwo small columns, one fitted above the other, with 14.5-mmstandard ground-glass joints. Use a 100-mL dropping funnel,equipped with a Teflon stopcock, as the solvent reservoir. Fillthe upper column [150 × 12.5 mm (id), filling height max 9cm, bed height 50 to 60 mm; or 200 × 10 mm (id), fillingheight max 14 cm, bed height 80 to 90 mm], equipped witha 1-mm (id) capillary outlet, with Cation-Exchange Resin(Weak). Fill the lower column [175 mm × 10 mm (id), bedheight 60 mm], equipped with a 1-mm (id) capillary outletand a Teflon stopcock, with Cation-Exchange Resin (Strong).
Sample Preparation Dissolve 200 to 250 mg of sample,accurately weighed, in 3 mL of water. Quantitatively transferthe solution to the upper part of the combination column.Elute with water until a total of about 100 mL of water haspassed through the column. Disconnect the upper column,and elute the lower column with 0.5 N hydrochloric acid.Discard the first 10.0 mL of eluate, and subsequently collecta volume of 35 mL. Concentrate the solution to dryness at40° (15 mm Hg), then dissolve the syrup residue in 250�L of carbonyl-free methanol, and add 250 �L of DNPHHydrochloride Reagent. Transfer the reaction mixture (sam-ple) to a septum-capped vial, and store for 5 h at room temper-ature.
Procedure Prepare a series of THI–DNPH Standard Solu-tions serially diluted from the Stock THI–DNPH Solution.Pipet 1, 2, and 5 mL, respectively, of the Stock THI–DNPHSolution, into separate 10-mL volumetric flasks, and diluteto volume with absolute, carbonyl-free methanol. Prepare astandard curve by injecting 5 �L of the Stock THI–DNPHSolution, and the serially diluted THI–DNPH Standard Solu-tions into a 250-mm × 4-mm (id), 10-1m LiChrosorb RP-8HPLC column (Alltech Associates, Inc., or equivalent) fittedwith an ultraviolet detector set at 385 nm. The mobile phaseis 50:50 (v/v) methanol:0.1 M phosphoric acid. Inject 5 �Lof sample into the column. Adjustments in the mobile phasecomposition may be needed as column characteristics varyamong manufacturers. At a mobile phase flow rate of 2 mL/min and column dimensions of 250 × 4.6 mm, elute THI–DNPH at about 6.3 � 0.1 min. Measure the peak areas.Calculate the amount of THI in the sample from the standardcurve. (For THI limits greater than 25 mg/kg, prepare a seriesof Standard THI–DNPH Solutions in a range encompassingthe expected THI concentration in the sample.)
Caraway Oil
CAS: [8000-42-8]
DESCRIPTION
Caraway Oil occurs as a colorless to pale yellow liquid withthe characteristic odor and taste of caraway. It is a volatile
View IR
94 / Carbon, Activated / Monographs FCC V
oil distilled from the dried, ripe fruit of Carum carvi L. (Fam.Umbelliferae).
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 50.0%, by volume, of ketones ascarvone.Angular Rotation Between +70° and +80°.Refractive Index Between 1.484 and 1.488 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.900 and 0.910.
TESTS
Assay Determine as directed in the Neutral Sulfite Methodunder Aldehydes and Ketones, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 8 mL of 80% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers in acool place protected from light.
Carbon, Activated
DESCRIPTION
Carbon, Activated, occurs as a black substance, varying inparticle size from coarse granules to a fine powder. It is asolid, porous, carbonaceous material prepared by carbonizingand activating organic substances. The raw materials, whichinclude sawdust, peat, lignite, coal, cellulose residues, coconutshells, and petroleum coke, may be carbonized and activatedat a high temperature with or without the addition of inorganicsalts in a stream of activating gases such as steam or carbondioxide. Alternatively, carbonaceous matter may be treatedwith a chemical activating agent such as phosphoric acidor zinc chloride, and the mixture carbonized at an elevatedtemperature, followed by removal of the chemical activatingagent by water washing. Activated Carbon is insoluble inwater and in organic solvents.
Function Decolorizing agent; taste- and odor-removingagent; purification agent in food processing.
REQUIREMENTS
IdentificationA. Place about 3 g of powdered sample in a glass-stoppered
Erlenmeyer flask containing 10 mL of dilute hydrochloricacid (5%), boil for 30 s, and cool to room temperature. Add100 mL of iodine TS, stopper, and shake vigorously for 30s. Filter through Whatman No. 2 filter paper, or equivalent,discarding the first portion of filtrate. Compare 50 mL ofthe subsequent filtrate with a reference solution prepared bydiluting 10 mL of iodine TS to 50 mL with water, but nottreated with carbon. The color of the carbon-treated iodinesolution is no darker than that of the reference solution, indi-cating the adsorptivity of the sample.
B. Ignite a portion of the sample in air. Carbon monoxideand carbon dioxide are produced, and an ash remains.Cyanogen Compounds Passes test.Higher Aromatic Hydrocarbons Passes test.Iodine Number Not less than 400.Lead Not more than 10 mg/kg.Water Extractables Not more than 4.0%.The following additional Requirements should conform to therepresentations of the vendor: Loss on Drying and Residueon Ignition.
TESTS
Cyanogen Compounds Mix 5 g of sample with 50 mL ofwater and 2 g of tartaric acid, and distill the mixture, collecting25 mL of distillate below the surface of a mixture of 2 mLof 1 N sodium hydroxide and 10 mL of water contained ina small flask placed in an ice bath. Dilute the distillate to 50mL with water, and mix. Add 12 drops of ferrous sulfate TSto 25 mL of the diluted distillate, heat almost to boiling, cool,and add 1 mL of hydrochloric acid. No blue color is produced.Higher Aromatic Hydrocarbons Extract 1 g of samplewith 12 mL of cyclohexane in a continuous-extraction appara-tus for 2 h. Place the extract in a Nessler tube and a solutionof 100 �g of quinine sulfate in 1000 mL of 0.1 N sulfuricacid in a matching Nessler tube. The extract shows no morecolor or fluorescence than does the solution when observedunder ultraviolet light.Iodine Number1
Hydrochloric Acid Solution (5% by weight) Add 70 mLof concentrated hydrochloric acid to 550 mL of water, andmix well.
Potassium Iodate Solution (0.1000 N) Dry 4 or moregrams of primary standard-grade potassium iodate (KIO3) at110° � 5° for 2 h, and cool to room temperature in a desiccator.
1Portions of this test are adapted from ‘‘ASTM D 4607-94(1999)—Standard Test Method for Determination of Iodine Number of ActivatedCarbon.’’ The original ASTM method is available in its entirety fromASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428; phone:610-832-9585; fax: 610-832-9555; email: [email protected]; website:<www.astm.org>.
FCC V Monographs / Carbon, Activated / 95
Dissolve 3.5667 � 0.1 mg of the dry potassium iodate inabout 100 mL of water. Quantitatively transfer to a 1-L volu-metric flask, dilute to volume with water, and mix thoroughly.Store in a glass-stoppered bottle.
Starch Solution Mix 1.0 � 0.5 g of starch with 5 to 10mL of cold water to make a paste. Continue to stir whileadding an additional 25 � 5 mL of water to the starch paste.Pour the mixture, while stirring, into 1 L of boiling water,and boil for 4 to 5 min. Make this solution fresh daily.
Sodium Thiosulfate Solution (0.100 N) Dissolve 24.820g of sodium thiosulfate in approximately 75 � 25 mL offreshly boiled water, and add 0.10 � 0.01 g of sodium carbon-ate. Quantitatively transfer the mixture to a 1-L volumetricflask, and dilute to volume with water. Allow the solution tostand for a minimum of 4 days before standardizing. Storethe solution in an amber bottle.
To standardize the solution, perform the following in tripli-cate: Pipet 25.0 mL of 0.1000 N Potassium Iodate Solutioninto a wide-mouthed Erlenmeyer flask. Add 2.00 � 0.01 g ofpotassium iodide, and shake the flask to dissolve the potassiumiodide crystals. Pipet 5.0 mL of concentrated hydrochloricacid into the flask, and titrate the free iodine with SodiumThiosulfate Solution to a light yellow color. Add a few dropsof Starch Solution, and continue the titration until 1 dropproduces a colorless solution. Determine the Sodium Thiosul-fate Solution normality using the following formula:
(P × R)/S,
in which P is the volume, in milliliters, of 0.1000 N PotassiumIodate Solution; R is the normality of the 0.1000 N PotassiumIodate Solution; and S is the volume, in milliliters, of SodiumThiosulfate Solution. Average the three normality results; re-peat the test if the range of values exceeds 0.003 N.
Standard Iodine Solution (0.100 � 0.001 N) Transfer12.700 g of iodine and 19.100 g of potassium iodide (KI),accurately weighed, into a beaker, and mix. Add 2 to 5 mLof water, and stir well. While stirring, continue to add smallincrements, approximately 5 mL each, of water until the totalvolume is 50 to 60 mL. Allow the solution to stand a minimumof 4 h to ensure crystal dissolution, stirring occasionally.Quantitatively transfer the solution to a 1-L volumetric flask,and dilute to volume with water. The iodide-to-iodine weightratio must be 1.5:1. Store the solution in an amber bottle.
Note: Standardize this solution just before use.
To standardize this solution, perform the following in tripli-cate. Pipet 25.0 mL into a 250-mL wide-mouthed Erlenmeyerflask. Titrate with the standardized Sodium Thiosulfate Solu-tion until a light yellow color develops. Add a few drops ofStarch Solution, and continue the titration until 1 drop pro-duces a colorless solution. Determine the Iodine Solution nor-mality using the following formula:
(S × N1)/I,
in which S is the volume, in milliliters, of the standardizedSodium Thiosulfate Solution; N1 is the normality of the stan-dardized Sodium Thiosulfate Solution; and I is the volume,in milliliters, of Iodine Solution. Average the three normalityresults; repeat the test if the range of values exceeds 0.003
N. The standardized Iodine Solution concentration must be0.100 � 0.001 N. If it is not, repeat all of the steps startingfrom, ‘‘Transfer 12.700 g of iodine. . . .’’
Procedure This procedure applies to both powdered andgranular sample. A representative sample of powdered samplemay need additional grinding until 60 wt % (or more) passesthrough a 325-mesh screen and 95 wt % (or more) passesthrough a 100-mesh screen. Grind a representative samplesufficiently to pass through the screens as described above.Dry the ground sample, and cool to room temperature in adesiccator.
Three dosages of sample must be estimated to determinethe iodine number. Weigh the three dosages (M) of dry carbonto the nearest milligram. Transfer each to one of three clean,dry 250-mL Erlenmeyer flasks equipped with ground glassstoppers.
Pipet 10.0 mL of Hydrochloric Acid Solution into eachflask, stopper each flask, and swirl gently until the carbon iscompletely wetted. Loosen the stoppers to vent the flasks,place on a hot plate in a fume hood, and bring the contentsto a boil. Allow to boil gently for 30 � 2 s to remove anysulfur (which may interfere with the test results). Remove theflasks from the hot plate and cool to room temperature.
Standardize and then pipet 100.0 mL of Iodine Solutioninto each flask.
Note: Stagger the addition of standardized Iodine Solu-tion to the three flasks so that no delays are encounteredin handling.
Immediately stopper the flasks, and shake the contents vigor-ously for 30 � 1 s. Quickly filter each mixture by gravitythrough one sheet of folded filter paper (Whatman No. 2V,or equivalent) into one of three beakers.
Note: Prepare the filtration equipment in advance toavoid delays in filtering the samples.
For each filtrate, use the first 20 to 30 mL to rinse a pipet,and discard the rinse portions. Use clean beakers to collectthe remaining filtrates. Mix each filtrate by swirling the bea-ker, and pipet 50.0 mL of each filtrate into one of threeclean 250-mL Erlenmeyer flasks. Titrate each filtrate withstandardized Sodium Thiosulfate Solution until a pale yellowcolor develops. Add 2 mL of Starch Solution, and continuethe titration with standardized Sodium Thiosulfate Solutionuntil 1 drop produces a colorless solution. Record the volume(S) of standardized Sodium Thiosulfate Solution used.
Calculation The capacity of a carbon for any adsorbatedepends on the concentration of the adsorbate. The concentra-tions of the standard iodine solution and filtrate must be knownto determine an appropriate carbon weight to produce finalconcentrations agreeing with the definition of iodine number.The amount of sample to be used in the determination isgoverned by the activity of the sample. If filtrate normalities(C) are not within the range of 0.008 N to 0.040 N, repeatthe procedure using different sample weights.
Once filtrate normalities are set within the specified range,perform the following calculations for each carbon dosage:
A = (N2)(12693.0),
96 / Carbon Dioxide / Monographs FCC V
in which N2 is the normality of the standardized Iodine So-lution.
B = (N1)(126.93),
in which N1 is the normality of the standardized SodiumThiosulfate Solution.
Calculate the dilution factor (DF) using the equation
DF = (I + H)/F,
in which I is the volume, in milliliters, of Iodine Solutionused in the standardization procedure; H is the volume, inmilliliters, of Hydrochloric Acid Solution used; and F is thevolume, in milliliters, of filtrate used.
Calculate the weight, in milligrams, of iodine adsorbed pergram of sample (X/M) by the equation
X/M = [A − (DF)(B)(S)]/M,
in which S is the volume, in milliliters, of standardized SodiumThiosulfate Solution used, and M is the weight, in grams, ofthe sample.
Calculate the normality of the residual filtrate (C) asfollows:
C = (N1 × S)/F.
Using logarithmic paper, plot X/M (as the ordinate) versusC (as the abscissa) for each of the three carbon dosages.Calculate the least squares fit for the three points, and plot.The iodine number is the X/M value at a residual iodineconcentration (C) of 0.02 N. The regression coefficient forthe least squares fit should be greater than 0.995.
Carbon dosages may be estimated initially by using threevalues of C (usually 0.01, 0.02, and 0.03) as follows:
M = [A − (DF)(C)(126.93)(50)]/E,
in which M is the weight, in grams, of the carbon dosage andE is the nominal iodine number of the sample. If new carbondosages have been determined, repeat the Procedure and Cal-culations.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, testing a 20-mL portion of the filtrate obtained inthe test for Water Extractables (below) and using 10 �g oflead ion (Pb) in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 120° for 4 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 500-g sample.Water Extractables Transfer 5.00 g of sample into a 250-mL flask provided with a reflux condenser and a Bunsenvalve. Add 100 mL of water and several glass beads, andreflux for 1 h. Cool slightly, and filter through Whatman No.2, or equivalent, filter paper, discarding the first 10 mL offiltrate. Cool the subsequent filtrate to room temperature, andpipet 25.0 mL into a tared crystallization dish.
Note: Retain the remainder of the filtrate for theLead test.
Evaporate the filtrate in the dish to incipient dryness on a hotplate, never allowing the solution to boil. Dry for 1 h at 100°in a vacuum oven, cool, and weigh.
Packaging and Storage Store in well-closed containers.
Carbon Dioxide
CO2 Formula wt 44.01
INS: 290 CAS: [124-38-9]
DESCRIPTION
Carbon Dioxide occurs as a colorless gas. One liter of CarbonDioxide weighs about 1.98 g at 0° and a pressure of 760 mmHg. Under a pressure of about 59 atmospheres, it may becondensed to a liquid, a portion of which forms a white solid(‘‘dry ice’’) upon rapid vaporization. Solid Carbon Dioxideevaporates without melting upon exposure to air. One volumeof the gas dissolves in about 1 volume of water, forming asolution that is acid to litmus.
Function Propellant and aerating agent; carbonating agent;direct-contact freezing agent.
REQUIREMENTS
IdentificationA. Pass 100 � 5 mL of sample, released from the vapor
phase of the contents of the container, through a carbon diox-ide detector tube (see Detector Tubes under Solutions andIndicators) at the rate specified for the tube. The indicatorchange extends throughout the entire indicating range ofthe tube.
B. The sample, when passed through barium hydroxideTS, forms a precipitate that dissolves with effervescence inacetic acid.Assay Not less than 99.5% of CO2, by volume.Carbonyl Sulfide Not more than 0.5 ppm, by volume.Hydrogen Sulfide Not more than 0.5 ppm, by volume.Nitric Oxide (NO) and Nitrogen Dioxide (NO2) Not morethan 5 ppm total, by volume.Nonvolatile Hydrocarbons Not more than 10 mg/kg.Sulfur Dioxide Not more than 5 ppm, by volume.Volatile Hydrocarbons (as methane) Not more than0.005%, by volume.Water Passes test.
TESTS
Note: The following Tests are designed to reflect thequality of Carbon Dioxide in both its vapor and itsliquid phases, which are present in previously unopenedcylinders. Reduce the container pressure by means ofa regulator.
FCC V Monographs / Carbon Dioxide / 97
Withdraw the samples for the Tests with the leastpossible release of gas consistent with proper purgingof the sampling apparatus. Measure the gases with agas volume meter downstream from the detector tubesto minimize contamination of or changes to the samples.Perform Tests in the sequence in which they are listed.
The various detector tubes called for in the respectiveTests are listed under Detector Tubes in Solutions andIndicators.
Assay (Note: Sampling for this Assay may be done fromthe vapor phase for convenience, but this results in moreresidual volume. If the specification of 0.5 mL is exceededfrom the vapor phase, a liquid sample may be taken.) Assemblea 100-mL gas buret provided with a leveling bulb and a two-way stopcock to a gas absorption pipet of suitable capacityby connecting the pipet to one of the buret outlets. Fill theburet with slightly acidified water (turned pink with methylorange), and fill the pipet with potassium hydroxide solution(1:2). By manipulating the leveling bulb and leveling water,draw the potassium hydroxide solution to fill the pipet andcapillary connection up to the stopcock, and then fill the buretwith the leveling water, and draw it through the other stopcockopening in such a manner that all gas bubbles are eliminatedfrom the system. Draw into the buret 100.0 mL of sampletaken from the liquid phase as directed below in the test forNitric Oxide and Nitrogen Dioxide. By raising the levelingbottle, force the measured sample into the pipet. The absorp-tion may be facilitated by rocking the pipet or by flowing thesample between pipet and buret. Draw any residual gas intothe buret, and measure its volume. Not more than 0.5 mL ofgas remains.Carbonyl Sulfide
Standard Preparation Obtain a standard gas mixture of 50ppm carbonyl sulfide in helium from a specialty gas supplier.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a Sievers350 (or equivalent)1 Chemiluminescence Detector (SCD) anda 30-m × 0.53-mm id, 5-mm DB-5 capillary column (J&WScientific Company, or equivalent). Set the carrier gas, helium,at a head-pressure of 5 psig. Set the injection port at 100°,and the split ratio at 1:1. Set the column temperature at 30°.The retention time for carbonyl sulfide is approximately 3min. Operate the SCD with 190 mL/min of hydrogen and 396mL/min of air. Optimize the gas flows and probe position ofthe SCD for maximum sensitivity.
Procedure Inject, in triplicate, 5.00 mL of the StandardPreparation into the gas chromatograph, record the chromato-grams, and average the peak area responses. The relativestandard deviation does not exceed 5.0%.
Similarly, inject, in triplicate, 5.00 mL of sample, averagethe peak area responses, and calculate the ppm v/v in thesample by the equation
ppm = S(AU/AS),
1Any sulfur-selective detector may be used; e.g., electrolytic conductiv-ity, flame photometric, or sulfur chemiluminescence. The detector mustbe capable of detecting less than 0.1 ppm v/v of carbonyl sulfide witha signal-to-noise ratio of 10:1.
in which S is the calculated ppm of carbonyl sulfide in theStandard Preparation (approximately 0.5 ppm), AU is theaverage of the sample peak area responses, and AS is theaverage area of the Standard Preparation area responses.Hydrogen Sulfide Pass 50 mL of sample released from thevapor phase through a hydrogen sulfide detector tube (Dräger#672804, 0.5 to 15 ppm, or equivalent) at the rate specifiedfor the tube. The indicator change corresponds to not morethan 0.5 ppm, for the volume of carbon dioxide specified inthis test.Nitric Oxide (NO) and Nitrogen Dioxide (NO2) Positionthe sample container so that when its valve is opened, theliquid phase can be sampled (generally this requires that thecylinder be inverted). Attach a section of tubing long enoughto act as a vaporizer for the small quantity of liquid to besampled. Connect one end of a nitric oxide–nitrogen dioxidedetector tube (see Detector Tubes) to the tubing and the otherend to a gas flow meter. Pass 500 mL of the liquid samplethrough the tube at a suitable rate. No frost should reach thetube inlet from the expanding sample. The indicator changecorresponds to not more than 5 ppm.Nonvolatile Hydrocarbons Pass a sample of liquid CarbonDioxide from a storage container or sample cylinder througha commercial carbon dioxide snow horn directly into an open,clean container. Collect the resulting Carbon Dioxide snowin this container. Weigh 500 g of this sample and transfer itinto a clean beaker. Allow the Carbon Dioxide solid to sublimecompletely, with a watch-glass placed over the beaker toprevent ambient contamination. Wash the beaker with a resi-due-free solvent, and transfer the solvent from the beaker toa clean, tared watch-glass or petri dish with two additionalrinses of the beaker with the solvent. Allow the solvent toevaporate, using heating to 104°, until the watch-glass or petridish is at a constant weight. Determine the weight of theresidue by difference. The weight of the residue does notexceed 5 mg (10 mg/kg).Sulfur Dioxide Pass 1050 � 50 mL of sample, taken fromthe liquid phase as described in the test for Nitric Oxide andNitrogen Dioxide, through a sulfur dioxide detector tube (seeDetector Tubes) at the rate specified for the tube. The indicatorchange corresponds to not more than 5 ppm.Volatile Hydrocarbons
Standard Preparation Obtain a standard gas mixture of50 ppm methane in helium from a specialty gas supplier.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flameionization detector and a 1.8-m × 3-mm (od) metal column,or equivalent, packed with 80- to 100-mesh HayeSep Q (orequivalent). Use helium as the carrier gas at a flow rate of30 mL/min. Maintain both the injector temperature and thedetector temperatures at 230°. Program the column tempera-ture according to the following steps: Hold it at 70° for 1min, then increase to 200° at a rate of 20°/min, and thenhold at 200° for 10 min. The parameters for the detector aresensitivity range: 10–12 A/mV; attenuation: 32. The concentra-tion of volatile hydrocarbons is reported in methane equiva-lents. The various gas chromatographic responses, excludingthe Carbon Dioxide response, are summed to yield the total
98 / Cardamom Oil / Monographs FCC V
volatile hydrocarbon concentration. The composition of hy-drocarbons present will vary from sample to sample. Typicalretention times are methane: 0.4 min; carbon dioxide: 0.8min; hexane: 14.4 min.
Procedure Inject in triplicate 1.00 mL of the StandardPreparation into the gas chromatograph, and average the peakarea responses. The relative standard deviation should notexceed 5.0%. Similarly, inject in triplicate 1.00 mL of sample,sum the average peak areas of the individual peaks, exceptfor the Carbon Dioxide peaks, and calculate the ppm v/v inthe sample by the equation
ppm = S(AU/AS),
in which S is the calculated ppm of methane in the StandardPreparation (approximately 50 ppm), AU is the sum of theaverages of the individual peak area responses in the sample,and AS is the average of the Standard Preparation area re-sponses.Water Pass 24,000 mL of the gas sample through a suitablewater-absorption tube (see Detector Tubes), not less than 100mm long, which previously has been flushed with about 500mL of sample and weighed. Regulate the flow so that about60 min will be required for passage of the gas. The gain inweight of the absorption tube does not exceed 1.0 mg.
Packaging and Storage Store in metal cylinders.
Cardamom Oil
CAS: [8000-66-6]
DESCRIPTION
Cardamom Oil occurs as a colorless or very pale yellow liquidwith the aromatic, penetrating, and somewhat camphoraceousodor of cardamom and a pungent, strongly aromatic taste. Itis the volatile oil distilled from the seed of Elettaria carda-momum (L.) Maton (Fam. Zingiberaceae). It is affected bylight. It is miscible with alcohol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between +22° and +44°.Refractive Index Between 1.462 and 1.466 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.917 and 0.947.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex Appendix IIB, using an Abbé or other refractometer ofequal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 5 mL of 70% alcohol. The solution may be clear or hazy.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers ina cool place protected from light.
CarmineCarminic Acid
OH O
O
CH3
OH
COOH
OH
HO
HO
HOH
H HO
H
HO
CH2OH
C22H20O13 Formula wt 492.39
INS: 120 CAS: [1390-65-4]
DESCRIPTION
Carmine occurs as bright red, friable pieces or as a dark redpowder. It is the aluminum or the calcium–aluminum lake,on an aluminum hydroxide substrate, of the coloring principlesobtained by an aqueous extraction of cochineal. Cochinealconsists of the dried female insects Dactylopius coccus costa(Coccus cacti L.), enclosing young larvae; the coloring princi-ples thus derived consist mainly of carminic acid (C22H20O13).It is soluble in alkali solutions, slightly soluble in hot water,and practically insoluble in cold water and in dilute acids.
Before use in food, Carmine must be pasteurized or other-wise treated to destroy all viable Salmonella microorganisms.According to the pertinent U.S. color additive regulation (21CFR 73.100), ‘‘. . . pasteurization or such other treatment isdeemed to permit the adding of safe and suitable substances(other than chemical preservatives) that are essential to themethod of pasteurization or other treatment used.’’
Carminic acid crystallizes from water as bright red crystalsthat darken at 130° and decompose at 250°; it is freely soluble
View IR
FCC V Monographs / Carnauba Wax / 99
in water, in alcohol, in ether, in concentrated sulfuric acid,and in solutions of alkali hydroxides; it is insoluble in petro-leum ether and in chloroform. Its aqueous solutions at pH 4.8are red-orange to yellow, and at 6.2 are dark red to violet.
Note: The specifications and tests in this monographrefer to Carmine without any added substances for pas-teurization or any other such treatment.
Function Color.
REQUIREMENTS
Identification Mix 333 mg of sample with 44 mL of water,0.15 mL of a 1:10 sodium hydroxide solution, and 0.2 mLof ammonium hydroxide, warm to dissolve, and dilute tovolume with water in a 500-mL volumetric flask. Pipet 10.0mL of this solution into a 250-mL volumetric flask, dilute tovolume with water, and mix. The resulting solution exhibitsabsorption maxima at 520 nm and 550 nm when determinedin a 1-cm cell with a suitable spectrophotometer against awater blank, and the absorbance at 520 nm is not less than 0.30.Assay Not less than 50.0% of carminic acid (C22H20O13),calculated on the dried basis.Arsenic Not more than 1 mg/kg.Ash Not more than 12.0%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 20.0%.Microbial Limits:
Salmonella Negative in 25 g.Protein Not more than 25%.
TESTS
Assay Dissolve about 0.100 g of sample (~52% carminicacid content), accurately weighed, in 30 mL of 2 N hydrochlo-ric acid, and heat to a boil for 30 s. After cooling, dilute toa volume of 1 L.
Note: If a black or brown precipitate forms, filter thesolution.
With a suitable spectrophotometer, determine the absorbanceof this solution in a 1-cm cell at the wavelength of maximumabsorbance (about 494 nm), using a 1:3 aqueous dilution of2 N hydrochloric acid as the blank. To obtain accurate results,the absorbance must be in the range of 0.650 to 0.750. Adjustthe starting weight as necessary to achieve this absorbance.Calculate the percent carminic acid in the sample taken bythe formula
100A/13.9W,
in which A is the absorbance of the sample solution and Wis the weight, in grams, of the sample taken.Arsenic Transfer 3.0 g of sample into a 500-mL Kjeldahlflask equipped with a steam trap, add 5 g of ferrous sulfateand 75 mL of hydrochloric acid, and mix. Connect the flaskwith the steam trap and with a condenser, the delivery tubeof which consists of a large-sized straight adapter and extendsto slightly above the bottom of a 500-mL Erlenmeyer flask
containing 100 mL of water. Begin heating the Kjeldahl flask,and collect about 40 mL of distillate in the Erlenmeyer flask.Pour the distillate mixture into a 600-mL beaker, add 20 mLof bromine water, and heat on a hot plate until the volumeis reduced to about 2 mL. Transfer the residual liquid into a125-mL arsine generator flask (see Appendix IIIB, Fig. 11)with the aid of 35 mL of water, and continue as directedin the Procedure under Arsenic Limit Test, Appendix IIIB,beginning with ‘‘Add 20 mL of 1:5 sulfuric acid. . . .’’Ash Transfer about 1 g of sample into a tared, previouslyignited and cooled porcelain crucible, and ignite red-hot witha Meker burner to constant weight.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 1-g sample at 135° for 3 h.Microbial Limits (Note: The current method for the follow-ing test may be found online at <www.cfsan.fda.gov/~ebam/bam-toc.html>):
SalmonellaProtein Determine the nitrogen content (N) of the sampleas directed in Method II under Nitrogen Determination, Ap-pendix IIIC. Calculate the protein content, in percent, by theformula
6.25 N/W)100,
in which 6.25 is the conversion factor from nitrogen to protein;N is the weight, in milligrams, of nitrogen; and W is theweight, in milligrams, of sample taken.
Packaging and Storage Store in well-closed containers ina cool, dry place.
Carnauba Wax
INS: 903 CAS: [8015-86-9]
DESCRIPTION
Carnauba Wax occurs as a hard, brittle substance with aresinous fracture and a color ranging from light brown to paleyellow. It is a purified wax obtained from the leaf buds andleaves of the Brazilian wax palm Copernicia cereferia (Ar-ruda) Mart. [synonym C. prunifera (Muell.)]. Its specific grav-ity is about 0.997. It is partially soluble in boiling alcohol, issoluble in chloroform and in ether, but is insoluble in water.
Function Anticaking agent; surface-finishing (glazing)agent; release agent; carrier for flavors.
REQUIREMENTS
Acid Value Between 2 and 7.Ester Value Between 71 and 88.
100 / L-Carnitine / Monographs FCC V
Lead Not more than 5 mg/kg.Melting Range Between 80° and 86°.Residue on Ignition Not more than 0.25%.Saponification Value Between 78 and 95.Unsaponifiable Matter Between 50.0% and 55.0%.
TESTS
Acid Value Determine as directed in Method I under AcidValue, Appendix VII.Ester Value Subtract the Acid Value from the Saponifica-tion Value (below) to obtain the Ester Value.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Melting Range Determine as directed in Procedure forClass II under Melting Range or Temperature, Appendix IIB.Residue on Ignition Heat a 2-g sample in a tared, open,porcelain or platinum dish over an open flame. It volatilizeswithout emitting an acrid odor. Ignite as directed under Resi-due on Ignition, Appendix IIC.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII, using about 5 g of sample,accurately weighed.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.
Packaging and Storage Store in well-closed containers.
L-Carnitine4-Amino-3-hydroxybutyric Acid Trimethylbetaine; Levocar-nitine; 4-Trimethylamino-3-hydroxybutyrate; (R)-3-Carboxy-2-hydroxy-N,N,N-trimethyl-1-propanaminium Hydroxide,Inner Salt
ON
CH3
H3C
H3C H OH O
C7H15NO3 Formula wt 161.20
CAS: [541-15-1]
DESCRIPTION
L-Carnitine occurs as white crystals or as a white, crystalline,hygroscopic powder. It is freely soluble in water, in alcohol, inalkaline solutions, and in dilute mineral acids. It is practicallyinsoluble in acetone and in ethyl acetate. It decomposes with-out melting at about 185° to 195°.
Function Nutrient.
REQUIREMENTS
IdentificationA. Dissolve about 1 g of sample in 10 mL of water and
10 mL of 1 N hydrochloric acid, and add 5 mL of sodiumtetraphenylborate TS. A white precipitate forms.
B. The infrared absorption spectrum of a potassium bro-mide dispersion of the sample, previously dried in vacuumat 60° for 5 h, exhibits maxima only at the same wavelengths asthose of a similar preparation of USP Levocarnitine ReferenceStandard.Assay Not less than 97.0% and not more than 103.0% ofC7H15NO3, calculated on the anhydrous basis.Chloride Not more than 0.4%.Lead Not more than 1 mg/kg.Optical (Specific) Rotation [�]D
20°: Between –29.0° and−32.0°, calculated on the anhydrous basis.pH Between 5.5 and 9.5, in a 1:20 aqueous solution.Potassium Not more than 0.2%.Residue on Ignition Not more than 0.5%.Sodium Not more than 0.1%.Water Not more than 4.0%.
TESTS
Assay Dissolve about 1.0 g of sample, accurately weighed,in water contained in a 250-mL flask.
Note: Avoid atmospheric moisture uptake duringweighing.
Titrate with 1.0 N hydrochloric acid to a potentiometricendpoint. Perform a blank determination (see General Provi-sions), and make any necessary correction. Each milliliter of1.0 N hydrochloric acid is equivalent to 161.2 mg ofC7H15NO3.Chloride
Test Solution Dissolve about 100 mg of sample, accu-rately weighed, in 30 to 40 mL of water, and mix. Add 10%nitric acid dropwise until the solution is neutral to litmus.Add an additional 1 mL of 10% nitric acid, and dilute withwater to a total volume of 50 mL.
Chloride Reference Solution Transfer by micropipet 0.56mL of 0.02 N hydrochloric acid solution to 30 to 40 mL ofwater in a 50-mL flask, add 1 mL of 10% nitric acid, anddilute with water to a volume of 50 mL.
Procedure Add 1 mL of 0.1 N silver nitrate to both theTest Solution and the Chloride Reference Solution. Mix, allowto stand for 5 min protected from direct sunlight, and comparethe two solutions. The turbidity of the Test Solution is notgreater than that of the Chloride Reference Solution.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 10 g of a previously dried sample in 100 mLof water.pH Determine as directed under pH Determination, Appen-dix IIB.
FCC V Monographs / �-Carotene / 101
Potassium (Note: The Standard Solution and the Test Solu-tions may be modified, if necessary, to obtain solutions ofsuitable concentrations adaptable to the linear or workingrange of the spectrophotometer.)
Standard Solution Transfer 5.959 g of potassium chloride,previously dried at 105° for 2 h and accurately weighed, intoa 250-mL volumetric flask, dilute to volume with water, andmix. This solution contains 12.5 mg of potassium per milliliter.Quantitatively dilute an accurately measured volume of thissolution with water, stepwise if necessary, to obtain a solutioncontaining 31.25 �g of potassium per milliliter.
Test Solutions Transfer 62.5 mg of sample into a 100-mL volumetric flask, dissolve in and dilute to volume withwater, and mix to obtain a stock solution. Place 0, 2.0, and4.0 mL of the Standard Solution into three separate 25-mLvolumetric flasks. Add 20.0 mL of the stock solution to eachflask, dilute to volume with water, and mix. These solutionscontain 0 (Test Solution A), 2.5 (Test Solution B), and 5.0(Test Solution C) �g/mL of potassium.
Procedure Concomitantly determine the absorbance val-ues of the Test Solutions at the potassium emission line at766.7 nm with a suitable atomic absorption spectrophotometerequipped with an air–acetylene flame, using water as theblank. Plot the absorbance values of the Test Solutions versustheir contents of potassium, in micrograms per milliliter; drawthe straight line best fitting the three points; and extrapolatethe line until it intersects with the concentration axis. Fromthe intercept, determine the amount, in micrograms, of potas-sium in each milliliter of Test Solution A. Calculate the percentpotassium in the portion of sample taken by multiplying theconcentration, in micrograms per milliliter, of potassiumfound in Test Solution A by 0.2.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Sodium (Note: The Standard Solution and the Test Solu-tions may be modified, if necessary, to obtain solutions ofsuitable concentrations adaptable to the linear or workingrange of the spectrophotometer.)
Standard Solution Transfer 6.355 g of sodium chloride,previously dried at 105° for 2 h and accurately weighed, intoa 250-mL volumetric flask, dilute to volume with water, andmix. This solution contains 10.0 mg of sodium per milliliter.Quantitatively dilute an accurately measured volume of thissolution with water, stepwise if necessary, to obtain a solutioncontaining 250 �g of sodium per milliliter.
Test Solutions Transfer 4.0 g of sample into a 100-mLvolumetric flask, dissolve in and dilute to volume with water,and mix to obtain a stock solution. Add 0, 2.0, and 4.0 mLof the Standard Solution to three separate 25-mL volumetricflasks. Add 20.0 mL of the stock solution to each flask, diluteto volume with water, and mix. These solutions contain 0 (TestSolution A), 20.0 (Test Solution B), and 40.0 (Test Solution C)�g/mL of sodium.
Procedure Concomitantly determine the absorbance val-ues of the Test Solutions at the sodium emission line at 589.0nm with a suitable atomic absorption spectrophotometerequipped with an air–acetylene flame, using water as theblank. Plot the absorbance values of the Test Solutions versus
their contents of sodium, in micrograms per milliliter; drawthe straight line best fitting the three points; and extrapolatethe line until it intersects with the concentration axis. Fromthe intercept, determine the amount, in micrograms, of sodiumin each milliliter of Test Solution A. Calculate the percentsodium in the portion of sample taken by multiplying theconcentration, in micrograms per milliliter, of sodium foundin Test Solution A by 0.003125.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers.
�-CaroteneCarotene
CH3H3C
CH3
CH3 CH3
CH3 CH3CH3H3C
H3C
C40H56 Formula wt 536.88
INS: 160a(ii) CAS: [7235-40-7]
DESCRIPTION
Beta-Carotene occurs as red crystals or as crystalline powder.It is insoluble in water and in acids and alkalies, but is solublein carbon disulfide and in chloroform. It is sparingly solublein ether, in solvent hexane, and in vegetable oils, and ispractically insoluble in methanol and in ethanol. It meltsbetween 176° and 182°, with decomposition.
Function Nutrient; color.
REQUIREMENTS
IdentificationA. Determine the absorbance of Sample Solution B (pre-
pared for the Assay) at 455 nm and at 483 nm. The ratio A455/A483 is between 1.14 and 1.18.
B. Determine the absorbance of Sample Solution B at 455nm and that of Sample Solution A at 340 nm. The ratio A455/A340 is not lower than 1.5.Assay Not less than 96.0% and not more than 101.0% ofC40H56, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.2%.Residue on Ignition Not more than 0.2%.
TESTS
Assay (Note: Carry out all work in low-actinic glasswareand in subdued light.)
102 / Carrot Seed Oil / Monographs FCC V
Sample Solution A Transfer about 50 mg of sample, accu-rately weighed, into a 100-mL volumetric flask, dissolve itin 10 mL of acid-free chloroform, immediately dilute to vol-ume with cyclohexane, and mix. Pipet 5 mL of this solutioninto a second 100-mL volumetric flask, dilute to volume withcyclohexane, and mix.
Sample Solution B Pipet 5 mL of Sample Solution A intoa 50-mL volumetric flask, dilute to volume with cyclohexane,and mix.
Procedure Determine the absorbance of Sample SolutionB in a 1-cm cell at the wavelength of maximum absorptionat about 455 nm, with a suitable atomic absorption spectropho-tometer, using cyclohexane as the blank. Calculate the quan-tity, in milligrams, of C40H56 in the sample taken by theformula
20,000A/250,
in which A is the absorbance of the solution, and 250 is theabsorptivity of pure �-Carotene.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in a vacuum over phospho-rus pentoxide at 40° for 4 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in a cool place in tight, light-resistant containers under inert gas.
Carrot Seed Oil
CAS: [8015-88-1]
FEMA: 2244
DESCRIPTION
Carrot Seed Oil occurs as a light yellow to amber liquidhaving a pleasant, aromatic odor. It is the volatile oil obtainedby steam distillation from the crushed seeds of Daucus carotaL. (Fam. Umbelliferae). It is soluble in most fixed oils, andis soluble, with opalescence, in mineral oil. It is practicallyinsoluble in glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 5.0.
Angular Rotation Between –4° and –30°.Refractive Index Between 1.483 and 1.493 at 20°.Saponification Value Between 9 and 58.Solubility in Alcohol Passes test.Specific Gravity Between 0.900 and 0.943.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed under Saponi-fication Value, Appendix VI, using about 5 g of sample,accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 90% alcohol. The solution may become opales-cent upon further dilution up to 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from glassor aluminum or that are lined with tin.
Cascarilla Oil
Sweetwood Bark Oil
CAS: [8007-06-5]
DESCRIPTION
Cascarilla Oil occurs as a light yellow to brown amber liquidwith a pleasant, spicy odor. It is the volatile oil obtained bysteam distillation of the dried bark of Croton cascarilla Benn.and of Croton eluteria Benn. (Fam. Euphorbiaceae). It issoluble in most fixed oils and in mineral oil, but it is practicallyinsoluble in glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseshown in the respective spectrum in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Between 3 and 10.Angular Rotation Between –1° and +8°.Ester Value after Acetylation Between 62 and 88.
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View IR
FCC V Monographs / Casein and Caseinate Salts / 103
Refractive Index Between 1.488 and 1.494 at 20°.Saponification Value Between 8 and 20.Solubility in Alcohol Passes test.Specific Gravity Between 0.892 and 0.914.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value after Acetylation Determine as directed underTotal Alcohols, Appendix VI, using about 2 g of the dried,acetylated sample oil, accurately weighed. Calculate the EsterValue after Acetylation by the formula
A × 28.05/B,
in which A is the number of milliliters of 0.5 N alcoholicpotassium hydroxide consumed in the saponification, and Bis the weight, in grams, of the acetylated sample oil.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed under Saponi-fication Value, Appendix VI, using 5 g of sample, accuratelyweighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 90% alcohol and remains in solution on dilutionto 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Requirements).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Casein and Caseinate Salts
CAS: [9000-71-9]
DESCRIPTION
Casein occurs as an off white to cream colored, granular orfine powder. It is derived from the coagulum formed bytreating skim milk with a food-grade acid (Acid Casein),enzyme (Rennet Casein), or other food-grade precipitatingagent. After the precipitation, Casein is separated from thesoluble milk fraction, washed, and dried. Chemically, Caseinis a mixture of at least 20 electrophoretically distinct phospho-proteins. The main fractions—designated �-casein, �-casein,and �-casein—are known to be mixtures, rather than singleproteins. Casein contains all the amino acids known to beessential for human nutrition. It is insoluble in water and
alcohol, but it can be dissolved by aqueous alkalies to formCaseinate Salts.
Caseinate Salts occur as white to cream colored granulesor powders. They are soluble or dispersible in water. Theyare prepared by treatment of Casein with food-grade alkalies,neutralizing agents, enzymes, buffers, or sequestrants. Com-mon counter-ions are NH4
+, Ca++, Mg++, K+, and Na+.
Function Binder; extender; clarifying agent; emulsifier; sta-bilizer.
REQUIREMENTS
Assay Acid Casein: Not less than 90.0% protein; RennetCasein: Not less than 86.0% protein; Caseinate Salts: Notless than 84.0% protein, calculated on the dried basis.Fat Not more than 2.25%.Free Acid (Casein only) Passes test.Lactose Not more than 2.0%.Lead Not more than 1 mg/kg.Loss on Drying Not more than 12.0%.
TESTS
Assay Determine as directed under Nitrogen Determination,Appendix IIIC. Calculate the percent protein (P) by theequation
P = N × 6.38,
in which N is the percent nitrogen.Fat Transfer 1 g of sample, accurately weighed, into a fat-extraction flask, add 10 mL of water, and shake until homoge-neous (warm if necessary). Add approximately 1 mL of ammo-nium hydroxide, and heat in a water bath for 15 min at 60°to 70°, shaking occasionally. Add 10 mL of alcohol, and mixwell. Add 25 mL of peroxide-free ether, stopper, and shakevigorously for 1 min; allow to cool, if necessary; add 25 mLof petroleum ether; and repeat the vigorous shaking. Allowthe layers to separate and clarify, or centrifuge to expeditethe process. Decant the organic layer into a suitable flask ordish, and repeat the extraction twice with 15 mL each ofether and petroleum ether for each extraction. Evaporate thecombined ether extractions on a steam bath, and dry theresidue to a constant weight at 102°, or 70° to 75° at lessthan 50 mm Hg. Calculate the percent fat (F) by the equation
F = (R × 100)/S,
in which R is the weight, in grams, of the residue, and S isthe weight, in grams, of the sample.Free Acid Transfer 10 g of finely ground sample, accuratelyweighed, into a 500-mL conical flask. Add 200 mL of freshlyboiled water maintained at 60°, swirl, and stopper. Place theflask in an 80° water bath for 30 min, shaking at 10-minintervals. Cool to room temperature, and filter. Transfer a100.0-mL portion of the clear filtrate, accurately weighed,into a 250-mL conical flask, add 0.5 mL of phenolphthaleinTS, and titrate with 0.1 N sodium hydroxide to a pink endpointthat persists for 30 s. Not more than 2.7 mL of 0.1 N sodiumhydroxide is consumed.
104 / Cassia Oil / Monographs FCC V
LactosePhenol Reagent Heat a mixture of 8 g of phenol and 2
g of water until the crystals dissolve.Lactose Solution Transfer approximately 2 g of lactose
monohydrate, accurately weighed, into a 100-mL volumetricflask; dissolve in and dilute to volume with water.
Sample Solution Transfer approximately 1 g of sample,accurately weighed, into a 150-mL beaker. If the sample isAcid Casein, add 0.10 g of sodium hydrogen carbonate. Ifthe sample is Rennet Casein, add 0.10 g of sodium tripolyphos-phate. Add 25 mL of water, and dissolve the sample by gentlyswirling while warming to 60° to 70° on a hot plate. Coolthe solution to ambient temperature, and add 15 mL of water,8 mL of 0.1 N hydrochloric acid, and 1 mL of a 10% solutionof acetic acid. Mix well by swirling, and after 5 min, add 1mL of 1 M sodium acetate, and mix well.
After the precipitate has settled, filter and discard the first5 mL of filtrate. Pipet 2 mL of the remaining filtrate into atest tube, add 0.2 mL of Phenol Reagent, and mix well. Add5 mL of sulfuric acid by using an automatic dispenser or byother means that permit mixing within 1 to 2 s. Ensure thatthe solution has been thoroughly mixed, and allow it to standfor 15 min, then cool to 20° in a water bath for 5 min.
Standard Solutions Transfer 10 mL of Lactose Solutioninto a 100-mL volumetric flask, dissolve in and dilute tovolume with water (Diluted Lactose Solution). Transfer, re-spectively, 1, 2, 3, and 4 mL of Diluted Lactose Solution intofour 100-mL volumetric flasks, and dilute to volume withwater. These dilutions (Standard Dilutions of Lactose) contain20, 40, 60, and 80 �g of lactose per milliliter of solution,respectively. Into each of five test tubes add, in sequence, 2mL of water and, respectively, 3 mL of each of the StandardDilutions of Lactose. Add Phenol Reagent and sulfuric acidto each test tube as described under Sample Solution.
Apparatus Use a suitable spectrophotometer capable ofoperating in the visible range.
Calibration Determine the absorbance of each StandardSolution in a 1-cm pathlength cell at 490 nm against the waterblank. Calculate the slope of the curve obtained by plottingabsorbance versus micrograms per milliliter of lactose. Theslope of the curve is the absorptivity (a) of the lactose–reagentproduct.
Procedure Determine the absorbance of the Sample Solu-tion at 490 nm against a blank prepared using identical re-agents.
Calculation Calculate the percent lactose (L) in the sam-ple by the equation
L = (A × 0.00475)/(a × m),
in which A is the absorbance of the Sample Solution at 490nm; the numerical factor accounts for dilution and conversionto percent from micrograms per milliliter; a is the absorptivitycalculated under Calibration; and m is the weight, in grams,of the sample taken.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.
Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 102° for 3 h.
Packaging and Storage Store in well-closed containers.
Cassia Oil
Cinnamon Oil
FEMA: 2258 CAS: [8007-80-5]
DESCRIPTION
Cassia Oil occurs as a yellow or brown liquid having thecharacteristic odor and taste of cassia cinnamon. It is thevolatile oil obtained by steam distillation from the leavesand twigs of Cinnamomum cassia Blume (Fam. Lauraceae),rectified by distillation, and consisting mainly of cinnamicaldehyde. Upon aging or exposure to air it darkens and thick-ens. It is soluble in glacial acetic acid and in alcohol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 80.0%, by volume, of total aldehydes.Angular Rotation Between –1° and +1°.Chlorinated Compounds Passes test.Refractive Index Between 1.602 and 1.614 at 20°.Rosin or Rosin Oils Passes test.Solubility in Alcohol Passes test.Specific Gravity Between 1.045 and 1.063.
TESTS
Assay Determine as directed in Neutral Sulfite Method un-der Aldehydes and Ketones, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Chlorinated Compounds Determine as directed underChlorinated Compounds, Appendix VI.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Rosin or Rosin Oils Shake a 2-mL sample in a test tubewith 5 to 10 mL of solvent hexane, allow the liquids toseparate, decant the hexane layer, which is just slightly col-ored, into another test tube, and shake it with an equal volumeof 1:1000 cupric acetate solution. The mixture does notturn green.
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FCC V Monographs / Cedar Leaf Oil / 105
Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight, light-resistantcontainers. Avoid exposure to excessive heat.
Castor Oil
Ricinus Oil
INS: 1503 CAS: [8001-79-4]
DESCRIPTION
Castor Oil occurs as a pale yellow or almost colorless, trans-parent, viscous liquid. It is the fixed oil obtained from the seedof Ricinus communis L. (Fam. Euphorbiaceae) and consistsmainly of the triglyceride of ricinoleic acid. It is soluble inalcohol, and is miscible with absolute alcohol, with glacialacetic acid, with chloroform, and with ether.
Function Antisticking agent; release agent; component ofprotective coatings.
REQUIREMENTS
IdentificationA. A sample is only partly soluble in solvent hexane (dis-
tinction from most other fixed oils), but it yields a clear liquidwith an equal volume of alcohol (foreign fixed oils).
B. Castor Oil exhibits the following composition profileof fatty acids determine as directed under Fatty Acid Composi-tion, Appendix VII.
Fatty Acid: 16:0 18:0 18:1 18:3 18:0 di-OHWeight % (Range): 0.9–1.6 1.0–1.8 3.7–6.7 0.2–0.6 0.4–1.3Fatty Acid: 18:1-OH 20:0Weight % (Range): 83.6–89.0 0.2–0.5
Free Fatty Acids Passes test.Hydroxyl Value Between 160 and 168.Iodine Value Between 83 and 88.Lead Not more than 0.1 mg/kg.Saponification Value Between 176 and 185.Specific Gravity Between 0.952 and 0.966.
TESTS
Free Fatty Acids Dissolve about 10 g of sample, accuratelyweighed, in 50 mL of a mixture of equal volumes of alcoholand ether (which has been neutralized to phenolphthalein with0.1 N sodium hydroxide) contained in a flask. Add 1 mL ofphenolphthalein TS, and titrate with 0.1 N sodium hydroxide
until the solution remains pink after shaking for 30 s. Notmore than 7 mL of 0.1 N sodium hydroxide is required fora 10.0-g sample.Hydroxyl Value Determine as directed in Method II underHydroxyl Value, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII, using about 300 mg of sample, accuratelyweighed.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII, using about 3 g of sample,accurately weighed.Specific Gravity Determine as directed under SpecificGravity, Appendix VII.
Packaging and Storage Store in tight containers, and avoidexposure to excessive heat.
Cedar Leaf Oil
Thuja Oil; White Cedar Leaf Oil
FEMA: 2267 CAS: [8007-20-3]
DESCRIPTION
Cedar Leaf Oil occurs as a colorless to yellow liquid havinga strong camphoraceous and sage odor. It is the volatile oilobtained by steam distillation from the fresh leaves and branchends of the eastern arborvitae, Thuja occidentalis L. (Fam.Cupressaceae). It is soluble in most fixed oils, in mineral oil,and in propylene glycol. It is practically insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 60.0% of ketones, calculated as thujone(C10H16O).Angular Rotation Between –7° and –14°.Refractive Index Between 1.456 and 1.460 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.906 and 0.916.
TESTS
Assay Accurately weigh about 1 g of sample, and determineas directed in the Hydroxylamine Method under Aldehydesand Ketones, Appendix VI, using 76.10 as the equivalencefactor (e) in the calculation.
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106 / Celery Seed Oil / Monographs FCC V
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 70% alcohol, occasionally becoming cloudy ondilution to 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from glassor that are lined with tin.
Celery Seed Oil
DESCRIPTION
Celery Seed Oil occurs as a yellow to green-brown liquidwith a pleasant, aromatic odor. It is the volatile oil obtainedby steam distillation of the fruit or seed of Apium graveolensL. It is soluble in most fixed oils with the formation of aflocculent precipitate, and in mineral oil with turbidity. Itis partly soluble in propylene glycol, but it is insoluble inglycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 4.5.Angular Rotation Between +48° and +78°.Refractive Index Between 1.480 and 1.490 at 20°.Saponification Value Between 25 and 65.Solubility in Alcohol Passes test.Specific Gravity Between 0.870 and 0.910.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.
Saponification Value Determine as directed under Saponi-fication Value, Appendix VI, using 5 g of sample, accuratelyweighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 8 mL of 90% alcohol, usually with turbidity.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Cellulose Gel
Cellulose, Microcrystalline
INS: 460 CAS: [9004-34-6]
DESCRIPTION
Cellulose Gel occurs as a fine, white or almost white powder.It is purified, partially depolymerized cellulose prepared bytreating alpha-cellulose, obtained as a pulp from fibrous plantmaterial, with mineral acids. It consists of free-flowing, nonfi-brous particles that may be compressed into self-binding tab-lets that disintegrate rapidly in water. It is insoluble in water,in dilute acids, in dilute sodium hydroxide solutions, and inmost organic solvents.
Function Anticaking agent; binding agent; dispersing agent.
REQUIREMENTS
IdentificationA. Sift 20 g of sample for 5 min on an air-jet sieve equipped
with a screen having 38-�m openings. If more than 5% isretained on the screen, mix 30 g of sample with 270 mL ofwater; otherwise, mix 45 g of sample with 255 mL of water.Mix for 5 min in a single-speed, high-speed (equal to or greaterthan 18,000 rpm) power blender (use a Waring Blender, Model700G, or equivalent) that has a clover-shaped jar design. Thejar and blades meet the following specifications: The jar hasa 7.0-cm id at the bottom and a 9.2-cm id at the top and anoverall height of 21.9 cm, and the four blades are arrangedso that two of the blades are pointed up and two are pointeddown. Transfer 100 mL of the dispersion into a 100-mLgraduated cylinder, and allow it to stand for 3 h. A white,opaque, bubble-free dispersion that does not form a superna-tant liquid at the surface is obtained.
B. Add a few drops of iodine TS to 20 mL of the dispersionobtained in Identification Test A, and mix. No purple to blueor blue color appears.Assay Not less than 97.0% and not more than 102.0% ofcarbohydrate, calculated as cellulose on the dried basis.
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FCC V Monographs / Cellulose Gum / 107
Lead Not more than 2 mg/kg.Loss on Drying Not more than 7.0%.pH Between 5.0 and 7.5.Residue on Ignition Not more than 0.05%.Water-Soluble Substances Not more than 0.24%.
TESTS
Assay With the aid of about 25 mL of water, transfer about125 mg of sample, accurately weighed, into a 300-mL Erlen-meyer flask. Add 50.0 mL of 0.5 N potassium dichromate,mix, then carefully add 100 mL of sulfuric acid, and heat toboiling. Remove the mixture from the heat, allow it to standat room temperature for 15 min, cool it in a water bath, andtransfer it into a 250-mL volumetric flask. Dilute almost tovolume with water, cool to 25°, then dilute to volume withwater, and mix. Titrate a 50.0-mL aliquot with 0.1 N ferrousammonium sulfate, using 2 or 3 drops of orthophenanthrolineTS as the indicator, and record the volume required, in millili-ters, as S. Perform a blank determination (see General Provi-sions), and record the volume of 0.1 N ferrous ammoniumsulfate required, in milliliters, as B. Calculate the percentcellulose in the sample by the formula
(B – S) × 338/W,
in which W is the weight, in milligrams, of sample taken,corrected for Loss on Drying (below).Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample to constant weight at 105°.pH Determine as directed under pH Determination, Appen-dix IIB, using the supernatant liquid from the following proce-dure: Shake about 5 g of sample with 40 mL of water for 20min, and centrifuge.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Water-Soluble Substances Shake 5 g of sample with 80mL of water for 10 min. Filter the mixture through WhatmanNo. 42, or equivalent, filter paper into a tared beaker, evaporatethe filtrate to dryness on a steam bath, dry at 105° for 1 h,cool, and weigh.
Packaging and Storage Store in well-closed containers.
Cellulose GumSodium Carboxymethylcellulose; CMC; Modified Cellulose
OH
H
CH2OR
H
ORH
OR HO
n
in which R = H or CH2COONa
INS: 466 CAS: [9004-32-4]
DESCRIPTION
Cellulose Gum occurs as a white to cream colored powderor as granules. The powder is hygroscopic. It readily dispersesin water to form colloidal solutions. It is insoluble in mostsolvents. A 1:100 aqueous suspension has a pH between 6.5and 8.5.
Function Thickener; stabilizer.
REQUIREMENTS
IdentificationA. While stirring to produce a uniform dispersion, add
about 1 g of powdered sample to 50 mL of warm water.Continue stirring until a colloidal solution is produced, andthen cool to room temperature. Save part of this solution forIdentification Test B. Add 10 mL of cupric sulfate TS to about10 mL of the solution. A fluffy, blue-white precipitate forms.
B. The solution from Identification Test A gives positivetests for Sodium, Appendix IIIA.Assay Not less than 99.5% and not more than 100.5% ofCellulose Gum, calculated on the dried basis.Degree of Substitution Not less than 0.2 and not more than1.50 carboxymethyl groups (—CH2COOH) per anhydroglu-cose unit after drying.Lead Not more than 3 mg/kg.Loss on Drying Not more than 10.0%.Sodium Not more than 12.4% after drying.Viscosity of a 2%, Weight in Weight, Solution Not lessthan 25 centipoises.
TESTS
Assay Calculate the percent Cellulose Gum by subtractingfrom 100 the percents of Sodium Chloride and Sodium Gly-colate determined as follows:
Sodium Chloride Transfer about 5 g of sample, accuratelyweighed, into a 250-mL beaker, add 50 mL of water and 5mL of 30% hydrogen peroxide, and heat on a steam bath for20 min, stirring occasionally to ensure complete dissolution.
108 / Cellulose Gum / Monographs FCC V
Cool, add 100 mL of water and 10 mL of nitric acid, andtitrate with 0.05 N silver nitrate to a potentiometric endpoint,using a silver/calomel (AgCl) electrode set, and stirring con-stantly. Calculate the percent sodium chloride in the sampleby the formula
(584.4 × V × N)/(100 – b)W,
in which 584.4 is an equivalence factor for sodium chloride;V and N represent the volume, in milliliters, and the normality,respectively, of the silver nitrate; b is the percent Loss onDrying, determined separately (below); and W is the weight,in grams, of the sample.
Sodium Glycolate Sample Solution Transfer about 500mg of sample, accurately weighed, into a 100-mL beaker,moisten thoroughly with 5 mL of glacial acetic acid, followedby 5 mL of water, and stir with a glass rod until solution iscomplete (usually about 15 min). While stirring, slowly add50 mL of acetone, then add 1 g of sodium chloride, and stirfor several minutes to ensure complete precipitation of theCellulose Gum. Filter through a soft, open-textured paper,previously wetted with a small amount of acetone, and collectthe filtrate in a 100-mL volumetric flask. Use an additional30 mL of acetone to facilitate transfer of the solids and towash the filter cake, then dilute to volume with acetone,and mix.
Standard Solution Prepare a series of Standard Solutionsas follows: Transfer 100 mg of glycolic acid, previously driedin a desiccator at room temperature overnight and accuratelyweighed, into a 100-mL volumetric flask, dissolve in anddilute to volume with water, and mix. Use this solution within30 days. Transfer 1.0, 2.0, 3.0, and 4.0 mL, respectively,of this solution into separate 100-mL volumetric flasks, addsufficient water to each flask to make 5 mL, then add 5 mLof glacial acetic acid, and dilute to volume with acetone.
Procedure Transfer 2.0 mL of the Sample Solution and 2.0mL of each Standard Solution into separate 25-mL volumetricflasks, and prepare a blank flask with 2.0 mL of a solutioncontaining 5% each of glacial acetic acid and water in acetone.Place the uncovered flasks in a boiling water bath for exactly20 min to remove the acetone, remove from the bath, andcool. Add to each flask 5.0 mL of 2,7-dihydroxynaphthaleneTS, mix thoroughly, add an additional 15 mL, and again mixthoroughly. Cover the mouth of each flask with a small pieceof aluminum foil. Place the flasks upright in a boiling waterbath for 20 min, then remove from the bath, cool, dilute tovolume with sulfuric acid, and mix.
Using a suitable spectrophotometer, determine the ab-sorbance of each solution at 540 nm against the blank, andprepare a standard curve using the absorbance obtained fromeach of the Standard Solutions.
Calculation From the standard curve and the absorbanceof the Sample Solution, determine the weight (w), in milli-grams, of glycolic acid in the sample, and calculate the percentsodium glycolate in the sample by the formula
(12.9 × w)/(100 – b)W,
in which 12.9 is a factor converting glycolic acid to sodiumglycolate; b is the percent Loss on Drying, determined sepa-rately; and W is the weight, in grams, of the sample.
Degree of Substitution Transfer about 200 mg of sample,previously dried at 105° to constant weight, and accuratelyweighed, into a 250-mL, glass-stoppered Erlenmeyer flask.Add 75 mL of glacial acetic acid, connect the flask with awater-cooled condenser, and reflux gently on a hot plate for2 h. Cool, transfer the solution to a 250-mL beaker with theaid of 50 mL of glacial acetic acid, and titrate with 0.1 Nperchloric acid in dioxane while stirring with a magneticstirrer.
Caution: Handle perchloric acid in an appropriatefume hood.
Determine the endpoint potentiometrically with a pH meterequipped with a standard glass electrode and a calomel elec-trode modified as follows: Discard the aqueous potassiumchloride solution contained in the electrode, rinse and fill withthe supernatant liquid obtained by shaking thoroughly 2 geach of potassium chloride and silver chloride (or silver oxide)with 100 mL of methanol, then add a few crystals of potassiumchloride and silver chloride (or silver oxide) to the electrode.
Record the milliliters of 0.1 N perchloric acid versus mV(0- to 700-mV range), and continue the titration to a fewmilliliters beyond the endpoint. Plot the titration curve, andread the volume (A), in milliliters, of 0.1 N perchloric acidat the inflection point.
Calculate the degree of substitution by the formula
(16.2 A/G)/[1.000 – (8.0 A/G)],
in which 16.2 is one-tenth of the molecular weight of oneanhydroglucose unit; A is the volume, in milliliters, of 0.1 Nperchloric acid required; G is the weight, in milligrams, ofthe sample taken; and 8.0 is one-tenth of the molecular weightof one sodium carboxymethyl group.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, using 2 g of sample and 6 �g of lead(Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample to constant weight at 105°.Sodium From the weight of the sample and the number ofmilliliters of 0.1 N perchloric acid consumed in the determina-tion of Degree of Substitution (above), calculate the percentsodium. Each milliliter of 0.1 N perchloric acid is equivalentto 2.299 mg of sodium (Na).Viscosity of a 2%, Weight in Weight, Solution Determineas directed under Viscosity of Cellulose Gum, Appendix IIB.
Packaging and Storage Store in well-closed containers.
FCC V Monographs / Cellulose, Powdered / 109
Cellulose, Powdered
H O
H
H
OH
H OH
CH2OH
OH HH
CH2OH
H OH
OH HH
O
n
INS: 460(ii) CAS: [9004-34-6]
DESCRIPTION
Cellulose, Powdered, occurs as a white substance and consistsof fibrous particles that may be compressed into self-bindingtablets that disintegrate rapidly in water. It exists in variousgrades, exhibiting degrees of fineness ranging from a dense,free-flowing powder to a coarse, fluffy, nonflowing material.It is purified, mechanically disintegrated cellulose preparedby processing bleached cellulose obtained as a pulp from suchfibrous materials as wood or cotton. It is insoluble in water,in dilute acids, and in nearly all organic solvents. It is slightlysoluble in 1 N sodium hydroxide.
Function Anticaking agent; binding agent; bulking agent;dispersing agent; filter aid; texturizing agent; thickening agent.
REQUIREMENTS
IdentificationA. Mix approximately 30 g of sample with 270 mL of
water in a high-speed (approximately 12,000 rpm) powerblender for 5 min. The mixture will be either a free-flowingsuspension or a heavy, lumpy suspension that flows poorly(if at all), settles only slightly, and contains many trapped airbubbles. The mixture is not slimy. If a free-flowing suspensionis obtained, transfer 100 mL of it into a 100-mL graduatedcylinder, and allow it to settle for 1 h. The solids settle in thecylinder and a supernatant liquid appears above the layer ofsample. (Save the mixture for Identification Tests D and E.)
B. Boil 10 g of sample with 90 mL of water for 5 min,filter while hot through ashless, fine quantitative paper (S &S 589 Blue Ribbon, or equivalent), and add 2 drops of iodineTS to the filtrate. The color does not change from yellow-red.
C. Add 2 to 5 mg of sample to 20 mL of a 0.1% solutionof anthrone in 75% sulfuric acid, and heat on a steam bath.The solution turns blue-green within 5 min.
D. Place a few drops of the stirred mixture from Identifica-tion Test A on a microscope slide, and insert a coverglass.Observe at 100 magnifications with a microscope. Fibers andfiber fragments are visible, regardless of the degree of finenessof the sample.
E. Dilute 10 mL of the stirred mixture from IdentificationTest A to 1000 mL with water, and filter 125 mL of the
dilution through a Büchner funnel using Whatman No. 4 filterpaper, or equivalent. Rinse the pad with 25 mL of acetone,and dry (paper included) at 105°. Transfer the powder toa tared weighing bottle, weigh, then transfer to a 50-mLErlenmeyer flask, and seal with a rubber stopper. Record theweight, in milligrams, of the sample as w. Prepare 0.167 Mand 1.0 M solutions of cupriethylenediamine (CED), determin-ing the volumes of each as follows: 0.12 × w equals themilliliters of 0.167 M CED to use, and 0.08 × w equals themilliliters of 1.0 M CED to use. Add a few 3-mm glass beadsand the calculated volume of 0.167 M CED, blow nitrogenover the surface of the solution, and shake for 2 min. Addthe calculated volume of 1.0 M CED, again introduce thenitrogen, and shake vigorously for at least 3 min. A dark bluesolution, clear under microscopic examination, appears.Assay Not less than 97.0% and not more than 102.0% ofcarbohydrate, calculated as Cellulose.Ash (Total) Not more than 0.3%.Chloride Not more than 0.05%.Lead Not more than 3 mg/kg.Loss on Drying Not more than 7.0%.pH Between 5.0 and 7.5.Sulfur (Total) Not more than 0.01%.Water-Soluble Substances Not more than 1.5%.
TESTS
Assay With the aid of about 25 mL of water, transfer about125 mg of sample, accurately weighed, into a 300-mL Erlen-meyer flask. Add 50.0 mL of 0.5 N potassium dichromate,mix, then carefully add 100 mL of sulfuric acid, and heat toboiling. Remove the flask from the heat, allow the solutionto stand at room temperature for 15 min, cool it in a waterbath, and transfer the solution to a 250-mL volumetric flask.Dilute with water almost to volume, cool to 25°, dilute tovolume with water, and mix. Titrate a 50-mL aliquot with0.1 N ferrous ammonium sulfate, using 2 or 3 drops of ortho-phenanthroline TS. Perform a blank determination (see Gen-eral Provisions), and make any necessary correction. Calcu-late the normality, N, of the ferrous ammonium sulfate solutionby the formula
(0.1 × 50)/B,
in which B is the volume, in milliliters, of ferrous ammoniumsulfate solution required in the blank titration. Calculate thepercent Cellulose in the sample by the formula
6.75(B – S) × N/2W,
in which S is the volume, in milliliters, of ferrous ammoniumsulfate solution used in the sample titration, and W is theweight, in grams, of the sample taken, corrected for moisturecontent (see Loss on Drying, below).Ash (Total) Heat 3 g of sample at 550° � 50° until com-pletely charred, then ignite at 800° � 25° until free fromcarbon, cool in a desiccator, and weigh.Chloride Transfer about 5 g of sample, accurately weighed,into a 500-mL conical flask, add 250 mL of water, and refluxthe mixture for 1 h. Filter through Whatman No. 4 filter paper,or equivalent, and reflux the sample with 200 mL of water
110 / Chamomile Oil, English Type / Monographs FCC V
for 30 min. Filter as before, and combine the filtrates and hotwater rinses. Add 1 mL of nitric acid, heat to boiling, andslowly add 5 mL of a 5% solution of silver nitrate. After theprecipitate has coagulated, cool, and filter through a sintered-glass filtering funnel. Wash with a 1:100 nitric acid solutionuntil free from silver nitrate, rinse with water, dry at 130°,and weigh. Perform a blank determination (see General Provi-sions) to obtain the corrected weight of the sample precipitate,each milligram of which is equivalent to 0.247 mg of chloride.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 3-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample to constant weight at 105°.pH Determine as directed under pH Determination, Appen-dix IIB, using the supernatant liquid from the following prepa-ration: Mix 10 g of sample with 90 mL of water, allow tostand with occasional stirring for 1 h.Sulfur (Total) Transfer about 5 g of sample, previouslydried at 105° to constant weight and accurately weighed, intoa 300-mL conical flask, and add 50 mL of 2:3 perchloricacid:nitric acid (v/v).
Caution: Handle perchloric acid in an appropriatefume hood.
Heat on a hot plate under a hood, and boil until all organicmatter has been destroyed and copious fumes of perchloricacid evolve. If the organic matter chars and cannot be de-stroyed quickly by further heating for a short time, add 10 to20 mL of the acid mixture, and continue the treatment untila clear, syrupy residue is obtained.
Note: All of the nitric acid must be driven from theflask, because it will otherwise form a double salt withthe barium sulfate formed later.
Allow the mixture to cool for a few min, then add 200 mLof hot water, and heat again to boiling. (If the mixture iscloudy, filter, and rinse the filter with a small amount of hotwater before boiling.) As soon as the mixture is boiling gently,carefully run in 20 mL of barium chloride TS, boil for a fewminutes longer, and allow to stand for at least 12 h on a steambath. Filter any barium sulfate onto an ashless filter paper,and rinse with five portions of boiling water to remove tracesof perchloric acid. Place the paper in a tared platinum dish,dry in an oven at 105°, and ignite at 800° � 25° for 1 h.Perform a blank determination (see General Provisions) toobtain the corrected weight of the sample precipitate, eachmilligram of which is equivalent to 0.137 mg of sulfur.Water-Soluble Substances Mix 6 g of sample with 90 mLof recently boiled and cooled water, and allow to stand withoccasional stirring for 10 min. Filter through Whatman No.2 filter paper, or equivalent, discard the first 10 mL of filtrate,and pass the filtrate through the same filter a second time, ifnecessary, to obtain a clear filtrate. Evaporate a 15-mL portionof the filtrate to dryness in a tared evaporating dish on a steambath, dry at 105° for 1 h, cool in a desiccator, and weigh.
Packaging and Storage Store in well-closed containers.
Chamomile Oil, English Type
CAS: [8015-92-7]
DESCRIPTION
Chamomile Oil, English Type, occurs as a light blue or lightgreen-blue liquid with a strong, aromatic odor, characteristicof the flowers. The color may change with age to green-yellowor yellow-brown. It is the oil obtained by steam distillation ofthe dried flowers of the so-called English or Roman Chamo-mile, Anthemis nobilis L. (Fam. Asteraceae). It is soluble inmost fixed oils, and it is almost completely soluble in mineraloil. It is soluble, with slight haziness, in propylene glycol,but it is insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Acid Value Not more than 15.0.Ester Value Between 250 and 310.Refractive Index Between 1.440 and 1.450 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.892 and 0.910.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Ester Value Determine as directed under Ester Value, Ap-pendix VI, using about 1 g of sample, accurately weighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 80% alcohol, sometimes with a slight precipitate.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
View IR
FCC V Monographs / Chlorine / 111
Chamomile Oil, German Type
Chamomile Oil, Hungarian Type
DESCRIPTION
Chamomile Oil, German Type, occurs as a deep blue or blue-green liquid with a strong, characteristic odor and a bitter,aromatic taste. When the oil is exposed to light or air, theblue color changes to green and finally to brown. It is the oilobtained by steam distillation of the flowers and stalks ofMatricaria chamomilla L. (Fam. Asteraceae). Upon cooling,the oil may become viscous. It is soluble in most fixed oilsand in propylene glycol. It is insoluble in glycerin and inmineral oil.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Between 5 and 50.Ester Value Not more than 40.Ester Value after Acetylation Between 65 and 155.Solubility in Alcohol Passes test.Specific Gravity Between 0.910 and 0.950.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Ester Value Determine as directed under Ester Value, Ap-pendix VI, using about 5 g of sample, accurately weighed.Ester Value after Acetylation Determine as directed underEster Value, Appendix VI, except acetylate a 10-mL sampleas directed under Total Alcohols, Appendix VI, and use about1.5 g of the dried, acetylated oil, accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. The oil does not usually dissolveclearly in 95% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Chlorine
Cl2 Formula wt 70.91
INS: 925 CAS: [7782-50-5]
DESCRIPTION
Chlorine occurs as a green-yellow gas, normally packaged asa liquid under pressure in containers approved by the U.S.Department of Transportation. At 60 °F, it has a vapor pressureof 70.91 psig. Its vapor density is about 2.5 times that of air.About 0.8 lb (0.362 kg) is soluble in 100 lb (45.4 kg) of waterat 60 °F under atmospheric pressure.
Caution: Chlorine gas is a respiratory irritant. Largeamounts cause coughing, labored breathing, and irrita-tion of the eyes. In extreme cases, the difficulty inbreathing may cause death due to suffocation. LiquidChlorine causes skin and eye burns on contact. (Safetyprecautions to be observed in handling the material arespecified in the Chlorine Manual, available from TheChlorine Institute, Inc., Suite 506, 2001 L Street, N.W.,Washington, D.C. 20036, <www.chl2.com>.)
Function Antimicrobial agent; bleaching agent; oxidizingagent.
REQUIREMENTS
Identification Cautiously pass a few milliliters of samplegas through 10 mL of 1 N sodium hydroxide that has pre-viously been chilled in an ice bath. The resulting solutiongives positive tests for Chloride, Appendix IIIA, and it darkensstarch iodide paper.Assay Not less than 99.5%, by volume.Lead Not more than 10 mg/kg.Mercury Not more than 1 mg/kg.Moisture Not more than 0.015%, by weight.Residue Not more than 0.015%, by weight, of nonvolatilematter.
TESTS
Assay Determine by ASTM Method E 412-93, ‘‘Assay ofLiquid Chlorine (Zinc Amalgam Method).’’
Sample Solution for the Determination of Lead andMercury Dissolve the residue obtained under Resi-due (below) in 2.5 mL of freshly prepared aqua regia,and dilute with water to a volume, in milliliters, equiva-lent to the weight, in grams, of the initial sample. Onemilliliter of the final dilution is equivalent to 1 g ofsample.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a 1.0-mL portion of the Sample Solution mixedwith 5 mL of water and 11 mL of 2.7 N hydrochloric acid,and 10 �g of lead (Pb) ion in the control.
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112 / Cholic Acid / Monographs FCC V
Mercury Determine as directed under Mercury Limit Test,Appendix IIIB, testing the Sample Preparation prepared asfollows: Transfer 2.0 mL of the Sample Solution into a 50-mL beaker, add 10 mL of water, 1 mL of 1:5 sulfuric acid,and 1 mL of a 1:25 solution of potassium permanganate, coverthe beaker with a watch glass, boil for a few seconds, and cool.Moisture and Residue Determine by ASTM Method E410-92, ‘‘Moisture and Residue in Liquid Chlorine.’’
Packaging and Storage Store in suitable pressure contain-ers, observing applicable U.S. Department of Transportationregulations pertaining to shipping containers.
Cholic AcidCholalic Acid; 3,7,12-Trihydroxycholanic Acid
HO OH
H3C
HO CH
CH3
CH2CH2COOH
H3C
C24H40O5 Formula wt 408.58
INS: 1000 CAS: [81-25-4]
DESCRIPTION
Cholic Acid occurs as colorless plates or as a white, crystallinepowder. One gram dissolves in about 30 mL of alcohol oracetone and in about 7 mL of glacial acetic acid. It is veryslightly soluble in water.
Function Emulsifier.
REQUIREMENTS
Identification Add 1 mL of a 1:100 furfural solution to 1mL of a 1:5000 solution in 50% acetic acid. Cool in an icebath for 5 min, add 15 mL of 1:2 sulfuric acid, mix, andwarm in a water bath at 70° for 10 min. Immediately cool inan ice bath, and stir for 2 min. A blue color appears.Assay Not less than 98.0% of C24H40O5, calculated on thedried basis.Lead Not more than 4 mg/kg.Loss on Drying Not more than 0.5%.Melting Range Between 197° and 202°.Optical (Specific) Rotation [�]D
25°: Not less than +37°, cal-culated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Transfer about 400 mg of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, add 20 mL ofwater and 40 mL of alcohol, cover with a watch glass, heatgently on a steam bath until dissolved, and cool. Add 5 dropsof phenolphthalein TS, and using a 10-mL microburet, titratewith 0.1 N sodium hydroxide to the first pink color thatpersists for 15 s. Perform a blank determination (see GeneralProvisions) and make any necessary correction. Each milliliterof 0.1 N sodium hydroxide is equivalent to 40.86 mg ofC24H40O5.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 140° under a vacuumof not more than 5 mm Hg for 4 h.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutionof sample in alcohol containing 200 mg of sample in each10 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in tight containers.
Choline Bitartrate(2-Hydroxyethyl)trimethylammonium-L-(+)-tartrate Salt
[HOCH2CH2N (CH3)3]C4H5O6
C9H19NO7 Formula wt 253.25
INS: 1001(v) CAS: [87-67-2]
DESCRIPTION
Choline Bitartrate occurs as a white, hygroscopic, crystallinepowder. It is freely soluble in water, slightly soluble in alcohol,and insoluble in ether and in chloroform.
Function Nutrient.
REQUIREMENTS
IdentificationA. Dissolve 500 mg of sample in 2 mL of water, add 3
mL of 1 N sodium hydroxide, and heat to boiling. The odorof trimethylamine is detectable.
B. Dissolve 500 mg of sample in 2 mL of iodine TS. Ared-brown precipitate forms immediately. Add 5 mL of 1 Nsodium hydroxide. The precipitate dissolves, and the solution
FCC V Monographs / Choline Chloride / 113
becomes clear yellow. Heat the solution. A pale yellow precip-itate forms.
C. Add 1 mL of a 1:100 aqueous solution and 2 mL of a1:50 solution of potassium ferrocyanide to 2 mL of cobaltouschloride TS. An emerald green color develops immediately.Assay Not less than 98.0% of C9H19NO7, calculated on theanhydrous basis.1,4-Dioxane Passes test.Lead Not more than 2 mg/kg.Optical (Specific) Rotation [�]D
25°: Between 17.5° and18.5°.Residue on Ignition Not more than 0.1%.Water Not more than 0.5%.
TESTS
Assay Transfer about 500 mg of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, add 50 mL ofglacial acetic acid, and warm on a steam bath until solutionis complete. Cool, add 2 drops of crystal violet TS, and titratewith 0.1 N perchloric acid in glacial acetic acid to a greenendpoint.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 25.36 mg of C9H19NO7.1,4-Dioxane Determine as directed under 1,4-Dioxane, Ap-pendix IIIB.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 400 mg of sample per milliliter of water.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Water Determine by drying a sample in a vacuum desiccatorover phosphorus pentoxide for 4 h, or as directed under WaterDetermination, Appendix IIB, using a 2-g sample dissolvedin 50 mL of methanol.
Packaging and Storage Store in tight containers.
Choline Chloride(2-Hydroxyethyl)trimethylammonium Chloride
HOCH2CH2N (CH3)3 Cl
C5H14ClNO Formula wt 139.65
INS: 1001(iii) CAS: [67-48-1]
DESCRIPTION
Choline Chloride occurs as colorless or white crystals or asa crystalline powder. It is hygroscopic, and is very soluble inwater and in alcohol.
Function Nutrient.
REQUIREMENTS
IdentificationA. A sample responds to Identification Tests A, B, and C
in the monograph for Choline Bitartrate.B. A 1:20 aqueous solution gives positive tests for Chlo-
ride, Appendix IIIA.Assay Not less than 98.0% and not more than 100.5% ofC5H14ClNO, calculated on the anhydrous basis.1,4-Dioxane Passes test.Lead Not more than 2 mg/kg.Residue on Ignition Not more than 0.05%.Water Not more than 0.5%.
TESTS
Assay Transfer about 300 mg of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, add 50 mL ofglacial acetic acid, and warm on a steam bath until solutionis complete. Cool, add 10 mL of mercuric acetate TS and 2drops of crystal violet TS, and titrate with 0.1 N perchloricacid in glacial acetic acid to a green endpoint.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.96 mg of C5H14ClNO.1,4-Dioxane Determine as directed under 1,4-Dioxane LimitTest, Appendix IIIB.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 4-g sample.Water Determine by drying a sample in a vacuum desiccatorover phosphorus pentoxide for 4 h, or determine as directedunder Water Determination, Appendix IIB.
Packaging and Storage Store in tight containers.
114 / Cinnamon Bark Oil, Ceylon Type / Monographs FCC V
Cinnamon Bark Oil, Ceylon Type
CAS: [8015-91-6]
FEMA: 2290
DESCRIPTION
Cinnamon Bark Oil, Ceylon Type, occurs as a yellow liquidwith an odor of cinnamon and a spicy burning taste. It is thevolatile oil obtained by steam distillation from the dried innerbark of the clipped cinnamon shrub Cinnamomum zeylanicumNees (Fam. Lauraceae). It is soluble in most fixed oils and inpropylene glycol. It is insoluble in glycerin and in mineral oil.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 55.0% and not more than 78.0% ofaldehydes, calculated as cinnamic aldehyde (C9H8O).Angular Rotation Between –2° and 0°.Refractive Index Between 1.573 and 1.591 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 1.010 and 1.030.
TESTS
Assay Determine as directed under Aldehydes, AppendixVI, using about 2.5 g of sample, accurately weighed, andusing 66.10 as the equivalence factor (e) in the calculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from glassor aluminum or that are lined with tin.
Cinnamon Leaf Oil
CAS: [8015-91-6]
FEMA: 2292
DESCRIPTION
Cinnamon Leaf Oil occurs as a light to dark brown liquidwith a spicy cinnamon–clove odor and taste. It is the volatileoil obtained by steam distillation from the leaves and twigsof the true cinnamon shrub Cinnamomum zeylanicum Nees(Fam. Lauraceae). The commercial oils, according to the geo-graphical origin, are designated as either Cinnamon Leaf Oil,Ceylon, or Cinnamon Leaf Oil, Seychelles, and the two typesdiffer in physical and chemical properties. Cinnamon LeafOil is soluble in most fixed oils and in propylene glycol. Itis soluble, with cloudiness, in mineral oil, but is insoluble inglycerin.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate whether it is the Ceylon or Seychellestype.Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Ceylon Type: Not less than 80.0% and not more than88.0%, by volume, of phenols; Seychelles Type: Not less than87.0% and not more than 96.0%, by volume, of phenols.Angular Rotation Ceylon Type: Between –2° and +1°; Sey-chelles Type: Between –2° and 0°.Refractive Index Ceylon Type: Between 1.529 and 1.537;Seychelles Type: Between 1.533 and 1.540 at 20°.Solubility in Alcohol Passes test.Specific Gravity Ceylon Type: Between 1.030 and 1.050;Seychelles Type: Between 1.040 and 1.060.
TESTS
Assay Determine as directed under Phenols, Appendix VI,using a measure of filtered sample prepared as follows: Shakea suitable quantity of the oil with about 2% of powderedtartaric acid, and filter.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. Ceylon Type: One milliliter of sam-ple dissolves in 1.5 mL of 70% alcohol. Seychelles Type: Onemilliliter of sample dissolves in 1 mL of 70% alcohol. Thesolutions may cloud upon further dilution.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
View IRView IR
FCC V Monographs / Citric Acid / 115
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from glassor aluminum or that are lined with tin.
Citric Acid
HO O
HO HOHOO O
C6H8O7 Formula wt, anhydrous 192.13C6H8O7·H2O Formula wt, monohydrate 210.14
INS: 330 CAS: anhydrous [77-92-9]CAS: monohydrate [5949-29-1]
DESCRIPTION
Citric Acid occurs as colorless, translucent crystals or as awhite, granular to fine, crystalline powder. It is anhydrous orcontains one molecule of water of hydration. The hydrousform is efflorescent in dry air. It is odorless and has a stronglyacid taste. One gram is soluble in about 0.5 mL of water, inabout 2 mL of alcohol, and in about 30 mL of ether.
Function Sequestrant; dispersing agent; acidifier; flavor-ing agent.
REQUIREMENTS
Labeling Indicate whether it is anhydrous or hydrous.Identification A 1:10 aqueous solution gives positive testsfor Citrate, Appendix IIIA.Assay Not less than 99.5% and not more than 100.5% ofC6H8O7, calculated on the anhydrous basis.Lead Not more than 0.5 mg/kg.Oxalate Passes test.Readily Carbonizable Substances Passes test.Residue on Ignition Not more than 0.05%.Tridodecylamine (for solvent-extracted Citric Acid only)Not more than 0.1 mg/kg.Water Anhydrous: Not more than 0.5%; Monohydrate: Notmore than 8.8%.
TESTS
Assay Dissolve about 3 g of sample, accurately weighed,in 40 mL of water, add phenolphthalein TS, and titrate with1 N sodium hydroxide. Each milliliter of 1 N sodium hydroxideis equivalent to 64.04 mg of C6H8O7.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.
Oxalate Neutralize 10 mL of a 1:10 aqueous solution with6 N ammonium hydroxide, add 5 drops of 2.7 N hydrochloricacid, cool, and add 2 mL of calcium chloride TS. No turbidityforms.Readily Carbonizable Substances Transfer 1.00 � 0.01 gof finely powdered sample into a 150-mm × 18-mm (od) tubepreviously rinsed with 10 mL of 98% sulfuric acid at 90° orused exclusively for this test. Add 10 � 0.1 mL of 98% sulfuricacid, carefully agitate the tube until solution is complete, andimmerse the tube in a water bath at 90° � 1° for 1 h. Occasion-ally remove the tube from the water bath, and carefully agitateit to ensure that the sample is dissolved and gaseous decompo-sition products are allowed to escape to the atmosphere. Coolthe tube to ambient temperature, carefully shake it to ensurethat all gases are removed, and using an adequate spectropho-tometer, measure the absorbance and transmission of the solu-tion at 470 nm in a 1-cm cell. The absorbance does not exceed0.52, and the transmission is equal to or exceeds 30%.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 4-g sample.Tridodecylamine (for solvent-extracted Citric Acid only)
Buffered Indicator Solution Prepare a mixture consistingof 700 mL of 0.1 M citric acid (anhydrous, reagent grade),200 mL of 0.2 M disodium phosphate, and 50 mL each of0.2% bromophenol blue and of 0.2% bromocresol green inspectrograde methanol.
No-Indicator Buffer Solution Prepare a mixture consistingof 700 mL of 0.1 M citric acid (anhydrous, reagent grade), 200mL of 0.2 M disodium phosphate, and 100 mL of spectrogrademethanol.
Amine Stock Solution Transfer between 40 and 45 mg oftridodecyl(trilauryl)amine, accurately weighed, into a 500-mLvolumetric flask, dilute to volume with isopropyl alcohol, andmix. Discard after 3 weeks.
Standard Amine Solution Using a graduated 5-mL pipet,transfer an amount of Amine Stock Solution equivalent to 400�g of tridodecylamine into a 100-mL volumetric flask, diluteto volume with isopropyl alcohol, and mix. Prepare this solu-tion fresh on the day of use.
Sample Solution Either dissolve 160 g of anhydrous sam-ple in 320 mL of water, or dissolve 174 g of monohydratesample in 306 mL of water.
Procedure Dissolve 160 g of anhydrous, reagent-gradecitric acid in 320 mL of water, and divide the solution equallybetween two 250-mL separators, S1 and S2. Add 5 mL of No-Indicator Buffer Solution to S1. Add 2.0 mL of StandardAmine Solution and 5 mL of Buffered Indicator Solution toS2. Divide the Sample Solution equally between two additional250-mL separators, S3 and S4. Add 5 mL of No-IndicatorBuffer Solution to S3, and 5 mL of Buffered Indicator Solutionto S4.
Add 20 mL of a spectrograde chloroform:n-heptane (1:1v/v) to each of the four separators, shake for 15 min on amechanical shaker, and allow the phases to separate for 45min. Drain all except the last few drops of the lower (aqueous)phases, and discard. Add 25 mL of 0.05 N sulfuric acid tothe organic phases in each separator, hand-shake for 30 s,and allow the phases to separate for 30 min. Drain all except
116 / Clary Oil / Monographs FCC V
the last few drops of the lower (organic) phases through dryWhatman No. 40, or equivalent, paper, and collect the aqueousfiltrates in separate, small, glass-stoppered containers.
Using a suitable spectrophotometer standardized beforeanalysis, determine the absorbance of each solution againstchloroform:heptane (1:1 v/v) in a 5-cm cell at 400 nm. Thenet absorbance of the sample (S4 – S3) is not greater than thatof the standard (S2 – S1).Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers.
Clary Oil
Clary Sage Oil
CAS: [8016-63-5]
DESCRIPTION
Clary Oil occurs as a pale yellow to yellow liquid with aherbaceous odor and a winy bouquet. It is the oil obtainedby steam distillation from the flowering tops and leaves ofthe clary sage plant, Salvia sclarea L. (Fam. Labiatae). It issoluble in most fixed oils, and in mineral oil up to 3 volumes,but it becomes opalescent on further dilution. It is insolublein glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 48.0% and not more than 75.0% ofesters, calculated as linalyl acetate (C12H20O2).Acid Value Not more than 2.5.Angular Rotation Between –6° and –20°.Refractive Index Between 1.458 and 1.473 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.886 and 0.929.
TESTS
Assay Determine as directed under Ester Determination,Appendix VI, using about 2 g of sample, accurately weighed,and using 98.15 as the equivalence factor (e) in the calculation.Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.
Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 90% alcohol, becoming opalescent on furtherdilution.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Clove Leaf Oil
CAS: [8015-97-2]
DESCRIPTION
Clove Leaf Oil occurs as a pale yellow liquid with a sharp,spicy, peppery odor and taste. It is the volatile oil obtainedby steam distillation of the leaves of Eugenia caryophyllataThunberg (Eugenia aromatica L. Baill.) (Fam. Myrtaceae).It is soluble in propylene glycol and in most fixed oils withslight opalescence, and it is relatively insoluble in glycerinand in mineral oil.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 84.0% and not more than 88.0%, byvolume, of phenols.Angular Rotation Between –2° and 0°.Refractive Index Between 1.531 and 1.535 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 1.036 and 1.046.
TESTS
Assay Determine as directed under Phenols, Appendix VI,using a measure of filtered sample prepared as follows: Shakea suitable quantity of sample with 2% powdered tartaric acidfor about 2 min, and filter. Modify the test by heating theflask in a boiling water bath for 10 min after shaking thesample with 1 N potassium hydroxide. Remove from theboiling water bath, cool, and proceed as directed.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.
View IR View IR
FCC V Monographs / Clove Stem Oil / 117
Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 70% alcohol. A slight opalescence may occurwhen additional solvent is added.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Clove Oil
Clove Bud OilCAS: [8000-34-8]
DESCRIPTION
Clove Oil occurs as a colorless or pale yellow liquid with asharp, spicy odor and taste. It is the volatile oil obtainedby steam distillation from the dried flowerbuds of Eugeniacaryophyllata Thunberg (Eugenia aromatica L. Baill.) (Fam.Myrtaceae). It darkens and thickens upon aging or exposureto air.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 85.0%, by volume, of phenols.Angular Rotation Between –1.5° and 0°.Phenols Passes test.Refractive Index Between 1.527 and 1.535 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 1.038 and 1.060.
TESTS
Assay Determine as directed under Phenols, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Phenols Shake 1 mL of sample with 20 mL of hot water.The water shows no more than a scarcely perceptible acidreaction with blue litmus paper. Cool the mixture, pass thewater layer through a wetted filter, and treat the clear filtratewith 1 drop of ferric chloride TS. The mixture has only atransient gray-green color, but not a blue or violet color.
Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in aluminum or tin- or epoxy-phenolic-lined, tight, light-resistant containers, and avoid ex-posure to excessive heat.
Clove Stem Oil
CAS: [8015-98-3]
DESCRIPTION
Clove Stem Oil occurs as a yellow to light brown liquid witha sharp, spicy odor and taste. It is the volatile oil obtainedby steam distillation from the dried stems of the buds ofEugenia caryophyllata Thunberg (Eugenia aromatica L.Baill.) (Fam. Myrtaceae). It is soluble in fixed oils and inpropylene glycol, but it is relatively insoluble in glycerin andin mineral oil.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 89.0% and not more than 95.0%, byvolume, of phenols.Angular Rotation Between –1.5° and 0°.Refractive Index Between 1.534 and 1.538 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 1.048 and 1.056.
TESTS
Assay Determine as directed under Phenols, Appendix VI,using a measure of filtered sample prepared as follows: Shakea suitable quantity of the sample oil with about 2% powderedtartaric acid for about 2 min, and filter. Modify the test byheating the flask in a boiling water bath for 10 min aftershaking the sample oil with 1 N potassium hydroxide. Removethe flask from the boiling water bath, cool, and proceed asdirected.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mL tube.
View IR
View IR
118 / Cocoa Butter Substitute / Monographs FCC V
Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Cocoa Butter Substitute
DESCRIPTION
Cocoa Butter Substitute occurs as a white, waxy solid that ispredominantly a mixture of triglycerides derived primarilyfrom palm, safflower, sunflower, or coconut oils. The resultingproducts may be used directly or with cocoa butter in allproportions for the preparation of coatings. In contrast tomany edible oils and hard butters, Cocoa Butter Substitutehas an abrupt melting range, changing from a rather firm,plastic solid below 32° to a liquid at about 33.8° to 35.5°.
Function Coating agent; texturizer.
REQUIREMENTS
Identification Cocoa Butter Substitute exhibits the follow-ing typical composition profile of fatty acids determined asdirected under Fatty Acid Composition, Appendix VII:
Fatty Acid: ≤12 12:0 14:0 16:0 16:1Weight % (Range): 0.0 0.0 0.0 21–24 0.0Fatty Acid: 18:0 18:1 18:2 ≥20Weight % (Range): 40–44 31–35 0.5–1.5 0.3–0.7
Color (AOCS-Wesson) Not more than 2.5 red.Free Fatty Acids (as oleic acid) Not more than 1.0%.Hexane Not more than 5 mg/kg.Iodine Value Between 30 and 33.Lead Not more than 0.1 mg/kg.Peroxide Value Not more than 10 meq/kg.Residual Catalyst (as F) Not more than 0.5 mg/kg.Total Glycerides Not less than 98.0% of total.
Monoglycerides Not more than 1.0%.Diglycerides Not more than 7.0%.Triglycerides Not less than 90.0%.
Unsaponifiable Matter Not more than 1.0%.Water Not more than 0.1%.
TESTS
Color (AOCS-Wesson) Determine as directed under Color(AOCS-Wesson), Appendix VII.
Free Fatty Acids (as oleic acid) Using the diglyceride frac-tion under Total Glycerides (below), determine as directedunder Free Fatty Acids, Appendix VII, except add 2 mL ofphenolphthalein TS, and titrate with the appropriate normalityof sodium hydroxide. Use the following equivalence factor(e) in the formula given in the procedure:
Free fatty acids as oleic acid, e = 28.2.
HexaneStandard Preparation Using a micropipet, transfer and
dissolve 34 �L of hexane in 45 g of cold-pressed cottonseedoil that has not been extracted with hexane. As directed underProcedure (below), analyze aliquots of 0.1, 0.25, 0.5, and 5.0mg; the aliquots correspond to 2, 5, 10, and 100 mg/kg,respectively, of residual hexane in a 25-mg sample.
Assay Preparation Pack the lower half of 8.5-cm × 9.5-mm (od) borosilicate glass tubing (inlet liner) with glass woolthat has been heated at 200° for 16 h to expel volatiles.Transfer 25 mg of sample, accurately weighed, into the glasstubing, and cover it with a small plug of treated glass wool.
Standard Curve Chromatograph aliquots of each Stan-dard Preparation as directed under Procedure. Measure thepeak areas for each Standard Preparation. Plot a standardcurve using the concentration, in milligrams per kilogram, ofeach Standard Preparation versus its corresponding peak area,and draw the best straight line. To ensure that the relativestandard deviation does not exceed 2.0%, chromatograph asufficient number of replicates of each Standard Preparation,and record the areas as directed under Procedure (below).
Procedure (See Chromatography, Appendix IIA.) Use asuitable gas chromatograph that is equipped with independentdual flame-ionization detectors and a 0.6-m × 6.35-mm (od)stainless-steel U-tube, or equivalent, packed with Porapak P,or equivalent. Maintain the inlet temperature at 110° and thedetectors at 200°. Hold the column oven initially at 70° for2 min followed by a linear temperature gradient at 5°/min to180° and a final hold at 180° for 10 min or until the columnis clean. Use helium as the carrier gas at a flow rate of 60mL/min, hydrogen as the fuel gas at a flow rate of 52 mL/min for each flame, and air as the scavenger gas for both flamesat a flow rate of 500 mL/min. Insert the Assay Preparation intothe inlet liner of the gas chromatograph, immediately sealingthe base of the inlet and the lower lip of the glass tubingwith a silicone O-ring (Applied Science Laboratories, Inc.,or equivalent) previously heated at 200° for 2 h to removevolatile impurities. Immediately close the inlet liner with theseptum and septum liner. Allow the carrier gas to flow throughthe Assay Preparation, chromatograph, and record the chro-matograms. Using the peak area of hexane eluting from theAssay Preparation at the same time as the Standard Prepara-tion, read directly from the Standard Curve the concentration,C, of hexane, in milligrams per kilogram, of the Assay Prepa-ration. Calculate the quantity of hexane, in milligrams perkilogram, in the sample taken by the formula
25C/W,
in which W is the weight, in milligrams, of the sample intro-duced into the gas chromatograph.Iodine Value Determine as directed under Iodine Value,Appendix VII.
FCC V Monographs / Coconut Oil (Unhydrogenated) / 119
Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 5-g sample.Peroxide Value Determine as directed in Method II underPeroxide Value, Appendix VII.Residual Catalyst (as F) Determine as directed in MethodI under Fluoride Limit Test, Appendix IIIB, beginning with‘‘. . . and 30 mL of water in a 125-mL distillation flask havinga side arm and trap,’’ using the ashed residue of the followingas the sample: Transfer 30 g of sample, accurately weighed,into a 250-mL distillation flask having a side arm and atrap. Connect the flask with a condenser, and fit it with athermometer and a capillary tube. Both of these should reachnearly to the bottom of the flask so that they extend into theliquid during the distillation. Add 0.2 g of silver sulfate, threeboiling beads, and 25 mL of 1:1 sulfuric acid:water to theflask. Connect a dropping funnel or a steam generator to thecapillary tube. Distill until the temperature reaches 135°. Then,through the capillary, add water from the funnel or introducesteam, as necessary, to maintain the temperature as close aspossible to 135° until 250 mL of distillate has been collectedin a beaker. Cool the distillate. Add 3 mL of 30% hydrogenperoxide to remove any sulfites, let it stand for 5 min, andevaporate the distillate in a dish containing 15 mL of saturatedcalcium hydroxide suspension. Ash the residue at 600° for 4 h.
The total volume of sodium fluoride TS required for thesolutions from both Distillate A and Distillate B should notexceed 0.75 mL.Total Glycerides Determine as directed under Total Mono-glycerides, Appendix VII, except save all three elution frac-tions to determine the percentages of Monoglycerides, Diglyc-erides, and Triglycerides.
Note: Use toluene instead of benzene.
The diglyceride fraction also contains free fatty acids, thepercentage of which is determined under Free Fatty Acids.Calculate the percentage of Total Glycerides (G), which isthe sum of the percentages of Monoglycerides, Diglycerides,and Triglycerides, by the following formulas:
M = WM100/WU,
D = (WD100/WU) – F,
T = WT100/WU,
G = M + D + T,
in which M is the percentage of monoglycerides; WM is theweight, in grams, of monoglycerides; WU is the weight, ingrams, of the sample taken; D is the percentage of diglycer-ides; WD is the weight, in grams, of diglycerides; F is thepercentage of free fatty acids; T is the percentage of triglycer-ides; and WT is the weight, in grams, of triglycerides.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB. However, in place of 35 to 40 mL of methanol,use 50 mL of a 1:1 solution of chloroform:methanol to dissolvethe sample.
Packaging and Storage Store in well-closed containers.
Coconut Oil (Unhydrogenated)
CAS: [8001-31-8]
DESCRIPTION
Coconut Oil (Unhydrogenated) occurs as a viscous, white tolight yellow-tan liquid. It is obtained from the kernel of thefruit of the coconut palm Cocos nucifera (Fam. Palmae). Thecrude oil obtained by mechanically pressing dried coconutmeat (copra) is refined, bleached, and deodorized to substan-tially remove free fatty acids, phospholipids, color, odor andflavor components, and other non-oil materials. Comparedwith many natural fats, Coconut Oil (Unhydrogenated) hasan abrupt melting range, changing from a rather firm, plasticsolid at about 21° or below to a liquid at about 27°.
Function Coating agent; emulsifying agent; texturizer.
REQUIREMENTS
Identification Coconut Oil (Unhydrogenated) exhibits thefollowing typical composition profile of fatty acids determinedas directed under Fatty Acid Composition, Appendix VII:
Fatty Acid: 6:0 8:0 10:0 12:0 14:0 16:0 16:1Weight % (Range): 0–0.8 5–9 4–8 44–52 15–21 8–11 0–1Fatty Acid: 18:0 18:1 18:2 20:0Weight % (Range): 1–4 5–8 0–2.5 0–0.4
Arsenic Not more than 0.5 mg/kg.Color (AOCS-Wesson) Not more than 20 yellow/2.0 red.Free Fatty Acids Oleic Acid: Not more than 0.1%; LauricAcid: Not more than 0.07%.Iodine Value Between 6 and 11.Lead Not more than 0.1 mg/kg.Melting Range Between 23.5° and 27°.Peroxide Value Not more than 10 meq/kg.Unsaponifiable Matter Not more than 1.5%.Water Not more than 0.1%.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared using 2 gof sample, accurately weighed. The absorbance caused by anyred color from the solution of the sample does not exceedthat produced by 1.0 mL of Standard Arsenic Solution (1 �gAs) when treated in the same manner and under the sameconditions as the sample.Color (AOCS-Wesson) Determine as directed under Color(AOCS-Wesson), Appendix VII.Free Fatty Acids Determine as directed under Free FattyAcids, Appendix VII, using the following equivalence factors(e) in the formula given in the procedure:
Free fatty acids as oleic acid, e = 28.2.
Free fatty acids as lauric acid, e = 20.0.
120 / Cognac Oil, Green / Monographs FCC V
Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Melting Range Determine as directed under Melting Range,Appendix VII.Peroxide Value Determine as directed in Method II underPeroxide Value, Appendix VII.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB. However, in place of 35 to 40 mL of methanol,use 50 mL of chloroform to dissolve the sample.
Packaging and Storage Store in well-closed containers.
Cognac Oil, Green
Wine Yeast Oil
CAS: [8016-21-5]
DESCRIPTION
Cognac Oil, Green, occurs as a green to blue-green liquidwith the characteristic aroma of cognac. It is the volatile oilobtained by steam distillation from wine lees. It is soluble inmost fixed oils and in mineral oil. It is very slightly solublein propylene glycol, and it is insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Between 32 and 70.Angular Rotation Between –1° and +2°.Ester Value Between 200 and 245.Refractive Index Between 1.427 and 1.430 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.864 and 0.870.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed in Ester Value underEsters, Appendix VI, using about 1 g of sample, accuratelyweighed.
Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 80% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers in acool place protected from light.
Copaiba Oil
CAS: [8013-97-6]
DESCRIPTION
Copaiba Oil occurs as a colorless to slightly yellow liquidwith the characteristic odor of copaiba balsam and an aromatic,slightly bitter and pungent taste. It is the volatile oil obtainedby steam distillation of copaiba balsam, an exudate from thetrunk of various South American species of Copaifera L.(Fam. Leguminosae). It is soluble in alcohol, in most fixedoils, and in mineral oil. It is insoluble in glycerin and practi-cally insoluble in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between –7° and –33°.Gurjun Oil Passes test.Refractive Index Between 1.493 and 1.500 at 20°.Specific Gravity Between 0.880 and 0.907.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Gurjun Oil Add 5 or 6 drops of sample to 10 mL of glacialacetic acid containing 5 drops of nitric acid. No purple colorappears within 2 min, indicating the absence of gurjun oil.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
View IR
View IR
FCC V Monographs / Copper Sulfate / 121
Copper Gluconate
CH2OH(CHOH)4COO2Cu
C12H22CuO14 Formula wt 453.84
CAS: [527-09-3]
DESCRIPTION
Copper Gluconate occurs as a fine, light blue powder. It isvery soluble in water, and is very slightly soluble in alcohol.
Function Nutrient.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution gives positive tests for Copper,
Appendix IIIA.B. Dissolve a quantity of sample in water, heating in a
water bath at 60° if necessary, to obtain a Test Solution con-taining 10 mg/mL. Similarly, prepare a Standard Solutionof USP Potassium Gluconate Reference Standard in water,diluting to 10 mg/mL. Apply separate 5-�L portions of theTest Solution and the Standard Solution on a suitable thin-layer chromatographic plate (see Thin-Layer Chromatogra-phy, Appendix IIA) coated with a 0.25-mm layer of chromato-graphic silica gel, and allow to dry. Develop the chromatogramin a solvent system consisting of a mixture of alcohol, water,ammonium hydroxide, and ethyl acetate (50:30:10:10) untilthe solvent front has moved about three-fourths of the lengthof the plate. Remove the plate from the chamber, and dry itat 110° for 20 min. Allow it to cool, and spray it with a sprayreagent prepared as follows: Dissolve 2.5 g of ammoniummolybdate in about 50 mL of 2 N sulfuric acid in a 100-mLvolumetric flask, add 1.0 g of ceric sulfate, swirl to dissolve,dilute with 2 N sulfuric acid to volume, and mix. After spray-ing, heat the plate at 110° for about 10 min. The principalspot obtained from the Test Solution corresponds in color,size, and Rf value to that obtained from the Standard Solution.Assay Not less than 98.0% and not more than 102.0% ofC12H22CuO14.Lead Not more than 5 mg/kg.Reducing Substances Not more than 1.0%.
TESTS
Assay Dissolve about 1.5 g of sample, accurately weighed,in 100 mL of water in a 250-mL Erlenmeyer flask, add 2 mLof glacial acetic acid and 5 g of potassium iodide, mix well,and titrate with 0.1 N sodium thiosulfate to a light yellowcolor. Add 2 g of ammonium thiocyanate, mix, then add 3mL of starch TS and continue titrating to a milk-white end-point. Each milliliter of 0.1 N sodium thiosulfate is equivalentto 45.38 mg of C12H22CuO14.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a 1-g sample in 25 mL of water, and 5 �g oflead (Pb) ion in the control.Reducing Substances Transfer about 1 g of sample, accu-rately weighed, into a 250-mL Erlenmeyer flask, dissolve itin 10 mL of water, add 25 mL of alkaline cupric citrate TS,and cover the flask with a small beaker. Boil gently for exactly5 min, and cool rapidly to room temperature. Add 25 mL ofa 1:10 solution of acetic acid, 10.0 mL of 0.1 N iodine, 10mL of 2.7 N hydrochloric acid, and 3 mL of starch TS, andtitrate with 0.1 N sodium thiosulfate to the disappearance ofthe blue color. Calculate the weight, in milligrams, of reducingsubstances (as D-glucose) by the formula
27(V1N1 – V2N2),
in which 27 is an empirically determined equivalence factorfor D-glucose; V1 and N1 are the volume and normality,respectively, of the iodine solution; and V2 and N2 are thevolume and normality, respectively, of the sodium thiosulfatesolution.
Packaging and Storage Store in well-closed containers.
Copper Sulfate
Cupric Sulfate
CuSO4 Formula wt, anhydrous 159.6CuSO4·5H2O Formula wt, pentahydrate 249.68
INS: 519 CAS: anydrous [7758-98-7]CAS: pentahydrate [7758-99-8]
DESCRIPTION
Copper Sulfate occurs as blue crystals, crystalline granules,or powder. It effloresces slowly in dry air and is freely solublein water, soluble in glycerin, and slightly soluble in alcohol.
Function Nutrient.
REQUIREMENTS
Identification A 1:20 solution gives positive tests for Cop-per and for Sulfate, Appendix IIIA.Assay Not less than 98.0% and not more than 102.0% ofCuSO4·5H2O.Iron Not more than 0.01%.Lead Not more than 4 mg/kg.Substances Not Precipitated by Hydrogen Sulfide Notmore than 0.3%.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in 50 mL of water, add 4 mL of glacial acetic acid and 3 g
122 / Coriander Oil / Monographs FCC V
of potassium iodide, mix well, and titrate with 0.1 N sodiumthiosulfate to a light yellow color. Add 2 g of ammoniumthiocyanate, mix, and then add 3 mL of starch TS, and continuetitrating to a milky white endpoint. Perform a blank titration(see General Provisions), and make any necessary correction.Each milliliter of 0.1 N sodium thiosulfate used is equivalentto 24.97 mg of CuSO4·5H2O.Iron Add 2 mL of hydrochloric acid and 0.1 mL of nitricacid to the residue from Substances Not Precipitated by Hy-drogen Sulfide (below), cover with a watch glass, and digeston a steam bath for 20 min. Remove the watch glass, andevaporate to dryness. Dissolve the residue in 1 mL of hydro-chloric acid, and dilute to 60 mL with water. Dilute 5 mL ofthis solution to 40 mL with water, add 2 mL of hydrochloricacid, and dilute to 50 mL with water. Add 40 mg of ammoniumperoxydisulfate crystals and 10 mL of ammonium thiocyanateTS, and mix thoroughly. Any red color produced within 1 hshall not exceed that produced by 0.033 mg of iron in anequal volume of solution containing the reagents used inthe test.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Substances Not Precipitated by Hydrogen Sulfide Dis-solve 5 g of sample in 200 mL of 1:100 sulfuric acid, heatto 70°, and pass hydrogen sulfide through the solution untilthe copper is completely precipitated. Dilute to 250 mL, mixthoroughly, allow the precipitate to settle, and filter. Evaporate200 mL of the filtrate to dryness in a tared dish, ignite at800° � 25° for 15 min, cool, and weigh.
Packaging and Storage Store in tight containers.
Coriander Oil
CAS: [8008-52-4]
DESCRIPTION
Coriander Oil occurs as a colorless or pale yellow liquid withthe characteristic odor and taste of coriander. It is the volatileoil obtained by steam distillation from the dried ripe fruit ofCoriandrum sativum L. (Fam. Umbelliferae).
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between +8° and +15°.Refractive Index Between 1.462 and 1.472 at 20°.
Solubility in Alcohol Passes test.Specific Gravity Between 0.863 and 0.875.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers pro-tected from light. Avoid exposure to excessive heat.
Corn Oil (Unhydrogenated)
CAS: [8001-30-7]
DESCRIPTION
Corn Oil (Unhydrogenated) occurs as an amber-colored oil.It is obtained from the corn plant Zea mays (Fam. Gramineae),usually by solvent extraction of the corn germ. It is refined,bleached, and deodorized to substantially remove free fattyacids, phospholipids, color, odor and flavor components, andother non-oil materials. It is a liquid at 21° to 27°, but tracesof wax, unless they are removed by winterization, may causethe oil to cloud when cooled to low temperature. It is freefrom visible foreign material (other than wax) at 21° to 27°.
Function Coating agent; emulsifying agent; texturizer.
REQUIREMENTS
Identification Corn Oil exhibits the following typical com-position profile of fatty acids determined as directed underFatty Acid Composition, Appendix VII:
Fatty Acid: <14 14:0 16:0 16:1 18:0 18:1 18:2Weight % (Range): <0.1 <1.0 8.0–19 <0.5 0.5–4.0 19–50 38–65Fatty Acid: 18:3 20:0 20:1 22:0 22:1 24:0Weight % (Range): <2.0 <1.0 <0.5 <0.3 <0.1 <0.4
Arsenic Not more than 0.5 mg/kg.Color (AOCS-Wesson) Not more than 5.0 red.Free Fatty Acids (as oleic acid) Not more than 0.1%.Iodine Value Between 120 and 130.
View IR
FCC V Monographs / Cottonseed Oil (Unhydrogenated) / 123
Lead Not more than 0.1 mg/kg.Linolenic Acid Not more than 2.0%.Peroxide Value Not more than 10 meq/kg.Unsaponifiable Matter Not more than 1.5%.Water Not more than 0.1%.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds, and 4 g of sample, accuratelyweighed. The absorbance caused by any red color from thesolution of the sample does not exceed that produced by 2.0mL of Standard Arsenic Solution (2 �g As) when treated inthe same manner and under the same conditions as the sample.Color (AOCS-Wesson) Determine as directed under Color(AOCS-Wesson), Appendix VII.Free Fatty Acids (as oleic acid) Determine as directed underFree Fatty Acids, Appendix VII, using the following equiva-lence factor (e) in the formula given in the procedure:
Free fatty acids as oleic acid, e = 28.2.
Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Linolenic Acid Determine as directed under Fatty AcidComposition, Appendix VII.Peroxide Value Determine as directed in Method II underPeroxide Value, Appendix VII.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB. However, in place of 35 to 40 mL of methanol,use 50 mL of chloroform to dissolve the sample.
Packaging and Storage Store in well-closed containers.
Costus Root Oil
CAS: [8023-88-9]
DESCRIPTION
Costus Root Oil occurs as a light yellow to brown, viscousliquid with a peculiar, persistent odor reminiscent of violet,orris, and vetivert. It is the volatile oil obtained by steamdistillation from the dried, triturated roots of the herbaceousperennial plant Saussurea lappa Clarke (Fam. Compositae)or by a solvent extraction procedure followed by vacuumdistillation of the resinoid extract. It is soluble in most fixed
oils and in mineral oil. It is insoluble in glycerin and inpropylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 42.Angular Rotation Between +10° and +36°.Ester Value Between 90 and 150.Refractive Index Between 1.512 and 1.523 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.995 and 1.039.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed under Ester Value, Ap-pendix VI, using about 1 g of sample, accurately weighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 90% alcohol, but the solution becomes cloudyupon further dilution, and paraffin crystals may occasionallyseparate.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Cottonseed Oil (Unhydrogenated)
CAS: [8001-29-4]
DESCRIPTION
Cottonseed Oil (Unhydrogenated) occurs as a dark red-brownoil. It is obtained from the seed of the cotton plant Gossypiumhirsutum (American) or Gossypium barbadense (Egyptian)by mechanical expression or solvent extraction. It is refined,bleached, and deodorized to substantially remove free fattyacids, phospholipids, color, odor and flavor components, andmiscellaneous other non-oil materials. It is liquid at 21° to 27°,clouds at 21°, and partially solidifies at storage temperatures
View IR
124 / Cubeb Oil / Monographs FCC V
below 10° to 16°. It is free from visible foreign material at23° to 27°.
Function Cooking or salad oil; component of margarine orshortening; tenderizer; carrier; stabilizer; thickener; coatingagent; texturizer.
REQUIREMENTS
Identification Cottonseed Oil (Unhydrogenated) exhibitsthe following composition profile of fatty acids determinedas directed under Fatty Acid Composition, Appendix VII:
Fatty Acid: <14:0 14:0 16:0 16:1 18:0 18:1 18:2Weight % (Range): <0.1 0.5–2.017–29 <1.5 1.0–4.0 13–44 40–63Fatty Acid: 18:3 20:0 20:1 22:0 22:1 24:0Weight % (Range): 0.1–2.1 <0.5 <0.5 <0.5 <0.5 <0.5
Color (AOCS-Wesson) Not more than 70 yellow/4.5 red.Free Fatty Acids (as oleic acid) Not more than 0.1%.Iodine Value Between 99 and 119.Lead Not more than 0.1 mg/kg.Linolenic Acid Not more than 2.1%.Peroxide Value Not more than 10 meq/kg.Unsaponifiable Matter Not more than 1.5%.Water Not more than 0.1%.
TESTS
Color (AOCS-Wesson) Determine as directed under Color(AOCS-Wesson), Appendix VII.Free Fatty Acids (as oleic acid) Determine as directed underFree Fatty Acids, Appendix VII, using the following equiva-lence factor (e) in the formula given in the procedure:
Free fatty acids as oleic acid, e = 28.2.
Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Linolenic Acid Determine as directed under Fatty AcidComposition, Appendix VII.Peroxide Value Accurately weigh about 10 g of sample,add 30 mL of a 3:2 mixture of glacial acetic acid:chloroform,and mix. Add 1 mL of a saturated solution of potassiumiodide, and mix for 1 min. Add 100 mL of water, begintitrating immediately with 0.05 N sodium thiosulfate, addingstarch TS as the endpoint is approached, and continue thetitration until the blue starch color has just disappeared. Per-form a blank determination (see General Provisions), andmake any necessary correction. Calculate the peroxide value,as milliequivalents of peroxide per kilogram of sample, bythe formula
S × N × 1000/W,
in which S is the net volume, in milliliters, of sodium thiosul-fate solution required for the sample; N is the exact normalityof the sodium thiosulfate solution; and W is the weight, ingrams, of sample taken.
Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB. However, in place of 35 to 40 mL of methanoluse 50 mL of chloroform to dissolve the sample.
Packaging and Storage Store in well-closed containers.
Cubeb Oil
CAS: [8007-87-2]
DESCRIPTION
Cubeb Oil occurs as a colorless or light green to blue-green liquid with a spicy odor and a slightly acrid taste.It is the volatile oil obtained by steam distillation from themature, unripe, sun-dried fruit of the perennial vine Pipercubeba L. (Fam. Piperaceae). It is soluble in most fixedoils and in mineral oil, but it is insoluble in glycerin andpropylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Acid Value Not more than 2.0.Angular Rotation Between –12° and –43°.Refractive Index Between 1.492 and 1.502 at 20°.Saponification Value Not more than 8.Solubility in Alcohol Passes test.Specific Gravity Between 0.898 and 0.928.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed under Saponi-fication Value, Appendix VI, using about 5 g of sample,accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 10 mL of 90% alcohol.
View IR
FCC V Monographs / Curdlan / 125
Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Cumin Oil
CAS: [8014-13-9]
DESCRIPTION
Cumin Oil occurs as a light yellow to brown liquid with astrong and somewhat disagreeable odor. It is the volatile oilobtained by steam distillation from the plant Cuminum cymi-num L. (Fam. Umbelliferae). It is relatively soluble in mostfixed oils and in mineral oil. It is very soluble in glycerinand in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Assay Not less than 45.0% and not more than 54.0% ofaldehydes, calculated as cuminaldehyde (C10H12O).Angular Rotation Between +3° and +8°.Refractive Index Between 1.500 and 1.506 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.905 and 0.925.
TESTS
Assay Determine as directed under Aldehydes, AppendixVI, using about 1 g of sample, accurately weighed, and 74.10as the equivalence factor (e) in the calculation. Allow themixture to stand for 30 min at room temperature before ti-trating.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 8 mL of 80% alcohol. The solution may become hazy onthe addition of more alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
CurdlanBeta-1,3-glucan
O O O
OH OH OH
OH
OH OH OH
OHO O
CH2OH CH2OH CH2OH
n
(C6H10O5)n
CAS: [54724-00-4]
DESCRIPTION
Curdlan occurs as a white to nearly white powder. It is ahigh-molecular-weight polymer of glucose (�-1,3-glucan)produced by pure-culture fermentation of a carbohydrate bya nonpathogenic and nontoxigenic strain of Agrobacteriumbiobar 1 (formerly Alcaligenes faecalis var. myxogenes) orAgrobacterium radiobacter. Curdlan consists of �-(1,3)-linked glucose residues and has the unusual property of form-ing an elastic gel when its aqueous suspension is heated to atemperature above 54°. It is insoluble in water, but is solublein alkaline solutions.
Function Firming agent; gelling agent; stabilizer; thickener.
REQUIREMENTS
IdentificationA. Add 5 mL of sulfuric acid TS to 10 mL of a 2% aqueous
suspension of sample, heat in a boiling water bath for 30 min,and cool. Neutralize the mixture with barium carbonate, andcentrifuge it at 900 g for 10 min. Add 1 mL of the supernatantto 5 mL of hot alkaline cupric tartrate TS. A copious redprecipitate of cuprous oxide forms.
B. Heat a 2% aqueous suspension of sample in a boilingwater bath for 10 min, and cool. A firm gel forms.
C. Suspend 0.2 g of sample in 5 mL of water, add 1 mLof 3 N sodium hydroxide, and shake. The sample dissolves.Assay Not less than 80% (calculated as anhydrous glucose).Gel Strength (2% aqueous suspension) Not less than 600g/cm2.Lead Not more than 0.5 mg/kg.Loss on Drying Not more than 10%.Microbial Limits
Aerobic Plate Count Not more than 1000 CFU per gram.E. coli Negative in 1 g.
View IR
126 / beta-Cyclodextrin / Monographs FCC V
Nitrogen Not more than 0.3%.pH (1% aqueous suspension) Between 6.0 and 7.5.Residue on Ignition Not more than 6%.
TESTS
AssaySample Solution Transfer about 100 mg of sample, accu-
rately weighed, into a 100-mL volumetric flask, and dissolvein about 90 mL of 0.1 N sodium hydroxide. Add 0.1 N sodiumhydroxide to volume, and mix well. Transfer 5 mL of thesolution into a 100-mL volumetric flask, add water to volume,and mix well. Add 1 mL of a 5:100 solution of reagent-gradephenol and 5 mL of sulfuric acid TS to 1 mL of the solution,shake vigorously, and cool in ice-cold water. Prepare a blankand a Reference Standard Solution in the same manner, using0.1 mL of water and 100 mg of reagent-grade glucose, respec-tively.
Procedure Determine the absorbance of the Sample Solu-tion and the Reference Standard Solution in 1-cm cells at 490nm with a suitable spectrophotometer, using the blank solutionto zero it.
Calculation Calculate the percent Curdlan in the sampletaken using the following equation:
Curdlan (%) = (A/AR) × (0.9 × WR/W) × 100,
in which A is the absorbance of the Sample Solution; AR isthe absorbance of the Reference Standard Solution; 0.9 isthe molecular weight of anhydrous glucose divided by themolecular weight of glucose; WR is the weight, in milligrams,of the reagent-grade glucose used to make the ReferenceStandard Solution; and W is the weight, in milligrams, of thesample.Gel Strength (2% aqueous suspension)
Procedure Place 200 mg of sample into the tube of aPotter-Elvehjem homogenizer, add 10 mL of water, and ho-mogenize at about 1500 g for 5 min. Transfer the suspensioninto a 16-mm × 150-mm test tube, de-aerate in vacuum for3 min, and heat in a boiling water bath for 10 min to form agel. Cool in running water, let it stand for 30 min, and removethe gel from the test tube. Accurately cut the gel at distancesof 20 mm and 30 mm from the bottom to obtain a section 10mm long. Determine the gel strength using a Rheo MeterModel CR-200D (Sun Scientific Co., Ltd., Japan; Load cell:1000 g; set to a measurement mode 4) or an equivalent instru-ment capable of uniaxial compression and having a load cellsensitivity of 500 to 1000 g. Use a cylindrical stainless steelplunger with a 0.5-cm diameter. Lower the plunger into thegel at 250 mm/min. The resulting force-time curve is recordedand used for gel strength calculation.
Calculation Calculate gel strength by the followingequation:
Gel strength (g force/cm2) = f/0.196 cm2,
in which f is the force on the force-time curve that shows asharp yielding downward trend associated with rupture of thegel, and 0.196 is the area, in centimeters squared, of theplunger.
Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in a vacuum for 5 h at 60°.Microbial Limits (Note: Current methods for the followingtests may be found online at <www.cfsan.fda.gov/~ebam/bam-toc.html>):
Aerobic Plate CountE. coli
Nitrogen Determine as directed in Method II under NitrogenDetermination, Appendix IIIC, using a 1-g sample.pH (1% aqueous suspension) Determine as directed underpH Determination, Appendix IIB, using a 1% aqueous sus-pension.Residue on Ignition Determine as directed for Method Iunder Residue on Ignition, Appendix IIC, using a 1-g sample.
Packaging and Storage Store in airtight containers.
beta-Cyclodextrin
�-Cyclodextrin; BCD
(C6H10O5)7 Formula wt 1135.0
INS: 459 CAS: [7585-39-9]
DESCRIPTION
Beta-Cyclodextrin occurs as a white, fine, crystalline solid,frequently a fine, crystalline powder. It is a nonreducing cycliccompound consisting of seven alpha-(1,4) linked D-glucopyra-nosyl units. It is slightly soluble in water.
Function Encapsulating agent; stabilizer.
REQUIREMENTS
IdentificationA. The infrared absorption spectrum of a potassium bro-
mide dispersion of sample exhibits relative maxima at thesame wavelengths as those of a similar preparation of USPbeta-Cyclodextrin Reference Standard.
B. The retention time of the major peak in the chromato-gram of Assay Preparation corresponds to that in the chroma-togram of Standard Preparation, obtained as directed in theAssay (below).Assay Not less than 98.0% and not more than 101.0% of(C6H10O5)7 as beta-Cyclodextrin, calculated on the anhy-drous basis.Lead Not more than 1 mg/kg.Optical (Specific) Rotation [�]D
20°: Between +160° and+164°, calculated on the anhydrous basis.
FCC V Monographs / beta-Cyclodextrin / 127
Reducing Sugars (dextrose equivalent) Not more than1.0%, calculated on the anhydrous basis.Residue on Ignition Not more than 0.1%.Toluene Not more than 1 mg/kg.Trichloroethylene Not more than 1 mg/kg.Water Not more than 14.0%.
TESTS
AssayMobile Phase Prepare a filtered and degassed 65:35 mix-
ture of acetonitrile:water.Internal Standard Solution Transfer 2.0 g of glycerol to
a 100-mL volumetric flask. Dilute to volume with water, andmix. Filter through a 0.45-�m membrane filter. Use fresh orstore in a freezer, thaw in hot water, and mix.
Standard Preparation Transfer 100 mg of USP beta-Cyclodextrin Reference Standard, accurately weighed, into a10-mL volumetric flask. Dilute to volume with water, andmix. Use fresh or store in a freezer, thaw in hot water, andmix. Mix 1.0 mL of this solution with 1.0 mL of InternalStandard Solution.
System Suitability Preparation Transfer 50 mg each ofUSP alpha-Cyclodextrin Reference Standard and USP beta-Cyclodextrin Reference Standard, accurately weighed, into a10-mL volumetric flask. Dilute to volume, and mix. Filterthrough a 0.45-�m membrane filter.
Assay Preparation Transfer 1 g of beta-Cyclodextrin, ac-curately weighed, into a 100-mL volumetric flask, dilute tovolume with water, and mix. Filter this solution through a0.45-�m membrane filter. Mix 1.0 mL of the filtered solutionwith 1.0 mL of Internal Standard Solution.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a liquid chromatograph equipped with a refrac-tive index detector that can be maintained at a constant temper-ature of 25°, a 25-cm × 4.6-mm (id) column packed with 10-�m porous silica gel bonded with aminopropylsilane (Alltech35643, or equivalent), and a guard column that contains thesame packing. Maintain the column at a constant temperatureof 25° � 2°, and the flow rate at about 2.0 mL/min. Inject20 �L of System Suitability Preparation into the chromato-graph, and record the peak responses as directed under Proce-dure. The relative standard deviation for replicate injectionsis not more than 2.0%, and the alpha-Cyclodextrin and beta-Cyclodextrin peaks exhibit baseline separation, the relativeretention times being about 0.8 and 1.0, respectively.
Procedure Separately inject about 20 �L of Assay Prepa-ration and Standard Preparation into the chromatograph, rec-ord the chromatograms, and measure the responses for themajor peaks. Calculate the quantity, in milligrams, of(C6H10O5)7 in the portion of beta-Cyclodextrin taken by theformula
100 × C × (RU/RS),
in which C is the concentration, in milligrams per milliliter,of anhydrous beta-Cyclodextrin in the Standard Preparation,as determined from the concentration of USP beta-Cyclodex-trin Reference Standard corrected for moisture content by atitrimetric water determination, and RU and RS are the peak
response ratios of the beta-Cyclodextrin peak to the internalstandard peak obtained from the Assay Preparation and theStandard Preparation, respectively.Lead Determine as directed in Method II for the FlameAbsorption Spectrophotometric Method under Lead LimitTest, Appendix IIIB, using a 10-g sample.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 1 g of cyclodextrin dissolved in 100 mL of water.Reducing Sugars Determine as directed under ReducingSugars Assay, Appendix X, using 60 to 120 mg of sample,accurately weighed.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, using a 1- to 2-g sample.Toluene
Toluene Stock Solution Transfer 100 mg, accuratelyweighed, or 115.3 �L, accurately measured, of toluene into a100-mL volumetric flask, and dilute to volume with methanol.This solution contains 1000 �g of toluene in each milliliter.
Toluene Standard Solutions Prepare five working stan-dard solutions with concentrations of 10, 50, 100, 250, and500 �g/mL by quantitatively diluting Toluene Stock Solutionwith methanol.
�,�,�-Trifluorotoluene Stock Solution Transfer 80 mg,accurately weighed, or 67.3 �L, accurately measured, of�,�,�-trifluorotoluene into a 100-mL volumetric flask, anddilute to volume with methanol. The resulting solution con-tains 800 �g of �,�,�-trifluorotoluene in each milliliter.
�,�,�-Trifluorotoluene Standard Solutions Prepare threeworking standard solutions with concentrations of 80, 400, and800 �g/mL by quantitatively diluting �,�,�-TrifluorotolueneStock Solution with methanol. These are the surrogate stan-dards.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph connected to a purge andtrap apparatus (see below). The gas chromatograph is equippedwith a photoionization detector and a 30-m × 0.53-mm (id)fused silica open tubular column, or equivalent, with a 1.5-�mcrossbonded 5% diphenyl, 95% dimethyl polysiloxane (RestekRTX-5, or equivalent) stationary phase. Use ultra-high-purityhelium as the carrier gas at a flow rate of 20 mL/min. Programthe column temperature according to the following steps: Holdit at 40° for 2 min, then increase it to 180° at a rate of 20°/min,and hold it at 180° for 2 min.
Purge andTrap Apparatus Thepurging apparatususes dis-posable 15- × 150-mm test tubes. Use ultra-high-purity heliumto purge the sample for 15 min at a flow rate of 60 mL/min.Maintain at 100° all lines that the sample vapor passes throughin the purge module.
The trap consists of a 30.5-cm × 2.7-mm (id) stainless steeltube, with apacking of a porous polymerbased on 2,6-diphenyl-p-phenylene oxide (Tenax, or equivalent). The length of thepacking in the tube is 24 cm. The entire void volume of the trapis at the vented end of the trap column. Maintain the trap at 100°.Recondition the trap for a subsequent run by baking it for 5 minat 190°.
128 / beta-Cyclodextrin / Monographs FCC V
ProcedureCalibration Place an empty purge tube into the purging
apparatus. Fill a 5-mL syringe with 0.5 N sodium hydroxide,and inject into this solution an accurately measured 5-�Laliquot of 10 �g/mL Toluene Standard Solution. Introducethis solution into the purge tube. Start the instrument for therun, automated if desired, by purging for 15 min, then heatingthe trap at 180° for 3 min. Repeat this sequence for each of theToluene Standard Solutions and the �,�,�-TrifluorotolueneStandard Solutions.
Plot standard curves of the standard concentration (CS), inmicrograms per milliliter, versus detector response (rS) forthe Toluene Standard Solutions and �,�,�-TrifluorotolueneStandard Solutions.
Determination To prepare the Sample Preparation, trans-fer 500 mg of sample, accurately weighed, and about 0.1 gof salicylic acid into a purge tube, and attach the purge tubeto the purging apparatus. Add 5 �L of 400-�g/mL �,�,�-Trifluorotoluene Standard Solution to a 5-mL syringe filledwith 0.5 N sodium hydroxide. Add the contents of the syringeto the sample in the purge apparatus, and start the run asdescribed under Calibration. The procedure is valid only whenthe detector response of the surrogate standard in the SamplePreparation is within � 15% of the value from the standardcurve for �,�,�-trifluorotoluene. Calculate the concentration,in micrograms per gram (numerically equivalent to milligramsper kilogram), of toluene in the sample taken by the formula
5CS/WS,
in which CS is the concentration, in micrograms per milliliter,of toluene from the toluene standard curve based on the detec-tor response for toluene obtained from the Sample Prepara-tion, and WS is the weight, in grams, of sample taken forthe assay.Trichloroethylene
Purge and Trap Apparatus The apparatus1 comprisesthree sections: the sample purge, the trap, and the desorber.The sample purge accepts 5-mL samples with a water columnnot less than 3 cm deep, and the gaseous headspace betweenthe water column and the trap has a total volume of not morethan 15 mL. The purge gas is passed through the water columnas finely divided bubbles with a diameter of less than 3 mmat the origin and is introduced not more than 5 mm from thebase of the water column.
Use a trap not shorter than or narrower than 25 cm × 2.67mm (id). Pack the trap to contain the indicated minimumlengths of adsorbents in the following order, beginning at thetrap inlet: 7.7 cm of 2,6-diphenylene oxide polymer (TENAXGC, or equivalent), 7.7 cm of silica gel, and 7.7 cm of coconutcharcoal.
The desorber is capable of rapidly heating the trap to 250°,which is the maximum temperature to be used.
Condition the assembled trap before use at 225° overnightwith an inert gas at a flow rate of not less than 20 mL/min.Before daily use, condition the trap for 15 min at 225°.
1The apparatus used is based on that described in the U.S. Environmen-tal Protection Agency Test Method for Purgeable Halocarbons—Method 601.
Standard Solution Transfer 50 mg of reagent-grade tri-chloroethylene, accurately weighed, to a 50-mL volumetricflask. Dilute with methanol to volume, and mix.
Diluted Standard Solution Accurately transfer 0.5, 1.0,2.0, 3.0, and 5.0 mL of the Standard Solution into five 50-mL volumetric flasks, and dilute to volume with water. TheseDiluted Standard Solutions correspond to trichloroethyleneconcentrations of 10.2, 20.4, 40.8, 61.2, and 102 ng/�L.
Chromatographic System (See Chromatography, Appen-dix IIA.) Connect the purge and trap apparatus to the gaschromatograph with a flame-ionization detector. Use a gaschromatograph equipped with a 30-m × 0.32-mm (id) capillarycolumn coated with a 1-�m film thickness of dimethylpolysi-loxane oil (such as DB-1, OV-1, or equivalent). Hold thecolumn temperature initially at 40° for 3 min, then programit to rise to 220° at 4°/min. Set the detector temperature to280°. Use helium as the carrier gas, and nitrogen as the purgegas, at a flow rate of 40 mL/min.
Procedure Introduce exactly 20 �L of each Diluted Stan-dard Solution on the inner wall of the sample purge. Desorbaccording to equipment instructions, and record the peak areas.Prepare a calibration graph by plotting the peak area responsesversus the weight of trichloroethylene introduced into thepurge.
Introduce about 250 mg of sample (W) accurately weighed,on the fritted sparger of the sample purge. Purge and desorbaccording to equipment instructions. Record the peak area oftrichloroethylene, and read the corresponding weight (X) oftrichloroethylene from the calibration curve. Calculate theamount of trichloroethylene by the formula
trichloroethylene (mg/kg) = X (ng)/W (mg).
Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers in a dryplace.
FCC V Monographs / gamma-Cyclodextrin / 129
gamma-Cyclodextrin�-Cyclodextrin; gamma-CD; Cyclooctaamylose;Cyclomaltooctaose
O
O
O
O
O
O
O
O
CH2 OH
CH
2 OH
CH2OH
CH 2OH
HO
H2 C HOH
2 CHOH2C
HOH 2C
OHOH
OH
OH
OH
OH
OH OH
OH
OH
OHOH
OHOH
OHOH
O
O
O
O
O
O
O
O
(C6H10O5)8 Formula wt 1297.14
CAS: [17465-86-0]
DESCRIPTION
Gamma-Cyclodextrin occurs as a white or almost white crys-talline solid. It is a nonreducing cyclic saccharide consistingof eight �-1,4-linked D-glucopyranosyl units manufacturedby the action of cyclomaltodextrin glucanotransferase on hy-drolyzed starch followed by purification of the gamma-Cyclo-dextrin. It is freely soluble in water and is very slightly solublein ethanol.
Function Stabilizer; emulsifier; carrier.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits maxima at the same wavelengths as those of atypical spectrum as shown in the section on Infrared Spectra,using the same test conditions as specified therein.Assay Not less than 98.0% as (C6H10O5)8, calculated on theanhydrous basis.Iodine Reaction Passes test.Lead Not more than 1 mg/kg.Optical (Specific) Rotation [�]D
25°: Between +174° and+180° in a 1% solution.Reducing Sugars (as glucose) Not more than 0.5%.Residue on Ignition Not more than 0.1%.Volatile Organic Compounds Not more than 20 mg/kg.Water Not more than 11.0%.
TESTS
AssaySample Solution Transfer 1.0 g of sample, accurately
weighed, into a 100-mL flask, dilute to volume with water,and mix.
Chromatographic System Determine as directed underChromatography, Appendix IIA, but use a liquid chromato-graph equipped with a differential refractometer detector anda 30-cm × 7.8-mm (id) column packed with 25-�m diameterbeads of silver bonded to sulfonated divinyl benzene–styrenecopolymer (Aminex HPX-42A, Bio-Rad Laboratories, orequivalent). Maintain the column at a constant temperatureof 65° � 10°, and the flow rate at 0.3 to 1.0 mL/min. Usedeionized water as the mobile phase.
Procedure Inject about 20 �L of the Sample Solution intothe chromatograph, record the chromatogram, and measurethe responses for all peaks.
Calculation Calculate the content of gamma-Cyclodextrinin the sample by the peak area percentage method using thefollowing equation:
A = (B/C) × 100,
in which A is the percentage of gamma-Cyclodextrin in thesample, B is the peak area of gamma-Cyclodextrin in thechromatogram, and C is the sum of the areas of all peaksrecorded in the chromatogram.Iodine Reaction Place 0.2 g of sample in a test tube, andadd 2 mL of a 0.1 N iodine solution. Heat the mixture in awater bath, and allow to cool at room temperature. A clear,brown solution forms.Lead Reflux about 5 g of sample, accurately weighed, with30 mL of nitric acid for 1 h. Remove the reflux condenser,and attach a condenser to the flask. Continue to heat, andcollect the distilled nitric acid. Allow the residue to cool, add20 mL of water, and allow it to cool again. Add 2 mL oforthophosphoric acid, dilute to 100 mL with water, and deter-mine the lead content of the solution as directed for MethodI in Atomic Absorption Spectrophotometric Graphite FurnaceMethod under Lead Limit Test, Appendix IIIB.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB.Reducing Sugars (as glucose) Transfer about 1 g of sample,accurately weighed, into a 250-mL Erlenmeyer flask, dissolvein 10 mL of water, add 25 mL of alkaline cupric citrate TS,and cover the flask with a small beaker. Boil gently for exactly5 min, and cool rapidly to room temperature. Add 25 mL of10% acetic acid solution, 10.0 mL of 0.1 N iodine, 10 mL ofdilute hydrochloric acid TS, and 3 mL of starch TS, and titratewith 0.1 N sodium thiosulfate to the disappearance of the bluecolor. Calculate the content of reducing substances (as D-glucose) (R) by the equation
%R = [(V1N1 − V2N2) × 2.7]/W,
in which V1 and N1 are the volume, in milliliters, and thenormality, respectively, of the iodine solution; V2 and N2 arethe volume, in milliliters, and the normality, respectively, ofthe sodium thiosulfate solution; 2.7 is an empirically deter-mined equivalence factor for D-glucose; and W is the weight,in grams, of the sample.Residue on Ignition Determine as directed in Method I (forSolids) under Residue on Ignition (Sulfated Ash), Appendix IIC.Volatile Organic Compounds Dissolve 50 g of sample inabout 700 mL of water in a 1-L round-bottom flask, and add amagnetic stirrer. Attach the flask to the lower part of a Bleidner
View IR
130 / L-Cysteine Monohydrochloride / Monographs FCC V
apparatus (see Figure 1), and connect a 100-mL round-bottomflask containing about 70 mL of hexane and a few boiling stonesto the other side of the apparatus. Fill the Bleidner apparatuswith equal amounts of water and hexane, and place a refluxcondenser on the top. Heat both flasks with heating mantels toboiling. Using the magnetic stirrer, stir the contents of the 1-Lflask well. Keep the content of the two flasks boiling for 8 h.After cooling, remove the 100-mL flask, transfer the contentsto a 100-mL volumetric flask, and fill that flask to volume withhexane.
Analyze the hexane solution as directed under Gas Chroma-tography, Appendix IIA, with the following conditions: Usea gas chromatograph equipped with a flame-ionization detec-tor and a 30-m × 0.32-mm (id) column with a stationaryphase consisting of 0.25-�m, cross-bonded, 95% dimethyl5% diphenyl polysiloxane (JBW Scientific DB-5.625, orequivalent). Set the injector to 280°, and hold the temperatureat 70° for 4 min, and then increase it to 250° at intervals of10° per min. Use nitrogen gas as a carrier with a flow rateof 70 mL per minute. The detection is FID at 280°.
Calculate the area(s) under the peak for each volatile organiccompound, and convert it to milligrams per kilograms ofgamma-Cyclodextrin using the response factor for 8-cyclo-hexadecen-1-one. The response factor is determined from acalibration curve using 8-cyclohexadecen-1-one concentra-tions of 0.1 to 6 mg/100 mL of hexane.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers in a dryplace.
L-Cysteine MonohydrochlorideL-2-Amino-3-mercaptopropanoic Acid Monohydrochloride
C3H7NO2S·HCl Formula wt, anhydrous 157.62C3H7NO2S·HCl·H2O Formula wt, monohydrate 175.63
CAS: anhydrous [52-89-1]INS: 920 CAS: monohydrate [7048-04-6]
DESCRIPTION
L-Cysteine Monohydrochloride occurs as a white, crystallinepowder. It is freely soluble in water and in alcohol. Theanhydrous form melts with decomposition at about 175°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits maxima only at the same wavelengths as those
of a typical spectrum as shown in the section on InfraredSpectra, using the same conditions as specified therein.Assay Not less than 98.0% and not more than 101.5% ofC3H7NO2S·HCl, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not less than 8.0% and not more than 12.0%.Optical (Specific) Rotation [�]D
20°: Between +5.0° and+8.0°; or [�]D
25°: Between +4.9° and +7.9°, calculated on thedried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Transfer about 300 mg of sample, previously driedas directed under Loss on Drying (below) and accuratelyweighed, into a 250-mL glass-stoppered flask. Add 20 mLof water, 4 g of potassium iodide, 5 mL of 2.7 N hydrochloricacid, and 25.0 mL of 0.1 N iodine. Stopper the flask, allowthe mixture to stand for 30 min in a dark place, and titratethe excess iodine with 0.1 N sodium thiosulfate. Perform ablank determination (see General Provisions), and make anynecessary correction. Each milliliter of 0.1 N iodine is equiva-lent to 15.76 mg of C3H7NO2S·HCl.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at room temperature for24 h in a vacuum desiccator using a suitable desiccant andmaintaining a pressure of not more than 5 mm Hg.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 8 g of undried sample in sufficient 1 N hydrochloricacid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
L-Cystine3,3’-Dithiobis(2-aminopropanoic acid)
HOOCCH(NH2)CH2SSCH2CH(NH2)COOH
C6H12N2O4S2 Formula wt 240.30
INS: 921 CAS: [56-89-3]
DESCRIPTION
L-Cystine occurs as colorless to white crystals. It is solublein diluted mineral acids and in alkaline solutions. It is veryslightly soluble in water and in alcohol.
Function Nutrient.
View IR
View IR
FCC V Monographs / Decanoic Acid / 131
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H12N2O4S2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.2%.Optical (Specific) Rotation [�]D
20°: Between −215° and−225°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Determine as directed under Nitrogen Determination,Appendix IIIC, using a 200-mg sample. Percent L-Cystineequals percent N × 8.58.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 2 g of a previously dried sample in sufficient 1 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in well-closed containers.
Dammar Gum
Dammar Resin; Damar Gum; Damar Resin; Dammar
CAS: [9000-16-2]
DESCRIPTION
Crude Dammar Gum occurs as irregular, white to yellow tobrown tears, fragments, or powder, sometimes admixed withfragments of bark. Refined grades are white to yellow andare free of fragments of ligneous matter. Dammar Gum is thedried exudate from trees of the Agathis, Hopea, or Shoreagenera. It consists of a complex mixture of acidic and neutralterpenoid compounds together with polysaccharide material.It is insoluble in water and in ethanol and is soluble in tolueneand in limonene. A chloroform solution of Dammar Gum isdextrorotatory.
Function Stabilizer; glazing agent.
REQUIREMENTS
Identification Prepare a 10% solution of sample in chloro-form, and spot 20 �L of it on a thin-layer plate coated with
a 0.2-mm layer of silica (Merck F254, or equivalent) in apreviously equilibrated chamber. Elute with a 30:25 mixtureof diethyl ether:heptane. In a suitable fume hood, spray theplate with sulfuric acid, and dry it at 180° for 3 min. Twodark spots are observed at Rf with values of 0.8 and 0.7, andwith the ratio of the faster-moving spot to the second spotbeing about 1.1.Acid Number Between 20 and 40.Ash (Total) Not more than 0.5%.Iodine Value Between 10 and 40.Lead Not more than 5 mg/kg.Loss on Drying Not more than 6.0%.Melting Range Between 90° and 95°.Softening Point Between 86° and 90°.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX, but modified as follows: Add 30 mL of tolueneand 30 mL of neutral ethanol to an accurately weighed 5-gsample. Titrate with 0.5 N alcoholic potassium hydroxide,using phenolphthalein TS as the indicator.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 18 h.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Softening Point Determine as directed for the Ring-and-Ball Method under Softening Point, Appendix IX.
Packaging and Storage Store in well-closed containers.
Decanoic AcidCapric Acid
CH3(CH2)8COOH
C10H20O2 Formula wt 172.27
CAS: [334-48-5]
FEMA: 2364
DESCRIPTION
Decanoic Acid occurs as white crystals having a sour, fatty,rancid odor. It is soluble in most organic solvents and practi-cally insoluble in water.
View IR
132 / Dehydroacetic Acid / Monographs FCC V
Function Component in the manufacture of other food-grade additives; defoaming agent; flavoring agent.
REQUIREMENTS
Acid Value Between 320 and 329.Iodine Value Not more than 0.6.Residue on Ignition Not more than 0.1%.Titer (Solidification Point) Between 27° and 32°.Unsaponifiable Matter Not more than 0.2%.Water Not more than 0.2%.
TESTS
Acid Value Determine as directed in Method I under AcidValue, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 10-g sample.Titer (Solidification Point) Determine as directed under So-lidification Point, Appendix IIB.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Dehydroacetic Acid3-Acetyl-6-methyl-1,2-pyran-2,4(3H)-dione;Methylacetopyronone
OH3C O
COCH3
O
C8H8O4 Formula wt 168.15
CAS: [520-45-6]
DESCRIPTION
Dehydroacetic Acid occurs as a white or nearly white, crystal-line powder. It is soluble in aqueous solutions of fixed alkalies,and is very slightly soluble in water. One gram of sampledissolves in about 35 mL of alcohol and in 5 mL of acetone.
Function Antimicrobial agent; preservative.
REQUIREMENTS
Identification The infrared absorption spectrum of a potas-sium bromide dispersion of the sample exhibits relative max-ima at the same wavelengths as those of a similar preparationof USP Dehydroacetic Acid Reference Standard.Assay Not less than 98.0% and not more than 100.5% ofC8H8O4, calculated on the dried basis.Lead No more than 0.5 mg/kg.Loss on Drying Not more than 1%.Melting Range Between 109° and 111°.Residue on Ignition Not more than 0.1%.
TESTS
Assay Transfer about 500 mg of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, dissolve it in 75mL of neutral alcohol, add phenolphthalein TS, and titratewith 0.1 N sodium hydroxide to a pink endpoint that persistsfor at least 30 s. Each milliliter of 0.1 N sodium hydroxideis equivalent to 16.82 mg of C8H8O4.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 80° for 4 h.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in well-closed containers.
Desoxycholic AcidDeoxycholic Acid; 13�,12�-Dihydroxycholanic Acid
HO
CH
CH3
CH2CH2COOH
CH3
CH3
OH
C24H40O4 Formula wt 392.58
CAS: [83-44-3]
DESCRIPTION
Desoxycholic Acid occurs as a white, crystalline powder. Itis practically insoluble in water, slightly soluble in chloroform
FCC V Monographs / Dexpanthenol / 133
and in ether, soluble in acetone and in solutions of alkalihydroxides and carbonates, and freely soluble in alcohol.
Function Emulsifier.
REQUIREMENTS
Identification Add 2 drops of benzaldehyde and 3 drops of75% sulfuric acid to about 10 mg of sample, heat at 50° for5 min, and then add 10 mL of glacial acetic acid. A greencolor appears. (Cholic acid produces a brown color.)Assay Not less than 98.0% and not more than 102.0% ofC24H40O4, calculated on the dried basis.Lead Not more than 4 mg/kg.Loss on Drying Not more than 1%.Melting Range Between 172° and 175°.Residue on Ignition Not more than 0.2%.
TESTS
Assay Transfer about 500 mg of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, and add 20 mL ofwater and 40 mL of alcohol. Cover the flask with a watchglass, heat the mixture gently on a steam bath until the sampleis dissolved, and allow the mixture to cool to room tempera-ture. Add a few drops of phenolphthalein TS to the solution,and titrate with 0.1 N sodium hydroxide to a pink endpointthat persists for 15 s. Each milliliter of 0.1 N sodium hydroxideis equivalent to 39.26 mg of C24H40O4.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 140° under a vacuumof not more than 5 mm Hg for 4 h.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in tight containers.
DexpanthenolD(+)-Pantothenyl Alcohol; Panthenol
HO
HN OH
H3C CH3
HO H
O
C9H19NO4 Formula wt 205.25
CAS: [81-13-0]
DESCRIPTION
Dexpanthenol occurs as a clear, viscous, somewhat hygro-scopic liquid. It is the dextrorotatory isomer of the alcohol
analogue of pantothenic acid. Some crystallization may occuron standing. It is freely soluble in water, in alcohol, in metha-nol, and in propylene glycol. It is soluble in chloroform andin ether, and is slightly soluble in glycerin. Its solutions arealkaline to litmus.
Function Nutrient.
REQUIREMENTS
IdentificationA. Add 5 mL of 1 N sodium hydroxide and 1 drop of
cupric sulfate TS to 1 mL of a 10% aqueous solution, andshake vigorously. A deep-blue color develops.
B. Add 1 mL of 1 N hydrochloric acid to 1 mL of a 1%aqueous solution, and heat on a steam bath for about 30 min.Cool, add 100 mg of hydroxylamine hydrochloride, mix, andadd 5 mL of 1 N sodium hydroxide. Allow to stand for 5min, then adjust the pH to within a range of 2.5 to 3.0 with1 N hydrochloric acid, and add 1 drop of ferric chloride TS.A purple-red color develops.
C. The infrared absorption spectrum of a film of the sampleexhibits maxima only at the same wavelengths as those of asimilar preparation of USP Dexpanthenol Reference Standard.Assay Not less than 98.0% and not more than 102.0% ofC9H19NO4, calculated on the anhydrous basis.Aminopropanol Not more than 1%.Lead Not more than 5 mg/kg.Optical (Specific) Rotation [�]D
25°: Between +29.0° and+31.5°.Refractive Index Between 1.495 and 1.502 at 20°.Residue on Ignition Not more than 0.1%.Water Not more than 1%.
TESTS
Assay Transfer about 400 mg of sample, accuratelyweighed, into a 300-mL reflux flask fitted with a standard-taper glass joint, add 50.0 mL of 0.1 N perchloric acid inglacial acetic acid, and reflux for 5 h.
Caution: Handle perchloric acid in an appropriatefume hood.
Cool, covering the condenser with foil to prevent contamina-tion by moisture, and rinse the condenser with glacial aceticacid. Add 5 drops of crystal violet TS, and titrate with 0.1 Npotassium acid phthalate in glacial acetic acid to a blue-green endpoint. Perform a blank determination (see GeneralProvisions), and make any necessary correction. Each millili-ter of 0.1 N perchloric acid is equivalent to 20.53 mg ofC9H19NO4.Aminopropanol Transfer about 5 g of sample, accuratelyweighed, into a 50-mL flask, and dissolve in 10 mL of water.Add bromothymol blue TS, and titrate with 0.1 N sulfuricacid from a microburet to a yellow endpoint. Each milliliterof 0.1 N sulfuric acid is equivalent to 7.5 mg of aminopropanol.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.
134 / Dextrin / Monographs FCC V
Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 500 mg of sample, calculated on the anhydrousbasis, in each 10 mL of water.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers.
Dextrin
INS: 1400 CAS: [9004-53-9]
DESCRIPTION
Dextrin occurs as free-flowing white, yellow, or brown pow-ders and consist chiefly of polygonal, rounded, or oblongor truncated granules. Dextrin is partially hydrolyzed starchconverted by heat alone, or by heating in the presence ofsuitable food-grade acids and buffers, from any of severalgrain- or root-based unmodified native starches (e.g., corn,waxy maize, high-amylose maize, milo, waxy milo, potato,arrowroot, wheat, rice, tapioca, sago, etc.). Dextrin is partiallyto completely soluble in water.
Function Thickener; colloidal stabilizer; binder; surface-finishing agent.
REQUIREMENTS
Labeling Indicate the presence of sulfur dioxide if the resid-ual concentration is greater than 10 mg/kg.Identification Suspend about 1 g of sample in 20 mL ofwater, and add a few drops of iodine TS. A dark blue to red-brown color appears.Chloride Not more than 0.2%.Crude Fat Not more than 1.0%.Lead Not more than 1 mg/kg.Loss on Drying Not more than 13.0%.Protein Not more than 1.0%.Reducing Sugars Not more than 18.0% (expressed as D-glucose), calculated on the dried basis.Residue on Ignition Not more than 0.5%.Sulfur Dioxide Not more than 0.005%.
TESTS
Chloride Dissolve 1 g of sample in 25 mL of boilingwater, cool, dilute to 100 mL with water, and filter. Add
24 mL of water, 2 mL of nitric acid, and 1 mL of silvernitrate TS to 1 mL of the filtrate. Any turbidity produceddoes not exceed that shown in a control containing 20 �gof chloride ion.Crude Fat Determine as directed under Crude Fat, Appen-dix X.Lead Transfer 4.0 g of sample to an evaporating dish,add 4 mL of sulfuric acid solution (1:4), distributing itevenly throughout the sample, and evaporate most of thewater on a steam bath. Char and dehydrate the sample byheating on a hot plate, while at the same time, heatingwith an infrared lamp from above, and then heat in amuffle furnace at 500° until the residue is free from carbon.Remove the dish from the furnace, cool, and cautiouslywash down the inside of the dish with water. Add 1 mLof 1 N hydrochloric acid, evaporate to dryness on a steambath, then add 2 mL of 1 N hydrochloric acid, and heatbriefly, while stirring, on a steam bath. Quantitativelytransfer the solution into a separator with the aid of smallquantities of water, and neutralize with 1 N ammoniumhydroxide. This Sample Solution meets the requirements ofthe Lead Limit Test, Appendix IIIB, using 4 �g of lead(Pb) ion in the control.Loss on Drying Determine as directed under Loss onDrying, Appendix IIC, drying a 5-g sample in a vacuumoven, not exceeding 100 mm Hg, at 120° for 4 h.Protein Transfer about 10 g of sample, accurately weighed,into an 800-mL Kjeldahl flask, and add 10 g of anhydrouspotassium or sodium sulfate, 300 mg of copper selenite ormercuric oxide, and 60 mL of sulfuric acid. Gently heatthe mixture, keeping the flask inclined at about a 45° angle,and after frothing has ceased, boil briskly until the solutionhas remained clear for about 1 h. Cool, add 30 mL ofwater, mix, and cool again. Cautiously pour about 75 mL(or enough to make the mixture strongly alkaline) of sodiumhydroxide solution (2:5) down the inside of the flask sothat it forms a layer under the acid solution, and then adda few pieces of granular zinc. Immediately connect theflask to a distillation apparatus consisting of a Kjeldahlconnecting bulb and a condenser, the delivery tube of whichextends well beneath the surface of an accurately measuredexcess of 0.1 N sulfuric acid contained in a 50-mL flask.Gently rotate the contents of the Kjeldahl flask to mix,and distill until all ammonia has passed into the absorbingacid solution (about 250 mL of distillate). Add 0.25 mLof methyl red–methylene blue TS to the receiving flask,and titrate the excess acid with 0.1 N sodium hydroxide.Perform a blank determination, substituting pure sucrose ordextrose for the sample, and make any necessary correction(see General Provisions). Each mL of 0.1 N sulfuric acidconsumed is equivalent to 1.401 mg of nitrogen (N).Calculate the percent N in the sample, and then calculatethe percent protein by multiplying the percent N by 6.25,in the case of starches obtained from corn, or by 5.7, inthe case of starches obtained from wheat. Other factorsmay be applied as necessary for starches obtained fromother sources.
FCC V Monographs / Dextrose / 135
Reducing Sugars Transfer about 10 g of sample, accu-rately weighed, into a 200-mL collecting flask, dilute tovolume with water, shake for 30 min, and filter throughWhatman No. 1 filter paper, or equivalent, collecting thefiltrate in a clean, dry flask. Pipet 10 mL each of Fehling’sSolution A and Fehling’s Solution B (see Cupric TartrateTS, Alkaline, in the section on General Tests and Assays,Solutions and Indicators) into a 250-mL Erlenmeyer flask,add 20.0 mL of the sample filtrate and 10 mL of water,and mix. Add two small glass beads, cover the mouth ofthe flask with a small glass funnel or glass bulb, and heaton a hot plate adjusted to bring the solution to a boil in3 min. Continue boiling for exactly 2 min (total heatingtime, 5 min), and then quickly cool to room temperaturein an ice bath or in a cold running-water bath. Add 10mL each of 30% potassium iodide solution and 28% sulfuricacid, and titrate immediately with 0.1 N sodium thiosulfate.Near the endpoint, add 1 mL of starch TS, and continuetitrating carefully, while agitating the solution continuously,until the blue color is discharged. Record the volume, inmL, of 0.1 N sodium thiosulfate required as S. Conducttwo reagent blank determinations in the same manner,substituting water for the sample filtrate, and record theaverage volume, in mL, of the blanks as B. Obtain the TiterDifference, expressed as mL of 0.1 N sodium thiosulfate, bysubtracting S from B. Determine the weight, in mg, ofreducing sugars, expressed as D-glucose (dextrose), by refer-ence to the table below entitled Conversion of Titer Differ-ence to Reducing Sugars Content, and record this value asR. Calculate the percentage of reducing sugars, as D-glucose,on the dried basis, by the formula
(R × 200 × 100)/(W × 20 × 1000),
in which W is the weight, in g, of sample taken, correctedfor Loss on Drying.
Conversion of Titer Difference to Reducing Sugars Contenta
TiterDifference(mL) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Reducing Sugar (as Dextrose) (mg)
0.0 0.0 0.3 0.7 1.0 1.3 1.6 1.9 2.2 2.5 2.81.0 3.2 3.5 3.8 4.1 4.4 4.7 5.0 5.3 5.6 5.92.0 6.4 6.6 6.9 7.2 7.5 7.8 8.1 8.5 8.8 9.13.0 9.4 9.8 10.1 10.4 10.7 11.0 11.4 11.7 12.0 12.34.0 12.6 13.0 13.3 13.6 14.0 14.3 14.6 15.0 15.3 15.6
5.0 15.9 16.3 16.6 16.9 17.2 17.6 17.9 18.2 18.5 18.96.0 19.2 19.5 19.8 20.1 20.5 20.8 21.1 21.4 21.8 22.17.0 22.4 22.7 23.0 23.3 23.7 24.0 24.3 24.6 24.9 25.28.0 25.6 25.9 26.2 26.6 26.9 27.3 27.6 28.0 28.3 28.69.0 28.9 29.3 29.6 30.0 30.3 30.6 31.0 31.3 31.6 31.9
10.0 32.3 32.7 33.0 33.3 33.7 34.0 34.3 34.6 35.0 35.311.0 35.7 36.0 36.3 36.7 37.0 37.3 37.6 38.0 38.3 38.712.0 39.0 39.3 39.6 40.0 40.3 40.6 41.0 41.3 41.7 42.013.0 42.4 42.8 43.1 43.4 43.7 44.1 44.4 44.8 45.2 45.514.0 45.8 46.2 46.5 46.9 47.2 47.6 47.9 48.3 48.6 48.9
Conversion of Titer Difference to Reducing Sugars Contenta (Cont.)
TiterDifference(mL) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Reducing Sugar (as Dextrose) (mg)
15.0 49.3 49.6 49.9 50.3 50.7 51.1 51.4 51.7 52.1 52.416.0 52.8 53.2 53.5 53.9 54.2 54.5 54.9 55.3 55.6 56.017.0 56.3 56.7 57.0 57.3 57.7 58.1 58.4 58.8 59.1 59.518.0 59.8 60.1 60.5 60.9 61.2 61.5 61.9 62.3 62.6 63.019.0 63.3 63.6 64.0 64.3 64.7 65.0 65.4 65.8 66.1 66.5
20.0 66.9 67.2 67.6 68.0 68.4 68.8 69.1 69.5 69.9 70.321.0 70.7 71.1 71.5 71.9 72.2 72.6 73.0 73.4 73.7 74.122.0 74.5 74.9 75.3 75.7 76.1 76.5 76.9 77.3 77.7 78.123.0 78.5 78.9 79.3 79.7 80.1 80.5 80.9 81.3 81.7 82.124.0 82.6 83.0 83.4 83.8 84.2 84.6 85.0 85.4 85.8 86.2
25.0 86.6 87.0 87.4 87.8 88.2 88.6 89.0 89.4 89.8 90.226.0 90.7 91.1 91.5 91.9 92.3 92.7 93.1 93.5 93.9 94.327.0 94.8
aUse of this table presumes the ability of the analyst to duplicate exactlythe conditions under which the data were developed. The risk of error canbe avoided by careful duplicate standardization with known quantities ofpure dextrose (five samples, ranging from 10 to 70 mg). A plot of TiterDifference versus mg of dextrose is slightly curvilinear, passing throughthe origin. If use of a standardization curve is adopted, the thiosulfatesolution need not be standardized. Some additional increase in accuracyresults from use of a 0.065 N sodium thiosulfate solution, which increasesthe blank titer to about 44 to 45 mL.
Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 5-g sample.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X.
Packaging and Storage Store in well-closed containers.
DextroseD-Glucose; Glucose; Corn Sugar
HO
HO
OH
OH
OH·H2O
C6H12O6 Formula wt 180.16
CAS: [50-99-7]
DESCRIPTION
Dextrose occurs as white, crystalline granules or as a granularpowder. It is purified and crystallized D-glucose. It is anhy-drous or contains one molecule of water of crystallization. It
136 / Diacetyl Tartaric Acid Esters of Mono- and Diglycerides / Monographs FCC V
is freely soluble in water, very soluble in boiling water, andslightly soluble in alcohol.
Function Nutritive sweetener; humectant; texturizing agent.
REQUIREMENTS
Labeling Indicate the presence of sulfur dioxide if the resid-ual concentration is greater than 10 mg/kg.Identification Add a few drops of a 1:20 aqueous solutionto 5 mL of hot alkaline cupric tartrate TS. A copious redprecipitate of cuprous oxide forms.Assay Not less than 99.5% and not more than 100.5% ofreducing sugar content (dextrose equivalent), expressed as D-glucose, calculated on the dried basis.Arsenic Not more than 1 mg/kg.Chloride Not more than 0.018%.Lead Not more than 0.1 mg/kg.Loss on Drying Anhydrous: Not more than 2.0%; Monohy-drate: Not more than 10.0%.Optical (Specific) Rotation [�]D
25°: Between +52.6° and+53.2° after drying.Residue on Ignition Not more than 0.1%.Starch Passes test.Sulfur Dioxide Not more than 0.002%.
TESTS
Assay Determine as directed under Reducing Sugars Assay,Appendix X.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared using a 1-g sample, and 1 mL of Standard Arsenic Solution in thecontrol (1 �g As).Chloride A 2.0-g sample shows no more chloride than cor-responds to 0.50 mL of 0.020 N hydrochloric acid.Lead Determine as directed for Method I in Atomic Absorp-tion Spectrophotometric Graphite Furnace Method underLead Limit Test, Appendix IIIB, using a 5-g sample.Loss on Drying Using Loss on Drying, Appendix IIC, asa guide, dry 10 g of anhydrous sample or 5 g of monohydratesample for 2 h at 70° in a vacuum oven not exceeding 50mm Hg, cool in a desiccator for 30 min, and weigh. Dry forsuccessive 1-h intervals until the weight change is less than2 mg.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 10 g of a previously dried sample and 0.2 mL of6 N ammonium hydroxide in sufficient water to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 10-g sample.Starch Add 1 drop of iodine TS to 1 g of sample dissolvedin 10 mL of water. A yellow color indicates the absence ofsoluble starch.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X, using a 75-g sample.
Packaging and Storage Store in tight containers in a dryplace.
Diacetyl Tartaric Acid Esters of Mono- andDiglyceridesDATEM
CAS: [91052-83-4]INS: 472e CAS: [100085-39-0]
DESCRIPTION
Diacetyl Tartaric Acid Esters of Mono- and Diglycerides occurover a range in appearance from sticky, viscous liquidsthrough a fatlike consistency to a waxy solid, depending onthe iodine value of the oils or fats used in their manufacture.They are the reaction product of partial glycerides of edibleoils, fats, or fat-forming fatty acids with diacetyl tartaric anhy-dride. The diacetyl tartaroyl esters are miscible in all propor-tions with oils and fats. They are soluble in most commonfat solvents, in methanol, in acetone, and in ethyl acetate, butare insoluble in other alcohols, in acetic acid, and in water.They are dispersible in water and resistant to hydrolysis formoderate periods of time. The pH of a 3% dispersion in wateris between 2 and 3.
Function Emulsifier.
REQUIREMENTS
Identification Add, dropwise, lead acetate TS to a solutionof 500 mg of sample in 10 mL of methanol. A white, floccu-lent, practically insoluble precipitate forms.Assay for Tartaric Acid Between 17.0 and 20.0 g of tartaricacid (C4H6O6) per 100 g of sample after saponification.Acetic Acid Between 14.0 and 17.0 g of acetic acid(CH3COOH) per 100 g of sample after hydrolysis.Acid Value Between 62 and 76.Fatty Acids (Total) Not less than 56.0 g of total fatty acidsper 100 g of sample after hydrolysis.Glycerin Not less than 12.0 g of glycerin (C3H8O3) per 100g of sample after hydrolysis.Lead Not more than 2 mg/kg.Residue on Ignition Not more than 0.01%.Saponification Value Between 380 and 425.
TESTS
Assay for Tartaric AcidStandard Reference Curve Transfer 100 mg of reagent-
grade tartaric acid, accurately weighed, into a 100-mL volu-metric flask, dissolve it in about 90 mL of water, add waterto volume, and mix well. Transfer 3.0-, 4.0-, 5.0-, and 6.0-mL portions into separate 19- × 150-mm matched cuvettes,and add sufficient water to make 10.0 mL. Add 4.0 mL of afreshly prepared 1:20 sodium metavanadate solution and 1.0mL of glacial acetic acid to each cuvette.
Note: Use these solutions within 10 min after colordevelopment.
Prepare a blank in the same manner, using 10 mL of waterin place of the tartaric acid solutions. Set a suitable spectropho-
FCC V Monographs / Diatomaceous Earth / 137
tometer or a photoelectric colorimeter equipped with a 520-nm filter at zero with the blank, and then determine the ab-sorbance of the four solutions of tartaric acid at 520 nm. Fromthe data thus obtained, prepare a reference curve by plottingthe absorbances on the ordinate against the correspondingquantities, in milligrams, of tartaric acid on the abscissa.
Assay Preparation Transfer about 4 g of sample, accu-rately weighed, into a 250-mL Erlenmeyer flask, and add 80mL of 0.5 N potassium hydroxide and 0.5 mL of phenolphtha-lein TS. Connect an air condenser at least 65 cm long to theflask, and heat the mixture on a hot plate for about 2.5 h.Remove the air condenser and add approximately 10% phos-phoric acid to the hot mixture until it is definitely acid tocongo red test paper. Reconnect the air condenser, and heatuntil the fatty acids are liquified and clear. Cool, and transferthe mixture into a 250-mL separator with the aid of smallportions of water and hexane. Extract the liberated fatty acidswith three successive 25-mL portions of hexane, and collectthe extracts in a second separator. Wash the combined hexaneextracts with two 25-mL portions of water, and add the wash-ings to the separator containing the water layer. Retain thecombined hexane extracts for the determination of total fattyacids. Transfer the contents of the first separator to a 250-mL beaker, heat on a steam bath to remove traces of hexane,filter through acid-washed, fine-texture filter paper into a 500-mL volumetric flask, and finally dilute to volume with water(Solution I). Pipet 25.0 mL of this solution into a 100-mLvolumetric flask, and dilute to volume with water (SolutionII). Retain the rest of Solution I for the determination ofGlycerin (below).
Procedure Transfer 10.0 mL of Solution II into a 19- ×150-mm cuvette, and continue as directed under StandardReference Curve, beginning with ‘‘add 4.0 mL of a freshlyprepared 1:20 sodium metavanadate solution. . . .’’ From thereference curve, determine the weight, in milligrams, of tar-taric acid in the final dilution, multiply this by 20, and dividethe result by the weight of the original sample to obtain thepercentage of tartaric acid.Acetic Acid Determine as directed under Volatile Acidity,Appendix VII, using a 4-g sample, accurately weighed, and30.03 as the equivalence factor (e).Acid Value Transfer about 1 g of sample, accuratelyweighed, into a 125-mL Erlenmeyer flask. Prepare a solventby mixing 1 volume of hexane with 4 volumes of methanol,adding phenol red TS, and neutralizing, if necessary. Dissolvethe sample in about 25 mL of this solvent by gently warming,if necessary. Titrate the solution with 0.1 N methanolic potas-sium hydroxide to a light red endpoint. Perform a blank deter-mination (see General Provisions) using a 25-mL portion ofthe solvent, and make any necessary correction. Calculate theacid value by the formula
56.1V × N/W,
in which V is the volume, in milliliters, of the methanolicpotassium hydroxide and N is the normality; and W is theweight, in grams, of the sample taken.Fatty Acids (Total) Dry the combined hexane extracts offatty acids obtained in the Assay for Tartaric Acid by shakingwith a few grams of anhydrous sodium sulfate. Filter the
solution into a tared, 250-mL beaker, evaporate the hexaneon a steam bath, cool, and weigh.Glycerin Prepare periodic acid solution by dissolving 2.7g of periodic acid (H5IO6) in 50 mL of water, adding 950mL of glacial acetic acid, and mixing thoroughly (protect thissolution from light). Transfer 5.0 mL of Solution I, preparedin the Assay for Tartaric Acid (above), into a 250-mL glass-stoppered Erlenmeyer or iodine flask. Add 15 mL of glacialacetic acid and 25.0 mL of periodic acid solution to the flask,shake the mixture for 1 or 2 min, allow it to stand for 15min, add 15 mL of a 15:100 potassium iodide solution and15 mL of water, swirl, let it stand for 1 min, and then titratethe liberated iodine with 0.1 N sodium thiosulfate, using starchTS as the indicator. Perform a Residual Blank Titration (seeGeneral Provisions) using water in place of sample, and makeany necessary correction. The corrected volume is the numberof milliliters of 0.1 N sodium thiosulfate required for theglycerin and the tartaric acid in the sample represented bythe 5 mL of Solution I. From the percentage determined inthe Assay for Tartaric Acid, calculate the volume of 0.1 Nsodium thiosulfate required for the tartaric acid in the titration.The difference between the corrected volume and the calcu-lated volume required for the tartaric acid is the number ofmilliliters of 0.1 N sodium thiosulfate consumed because ofthe glycerin in the sample. One milliliter of 0.1 N sodiumthiosulfate is equivalent to 2.303 mg of glycerin and to 7.505mg of tartaric acid.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 10-g sample.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 10-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII, using about 2 g of sample,accurately weighed.
Note: Add 5 to 10 mL of water to samples and blanksbefore saponification; otherwise, sufficient salts precip-itate during saponification to cause serious bumpingand spattering.
Packaging and Storage Store in well-closed containers.
Diatomaceous Earth
Diatomaceous Silica; Diatomite; D.E.
CAS: natural powder and calcined powder [61790-53-2]
CAS: flux-calcined powder [68855-54-9]
DESCRIPTION
Diatomaceous Earth occurs as a powder of varying colorsconsisting of processed siliceous skeletons of diatoms. Thenatural powder (gray to off white) is air dried and classified
138 / Dilauryl Thiodipropionate / Monographs FCC V
by particle size; the calcined powder (pink to buff-colored)is air dried, classified, calcined at a high temperature (815°to 982°), and again classified; the flux-calcined powder (white)is air dried, classified, calcined in the presence of a suitableflux (generally soda ash or other alkaline salt), and againclassified; and the acid-washed powder is any of the precedingpowders having been further purified by washing in acid andrinsing with water. It is insoluble in water, in acids (excepthydrofluoric), and in dilute alkalis.
Function Filter aid in food processing.
REQUIREMENTS
Identification When the powder is examined with a 100-to 200-power microscope, typical diatom shapes are observed.Arsenic Not more than 10 mg/kg.Lead Not more than 10 mg/kg.Loss on Drying Natural and acid-washed powders: Notmore than 10.0%; Calcined and flux-calcined powders: Notmore than 3.0%.Loss on Ignition Natural Powders: Not more than 7.0%,calculated on the dried basis; Calcined and Flux-CalcinedPowders: Not more than 0.5%, calculated on the dried basis.Nonsiliceous Substances Not more than 25.0%, calculatedon the dried basis.pH of Filtrate Natural, Calcined, or Acid-Washed Powders:Between 5.0 and 10.0; Flux-Calcined Powders: Between 8.0and 11.0.
TESTS
Arsenic Transfer 10.0 g of sample into a 250-mL beaker,add 50 mL of 0.5 N hydrochloric acid, cover with a watchglass, and heat at 70° for 15 min. Cool, and decant througha Whatman No. 3 filter paper, or equivalent, into a 100-mLvolumetric flask. Wash the slurry with three 10-mL portionsof hot water and wash the filter paper with 15 mL of hotwater, dilute to volume with water, and mix. A 3.0-mL portionof this solution meets the requirements of the Arsenic LimitTest, Appendix IIIB.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a 10.0-mL portion of the solution preparedunder Arsenic Test, above, and 10 �g of lead (Pb) ion in thecontrol.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Loss on Ignition Accurately weigh about 1 g of sample,and ignite at 800° to constant weight in a suitable, taredcrucible.Nonsiliceous Substances Transfer about 200 mg of sample,accurately weighed, into a tared platinum crucible, add 5 mLof hydrofluoric acid and 2 drops of 1:2 sulfuric acid, andevaporate gently to dryness. Cool, add 5 mL of hydrofluoricacid, evaporate again to dryness, and then ignite to constantweight.pH of Filtrate Boil 10 g of sample with 100 mL of waterfor 30 min, make up to 100 mL with water, and filter through
a fine-porosity sintered-glass funnel. Determine the pH of thefiltrate as described under pH Determination, Appendix IIB.
Packaging and Storage Store in well-closed containers.
Dilauryl Thiodipropionate
(C12H25OOCCH2CH2)2S
C30H58O4S Formula wt 514.85
INS: 389 CAS: [123-28-4]
DESCRIPTION
Dilauryl Thiodipropionate occurs as white, crystalline flakes.It is soluble in most organic solvents, but insoluble in water.
Function Antioxidant.
REQUIREMENTS
Identification Dilauryl Thiodipropionate may be identifiedby its solidification point, as determined under SolidificationPoint (below).Assay Not less than 99.0% and not more than 100.5% ofC30H58O4S.Acidity (as thiodipropionic acid) Not more than 0.2% of3,3′-thiodipropionic acid.Lead Not more than 10 mg/kg.Solidification Point Not below 40°.
TESTS
Assay Transfer about 700 mg of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, and add 100 mLof glacial acetic acid and 50 mL of alcohol. Heat the mixtureat a temperature of about 40° until the sample is completelydissolved, then add 3 mL of hydrochloric acid and 4 dropsof p-ethoxychrysoidin TS, and immediately titrate the solutionwith 0.1 N bromine. When the endpoint is approached (pinkcolor), add 4 more drops of the indicator solution and continuethe titration, dropwise, to a color change from red to paleyellow. Perform a blank determination (see General Provi-sions), and make any necessary correction. Each milliliter of0.1 N bromine is equivalent to 25.74 mg of C30H58O4S. Multi-ply the percentage of thiodipropionic acid, determined in theAcidity test (below), by 2.89, and subtract this value from thepercentage of Dilauryl Thiodipropionate calculated from thetitration. The difference is the percent purity of C30H58O4S.Acidity (as thiodipropionic acid) Transfer about 2 g of sam-ple, accurately weighed, into a 250-mL Erlenmeyer flask.Dissolve the sample in 50 mL of a mixture comprising 1part methyl alcohol and 3 parts benzene, add 5 drops ofphenolphthalein TS, and titrate with 0.1 N alcoholic potassium
FCC V Monographs / Dill Seed Oil, Indian Type / 139
hydroxide. Each milliliter of 0.1 N alcoholic potassium hy-droxide is equivalent to 8.91 mg of 3,3′-thiodipropionic acid.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Solidification Point Determine as directed under Solidifica-tion Point, Appendix IIB.
Packaging and Storage Store in well-closed containers.
Dill Seed Oil, European Type
DESCRIPTION
Dill Seed Oil, European Type, occurs as a pale yellow to lightyellow liquid with a caraway odor and flavor. It is the volatileoil obtained by steam distillation from the crushed, dried fruitor seeds of Anethum graveolens L. (Fam. Umbelliferae). Itis soluble in most fixed oils and in mineral oil. It is soluble,with slight opalescence, in propylene glycol, but it is practi-cally insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 42.0% and not more than 60.0%, byvolume, of ketones as carvone.Angular Rotation Between +70° and +82°.Refractive Index Between 1.483 and 1.490 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.890 and 0.915.
TESTS
Assay Determine as directed in the Neutral Sulfite Methodunder Aldehydes and Ketones, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 80% alcohol, with slight opalescence that maynot disappear on dilution to as much as 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Dill Seed Oil, Indian Type
Dill Seed Oil, Indian; Dill Oil, Indian Type
DESCRIPTION
Dill Seed Oil, Indian Type, occurs as a light yellow to lightbrown liquid with a rather harsh, caraway odor and flavor. Itis the volatile oil obtained by steam distillation from thecrushed mature fruit of Indian Dill, Anethum sowa D.C. (Fam.Umbelliferae). It is soluble in most fixed oils and in mineraloil, occasionally with slight opalescence. It is sparingly solublein propylene glycol and practically insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 20.0% and not more than 30.0%, byvolume, of ketones as carvone.Angular Rotation Between +40° and +58°.Refractive Index Between 1.486 and 1.495 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.925 and 0.980.
TESTS
Assay Determine as directed in the Neutral Sulfite Methodunder Aldehydes and Ketones, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 90% alcohol and remains clear on dilution.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
View IR
View IR
140 / Dillweed Oil, American Type / Monographs FCC V
Dillweed Oil, American TypeDill Oil; Dill Herb Oil, American Type
CAS: [8006-75-5]
DESCRIPTION
Dillweed Oil, American Type, occurs as a light yellow toyellow liquid. It is the volatile oil obtained by steam distillationfrom the freshly cut stalks, leaves, and seeds of the plantAnethum graveolens L. (Fam. Umbelliferae). It is soluble inmost fixed oils and in mineral oil. It is soluble, usually withopalescence or turbidity, in propylene glycol, but it is practi-cally insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 28.0%, except in specific cases (seeNote, below), and not more than 45.0%, by volume, of ketonesas carvone.
Note: Oil obtained from early season distillation mayshow a carvone content as low as 25.0% and a corres-pondingly lower specific gravity, lower refractive in-dex, and higher angular rotation.
Angular Rotation Between +84° and +95° (see Note,above).Refractive Index Between 1.480 and 1.485 at 20° (see Note,above).Solubility in Alcohol Passes test.Specific Gravity Between 0.884 and 0.900 (see Note,above).
TESTS
Assay Determine as directed in the Neutral Sulfite Methodunder Aldehydes and Ketones, Appendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 1 mL of 90% alcohol, frequently with opalescence thatmay not disappear on dilution to as much as 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Dimethyl DicarbonateDicarbonic Acid; Dimethyl Ester; Dimethyl Pyrocarbonate;DMDC
H3CO O OCH3
O O
C4H6O5 Formula wt 134.09
INS: 242 CAS: [4525-33-1]
DESCRIPTION
Dimethyl Dicarbonate occurs as a clear, colorless liquid. Itssolubility in water is 35 g/L at 20° with decomposition, itsmelting point is about 17°, and its flash point is 85°. It reactsquantitatively with water, producing carbon dioxide andmethanol.
Caution: Dimethyl Dicarbonate is toxic if inhaled.
Function Preservative; antimicrobial.
REQUIREMENTS
Identification The infrared absorption spectrum of a neatdispersion of the sample between two sodium chloride platesexhibits relative maxima at the same wavelengths as those ofa typical spectrum as shown in the section on Infrared Spectra,using the same test conditions as specified therein.Assay Not less than 99.8% and not more than 101.5% ofC4H6O5.Dimethyl Carbonate Not more than 0.2%.Lead Not more than 1 mg/kg.
TESTS
Assay1 N Di-n-butylamine Transfer 12.93 g of di-n-butylamine
into a 100-mL volumetric flask, dilute to volume with toluene,and mix.
Procedure Transfer about 2 g of sample, accuratelyweighed, into a 250-mL beaker, and dissolve it in 100 mLof acetone. Add 25 mL of 1 N Di-n-butylamine by pipet,allow the mixture to stand for 5 min, and titrate with 1 Nhydrochloric acid, determining the endpoint potentiomet-rically. Perform a blank titration (see General Provisions),and make any necessary correction. Calculate the percentDimethyl Dicarbonate in the sample taken by the formula
100(B − A)0.134/W,
in which B and A are the volumes, in milliliters, of hydrochlo-ric acid used for the blank and the sample, respectively; 0.134is the milliequivalent weight of Dimethyl Dicarbonate; andW is the weight, in milligrams, of sample taken.
View IR
View IR
FCC V Monographs / Dioctyl Sodium Sulfosuccinate / 141
Dimethyl Carbonate (Note: Conduct this procedure with-out delays.)
Internal Standard Solution Dissolve about 100 mg ofmethyl isobutylketone, accurately weighed, in 10 mL of meth-anol contained in a 100-mL volumetric flask, dilute to volumewith methanol, and mix.
Standard Solution Transfer about 20 mg of 99% dimethylcarbonate (Aldrich, or equivalent), accurately weighed, intoa 10-mL volumetric flask, dilute to volume with InternalStandard Solution, and mix.
Test Solution Transfer about 10 g of sample, accuratelyweighed, into a 10-mL volumetric flask, dilute to volumewith Internal Standard Solution, and mix.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flame-ionization detector and containing a 50-m × 0.3-mm (id) capil-lary column coated with SE 30-D, or equivalent, and maintainthe column, initially at 30° for a 5-min hold time, followedby a linear temperature gradient of 40° per minute to a finaltemperature of 120° held for 5 min. Use helium as the carriergas at a flow rate of 11 mL/min, and use hydrogen as thefuel gas at a flow rate of 35 mL/min. Chromatograph fivereplicate injections of the Standard Solution. The relativestandard deviation is not greater than 2.0%.
Procedure Using a metal-free syringe, separately injectsuitable portions (about 5 �L) of the Standard Solution andthe Test Solution into the gas chromatograph, and recordthe chromatograms. Measure the peak area ratio of dimethylcarbonate to that of the internal standard obtained with theStandard Solution. Similarly, measure the same peak arearatio for the Test Solution. The ratio is equal to or smallerthan that obtained with the Standard Solution.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.
Packaging and Storage Store in the original container ina cool (about 20°), dry, and well-ventilated area. Do notrepackage because it is particularly susceptible to contamina-tion by water.
DimethylpolysiloxaneDimethyl Silicone; Polydimethylsiloxane
CAS: [9006-65-9]
DESCRIPTION
Dimethylpolysiloxane occurs as a clear, colorless, viscousliquid. It is a mixture of fully methylated linear siloxanepolymers containing repeating units of the formula (CH3)2SiOthat are terminated with trimethylsiloxy end-blocking unitsof the formula (CH3)3SiO—. It is soluble in most aliphaticand aromatic hydrocarbon solvents, but it is insoluble in water.
Note: Dimethylpolysiloxane is frequently used in com-merce as such, or as a liquid containing silica (usually4% to 5%), which must be removed by high-speed
centrifugation (about 20,000 rpm) before testing theDimethylpolysiloxane for Identification, Refractive In-dex, Specific Gravity, and Viscosity. This monographdoes not apply to aqueous emulsions containing emulsi-fying agents and preservatives in addition to silica.
Function Defoaming agent.
REQUIREMENTSIdentification The infrared absorption spectrum of a neatdispersion of the sample between two sodium chloride platesexhibits relative maxima at the same wavelengths as those ofa similar preparation of USP Dimethylpolysiloxane ReferenceStandard.Lead Not more than 5 mg/kg.Loss on Heating Not more than 18.0%.Refractive Index Between 1.4000 and 1.4050.Specific Gravity Between 0.96 and 0.98.Viscosity Between 300 and 1500 centistokes.
TESTSLead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Heating Transfer 15 g of sample, accuratelyweighed, into an open, tared aluminum cup having an internalsurface area of about 30 cm2, weigh the cup and its contents,heat for 4 h at 200° in a circulating air oven, cool, andweigh again.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Specific Gravity Determine by any reliable method (seeGeneral Provisions).Viscosity Determine as directed under Viscosity of Dimeth-ylpolysiloxane, Appendix IIB.
Packaging and Storage Store in tightly closed containers.
Dioctyl Sodium SulfosuccinateDOSS; Docusate Sodium
H3C OO
H3CO
SO3Na
CH3
CH3
O
C20H37NaO7S Formula wt 444.56
INS: 480 CAS: [577-11-7]
DESCRIPTION
Dioctyl Sodium Sulfosuccinate occurs as a white, waxlike,plastic solid. One gram of sample dissolves slowly in about
142 / Dioctyl Sodium Sulfosuccinate / Monographs FCC V
70 mL of water. It is freely soluble in alcohol and in glycerin,and it is very soluble in solvent hexane.
Function Emulsifier; wetting agent.
REQUIREMENTS
Identification The infrared absorption spectrum of a filmof the sample between two salt plates exhibits relative maximaat the same wavelengths as those of a similar preparation ofUSP Docusate Sodium Reference Standard.Assay Not less than 98.5% of C20H37NaO7S, calculated onthe dried basis.Bis(2-ethylhexyl)maleate Not more than 0.4%.Clarity of Solution Passes test.Lead Not more than 2 mg/kg.Loss on Drying Not more than 2.0%.Residue on Ignition Between 15.5% and 16.2%.
TESTS
AssaySample Solution Transfer about 3.8 g of sample, accu-
rately weighed, into a 50-mL volumetric flask, dissolve inand dilute to volume with chloroform, and mix.
Tetra-n-butylammonium Iodide Solution Transfer 1.250g of tetra-n-butylammonium iodide into a 500-mL volumetricflask, dilute to volume with water, and mix.
Salt Solution Dissolve 100 g of anhydrous sodium sulfateand 10 g of sodium carbonate in sufficient water to make1000.0 mL.
Procedure Pipet 10.0 mL of the Sample Solution into a250-mL flask, and add 40 mL of chloroform, 50 mL of SaltSolution, and 10 drops of bromophenol blue TS. Titrate withTetra-n-butylammonium Iodide Solution to the first appear-ance of a blue color in the chloroform layer after vigorousshaking. Calculate the percent C20H37NaO7S by the formula
(V × 1.250 × 444.6 × 10)/(W × 369.4),
in which V is the volume, in milliliters, of Tetra-n-butylam-monium Iodide Solution required; 444.6 is the approximatemolecular weight of Dioctyl Sodium Sulfosuccinate; W is theweight, in grams, of the sample taken; and 369.4 is the molecu-lar weight of tetra-n-butylammonium iodide.Bis(2-ethylhexyl)maleate
Supporting Electrolyte Dissolve 21.2 g of anhydrous lith-ium perchlorate (LiClO4) in 175 mL of water contained in a250-mL beaker. Adjust the pH of this solution to 3.0 byadding, dropwise, glacial acetic acid (usually 1 or 2 drops issufficient), using a suitable pH meter. Quantitatively transferthe solution into a 200-mL volumetric flask, dilute to volumewith water, and mix.
Standard Solution Transfer 100 to 110 mg of USP Bis(2-ethylhexyl)maleate Reference Standard, accurately weighed,into a 100-mL volumetric flask. Record the exact weight tothe nearest 0.1 mg as WA. Add 60 to 70 mL of isopropylalcohol, swirl to dissolve, then dilute to volume with water,and mix.
Sample Stock Solution Transfer 12.5 g of sample, accu-rately weighed, into a 150-mL beaker. Record the exact weightto the nearest 0.1 mg as WS. Add 80 to 90 mL of isopropylalcohol, and stir with a glass stirring rod until the sample isdissolved. Quantitatively transfer this solution, with the aidof isopropyl alcohol, into a 250-mL volumetric flask, thendilute to volume with isopropyl alcohol, and mix.
Test Solution A Pipet 50.0 mL of Sample Stock Solutionand 20.0 mL of Supporting Electrolyte into a 100-mL volumet-ric flask. Dilute with isopropyl alcohol to within 15 mm ofthe graduated volume line, stopper, shake to facilitate solution,and set aside for 2 min. Dilute to volume with isopropylalcohol, and mix. A completely clear solution is obtained.
Test Solution B Pipet 50.0 mL of Sample Stock Solution,10.0 mL of Standard Solution, and 20.0 mL of SupportingElectrolyte into a 100-mL volumetric flask, and complete thepreparation as described for Test Solution A.
Blank Pipet 20.0 mL of the Supporting Electrolyte intoa 100-mL volumetric flask, dilute to volume with isopropylalcohol, and mix.
Procedure Rinse a polarographic H-cell several timeswith small portions of Test Solution A, then fill the cell half-full with the same solution, place a paper tissue in the top ofthe sample side of the cell, and pass a moderate stream ofsaturated nitrogen through the solution for 15 min.
Note: First saturate the nitrogen by passing it througha suitable scrubber containing isopropyl alcohol.
After 15 min, divert the nitrogen stream over the surface ofthe solution, and remove the tissue from the cell.
Set the polarizing voltage of a suitable, previously cali-brated polarograph (Metrohm Polarocord E-261, or equiva-lent) at –1.3 V. Adjust the current sensitivity to the lowestrange (most sensitive) at which the current oscillations willremain on scale. Record the polarogram, scanning a voltagerange of –0.9 V to –1.5 V at this sensitivity and using asaturated calomel electrode as the reference electrode. Recordthe average oscillations, in millimeters, at –1.3 V as A, andthose at –1.0 V as B.
Note: If a manual polarograph is used, record the aver-age oscillations of the solutions at –1.3 V and –1.0 V,respectively.
Repeat the entire procedure using Test Solution B, recordingthe average oscillations at –1.3 V as D, and those at –1.0 Vas E. Similarly, repeat the entire procedure using the Blank,recording the average oscillations at –1.3 V as G, and thoseat –1.0 V as H.
Calculation Make the following preliminary calculationsto obtain C (net diffusion current of Test Solution A); F (netdiffusion current of Test Solution B); I (net current introducedby the Blank); J (diffusion current due to added maleate); andK (diffusion current due to originally present maleate):
C = (A – B) × S1,
F = (D – E) × S2,
I = (G – H) × S3,
J = F – C,
K = C – I,
FCC V Monographs / Disodium EDTA / 143
in which S1, S2, and S3 represent the current sensitivitiesused for Test Solution A, Test Solution B, and the Blank,respectively.
Finally, calculate the percent bis(2-ethylhexyl)maleate inthe original sample taken by the formula
(K × 50WA)/(J × WS).
Clarity of Solution Dissolve 25 g of sample in 94 mL ofalcohol. The solution does not develop a haze within 24 h.Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers.
Disodium EDTADisodium Ethylenediaminetetraacetate; Disodium(Ethylenedinitrilo)tetraacetate; Disodium Edetate
H2C
COOH
NCH2CH2
N
CH2
HOOC
NaOOCH2C CH2COONa
2H2O
C10H14N2Na2O8·2H2O Formula wt 372.24
INS: 386 CAS: [6381-92-6]
DESCRIPTION
Disodium EDTA occurs as a white, crystalline powder. It issoluble in water.
Function Preservative; sequestrant; stabilizer.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution responds to the flame test for
Sodium, Appendix IIIA.B. Add 2 drops of ammonium thiocyanate TS and 2 drops
of ferric chloride TS to 5 mL of water in a test tube. Addabout 50 mg of sample to the deep red solution so obtained,and mix. The deep red color disappears.
C. The infrared absorption spectrum of a potassium bro-mide dispersion of sample exhibits relative maxima at thesame wavelengths as those of a similar preparation of USPEdetate Disodium Reference Standard.
Assay Not less than 99.0% and not more than 101.0% ofC10H14N2Na2O8·2H2O.Calcium Negative.Lead Not more than 10 mg/kg.Nitrilotriacetic Acid Not more than 0.1%.pH of a 1:100 Solution Between 4.3 and 4.7.
TESTS
AssayAssay Preparation Transfer about 5 g of sample, accu-
rately weighed, into a 250-mL volumetric flask, dissolve inwater, dilute to volume, and mix.
Procedure Place about 200 mg of chelometric standardcalcium carbonate, accurately weighed, in a 400-mL beaker,add 10 mL of water, and swirl to form a slurry. Cover thebeaker with a watch glass, and introduce 2 mL of 2.7 Nhydrochloric acid from a pipet inserted between the lip of thebeaker and the edge of the watch glass. Swirl the contents ofthe beaker to dissolve the calcium carbonate. Wash down thesides of the beaker, the outer surface of the pipet, and thewatch glass, and dilute to about 100 mL with water. Whilestirring, preferably with a magnetic stirrer, add about 30 mLof the Assay Preparation from a 50-mL buret, then add 15mL of 1 N sodium hydroxide and 300 mg of hydroxy naphtholblue indicator, and continue the titration to a blue endpoint.Calculate the weight, in mg, of C10H14N2Na2O8·2H2O in thesample taken by the formula
929.8(W/V),
in which W is the weight, in mg, of calcium carbonate, andV is the volume, in mL, of the Assay Preparation consumedin the titration.Calcium Add 2 drops of methyl red TS to a 1:20 aqueoussolution of the sample, and neutralize with 6 N ammoniumhydroxide. Add 3 N hydrochloric acid dropwise until thesolution is just acid, and then add 1 mL of ammonium oxalateTS. No precipitate forms.Lead A Sample Solution prepared as directed for organiccompounds meets the requirements of the Lead Limit Test,Appendix IIIB, using 10 �g of lead (Pb) ion in the control.Nitrilotriacetic Acid
Mobile Phase Add 10 mL of a 1:4 solution of tetrabuty-lammonium hydroxide in methanol to 200 mL of water, andadjust with 1 M phosphoric acid to a pH of 7.5 � 0.1. Transferthe solution to a 1000-mL volumetric flask, add 90 mL ofmethanol, dilute with water to volume, mix, filter through amembrane filter (0.5-�m or finer porosity), and de-gas.
Cupric Nitrate Solution Prepare an aqueous solution con-taining about 10 mg of cupric nitrate per milliliter.
Stock Standard Solution Transfer about 100 mg of nitrilo-triacetic acid, accurately weighed, to a 10-mL volumetricflask, add 0.5 mL of ammonium hydroxide, and mix. Diluteto volume, and mix.
Standard Preparation Transfer 1.0 g of sample to a 100-mL volumetric flask, add 100 �L of Stock Standard Solution,dilute to volume with Cupric Nitrate Solution, and mix. Soni-cate, if necessary, to achieve complete solution.
144 / Disodium Guanylate / Monographs FCC V
Test Preparation Transfer 1.0 g of sample to a 100-mLvolumetric flask, dilute with Cupric Nitrate Solution to vol-ume, and mix. Sonicate, if necessary, to achieve completesolution.
Chromatographic System Set up the system with refer-ence to High-Performance Liquid Chromatography underChromatography, Appendix IIA. The HPLC chromatographhas a 254-nm detector and a 4.6-mm × 15-cm column thatcontains 5- to 10-mm porous microparticles of silica bondedto octylsilane (Zorbax 8, or equivalent). The flow rate is about2 mL/min. Chromatograph three replicate injections of theStandard Preparation, and record the peak responses as di-rected under Procedure. The relative standard deviation isnot more than 2.0%, and the resolution factor between nitrilo-triacetic acid and Disodium EDTA is not less than 4.0.
Procedure Separately inject equal volumes (about 50 �L)of the Standard Preparation and the Test Preparation intothe chromatograph, record the chromatograms, and measurethe responses for the major peaks. The retention times areabout 3.5 min for nitrilotriacetic acid and 9 min for DisodiumEDTA. The response of the nitrilotriacetic acid peak of theTest Preparation does not exceed the difference between thenitrilotriacetic acid peak responses obtained from the StandardPreparation and the Test Preparation.pH of a 1:100 Solution Determine as directed under pHDetermination, Appendix IIB.
Packaging and Storage Store in well-closed containers.
Disodium GuanylateDisodium 5′-Guanylate; Disodium Guanosine-5′-monophosphate
N
NN
N
OH
H
OH
H
HO
H
H
Na2O3POCH2 O NH2
C10H12N5Na2O8P·xH2O Formula wt, anhydrous 407.19
INS: 627 CAS: [5550-12-9]
DESCRIPTION
Disodium Guanylate occurs as colorless or white crystals, oras a white, crystalline powder. It contains approximately seven
molecules of water of crystallization. It is soluble in water,sparingly soluble in alcohol, and practically insoluble in ether.
Function Flavor enhancer.
REQUIREMENTS
Identification The ultraviolet absorption spectrum of a1:50,000 solution in 0.01 N hydrochloric acid exhibits anabsorbance maximum at 256 � 2 nm.Assay Not less than 97.0% and not more than 102.0% ofC10H12N5Na2O8P, calculated on the dried basis.Amino Acids Passes test.Ammonium Salts Passes test.Clarity and Color of Solution Passes test.Lead Not more than 5 mg/kg.Loss on Drying Not more than 25.0%.Other Nucleotides Passes test.pH of a 1:20 Solution Between 7.0 and 8.5.
TESTS
Assay Transfer about 500 mg of sample, accuratelyweighed, into a 1000-mL volumetric flask, dissolve in anddilute to volume with 0.01 N hydrochloric acid, and mix.Transfer 10.0 mL of this solution into a 250-mL volumetricflask, dilute to volume with 0.01 N hydrochloric acid, andmix. Using a suitable spectrophotometer and 0.01 N hydro-chloric acid as the blank, determine the absorbance of thissolution and of a similarly prepared solution of USP DisodiumGuanylate Reference Standard, at a concentration of 20 �g/mL, in 1-cm cells, at the maximum at about 260 nm. Calculatethe quantity, in milligrams, of C10H12N5Na2O8P in the sampletaken by the formula
25C × AU/AS,
in which C is the exact concentration, in micrograms permilliliter, of the Reference Standard solution; AU is the ab-sorbance of the Sample Solution; and AS is the absorbance ofthe Reference Standard solution.Amino Acids Add 1 mL of ninhydrin TS to 5 mL of a1:1000 aqueous solution, and heat for 3 min. No color appears.Ammonium Salts Transfer about 100 mg of sample into asmall test tube, and add 50 mg of magnesium oxide and 1mL of water. Moisten a piece of red litmus paper with water,suspend it in the tube, cover the mouth of the tube, and heatin a water bath for 5 min. The litmus paper does not changeto blue.Clarity and Color of Solution A 100-mg portion of sampledissolved in 10 mL of water is colorless and shows no morethan a trace of turbidity.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 120° for 4 h.Other Nucleotides Prepare a strip of Whatman No. 2, orequivalent, filter paper about 20 × 40 cm, and draw a lineacross the narrow dimension about 5 cm from one end. Using
FCC V Monographs / Disodium Inosinate / 145
a micropipet, apply 10 �L of a 1:100 aqueous solution onthe center of the line, and dry the paper in air.
Fill the trough of an apparatus suitable for descendingchromatography (see Chromatography, Appendix IIA) witha 160:3:40 solution of saturated ammonium sulfate solu-tion:tert-butyl alcohol:0.025 N ammonia, and suspend the stripin the chamber, placing the end of the strip in the trough ata distance about 1 cm from the pencil line. Seal the chamber,and allow the chromatogram to develop until the solvent frontdescends to a distance about 30 cm from the starting line.Remove the strip from the chamber, dry in air, and observeunder shortwave (254 nm) ultraviolet light in the dark. Onlyone spot is visible.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 120° for 4 h.pH of a 1:20 Solution Determine as directed under pHDetermination, Appendix IIB.
Packaging and Storage Store in well-closed containers.
Disodium InosinateDisodium 5′-Inosinate; Disodium Inosine-5′-monophosphate
N
NN
N
OH
H
OH
H
HO
H
H
Na2O3POCH2 O
C10H11N4Na2O8P·xH2O Formula wt, anhydrous 392.17
INS: 631 CAS: [4691-65-0]
DESCRIPTION
Disodium Inosinate occurs as colorless or white crystals oras a white, crystalline powder. It contains approximately 7.5molecules of water of crystallization. It is soluble in water,sparingly soluble in alcohol, and practically insoluble in ether.
Function Flavor enhancer.
REQUIREMENTS
Identification The ultraviolet absorption spectrum of a1:50,000 solution in 0.01 N hydrochloric acid exhibits anabsorbance maximum at 250 � 2 nm. The ratio A250/A260 isbetween 1.55 and 1.65, and the ratio A280/A260 is between0.20 and 0.30.Assay Not less than 97.0% and not more than 102.0% ofC10H11N4Na2O8P, calculated on the anhydrous basis.
Amino Acids Passes test.Ammonium Salts Passes test.Clarity and Color of Solution Passes test.Lead Not more than 5 g/kg.Other Nucleotides Passes test.pH of a 1:20 Solution Between 7.0 and 8.5.Water Not more than 28.5%.
TESTS
Assay Transfer about 500 mg of sample, accuratelyweighed, into a 1000-mL volumetric flask, dissolve in anddilute to volume with 0.01 N hydrochloric acid, and mix.Transfer 10.0 mL of this solution into a 250-mL volumetricflask, dilute to volume with 0.01 N hydrochloric acid, andmix. Using a suitable spectrophotometer and 0.01 N hydro-chloric acid as the blank, determine the absorbance of thissolution and of a similarly prepared solution of USP DisodiumInosinate Reference Standard in 1-cm cells with the maximumat about 250 nm. Calculate the quantity, in milligrams, ofC10H11N4Na2O8P in the sample taken by the formula
25C × AU/AS,
in which C is the exact concentration, in micrograms permilliliter, of the Reference Standard solution; AU is the ab-sorbance of the Sample Solution; and AS is the absorbance ofthe Reference Standard solution.Amino Acids Add 1 mL of ninhydrin TS to 5 mL of a1:1000 aqueous solution. No color appears.Ammonium Salts Transfer about 100 mg of sample into asmall test tube, and add 50 mg of magnesium oxide and 1mL of water. Moisten a piece of red litmus paper with water,suspend it in the tube, cover the mouth of the tube, and heatin a water bath for 5 min. The litmus paper does not changeto blue.Clarity and Color of Solution A 500-mg portion of sampledissolved in 10 mL of water is colorless and shows no morethan a trace of turbidity.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Other Nucleotides Prepare a strip of Whatman No. 2, orequivalent, filter paper about 20 × 40 cm, and draw a lineacross the narrow dimension about 5 cm from one end. Usinga micropipet, apply 10 �L of a 1:100 aqueous solution onthe center of the line, and dry the paper in air.
Fill the trough of an apparatus suitable for descendingchromatography (see Chromatography, Appendix IIA) with a160:3:40 mixture of saturated ammonium sulfate solution:tert-butyl alcohol:0.025 N ammonia, and suspend the strip in thechamber, placing the end of the strip in the trough at a distanceabout 1 cm from the pencil line. Seal the chamber, and allowthe chromatogram to develop until the solvent front moves adistance about 30 cm from the starting line. Remove the stripfrom the chamber, dry in air, and observe under shortwave(254 nm) ultraviolet light in the dark. Only one spot is visible.pH of a 1:20 Solution Determine as directed under pHDetermination, Appendix IIB.
146 / Enzyme-Modified Fats / Monographs FCC V
Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Enzyme-Modified Fats
DESCRIPTION
Enzyme-Modified Fats occur as light to medium tan liquids,pastes, or powders with a strong fatty acid odor and flavor.They are produced by enzyme lipolysis of fats obtained frommilk, refined beef fat, or steam-rendered chicken fat, usingsuitable food-grade enzymes. Enzyme-modified milkfat maybe prepared from milk, concentrated milk, dry whole milk,cream, concentrated cream(s), dry cream, butter, butter oil,dried butter, or anhydrous milkfat. For enzyme-modified milk-fat, optional dairy ingredients such as skim milk, concentratedskim milk, nonfat dry milk, buttermilk, concentrated butter-milk, dried buttermilk, liquid whey, concentrated whey, anddried whey may be used to adjust the concentration of theflavors. Fat emulsions are reacted with suitable food-gradeenzymes under controlled conditions to increase the flavorcomponents. Thermoprocessing is then used to destroy theenzyme activity and provide acceptable microbiological qual-ity. Suitable preservatives, emulsifiers, buffers, stabilizers,and antioxidants as well as sodium chloride may be added.The resulting product is concentrated or dried.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate the Acid Value.Identification A sample has a very strong fatty acid odor.Acid Value Not less than 98.0% and not more than 102.0%of the labeled value.Lead Not more than 1 mg/kg.Loss on Drying Not more than 4.0% for the dry product.Microbial Limits:
Aerobic Plate Count Not more than 10,000 CFU pergram.
Coliforms Not more than 10 CFU per gram.Salmonella Negative in 25 g.Staphylococcal Enterotoxins Negative in 1 g.Staphylococcus aureus Not more than 100 CFU per
gram.Yeasts and Molds Not more than 10 CFU per gram.
TESTS
Acid Value Determine as directed in Method II under AcidValue, Appendix VII, using a 5-g sample.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 48 h.Microbial Limits (Note: Current methods for the followingtests may be found online at <www.cfsan.fda.gov/~ebam/bam-toc.html>):
Aerobic Plate CountColiformsSalmonellaStaphylococcal EnterotoxinsStaphylococcus aureusYeasts and Molds
Packaging and Storage Store in tight containers in acool place.
Enzyme Preparations
DESCRIPTION
Enzyme Preparations used in food processing are derivedfrom animal, plant, or microbial sources (see Classification,below). They may consist of whole cells, parts of cells, orcell-free extracts of the source used, and they may contain oneactive component or, more commonly, a mixture of several, aswell as food-grade diluents, preservatives, antioxidants, andother substances consistent with good manufacturing prac-tices.
The individual preparations usually are named accordingto the substance to which they are applied, such as Proteaseor Amylase. Traditional names such as Malt, Pepsin, andRennet also are used, however.
The color of the preparations—which may be liquid, semi-liquid, or dry—may vary from virtually colorless to darkbrown. The active components consist of the biologicallyactive proteins, which are sometimes conjugated with metals,carbohydrates, and/or lipids. Known molecular weights ofthe active components range from approximately 12,000 toseveral hundred thousand.
The activity of enzyme preparations is measured accordingto the reaction catalyzed by individual enzymes (see below)and is usually expressed in activity units per unit weight ofthe preparation. In commercial practice (but not for Food
FCC V Monographs / Enzyme Preparations / 147
Chemicals Codex purposes), the activity of the product issometimes also given as the quantity of the preparation to beadded to a given quantity of food to achieve the desired effect.
Additional information relating to the nomenclature andthe sources from which the active components are derived isprovided under Enzyme Assays, Appendix V.
Function Enzyme (see discussion under Classification,below).
CLASSIFICATION
Animal-Derived Preparations
Catalase, Bovine Liver Produced as partially purified liq-uid or powdered extracts from bovine liver. Major activeprinciple: catalase. Typical application: used in the manufac-ture of certain cheeses.
Chymotrypsin Obtained from purified extracts of bovine orporcine pancreatic tissue. Produced as white to tan, amorphouspowders soluble in water, but practically insoluble in alcohol,in chloroform, and in ether. Major active principle: chymotryp-sin. Typical application: used in the hydrolysis of protein.
Lipase, Animal Obtained from the edible forestomach tis-sue of calves, kids, or lambs; and from animal pancreatictissue. Produced as purified edible tissue preparations or asaqueous extracts dispersible in water, but insoluble in alcohol.Major active principle: lipase. Typical applications: used inthe manufacture of cheese and in the modification of lipids.
Lysozyme Obtained from extracts of purified chicken eggwhites. Generally prepared and used in the hydrochloride formas a white powder. Major active principle: lysozyme. Typicalapplication: used as an antimicrobial in food processing.
Pancreatin Obtained from porcine or bovine (ox) pancreatictissue. Produced as a white to tan, water-soluble powder.Major active principles: (1) �-amylase; (2) protease; and (3)lipase. Typical applications: used in the preparation of pre-cooked cereals, infant foods, and protein hydrolysates.
Pepsin Obtained from the glandular layer of hog stomach.Produced as a white to light tan, water-soluble powder; amberpaste; or clear, amber to brown, aqueous liquids. Major activeprinciple: pepsin. Typical applications: used in the preparationof fishmeal and other protein hydrolysates and in the clottingof milk in manufacture of cheese (in combination with rennet).
Phospholipase A2 Obtained from porcine pancreatic tissue.Produced as a white to tan powder or pale to dark yellowliquid. Major active principle: phospholipase A2. Typical ap-plication: used in the hydrolysis of lecithins.
Rennet, Bovine Aqueous extracts made from the fourthstomach of bovines. Produced as a clear, amber to dark brownliquid or a white to tan powder. Major active principle: prote-ase (pepsin). Typical application: used in the manufacture of
cheese. Similar preparations may be made from the fourthstomach of sheep or goats.
Rennet, Calf Aqueous extracts made from the fourth stom-ach of calves. Produced as a clear, amber to dark brown liquidor a white to tan powder. Major active principle: protease(chymosin). Typical application: used in the manufacture ofcheese. Similar preparations may be made from the fourthstomach of lambs or kids.
Trypsin Obtained from purified extracts of porcine or bo-vine pancreas. Produced as white to tan, amorphous powderssoluble in water, but practically insoluble in alcohol, in chloro-form, and in ether. Major active principle: trypsin. Typicalapplications: used in baking, in the tenderizing of meat, andin the production of protein hydrolysates.
Plant-Derived Preparations
Amylase Obtained from extraction of ungerminated barley.Produced as a clear, amber to dark brown liquid or a whiteto tan powder. Major active principle: �-amylase. Typicalapplications: used in the production of alcoholic beveragesand sugar syrups.
Bromelain The purified proteolytic substance derived fromthe pineapples Ananas comosus and Ananas bracteatus L.(Fam. Bromeliaceae). Produced as a white to light tan, amor-phous powder soluble in water (the solution is usually color-less to light yellow and somewhat opalescent), but practicallyinsoluble in alcohol, in chloroform, and in ether. Major activeprinciple: bromelain. Typical applications: used in the chill-proofing of beer, in the tenderizing of meat, in the preparationof precooked cereals, in the production of protein hydroly-sates, and in baking.
Ficin The purified proteolytic substance derived from thelatex of Ficus sp. (Fam. Moraceae), which include a varietyof tropical fig trees. Produced as a white to off white powdercompletely soluble in water. (Liquid fig latex concentratesare light to dark brown.) Major active principle: ficin. Typicalapplications: used in the chillproofing of beer, in the tenderiz-ing of meat, and in the conditioning of dough in baking.
Malt The product of the controlled germination of barley.Produced as a clear amber to dark brown liquid preparationsor as a white to tan powder. Major active principles: (1) �-amylase and (2) �-amylase. Typical applications: used inbaking; used in the manufacture of alcoholic beverages andof syrups.
Papain The purified proteolytic substance derived from thefruit of the papaya Carica papaya L. (Fam. Caricaceae). Pro-duced as a white to light tan, amorphous powder or a liquidsoluble in water (the solution is usually colorless or lightyellow and somewhat opalescent), but practically insolublein alcohol, in chloroform, and in ether. Major active principles:(1) papain and (2) chymopapain. Typical applications: usedin the chillproofing of beer, in the tenderizing of meat, in the
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preparation of precooked cereals, and in the production ofprotein hydrolysates.
Microbially Derived Preparations
�-Acetolactatedecarboxylase (Bacillus subtilis containing aBacillus brevis gene) Produced as a brown liquid by con-trolled fermentation using the modified Bacillus subtilis. Solu-ble in water (the solution is usually a light yellow to brown).Major active principle: decarboxylase. Typical application:used in the preparation of beer.
Aminopeptidase, Leucine (Aspergillus niger var., Aspergil-lus oryzae var., and other microbial species) Produced as alight tan to brown powder or as a brown liquid by controlledfermentation using Aspergillus niger var., Aspergillus oryzaevar., or other microbial species. The powder is soluble inwater (the solution is usually light yellow to brown). Majoractive principles: (1) aminopeptidase, (2) protease, and (3)carboxypeptidase activities in varying amounts. Typical appli-cations: used in the preparation of protein hydrolysates andin the development of flavors in processed foods.
Carbohydrase (Aspergillus niger var., including Aspergillusaculeatus) Produced as an off white to tan powder or atan to dark brown liquid by controlled fermentation usingAspergillus niger var. (including Aspergillus aculeatus). Solu-ble in water (the solution is usually light yellow to darkbrown), but practically insoluble in alcohol, in chloroform,and in ether. Major active principles: (1) �-amylase, (2) pec-tinase (a mixture of enzymes, including pectin depolymerase,pectin methyl esterase, pectin lyase, and pectate lyase), (3)cellulase, (4) glucoamylase (amyloglucosidase), (5) amylo-1,6-glucosidase, (6) hemicellulase (a mixture of enzymes,including poly(galacturonate) hydrolase, arabinosidase,mannosidase, mannanase, and xylanase), (7) lactase, (8) �-glucanase, (9) �-D-glucosidase, (10) pentosanase, and (11)�-galactosidase. Typical applications: used in the preparationof starch syrups and dextrose, alcohol, beer, ale, fruit juices,chocolate syrups, bakery products, liquid coffee, wine, dairyproducts, cereals, and spice and flavor extracts.
Carbohydrase (Aspergillus oryzae var.) Produced as an offwhite to tan, amorphous powder or a liquid by controlledfermentation using Aspergillus oryzae var. Soluble in water(the solution is usually light yellow to dark brown), but practi-cally insoluble in alcohol, in chloroform, and in ether. Majoractive principles: (1) �-amylase, (2) glucoamylase (amyloglu-cosidase), and (3) lactase. Typical applications: used in thepreparation of starch syrups, alcohol, beer, ale, bakery prod-ucts, and dairy products.
Carbohydrase (Bacillus acidopullulyticus) Produced as anoff white to brown, amorphous powder or a liquid by con-trolled fermentation using Bacillus acidopullulyticus. Solublein water (the solution is usually light yellow to dark brown),but practically insoluble in alcohol, in chloroform, and inether. Major active principle: pullulanase. Typical applica-tions: used in the hydrolysis of amylopectins and otherbranched polysaccharides.
Carbohydrase (Bacillus stearothermophilus) Produced asan off white to tan powder or a light yellow to dark brownliquid by controlled fermentation using Bacillus stearother-mophilus. Soluble in water, but practically insoluble in alco-hol, in ether, and in chloroform. Major active principle:�-amylase. Typical applications: used in the preparation ofstarch syrups, alcohol, beer, dextrose, and bakery products.
Carbohydrase (Candida pseudotropicalis) Produced as anoff white to tan, amorphous powder or a liquid by controlledfermentation using Candida pseudotropicalis. Soluble inwater (the solution is usually light yellow to dark brown) butinsoluble in alcohol, in chloroform, and in ether. Major activeprinciple: lactase. Typical applications: used in the manufac-ture of candy and ice cream and in the modification of dairyproducts.
Carbohydrase (Kluyveromyces marxianus var. lactis) Pro-duced as an off white to tan, amorphous powder or a liquidby controlled fermentation using Kluyveromyces marxianusvar. lactis. Soluble in water (the solution is usually lightyellow to dark brown), but insoluble in alcohol, in chloroform,and in ether. Major active principle: lactase. Typical applica-tions: used in the manufacture of candy and ice cream andin the modification of dairy products.
Carbohydrase (Mortierella vinaceae var. raffinoseuti-lizer) Produced as an off white to tan powder or as pelletsby controlled fermentation using Mortierella vinaceae var.raffinoseutilizer. Soluble in water (pellets may be insolublein water), but practically insoluble in alcohol, in chloroform,and in ether. Major active principle: �-galactosidase. Typicalapplication: used in the production of sugar from sugar beets.
Carbohydrase (Rhizopus niveus) Produced as an off whiteto brown, amorphous powder or a liquid by controlled fermen-tation using Rhizopus niveus. Soluble in water (the solutionis usually light yellow to dark brown), but practically insolublein alcohol, in chloroform, and in ether. Major active principles:(1) �-amylase and (2) glucoamylase. Typical application: usedin the hydrolysis of starch.
Carbohydrase (Rhizopus oryzae var.) Produced as a pow-der or a liquid by controlled fermentation using Rhizopusoryzae var. Soluble in water, but practically insoluble in alco-hol, in chloroform, and in ether. Major active principles: (1)�-amylase, (2) pectinase, and (3) glucoamylase (amylogluco-sidase). Typical applications: used in the preparation of starchsyrups and fruit juices, vegetable purees, and juices and inthe manufacture of cheese.
Carbohydrase (Saccharomyces species) Produced as awhite to tan, amorphous powder by controlled fermentationusing a number of species of Saccharomyces traditionallyused in the manufacture of food. Soluble in water (the solutionis usually light yellow), but practically insoluble in alcohol,in chloroform, and in ether. Major active principles: (1) in-vertase and (2) lactase. Typical applications: used in the man-ufacture of candy and ice cream and in the modification ofdairy products.
FCC V Monographs / Enzyme Preparations / 149
Carbohydrase [(Trichoderma longibrachiatum var.) (for-merly reesei)] Produced as an off white to tan, amorphouspowder or as a liquid by controlled fermentation using Tri-choderma longibrachiatum var. Soluble in water (the solutionis usually tan to brown), but practically insoluble in alcohol,in chloroform, and in ether. Major active principles: (1) cellu-lase, (2) �-glucanase, (3) �-D-glucosidase, (4) hemicellulase,and (5) pentosanase. Typical applications: used in the prepara-tion of fruit juices, wine, vegetable oils, beer, and bakedgoods.
Carbohydrase (Bacillus subtilis containing a Bacillus mega-terium �-amylase gene) Produced as an off white to brown,amorphous powder or liquid by controlled fermentation usingthe modified Bacillus subtilis. Soluble in water (the solutionis usually light yellow to dark brown), but practically insolublein alcohol, in chloroform, and in ether. Major active principle:�-amylase. Typical applications: used in the preparation ofstarch syrups, alcohol, beer, and dextrose.
Carbohydrase (Bacillus subtilis containing a Bacillus stearo-thermophilus �-amylase gene) Produced as an off white tobrown, amorphous powder or a liquid by controlled fermenta-tion using the modified Bacillus subtilis. Soluble in water (thesolution is usually light yellow to dark brown), but practicallyinsoluble in alcohol, in chloroform, and in ether. Major activeprinciple: maltogenic amylase. Typical applications: used inthe preparation of starch syrups, dextrose, alcohol, beer, andbaked goods.
Carbohydrase and Protease, Mixed (Bacillus licheniformisvar.) Produced as an off white to brown, amorphous powderor as a liquid by controlled fermentation using Bacillus licheni-formis var. Soluble in water (the solution is usually lightyellow to dark brown), but practically insoluble in alcohol,in chloroform, and in ether. Major active principles: (1) �-amylase and (2) protease. Typical applications: used in thepreparation of starch syrups, alcohol, beer, dextrose, fishmeal,and protein hydrolysates.
Carbohydrase and Protease, Mixed (Bacillus subtilis var.including Bacillus amyloliquefaciens) Produced as an offwhite to tan, amorphous powder or as a liquid by controlledfermentation using Bacillus subtilis var. Soluble in water (thesolution is usually light yellow to dark brown), but practicallyinsoluble in alcohol, in chloroform, and in ether. Major activeprinciples: (1) �-amylase, (2) �-glucanase, (3) protease, and(4) pentosanase. Typical applications: used in the preparationof starch syrups, alcohol, beer, dextrose, bakery products, andfishmeal; in the tenderizing of meat; and in the preparationof protein hydrolysates.
Catalase (Aspergillus niger var.) Produced as an off whiteto tan, amorphous powder or as a liquid by controlled fermen-tation using Aspergillus niger var. Soluble in water (the solu-tion is usually tan to brown), but practically insoluble inalcohol, in chloroform, and in ether. Major active principle:catalase. Typical applications: used in the manufacture ofcheese, egg products, and soft drinks.
Catalase (Micrococcus lysodeikticus) Produced by con-trolled fermentation using Micrococcus lysodeikticus. Solublein water (the solution is usually light yellow to dark brown),but practically insoluble in alcohol, in chloroform, and inether. Major active principle: catalase. Typical application:used in the manufacture of cheese, egg products, and softdrinks.
Chymosin (Aspergillus niger var. awamori, Escherichia coliK-12, and Kluyveromyces marxianus, each microorganismcontaining a calf prochymosin gene) Produced as a whiteto tan, amorphous powder or as a light yellow to brown liquidby controlled fermentation using the above-named geneticallymodified microorganisms. The powder is soluble in water,but practically insoluble in alcohol, in chloroform, and inether. Major active principle: chymosin. Typical application:used in the manufacture of cheese and in the preparation ofmilk-based desserts.
Glucose Isomerase (Actinoplanes missouriensis, Bacillus co-agulans, Streptomyces olivaceus, Streptomyces olivochromo-genes, Microbacterium arborescens, Streptomyces rubigino-sus var., or Streptomyces murinus) Produced as an off whiteto tan, brown, or pink, amorphous powder, granules, or liquidby controlled fermentation using any of the above-namedorganisms. The products may be soluble in water, but practi-cally insoluble in alcohol, in chloroform, and in ether; or ifimmobilized, may be insoluble in water and partially solublein alcohol, in chloroform, and in ether. Major active principle:glucose (or xylose) isomerase. Typical applications: used inthe manufacture of high-fructose corn syrup and other fructosestarch syrups.
Glucose Oxidase (Aspergillus niger var.) Produced as ayellow to brown solution or as a yellow to tan or off whitepowder by controlled fermentation using Aspergillus nigervar. Soluble in water (the solution is usually light yellow tobrown), but practically insoluble in alcohol, in chloroform,and in ether. Major active principles: (1) glucose oxidase and(2) catalase. Typical applications: used in the removal ofsugar from liquid eggs and in the deoxygenation of citrusbeverages.
Lipase (Aspergillus niger var.) Produced as an off whiteto tan, amorphous powder by controlled fermentation usingAspergillus niger var. Soluble in water (the solution is usuallylight yellow), but practically insoluble in alcohol, in chloro-form, and in ether. Major active principle: lipase. Typicalapplication: used in the hydrolysis of lipids (e.g., fish oilconcentrates and cereal-derived lipids).
Lipase (Aspergillus oryzae var.) Produced as an off white totan, amorphous powder or a liquid by controlled fermentationusing Aspergillus oryzae var. Soluble in water (the solutionis usually light yellow), but practically insoluble in alcohol,in chloroform, and in ether. Major active principle: lipase.Typical applications: used in the hydrolysis of lipids (e.g.,fish oil concentrates) and in the manufacture of cheese andcheese flavors.
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Lipase (Candida rugosa; formerly Candida cylindra-cea) Produced as an off white to tan powder by controlledfermentation using Candida rugosa. Soluble in water, butpractically insoluble in alcohol, in chloroform, and in ether.Major active principle: lipase. Typical applications: used inthe hydrolysis of lipids, in the manufacture of dairy productsand confectionery goods, and in the development of flavor inprocessed foods.
Lipase [Rhizomucor (Mucor) miehei] Produced as an offwhite to tan powder or as a liquid by controlled fermentationusing Rhizomucor miehei. Soluble in water (the solution isusually light yellow to dark brown), but practically insolublein alcohol, in chloroform, and in ether. Major active principle:lipase. Typical applications: used in the hydrolysis of lipids,in the manufacture of cheese, and in the removal of haze infruit juices.
Phytase (Aspergillus niger var.) Produced as an off whiteto brown powder or as a tan to dark brown liquid by controlledfermentation using Aspergillus niger var. Soluble in water,but practically insoluble in alcohol, in chloroform, and inether. Major active principles: (1) 3-phytase and (2) acidphosphatase. Typical applications: used in the production ofsoy protein isolate and in the removal of phytic acid fromplant materials.
Protease (Aspergillus niger var.) Produced by controlledfermentation using Aspergillus niger var. The purified enzymeoccurs as an off white to tan, amorphous powder. Soluble inwater (the solution is usually light yellow), but practicallyinsoluble in alcohol, in chloroform, and in ether. Major activeprinciple: protease. Typical application: used in the produc-tion of protein hydrolysates.
Protease (Aspergillus oryzae var.) Produced by controlledfermentation using Aspergillus oryzae var. The purified en-zyme occurs as an off white to tan, amorphous powder. Solublein water (the solution is usually light yellow), but practicallyinsoluble in alcohol, in chloroform, and in ether. Major activeprinciple: protease. Typical applications: used in the chill-proofing of beer, in the production of bakery products, in thetenderizing of meat, in the production of protein hydrolysates,and in the development of flavor in processed foods.
Rennet, Microbial (nonpathogenic strain of Bacillus ce-reus) Produced as a white to tan, amorphous powder or alight yellow to dark brown liquid by controlled fermentationusing Bacillus cereus. Soluble in water, but practically insolu-ble in alcohol, in chloroform, and in ether. Major active princi-ple: protease. Typical application: used in the manufactureof cheese.
Rennet, Microbial (Endothia parasitica) Produced as anoff white to tan, amorphous powder or as a liquid by controlledfermentation using nonpathogenic strains of Endothia parasit-ica. The powder is soluble in water (the solution is usuallytan to dark brown), but practically insoluble in alcohol, inchloroform, and in ether. Major active principle: protease.Typical application: used in the manufacture of cheese.
Rennet, Microbial [Rhizomucor (Mucor) sp.] Produced asa white to tan, amorphous powder by controlled fermentationusing Rhizomucor miehei, or pusillus var. Lindt. The powderis soluble in water (the solution is usually light yellow), butpractically insoluble in alcohol, in chloroform, and in ether.Major active principle: protease. Typical application: used inthe manufacture of cheese.
Transglutaminase (Streptoverticillium mobaraense var.)Produced as an off white to weak yellow-brown, amorphouspowder by controlled fermentation using Streptoverticilliummobaraense var. Soluble in water but practically insoluble inalcohol, in chloroform, and in ether. Major active principle:transglutaminase. Typical applications: used in the processingof meat, poultry, and seafood; production of yogurt, certaincheeses, and frozen desserts; and manufacture of pasta prod-ucts and noodles, baked goods, meat analogs, ready-to-eatcereals, and other grain-based foods.
REACTIONS CATALYZED
Note: The reactions catalyzed by any given active com-ponent are essentially the same, regardless of the sourcefrom which that component is derived.
�-Acetolactatedecarboxylase Decarboxylation of �-aceto-lactate to acetoin.
Aminopeptidase, Leucine Hydrolysis of N-terminal aminoacid, which is preferably leucine, but may be other aminoacids, from proteins and oligopeptides, yielding free aminoacids and oligopeptides of lower molecular weight.
�-Amylase Endohydrolysis of �-1,4-glucan bonds in poly-saccharides (starch, glycogen, etc.), yielding dextrins and ol-igo- and monosaccharides.
�-Amylase Hydrolysis of �-1,4-glucan bonds in polysac-charides (starch, glycogen, etc.), yielding maltose and beta-limit dextrins.
Bromelain Hydrolysis of polypeptides, amides, and esters(especially at bonds involving basic amino acids, leucine, orglycine), yielding peptides of lower molecular weight.
Catalase 2H2O2→ O2 + 2H2O.
Cellulase Hydrolysis of �-1,4-glucan bonds in such poly-saccharides as cellulose, yielding �-dextrins.
Chymosin (calf and fermentation derived) Cleaves a singlebond in kappa casein.
Ficin Hydrolysis of polypeptides, amides, and esters (espe-cially at bonds involving basic amino acids, leucine, or gly-cine), yielding peptides of lower molecular weight.
�-Galactosidase Hydrolysis of terminal nonreducing �-D-galactose residues in �-D-galactosides.
�-Glucanase Hydrolysis of �-1,3- and �-1,4-linkages in �-D-glucans, yielding oligosaccharides and glucose.
FCC V Monographs / Enzyme Preparations / 151
Glucoamylase (amyloglucosidase) Hydrolysis of terminal�-1,4- and �-1,6-glucan bonds in polysaccharides (starch,glycogen, etc.), yielding glucose (dextrose).
Glucose Isomerase (xylose isomerase) Isomerization ofglucose to fructose, and xylose to xylulose.
Glucose Oxidase �-D-glucose + O2 → D-glucono-�-lactone+ H2O2.
�-D-Glucosidase Hydrolysis of terminal, nonreducing �-D-glucose residues with the release of �-D-glucose.
Hemicellulase Hydrolysis of �-1,4-glucans, �-L-arabino-sides, �-D-mannosides, 1,3-�-D-xylans, and other polysaccha-rides, yielding polysaccharides of lower molecular weight.
Invertase (�-fructofuranosidase) Hydrolysis of sucrose toa mixture of glucose and fructose (invert sugar).
Lactase (�-galactosidase) Hydrolysis of lactose to a mixtureof glucose and galactose.
Lysozyme Hydrolysis of cell-wall polysaccharides of vari-ous bacterial species leading to the breakdown of the cell wallmost often in Gram-positive bacteria.
Maltogenic Amylase Hydrolysis of �-1,4-glucan bonds.
Lipase Hydrolysis of triglycerides of simple fatty acids,yielding mono- and diglycerides, glycerol, and free fatty acids.
Pancreatin�-Amylase Hydrolysis of �-1,4-glucan bonds.Protease Hydrolysis of proteins and polypepticles.Lipase Hydrolysis of triglycerides of simple fatty acids.
PectinasePectate lyase Hydrolysis of pectate to oligosaccharides.Pectin depolymerase Hydrolysis of 1,4-galacturonide
bonds.Pectin lyase Hydrolysis of oligosaccharides formed by
pectate lyase.Pectinesterase Demethylation of pectin.
Pepsin Hydrolysis of polypeptides, including those withbonds adjacent to aromatic or dicarboxylic L-amino acid resi-dues, yielding peptides of lower molecular weight.
Phospholipase A2 Hydrolysis of lecithins and phosphatidyl-choline, producing fatty acid anions.
Phytase3-Phytase myo-Inositol hexakisphosphate + H2O → 1,2,4,
5,6-pentakisphosphate + orthophosphate.Acid Phosphatase Orthophosphate monoester + H2O →
an alcohol + orthophosphate.
Protease (generic) Hydrolysis of polypeptides, yieldingpeptides of lower molecular weight.
Pullulanase Hydrolysis of 1,6-�-D-glycosidic bonds onamylopectin and glycogen and in �- and �-limit dextrins,yielding linear polysaccharides.
Rennet (bovine and calf) Hydrolysis of polypeptides; speci-ficity may be similar to pepsin.
Transglutaminase Binding of proteins.
Trypsin Hydrolysis of polypeptides, amides, and esters atbonds involving the carboxyl groups of L-arginine and L-lysine, yielding peptides of lower molecular weight.
GENERAL REQUIREMENTS
Enzyme preparations are produced in accordance with goodmanufacturing practices. Regardless of the source of deriva-tion, they should cause no increase in the total microbialcount in the treated food over the level accepted for therespective food.
Animal tissues used to produce enzymes must comply withthe applicable U.S. meat inspection requirements and mustbe handled in accordance with good hygienic practices.
Plant material used to produce enzymes or culture mediaused to grow microorganisms consist of components that leaveno residues harmful to health in the finished food under normalconditions of use.
Preparations derived from microbial sources are producedby methods and under culture conditions that ensure a con-trolled fermentation, thus preventing the introduction of mi-croorganisms that could be the source of toxic materials andother undesirable substances.
The carriers, diluents, and processing aids used to producethe enzyme preparations shall be substances that are accept-able for general use in foods, including water and substancesthat are insoluble in foods but removed from the foods afterprocessing.
Although limits have not been established for mycotoxins,appropriate measures should be taken to ensure that the prod-ucts do not contain such contaminants.
ADDITIONAL REQUIREMENTS
Assay Not less than 85.0% and not more than 115.0% ofthe declared units of enzyme activity.Lead Not more than 5 mg/kg.Microbial Limits:
Coliforms Not more than 30 CFU per gram.Salmonella Negative in 25 g.
TESTS
Assay The following procedures, which are included underEnzyme Assays, Appendix V, are provided for applicationas necessary in determining compliance with the declared
152 / Erythorbic Acid / Monographs FCC V
representations for enzyme activity:1 Acid Phosphatase Activ-ity, �-Amylase Activity (Nonbacterial); Bacterial �-AmylaseActivity (BAU); Catalase Activity; Cellulase Activity; Chy-motrypsin Activity; Diastase Activity (Diastatic Power); �-Galactosidase Activity, �-Glucanase Activity; GlucoamylaseActivity (Amyloglucosidase Activity); Glucose Isomerase Ac-tivity; Glucose Oxidase Activity; �-D-Glucosidase Activity;Hemicellulase Activity; Invertase Activity; Lactase (Neutral)(�-Galactosidase) Activity; Lactase (Acid) (�-Galactosidase)Activity; Lipase Activity; Lipase/Esterase (Forestomach) Ac-tivity; Maltogenic Amylase Activity; Milk-Clotting Activity;Pancreatin Activity; Pepsin Activity; Phospholipase Activity;Phytase Activity; Plant Proteolytic Activity; Proteolytic Activ-ity, Bacterial (PC); Proteolytic Activity, Fungal (HUT); Pro-teolytic Activity, Fungal (SAP); Pullulanase Activity; andTrypsin Activity.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Microbial Limits (Note: Current methods for the followingtests may be found online at <www.cfsan.fda.gov/~ebam/bam-toc.html>):
ColiformsSalmonella
Packaging and Storage Store in tight containers in a cool,dry place.
Erythorbic AcidD-Araboascorbic Acid
O
OH OH
OHOCH2C
H
OH
C6H8O6 Formula wt 176.13
INS: 315 CAS: [89-65-6]
DESCRIPTION
Erythorbic Acid occurs as white or slightly yellow crystalsor powder. It gradually darkens when exposed to light. In thedry state, it is reasonably stable in air, but in solution, itrapidly deteriorates in the presence of air. It melts between
1Because of the varied conditions under which pectinases are employed,and because laboratory hydrolysis of a purified pectin substrate does notcorrelate with results observed with the natural substrates under useconditions, pectinase suppliers and users should develop their own assayprocedures that would relate to the specific application under consider-ation.
164° and 171° with decomposition. One gram is soluble inabout 2.5 mL of water and in about 20 mL of alcohol. It isslightly soluble in glycerin.
Function Preservative; antioxidant.
REQUIREMENTS
IdentificationA. Add a few drops of sodium nitroferricyanide TS to 2 mL
of a 1:50 aqueous solution, then add 1 mL of approximately0.1 N sodium hydroxide. A transient blue color immediatelyappears.
B. Dissolve about 15 mg of sample in 15 mL of a 1:20trichloroacetic acid solution, add about 200 mg of activatedcharcoal, and shake the mixture vigorously for 1 min. Filterthrough a small fluted filter, refiltering if necessary to obtaina clear filtrate. Add 1 drop of pyrrole to 5 mL of the clearfiltrate, agitate the mixture until the pyrrole is dissolved, thenheat in a water bath at 50°. A blue color appears.Assay Not less than 99.0% and not more than 100.5% ofC6H8O6, calculated on the dried basis.Lead Not more than 2 mg/kg.Loss on Drying Not more than 0.4%.Optical (Specific) Rotation [�]D
25°: Between −16.5° and−18.0°.Residue on Ignition Not more than 0.3%.
TESTS
Assay Dissolve about 400 mg of sample, accuratelyweighed, in a 100:25 (v/v) mixture of recently boiled andcooled water:2 N sulfuric acid. Titrate the solution immedi-ately with 0.1 N iodine, adding starch TS near the endpoint.Each milliliter of 0.1 N iodine is equivalent to 8.806 mg ofC6H8O6.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample under reduced pressureover silica gel for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using the followingsolution: Transfer about 2.5 g of sample, accurately weighed,into a 25-mL volumetric flask, dissolve it in about 20 mL ofwater, and dilute to volume.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in tight, light-resistant con-tainers.
FCC V Monographs / Erythritol / 153
Erythritol1,2,3,4-Butanetetrol; meso-Erythritol
HOOH
H OH
H OH
C4H10O4 Formula wt 122.12
CAS: [149-32-6]
DESCRIPTION
Erythritol occurs as white crystals. It is obtained from thefermentation broth of the yeasts Moniliella pollinis or Tri-chosporonoides megachiliensis. It is stable to heat and isnonhygroscopic. It is soluble in water and is slightly solublein alcohol. Erythritol melts between 119° and 123°.
Function Flavor enhancer; humectant; nutritive sweetener;texturizing agent; stabilizer.
REQUIREMENTS
Identification The retention time of the major peak in thechromatogram of the Assay Preparation corresponds to thatin the chromatogram of the Standard Preparation obtainedin the Assay.Assay Not less than 99.5% and not more than 100.5% ofC4H10O4, calculated on the dried basis.Lead Not more than 1 mg/kg.Loss on Drying Not more than 0.2%.Reducing Sugars (as glucose) Not more than 0.3%.Residue on Ignition Not more than 0.1%.Ribitol and Glycerol Not more than 0.1%.
TESTS
AssayMobile Phase Use twice-distilled water.Standard Preparation Transfer 500 mg of Erythritol Stan-
dard1 and 50 mg each of reagent-grade glycerol and ribitol,accurately weighed, into a 100-mL volumetric flask. Diluteto volume with Mobile Phase, and mix. Save this preparationfor the Ribitol and Glycerol Test.
Assay Preparation Transfer 4.0 g of sample, accuratelyweighed, into a 25-mL volumetric flask. Add Mobile Phaseto volume, and mix. Filter through a 0.45-�m filter beforeinjecting into the chromatograph. Save this preparation forthe Ribitol and Glycerol Test.
1Available from Cerestar, EBS Vilvoorde R&D Centre, Centre of Ex-pertise Fermentation, Havenstraat 84, 1800 Vilvoorde, Belgium; Mitsubi-shi Chemical Corporation, Specialty Chemicals Company, IntermediateChemicals Department, 5-2 Marunouchi 2-chome, Chiyoda-ku, Tokyo100-0005, Japan; or Nikken Chemicals Co., Ltd., Development Depart-ment, Sumitomo-Tsukiji Bldg., No. 4-14, Tsukiji 5-chome, Chuo-ku,Tokyo 104-0045, Japan.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a high-performance liquid chromatographequipped with a constant-flow, pulseless pump and fitted witha sensitive differential refractive index detector. The columnis packed with a strong cation exchange resin in the hydrogenform consisting of a macroreticular sulfonated polystyrenedivinylbenzene and an 8% crosslinked copolymer, such asMCI-CKO8SH or Shodex KC811 (Mitsubishi Chemical Corp.and Showa Denko, Ltd.), or equivalent. The flow rate is about0.6 mL/min, and the maximum pressure of the system is about1500 psi.
Procedure Separately inject equal volumes of about 10�L each of the Standard Preparation, followed by the AssayPreparation, into the chromatograph, and record the peakresponses over a period of 60 min. The relative retention timesare 1.0 for Erythritol, 1.1 for glycerol, and 0.9 for ribitol.Calculate the percentage of Erythritol in the sample taken bythe equation
% Erythritol = 100E/T,
in which E is the area response of the Erythritol peak, and Tis the total area responses in the chromatogram obtained withthe Assay Preparation.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Reducing Sugars (as glucose) Dissolve about 500 mg ofsample, accurately weighed, in 2 mL of water in a 20-mLflask, and mix. Transfer 2 mL of a glucose solution containing0.75 mg/mL into another flask. Add 1 mL of Fehling’s Solu-tion A and of Fehling’s Solution B (see Cupric Tartrate TS,Alkaline, under Solutions and Indicators) to each flask, heatto boiling, and cool. The sample solution is less turbid thanthe glucose solution, which forms a red-brown precipitate.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Ribitol and Glycerol Determine as directed in the Assay.Identify the peak area responses for glycerol and ribitol inthe chromatogram of the Assay Preparation by comparisonwith the chromatogram of the Standard Preparation, andcalculate the percentage of glycerol and ribitol by the equa-tions
% glycerol = 100G/T,
% ribitol = 100R/T,
in which G is the area response of the glycerol peak, R is thearea response of the ribitol peak, and T is the total arearesponses in the chromatogram obtained with the Assay Prepa-ration. The sum of glycerol and ribitol is not more than 0.1%.
Packaging and Storage Store in well-closed containers.
154 / Erythrosine / Monographs FCC V
Erythrosine1
CI Food Red 14; CI 45430; Class: Xanthene
I
NaO
I
O
C
I
O
ICOONa
C20H6O5I4Na2 Formula wt 879.86
INS: 127 CAS: [16423-68-0]
DESCRIPTION
Erythrosine occurs as a brown powder or granules. It isprincipally the disodium salt of the monohydrate of 9-(o-carboxy-phenyl)-6-hydroxy-2,4,5,7-tetraiodo-3H-xanthen-3-one. It dissolves in water to give a solution red at neutrality,with a yellow-brown precipitate in acid, and with a red precipi-tate in base. When dissolved in concentrated sulfuric acid, ityields a brown-yellow solution that evolves iodine and aprecipitate of the free acid when heated. It is insoluble inethanol.
Function Color.
REQUIREMENTS
Identification An aqueous solution containing 2.8 mg ofsample per liter exhibits absorbance intensities (A) and wave-length maxima as follows: in neutral (pH = 7) and alkaline(pH = 13) solutions, A = 0.32 at 527 nm with a shoulder at490 nm. In acid solution, a yellow-brown precipitate forms.Assay Not less than 87.0% total coloring matter.Arsenic Not more than 3 mg/kg.Ether Extracts (combined) Not more than 0.2%.Lead Not more than 10 mg/kg.Loss on Drying (Volatile Matter) at 135°, Chlorides, andSulfates (as sodium salts) Not more than 13.0% in combi-nation.Subsidiary Colors
Monoiodofluoresceins Not more than 1.0%.Other Lower Iodinated Fluoresceins Not more than 9.0%.
1To be used or sold for use to color food that is marketed in the UnitedStates, this color additive must be from a batch that has been certifiedby the U.S. Food and Drug Administration (FDA). If it is not from anFDA-certified batch, it is not a permitted color additive for food use inthe United States, even if it is compositionally equivalent. The nameFD&C Red No. 3 can be applied only to FDA-certified batches of thiscolor additive. Erythrosine is a common name given to the uncertifiedcolorant. See the monograph entitled FD&C Red No. 3 for directions forproducing an FDA-certified batch.
Uncombined Intermediates and Products of Side Reac-tions
2-(2′,4′-Dihydroxy-3′,5′-diiodobenzoyl)benzoic Acid Notmore than 0.2%.
Sodium Iodide Not more than 0.4%.Triiodoresorcinol Not more than 0.2%.Unhalogenated Intermediates Total not more than 0.1%.
Water-Insoluble Matter Not more than 0.2%.
TESTS
Assay Determine the total color strength as the weight per-cent of the sample using Methods I and III in Total Colorunder Colors, Appendix IIIC. Express the Total Color as theaverage of the two results.
Method I (Sample Preparation) Transfer 75 to 100 mgof sample, accurately weighed, into a 1-L volumetric flask;dissolve in and dilute to volume with water. The absorptivity(a) for Erythrosine is 0.110 mg/L/cm at 527 nm.
Method III (Sample Preparation) Proceed as directed forMethod III in Total Color under Colors, Appendix IIIC. Thegravimetric conversion factor (F) for Erythrosine is 1.074.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Chlorides Determine as directed in Sodium Chloride underColors, Appendix IIIC.Ether Extracts Determine as directed in Ether Extractsunder Colors, Appendix IIIC, using a solution with a pH ofnot less than 7.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Loss on Drying (Volatile Matter) at 135° Determine asdirected in Loss on Drying (Volatile Matter) under Colors,Appendix IIIC.Subsidiary Colors
Solvent System Use a solvent system composed of 95 mLof acetone, 25 mL of chloroform, 10 mL of butylamine, and10 mL of water.
Sample Solution Transfer approximately 2 g of sample,accurately weighed, into a 100-mL volumetric flask. Fill theflask about ¾-full with water, incubate in the dark for 1 h;dilute to volume with water, and mix well.
Procedure Spot 0.1 mL of Sample Solution in a line acrossa 20- × 20-cm glass plate coated with a 0.25-mm layer ofSilica Gel G, approximately 3 cm from the bottom edge.Allow the plate to dry for about 20 min in the dark, thendevelop with the Solvent System in an unlined tank equilibratedfor at least 20 min before inserting the plate. Allow the solventfront to reach to within about 3 cm of the top of the plate.Dry the developed plate in the dark.
Scrape off each subsidiary color and extract with 3- to 5-mL portions of 50% aqueous ethanol until no color remainson the gel by visual inspection. Dilute each sample to 13 to15 mL, add a few drops of ammonium hydroxide, and recordthe final volume. Repeat this procedure for the band of Eryth-rosine using 10- to 20-mL portions of 50% ethanol, and dilutethe eluant to 250 mL in a volumetric flask after adding enough
FCC V Monographs / Ethoxylated Mono- and Diglycerides / 155
ammonium hydroxide to make the solution slightly alkaline.The approximate band positions (Rf), wavelengths of maximalabsorbance (�), and absorptivities (a) are as follows:
Color Rf � a
Unknown 0.84 524 0.110Red-3 0.84 526 0.1102,4,7 0.76 521 0.1402,4,5 0.67 521 0.1162,4/2,5 0.45 513 0.145Unknown 0.45 524 0.110
Record the spectrum of each solution between 400 and600 nm, and calculate the quantity, in percent (P), of eachsubsidiary color by the equation
P = (A × V × 100)/(a × W × b),
in which A is the absorbance at the wavelength maximum; Vis the volume, in milliliters, of the solution; a is the absorptiv-ity, in milligrams per liter per centimeter, as given above; Wis the weight, in milligrams, of the sample; and b is the path-length, in centimeters, of the cell.Sulfates Determine as directed in Sodium Sulfate under Col-ors, Appendix IIIC.Uncombined Intermediates and Products of Side Reac-tions Determine as directed for Method I in UncombinedIntermediates and Products of Side Reactions under Colors,Appendix IIIC, using the following Sample Solution: Transfer2 g of sample, accurately weighed, into a 100-mL volumetricflask; dissolve in and dilute to volume with water. Calculatethe concentrations of 2-(2,4-dihydroxy-3,5-diiodobenzoyl)benzoic acid, iodine, phthalic acid, sodium iodide, and triio-doresorcinol, using the following absorptivities:
2-(2,4-Dihydroxy-3,5-diiodobenzoyl)benzoic Acid: a =0.047 mg/L/cm at 348 nm (alkaline solution).
Iodine: a = 0.082 mg/L/cm at 245 nm (acidic solution).Phthalic Acid: a = 0.045 mg/L/cm at 228 nm (acidic so-
lution).Sodium Iodide: a = 0.091 mg/L/cm at 220 nm (acidic so-
lution).Triiodoresorcinol: a = 0.079 mg/L/cm at 223 nm (acidic
solution).Water-Insoluble Matter Determine as directed in Water-Insoluble Matter under Colors, Appendix IIIC.
Packaging and Storage Store in well-closed containers.
Ethoxylated Mono- and DiglyceridesPolyoxyethylene (20) Mono- and Diglycerides of FattyAcids; Polyglycerate (60)
INS: 488
DESCRIPTION
Ethoxylated Mono- and Diglycerides occur as a pale, slightlyyellow-colored, oily liquid or semigel. They are a mixture of
stearate, palmitate, and lesser amounts of myristate partialesters of glycerin condensed with approximately 20 molesof ethylene oxide per mole of alpha-monoglyceride reactionmixture, having an average molecular weight of 535 (� 10%).They are soluble in water, in alcohol, and in xylene. Theyare partially soluble in mineral oil and in vegetable oils.
Note: If the product is manufactured by direct esterifica-tion of glycerin with a mixture of primary stearic, pal-mitic, and myristic acids, then the intermediate product(before reaction with ethylene oxide) has an acid valueof not greater than 0.3 and a water content of not greaterthan 0.2%.
Function Dough conditioner; emulsifier.
REQUIREMENTS
IdentificationA. Add 5 mL of 1 N sodium hydroxide to 5 mL of a 1:20
aqueous solution, boil for a few min, cool, and acidify with2.7 N hydrochloric acid. The solution is strongly opalescent.
B. Make a 46:54 (v/v) mixture of sample:water at 40° orcooler. A gelatinous mass forms.Acid Value Not more than 2.1,4-Dioxane Passes test.Hydroxyl Value Between 65 and 80.Lead Not more than 1 mg/kg.Oxyethylene Content (apparent) Not less than 60.5% andnot more than 65.0%, calculated as ethylene oxide (C2H4O),on the anhydrous basis.Saponification Value Between 65 and 75.Stearic, Palmitic, and Myristic Acids Between 31 and 33g per 100 g of sample.Water Not more than 1%.
TESTS
Acid Value Determine as directed in Method II under AcidValue, Appendix VII.1,4-Dioxane Determine as directed under 1,4-Dioxane LimitTest, Appendix IIIB.Hydroxyl Value Determine as directed in Method II underHydroxyl Value, Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Oxyethylene Content (apparent) Determine as directed un-der Oxyethylene Determination, Appendix VII, using 70 mgof sample, accurately weighed.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII, using about 6 g of sample,accurately weighed.Stearic, Palmitic, and Myristic Acids Transfer about 25g of sample, accurately weighed, into a 500-mL round-bottomboiling flask, add 250 mL of alcohol and 7.5 g of potassiumhydroxide, and mix. Connect a suitable condenser to the flask,reflux the mixture for 1 to 2 h, then transfer to an 800-mLbeaker, rinsing the flask with about 100 mL of water and
156 / Ethoxyquin / Monographs FCC V
adding the washings to the beaker. Heat on a steam bath toevaporate the alcohol, adding water occasionally to replacethe alcohol, and evaporate until the odor of alcohol can nolonger be detected. Use hot water to adjust the final volumeto about 250 mL. Neutralize the soap solution with 1:2 sulfuricacid; add 10% in excess; and while stirring, heat until thefatty acid layer separates. Transfer the fatty acids into a 500-mL separator, wash with three or four 20-mL portions of hotwater, and combine the washings with the original aqueouslayer from the saponification. Extract the combined aqueouslayer with three 50-mL portions of petroleum ether, add theextracts to the fatty acid layer, evaporate to dryness in a tareddish, cool, and weigh. The product so obtained has an AcidValue between 199 and 211 (Method I, Appendix VII) and aSolidification Point ≥ 50° (Appendix IIB).Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Ethoxyquin6-Ethoxy-1,2-dihydro-2,2,4-trimethylquinoline
NH
C2H5O
CH3
CH3
CH3
C14H19NO Formula wt, monomer 217.31
INS: 324 CAS: [91-53-2]
DESCRIPTION
Ethoxyquin occurs as a clear yellow to red liquid that maydarken with age without affecting its antioxidant activity.It is a mixture consisting predominantly of the monomer(C14H19NO). It also contains dimers and other polymers ofC14H19NO. Its specific gravity is about 1.02, and its refractiveindex is about 1.57.
Function Antioxidant.
REQUIREMENTS
Identification A solution of 1 mg of sample in 10 mL ofacetonitrile exhibits a strong fluorescence when viewed undershort-wavelength ultraviolet light.Assay Not less than 91.0% of C14H19NO.Lead Not more than 2 mg/kg.p-Phenetidine Not more than 3.0%.Ethoxyquin-Related Impurities (low-boiling monomers andhigh-boiling dimers, trimers, and oligomers of Ethoxyquin)Not more than 8.0%.
TESTS
Assay Transfer about 200 mg of sample, accuratelyweighed, into a 150-mL beaker containing 50 mL of glacialacetic acid, and immediately titrate with 0.1 N perchloric acidin glacial acetic acid, determining the endpoint potentiomet-rically.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 21.73 mg of C14H19NO.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.p-Phenetidine
Standard Preparation (Caution: Perform all steps in afume hood and away from a source of ignition. Wear appro-priate protective equipment, including gloves.) (Note: Boththe p-phenetidine and the diphenyl ether must be of knownpurity. Unless the reagent supplier’s reported purity is certifiedquantitative and traceable, determine the purity of a reagentstandard by conducting an area percent profile by injecting0.1 �L on the same column and at conditions analogous tothose described below. The area percent corresponding to thestandard in the chromatograph represents its purity.) Transferapproximately 200 mg of p-phenetidine and approximately200 mg of diphenyl ether, both accurately weighed, into a 4-dram bottle, add 10 mL of toluene and 5 drops of 10% sodiumhydroxide, cap, and shake vigorously to dissolve. Prepare intriplicate.
Sample Preparation Transfer about 0.1 g of sample, accu-rately weighed, into a 4-dram vial. Add between 0.010 to0.015 g of diphenyl ether, accurately weighed, 10 mL oftoluene, and 5 drops of 10% sodium hydroxide solution; capthe vial; and shake well. Allow the vial to stand until thecaustic layer settles to the bottom, and filter the neutralizedsample through a 0.45-�m polytetrafluoroethylene (PTFE)filter, or equivalent.
Apparatus (See Chromatography, Appendix IIA.) Use asuitable gas chromatograph (HP 6890, or equivalent) equippedwith a split injector port, a flame-ionization detector (FID),and a 30-m × 0.25-mm (od) GC capillary column (DB-5MS,or equivalent) having a film thickness of 0.25 �m.
Operating Conditions The operating parameters mayvary, depending on the particular instrument used, but a suit-able chromatogram may be obtained using the following con-ditions: initial temperature, 50°; initial hold time, 1 min; pro-gram rate, 10°/min; final temperature, 280°; final hold time,10 min; injector temperature, 250°; detector temperature,280°. Use helium as the carrier gas, at a column flow rate of2.3 mL/min and a makeup flow rate of 50 mL/min. Set thehydrogen and air flows to the burner at 45 mL/min and 450mL/min, respectively. Use a split flow rate of 232 mL/minand a total flow rate of 237 mL/min.
Calibration and Standardization Inject the StandardPreparation into the chromatograph. Calculate the p-pheneti-dine factor (F) by the formula
FCC V Monographs / Ethyl Alcohol / 157
(ADE × WPF × PPF)/(APF × WDE × PDE),
in which ADE and APF are the area responses for diphenylether and p-phenetidine, respectively; WPF and WDE are theweights, in grams, of p-phenetidine and diphenyl ether, respec-tively; and PPF and PDE are the purities of p-phenetidine anddiphenyl ether, respectively.
Procedure Inject 1.0 �L of sample into the gas chromato-graph, and calculate the content of p-phenetidine, in percent,by the formula
(AP × WD × F)/(WS × AD),
in which AP is the area of the p-phenetidine peak; WD is theweight, in grams, of diphenyl ether in the Sample Preparation;F is the p-phenetidine factor; WS is the weight, in grams, ofthe sample taken; and AD is the area of the diphenyl ether peak.Ethoxyquin-Related Impurities Calculate the quantity, inpercent, of related impurities by the formula
100 − (% Assay + % p-Phenetidine).
Packaging and Storage Store in tightly closed carbon steelor black iron (not rubber, neoprene, or nylon) containers orin polypropylene or polyethylene drums or lined drums ina cool, dark place. Prolonged exposure to sunlight causespolymerization.
Ethyl AlcoholAlcohol; Ethanol
C C OH
H
H
H
H
H
C2H6O Formula wt 46.07
CAS: [64-17-5]
DESCRIPTION
Ethyl Alcohol occurs as a clear, colorless, mobile liquid. Itis miscible with water, with ether, and with chloroform. Itboils at about 78° and is flammable. Its refractive index at20° is about 1.364.
Note: This monograph applies only to undenaturedethyl alcohol.
Function Extraction solvent; carrier solvent.
REQUIREMENTS
Assay Not less than 94.9% by volume (92.3% by weight)of C2H6O.Acidity (as acetic acid) Not more than 0.003%.
Alkalinity (as NH3) Not more than 3 mg/kg.Fusel Oil Passes test.Ketones, Isopropyl Alcohol Passes test.Lead Not more than 0.5 mg/kg.Methanol Passes test.Nonvolatile Residue Not more than 0.003%.Solubility in Water Passes test.Substances Darkened by Sulfuric Acid Passes test.Substances Reducing Permanganate Passes test.
TESTS
Assay The specific gravity of a sample, determined by anyreliable method (see General Provisions), is not greater than0.8096 at 25°/25° (equivalent to 0.8161 at 15.56°/15.56°).Acidity Transfer 10 mL of sample to a glass-stoppered flaskcontaining 25 mL of water, add 0.5 mL of phenolphthaleinTS, and then add 0.02 N sodium hydroxide to the first appear-ance of a pink color that persists after shaking for 30 s.Add 25 mL of sample, mix, and titrate with 0.02 N sodiumhydroxide until the pink color is restored. Not more than 0.5mL of 0.02 N sodium hydroxide is required to restore thepink color.Alkalinity Add 2 drops of methyl red TS to 25 mL of water,add 0.02 N sulfuric acid until a red color just appears, thenadd 25 mL of sample, and mix. Not more than 0.2 mL of0.02 N sulfuric acid is required to restore the red color.Fusel Oil Mix 10 mL of sample with 1 mL of glycerin and1 mL of water, and allow to evaporate from a piece of clean,odorless, absorbent paper. No foreign odor is perceptible whenthe last traces of alcohol leave the paper.Ketones, Isopropyl Alcohol Transfer 1 mL of sample, 3mL of water, and 10 mL of mercuric sulfate TS to a test tube;mix; and heat in a boiling water bath. No precipitate formswithin 3 min.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 10-g sample.Methanol Transfer 1 drop of sample to a test tube, add 1drop of 1:20 phosphoric acid and 1 drop of 1:20 potassiumpermanganate solution; mix; and allow to stand for 1 min.Add, dropwise, 1:10 sodium bisulfite solution until the per-manganate color disappears. If a brown color remains, add 1drop of the phosphoric acid solution. Add 5 mL of freshlyprepared chromotropic acid TS to the colorless solution, andheat it in a water bath at 60° for 10 min. No violet colorappears.Nonvolatile Residue Evaporate 125 mL (about 100 g) ofsample to dryness in a tared dish on a steam bath, dry theresidue at 105° for 30 min, cool, and weigh.Solubility in Water Transfer 50 mL of sample to a 100-mL glass-stoppered graduate, dilute to 100 mL with water,and mix. Place the graduate in a water bath maintained at10°, and allow it to stand for 30 min. No haze or turbiditydevelops.Substances Darkened by Sulfuric Acid Transfer 10 mLof sulfuric acid into a small Erlenmeyer flask, cool to 10°,and with constant agitation, add 10 mL of sample, dropwise.
158 / Ethyl Cellulose / Monographs FCC V
The mixture is colorless or has no more color than either theacid or the sample before mixing.Substances Reducing Permanganate Transfer 20 mL ofsample, previously cooled to 15°, to a glass-stoppered cylin-der, add 0.1 mL of 0.1 N potassium permanganate, mix, andallow to stand for 5 min. The pink color does not entirelydisappear.
Packaging and Storage Store in tight containers, remotefrom fire.
Ethyl CelluloseModified Cellulose, EC
INS: 462 CAS: [9004-57-3]
DESCRIPTION
Ethyl Cellulose occurs as a free-flowing, white to light tanpowder. It is heat-labile, and exposure to high temperatures(240°) causes color degradation and loss of properties. It ispractically insoluble in water, in glycerin, and in propyleneglycol, but is soluble in varying proportions in certain organicsolvents, depending on the ethoxyl content. Ethyl Cellulosecontaining less than 46% to 48% of ethoxyl groups is freelysoluble in tetrahydrofuran, in methyl acetate, in chloroform,and in aromatic hydrocarbon–alcohol mixtures. Ethyl Cellu-lose containing 46% to 48% or more of ethoxyl groups is freelysoluble in alcohol, in methanol, in toluene, in chloroform, andin ethyl acetate. A 1:20 aqueous suspension is neutral tolitmus.
Function Protective coating; binder; filler.
REQUIREMENTS
Identification Dissolve 5 g of sample in 95 g of an 80:20(w/w) mixture of toluene:ethanol. A clear, stable, slightlyyellow solution forms. Pour a few milliliters of the solutiononto a glass plate, and allow the solvent to evaporate. A thick,tough, clear, flammable film remains.Assay Not less than 44.0% and not more than 50.0% ofethoxyl groups (—OC2H5) after drying (equivalent to notmore than 2.6 ethoxyl groups per anhydroglucose unit).Lead Not more than 3 mg/kg.Loss on Drying Not more than 3%.Residue on Ignition Not more than 0.4%.Viscosity Not less than 90% and not more than 110% ofthe viscosity stated on the label as 10 centipoises or more;not less than 80% and not more than 120% of the viscositystated on the label as 10 centipoises or fewer.
TESTS
Assay Place about 50 mg of sample, previously dried at105° for 2 h, in a tared gelatin capsule, accurately weigh,
transfer the capsule and its contents into the boiling flask ofa methoxyl determination apparatus, and proceed as directedunder Methoxyl Determination, Appendix IIIC. Each milliliterof 0.1 N sodium thiosulfate is equivalent to 751 �g of ethoxylgroups (—OC2H5).Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared from a 2-g sampleas directed for organic compounds, and 6 �g of lead (Pb) ionin the control.
Alternatively, determine as directed for Flame Atomic Ab-sorption Spectrophotometric Method under Lead Limit Test,Appendix IIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Viscosity
Solvent Systems For Ethyl Cellulose containing less than46% to 48% of ethoxyl groups, prepare a solvent consisting ofa 60:40 (w/w) mixture of toluene:alcohol; for Ethyl Cellulosecontaining 46% to 48% or more of ethoxyl groups, preparea solvent consisting of an 80:20 (w/w) mixture of toluene:al-cohol.
Procedure Transfer 5.0 g of sample, previously dried at105° for 2 h and accurately weighed, into a bottle containing95 � 0.5 g of the appropriate solvent system. Shake or tumblethe bottle until the sample is completely dissolved, and thenadjust the temperature of the solution to 25° � 0.1°. Determinethe viscosity as directed under Viscosity of Methylcellulose,Appendix IIB, but make all determinations at 25° instead ofat 20°.
Packaging and Storage Store in well-closed containers.
Ethylene Dichloride1,2-Dichloroethane
C C
H
H
Cl
H
H
Cl
C2H4Cl2 Formula wt 98.96
CAS: [107-06-2]
DESCRIPTION
Ethylene Dichloride occurs as a clear, colorless, flammable,oily liquid. It is slightly soluble in water, and is soluble inalcohol, in ether, and in acetone. Its refractive index at 20°is about 1.445.
Function Extraction solvent.
FCC V Monographs / Eucalyptus Oil / 159
REQUIREMENTS
Acidity (as HCl) Not more than 10 mg/kg.Distillation Range Between 82° and 85°.Free Halogens Passes test.Lead Not more than 1 mg/kg.Nonvolatile Residue Not more than 0.002%.Specific Gravity Between 1.245 and 1.255.Water Not more than 0.03%.
TESTS
Acidity (as HCl) Transfer 25 mL of alcohol to a 100-mLglass-stoppered flask, add 2 drops of phenolphthalein TS, andtitrate with 0.01 N sodium hydroxide to the first appearanceof a slight pink color. Add 25 mL of sample, mix, and titratewith 0.01 N sodium hydroxide until the faint pink color isrestored. Not more than 0.85 mL of 0.01 N sodium hydroxideis required to restore the pink color.Distillation Range Determine as directed under DistillationRange, Appendix IIB.Free Halogens Mix 10 mL of sample with 10 mL of 10%potassium iodide solution and 1 mL of starch TS. Shake themixture vigorously for 2 min. A blue color does not appearin the water layer.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 10-g sample.Nonvolatile Residue Evaporate 80 mL (about 100 g) ofsample to dryness in a tared dish on a steam bath, dry theresidue at 105° for 30 min, cool, and weigh.
Caution: Use an appropriate fume hood.
Specific Gravity Determine by any reliable method (seeGeneral Provisions).Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers.
Ethyl Maltol2-Ethyl-3-hydroxy-4-pyrone
O C2H5
OH
O
C7H8O3 Formula wt 140.14
INS: 637 CAS: [4940-11-8]
FEMA: 3487
DESCRIPTION
Ethyl Maltol occurs as a white, crystalline powder having acotton-candy odor and a sweet, fruitlike flavor in dilute solu-
tion. One gram dissolves in about 55 mL of water, 10 mL ofalcohol, 17 mL of propylene glycol, and 5 mL of chloroform.It melts at about 90°.
Function Flavoring agent; flavor enhancer.
REQUIREMENTS
Identification The infrared absorption spectrum of a 1:50solution in chloroform, determined in a 0.1-mm cell, exhibitsrelative maxima at the same wavelengths as those of USPEthyl Maltol Reference Standard, similarly prepared.Assay Not less than 99.0% of C7H8O3, calculated on theanhydrous basis.Residue on Ignition Not more than 0.2%.Water Not more than 0.5%.
TESTS
AssayStandard Solution Dissolve about 50 mg of USP Ethyl
Maltol Reference Standard, accurately weighed, in sufficient0.1 N hydrochloric acid to make 250.0 mL, and mix. Transfer5.0 mL of this solution into a 100-mL volumetric flask, diluteto volume with 0.1 N hydrochloric acid, and mix.
Assay Solution Dissolve about 50 mg of sample, accu-rately weighed, in sufficient 0.1 N hydrochloric acid to make250.0 mL, and mix. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, dilute to volume with 0.1 N hydrochloricacid, and mix.
Procedure Using a suitable spectrophotometer and 0.1 Nhydrochloric acid as the blank, determine the absorbance ofeach solution in a 1-cm cell at the wavelength of maximumabsorption (about 276 nm). Calculate the quantity, in milli-grams, of C7H8O3 in the sample taken by the formula
5C(AU/AS),
in which C is the concentration, in micrograms per milliliter,of USP Ethyl Maltol Reference Standard in the StandardSolution; AU is the absorbance of the Assay Solution; and AS
is the absorbance of the Standard Solution.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers.
Eucalyptus OilCAS: [8000-48-4]
DESCRIPTION
Eucalyptus Oil occurs as a colorless or pale yellow liquid witha characteristic, aromatic, somewhat camphoraceous odor and
View IR
160 / Fast Green / Monographs FCC V
a pungent, spicy, cooling taste. It is the volatile oil obtained bysteam distillation from the fresh leaves of Eucalyptus globulusLabillardiere and other species of Eucalyptus L’Heritier (Fam.Myrtaceae).
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 70.0% of cineole (C10H18O).Phellandrene Passes test.Refractive Index Between 1.458 and 1.470 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.905 and 0.925.
TESTS
Assay Transfer about 3 g of sample, previously dried withanhydrous sodium sulfate and accurately weighed, into a 25-× 150-mm test tube. Add 2.100 g of melted o-cresol that ispure and dry, with a solidification point of 30° or higher, tothe sample.
Note: Moisture in the o-cresol may cause low results.
Stir the mixture with a thermometer (see Thermometers, Ap-pendix I) to induce crystallization, and note the highest tem-perature reading obtained. Warm the tube gently until thecontents are completely melted, then insert the test tube intoan apparatus assembled as directed under Solidification Point,Appendix IIB. Allow the mixture to cool slowly until crystalli-zation starts, or until the temperature has fallen to the pointnoted above. Stir the mixture vigorously with the thermometer,rubbing the sides of the test tube with an up and down motionto induce crystallization. Continue the stirring and rubbinguntil the temperature no longer rises. Record the highest tem-perature obtained as the solidification point. Repeat the proce-dure until two results agreeing within 0.1° are obtained. Calcu-late the percentage of cineole from the Percentage of Cineoletable, Appendix VI.Phellandrene Mix 2.5 mL of sample with 5 mL of solventhexane, add 5 mL of a solution of sodium nitrite (made bydissolving 5 g of sodium nitrite in 8 mL of water), and gradu-ally add 5 mL of glacial acetic acid. No crystals form in themixture within 10 min.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 5 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in well-filled, tight containersin a cool place protected from light.
Fast Green1
Fast Green FCF; CI 42053; Class: Triphenylmethane
HO
SO3NaNCH2
NCH2
C2H5
C2H5SO3Na
SO3
C37H34N2O10S3Na2 Formula wt 808.86
INS: 143 CAS: [2353-45-9]
DESCRIPTION
Fast Green occurs as a red to brown-violet powder or granules.It is principally the inner disodium salt of N-ethyl-N-[4-[[4-[ethyl[(3-sulfophenyl)methyl]amino]phenyl](4-hydroxy-2-sulfophenyl)methylene]-2,5-cyclohexadien-1-ylidene]-3-sul-fobenzenemethanaminium hydroxide. It dissolves in water togive a solution blue-green at neutrality, green in acid, and blue-violet inbase. When dissolved in sulfuric acid, it yieldsa brown-orange solution that turns green when diluted with water. Whenheated to 130° with triacetin and an excess of acetic anhydride,acetylation of its phenolic hydroxyl group causes a color changefrom green to light blue. It is slightly soluble in ethanol.
Function Color.
REQUIREMENTS
Identification A freshly prepared aqueous solution con-taining 5 mg of sample per liter exhibits absorbance intensities(A) and wavelength maxima as follows: at pH 7, A = 0.80 at624 nm, and A = 0.08 at 423 nm; at pH 1, A = 0.83 at 625nm, and A = 0.09 at 423 nm; and at pH 13, A = 0.74 at 610 nm.Assay Not less than 85.0% total coloring matter.Arsenic Not more than 3 mg/kg.Chromium Not more than 0.005%.Ether Extracts (combined) Not more than 0.4%.Lead Not more than 10 mg/kg.Leuco Base Not more than 5.0%.Loss on Drying (Volatile Matter) at 135°, Chlorides andSulfates (as sodium salts) Not more than 15.0% in combi-nation.
1To be used or sold for use to color food that is marketed in the UnitedStates, this color additive must be from a batch that has been certifiedby the U.S. Food and Drug Administration (FDA). If it is not from anFDA-certified batch, it is not a permitted color additive for food use inthe United States, even if it is compositionally equivalent. The nameFD&C Green No. 3 can be applied only to FDA-certified batches of thiscolor additive. Fast Green is a common name given to the uncertifiedcolorant. See the monograph entitled FD&C Green No. 3 for directionsfor producing an FDA-certified batch.
FCC V Monographs / Fast Green / 161
Subsidiary Colors Not more than 6.0%, total, of isomericinner salt disodium salt of N-ethyl-N-[4-[[4-[ethyl[(3-sulfo-phenyl)methyl]amino]phenyl](4-hydroxy-2-sulfophenyl)-methylene]-2,5-cyclohexadien-1-ylidene]-4-sulfobenzene-methanaminium hydroxide, N-ethyl-N-[4-[[4-[ethyl[(4-sulfo-phenyl)methyl]amino]phenyl](4-hydroxy-2-sulfophenyl)-methylene]-2,5-cyclohexadien-1-ylidene]-4-sulfobenzene-methanaminium hydroxide, and N-ethyl-N-[4-[[4-[ethyl[(2-sulfophenyl)methyl]amino]phenyl](4-hydroxy-2-sulfophe-nyl)methylene]-2,5-cyclohexadiene-1-ylidene]-3-sulfoben-zenemethanaminium hydroxide.Uncombined Intermediates and Products of Side Reactions
Sum of 3- and 4-[[Ethyl(4-sulfophenyl)amino]methyl]ben-zenesulfonic Acid Disodium Salts Not more than 0.3%.
Sum of 2-, 3-, and 4-Formyl Benzenesulfonic Acids, SodiumSalts Not more than 0.5%.
2-Formyl-5-hydroxybenzenesulfonic Acid, Sodium SaltNot more than 0.5%.Water-Insoluble Matter Not more than 0.2%.
TESTS
Assay Determine the total color strength as the weight per-cent of the sample using Methods I and II in Total Colorunder Colors, Appendix IIIC. Express the Total Color as theaverage of the two results.
Method I (Sample Preparation) Transfer 50 to 75 mgof sample, accurately weighed, into a 1-L volumetric flask;dissolve in and dilute to volume with water. The absorptivity(a) for Fast Green is 0.156 mg/L/cm at 625 nm.
Method II (Sample Preparation) Transfer approximately0.5 g of sample, accurately weighed, into the titration flask.The stoichiometric factor (Fs) for Fast Green is 2.47.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Chloride Determine as directed in Sodium Chloride underColors, Appendix IIIC.Chromium Determine as directed in Chromium under Col-ors, Appendix IIIC.Ether Extracts Determine as directed in Ether Extractsunder Colors, Appendix IIIC.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Leuco Base Determine as directed in Leuco Base underColors, Appendix IIIC, using the following Sample Solution:Transfer approximately 130 mg of sample, accuratelyweighed, into a 1-L volumetric flask; dissolve in and diluteto volume with water.Loss on Drying (Volatile Matter) at 135°, Chlorides andSulfates (as sodium salts) Determine as directed in Loss onDrying (Volatile Matter) under Colors, Appendix IIIC.Subsidiary Colors
Solvent System Use a solvent system composed of 50 mLof acetonitrile, 50 mL of isoamyl alcohol, 15 mL of 2-buta-none, 10 mL of water, and 5 mL of ammonium hydroxide.
Sample Solution Transfer approximately 1 g of sample,accurately weighed, into a 100-mL volumetric flask. Fill the
flask about ¾ full with water, and incubate in the dark for 1h; dilute to volume with water, and mix well.
Procedure Spot 0.1 mL of the Sample Solution in a lineacross a 20- × 20-cm glass plate coated with a 0.25-mm layerof Silica Gel G, approximately 3 cm from the bottom edge.Allow the plate to dry for about 20 min in the dark, thendevelop with the Solvent System in an unlined tank equilibratedfor at least 20 min before the plate is inserted. Allow thesolvent front to reach within about 3 cm of the top of theplate. Dry the developed plate in the dark.
When the plate has dried, scrape off all the colored bandsabove the Fast Green, which remains close to the origin, intoa 30-mL beaker. Extract the subsidiary colors with three 6-mL portions of 95% ethanol, or until no color remains on thegel by visual inspection. Record the volume of ethanol usedand the spectrum of the extract between 400 and 700 nm.Calculate the percent of subsidiary colors (P) by the equation
P = (A × V × 100)/(a × W × b),
in which A is the absorbance at the wavelength maximum; Vis the volume, in milliliters, of the extract; a is the absorptivity(0.126 mg/L/cm); W is the weight, in milligrams, of the sam-ple; and b is the path-length, in centimeters, of the cell.Sulfate Determine as directed in Sodium Sulfate under Col-ors, Appendix IIIC.Uncombined Intermediates and Products of Side Reac-tions Determine as directed for Method I under UncombinedIntermediates and Products of Side Reactions under Colors,Appendix IIIC, except use the following as the Sample Solu-tion: Transfer approximately 2 g of sample, accuratelyweighed, into a 100-mL volumetric flask; dissolve in anddilute to volume with water. Replace the Calculation withthe following: Calculate the amounts of intermediates andother products present using the following absorptivities afteridentifying the unknowns by comparing their spectra withstandards:
4-Hydroxy-2-sulfobenzaldehyde: a = 0.080 mg/L/cm at 335nm (alkaline solution).
m-Sulfobenzaldehyde: a = 0.495 mg/L/cm at 246 nm (acidicsolution).
N-Ethyl-N-(3-sulfobenzyl)-sulfanilic Acid: a = 0.078 mg/L/cm at 277 nm (alkaline solution).Water-Insoluble Matter Determine as directed in Water-Insoluble Matter under Colors, Appendix IIIC.
Packaging and Storage Store in well-closed containers.
162 / FD&C Blue No. 1 / Monographs FCC V
FD&C Blue No. 11
Brilliant Blue FCF;2 CI 42090;1 Class: Triphenylmethane
SO3
C
N
C2H5
CH2
N
C2H5
CH2
SO3Na
SO3Na
C37H34N2O9S3Na2 Formula wt 792.86
INS: 133 CAS: [3844-45-9]
DESCRIPTION
FD&C Blue No. 1 is principally the disodium salt of ethyl[4-[p-[ethyl(m-sulfobenzyl)amino]-�-(o-sulfophenyl)benzyli-dene]-2,5-cyclohexadien-1-ylidene](m-sulfobenzyl)ammo-nium hydroxide inner salt, with smaller amounts of the isomericdisodium salts of ethyl[4-[p-[ethyl(p-sulfobenzyl)amino]-�-(o-sulfophenyl)benzylidene]-2,5-cyclohexadien-1-ylidene](p-sulfobenzyl)ammonium hydroxide inner salt and ethyl[4-[p-[ethyl(o-sulfobenzyl)amino]-�-(o-sulfophenyl)benzyli-dene]-2,5-cyclohexadien-1-ylidene](o-sulfobenzyl)ammo-nium hydroxide inner salt.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Blue No. 1 dissolved in 0.04 N aqueous ammoniumacetate has a wavelength maximum of 630 nm, with an absorp-tivity of 0.164 L/(mg·cm).Arsenic (as As) Not more than 3 mg/kg.Chromium (as Cr) Not more than 0.005%.Manganese (as Mn) Not more than 0.01%.Ether Extracts3 (combined) Not more than 0.4%.Lead (as Pb) Not more than 10 mg/kg.Leuco Base Not more than 5%.Subsidiary Colors Not more than 6.0%.Total Color Not less than 85.0%.Uncombined Intermediates and Products of Side Reac-tions
o-, m-, and p-Sulfobenzaldehydes Not more than 1.5%,combined.
1To be used or sold in the United States, this color additive must bebatch certified by the U.S. Food and Drug Administration. The monographtitle is the name of the color additive only after batch certification hasbeen completed.
2Generic designations; not synonyms for certified batches of coloradditive.
3Not required for certification in the United States.
N-Ethyl-N-(m-sulfobenzyl)sulfanilic Acid Not morethan 0.3%.Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 15.0% in combination.Water-Insoluble Matter Not more than 0.2%.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, 5100 Paint Branch Parkway, CollegePark, Maryland 20740.
Packaging and Storage Store in well-closed containers.
FD&C Blue No. 21
Indigotine;2 Indigo Carmine;2 CI 73015;2 Class: Indigoid
C CN C
C
O
H
N
H
O
NaO3S
SO3Na
C16H8N2O8S2Na2 Formula wt 466.36
INS: 132 CAS: [860-22-0]
DESCRIPTION
FD&C Blue No. 2 is principally the disodium salt of 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid, with smaller amounts of thedisodium salt of 2-(1,3-dihydro-3-oxo-7-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid and thesodium salt of 2-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Blue No. 2 dissolved in 0.04 N aqueous ammoniumacetate has a wavelength maximum of 610 nm, with an absorp-tivity of 0.0478 L/(mg·cm).
FCC V Monographs / FD&C Green No. 3 / 163
Arsenic (as As) Not more than 3 mg/kg.Ether Extracts3 (combined) Not more than 0.4%.Lead (as Pb) Not more than 10 mg/kg.Mercury (as Hg) Not more than 1 mg/kg.Subsidiary and Isomeric Colors
Disodium Salt of 2-(1,3-Dihydro-3-oxo-7-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic Acid Notmore than 18%.
Sodium Salt of 2-(1,3-Dihydro-3-oxo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic Acid Not more
than 2%.Total Color Not less than 85%.Decomposition Products
Isatin-5-sulfonic Acid Not more than 0.4%.5-Sulfoanthranilic Acid Not more than 0.2%.
Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 15% in combination.Water-Insoluble Matter Not more than 0.4%.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, U.S. Food and Drug Administration,5100 Paint Branch Parkway, College Park, Maryland 20740.
Packaging and Storage Store in well-closed containers.
FD&C Green No. 31
Fast Green FCF;2 CI 42053;2 Class: Triphenylmethane
C
N
C2H5
CH2
N CH2
SO3Na
SO3
HO C2H5
SO3Na
C37H34N2O10S3Na2 Formula wt 808.86
INS: 143 CAS: [2353-45-9]
DESCRIPTION
FD&C Green No. 3 is principally the inner salt disodium saltof N-ethyl-N-[4-[[4-[ethyl[(3-sulfophenyl)methyl]amino]-phenyl](4-hydroxy-2-sulfophenyl)methylene]-2,5-cyclohex-adien-1-ylidene]-3-sulfobenzenemethanaminium hydroxide,with smaller amounts of the isomeric inner salt disodium saltof N-ethyl-N-[4-[[4-[ethyl[(3-sulfophenyl)methyl]amino]-phenyl](4-hydroxy-2-sulfophenyl)methylene]-2,5-cyclohex-adien-1-ylidene]-4-sulfobenzenemethanaminium hydroxide;of N-ethyl-N-[4-[[4-[ethyl[(4-sulfophenyl)methyl]amino]-phenyl](4-hydroxy-2-sulfophenyl)methylene]-2,5-cyclohex-adien-1-ylidene]-4-sulfobenzenemethanaminium hydroxide;and of N-ethyl-N-[4-[[4-[ethyl[(2-sulfophenyl)methyl]am-ino]phenyl](4-hydroxy-2-sulfophenyl)methylene]-2,5-cyclo-hexadien-1-ylidene]-3-sulfobenzenemethanaminium hy-droxide.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Green No. 3 dissolved in 0.04 N aqueous ammoniumacetate has a wavelength maximum of 625 nm, with an absorp-tivity of 0.156 L/(mg·cm).Arsenic (as As) Not more than 3 mg/kg.Chromium (as Cr) Not more than 0.005%.Ether Extracts3 (combined) Not more than 0.4%.Lead (as Pb) Not more than 10 mg/kg.Leuco Base Not more than 5%.Mercury (as Hg) Not more than 1 mg/kg.Subsidiary Colors Not more than 6%.Total Color Not less than 85%.
1To be used or sold in the United States, this color additive must bebatch certified by the U.S. Food and Drug Administration. The monographtitle is the name of the color additive only after batch certification hasbeen completed.
2Generic designations; not synonyms for certified batches of coloradditive.
3Not required for certification in the United States.
164 / FD&C Red No. 3 / Monographs FCC V
Uncombined Intermediates and Products of Side Reac-tions
Sum of 3- and 4-[[Ethyl(4-sulfophenyl)amino]methyl]ben-zenesulfonic Acid, Disodium Salts Not more than 0.3%.
Sum of 2-, 3-, and 4-Formylbenzenesulfonic Acids, SodiumSalts Not more than 0.5%.
2-Formyl-5-hydroxybenzenesulfonic Acid, Sodium SaltNot more than 0.5%.Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 15.0% in combination.Water-Insoluble Matter Not more than 0.2%.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, 5100 Paint Branch Parkway, CollegePark, Maryland 20740.
Packaging and Storage Store in well-closed containers.
FD&C Red No. 31
Erythrosine;2 CI 45430;2 Class: Xanthene
I
NaO
I
O
C
I
O
ICOONa
C20H6O5I4Na2 Formula wt 879.86
INS: 127 CAS: [16423-68-0]
DESCRIPTION
FD&C Red No. 3 is principally the monohydrate of 9-(o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetraiodo-3H-xanthen-3-
1To be used or sold in the United States, this color additive must bebatch certified by the U.S. Food and Drug Administration. The monographtitle is the name of the color additive only after batch certification hasbeen completed.
2Generic designations; not synonyms for certified batches of coloradditive.
one disodium salt, with smaller amounts of lower iodinatedfluoresceins.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Red No. 3 dissolved in 0.05% aqueous ammoniumhydroxide has a wavelength maximum of 527 nm, with anabsorptivity of 0.110 L/(mg·cm).Arsenic (as As) Not more than 3 mg/kg.Ether Extracts3 (combined) Not more than 0.2%.Lead (as Pb) Not more than 10 mg/kg.Subsidiary Colors
Monoiodofluoresceins Not more than 1.0%.Other Lower Iodinated Fluoresceins Not more than 9.0%.
Total Color Not less than 87.0%.Uncombined Intermediates and Products of Side Reac-tions
2- (2′,4′ -Dihydroxy-3′,5′ -diiodobenzoyl)benzoic AcidNot more than 0.2%.
Sodium Iodide Not more than 0.4%.Triiodoresorcinol Not more than 0.2%.Unhalogenated Intermediates, Total Not more than 0.1%.
Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 13% in combination.Water-Insoluble Matter Not more than 0.2%.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, 5100 Paint Branch Parkway, CollegePark, Maryland 20740.
Packaging and Storage Store in well-closed containers.
3Not required for certification in the United States.
FCC V Monographs / FD&C Yellow No. 5 / 165
FD&C Red No. 401
Allura Red AC;2 CI 16035;2 Class: Monoazo
SO3Na
OH
N N
OCH3
SO3Na
CH3
C18H14N2O8S2Na2 Formula wt 496.43
INS: 129 CAS: [25956-17-6]
DESCRIPTION
FD&C Red No. 40 is principally the disodium salt of 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naph-thalenesulfonic acid.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Red No. 40 dissolved in 0.04 N aqueous ammoniumacetate has a wavelength maximum of 500 nm, with an absorp-tivity of 0.052 L/(mg·cm).Arsenic (as As) Not more than 3 mg/kg.Lead (as Pb) Not more than 10 mg/kg.Subsidiary Colors
Disodium Salt of 6-Hydroxy-5-[(2-methoxy-5-methyl-4-sul-fophenyl)azo]-8-(2-methoxy-5-methyl-4-sulfophenoxy)-2-naphthalenesulfonic Acid Not more than 1.0%.
Higher Sulfonated Subsidiary Colors (as sodium salts)Not more than 1.0%.
Lower Sulfonated Subsidiary Colors (as sodium salts)Not more than 1.0%.Total Color Not less than 85.0%.Uncombined Intermediates and Products of Side Reac-tions
4-Amino-5-methoxy-o-toluenesulfonic Acid Not morethan 0.2%.
Disodium Salt of 6,6′-Oxybis(2-naphthalenesulfonic Acid)Not more than 1.0%.
Sodium Salt of 6-Hydroxy-2-naphthalenesulfonic AcidNot more than 0.3%.Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 14.0% in combination.Water-Insoluble Matter Not more than 0.2%.
1To be used or sold in the United States, this color additive must bebatch certified by the U.S. Food and Drug Administration. The monographtitle is the name of the color additive only after batch certification hasbeen completed.
2Generic designations; not synonyms for certified batches of coloradditive.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, 5100 Paint Branch Parkway, CollegePark, Maryland 20740.
Packaging and Storage Store in well-closed containers.
FD&C Yellow No. 51
Tartrazine;2 CI 19140;2 Class: Pyrazolone
NaO3S N N C
CHON
SO3Na
N
C COONa
C16H9N4O9S2Na3 Formula wt 534.37
INS: 102 CAS: [1934-21-0]
DESCRIPTION
FD&C Yellow No. 5 is principally the trisodium salt of 4,5-dihydro-5-oxo-1-(4-sulfophenyl)-4-[4-sulfophenyl-azo]-1H-pyrazole-3-carboxylic acid.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Yellow No. 5 dissolved in 0.04 N aqueous ammo-nium acetate has a wavelength maximum of 428 nm, with anabsorptivity of 0.053 L/(mg·cm).Arsenic (as As) Not more than 3 mg/kg.Ether Extracts3 (combined) Not more than 0.2%.Lead (as Pb) Not more than 10 mg/kg.
3Not required for certification in the United States.
166 / FD&C Yellow No. 6 / Monographs FCC V
Mercury (as Hg) Not more than 1 mg/kg.Total Color Not less than 87%.Uncombined Intermediates and Products of Side Reac-tions
4,4′-[4,5-Dihydro-5-oxo-4-[(4-sulfophenyl)-hydrazono]-1H-pyrazol-1,3-diyl]bis[benzenesulfonic Acid], TrisodiumSalt Not more than 1%.
4-[(4′,5-Disulfo[1,1′-biphenyl]-2-yl)hydrazono]-4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylicAcid, Tetrasodium Salt Not more than 1%.
Ethyl or Methyl 4,5-Dihydro-5-oxo-1-(4-sulfophenyl)-4-[(4-sulfophenyl)hydrazono]-1H-pyrazole-3-carboxylate, Di-sodium Salt Not more than 1%.
Sum of 4,5-Dihydro-5-oxo-1-phenyl-4-[(4-sulfophenyl)-azo]-1H-pyrazole-3-carboxylic Acid, Disodium Salt, and 4,5-Dihydro-5-oxo-4-(phenylazo)-1-(4-sulfophenyl)-1H-pyra-zole-3-Carboxylic Acid, Disodium Salt Not more than 0.5%.
4-Aminobenzenesulfonic Acid, Sodium Salt Not morethan 0.2%.
4,5-Dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carb-oxylic Acid, Disodium Salt Not more than 0.2%.
Ethyl or Methyl 4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylate, Sodium Salt Not more than 0.1%.
4,4′-(1-Triazene-1,3-diyl)bis[benzenesulfonic acid], Diso-dium Salt Not more than 0.05%.
4-Aminoazobenzene Not more than 75 �g/kg.4-Aminobiphenyl Not more than 5 �g/kg.Aniline Not more than 100 �g/kg.Azobenzene Not more than 40 �g/kg.Benzidine Not more than 1 �g/kg.1,3-Diphenyltriazene Not more than 40 �g/kg.
Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 13% in combination.Water-Insoluble Matter Not more than 0.2%.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, 5100 Paint Branch Parkway, CollegePark, Maryland 20740.
Packaging and Storage Store in well-closed containers.
FD&C Yellow No. 61
Sunset Yellow FCF;2 CI 15985;2 Class: Monoazo
SO3Na
OH
NNNaO3S
C16H10N2O7S2Na2 Formula wt 452.37
INS: 110 CAS: [2783-94-0]
DESCRIPTION
FD&C Yellow No. 6 is principally the disodium salt of 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid.The trisodium salt of 3-hydroxy-4-[(4-sulfophenyl)azo]-2,7-naphthalenedisulfonic acid may be added in small amounts.
Function Color.
REQUIREMENTS
Identification The visible absorption spectrum of a sampleof FD&C Yellow No. 6 dissolved in 0.04 N aqueous ammo-nium acetate has a wavelength maximum of 484 nm, with anabsorptivity of 0.054 L/(mg·cm).Arsenic (as As) Not more than 3 mg/kg.Ether Extracts3 (combined) Not more than 0.2%.Lead (as Pb) Not more than 10 mg/kg.Mercury (as Hg) Not more than 1 mg/kg.Total Color Not less than 87%.Uncombined Intermediates and Products of Side Reac-tions
4-Aminoazobenzene Not more than 50 �g/kg.4-Aminobiphenyl Not more than 15 �g/kg.Aniline Not more than 250 �g/kg.Azobenzene Not more than 200 �g/kg.Benzidine Not more than 1 �g/kg.1,3-Diphenyltriazene Not more than 40 �g/kg.1-(Phenylazo)-2-naphthalenol Not more than 10 mg/kg.Sodium Salt of 4-Aminobenzenesulfonic Acid Not more
than 0.2%.Sodium Salt of 6-Hydroxy-2-naphthalenesulfonic Acid
Not more than 0.3%.Disodium Salt of 6,6′-Oxybis[2-naphthalenesulfonic Acid]
Not more than 1%.
1To be used or sold in the United States, this color additive must bebatch certified by the U.S. Food and Drug Administration. The monographtitle is the name of the color additive only after batch certification hasbeen completed.
2Generic designations; not synonyms for certified batches of coloradditive.
3Not required for certification in the United States.
FCC V Monographs / Ferric Ammonium Citrate, Brown / 167
Disodium Salt of 4,4′-(1-Triazene-1,3-diyl)bis[benzenesul-fonic Acid] Not more than 0.1%.
Sum of the Sodium Salt of 6-Hydroxy-5-(phenylazo)-2-naphthalenesulfonic Acid and the Sodium Salt of 4-[(2-Hy-droxy-1-naphthalenyl)azo]benzenesulfonic Acid Not morethan 1%.
Sum of the Trisodium Salt of 3-Hydroxy-4-[(4-sulfophe-nyl)azo]-2,7-naphthalenedisulfonic Acid and Other HigherSulfonated Subsidiaries Not more than 5%.Volatile Matter (at 135°) and Chlorides and Sulfates (assodium salts) Not more than 13% in combination.Water-Insoluble Matter Not more than 0.2%.
TESTS
FDA-certifiable color additives are batch certified by theUnited States Food and Drug Administration using analyticalchemistry methods developed for this purpose by the FDA.The color additive regulations are described in Title 21, Parts70 to 82, of the United States Code of Federal Regulations(21 CFR Parts 70 to 82). The batch certification process isdescribed in 21 CFR Part 80. Current certification analyticalmethods are available from the Office of Cosmetics and Col-ors, Colors Certification Branch (HFS-107), U.S. Food andDrug Administration, 5100 Paint Branch Parkway, CollegePark, Maryland 20740.
Packaging and Storage Store in well-closed containers.
Fennel OilCAS: [8006-84-6]
DESCRIPTION
Fennel Oil occurs as a colorless or pale yellow liquid withthe characteristic odor and taste of fennel. It is the volatileoil obtained by steam distillation from the dried ripe fruit ofFoeniculum vulgare Miller (Fam. Umbelliferae).
Note: If solid material has separated, carefully warmthe sample until it is completely liquefied, and mix itbefore using.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between +12° and +24°.Refractive Index Between 1.532 and 1.543 at 20°.Solidification Point Not lower than 3°.
Solubility in Alcohol Passes test.Specific Gravity Between 0.953 and 0.973.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solidification Point Determine as directed under Solidifica-tion Point, Appendix IIB.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 1 mL of 90% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers in acool place protected from light.
Ferric Ammonium Citrate, BrownIron Ammonium Citrate
INS: 381 CAS: [1185-57-5]
DESCRIPTION
Ferric Ammonium Citrate, Brown, occurs as thin, transparentbrown, red-brown, or garnet red scales or granules, or as abrown-yellow powder. It is a complex salt of undeterminedstructure, composed of iron, ammonia, and citric acid. It isvery soluble in water, but is insoluble in alcohol. The pH ofa 1:20 aqueous solution is about 5.0 to 8.0. It is deliquescentin air and is affected by light.
Function Nutrient.
REQUIREMENTS
Identification
A. A 500-mg sample, when ignited, chars and leaves aresidue of iron oxide.
B. Add 0.3 mL of potassium permanganate TS and 4 mLof mercuric sulfate TS to 5 mL of a 1:10 aqueous solution,and heat the mixture to boiling. A white precipitate forms.
C. Dissolve about 500 mg of sample in 5 mL of water, andadd 5 mL of 1 N sodium hydroxide. A red-brown precipitateforms, and ammonia is evolved when the mixture is heated.Assay Not less than 16.5% and not more than 18.5% ofiron (Fe).Ferric Citrate Passes test.Lead Not more than 2 mg/kg.
View IR
168 / Ferric Ammonium Citrate, Brown / Monographs FCC V
Mercury Not more than 1 mg/kg.Oxalate Passes test.Sulfate Not more than 0.3%.
TESTS
Assay Transfer about 1 g of sample, accurately weighed,into a 250-mL glass-stoppered Erlenmeyer flask, and dissolvein 25 mL of water and 5 mL of hydrochloric acid. Add 4 gof potassium iodide, stopper, and allow to stand protectedfrom light for 15 min. Add 100 mL of water, and titrate theliberated iodine with 0.1 N sodium thiosulfate, using starch TSas the indicator. Perform a blank determination (see GeneralProvisions), and make any necessary correction. Each millili-ter of 0.1 N sodium thiosulfate is equivalent to 5.585 mg ofiron (Fe).Ferric Citrate Add potassium ferrocyanide TS to a 1:100aqueous solution. No blue precipitate forms.Lead (Note: The following method has been found to besatisfactory when the particular atomic absorption spectropho-tometer specified is used. The method may be modified asnecessary for use with other suitable atomic absorption spec-trophotometers capable of determining lead in the sample atthe limit specified.)
Lead Nitrate Stock Solution Dissolve 159.8 mg of ACSreagent-grade lead nitrate [Pb(NO3)2] in 100 mL of watercontaining 1 mL of nitric acid, dilute to 1000.0 mL with water,and mix. Prepare and store this solution in glass containers thatare free from lead salts.
Standard Preparation Transfer 2.0 mL of Lead NitrateStock Solution into a 500-mL volumetric flask, dilute to vol-ume with water, and mix. This solution should be preparedon the day of use. Each milliliter contains the equivalent of0.4 �g of lead ion (Pb).
Sample Preparation Transfer about 20 g of sample, accu-rately weighed, into a 100-mL volumetric flask (previouslyrinsed with nitric acid and water), dissolve in a mixture of50 mL of water and 1 mL of nitric acid, dilute to volumewith water, and mix.
Procedure Use a Perkin-Elmer 403 atomic absorptionspectrophotometer equipped with a deuterium arc backgroundcorrector, a digital readout device, and a burner head capableof handling 20% solids content. Blank the instrument withwater following the manufacturer’s operating instructions. As-pirate a portion of the Standard Preparation, and record theabsorbance as AS; then aspirate a portion of the Sample Prepa-ration, and record the absorbance as AU. Calculate the leadcontent, in milligrams per kilogram, of the sample taken bythe formula
100 × (C/W) × (AU/AS),
in which C is the concentration, in micrograms per milliliter,of lead in the Standard Preparation, and W is the weight, ingrams, of the sample taken.Mercury
Standard Preparations Prepare a solution containing 1 �gof mercury per milliliter as directed for Standard Preparationunder Mercury Limit Test, Appendix IIIB. Pipet 0.25, 0.50,1.0, and 3.5 mL of this solution, respectively, into each of
four glass-stoppered bottles of about 300-mL capacity, suchas BOD (biological oxygen demand) bottles. Dilute the con-tents of each bottle to 100 mL with water, and mix. Thesesolutions contain the equivalent of 0.25, 0.50, 1.0, and 3.5mg/kg of mercury, respectively.
Sample Preparation Transfer 1.000 g of sample into a200-mL screw-cap centrifuge bottle, and add 5 mL of nitricacid and 5 mL of hydrochloric acid. Close the bottle tightlywith a Teflon-lined screw-cap, digest on a steam bath for1 h, and cool. Quantitatively transfer into a suitable glass-stoppered bottle (see Standard Preparations), dilute to 100mL with water, and bubble air through the sample for 2 min.Prepare a reagent blank in the same manner.
10% Stannous Chloride Solution Dissolve 20 g of stan-nous chloride (SnCl2·2H2O) in 40 mL of warm hydrochloricacid, and dilute with 160 mL of water. Prepare fresh eachweek.
Procedure (Note: The Apparatus and Procedure de-scribed under Mercury Limit Test, Appendix IIIB, may besuitably modified for this determination.) Use a suitableatomic absorption spectrophotometer assembly designed formercury analysis, such as the Coleman MAS-50 MercuryAnalyzer. Add 5 mL of 10% Stannous Chloride Solution tothe solution to be tested, and immediately insert the bubblerof the mercury analysis apparatus. Obtain the absorbancereading by following the instrument manufacturer’s operatinginstructions. Correct the sample readings for the reagent blank,and determine the mercury concentration of the Sample Prepa-ration from a standard curve prepared by plotting the readingsobtained with the Standard Preparations against mercury con-centration, in milligrams per kilogram.Oxalate Transfer 1 g of sample into a 125-mL separator,dissolve in 10 mL of water, add 2 mL of hydrochloric acid,and extract successively with one 50-mL portion and one 20-mL portion of ether. Transfer the combined ether extracts toa 150-mL beaker, add 10 mL of water, and remove the etherby evaporation on a steam bath. Add 1 drop of glacial aceticacid and 1 mL of a 1:20 calcium acetate solution to the residualaqueous solution. No turbidity develops within 5 min.Sulfate Dissolve 100 mg of sample in 2.7 N hydrochloricacid, and dilute to 30 to 40 mL with water. Proceed as directedin the Sulfate Limit Test under Chloride and Sulfate LimitTests, Appendix IIIB, beginning with the addition of 3 mLof barium chloride TS. Any turbidity produced does not ex-ceed that shown in a control containing 300 �g of sulfate(SO4).
Packaging and Storage Store in tight, light-resistant con-tainers in a cool place.
FCC V Monographs / Ferric Phosphate / 169
Ferric Ammonium Citrate, GreenIron Ammonium Citrate
INS: 381 CAS: [1185-57-5]
DESCRIPTION
Ferric Ammonium Citrate, Green, occurs as thin, transparentgreen scales, as granules, as a powder, or as transparent greencrystals. It is a complex salt of undetermined structure, com-posed of iron, ammonia, and citric acid. It is very soluble inwater, but is insoluble in alcohol. Its solutions are acid tolitmus. It may deliquesce in air and is affected by light.
Function Nutrient; anticaking agent for sodium chloride.
REQUIREMENTS
Identification A sample responds to the Identification Testsin the monograph for Ferric Ammonium Citrate, Brown.Assay Not less than 14.5% and not more than 16.0% ofiron (Fe).Ferric Citrate Passes test.Lead Not more than 2 mg/kg.Mercury Not more than 1 mg/kg.Oxalate Passes test.Sulfate Not more than 0.3%.
TESTS
Determine as directed for the respective tests in the monographfor Ferric Ammonium Citrate, Brown.
Packaging and Storage Store in tight, light-resistant con-tainers in a cool place.
Ferric CitrateFeC6H5O7·xH2O Formula wt, anhydrous 244.95
CAS: anhydrous [2338-05-8]
DESCRIPTION
Ferric Citrate occurs as brown granules or as thin, transparent,garnet red scales. It is more readily soluble in hot water thanin cold, but it is insoluble in alcohol.
Function Nutrient.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Ferric Iron and for Citrate, Appendix IIIA.
Assay Not less than 16.5% and not more than 18.5% offerric Fe.Alkali Citrate Negative.Ammonia Negative.Chloride Negative.Lead Not more than 2 mg/kg.Sulfate Negative.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in a mixture of 5 mL of hydrochloric acid and 25 mL ofwater contained in a glass-stoppered flask, warming to aiddissolution, if necessary. Cool, add 4 g of potassium iodide,insert the stopper in the flask, and allow the solution to standfor 15 min. Dilute with 100 mL of water, and titrate theliberated iodine with 0.1 N sodium thiosulfate, adding 3 mLof starch TS as the endpoint is approached. Perform a blankdetermination (see General Provisions), and make any neces-sary correction. Each milliliter of 0.1 N sodium thiosulfate isequivalent to 5.585 mg of Fe.Alkali Citrate Ignite about 500 mg of sample until it isthoroughly charred, cool, and add 2 mL of hot water. Thewater is neutral or shows only a slight alkaline reaction tolitmus.Ammonia Heat 500 mg of sample with 5 mL of 1 N sodiumhydroxide. The odor of ammonia is not perceptible.Chloride Heat 1 g of sample with 25 mL of water and 2mL of nitric acid until the sample dissolves. Cool, dilute withwater to 100 mL, and mix. Add 1 mL of silver nitrate TS to10 mL of the solution. No turbidity immediately develops.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 1-g sample.Sulfate Add 1 mL of barium chloride TS to 10 mL of thesolution obtained in the test for Chloride (above). No turbiditydevelops within 15 s.
Packaging and Storage Store in well-closed containers.
Ferric PhosphateIron Phosphate; Ferric Orthophosphate
FePO4·xH2O Formula wt, anhydrous 150.82
CAS: [10045-86-0]
DESCRIPTION
Ferric Phosphate occurs as a yellow-white to buff coloredpowder. It contains from one to four molecules of water ofhydration. It is insoluble in water and in glacial acetic acid,but is soluble in mineral acids.
170 / Ferric Phosphate / Monographs FCC V
Function Nutrient.
REQUIREMENTS
Identification Dissolve 1 g of sample in 5 mL of 1:2 hydro-chloric acid, and add an excess of 1 N sodium hydroxide. Ared-brown precipitate forms. Boil the mixture, filter to removethe iron, and strongly acidify a portion of the filtrate withhydrochloric acid. Cool, mix with an equal volume of magne-sia mixture TS, and treat with a slight excess of 6 N ammoniumoxide. An abundant white precipitate forms. This precipitate,after being washed, turns green-yellow when treated with afew drops of silver nitrate TS.Assay Not less than 26.0% and not more than 32.0% of Fe.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 4 mg/kg.Loss on Ignition Not more than 32.5%.Mercury Not more than 3 mg/kg.
TESTS
Assay Dissolve about 3.5 g of sample, accurately weighed,in 75 mL of 1:2 hydrochloric acid, heat to boiling, and boilfor about 5 min. Cool, transfer into a 100-mL volumetricflask, dilute to volume with the dilute hydrochloric acid, andmix. Add 100 mL of the dilute hydrochloric acid to 25.0 mLof this solution, boil again for 5 min, and add, dropwise andwhile stirring, stannous chloride TS to the boiling solutionuntil the iron is just reduced as indicated by the disappearanceof the yellow color. Add 2 drops in excess (but no more) ofthe stannous chloride TS, dilute with about 50 mL of water,and cool to room temperature. While stirring vigorously, add15 mL of a saturated solution of mercuric chloride, and thenallow to stand for 5 min. Add 15 mL of a sulfuric acid–phosphoric acid mixture, prepared by slowly adding 75 mLof sulfuric acid to 300 mL of water, cooling, adding 75 mLof phosphoric acid, and then diluting to 500 mL with water.Mix, add 0.5 mL of barium diphenylamine sulfonate TS,and titrate with 0.1 N potassium dichromate to a red-violetendpoint. Each milliliter of 0.1 N potassium dichromate isequivalent to 5.585 mg of Fe.Arsenic Assemble the special distillation apparatus asshown in Fig. 13 under Arsenic Limit Test, Appendix IIIB.
Sample Solution Transfer 2 g of sample, 50 mL of hydro-chloric acid, and 5 g of cuprous chloride into the distillingflask (B). Reassemble the distillation apparatus and applygentle suction to flask F to produce a continuous stream ofbubbles. Heat the solution in flask B to boiling and distilluntil between 30 and 35 mL of distillate has been collectedin flask D. Quantitatively transfer the distillate to a 100-mLvolumetric flask with the aid of water, dilute to volume withwater, and mix.
Standard Solution Prepare this solution in the same man-ner as the Sample Solution, but use 6.0 mL of Standard ArsenicSolution (see Arsenic Limit Test, Appendix IIIB) in place ofthe sample.
Blank Solution Prepare this solution in the same manneras the Sample Solution, but use 6.0 mL of water in place ofthe sample.
Procedure Transfer 50.0 mL of the Sample Solution intothe generator flask, add 2 mL of a 15:100 solution of potassiumiodide, and continue as directed in the Procedure under Arse-nic Limit Test, Appendix IIIB, beginning with ‘‘[add] 0.5 mLof Stannous Chloride Solution, and mix. . . .’’ Modify theProcedure by using 5.0 g of Devarda’s metal in place of the3.0 g of 20-mesh granular zinc, and maintain the temperatureof the reaction mixture in the generator flask between 25°and 27°. Treat 50.0 mL each of the Standard Solution and ofthe Blank Solution in the same manner and under the sameconditions. Determine the absorbance at 525 nm produced byeach solution as directed under Procedure.
Calculation Calculate the arsenic content (in milligramsper kilogram) of the sample by the formula
3 × (AU − AB)/(AS − AB),
in which AU is the absorbance produced by the Sample Solu-tion, AB is the absorbance produced by the Blank Solution,and AS is the absorbance produced by the Standard Solution.
Note: If AB exceeds 0.300, different samples of reagent-grade cuprous chloride and Devarda’s metal should betested for arsenic content by the procedure describedherein, and lots of these reagents should be selectedthat will give blank readings that do not exceed 0.300.
Fluoride Determine as directed under Fluoride Limit Test,Appendix IIIB, using a 1.0-g sample, accurately weighed.Lead (Note: In preparing all aqueous solutions and in rins-ing glassware before use, use water that has been passedthrough a strong-acid, strong-base, mixed-bed ion-exchangeresin before use. Select all reagents to have as low a leadcontent as practicable, and store all reagent solutions in con-tainers of borosilicate glass. Clean glassware before use bysoaking in warm 8 N nitric acid for 30 min and by rinsingwith deionized water.)
Ascorbic Acid–Sodium Iodide Solution Dissolve 20 g ofascorbic acid and 38.5 g of sodium iodide in water in a 200-mL volumetric flask, dilute with water to volume, and mix.
Trioctylphosphine Oxide Solution (Caution: This solu-tion causes irritation. Avoid contact with eyes, skin, and cloth-ing. Take special precautions in disposing of unused portionsof solutions to which this reagent is added.) Dissolve 5.0 gof trioctylphosphine oxide in 4-methyl-2-pentanone in a 100-mL volumetric flask, dilute with the same solvent to volume,and mix.
Lead Nitrate Stock Solution Dissolve 159.8 mg of ACSreagent-grade lead nitrate [Pb(NO3)2] in 100 mL of watercontaining 1 mL of nitric acid, dilute with water to 1000.0mL, and mix. Prepare and store this solution in glass containersthat are free from lead salts.
Standard Preparation and Blank Transfer 5.0 mL of LeadNitrate Stock Solution to a 100-mL volumetric flask, dilutewith water to volume, and mix. Transfer 2.0 mL of the re-sulting solution to a 50-mL volumetric flask. Add 10 mL of9 N hydrochloric acid and about 10 mL of water to thisvolumetric flask and to a second, empty 50-mL volumetric
FCC V Monographs / Ferric Phosphate / 171
flask (Blank). Add 20 mL of Ascorbic Acid–Sodium IodideSolution and 5.0 mL of Trioctylphosphine Oxide Solution toeach flask, shake for 30 s, and allow the layers to separate.Add water to bring the organic solvent layer into the neck ofeach flask, shake again, and allow the layers to separate.The organic solvent layers are the Blank and the StandardPreparation, and they contain 0.0 and 2.0 �g of lead permilliliter, respectively.
Test Preparation Add 2.5 g of sample, 10 mL of 9 Nhydrochloric acid, about 10 mL of water, 20 mL of AscorbicAcid–Sodium Iodide Solution, and 5.0 mL of Trioctylphos-phine Oxide Solution to a 50-mL volumetric flask, shake for30 s, and allow the layers to separate. Add water to bring theorganic solvent layer into the neck of the flask, shake again,and allow the layers to separate. The organic solvent layer isthe Test Preparation.
Procedure Concomitantly determine the absorbance ofthe Blank, the Standard Preparation, and the Test Preparationat the lead emission line at 283.3 nm, with a suitable atomicabsorption spectrophotometer equipped with a lead hollow-cathode lamp and an air–acetylene flame, using 4-methyl-2-pentanone to set the instrument to zero. In a suitable analysis,the absorbance of the Blank is not greater than 20% of thedifference between the absorbance of the Standard Prepara-tion and the absorbance of the Blank. The absorbance ofthe Test Preparation does not exceed that of the StandardPreparation.Loss on Ignition Ignite a sample at 800° for 1 h.Mercury
Standard Preparations Dissolve 338.5 mg of mercuricchloride, in about 200 mL of water in a 250-mL volumetricflask, add 14 mL of 1:2 sulfuric acid, dilute to volume withwater, and mix. Pipet 10.0 mL of this solution into a 1000-mL volumetric flask containing about 800 mL of water and56 mL of 1:2 sulfuric acid, dilute to volume with water, andmix. Pipet 10.0 mL of the second solution into a second 1000-mL volumetric flask containing 800 mL of water and 56 mLof 1:2 sulfuric acid, dilute to volume with water, and mix.Each milliliter of this diluted stock solution contains 0.1 �gof mercury. Pipet 1.25, 2.50, 5.00, 7.50, and 10.00 mL of thelast solution (equivalent to 0.125, 0.250, 0.500, 0.750, and1.00 �g of mercury, respectively) into five separate 150-mLbeakers. Add 25 mL of aqua regia to each beaker, cover withwatch glasses, heat just to boiling, simmer for about 5 min,and cool to room temperature. Transfer the solutions intoseparate 250-mL volumetric flasks, dilute to volume withwater, and mix. Transfer a 50.0-mL aliquot from each solutioninto five separate 150-mL beakers, and add 1.0 mL of 1:5sulfuric acid and 1.0 mL of a filtered solution of 1:25 potas-sium permanganate solution to each. Heat the solutions justto boiling, simmer for about 5 min, and cool.
Sample Preparation Transfer 5.00 g of sample into a 150-mL beaker, add 25 mL of aqua regia, cover with a watchglass, and allow to stand at room temperature for about 5min. Heat just to boiling, allow to simmer for about 5 min,and cool. Transfer the solution into a 250-mL volumetricflask, dilute to volume with water, and mix.
Note: Disregard any undissolved material that may bepresent.
Transfer a 50.0-mL aliquot of this solution into a 150-mLbeaker, and add 1.0 mL of 1:5 sulfuric acid and 1.0 mL of afiltered solution of 1:25 potassium permanganate. Heat thesolution just to boiling, simmer for about 5 min, and cool.Prepare a reagent blank in the same manner.
Apparatus Use a Mercury Detection Instrument as de-scribed and an Aeration Apparatus as shown in Fig. 16 underMercury Limit Test, Appendix IIIB. For the purposes of thetest described in this monograph, the Techtron AA-1000atomic absorption spectrophotometer, equipped with a 10-cmsilica absorption cell (Beckman Part No. 75144, or equivalent)and coupled with a strip chart recorder (Varian Series A-25,or equivalent), is satisfactory.
Procedure Assemble the Aeration Apparatus as shownin Fig. 16 under Mercury Limit Test, Appendix IIIB. Usemagnesium perchlorate as the absorbent in the absorption cell(e), fill gas washing bottle c with 60 mL of water, and placestopcock b in the bypass position. Connect the assembly tothe 10-cm absorption cell (analogous to f in the figure) of thespectrophotometer, and adjust the air or nitrogen flow rate sothat, in the following procedure, maximum absorption andreproducibility are obtained without excessive foaming inthe test solution. Obtain a baseline reading at 253.7 nm byfollowing the equipment manufacturer’s operating instruc-tions. Using the Techtron AA-1000 spectrophotometer, thefollowing conditions are suitable: slit width: 2 Å; lamp current:3 mA; and scale expansion: × 1. With the strip chart recorder,set the chart speed at 25 in./h and the span at 2 mV. Precondi-tion the apparatus by an appropriate modification of the proce-dures described below for treatment of the test solutions.
Note: The fritted bubbler in gas washing bottle c shouldbe kept immersed in water between determinations.After each determination, wash the bubbler with astream of water.
Treat the blank, each of the Standard Preparations, andthe Sample Preparation as follows: Transfer the solution tobe tested into a 125-mL gas-washing bottle (c), using a fewdrops of 1:10 hydroxylamine hydrochloride solution to re-move any manganese hydroxide from the beaker. Dilute toabout 55 mL with water, and add a magnetic stirring bar.Discharge the permanganate color by adding dropwise thehydroxylamine hydrochloride solution, swirling after eachdrop is added. Add 15.0 mL of 10% stannous chloride solution[prepared by dissolving 20 g of stannous chloride(SnCl2·2H2O) in 40 mL of warm hydrochloric acid and dilut-ing with 160 mL of water], and immediately connect gas-washing bottle c to the aeration apparatus. Switch on themagnetic stirrer, turn stopcock b from the bypass to the aerat-ing position, and obtain the absorbance reading. Disconnectbottle c from the aeration apparatus, discard the solution justtested, wash bottle c and the fritted bubbler with water, andrepeat the procedure with the remaining solutions. Correctthe sample readings for the reagent blank, and determinethe mercury concentration of the Sample Preparation from astandard curve prepared by plotting the readings obtained with
172 / Ferric Pyrophosphate / Monographs FCC V
the Standard Preparations against mercury concentration, inmilligrams per kilogram, suitable adjustments being made fordilution factors.
Packaging and Storage Store in well-closed containers.
Ferric PyrophosphateIron Pyrophosphate
Fe4(P2O7)3·xH2O Formula wt, anhydrous 745.22
CAS: [10058-44-3]
DESCRIPTION
Ferric Pyrophosphate occurs as a tan or yellow-white powder.It is insoluble in water, but is soluble in mineral acids.
Function Nutrient.
REQUIREMENTS
Identification Dissolve 500 mg of sample in 5 mL of 1:2hydrochloric acid, and add an excess of 1 N sodium hydroxide.A red-brown precipitate forms. Allow the solution to standfor several minutes, and then filter, discarding the first fewmilliliters. Add 1 drop of bromophenol blue TS to 5 mL ofthe clear filtrate, and titrate with 1 N hydrochloric acid to agreen color. Add 10 mL of a 1:8 solution of zinc sulfate, andreadjust the pH to 3.8 (green color). A white precipitate forms(distinction from orthophosphates).Assay Not less than 24.0% and not more than 26.0% of Fe.Arsenic Not more than 3 mg/kg.Lead Not more than 4 mg/kg.Loss on Ignition Not more than 20.0%.Mercury Not more than 3 mg/kg.
TESTS
Assay Determine as directed under Assay in the monographfor Ferric Phosphate.Arsenic Prepare and test a 2-g sample as directed underArsenic in the monograph for Ferric Phosphate.Lead Determine as directed under Lead in the monographfor Ferrous Gluconate, using 2.5 g of sample in the TestPreparation.Loss on Ignition Determine as directed under Loss on Igni-tion, Appendix IIC, igniting a sample at 800° for 1 h.Mercury Determine as directed under Mercury in the mono-graph for Ferric Phosphate.
Packaging and Storage Store in well-closed containers.
Ferrous CitrateFeC6H6O7 Formula wt 245.95
CAS: [23383-11-1]
DESCRIPTION
Ferrous Citrate occurs as a slightly gray-green powder or aswhite crystals.
Function Nutrient.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Ferrous Iron and for Citrate, Appendix IIIA.Assay Not less than 22.0% of ferrous Fe.Chloride Not more than 0.2%.Ferric Iron Not more than 3.0%.Lead Not more than 2 mg/kg.Sulfate Not more than 0.06%.
TESTS
Assay Dissolve about 0.4 g of sample, accurately weighed,in 20 mL of 16:100 sulfuric acid, add 5 mL of 85% phosphoricacid, dilute with approximately 50 mL of water, and immedi-ately titrate with 0.1 N ceric sulfate, using orthophenanthrolineTS as the indicator. Perform a blank determination (see Gen-eral Provisions), and make any necessary correction. Eachmilliliter of 0.1 N ceric sulfate is equivalent to 5.585 mg of Fe.Chloride Heat 100 mg of sample, accurately weighed, with25 mL of water and 2 mL of nitric acid until the sampledissolves. Cool, dilute to 100 mL with water, and mix. Take10 mL of this solution, and dilute to 30 to 40 mL with water.Proceed as directed in the Chloride Limit Test under Chlorideand Sulfate Limit Tests, Appendix IIIB, beginning with ‘‘add1 mL of silver nitrate TS. . . .’’ Any turbidity produced doesnot exceed that shown in a control containing 20 �g of chlo-ride (Cl).Ferric Iron Dissolve about 2 g of sample, accuratelyweighed, in a mixture of 100 mL of water and 10 mL ofhydrochloric acid contained in a 250-mL glass-stopperedflask, add 3 g of potassium iodide, shake well, and allow themixture to stand in the dark for 5 min. Titrate any liberatediodine with 0.1 N sodium thiosulfate, using starch TS asthe indicator. Perform a blank determination (see GeneralProvisions), and make any necessary correction. Each millili-ter of 0.1 N sodium thiosulfate is equivalent to 5.585 mg offerric iron.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 1-g sample.Sulfate Dissolve 500 mg of sample, accurately weighed, in1 mL of 2.7 N hydrochloric acid, and dilute to 30 to 40 mLwith water. Proceed as directed in the Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB, beginningwith ‘‘add 3 mL of barium chloride TS. . . .’’ Any turbidity
FCC V Monographs / Ferrous Fumarate / 173
produced does not exceed that shown in a control containing300 �g of sulfate (SO4).
Packaging and Storage Store in well-closed containers.
Ferrous FumarateIron (II) Fumarate
OO
O
O
Fe
C4H2FeO4 Formula wt 169.90
CAS: [141-01-5]
DESCRIPTION
Ferrous Fumarate occurs as a red-orange to red-brown powder.It may contain soft lumps that produce a yellow streak whencrushed. It is soluble in water and in alcohol.
Function Nutrient.
REQUIREMENTS
IdentificationA. Add 25 mL of 1:2 hydrochloric acid to about 1.5 g of
sample, and dilute to 50 mL with water. Heat to effect com-plete solution; then cool; filter on a fine-porosity, sintered-glass crucible; wash the precipitate with 2:100 hydrochloricacid, saving the filtrate for Identification Test B; and dry theprecipitate at 105°. Add 3 mL of water and 7 mL of 1 Nsodium hydroxide to 400 mg of the dried precipitate, and stiruntil solution is complete. Add, dropwise, 2.7 N hydrochloricacid until the solution is just acid to litmus; add 1 g of p-nitrobenzyl bromide and 10 mL of alcohol; and reflux themixture for 2 h. Cool, filter, and wash the precipitate withtwo small portions of a 2:1 alcohol:water mixture, followedby two small portions of water. The precipitate, recrystallizedfrom hot alcohol and dried at 105°, melts at about 152° (seeMelting Range or Temperature, Appendix IIB).
B. A portion of the filtrate obtained in Identification TestA gives positive tests for Iron, Appendix IIIA.Assay Not less than 97.0% and not more than 101.0% ofC4H2FeO4, calculated on the dried basis.Ferric Iron Not more than 2.0%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 1.5%.Mercury Not more than 3 mg/kg.Sulfate Not more than 0.2%.
TESTS
Assay Transfer about 500 mg of sample, accuratelyweighed, into a 500-mL Erlenmeyer flask, add 25 mL of2:5 hydrochloric acid, and heat to boiling. Add, dropwise, asolution of 5.6 g of stannous chloride in 50 mL of 3:10hydrochloric acid until the yellow color disappears, and thenadd 2 drops in excess. Cool the solution in an ice bath toroom temperature, add 8 mL of mercuric chloride TS, andallow to stand for 5 min. Add 200 mL of water, 25 mL of1:2 sulfuric acid, and 4 mL of phosphoric acid; then addorthophenanthroline TS; and titrate with 0.1 N ceric sulfate.Each milliliter of 0.1 N ceric sulfate is equivalent to 16.99mg of C4H2FeO4.Ferric Iron Transfer 2 g of sample into a 250-mL glass-stoppered Erlenmeyer flask, add 25 mL of water and 4 mLof hydrochloric acid, and heat on a hot plate until solution iscomplete. Stopper the flask, and cool to room temperature.Add 3 g of potassium iodide, stopper, swirl to mix, and allowto stand in the dark for 5 min. Remove the stopper, add 75mL of water, and titrate with 0.1 N sodium thiosulfate, addingstarch TS near the endpoint. Not more than 7.16 mL of 0.1N sodium thiosulfate is consumed.Lead (Note: When preparing all aqueous solutions and rins-ing glassware before use, employ water that has been passedthrough a strong-acid, strong-base, mixed-bed ion-exchangeresin before use. Select all reagents to have as low a contentof lead as practicable, and store all reagent solutions in con-tainers of borosilicate glass. Clean glassware before use bysoaking in warm 8 N nitric acid for 30 min and by rinsingwith deionized water.)
Ascorbic Acid–Sodium Iodide Solution Transfer 20 g ofascorbic acid and 38.5 g of sodium iodide into a 200-mLvolumetric flask, dissolve in and dilute to volume with water,and mix.
Trioctylphosphine Oxide Solution (Caution: This reagentcauses irritation. Avoid contact with eyes, skin, and clothing.Take special precautions in disposing of unused portions ofsolutions to which this reagent is added.) Transfer 5.0 g oftrioctylphosphine oxide into a 100-mL volumetric flask. Dis-solve in and dilute to volume with 4-methyl-2-pentanone,and mix.
Lead Nitrate Stock Solution (100 �g/mL) Dissolve 159.8mg of ACS reagent-grade lead nitrate [Pb(NO3)2] in 100 mLof water containing 1 mL of nitric acid, dilute with water to1000.0 mL, and mix. Prepare and store this solution in glasscontainers that are free from lead salts.
Standard Preparation and Blank Preparation Transfer1.0 mL of Lead Nitrate Stock Solution to a 100-mL volumetricflask, dilute with water to volume, and mix. Transfer 2.0 mLof the resulting solution to a 50-mL beaker. Add 6 mL ofnitric acid and 10 mL of perchloric acid each both to thebeaker and to a second, empty beaker acting as the Blank,and evaporate in a fume hood to dryness.
Caution: Handle perchloric acid in an appropriatefume hood.
Cool, dissolve the residues in 10 mL of 9 N hydrochloricacid, and transfer the solutions, with the aid of about 10 mL
174 / Ferrous Gluconate / Monographs FCC V
of water, to separate 50-mL volumetric flasks. Add 20 mLof Ascorbic Acid–Sodium Iodide Solution and 5.0 mL of Trioc-tylphosphine Oxide Solution to each flask, shake for 30 s, andallow the layers to separate. Add water to bring the organicsolvent layer into the neck of each flask, shake again, andallow the layers to separate. The organic solvent layers arethe Blank Preparation and the Standard Preparation, andthey contain 0.0 and 0.4 �g of lead per milliliter, respectively.
Test Preparation Transfer 1.0 g of sample to a 50-mLbeaker, and add 6 mL of nitric acid and 10 mL of perchloricacid. Cover the beaker with a ribbed watch glass, and heatin a fume hood until completely dry. Cool, dissolve the residuein 10 mL of 9 N hydrochloric acid. Transfer the beaker’scontents, with the aid of about 10 mL of water, to a 50-mLvolumetric flask. Add 20 mL of Ascorbic Acid–Sodium IodideSolution and 5.0 mL of Trioctylphosphine Oxide Solution,shake for 30 s, and allow to separate. Add water to bring theorganic solvent layer into the neck of the flask, shake again,and allow to separate. The organic solvent layer is the TestPreparation.
Procedure Concomitantly determine the absorbance ofthe Blank Preparation, the Standard Preparation, and theTest Preparation at the lead emission line at 283.3 nm witha suitable atomic absorption spectrophotometer equipped witha lead hollow-cathode lamp and an air–acetylene flame, using4-methyl-2-pentanone to set the instrument to zero. In a suit-able analysis, the absorbance of the Blank Preparation is notgreater than 20% of the difference between the absorbanceof the Standard Preparation and that of the Blank Preparation.The absorbance of the Test Preparation does not exceed thatof the Standard Preparation.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 16 h.Mercury Determine as directed in Method II under MercuryLimit Test, Appendix IIIB.Sulfate Mix 1 g of sample with 100 mL of water in a 250-mL beaker, and heat on a steam bath, adding hydrochloricacid, dropwise, until complete solution is effected (about 2mL of the acid will be required). Filter the solution, if neces-sary, and dilute the clear solution or filtrate to 100 mL withwater. Heat to boiling, add 10 mL of barium chloride TS,warm on a steam bath for 2 h, cover, and allow to standovernight. If crystals of ferrous fumarate form, warm on asteam bath to dissolve them, then filter through paper, washthe residue with hot water, and transfer the paper containingthe residue to a tared crucible. Char the paper, without burning,and ignite the crucible and its contents at 600° to constantweight. Each milligram of the residue is equivalent to 0.412mg (412 �g) of sulfate (SO4).
Packaging and Storage Store in well-closed containers.
Ferrous GluconateIron (II) Gluconate
HOH2C C C C C
H H OH H
OHHOHOH
COO
2
Fe2
C12H22FeO14·2H2O Formula wt 482.18
INS: 579 CAS: [299-29-6]
DESCRIPTION
Ferrous Gluconate occurs as a fine, yellow-gray or pale green-yellow powder or granules. One gram dissolves in about 10mL of water with slight heating. It is practically insoluble inalcohol. A 1:20 aqueous solution is acid to litmus.
Function Nutrient; color adjunct.
REQUIREMENTS
IdentificationA. Dissolve a quantity of sample in water, heating in a water
bath at 60° if necessary, to obtain a Test Solution containing 10mg/mL. Similarly, prepare a Standard Solution of USP Fer-rous Gluconate Reference Standard in water, diluting to 10mg/mL. Apply separate 5-�L portions of the Test Solutionand the Standard Solution on a suitable thin-layer chromato-graphic plate (see Thin-Layer Chromatography, AppendixIIA) coated with a 0.25-mm layer of chromatographic silicagel, and allow to dry. Develop the chromatogram in a solventsystem consisting of a mixture of alcohol, water, ammoniumhydroxide, and ethyl acetate (50:30:10:10) until the solventfront has moved about three-fourths of the length of the plate.Remove the plate from the chamber, and dry at 110° for 20min. Allow to cool, and spray with a spray reagent preparedas follows: Dissolve 2.5 g of ammonium molybdate in about50 mL of 2 N sulfuric acid in a 100-mL volumetric flask,add 1.0 g of ceric sulfate, swirl to dissolve, dilute with 2 Nsulfuric acid to volume, and mix. After spraying, heat theplate at 110° for about 10 min. The principal spot obtainedfrom the Test Solution corresponds in color, size, and Rf valueto that obtained from the Standard Solution.
B. A 1:20 aqueous solution gives positive tests for FerrousSalts (Iron), Appendix IIIA.Assay Not less than 97.0% and not more than 102.0% ofC12H22FeO14, calculated on the dried basis.Chloride Not more than 0.07%.Ferric Iron Not more than 2.0%.Lead Not more than 2 mg/kg.Loss on Drying Between 6.5% and 10.0%.Mercury Not more than 3 mg/kg.Oxalic Acid Passes test.Reducing Sugars Passes test.Sulfate Not more than 0.1%.
FCC V Monographs / Ferrous Gluconate / 175
TESTS
Assay Dissolve about 1.5 g of sample, accurately weighed,in a mixture of 75 mL of water and 15 mL of 2 N sulfuricacid in a 300-mL Erlenmeyer flask, and add 250 mg of zincdust. Close the flask with a stopper containing a Bunsen valve,allow to stand at room temperature for 20 min, then filterthrough a sintered-glass filter crucible containing a thin layerof zinc dust, and wash the crucible and contents with 10mL of 2 N sulfuric acid, followed by 10 mL of water. Addorthophenanthroline TS, and titrate the filtrate in the suctionflask immediately with 0.1 N ceric sulfate. Perform a blankdetermination (see General Provisions), and make any neces-sary correction. Each milliliter of 0.1 N ceric sulfate is equiva-lent to 44.62 mg of C12H22FeO14.Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB. Dis-solve 1 g of sample in 100 mL of water. Any turbidity pro-duced by a 10-mL portion of this solution does not exceedthat shown in a control containing 70 �g of chloride (Cl) ion.Ferric Iron Dissolve about 5 g of sample, accuratelyweighed, in a mixture of 100 mL of water and 10 mL ofhydrochloric acid in a 250-mL glass-stoppered flask, add 3g of potassium iodide, shake well, and allow to stand in thedark for 5 min. Titrate any liberated iodine with 0.1 N sodiumthiosulfate, using starch TS as the indicator. Each milliliterof 0.1 N sodium thiosulfate is equivalent to 5.585 mg offerric iron.Lead (Note: When preparing all aqueous solutions and rins-ing glassware before use, employ water that has been passedthrough a strong-acid, strong-base, mixed-bed ion-exchangeresin before use. Select all reagents to have as low a contentof lead as practicable, and store all reagent solutions in con-tainers of borosilicate glass. Clean glassware before use bysoaking in warm 8 N nitric acid for 30 min and by rinsingwith deionized water.)
Ascorbic Acid–Sodium Iodide Solution Transfer 20 g ofascorbic acid and 38.5 g of sodium iodide into a 200-mLvolumetric flask, dissolve in and dilute to volume with water,and mix.
Trioctylphosphine Oxide Solution (Caution: This reagentcauses irritation. Avoid contact with eyes, skin, and clothing.Take special precautions in disposing of unused portions ofsolutions to which this reagent is added.) Transfer 5.0 g oftrioctylphosphine oxide to a 100-mL volumetric flask. Dis-solve in and dilute to volume with 4-methyl-2-pentanone,and mix.
Lead Nitrate Stock Solution (100 �g/mL) Transfer 159.8mg of reagent-grade lead nitrate [Pb(NO3)2] to a 1000-mLvolumetric flask, dissolve it in 100 mL of water containing1 mL of nitric acid, and dilute to volume with water.
Standard Preparation and Blank Preparation Transfer1.0 mL of Lead Nitrate Stock Solution into a 100-mL volumet-ric flask, dilute with water to volume, and mix. Transfer 2.0mL of the resulting solution into a 50-mL volumetric flask.Add 10 mL of 9 N hydrochloric acid and about 10 mL ofwater to both the volumetric flask and a second, empty, 50-mL volumetric flask (Blank Preparation). Add 20 mL ofAscorbic Acid–Sodium Iodide Solution and 5.0 mL of Trioctyl-
phosphine Oxide Solution to each flask, shake for 30 s, andallow the layers to separate. Add water to bring the organicsolvent layer into the neck of each flask, shake again, andallow the layers to separate. The organic solvent layers arethe Blank Preparation and the Standard Preparation, andthey contain 0.0 and 0.4 �g of lead per milliliter, respectively.
Test Preparation Add 1.0 g of sample, 10 mL of 9 Nhydrochloric acid, about 10 mL of water, 20 mL of AscorbicAcid–Sodium Iodide Solution, and 5.0 mL of Trioctylphos-phine Oxide Solution to a 50-mL volumetric flask, shake for30 s, and allow the layers to separate. Add water to bring theorganic solvent layer into the neck of the flask, shake again,and allow the layers to separate. The organic solvent layer isthe Test Preparation.
Procedure Concomitantly determine the absorbance ofthe Blank Preparation, the Standard Preparation, and theTest Preparation at the lead emission line at 283.3 nm, witha suitable atomic absorption spectrophotometer equipped witha lead hollow-cathode lamp and an air–acetylene flame, using4-methyl-2-pentanone to set the instrument to zero. In a suit-able analysis, the absorbance of the Blank Preparation is notgreater than 20% of the difference between the absorbanceof the Standard Preparation and the absorbance of the BlankPreparation. The absorbance of the Test Preparation doesnot exceed that of the Standard Preparation.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 16 h.Mercury Determine as directed in Method II under MercuryLimit Test, Appendix IIIB.Oxalic Acid Dissolve 1 g of sample in 10 mL of water,add 2 mL of hydrochloric acid, transfer to a separator, andextract successively with 50 and 20 mL of ether. Combinethe ether extracts, add 10 mL of water, and evaporate theether on a steam bath. Add 1 drop of acetic acid (36%) and1 mL of a 1:20 calcium acetate solution. No turbidity formswithin 5 min.Reducing Sugars Dissolve 500 mg of sample in 10 mL ofwater, warm, and make the solution alkaline with 1 mL of 6N ammonium hydroxide. Pass hydrogen sulfide gas into thesolution to precipitate the iron, and allow the mixture to standfor 30 min to coagulate the precipitate. Filter, and wash theprecipitate with two successive 5-mL portions of water. Acid-ify the combined filtrate and washings with hydrochloric acid,and add 2 mL of 2.7 N hydrochloric acid in excess. Boil thesolution until the vapors no longer darken lead acetate paper,and continue to boil, if necessary, until the solution has beenconcentrated to about 10 mL. Cool, add 5 mL of sodiumcarbonate TS and 20 mL of water, filter, and adjust the volumeof the filtrate to 100 mL with water. Add 2 mL of alkalinecupric tartrate TS to 5 mL of filtrate, and boil for 1 min. Nored precipitate forms within 1 min.Sulfate Determine as directed in the Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 200-mg sample does not exceed that shownin a control containing 200 �g of sulfate (SO4).
Packaging and Storage Store in tight containers.
176 / Ferrous Glycinate / Monographs FCC V
Ferrous GlycinateDiaquo bis(glycinato) iron (II); Ferrous Bisglycinate
O
H2O
NH2O
H2OO
Fe
NH2
O
H2O
OH2
O
NH2
O
O
NH2
O
Fe
cis trans
Fe(OH2)2(OOCCH2NH2)2 Formula wt 239.99
CAS: [20150-34-9]
DESCRIPTION
Ferrous Glycinate occurs as a fine, free-flowing powder. Ithas an octahedral structure with two water molecules and twochelated glycinate ions coordinated to the central ferrous iron.
Function Source of dietary iron.
REQUIREMENTS
Identification A sample gives a positive test for Iron (Fer-rous Salts), Appendix IIIA.Assay Not less than 97.0% and not more than 102.0% ofFe(OH2)2(OOCCH2NH2)2, calculated on the dried basis.Ferric Iron Not more than 2%.Lead Not more than 1 mg/kg.Loss on Drying Not more than 7%.Nitrogen Between 10% and 12%.Total Iron Not less than 20% and not more than 22%.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in a mixture of 150 mL of water and 10 mL of sulfuric acidin a 300-mL flask. Add 1 drop of orthophenanthroline TS,and immediately titrate with 0.1 N ceric sulfate prepared asindicated in Volumetric Solutions under Solutions and Indica-tors. Perform a blank determination (see General Provisions),and make any necessary correction. Each milliliter of 0.1 Nceric sulfate is equivalent to 24.00 mg of Fe(OH2)2
(OOCCH2NH2)2.Ferric Iron Dissolve about 5 g of sample, accuratelyweighed, in a mixture of 100 mL of water and 10 mL ofhydrochloric acid in a 250-mL glass-stoppered flask. Add 3g of potassium iodide, shake well, and allow to stand in thedark for 5 min. Titrate any liberated iodine with 0.1 N sodiumthiosulfate, using starch TS as the indicator. Each milliliterof 0.1 N sodium thiosulfate is equivalent to 5.585 mg offerric iron.
Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, using a10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Nitrogen
Equipment Use a LECO CNS 2000, or equivalent, instru-ment capable of analyzing for carbon, nitrogen, and sulfursimultaneously. The instrument consists of an autosampler, acombustion furnace, and a computer system for determinationand calculations required for operation. Before calibrating theinstrument, perform appropriate combustion, helium gas line,and ballast leak checks of the system, and correct any detectedleaks. Analyze about ten blanks through the system. Checkand monitor that the results for carbon, nitrogen, and sulfurare constant. (Inconsistent blank values indicate problemswith the instrument that must be corrected.) Use the resultsof these blank analyses to zero the instrument, then calibrateit by analyzing at least five 0.2-g samples of sulfamethazine,and verify that the results for carbon (51.7%), nitrogen(20.13%), and sulfur (11.52%) are within �10% of actualvalues. Use these values to drift-correct the instrument, thuscompleting calibration. Analyze at least two more samples ofsulfamethazine, and verify that the results for carbon, nitrogen,and sulfur are within �10% of actual values.
Procedure Place 0.2 g of sample into a ceramic weighboat. Mix in a small amount of comcat (100 g of tungsticanhydride and 15 g of 97% lithium metaphosphate) to facilitatecombustion. Analyze the sample through the system.Total Iron
Sample Digest Mix the sample thoroughly to achieve ho-mogeneity. Some samples may require additional grinding toattain homogeneity. Do so by placing small amounts of sampleinto a clean, dry laboratory grinder and grinding until thesample has attained the desired level of homogeneity. Place0.500 g of sample, accurately weighed, into an appropriatedigestion vessel. Add 5 mL of concentrated nitric acid, mixthe slurry, and cover the vessel with a watch glass or vaporrecovery device. Heat the sample to 95° � 5° for 30 to 40min without boiling. If brown fumes evolve after heating forthe allotted time, indicating that the nitric acid has incom-pletely oxidized the sample, add 2 mL of concentrated nitricacid repeatedly, with heating for 15 to 20 min, until no brownfumes evolve. Heat the sample digest until the volume hasbeen reduced to about 3 mL, ensuring that the bottom of thevessel is covered with the sample digest at all times. Removethe vessel from the heating source, and allow its contents tocool thoroughly. Add 2 mL of concentrated hydrochloric acidto the sample digest, and cover with a watch glass. Place thevessel on the heating source, and reflux the sample digest at95° � 5° for 15 to 20 min. Before removing the vessel fromthe heating source, be sure the evolving vapor is clear. Allowthe sample digest to cool to room temperature, and dilute itto 50 mL with water. Add 3 g of potassium iodide, shakewell, and allow to stand in the dark for 5 min. Titrate anyliberated iodine with 0.1 N sodium thiosulfate, using starch
FCC V Monographs / Ferrous Lactate / 177
TS as the indicator. Each milliliter of 0.1 N sodium thiosulfateis equivalent to 5.585 mg of iron.
Packaging and Storage Store in tight containers.
Ferrous LactateIron (II) Lactate; Iron (II) 2-Hydroxypropionate
CH3CH(OH)COO 2 Fe
C6H10FeO6·xH2O Formula wt, anhydrous 233.99
INS: 585 CAS: [5905-52-2]
DESCRIPTION
Ferrous Lactate occurs as a green-white powder or crystals.The levo enantiomer occurs as the dihydrate, and the racemicmixture occurs as the trihydrate. It is sparingly soluble inwater and practically insoluble in ethanol. A 1:50 aqueoussolution has a pH between 5 and 6.
Function Nutrient.
REQUIREMENTS
Labeling Indicate the state of hydration.Identification A 1:50 aqueous solution gives positive testsfor Lactate and for Iron (Ferrous Salts), Appendix IIIA.Assay Not less than 97.0% and not more than 100.5% ofC6H10FeO6, calculated on the anhydrous basis.Chloride Not more than 0.1%.Ferric Iron Not more than 0.2%.Lead Not more than 1 mg/kg.Optical (Specific) Rotation Dihydrate: [�]D
20°: Between+6.0° and +11.0°, calculated on the anhydrous basis.Oxalic Acid Passes test.Sulfate Not more than 0.1%.Water Dihydrate: Between 12.0% and 14.0%; Trihydrate:Between 18.0% and 20.0%.
TESTS
Assay Dissolve about 800 mg of sample, accuratelyweighed, in a mixture of 150 mL of water and 10 mL ofsulfuric acid contained in a 300-mL flask. Add 5 mL ofphosphoric acid, and cool to room temperature if necessary.Add 1 drop of orthophenanthroline TS, and immediately titratewith 0.1 N ceric sulfate. Perform a blank determination (seeGeneral Provisions), and make any necessary correction. Eachmilliliter of 0.1 N ceric sulfate is equivalent to 23.40 mg ofC6H10FeO6.Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIA, using
1 g of sample, accurately weighed, in 100 mL of water. Anyturbidity produced by a 10-mL portion of this solution doesnot exceed that shown in a control containing 100 �g ofchloride (Cl) ion. (Save the remaining Sample Solution forthe Sulfate Test, below.)Ferric Iron Dissolve about 5 g of sample, accuratelyweighed, in a mixture of 100 mL of water and 10 mL ofhydrochloric acid contained in a 250-mL glass-stopperedflask, add 3 g of potassium iodide, shake well, and allow themixture to stand in the dark for 5 min. Titrate any liberatediodine with 0.1 N sodium thiosulfate, using starch TS asthe indicator. Each milliliter of 0.1 N sodium thiosulfate isequivalent to 5.585 mg of ferric iron.Lead (Note: In preparing all aqueous solutions and for rins-ing glassware before use, employ water that has been passedthrough a strong-acid, strong-base, mixed-bed ion-exchangeresin before use. Select all reagents to have as low a contentof lead as practicable, and store all reagent solutions in con-tainers of borosilicate glass. Clean glassware before use bysoaking it in warm 8 N nitric acid for 30 min and then rinsingit with deionized water.)
Standard Lead Solution Prepare all lead solutions in 0.1%nitric acid. Use a single-element 1000 �g/mL lead stock solu-tion to prepare (weekly) an intermediate stock solution (1 �g/mL). Prepare (daily) a Standard Lead Solution (10 ng/mL)by diluting the intermediate stock solution 1:100 with 0.1 Nnitric acid.
Modifier Working Solution Weigh an amount of palla-dium nitrate equivalent to 1 g of palladium, and dilute to 100mL with 15% nitric acid to make a stock solution. Just beforeuse, prepare a Modifier Working Solution by diluting the stocksolution 1:10 with water.
Blank Solution Use 0.1% nitric acid.Sample Preparation Dissolve 2 g of sample, accurately
weighed, in 5 mL of water and 10 mL of 10% nitric acid.Dilute to 100.0 mL with water.
Procedure Use the following furnace program with a suit-able graphite furnace atomic absorption spectrophotometerset at 283.3 nm and equipped with an autosampler:
Temperature Time Gas flow (argon)(°C) (s) (L/min)
85 5.0 3.095 40.0 3.0
120 10.0 3.0300 30.0 3.0900 5.0 3.0900 1.0 3.0900 2.0 0.0
2100 0.6 0.02100 2.0 0.02800 3.0 3.0
Separately inject the following solution mixtures:Solution A: 30 �L of the Blank Solution and 5 �L of theModifier Working Solution;
178 / Ferrous Sulfate / Monographs FCC V
Solution B: 10 �L of the Standard Lead Solution, 10 �L ofthe Sample Preparation, 10 �L of the Blank Solution, and 5�L of the Modifier Working Solution;Solution C: 20 �L of the Standard Lead Solution, 10 �L ofthe Sample Preparation, and 5 �L of the Modifier WorkingSolution;Solution D: 10 �L of the Sample Preparation, 20 �L of theBlank Solution, and 5 �L of the Modifier Working Solution.
Calculate the blank-corrected absorbances of Solutions B,C, and D by subtracting from each the absorbance measuredfor Solution A. Plot the blank-corrected absorbances of Solu-tions B, C, and D (y-axis) versus the quantity of lead, innanograms, added to each solution (x-axis). These are equalto 0.1, 0.2, and 0 ng, respectively. Draw the best straight linethrough the points. Extrapolate the line to the x-axis interceptto obtain the quantity C, in nanograms, of lead in 10 �L ofthe Sample Solution. Calculate the concentration, in milli-grams per kilogram, of lead in the sample taken by the formula
10C/W,
in which W is the weight, in grams, of the sample taken.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 2 g of sample in 100 mL of oxygen-free water.Oxalic Acid Dissolve 1 g of sample in 10 mL of water and2 mL of hydrochloric acid, transfer to a separator, and extractwith two 35-mL portions of ether. Evaporate the combinedether extracts in a rotary evaporator or on a steam bath.Dissolve any residue in 10 mL of water, add 1 mL of glacialacetic acid and 1 mL of a 1:20 solution of calcium acetate.No turbidity develops in 5 min.Sulfate Determine as directed in the Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 20-mL portion of the solution prepared for theChloride Test (above) does not exceed that shown in a controlcontaining 200 �g of sulfate (SO4).Water Determine as directed under Water Determination,Appendix IIB, at 50°, using 100 mg of sample dissolved ina freshly prepared mixture of 20 mL of methanol and 20 mLof formamide.
Packaging and Storage Store in tight containers.
Ferrous SulfateFeSO4·7H2O Formula wt 278.02
CAS: [7782-63-0]
DESCRIPTION
Ferrous Sulfate occurs as pale, blue-green crystals or granulesthat are efflorescent in dry air. In moist air, it oxidizes readilyto form a brown-yellow, basic ferric sulfate. A 1:10 aqueoussolution has a pH of about 3.7. One gram dissolves in 1.5
mL of water at 25° and in 0.5 mL of boiling water. It isinsoluble in alcohol.
Function Nutrient.
REQUIREMENTS
Identification A sample gives positive tests for FerrousSalts (Iron) and for Sulfate, Appendix IIIA.Assay Not less than 99.5% and not more than 104.5% ofFeSO4·7H2O.Lead Not more than 2 mg/kg.Mercury Not more than 1 mg/kg.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in a mixture of 25 mL of 2 N sulfuric acid and 25 mL ofrecently boiled and cooled water, and immediately titrate with0.1 N ceric sulfate, using orthophenanthroline TS as the indica-tor. Perform a blank determination (see General Provisions),and make any necessary correction. Each milliliter of 0.1 Nceric sulfate is equivalent to 27.80 mg of FeSO4·7H2O.Lead Determine as directed in the monograph for FerrousGluconate.Mercury Determine as directed in Method II under MercuryLimit Test, Appendix IIIB.
Packaging and Storage Store in tight containers.
Ferrous Sulfate, DriedFeSO4·xH2O Formula wt, anhydrous 151.91
CAS: [7720-78-7]
DESCRIPTION
Ferrous Sulfate, Dried, occurs as a gray-white to buff coloredpowder consisting primarily of FeSO4·H2O, with varyingamounts of FeSO4·4H2O. It dissolves slowly in water, but isinsoluble in alcohol.
Function Nutrient.
REQUIREMENTS
Identification A sample gives positive tests for FerrousSalts (Iron) and for Sulfate, Appendix IIIA.Assay Not less than 86.0% and not more than 89.0% ofFeSO4.Insoluble Substances Not more than 0.05%.Lead Not more than 2 mg/kg.Mercury Not more than 1 mg/kg.
FCC V Monographs / Fir Needle Oil, Siberian Type / 179
TESTS
Assay Determine as directed under Assay in the monographfor Ferrous Sulfate. Each milliliter of 0.1 N ceric sulfate isequivalent to 15.19 mg of FeSO4.Insoluble Residue Dissolve 2 g of sample in 20 mL offreshly boiled 1:100 sulfuric acid, heat to boiling, and thendigest in a covered beaker on a steam bath for 1 h. Filterthrough a tared filtering crucible, wash thoroughly, and dryat 105°. The weight of the insoluble residue does not exceed1 mg.Lead Determine as directed in the monograph for FerrousGluconate.Mercury Determine as directed in Method II under MercuryLimit Test, Appendix IIIB.
Packaging and Storage Store in tight containers.
Fir Needle Oil, Canadian TypeBalsam Fir Oil
DESCRIPTION
Fir Needle Oil, Canadian Type, occurs as a colorless to faintlyyellow liquid with a pleasant, balsamic odor. It is the volatileoil obtained by steam distillation from needles and twigs ofAbies balsamea L., Mill (Fam. Pinaceae). It is soluble in mostfixed oils and in mineral oil. It is slightly soluble in propyleneglycol, but it is insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 8.0% and not more than 16.0% of esters,calculated as bornyl acetate (C12H20O2).Angular Rotation Between −19° and −24°.Refractive Index Between 1.473 and 1.476 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.872 and 0.878.
TESTS
Assay Measure about 5 g of sample, accurately weighed,and proceed as directed in Ester Determination under Esters,Appendix VI, using 98.15 as the equivalence factor (e) in thecalculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.
Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 4 mL of 90% alcohol, occasionally with haziness.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Fir Needle Oil, Siberian TypePine Needle Oil
DESCRIPTION
Fir Needle Oil, Siberian Type, occurs as an almost colorlessor faintly yellow liquid with a piney, balsamic odor. It is thevolatile oil obtained by steam distillation from needles andtwigs of Abies sibirica Lebed. (Fam. Pinaceae). It is solublein most fixed oils and in mineral oil. It is insoluble in glycerinand in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 32.0% and not more than 44.0% ofesters, calculated as bornyl acetate (C12H20O2).Angular Rotation Between −33° and −45°.Refractive Index Between 1.468 and 1.473 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.898 and 0.912.
TESTS
Assay Measure about 2 g of sample, accurately weighed,and proceed as directed in Ester Determination under Esters,Appendix VI, using 98.15 as the equivalence factor (e) in thecalculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 1 mL of 90% alcohol. Occasionally the solution may be-come hazy on further dilution.
View IR
View IR
180 / Folic Acid / Monographs FCC V
Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Folic AcidN- [4-[[(2-Amino-1,4-dihydro-4-oxo-6-pteridinyl) methyl]amino]benzoyl]-L-glutamic Acid; N-[p-[[(2-Amino-4-hydroxy-6-pteridinyl)methyl]amino]benzoyl]glutamic Acid; Pteroylglu-tamic Acid.
N
HN
N
NHN
HN
O
O HO
OH
O
H2N
C19H19N7O6 Formula wt 441.40
CAS: [59-30-3]
DESCRIPTION
Folic Acid occurs as yellow or yellow-orange crystals orcrystalline powder. About 1.6 mg dissolves in 1 mL of water.It is insoluble in acetone, in alcohol, in chloroform, and inether, but dissolves in solutions of alkali hydroxides and car-bonates. The pH of a suspension of 1 g in 10 mL of wateris between 4.0 and 4.8.
Function Nutrient.
REQUIREMENTS
Identification The ultraviolet absorption spectrum of a1:100,000 aqueous solution in 1:250 sodium hydroxide solu-tion exhibits maxima and minima at the same wavelengthsas those of a similar solution of USP Folic Acid ReferenceStandard, concomitantly measured. The ratio A256/A365 is be-tween 2.80 and 3.00.Assay Not less than 95.0% and not more than 102.0% ofC19H19N7O6, calculated on the anhydrous basis.Lead Not more than 2 mg/kg.Residue on Ignition Not more than 0.3%.Water Not more than 8.5%.
TESTS
AssayStandard Solution Accurately weigh about 30 mg of USP
Folic Acid Reference Standard, corrected for water content,
and dissolve in an aqueous solvent containing 2 mL of ammo-nium hydroxide and 1 g of sodium perchlorate per 100 mLof solvent. Using the same solvent, adjust the volume quantita-tively, according to the injection size to be used in the Proce-dure, so that between 5 and 20 �g of Folic Acid is chromato-graphed.
Sample Solution Prepare as directed for the Standard So-lution, using an accurately weighed quantity of sample inplace of the USP Folic Acid Reference Standard.
Mobile Phase Transfer 35.1 g of sodium perchlorate, 1.40g of monobasic potassium phosphate, 7.0 mL of 1 N potassiumhydroxide, and 40 mL of methanol to a 1000-mL volumetricflask, dilute to volume with water, and mix. Adjust the pHto 7.2 with 1 N potassium hydroxide.
Note: The methanol concentration may be varied tomeet system suitability requirements and to provide asuitable resolution (R) for the System Suitability So-lution.
System Suitability Solution Using an aqueous solvent con-taining 2 mL of ammonium hydroxide and 1 g of sodiumperchlorate per 100 mL of solvent, prepare a solution con-taining about 1 mg/mL each of USP Folic Acid ReferenceStandard and USP Calcium Formyltetrahydrofolate AuthenticSubstance.
Note: Before injection, filter all injection solutionsthrough a membrane filter of 1-�m porosity or finer.
Procedure (See Chromatography, Appendix IIA.) Usea high-performance liquid chromatograph equipped with anultraviolet detector that measures absorption at 254 nm and a25- to 30-cm × 4-mm (id) stainless-steel column, or equivalent,packed with octadecyl silane chemically bonded to poroussilica or ceramic microparticles 5 to 10 �m in diameter, orequivalent. Maintain the mobile phase at a pressure and flowrate capable of giving the required resolution (see below).Inject a volume, up to 25 �L, of the System Suitability Solutionin a similar manner. Calculate the resolution, R (≥3.6), be-tween calcium formyltetrahydrofolate and Folic Acid by theequation
R = 2(t2 − t1)/(W2 + W1),
in which t2 and t1 are the retention times of the two compo-nents, and W2 and W1, are the corresponding widths at thebases of the peaks obtained by extrapolating the relativelystraight sides of the peaks to the baseline.
Chromatograph five injections of equal volume, up to 25�L, of the Standard Solution, and measure the peak response.The relative standard deviation, calculated by the formula
100 × (standard deviation/mean peak response)
for the peak response does not exceed 2%.Introduce volumes of the Sample Solution equal to those
used for the Standard Solution into the chromatograph. Mea-sure the responses for the major peaks obtained with theSample Solution and the Standard Solution. Calculate thequantity, in milligrams, of C19H19N7O6 in the sample takenby the formula
VC × (PU/PS)/w × 100,
FCC V Monographs / Food Starch, Modified / 181
in which V is the volume, in milliliters, of the Sample Solution;C is the concentration, in milligrams per milliliter, of USPFolic Acid Reference Standard in the Standard Solution; PU
and PS are the peak responses of the solutions from the SampleSolution and the Standard Solution, respectively; w is theweight, in milligrams, of the sample taken; and 100 is theconversion factor for percent.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, using a 1-g sample.Water Determine as directed under Water Determination,Appendix IIB, using a 200-mg sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
Food Starch, ModifiedModified Food Starch; Food Starch–Modified
DESCRIPTION
Food Starch, Modified, usually occurs as white or nearly whitepowders; as intact granules; and if pregelatinized (that is,subjected to heat treatment in the presence of water), as flakes,amorphous powders, or coarse particles. Modified foodstarches are products of the treatment of any of several grain-or root-based native starches (for example, corn, sorghum,wheat, potato, tapioca, and sago), with small amounts of cer-tain chemical agents, which modify the physical characteris-tics of the native starches to produce desirable properties.
Starch molecules are polymers of anhydroglucose andoccur in both linear and branched form. The degree ofpolymerization and, accordingly, the molecular weight ofthe naturally occurring starch molecules vary radically.Furthermore, they vary in the ratio of branched-chain poly-mers (amylopectin) to linear-chain polymers (amylose), bothwithin a given type of starch and from one type to another.These factors, in addition to any type of chemical modifica-tion used, affect the viscosity, texture, and stability of thestarch sols significantly.
Starch is chemically modified by mild degradation reac-tions or by reactions between the hydroxyl groups of thenative starch and the reactant selected. One or more of thefollowing processes are used: mild oxidation (bleaching),moderate oxidation, acid and/or enzyme depolymerization,monofunctional esterification, polyfunctional esterification(cross-linking), monofunctional etherification, alkaline gela-tinization, and certain combinations of these treatments.These methods of preparation can be used as a basisfor classifying the starches thus produced (see AdditionalRequirements, below). Generally, however, the products arecalled Modified Food Starch, or Food Starch–Modified.
Modified food starches are insoluble in alcohol, in ether,and in chloroform. If not pregelatinized, they are practicallyinsoluble in cold water. Upon heating in water, the granulesusually begin to swell at temperatures between 45° and80°, depending on the botanical origin and the degree ofmodification. They gelatinize completely at higher tempera-tures. Pregelatinized starches hydrate in cold water.
Function Thickener; colloidal stabilizer; binder.
REQUIREMENTS
Labeling Indicate the presence of sulfur dioxide if the resid-ual concentration is greater than 10 mg/kg.Identification
A. Suspend about 1 g of sample in 20 mL of water, andadd a few drops of iodine TS. A dark blue to red color appears.
B. Place about 2.5 g of sample in a boiling flask, add 10mL of 3% hydrochloric acid and 70 mL of water, mix, refluxfor about 3 h, and cool. Add 0.5 mL of the resulting solutionto 5 mL of hot alkaline cupric tartrate TS. A copious redprecipitate forms.
C. Examine a portion of the sample with a polarizing micro-scope in polarized light under crossed Nicol prisms. The typi-cal polarization cross is observed, except in the case of pregel-atinized starches.Crude Fat Not more than 0.15%.Lead Not more than 1 mg/kg.Loss on Drying Cereal Starch: Not more than 15.0%; Po-tato Starch: Not more than 21.0%; Sago and Tapioca Starch:Not more than 18.0%.pH of Dispersions Between 3.0 and 9.0.Protein Not more than 0.5%; except in modified high-amy-lose starches, not more than 1%.Sulfur Dioxide Not more than 0.005%.
ADDITIONAL REQUIREMENTS
The modified food starches listed below according to methodof preparation must meet all of the above Requirements inaddition to the specified methods of Treatment (the reagentthat, if not specifically limited, should not exceed the amountreasonably required to accomplish the intended modification)and any requirements for Residuals Limitation.
Alkaline Gelatinization (Gelatinized Starch)
Treatment to ProduceGelatinized Starch Residuals Limitation
Sodium hydroxide, not to ex- —ceed 1%
Depolymerization (Thin-Boiling, or Acid-Modified Starch)This treatment results in partial depolymerization, causing areduction in viscosity. Any of these treatments may be usedin combination with the other treatments that follow.
182 / Food Starch, Modified / Monographs FCC V
Treatment to ProduceThin-Boiling Starch Residuals Limitation
Hydrochloric acid and/or sulfu- —ric acid
Alpha-amylase enzyme The enzyme must be generallyrecognized as safe or ap-proved as a food additive forthis purpose. The resultingnonsweet nutritive saccharidepolymer has a dextrose equiv-alent of less than 20
Etherification and Esterification (Starch Ether-Esters)
Treatment to Produce Hydroxy-propyl Distarch Phosphate Residuals Limitation
Phosphorus oxychloride, not to Not more than 3 mg/kg ofexceed 0.1%, and propylene residual propylene chloro-oxide, not to exceed 10% hydrin
Etherification with Oxidation (Oxidized Starch Ethers)
Treatment to Produce OxidizedHydroxypropyl Starch Residuals Limitation
Chlorine, as sodium hypochlo- Not more than 1 mg/kg of re-rite, not to exceed 0.055 lb. sidual propylene chloro-(25 g) of chlorine per lb. hydrin(454 g) of dry starch; activeoxygen obtained from hydro-gen peroxide, not to exceed0.45%; and propylene oxide,not to exceed 25%
Mild Oxidation (Bleached Starch) The starches resultingfrom mild oxidation are not altered chemically; in all cases,extraneous color bodies are oxidized, solubilized, and re-moved by washing and filtration. These treatments may beused in combination with the other forms of treatment listedin this section.
Treatment to Produce BleachedStarch Residuals Limitation
Active oxygen obtained from —hydrogen peroxide, and/orperacetic acid, not to exceed0.45% of active oxygen
Ammonium persulfate, not to —exceed 0.075%, and sulfur di-oxide, not to exceed 0.05%
Chlorine, as sodium hypochlo- —rite, not to exceed 0.0082 lb.(3.72 g) of chlorine per lb.(454 g) of dry starch
Chlorine, as calcium hypochlo- —rite, not to exceed 0.036% ofdry starch
Potassium permanganate, not to Not more than 0.005% of resid-exceed 0.2% ual manganese (as Mn)
Sodium chlorite, not to exceed —0.5%
Moderate Oxidation (Oxidized Starch) The maximumspecified treatment introduces about 1 carboxyl group per 28anhydroglucose units. The starch is whitened, and its molecu-lar weight and viscosity are reduced.
Treatment to Produce OxidizedStarch Residuals Limitation
Chlorine, as sodium hypochlo- —rite, not to exceed 0.055 lb.(25 g) of chlorine per lb.(454 g) of dry starch
Monofunctional and/or Polyfunctional Esterification(Starch Esters) The starch esters are named individually,depending on the method of preparation.
Treatment to Produce StarchAcetate Residuals Limitation
Acetic anhydride or vinyl ac- Not more than 2.5% of acetyletate groups introduced into fin-
ished product
Treatment to Produce Ace-tylated Distarch Adipate Residuals Limitation
Adipic anhydride, not to ex- Not more than 2.5% of acetylceed 0.12%, and acetic anhy- groups introduced into fin-dride ished product
Treatment to Produce StarchPhosphate Residuals Limitation
Monosodium orthophosphate Not more than 0.4% of residualphosphate (calculated as P)
Treatment to Produce StarchOctenyl Succinate Residuals Limitation
Octenyl succinic anhydride, not —to exceed 3%, followed bytreatment with alpha-amylaseenzyme
Treatment to Produce StarchSodium Octenyl Succinate Residuals Limitation
Octenyl succinic anhydride, not —to exceed 3%
Treatment to Produce StarchAluminum Octenyl Succinate Residuals Limitation
Octenyl succinic anhydride, not —to exceed 2%, and aluminumsulfate, not to exceed 2%
Treatment to Produce DistarchPhosphate Residuals Limitation
Phosphorus oxychloride, not to —exceed 0.1%
Sodium trimetaphosphate Not more than 0.04% of resid-ual phosphate (calculated asP)
FCC V Monographs / Food Starch, Unmodified / 183
Treatment to Produce Phos-phated Distarch Phosphate Residuals Limitation
Sodium tripolyphosphate and Not more than 0.4% of residualsodium trimetaphosphate phosphate (calculated as P)
Treatment to ProduceAcetylated Distarch Phosphate Residuals Limitation
Phosphorus oxychloride, not to Not more than 2.5% of acetylexceed 0.1%, followed by groups introduced intoeither acetic anhydride, not finished productto exceed 8%, or vinylacetate, not to exceed 7.5%
Treatment to Produce StarchSodium Succinate Residuals Limitation
Succinic anhydride, not to —exceed 4%
Monofunctional Etherification
Treatment to ProduceHydroxypropyl Starch Residuals Limitation
Propylene oxide, not to exceed Not more than 1 mg/kg of25% residual propylene chloro-
hydrin
TESTS
Crude Fat Determine as directed under Crude Fat, Appen-dix X.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 5-g sample in a vacuum oven,not exceeding 100 mm Hg, at 120° for 4 h.pH of Dispersions Determine as directed under pH Deter-mination, Appendix IIB, using the following suspension: Mix20 g of sample with 80 mL of water, and agitate continuouslyat a moderate rate for 5 min. (For pregelatinized starches,suspend 3 g of sample in 97 mL of water.)
Note: The water used for sample dispersion shouldrequire not more than 0.05 mL of 0.1 N acid or alkaliper 200 mL of sample to obtain the methyl red orphenolphthalein endpoint, respectively.
Protein Transfer about 10 g of sample, accurately weighed,into an 800-mL Kjeldahl flask, and add 10 g of anhydrouspotassium sulfate or anhydrous sodium sulfate, 300 mg ofcopper selenite or mercuric oxide, and 60 mL of sulfuric acid.Gently heat the mixture, keeping the flask inclined at abouta 45° angle, and after frothing has ceased, boil briskly untilthe solution remains clear for about 1 h. Cool, add 30 mL ofwater, mix, and cool again. Cautiously pour about 75 mL (orenough to make the mixture strongly alkaline) of a 2:5 aqueoussolution of sodium hydroxide down the inside of the flask sothat it forms a layer under the acid solution, and then add afew pieces of granular zinc. Immediately connect the flask toa distillation apparatus consisting of a Kjeldahl connecting
bulb and a condenser, the delivery tube of which extends wellbeneath the surface of an accurately measured excess of 0.1N sulfuric acid contained in a 50-mL flask. Gently rotate thecontents of the Kjeldahl flask to mix, and distill until allammonia has passed into the absorbing acid solution (about250 mL of distillate). Titrate the excess acid with 0.1 N sodiumhydroxide, using 0.25 mL of methyl red–methylene blue TSas the indicator. Perform a blank determination (see GeneralProvisions), substituting pure sucrose or dextrose for the sam-ple, and make any necessary correction. Each milliliter of 0.1N sulfuric acid consumed is equivalent to 1.401 mg of nitro-gen. Calculate the percent of nitrogen in the sample, and thencalculate the percent of protein in starches obtained from cornby multiplying the percent of nitrogen by 6.25, or in starchesobtained from wheat, by 5.7. Other factors may be appliedas necessary for starches obtained from other sources.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X.
TESTS (ADDITIONAL REQUIREMENTS)
Acetyl Groups Determine the content of acetyl groups instarch acetate, acetylated distarch adipate, and acetylateddistarch phosphate as directed under Acetyl Groups, Appen-dix X.Manganese Determine the residual manganese in bleachedstarch prepared with potassium permanganate as directedunder Manganese, Appendix IIIB.Phosphate Determine the residual phosphate (calculated asP) in starch phosphate, distarch phosphate, and phosphateddistarch phosphate as directed under Phosphorus, Appen-dix IIIB.Propylene Chlorohydrin Determine the residual propylenechlorohydrin in hydroxypropyl starch, hydroxypropyl starchphosphate, and oxidized hydroxypropyl starch as directed un-der Propylene Chlorohydrin, Appendix X.
Packaging and Storage Store in well-closed containers.
Food Starch, Unmodified
DESCRIPTION
Food Starch, Unmodified, occurs as white or nearly whitepowders; as intact granules; and if pregelatinized, as flakes,powders, or coarse particles. Food starches are extracted fromany of several grain or root crops, including corn (maize),sorghum, wheat, potato, tapioca, sago, and arrowroot andhybrids of these crops such as waxy maize and high-amylosemaize. They are chemically composed of either one or amixture of two glucose polysaccharides (amylose and amylo-pectin), the composition and relative proportions of which arecharacteristic of the plant source. Food starches are generallyproduced by extraction from the plant source using wet milling
184 / Formic Acid / Monographs FCC V
processes in which the starch is liberated by grinding aqueousslurries of the raw material. The extracted starch may besubjected to other nonchemical treatments such as purifica-tion, extraction, physical treatments, dehydration, heating, andminor pH adjustment during further processing steps. Foodstarch may be pregelatinized by heat treatment in the presenceof water or made cold-water swelling.
Food starches are insoluble in alcohol, in ether, and inchloroform. If they are not treated to be pregelatinized orcold-water swelling, then they are practically insoluble in coldwater. Pregelatinized and cold-water swelling starches hydratein cold water. When heated in water, the granules usuallybegin to swell at temperatures between 45° and 80°, dependingon the botanical origin of the starch. They gelatinize com-pletely at higher temperatures.
Function Thickener; colloidal stabilizer; binder.
REQUIREMENTS
Labeling Indicate the presence of sulfur dioxide if the resid-ual concentration is greater than 10 mg/kg.Identification
A. Suspend about 1 g of sample in 20 mL of water, andadd a few drops of iodine TS. A dark blue to red color appears.
B. Place about 2.5 g of sample in a boiling flask, add 10mL of 3% hydrochloric acid and 70 mL of water, mix, refluxfor about 3 h, and cool. Add 0.5 mL of the resulting solutionto 5 mL of hot alkaline cupric tartrate TS. A copious, redprecipitate forms.
C. Examine a portion of sample with a polarizing micro-scope in polarized light under crossed Nicol prisms. The typi-cal polarization cross is observed, except in the case of pregel-atinized starches.Crude Fat Not more than 0.15%.Lead Not more than 1 mg/kg.Loss on Drying Cereal Starch: Not more than 15.0%; Po-tato Starch: Not more than 21.0%; Sago and Tapioca Starch:Not more than 18.0%.pH of Dispersions Between 3.0 and 9.0.Protein Not more than 0.5%; except in high-amylose andother hybrid starches, not more than 1%.Sulfur Dioxide Not more than 0.005%.
TESTS
Crude Fat Determine as directed under Crude Fat, Appen-dix X.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 5-g sample in a vacuum oven,not exceeding 100 mm Hg, at 120° for 4 h.pH of Dispersions Determine as directed under pH Deter-mination, Appendix IIB, using the following suspension: Mix20 g of sample with 80 mL of water, and agitate continuouslyat a moderate rate for 5 min. (For pregelatinized starches,suspend 3 g of sample in 97 mL of water.)
Note: The water used for sample dispersion shouldrequire not more than 0.05 mL of 0.1 N acid or alkaliper 200 mL of sample to obtain the methyl red orphenolphthalein endpoint, respectively.
Protein Transfer about 10 g of sample, accurately weighed,into an 800-mL Kjeldahl flask, and add 10 g of anhydrouspotassium sulfate or anhydrous sodium sulfate, 300 mg ofcopper selenite or mercuric oxide, and 60 mL of sulfuric acid.Gently heat the mixture, keeping the flask inclined at abouta 45° angle, and after frothing has ceased, boil briskly untilthe solution remains clear for about 1 h. Cool, add 30 mL ofwater, mix, and cool again. Cautiously pour about 75 mL (orenough to make the mixture strongly alkaline) of a 2:5 aqueoussolution of sodium hydroxide down the inside of the flask sothat it forms a layer under the acid solution, and then add afew pieces of granular zinc. Immediately connect the flask toa distillation apparatus consisting of a Kjeldahl connectingbulb and a condenser, the delivery tube of which extends wellbeneath the surface of an accurately measured excess of 0.1N sulfuric acid contained in a 50-mL flask. Gently rotate thecontents of the Kjeldahl flask to mix, and distill until allammonia has passed into the absorbing acid solution (about250 mL of distillate). Titrate the excess acid with 0.1 N sodiumhydroxide, using 0.25 mL of methyl red–methylene blue TSas the indicator. Perform a blank determination (see GeneralProvisions), substituting pure sucrose or dextrose for the sam-ple, and make any necessary correction. Each milliliter of 0.1N sulfuric acid consumed is equivalent to 1.401 mg of nitro-gen. Calculate the percent nitrogen in the sample, and thencalculate the percent protein in starches obtained from cornby multiplying the percent of nitrogen by 6.25, or in starchesobtained from wheat, by 5.7. Other factors may be appliedas necessary for starches obtained from other sources.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X, using a 25-g sample.
Packaging and Storage Store in well-closed containers.
Formic Acid
HCOOH
CH2O2 Formula wt 46.03
INS: 236 CAS: [64-18-6]
FEMA: 2487
DESCRIPTION
Formic Acid occurs as a clear, colorless, highly corrosiveliquid with a characteristic, pungent odor. It is miscible with
FCC V Monographs / Fructose / 185
water, with alcohol, with glycerin, and with ether. Its specificgravity is about 1.20.
Function Flavoring adjunct; preservative.
REQUIREMENTS
IdentificationA. Add 2 mL of mercuric chloride TS to 5 mL of sample,
and warm the mixture. A white precipitate of mercurous chlo-ride forms.
B. Neutralize 1 mL of sample with sodium hydroxide TS,and then add 2 drops in excess and 1 mL of ferric chlorideTS. A deep, red-orange color appears that turns to yellow-orange on the addition of mineral acids.
C. Place 2 mL of sample in a test tube, add 5 mL of sulfuricacid, and test the gas evolved with a lighted splinter. A blueflame characteristic of carbon monoxide is produced.Assay Not less than 85.0% of CH2O2.Acetic Acid Not more than 0.4%.Dilution Test Passes test.Sulfate Not more than 0.004%.
TESTS
Assay Tare a small glass-stoppered Erlenmeyer flask con-taining about 15 mL of water. Transfer about 1.5 mL ofsample into the flask, and weigh. Dilute the solution to 50mL with water, add phenolphthalein TS, and titrate with 1 Nsodium hydroxide. Each milliliter of 1 N sodium hydroxideis equivalent to 46.03 mg of CH2O2.Acetic Acid Dilute 1 mL of sample to 100 mL with water,transfer 50 mL of this solution into a 250-mL boiling flask,and add 5 g of yellow mercuric oxide. While continuouslystirring, boil the mixture under a reflux condenser for 2 h,cool, filter, and wash the residue with about 25 mL of water.Add phenolphthalein TS to the combined filtrate and wash-ings, and titrate with 0.02 N sodium hydroxide. Not morethan 2.0 mL of 0.02 N sodium hydroxide is required to producea pink color.Dilution Test Dilute 1 volume of sample with 3 volumesof water. No turbidity develops within 1 h.Sulfate Add about 10 mg of sodium carbonate to 2.1 mL(2.5 g) of sample contained in a beaker, and evaporate todryness on a steam bath. Any turbidity produced by the residuedoes not exceed that shown in a control containing 100 �gof sulfate (SO4).
Packaging and Storage Store in tight containers.
FructoseD-Fructose; Levulose; Fruit Sugar
O
CH2OH
OH
HO
OHHO
C6H12O6 Formula wt 180.16
CAS: [57-48-7]
DESCRIPTION
Fructose occurs as white, hygroscopic, purified crystals or asa purified crystalline powder. It is a natural constituent offruit, and is obtained from glucose in corn syrup by the useof glucose isomerase. Its density is about 1.6. It is soluble inmethanol and in ethanol, freely soluble in water, and insolublein ether.
Function Nutritive sweetener.
REQUIREMENTS
IdentificationA. Add a few drops of a 1:10 aqueous solution to 5 mL
of hot alkaline cupric tartrate TS. A copious red precipitateof cuprous oxide is formed.
B. The infrared absorption spectrum of a potassium bromidedispersion of sample, previously dried, exhibits maxima onlyat the same wavelengths as those of a similar preparation ofUSP Fructose Reference Standard.Assay Not less than 98.0% and not more than 102.0% ofC6H12O6 after drying.Chloride Not more than 0.018%.Glucose Not more than 0.5%.Hydroxymethylfurfural Not more than 0.1%, calculatedon the dried basis.Lead Not more than 0.1 mg/kg.Loss on Drying Not more than 0.5%.Residue on Ignition Not more than 0.5%.Sulfate Not more than 0.025%.
TESTS
Assay Transfer about 10 g of sample, previously dried invacuum at 70° for 4 h and accurately weighed, into a 100-mL volumetric flask, dissolve in 50 mL of water, add 0.2 mLof 15.2 N ammonium hydroxide, dilute to volume with water,and mix. After 30 min, determine the angular rotation [seeOptical (Specific) Rotation, Appendix IIB] in a 100- or 200-mm tube at 25° with the sodium D line. The observed rotation,in degrees (absolute value), multiplied by 1.124 (or 0.562 forthe 200-mm tube), represents the weight, in grams, of Fructosein the sample taken.
186 / Fumaric Acid / Monographs FCC V
Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB. Anyturbidity produced by a 2-g sample does not exceed that shownin a control containing 0.5 mL of 0.02 N hydrochloric acid.Glucose
0.1 M Acetate Buffer Dissolve 13.608 g of sodium acetatetrihydrate in sufficient water to make 1000 mL, add 2.7 mLof acetic acid, and adjust the pH to 5.5 with glacial aceticacid or sodium acetate.
Reagent Solution Dissolve 40 mg of o-dianisidine dihy-drochloride, 40 mg of horseradish peroxidase (WorthingtonBiochemical Co., Freehold, NJ, or equivalent), and 0.4 mLof purified glucose oxidase (1000 glucose oxidase units permilliliter, Miles Laboratories, Inc., or equivalent) in 0.1 MAcetate Buffer, and dilute to 100 mL with 0.1 M AcetateBuffer.
Note: Commercially available preparations containingthe reagents in the proper proportions may also be used.
Glucose Standard Solution Transfer about 300 mg, accu-rately weighed, of USP Dextrose Reference Standard, pre-viously dried in vacuum at 70° for 4 h, into a 1000-mLvolumetric flask, dissolve in and dilute to volume with water,and mix. Allow to stand for 2 h to allow mutarotation tooccur, then transfer 20.0 mL to a 100-mL volumetric flask,dilute to volume with water, and mix. Prepare fresh on theday of use.
Sample Preparation Transfer 14 g of sample, accuratelyweighed, into a 100-mL volumetric flask, dissolve in anddilute to volume with water, and mix. Transfer 20.0 mL intoa second 100-mL volumetric flask, dilute to volume withwater, and mix.
Procedure Pipet 2 mL each of the Sample Preparation,the Glucose Standard Solution, and water into separate 150-× 18-mm test tubes. Heat the tubes for 5 min in a water bathmaintained at 30°. At zero time and after 30 and 60 s, add1.0 mL of the Reagent Solution to the first, second, and thirdtubes, respectively, mix the contents of the tubes, and allowthem to react for exactly 30 min from zero time. Immediatelystop the reaction in the first tube by adding 10.0 mL of 25%sulfuric acid. Similarly, add 10.0 mL of 25% sulfuric acid tothe remaining tubes after they have reacted for exactly 30min. Mix the contents of each tube, and cool them to roomtemperature. Using a suitable spectrophotometer, determinethe absorbance values of the mixtures obtained from the Sam-ple Preparation and from the Glucose Standard Solution at540 nm versus the mixture obtained from the Reagent Solutionin the reference cell. Calculate the percentage of glucose inthe sample by the formula
(50C/W) × AU/AS,
in which C is the exact concentration, in milligrams per millili-ter, of the Glucose Standard Solution; W is the weight, ingrams, of sample taken; AU is the absorbance of the mixtureobtained from the Sample Preparation; and AS is the ab-sorbance of the mixture obtained from the Glucose StandardSolution.Hydroxymethylfurfural Transfer approximately 1 g ofsample, accurately weighed, into a 100-mL volumetric flask,
dilute to volume with water, and mix. Read the absorbanceof this solution against a water blank at 283 nm in a 1-cmquartz cell in a spectrophotometer. Calculate the percentageof 5-hydroxymethylfurfural (HMF) by the following equation:
% HMF = (0.749 × A)/C,
in which A is the absorbance of the sample solution, and Cis the concentration, in milligrams per milliliter, of the samplesolution corrected for ash and moisture.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 5-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in vacuum at 70° for 4 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Sulfate Determine as directed in the Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 2-g sample does not exceed that shown in acontrol containing 0.5 mL of 0.02 N sulfuric acid.
Packaging and Storage Store in tight containers protectedfrom humidity.
Fumaric Acid(E)-Butenedioic Acid; trans-1,2-Ethylenedicarboxylic Acid
HOOCCH
HCCOOH
C4H4O4 Formula wt 116.07
INS: 297 CAS: [110-17-8]
FEMA: 2488
DESCRIPTION
Fumaric Acid occurs as white granules or as a crystallinepowder. A 1:30 aqueous solution has a pH of 2.0 to 2.5. Itis soluble in alcohol, slightly soluble in water and in ether,and very slightly soluble in chloroform.
Function Acidifier; flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of a potas-sium bromide dispersion of the sample exhibits maxima onlyat the same wavelengths as those of a similar preparation ofUSP Fumaric Acid Reference Standard.Assay Not less than 99.5% and not more than 100.5% ofC4H4O4, calculated on the anhydrous basis.Lead Not more than 2 mg/kg.Maleic Acid Not more than 0.1%.
FCC V Monographs / Furcelleran / 187
Residue on Ignition Not more than 0.1%.Water Not more than 0.5%.
TESTS
Assay Transfer about 1 g of sample, accurately weighed,into a 250-mL Erlenmeyer flask, add 50 mL of methanol, anddissolve the sample by warming gently on a steam bath.Cool, add phenolphthalein TS, and titrate with 0.5 N sodiumhydroxide to the first appearance of a pink color that persistsfor at least 30 s. Perform a blank determination (see GeneralProvisions), and make any necessary correction. Each millili-ter of 0.5 N sodium hydroxide is equivalent to 29.02 mg ofC4H4O4.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Maleic Acid
Mobile Phase Prepare 0.005 N sulfuric acid that has beensuitably filtered and degassed.
Standard Preparation Transfer about 1 mg of USP MaleicAcid Reference Standard, accurately weighed, into a 1000-mL volumetric flask, dilute to volume with Mobile Phase,and mix.
Test Preparation Transfer about 100 mg of sample, accu-rately weighed, into a 100-mL volumetric flask, dilute tovolume with Mobile Phase, and mix.
Resolution Solution Transfer 1 mg of USP Fumaric AcidReference Standard and 0.5 mg of USP Maleic Acid ReferenceStandard into a 100-mL volumetric flask, dilute to volumewith Mobile Phase, and mix.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a liquid chromatograph equipped with a 210-nm detector and a 22-cm × 4.6-mm column packed with astrong cation exchange resin consisting of sulfonated cross-linked styrene–divinylbenzene copolymer in the hydrogenform (Polypore H from Brownlee Laboratories, Inc., or equiv-alent). Set the flow rate at about 0.3 mL/min. Chromatographthe Resolution Solution, and record the peak responses. Theresolution, R, between the maleic acid and fumaric acid peaksis not less than 2.5, and the relative standard deviation of themaleic acid peak for replicate injections is not more than 2.0%.
Procedure Separately inject equal volumes (about 5 �L)of the Standard Preparation and the Test Preparation intothe chromatograph, record the chromatograms, and measurethe peak responses. The relative retention times are about 0.5for maleic acid and 1.0 for fumaric acid. Calculate the quantity,in milligrams, of maleic acid in the total weight of the sampletaken by the formula
100C(RU/RS),
in which C is the concentration, in milligrams per milliliter,of USP Maleic Acid Reference Standard in the StandardPreparation, and RU and RS are the responses of the maleicacid peaks obtained from the Test Preparation and the Stan-dard Preparation, respectively.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
FurcelleranDanish Agar
CAS: [9000-21-9]
DESCRIPTION
Furcelleran occurs as a brown or tan to white, coarse to finepowder. It is soluble in water at a temperature of about 80°,forming a viscous, clear or slightly opalescent solution thatflows readily. It disperses in water more readily if first moist-ened with alcohol, glycerin, or a saturated solution of sucrosein water.
Furcelleran is a hydrocolloid obtained from Furcellariafastigiata of the class Rhodophyceae (red seaweeds) by extrac-tion with water or aqueous alkali. It consists mainly of thepotassium, sodium, magnesium, calcium, and ammonium sul-fate esters of galactose and 3,6-anhydrogalactose copolymers.These hexoses are alternately linked �-1,3 and �-1,4 in thepolymer. The relative proportion of cations existing in Furcel-leran may be changed during processing to the extent thatone may become predominant.
The ester sulfate content of Furcelleran ranges from 8%to 20% (see Requirements, below). In addition, it containsinorganic salts that originate from the seaweed and the processof recovery from the extract. Furcelleran is recovered byalcohol precipitation, by potassium precipitation, or by freez-ing. The alcohols used during recovery and purification arerestricted to methanol, ethanol, and isopropanol.
Function Stabilizer; thickener; gelling agent.
REQUIREMENTS
IdentificationA. Add a 4-g sample to 200 mL of water, and heat the
mixture in a water bath at 80°, with constant stirring, untildissolved. Replace any water lost by evaporation, and allowthe solution to cool to room temperature. It becomes viscousand may form a gel.
B. Add 200 mg of potassium chloride to 50 mL of thesolution or gel obtained in Identification Test A, then reheat,mix well, and cool. A short-textured (‘‘brittle’’) gel forms.
C. Add 1 drop of a 1:100 solution of methylene blue to 5mL of the solution obtained in Identification Test A. A fibrousprecipitate forms.
D. Obtain the infrared absorption spectrum of the sampleby the following procedure: Prepare a 0.2% aqueous solution,cast films 0.0005 cm thick (when dry) on a suitable nonstick-
188 / Furcelleran / Monographs FCC V
ing surface such as Teflon, and obtain the infrared absorptionspectrum. (Alternatively, the spectrum may be obtained onpotassium bromide pellets if care is taken to avoid moisture).
Furcelleran has strong, broad absorption bands in the 1000to 1100 cm−1 region. The absorption maximum is 1065. Othercharacteristic absorption bands and their intensities relativeto the absorbance at 1050 cm−1 are as follows:
Wave Number Molecular Absorbance Relative to(cm−1) Assignment 1050 cm−1
1220–1260 ester sulfate 0.2–0.6928–933 3,6-anhydrogalactose 0.2–0.3840–850 galactose-4-sulfate 0.1–0.3
Acid-Insoluble Matter Not more than 1.0%.Arsenic Not more than 3 mg/kg.Ash (Acid-Insoluble) Not more than 1.0%.Ash (Total) Not more than 35.0%.Lead Not more than 5 mg/kg.Loss on Drying Not more than 12.0%.Solubility in Water Not more than 30 mL of water is re-quired to completely dissolve 1 g at 80°.Sulfate Between 8.0% and 20.0% on the dry weight basis.Viscosity of a 1.5% Solution Not less than 5 centipoisesat 75°.
TESTS
Acid-Insoluble Matter Transfer about 2 g of sample, accu-rately weighed, to a 250-mL beaker containing 150 mL ofwater and 1.5 mL of sulfuric acid. Cover with a watch glass,and heat on a steam bath for 6 h, rubbing down the wall ofthe beaker frequently with a rubber-tipped stirring rod, andreplacing any water lost by evaporation. Transfer about 500mg of a suitable filtering aid, accurately weighed, to thebeaker, and filter through a tared filtering crucible containinga 2.4-cm glass fiber filter. Wash the residue several timeswith hot water, dry at 105° for 3 h, cool in a desiccator, andweigh. The difference between the total weight and the sumof the weights of the filter aid, crucible, and glass fiber filteris the weight of the acid-insoluble matter.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, testing a Sample Solution prepared as directedfor organic compounds.Ash (Acid-Insoluble) Determine as directed under Ash(Acid-Insoluble), Appendix IIC.Ash (Total) Transfer about 2 g of sample, accuratelyweighed, into a previously ignited, tared, silica or platinumcrucible. Heat the sample with a suitable infrared heat lamp,increasing the intensity gradually, until the sample is com-pletely charred, and then continue for an additional 30 min.Transfer the crucible and charred sample into a muffle furnace,and ignite at about 550° for 1 h, then cool in a desiccator,and weigh. Repeat the ignition in the muffle furnace until aconstant weight is attained. If a carbon-free ash is not obtainedafter the first ignition, moisten the charred spots with a 1:10solution of ammonium nitrate, and dry under an infrared heatlamp before reigniting.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Solubility in Water Add 1 g of sample to 30 mL of coldwater, stir well, and heat to a temperature of 80° to completelydissolve the sample. The resulting solution, when maintainedat 80°, is uniformly viscous and clear or slightly opalescent.Sulfate Transfer about 500 mg of sample, previously driedat 105° for 12 h and accurately weighed, into a 100-mLKjeldahl flask. Add 10 mL of nitric acid, and heat gently for30 min, adding more of the acid, if necessary, to preventevaporation to dryness and to yield a volume of about 3 mLat the end of the heating. Cool the mixture to room tempera-ture, and decompose the excess nitric acid by adding formalde-hyde TS, dropwise, heating if necessary, until no brown fumesare evolved. Continue heating until the volume of the reactionmixture is reduced to about 5 mL, and then cool. Transferthe residue quantitatively, with the aid of water, into a 400-mL beaker, dilute it to about 100 mL, and filter, if necessary,to produce a clear solution. Dilute the solution to about 200mL, and add 1 mL of hydrochloric acid. Heat to boiling andwhile constantly stirring, add, dropwise, an excess (about 6mL) of hot barium chloride TS. Heat the mixture for 1 h ona steam bath, collect the precipitate of barium sulfate on afilter, wash it until it is free from chloride, dry, ignite, andweigh. The weight of the barium sulfate so obtained,multiplied by 0.4116, gives the equivalent of sulfate (SO4).Viscosity of a 1.5% Solution Transfer 7.5 g of sample intoa tared, 600-mL tall-form (Berzelius) beaker, and dispersewith agitation for 10 to 20 min in 450 mL of deionized water.Add sufficient water to bring the final weight to 500 g, andheat in a water bath, with continuous agitation, until a tempera-ture of 80° is reached (20 to 30 min). Add water to adjustfor loss by evaporation, cool to 76° to 77°, and place in aconstant-temperature bath at 75°. Preheat the bob and guardof a Brookfield LVF or LVT viscometer, or equivalent, toapproximately 75° in water, then dry the bob and guard andattach them to the viscometer, which should be equipped witha No. 1 spindle (19-mm diameter, approximately 65 mm long)capable of rotating at 30 rpm. Adjust the height of the bobin the sample solution, start the viscometer rotating at 30 rpm,and after six complete revolutions, take the reading on the 0to 100 scale. Record the results in centipoises by multiplyingthe reading by 2.
Note: Some samples may be too viscous to be readwhen a No. 1 spindle is used. Such samples obviouslypass the specification, but if a viscosity reading is de-sired for other reasons, use a No. 2 spindle, take thereading on the 0 to 100 scale, and multiply the readingby 10 to obtain the viscosity in centipoises, or read onthe 0 to 500 scale and multiply by 2. If the viscosityis very low, increased precision may be obtained byusing the Brookfield UL (ultra low) adapter, in whichcase the viscometer reading on the 0 to 100 scale should
FCC V Monographs / Gelatin / 189
be multiplied by 0.2 to obtain the viscosity in centi-poises.
Packaging and Storage Store in a well-closed container.
Garlic OilCAS: [8000-78-0]
DESCRIPTION
Garlic Oil occurs as a clear yellow to red-orange liquid witha strong, pungent odor and a flavor characteristic of garlic.It is the volatile oil obtained by steam distillation from thecrushed bulbs or cloves of the common garlic plant, Alliumsativum L. (Fam. Liliaceae). It is soluble in most fixed oilsand in mineral oil. It may be incompletely soluble in alcohol.It is insoluble in glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Refractive Index Between 1.550 and 1.580 at 20°.Specific Gravity Between 1.050 and 1.095.
TESTS
Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
GelatinFood-Grade Gelatin; Edible Gelatin
CAS: [9000-70-8]
DESCRIPTION
Gelatin is the product obtained from the acid, alkaline, orenzymatic hydrolysis of collagen, the chief protein component
of the skin, bones, and connective tissues of animals, includingfish and poultry. These animal sources shall not have beenexposed to pentachlorophenol.
Type A Gelatin is produced by the acid processing of collag-enous raw materials and exhibits an isoelectric point betweenpH 7 and pH 9. Type B Gelatin is produced by the alkalineor lime processing of collagenous raw materials and exhibitsan isoelectric point between pH 4.6 and pH 5.2. Mixtures ofTypes A and B as well as Gelatins produced by modificationsof the above mentioned processes may exhibit isoelectricpoints outside of the stated ranges.
Gelatin is a vitreous, brittle solid that is faintly yellow.When Gelatin granules are immersed in cold water, they hy-drate into discrete, swollen particles. On being warmed, Gela-tin disperses into the water, resulting in a stable suspension.Water solutions of Gelatin will form a reversible gel if cooledbelow the specific gel point of Gelatin. The gel point isdependent on the source of the raw material. Gelatin extractedfrom the tissues of warm-blooded animals will have a gelpoint in the range of 30° to 35°. Gelatin extracted from theskin of cold-water ocean fish will have a gel point in therange of 5° to 10°. Gelatin is soluble in aqueous solutions ofpolyhydric alcohols such as glycerin and propylene glycol. Itis insoluble in most organic solvents.
Function Firming agent; stabilizer and thickener; surface-active agent; surface-finishing agent.
REQUIREMENTS
IdentificationA. Dissolve 10 g of sample in 100 mL of hot water contained
in a suitable flask, and cool in a refrigerator at 2° for 24 h.A gel forms. Transfer the flask to a water bath heated to 60°.Within 30 min, when stirred, the gel reverts to the originalliquid state.
B. Add trinitrophenol TS or a 1:1.5 solution of potassiumdichromate, previously mixed with about one-fourth its vol-ume of 3 N hydrochloric acid, to a 1:100 aqueous solutionof sample. A yellow precipitate forms.Ash (Total) Not more than 3.0%.Chromium Not more than 10 mg/kg.Lead Not more than 1.5 mg/kg.Loss on Drying Not more than 15.0%.Microbial Limits:
E. coli Negative in 25 g.Salmonella Negative in 25 g.
Pentachlorophenol Limit Not more than 0.3 mg/kg.Protein The specification conforms to the representationsof the vendor.Sulfur Dioxide Not more than 0.005%.
TESTS
Ash (Total) Determine as directed under Ash (Total), Appen-dix IIC, but use a 5-g sample. Before ashing in a muffle furnaceat 500° to 550° for 15 to 20 h, add 1.5 to 2.0 g of paraffin to thesample, then heat the crucible on a low-flame hot plate or mufflefurnace until the sample is thoroughly charred.
View IR
190 / Gelatin / Monographs FCC V
ChromiumTest Solution Transfer 10 g of sample, accurately
weighed, into a 100-mL silica dish. Using a very low flame,heat the dish over a Bunsen burner, taking care that the sampledoes not swell over the lip of the dish or catch fire. Graduallyincrease the flame until the sample is completely charred,transfer it into a muffle furnace at 550°, and ash overnight.Cool to room temperature, add 10 mL of hydrochloric acidand 10 mL of nitric acid, and heat on a steam bath for 10min. Cool and transfer into a 25-mL volumetric flask, cau-tiously dilute to volume with water, and mix.
Chromium Stock Solution Transfer 192.3 mg of chro-mium trioxide, accurately weighed, into a 1000-mL volumet-ric flask, dissolve in 100 mL of water and 10 mL of nitricacid, dilute to volume with water, and mix. This solutioncontains 0.1 mg of chromium per milliliter. Transfer 100.0mL of the solution into a 1000-mL volumetric flask, diluteto volume with water, and mix. This solution contains 10 �gof chromium per milliliter.
Standard Preparations Transfer 10.0, 30.0, 50.0, and 70.0mL, respectively, of Chromium Stock Solution to separate,100-mL volumetric flasks, dilute to volume with water, andmix. The Standard Preparations contain, respectively, 1.0,3.0, 5.0, and 7.0 �g of chromium per milliliter.
Procedure Concomitantly determine the absorbances ofthe Standard Preparations and the Test Solution at the chro-mium emission line of 357.9 nm, with a suitable atomic ab-sorption spectrophotometer equipped with a chromium hol-low-cathode lamp and a slightly reducing air–acetylene flameusing water as the blank. Plot the absorbances of the StandardPreparations versus concentration, in micrograms per millili-ter, of chromium, and draw the straight line best fitting thefour plotted points. From the graph so obtained, determinethe concentration, CS, in micrograms per milliliter, of chro-mium in the Test Solution. Calculate the concentration ofchromium, in milligrams per kilogram, in the portion of sam-ple taken, by the formula
25CS/W,
in which W is the quantity, in grams, of the sample taken.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 5-g sample at 105° for 16 to 18h to constant weight.Microbial Limits (Note: Current methods for the followingtests may be found online at <www.cfsan.fda.gov/~ebam/bam-toc.html>):
E. coliSalmonella
PentachlorophenolPentachlorophenol (PCP) Stock Solution Transfer about
4.0 mg of PCP Reference Standard (Standard No. 5260, Pesti-cide Reference Standards Section, Environmental ProtectionAgency, Research Triangle Park, NC 27711, or equivalent,available from Aldrich Chemical Co.), accurately weighed,into a 1000-mL volumetric flask, dissolve in pesticide-grade
benzene, dilute to volume with benzene, and mix. Each millili-ter of this solution contains 4.0 �g of PCP.
PCP Standard Solutions Prepare a series of PCP StandardSolutions by serially diluting the PCP Stock Solution in hex-ane. Pipet 0.0, 1.0, 5.0, 10.0, 25.0, 50.0, and 100.0 mL, respec-tively, of PCP Stock Solution, into separate 1000-mL volumet-ric flasks, dilute to volume with hexane, and mix. The PCPStandard Solutions contain in each milliliter 0.0, 0.004, 0.020,0.040, 0.100, 0.200, and 0.400 �g of PCP, respectively.
Sample Preparation Transfer about 2 g of sample, accu-rately weighed, and 2.0 mL of water (to serve as the blank)into separate 25- × 150-mm screw-cap test tubes equippedwith Teflon-lined caps. Treat each in the following manner:Add 10 mL of 12 N sulfuric acid, close the tube, tighten thecap, and heat for 1 h in a fume hood in a water bath maintainedat 100°, removing the tube periodically and mixing the sampleby shaking. Remove the tube from the bath, and allow it tocool to room temperature. Add 10 mL of a 4:1 (v/v) solutionof hexane:isopropanol to the tube, and shake vigorously. Cen-trifuge for 2 min at 1000 × g in a suitable centrifuge (Interna-tional Equipment Co., or equivalent) with a head equippedto accommodate 25- × 150-mm test tubes. Use a Pasteur pipetto transfer the upper hexane layer to a second 25- × 150-mm test tube. Repeat the extraction and centrifugation twoadditional times, and combine the hexane extracts in the sec-ond test tube. Add 5.0 mL of 1.0 N potassium hydroxide tothe combined extracts, tighten the cap, shake the test tubevigorously, and centrifuge for 2 min at 1000 × g as before.Remove the upper layer with a Pasteur pipet, and discard.Add 10 mL of hexane to the test tube, tighten the cap, shakethe test tube vigorously, and centrifuge as before. Removethe upper layer with a Pasteur pipet, and discard. Add 5.0mL of 12 N sulfuric acid to the test tube, tighten the cap, andmix by carefully swirling the tube. Add 5.0 mL of hexane,tighten the cap, shake the test tube vigorously, centrifuge asbefore, and transfer the upper layer to a 10-mL volumetricflask. Repeat twice, using 2.0 mL of hexane each time, andtransfer the upper layer into the 10-mL volumetric flask. Diluteto volume with hexane.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a 63Nielectron capture detector and a 1.8-m × 4-mm (id) glass col-umn, or equivalent, containing 1% SP-1240DA on 100- to120-mesh Supelcoport (Supelco Inc.), or equivalent. Place asmall plug (2 to 3 mm) of phosphoric acid-washed glass woolin the detector end of the column. Use 5% methane in argonas the carrier gas, with a flow rate of 60 mL/min. Conditionthe column by purging with carrier gas at ambient temperaturefor 10 to 15 min; program the column oven to increase from70° to 190° at 1°/min, and hold the temperature at 190° for8 h while continuing to purge with carrier gas.
Caution: Use only recently prepared and thoroughlyconditioned columns; the appearance of ghost PCPpeaks may be noted following the injection of samplescontaining high levels of PCP; repeated injections ofsolvent may be necessary until ghost PCP peaks dis-appear.
FCC V Monographs / Gellan Gum / 191
For sample analyses, maintain the temperatures of the columnoven, injector port, and detector at 180°, 250°, and 350°,respectively. Adjust the electrometer to provide about half ofthe full-scale deflection when 0.1 ng of PCP is injected.
Procedure (Note: Inject each PCP Standard Solution andSample Preparation twice to ensure that consistent responsesare obtained. Following each injection of PCP Standard Solu-tions or Sample Preparation, rinse the syringe 10 times withhexane. After each injection of PCP Standard Solutions orSample Preparation, inject 5 �L of hexane onto the gaschromatograph, or equivalent, and record the chromatogram.If peaks are observed at the retention time for PCP, repeat thehexane injection until such peaks are no longer encountered.)Inject 5-�L portions of each of the PCP Standard Solutions(0.0, 0.02, 0.10, 0.20, 0.50, 1.0, and 2.0 ng, respectively) andthe Reagent Blank into the gas chromatograph sequentially,and record the chromatograms. Measure the areas under thePCP peaks and the peak heights for each of the PCP StandardSolutions (retention time for PCP should be about 10 min),corrected for the Reagent Blank. The maximum acceptableReagent Blank for satisfactory performance of the method is0.01 �g/g. Similarly, inject 5 �L of the Sample Preparationinto the gas chromatograph, and record the chromatogram.Measure the area under the PCP peak and the peak height,corrected for the Reagent Blank. Determine the amount ofPCP in the Sample Preparation by comparing the peak areaand height to the peak area and height obtained from injectionof known amounts of PCP Standard Solutions; to ensure validmeasurement of PCP in the Sample Preparation, the size ofthe PCP peak from the Sample Preparation and the standardsshould be within �10%. The Sample Preparation may requirefurther dilution. Designate as AS the amount of PCP, expressedin nanograms, in the aliquot of the Sample Preparation. Calcu-late the concentration of PCP, in micrograms per gram, inthe sample by the formula
5AS.
Protein Determine as directed under Nitrogen Determina-tion, Appendix IIIC, transferring 1 g of sample, accuratelyweighed, into a 500-mL Kjeldahl flask. Percent protein equalspercent N × 5.55.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X. However, instead of using a 50-g sample, dissolve a 20.0-g sample in 100 mL of a 5% alcoholin water mixture, and proceed as directed under Sample Intro-duction and Distillation.
Packaging and Storage Store in tight containers.
Gellan GumINS: 418 CAS: [71010-52-1]
DESCRIPTION
Gellan Gum occurs as an off white powder. It is a high-molecular-weight polysaccharide gum produced by fermenta-
tion of a carbohydrate with a pure culture of Pseudomonaselodea, purified by recovery with isopropyl alcohol, dried,and milled. It is a heteropolysaccharide comprising a tetrasac-charide repeating unit of one rhamnose, one glucuronic acid,and two glucose units. The glucuronic acid is neutralized tomixed potassium, sodium, calcium, and magnesium salts. Itmay contain acyl (glyceryl and acetyl) groups as the O-glyco-sidically linked esters. It is soluble in hot or cold deionizedwater.
Function Stabilizer; thickener.
REQUIREMENTS
IdentificationA. Prepare a 1% solution by dissolving 1 g of sample in
99 mL of deionized water. Using a motorized stirrer and apropeller-type stirring blade, stir the mixture for about 2 h.(Save part of this solution for Identification Test B). Draw asmall amount of the solution into a wide-bore pipet, andtransfer it into a solution of 10% calcium chloride. A tough,wormlike gel forms instantly.
B. Add 0.5 g of sodium chloride to the 1% deionized watersolution prepared for Identification Test A, heat the solutionto 80°, stirring constantly, and hold the temperature at 80°for 1 min. Stop heating and stirring the solution, and allowit to cool to room temperature. A firm gel forms.Assay A sample yields not less than 3.3% and not more than6.8% of carbon dioxide (CO2), calculated on the dried basis.Isopropyl Alcohol Not more than 0.075%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 15.0%.
TESTS
Assay Determine as directed under Alginates Assay, Appen-dix IIIC, but use about 1.2 g of undried sample, accuratelyweighed.Isopropyl Alcohol
IPA Standard Solution Transfer 500.0 mg of chromato-graphic-quality isopropyl alcohol into a 50-mL volumetricflask, dilute to volume with water, and mix. Pipet 10 mL ofthis solution into a 100-mL volumetric flask, dilute to volumewith water, and mix.
TBA Standard Solution Transfer 500.0 mg of chromato-graphic-quality tert-butyl alcohol into a 50-mL volumetricflask, dilute to volume with water, and mix. Pipet 10 mL ofthis solution into a 100-mL volumetric flask, dilute to volumewith water, and mix.
Mixed Standard Solution Pipet 4 mL each of the IPAStandard Solution and of the TBA Standard Solution into a125-mL, graduated Erlenmeyer flask, dilute to about 100 mLwith water, and mix. This solution contains approximately 40�g each of isopropyl alcohol and of tert-butyl alcohol permilliliter.
Sample Preparation Disperse 1 mL of a suitable antifoamemulsion, such as Dow-Corning G-10, or equivalent, in 200mL of water contained in a 1000-mL 24/40 round-bottomdistilling flask. Add about 5 g of sample, accurately weighed,
192 / Geranium Oil, Algerian Type / Monographs FCC V
and shake for 1 h on a wrist-action mechanical shaker. Connectthe flask to a fractionating column, and distill about 100 mL,adjusting the heat so that foam does not enter the column.Add 4.0 mL of TBA Standard Solution to the distillate toobtain the Sample Preparation.
Procedure (See Chromatoghaphy, Appendix IIA.) Injectabout 5 �L of the Mixed Standard Solution into a suitablegas chromatograph equipped with a flame-ionization detectorand a 1.8-m × 3.2-mm stainless steel column, or equivalent,packed with 80- to 100-mesh Porapak QS, or equivalent.Maintain the column at 165°. Set the temperature of both theinjection port and the detector to 200°. Use helium as thecarrier gas, flowing at 80 mL/min. The retention time ofisopropyl alcohol is about 2 min, and that of tert-butyl alcoholis about 3 min.
Determine the areas of the IPA and TBA peaks, and calculatethe response factor, f, by the formula
AIPA/ATBA,
in which AIPA is the area of the isopropyl alcohol peak, andATBA is the area of the tert-butyl alcohol peak.
Similarly, inject about 5 �L of the Sample Preparation, anddetermine the peak areas, recording the area of the isopropylalcohol peak as SIPA, and that of the tert-butyl alcohol peakas STBA. Calculate the isopropyl alcohol content, in milligramsper kilogram, in the sample taken by the formula
(SIPA × 4000)/(f × STBA × W),
in which W is the weight, in grams, of the sample taken.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, using 2 g of sample, and 4 �g of lead(Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2.5 h.
Packaging and Storage Store in well-closed containers.
Geranium Oil, Algerian TypeRose Geranium Oil, Algerian Type
CAS: [8000-46-2]
DESCRIPTION
Geranium Oil, Algerian Type, occurs as a light to deep yellowliquid with a characteristic odor resembling rose and geraniol.It is the oil obtained by steam distillation from the leavesof Pelargonium graveolens L’Her. (Fam. Geraniaceae). It issoluble in most fixed oils, and it is soluble, usually withopalescence, in mineral oil and in propylene glycol. It ispractically insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 13.0% and not more than 29.5% ofesters, calculated as geranyl tiglate (C15H24O2).Acid Value Between 1.5 and 9.5.Angular Rotation Between −7° and −13°.Ester Value after Acetylation Between 203 and 234.Refractive Index Between 1.464 and 1.472 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.886 and 0.898.
TESTS
Assay Determine as directed in Ester Value under Esters,Appendix VI, using about 6 g of sample, accurately weighed.The ester value multiplied by 0.422 equals the percentage ofgeranyl tiglate (C15H24O2).Acid Value Determine as directed under Acid Value, Ap-pendix VI, using about 5 g of sample, accurately weighed.Modify the procedure by using 15 mL of water, instead ofalcohol, as diluent and by agitating the mixture thoroughlyduring the titration to keep the oil in suspension.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value after Acetylation Determine as directed underTotal Alcohols, Appendix VI, using about 1.9 g of the ace-tylated sample oil, accurately weighed, for saponification.Calculate the ester value after acetylation by the formula
A × 28.05/B,
in which A is the number of milliliters of 0.5 N alcoholicpotassium hydroxide consumed in the saponification, and Bis the weight, in grams, of acetylated sample oil.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 70% alcohol, but on further dilution with thealcohol, opalescence may occur, sometimes followed by sepa-ration of paraffin particles.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
View IR
FCC V Monographs / Ginger Oil / 193
Gibberellic Acid2,4�,7-Trihydroxy-1-methyl-8-methylenegibb-3-ene-1,10-dicarboxylic Acid 1,4-�-Lactone
HOH
CH3 COOH
H
OH
CH2
O
CO
C19H22O6 Formula wt 346.38
CAS: [77-06-5]
DESCRIPTION
Gibberellic Acid occurs as a white to pale yellow, crystallinepowder. It melts at about 234°. It is slightly soluble in waterand is soluble in alcohol and in acetone.
Function Enzyme activator.
REQUIREMENTS
Identification Dissolve a few milligrams of sample in 2mL of sulfuric acid. A red solution having a green fluorescenceforms.Assay Not less than 90.0% of C19H22O6, calculated on thedried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 3.0%.Optical (Specific) Rotation [�]D
20°: Between +75.0° and+90.0°, calculated on the dried basis.
TESTS
AssayStandard Preparation Transfer an accurately weighed
quantity of Gibberellic Acid containing not less than 90% oftotal gibberellins as Gibberellic Acid (USP, or equivalent),equivalent to about 25 mg of pure Gibberellic Acid, into a50-mL volumetric flask, dissolve in and dilute to volume withmethanol, and mix. Transfer 10.0 mL of this solution into asecond 50-mL volumetric flask, dilute to volume with metha-nol, and mix.
Assay Preparation Transfer about 40 mg of sample, accu-rately weighed, into a 50-mL volumetric flask, dissolve inand dilute to volume with methanol, and mix. Transfer 10.0mL of this solution into a 100-mL volumetric flask, dilute tovolume with methanol, and mix.
Procedure Transfer 5.0 mL of the Assay Preparation intoa 25- × 200-mm glass-stoppered tube, and transfer 4.0-mL and5.0-mL portions of the Standard Preparation into separate,similar tubes. Place the tubes in a boiling water bath, evaporateto dryness, and then dry in an oven at 90° for 10 min. Removethe tubes from the oven, stopper, and allow to cool to room
temperature. Dissolve the residue in each tube in 10.0 mL of8:10 sulfuric acid, heat in a boiling water bath for 10 min,and then cool in a 10° water bath for 5 min. Determine theabsorbance of the solutions in 1-cm cells at 535 nm with asuitable spectrophotometer, using the dilute sulfuric acid asthe blank. Record the absorbance of the solution from theAssay Preparation as AU. Note the absorbance of the twosolutions prepared from the 4.0-mL and 5.0-mL aliquots ofthe Standard Preparation, and record the absorbance of thefinal solution giving the value nearest to that of the sampleas AS; record as V the volume of the aliquot used in preparingthis solution. Calculate the quantity, in milligrams, ofC19H22O6 in the sample taken by the formula
500C × (V/5) × (AU/AS),
in which C is the concentration, in milligrams per milliliter,of the Standard Preparation.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead ion (Pb) in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 100° in vacuum for 7 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutionin alcohol containing 100 mg of sample in each milliliter. Donot use heat in preparing the solution.
Packaging and Storage Store in well-closed containers.
Ginger OilCAS: [8007-08-7]
DESCRIPTION
Ginger Oil occurs as a light yellow to yellow liquid with thearomatic, characteristic odor of ginger. It is the volatile oilobtained by steam distillation of the dried ground rhizome ofZingiber officianale, Roscoe (Fam. Zingiberaceae). It is solu-ble in most fixed oils and in mineral oil. It is soluble, usuallywith turbidity, in alcohol, but it is insoluble in glycerin andin propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown under Infrared Spectra, usingthe same test conditions as specified therein.Angular Rotation Between −28° and −47°.Refractive Index Between 1.488 and 1.494 at 20°.Saponification Value Not more than 20.Specific Gravity Between 0.870 and 0.882.
View IR
194 / Glucono Delta-Lactone / Monographs FCC V
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed under Saponi-fication Value, Appendix VI, using about 5 g of sample,accurately weighed.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Glucono Delta-Lactone
O
O
OH
HO
HO
OH
C6H10O6 Formula wt 178.14
INS: 575 CAS: [90-80-2]
DESCRIPTION
Glucono Delta-Lactone occurs as a fine, white, crystallinepowder. It is freely soluble in water and is sparingly solublein alcohol. It decomposes at about 153°.
Function Acidifier; leavening agent; sequestrant.
REQUIREMENTS
Identification Dissolve a portion of sample in water, heat-ing at 60° if necessary, to obtain a test solution containing10 mg/mL. Similarly prepare a standard solution with thesame concentration using USP Potassium Gluconate Refer-ence Standard. Separately apply 5-�L portions of both solu-tions on a thin-layer chromatographic plate (see Chromatogra-phy, Appendix IIA) coated with a 0.25-mm layer ofchromatographic silica gel, and allow to dry. Develop thechromatogram in a solvent mixture of ethanol, water, ammo-nium hydroxide, and ethyl acetate (5:3:1:1) until the solventfront has moved three-fourths of the length of the plate. Re-move the plate from the chamber, dry at 110° for 20 min,and cool. Spray the plate with a reagent prepared as follows:Dissolve 2.5 g of ammonium molybdate in 50 mL of 2 Nsulfuric acid in a 100-mL volumetric flask, add 1.0 g of ceric
sulfate, swirl to dissolve, dilute to volume with 2 N sulfuricacid, and mix. Heat the plate at 110° for about 10 min. Theprincipal spot obtained from the sample solution correspondsin color, size, and Rf value to that obtained from the standardsolution.Assay Not less than 99.0% and not more than 100.5% ofC6H10O6.Lead Not more than 4 mg/kg.Reducing Substances (as D-glucose) Not more than 0.5%.
TESTS
Assay Dissolve about 600 mg of sample, accuratelyweighed, in 100 mL of water in a 300-mL Erlenmeyer flask,add 50.0 mL of 0.1 N sodium hydroxide, and allow to standfor 15 min. Add phenolphthalein TS, and titrate the excessalkali with 0.1 N hydrochloric acid. Perform a blank determi-nation (see General Provisions). Each milliliter of 0.1 N hy-drochloric acid is equivalent to 17.81 mg of C6H10O6.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 4 �g of lead (Pb) ion in the control.Reducing Substances Transfer 10.0 g of sample, accuratelyweighed, into a 400-mL beaker, dissolve the sample in 40mL of water, add a few drops of phenolphthalein TS, andneutralize with 1:2 sodium hydroxide solution. Dilute to 50.0mL with water, and add 50 mL of alkaline cupric tartrate TS.Heat the mixture over a Bunsen burner, regulating the flameso that boiling begins in 4 min, and continue the boiling forexactly 2 min. Filter through a sintered-glass filter crucible,wash the filter with 3 or more small portions of water, andplace the crucible in an upright position in the original beaker.Add 5 mL of water and 3 mL of nitric acid to the crucible,mix with a stirring rod to ensure complete solution of thecuprous oxide, and wash the solution into a beaker with severalmL of water. Add bromine TS (5 to 10 mL) to the beakeruntil the color becomes yellow, and dilute with water to about75 mL. Add a few glass beads, boil over a Bunsen burneruntil the bromine is completely removed, and cool. Slowlyadd ammonium hydroxide until a deep blue color appears,then adjust the pH to approximately 4 with glacial acetic acid,and dilute to about 100 mL with water. Add 4 g of potassiumiodide, and titrate with 0.1 N sodium thiosulfate, adding starchTS just before the endpoint is reached. Not more than 16.1mL of titrant is required.
Packaging and Storage Store in well-closed containers.
Glucose SyrupCorn Syrup
DESCRIPTION
Glucose Syrup occurs as a clear, white to light yellow, viscousliquid. It is a clarified, concentrated, aqueous solution of sac-
FCC V Monographs / Glucose Syrup, Dried / 195
charides obtained by the partial hydrolysis of edible starchby food-grade acids and/or enzymes. Depending on the degreeof hydrolysis, it contains varying amounts of D-glucose. Whenobtained from corn starch, it is commonly designated as cornsyrup. It is miscible in all proportions with water.
Function Nutritive sweetener.
REQUIREMENTS
Labeling Indicate the presence of sulfur dioxide if the resid-ual concentration is greater than 10 mg/kg.Identification Add a few drops of a 1:20 aqueous solutionto 5 mL of hot alkaline cupric tartrate TS. A red precipitateof cuprous oxide forms.Assay Not less than 20.0% reducing sugar content (dextroseequivalent) expressed as D-glucose, calculated on the driedbasis.Lead Not more than 0.1 mg/kg.Residue on Ignition Not more than 0.5%.Starch Passes test.Sulfur Dioxide Not more than 0.004%.Total Solids Not less than 70.0%.
TESTS
Assay Determine as directed under Reducing Sugars Assay,Appendix X.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 20-g sample.Starch Add 1 drop of iodine TS to 1 g of sample dissolvedin 10 mL of water. A yellow color indicates the absence ofsoluble starch.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X, using a 35-g sample.Total Solids Determine the refractive index of a sample at20° or 45°, and use the appropriate Glucose Syrup table underGlucose Syrup (Corn Syrup), Appendix X.
Packaging and Storage Store in tightly closed containersin a dry place.
Glucose Syrup, DriedDried Glucose Syrup; Glucose Syrup Solids
DESCRIPTION
Glucose Syrup, Dried, occurs as a white to light yellow powderor granules. It is a purified, concentrated mixture of nutritivesaccharides obtained by the hydrolysis of edible starch and bypartially drying the resulting solution (glucose syrup). When
obtained from corn starch, it is commonly designated driedcorn syrup or corn syrup solids. Depending on the degree ofhydrolysis, it contains varying amounts of D-glucose. It issoluble in water.
Function Nutritive sweetener.
REQUIREMENTS
Labeling Indicate the presence of sulfur dioxide if the resid-ual concentration is greater than 10 mg/kg.Identification Add a few drops of a 1:20 aqueous solutionto 5 mL of hot alkaline cupric tartrate TS. A red precipitateof cuprous oxide forms.Assay Not less than 20.0% of reducing sugar content (dex-trose equivalent) expressed as D-glucose, calculated on thedried basis.Lead Not more than 0.1 mg/kg.Residue on Ignition Not more than 0.5%.Starch Passes test.Sulfur Dioxide Not more than 0.004%.Total Solids Not less than 90.0% when the reducing sugarcontent is 88.0% or greater; not less than 93.0% when thereducing sugar content is between 20.0% and 88.0%.
TESTS
Assay Determine as directed under Reducing Sugars Assay,Appendix X.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample at 525° for2 h.Starch Add 1 drop of iodine TS to 1 g of sample dissolvedin 10 mL of water. A yellow color indicates the absence ofsoluble starch.Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X, using a 25-g sample.Total Solids Determine the water content of an accuratelyweighed sample as directed under Water Determination, Ap-pendix IIB. Calculate the percent Total Solids by the formula
100(WU − WW)/WU,
in which WU is the weight, in milligrams, of the sample taken,and WW is the weight, in milligrams, of water determined.
Packaging and Storage Store in tightly closed containersin a dry environment.
196 / L-Glutamic Acid / Monographs FCC V
L-Glutamic AcidGlutamic Acid; L-2-Aminopentanedioic Acid
HOOCCH2CH2CCOOH
H NH2
C5H9NO4 Formula wt 147.13
INS: 620 CAS: [56-86-0]
DESCRIPTION
L-Glutamic Acid occurs as a white, free-flowing, crystallinepowder. It is slightly soluble in water, forming acidic solu-tions. The pH of a saturated solution is about 3.2.
Function Salt substitute; nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC5H9NO4, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.1%.Optical (Specific) Rotation [�]D
20°: Between +31.5° and+32.5°, calculated on the dried basis.Residue on Ignition Not more than 0.3%.
TESTS
Assay Dissolve about 200 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid. Add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to a green endpoint or until the blue colordisappears completely.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 14.71 mg of C5H9NO4.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation [�]D
20°: Determine as directedunder Optical (Specific) Rotation, Appendix IIB, using a solu-tion containing 10 g of a previously dried sample in sufficient2 N hydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers.
L-Glutamic Acid Hydrochloride2-Aminopentanedioic Acid Hydrochloride
HOOCCH2CH2CCOOH·HCl
H NH2
C5H9NO4·HCl Formula wt 183.59
CAS: [138-15-8]
DESCRIPTION
L-Glutamic Acid Hydrochloride occurs as a white, crystallinepowder. One gram dissolves in about 3 mL of water. It isalmost insoluble in alcohol and in ether. Its solutions are acidto litmus.
Function Salt substitute; flavoring agent; nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5%C5H9NO4·HCl, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.5%.Optical (Specific) Rotation [�]D
20°: Between +25.2° and+25.8°, calculated on the dried basis.Residue on Ignition Not more than 0.25%.
TESTS
Assay Dissolve about 100 mg of sample, previously driedat 80° for 4 h and accurately weighed, in 0.5 mL of water,add exactly 15.0 mL of 0.1 N perchloric acid, and heat on awater bath for 30 min.
Caution: Handle perchloric acid in an appropriatefume hood.
After cooling, add 45 mL of glacial acetic acid, and titratethe excess perchloric acid with 0.1 N sodium acetate, determin-ing the endpoint potentiometrically. Perform a blank determi-nation (see General Provisions), and make any necessarycorrection. Each milliliter of 0.1 N perchloric acid is equivalentto 18.36 mg of C5H9NO4·HCl.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 80° for 4 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 10 g of sample in sufficient 2 N hydrochloric acidto make 100 mL.
View IR View IR
FCC V Monographs / Glutaraldehyde / 197
Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
L-GlutamineL-2-Aminoglutaramic Acid
H2NCOCH2CH2CCOOH
NH2H
C5H10N2O3 Formula wt 146.15
CAS: [56-85-9]
DESCRIPTION
L-Glutamine occurs as white crystals or a crystalline powder.It is soluble in water and practically insoluble in alcoholand in ether. Its solutions are acid to litmus. It melts withdecomposition at about 185°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits maxima only at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC5H10N2O3, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Optical (Specific) Rotation [�]D
20°: Between +6.3° and+7.3°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 150 mg of sample, previously driedat 105° for 3 h and accurately weighed, in 3 mL of formicacid and 50 mL of glacial acetic acid, and titrate with 0.1 Nperchloric acid, determining the endpoint potentiometrically.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 14.62 mg of C5H10N2O3.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.
Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 4 g of a previously dried sample in sufficient waterto make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
GlutaraldehydeGlutaral; 1,5-Pentanedial
C5H8O2 Formula wt 100.12
CAS: [111-30-8]
DESCRIPTION
Glutaraldehyde occurs as a clear, nearly colorless, aqueoussolution. It is miscible with water. The grades of Glutaralde-hyde suitable for food use usually have concentrations be-tween 15% and 50%.
Function Fixing agent in the immobilization of enzymepreparations; cross-linking agent for microencapsulating fla-voring substances; antimicrobial for sugar milling.
REQUIREMENTS
Labeling Indicate the concentration of Glutaraldehyde.Identification
2,4-Dinitrophenylhydrazine Reagent Add 4 mL of sulfu-ric acid to 0.8 g of 2,4-dinitrophenylhydrazine, then whileswirling, add 6 mL of water, dropwise. When dissolution isessentially complete, add 20 mL of alcohol, mix, and filter.The filtrate is the reagent.
Procedure Add 0.4 mL of sample to 20 mL of 2,4-Dinitro-phenylhydrazine Reagent. Mix by swirling, and allow themixture to stand for 5 min. Collect the precipitate on a filter,and rinse thoroughly with alcohol. Dissolve the precipitate in20 mL of hot ethylene dichloride, filter, and cool the filtratein an ice bath until crystallization occurs. Collect the precipi-tate on a filter. Redissolve the precipitate by refluxing with30 mL of acetone, filter, and cool the filtrate in an ice bathuntil crystallization occurs. Collect the precipitate on a filter.The 2,4-dinitrophenylhydrazone so obtained melts between185° and 195° (see Melting Range or Temperature, Appen-dix IIB).Assay Not less than 100.0% and not more than 105.0% ofthe labeled amount of C5H8O2.Lead Not more than 2 mg/kg.pH Between 3.1 and 4.5.
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198 / Glycerin / Monographs FCC V
TESTS
AssayHydroxylamine Hydrochloride Solution Prepare a 0.5 N
solution by dissolving 35.0 g of hydroxylamine hydrochloridein water contained in a 1-L volumetric flask, dilute to volumewith water, and mix.
Triethanolamine Solution Prepare a 0.5 N solution bytransferring 65 mL (74 g) of 98% triethanolamine into a 1-L volumetric flask, dilute to volume with water, and mix.
Procedure Adjust to pH 3.60 a volume of HydroxylamineHydrochloride Solution sufficient for analyzing both the blankand the sample. Using a suitable autotitrator, titrate with Tri-ethanolamine Solution.
Caution: The stirring rate is critical throughout theneutralization and analysis. When stirring is required,ensure adequate mixing without whipping air bubblesinto the solution. The stirring speed should be consistentfor both the sample and the blank.
Transfer 65.0 mL of the pH 3.60 Hydroxylamine Hydro-chloride Solution into each of two titration cups. Add a Teflon(or equivalent) stirrer to each cup. Using the autotitrator, add30.8 mL of the neutralized Triethanolamine Solution to eachtitration cup, cover, and mix. Using a weighing pipet, intro-duce into one of the cups a suitable portion of sample equiva-lent to about 300 mg of Glutaraldehyde. Mix the solutionsthoroughly, and allow the sample and blank to stand at roomtemperature for at least 60 min but not for more than 90 min.
Titrate the sample and the blank to pH 3.60 with 0.5 Nhydrochloric acid, determining the endpoint potentiomet-rically. Calculate the percentage, by weight, of C5H8O2 in thesample by the formula
[N(B – A)(0.05006)/W]100,
in which N is the normality of the hydrochloric acid; B andA are the volumes, in milliliters, of 0.5 N hydrochloric acidconsumed by the blank and the sample solutions, respectively;0.05006 is the milliequivalent weight, in grams per milliequiv-alent, of Glutaraldehyde; and W is the weight, in grams, ofsample taken.Lead Determine as directed in the Atomic Absorption Spec-trophotometric Method under Lead Limit Test, Appendix IIIB,using a 10-g sample.pH Determine as directed under pH Determination, Appen-dix IIB.
Packaging and Storage Store in tight, light-resistant con-tainers protected from heat.
GlycerinGlycerol
CH2OHCHOHCH2OH
C3H8O3 Formula wt 92.09
INS: 422 CAS: [56-81-5]
DESCRIPTION
Glycerin occurs as a clear, colorless, viscous liquid. It ishygroscopic, and its solutions are neutral. Glycerin is misciblewith water and with alcohol. It is insoluble in chloroform, inether, and in fixed and volatile oils.
Function Humectant; solvent; bodying agent; plasticizer.
REQUIREMENTS
Identification The infrared absorption spectrum of a thinfilm of sample exhibits a very strong, broad band at 2.7 �mto 3.3 �m; a strong doublet at about 3.4 �m; a maximum atabout 6.1 �m; a strong region of absorption between 6.7 �mand 8.3 �m, having maxima at about 7.1 �m, 7.6 �m, and8.2 �m, and a very strong region of bands at about 9.0 �m,9.6 �m, 10.1 �m, 10.9 �m, and 11.8 �m.
Note: Glycerin having a low water content may notexhibit a maximum at about 6.1 �m.
Assay Not less than 95.0% and not more than 100.5% ofC3H8O3.Chlorinated Compounds (as Cl) Not more than 0.003%.Color Passes test.Fatty Acids and Esters Passes test (limit about 0.1%, calcu-lated as butyric acid).Lead Not more than 1 mg/kg.Readily Carbonizable Substances Passes test.Residue on Ignition Not more than 0.01%.Specific Gravity Not less than 1.249.
TESTS
AssaySodium Periodate Solution Dissolve 60 g of sodium meta-
periodate (NaIO4) in sufficient water containing 120 mL of0.1 N sulfuric acid to make 1000 mL. Do not heat to dissolvethe periodate. If the solution is not clear, filter through asintered-glass filter. Store the solution in a glass-stoppered,light-resistant container. Test the suitability of this solutionas follows: Pipet 10 mL into a 250-mL volumetric flask, diluteto volume with water, and mix. Dissolve about 550 mg ofsample in 50 mL of water, and add 50 mL of the dilutedperiodate solution by pipet. For a blank, pipet 50 mL of thediluted periodate solution into a flask containing 50 mL ofwater. Allow the solutions to stand for 30 min, then add 5mL of hydrochloric acid and 10 mL of potassium iodide TS
FCC V Monographs / Glycerol Ester of Gum Rosin / 199
to each, and rotate to mix. Allow to stand for 5 min, add 100mL of water, and titrate with 0.1 N sodium thiosulfate, shakingcontinuously and adding starch TS near the endpoint. Theratio of the volume of 0.1 N sodium thiosulfate required forthe Glycerin:periodate mixture to that required for the blankshould be between 0.750 and 0.765.
Procedure Transfer about 400 mg of sample, accuratelyweighed, into a 600-mL beaker, dilute with 50 mL of water,add bromothymol blue TS, and acidify with 0.2 N sulfuricacid to a definite green or green-yellow color. Neutralize with0.05 N sodium hydroxide to a definite blue endpoint free ofgreen color. Prepare a blank containing 50 mL of water, andneutralize in the same manner. Pipet 50 mL of the SodiumPeriodate Solution into each beaker, mix by swirling gently,cover with a watch glass, and allow to stand for 30 min atroom temperature (not above 35°) in the dark or in subduedlight. Add 10 mL of a mixture consisting of equal volumesof ethylene glycol and water, and allow to stand for 20 min.Dilute each solution to about 300 mL with water, and titratewith 0.1 N sodium hydroxide to a pH of 8.1 � 0.1 for thesample and 6.5 � 0.1 for the blank, using a pH meter pre-viously calibrated with pH 4.0 Acid Phthalate Standard BufferSolution (see Solutions and Indicators). Each milliliter of0.1 N sodium hydroxide, after correction for the blank, isequivalent to 9.210 mg of Glycerin (C3H8O3).Chlorinated Compounds Transfer 5.0 g of sample into adry, 100-mL round-bottom, ground-joint flask, and add 15mL of morpholine to it. Connect the flask with a ground jointreflux condenser, and reflux the mixture gently for 3 h. Rinsethe condenser with 10 mL of water, receiving the washinginto the flask, and cautiously acidify with nitric acid. Transferthe solution to a suitable comparison tube, add 0.5 mL ofsilver nitrate TS, dilute with water to 50.0 mL, and mixthoroughly. Any turbidity does not exceed that produced by150 �g of chloride (Cl) in an equal volume of solution con-taining the quantities of reagents used in the test, omittingthe refluxing.Color The color of sample, when viewed downward againsta white surface in a 50-mL Nessler tube, is not darker thanthe color of a standard made by diluting 0.40 mL of ferricchloride CS with water to 50 mL and similarly viewed in aNessler tube of the same diameter and color as that containingthe sample.Fatty Acids and Esters Mix a 40.0-mL (50-g) sample with50 mL of recently boiled water and 5.0 mL of 0.5 N sodiumhydroxide. Boil the mixture for 5 min, cool, add phenolphtha-lein TS, and titrate the excess alkali with 0.5 N hydrochloricacid. More than 4 mL of 0.5 N hydrochloric acid is consumed.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Readily Carbonizable Substances Rinse a glass-stoppered25-mL cylinder with 95% sulfuric acid, and allow it to drainfor 10 min. Add 5 mL of sample and 5 mL of 95% sulfuricacid, shake vigorously for 1 min, and allow to stand for 1 h.The mixture is no darker than Matching Fluid H as describedunder Readily Carbonizable Substances, Appendix IIB.
Residue on Ignition Heat 50 g of sample in a tared, opendish, and ignite the vapors, allowing them to burn until thesample has been completely consumed. After cooling, moistenthe residue with 0.5 mL of sulfuric acid, and complete theignition by heating for 15-min periods at 800° � 25° toconstant weight.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in tight containers.
Glycerol Ester of Gum Rosin
DESCRIPTION
Glycerol Ester of Gum Rosin occurs as a hard, pale amber-colored resin (color N or paler as determined by ASTM Desig-nation D 509) produced by the esterification of pale gumrosin with food-grade glycerin and purified by steam stripping.It is soluble in acetone and in toluene, but is insoluble in water.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared absorption spectrum of a meltedsample on a potassium bromide plate exhibits relative maximaat the same wavelengths as those of a typical spectrum asshown in the section on Infrared Spectra, using the same testconditions as specified therein.Acid Number Between 3 and 9.Lead Not more than 1 mg/kg.Ring-and-Ball Softening Point 82° or higher.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV. This solution meets the requirementsof the Lead Limit Test, Appendix IIIB, using 5 �g of leadion (Pb) in the control.Ring-and-Ball Softening Point Determine as directed inthe Ring-and-Ball Method under Softening Point, AppendixIX.
Packaging and Storage Store in well-closed containers.
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200 / Glycerol Ester of Partially Dimerized Rosin / Monographs FCC V
Glycerol Ester of Partially Dimerized Rosin
DESCRIPTION
Glycerol Ester of Partially Dimerized Rosin occurs as a hard,pale, amber-colored resin (color M or paler as determined byASTM Designation D 509) produced by the esterification ofpartially dimerized rosin with food-grade glycerin, and puri-fied by steam stripping. It is soluble in acetone, but is insolublein water.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared absorption spectrum of a meltedsample on a potassium bromide plate exhibits relative maximaat the same wavelengths as those of a typical spectrum asshown in the section on Infrared Spectra, using the same testconditions as specified therein.Acid Number Between 3 and 8.Lead Not more than 1 mg/kg.Ring-and-Ball Softening Point 103° or higher.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV. This solution meets the requirementsof the Lead Limit Test, Appendix IIIB, using 5 �g of leadion (Pb) in the control.Ring-and-Ball Softening Point Determine as directed inthe Ring-and-Ball Method under Softening Point, AppendixIX.
Packaging and Storage Store in well-closed containers.
Glycerol Ester of Partially HydrogenatedGum Rosin
DESCRIPTION
Glycerol Ester of Partially Hydrogenated Gum Rosin occursas a medium-hard, pale amber-colored resin (color N or paleras determined by ASTM Designation D 509). It is producedby the esterification of partially hydrogenated gum rosin withfood-grade glycerin and purified by steam stripping. It issoluble in acetone and in toluene, but is insoluble in waterand in alcohol.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared absorption spectrum of a meltedsample on a potassium bromide plate exhibits relative maxima
at the same wavelengths as those of a typical spectrum asshown in the section on Infrared Spectra, using the same testconditions as specified therein.Acid Number Between 3 and 10.Drop Softening Point 79° or higher.Lead Not more than 1 mg/kg.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Drop Softening Point Determine as directed in the DropMethod under Softening Point, Appendix IX, using a bathtemperature of 100°.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV, using 5 g of sample. This solutionmeets the requirements of the Lead Limit Test, Appendix IIIB,using 5 �g of lead ion (Pb) in the control.
Packaging and Storage Store in well-closed containers.
Glycerol Ester of Partially HydrogenatedWood Rosin
DESCRIPTION
Glycerol Ester of Partially Hydrogenated Wood Rosin occursas a medium-hard, pale amber-colored resin (color N or paleras determined by ASTM Designation D 509). It is producedby the esterification of partially hydrogenated wood rosin withfood-grade glycerin and purified by steam stripping. It issoluble in acetone, but is insoluble in water and in alcohol.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared absorption spectrum of a disper-sion of the melted sample on a potassium bromide plate exhib-its relative maxima at the same wavelengths as those of atypical spectrum as shown in the section on Infrared Spectra,using the same test conditions as specified therein.Acid Number Between 3 and 10.Lead Not more than 1 mg/kg.Ring-and-Ball Softening Point 68° or higher.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV. This solution meets the requirementsof the Lead Limit Test, Appendix IIIB, using 5 �g of leadion (Pb) in the control.
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FCC V Monographs / Glycerol Ester of Wood Rosin / 201
Ring-and-Ball Softening Point Determine as directed inRing-and-Ball Method under Softening Point, Appendix IX,using a water bath.
Packaging and Storage Store in well-closed containers.
Glycerol Ester of Polymerized Rosin
DESCRIPTION
Glycerol Ester of Polymerized Rosin occurs as a hard, paleamber-colored resin (color M or paler as determined by ASTMDesignation D 509). It is produced by the esterification ofpolymerized rosin with food-grade glycerin and purified bysteam stripping. It is soluble in acetone, but is insoluble inwater and in alcohol.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared absorption spectrum of a disper-sion of the melted sample on a potassium bromide plate exhib-its relative maxima at the same wavelengths as those of atypical spectrum as shown in the section on Infrared Spectra,using the same test conditions as specified therein.Acid Number Between 3 and 12.Lead Not more than 1 mg/kg.Ring-and-Ball Softening Point 80° or higher.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV. This solution meets the requirementsof the Lead Limit Test, Appendix IIIB, using 5 �g of leadion (Pb) in the control.Ring-and-Ball Softening Point Determine as directed forRing-and-Ball Method under Softening Point, Appendix IX.
Packaging and Storage Store in well-closed containers.
Glycerol Ester of Tall Oil Rosin
DESCRIPTION
Glycerol Ester of Tall Oil Rosin occurs as a pale amber-colored resin (color N or paler as determined by ASTM Desig-nation D 509). It is produced by the esterification of tall oil
rosin with food-grade glycerin and purified by steam stripping.It is soluble in acetone, but is insoluble in water.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared spectrum of a melted sampleon a potassium bromide plate exhibits relative maxima at thesame wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Acid Number Between 2 and 12.Lead Not more than 1 mg/kg.Ring-and-Ball Softening Point 80° or higher.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV. This solution meets the requirementsof the Lead Limit Test, Appendix IIIB, using 5 �g of lead(Pb) ion in the control.Ring-and-Ball Softening Point Determine as directed inthe Ring-and-Ball Method under Softening Point, AppendixIX.
Packaging and Storage Store in well-closed containers.
Glycerol Ester of Wood Rosin
Ester Gum
INS: 445 CAS: [8050-30-4]
DESCRIPTION
Glycerol Ester of Wood Rosin occurs as a hard, pale amber-colored resin (color N or paler as determined by ASTM Desig-nation D 509). It is produced by the esterification of palewood rosin with food-grade glycerin. The rosin is obtainedby solvent extraction of aged pine stumps, followed by aliquid–liquid solvent refining process. When intended for usein chewing gum base, the product is usually purified by steamstripping, but when intended for use in adjusting the densityof citrus oils for beverages, it is purified by countercurrentsteam distillation. It is soluble in acetone, but it is insolublein water.
Function Masticatory substance in chewing gum base; bev-erage stabilizer.
REQUIREMENTS
Identification The infrared spectrum of a melted sampleon a potassium bromide plate exhibits relative maxima at the
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202 / Glyceryl Behenate / Monographs FCC V
same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Acid Number Between 3 and 9.Lead Not more than 1 mg/kg.Ring-and-Ball Softening Point 82° or higher.
TESTS
Acid Number Determine as directed under Acid Number,Appendix IX.Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV, using 3.3 �g of lead (Pb) ion in thecontrol.Ring-and-Ball Softening Point Determine as directed inthe Ring-and-Ball Method under Softening Point, AppendixIX.
Packaging and Storage Store in well-closed containers.
Glyceryl BehenateGlyceryl Tribehenate; Glyceryl Tridocosanoate; Tribehenoyl-sn-glycerol; Tridocosanoyl-sn-glycerol
C69H134O6 Formula wt 1059.83
CAS: [30233-64-8]
DESCRIPTION
Glyceryl Behenate occurs as a fine powder. It is a mixtureof fatty acid glycerides, primarily glyceryl esters of behenicacid, that melts at about 70°. It is soluble in chloroform andpractically insoluble in water and in alcohol.
Function Emulsifier; texturizer.
REQUIREMENTS
IdentificationA. (Caution: Ether is highly volatile and flammable. Its
vapor, when mixed with air and ignited, may explode.)Solvent Mixture Prepare a 96:4 chloroform:acetone
mixture.Standard Solution Prepare a 6% solution of USP Glyceryl
Behenate Reference Standard in chloroform.Test Solution Prepare a 6% solution of sample in chlo-
roform.Chromatographic Plates Use suitable thin-layer chro-
matographic plates (see Chromatography, Appendix IIA)coated with a 0.25-mm layer of chromatographic silica gel.Pretreat the plates by placing them in a chromatographicchamber saturated with ether. Remove the plates from thechamber, allow the ether to evaporate, and immerse them ina 2.5% solution of boric acid in alcohol. After about 1 min,
withdraw the plates, and allow them to dry at ambient tempera-ture. Heat to 110° for 30 min to activate the plates, and thenkeep them in a desiccator.
Procedure Apply 10 �L of Test Solution and 10 �L ofStandard Solution on one of the chromatographic plates. De-velop the chromatogram in the Solvent Mixture until the sol-vent front has moved about 12 cm. Remove the plate fromthe developing chamber and allow the solvent to evaporate.Spray the chromatogram with a 0.02% solution of dichloroflu-orescein in alcohol. Examine the spots under short-wavelengthultraviolet light. The Rf values of the spots obtained from theTest Solution correspond to those obtained from the StandardSolution.
B. (See Chromatography, Appendix IIA.) Dissolve about22 mg of sample in 1 mL of toluene in a screw-cap vial witha Teflon-lined septum. Add about 0.4 mL of 0.2 N methanolic(m-trifluoromethylphenyl) trimethylammonium hydroxide,attach the cap, and mix. Allow the vial to stand at roomtemperature for at least 30 min. Introduce a suitable volumeof the mixture into a gas chromatograph equipped with aflame-ionization detector and a 1.8-m × 4-mm (id) columnpacked with a 10% coating of 50% 3-cyanopropyl–50% phe-nylmethylsilicone (SP 2300, or equivalent) on a silanizedsiliceous earth support (Supelcoport, or equivalent) main-tained at a temperature of about 225°. Repeat the procedureusing USP Glyceryl Behenate Reference Standard in lieuof a sample. The retention time of the main peak in thechromatogram of a sample corresponds to that of the mainpeak in the chromatogram of the preparation of USP GlycerylBehenate Reference Standard. The ratio of the response ofthe main peak to the sum of all the responses is not lessthan 0.83.Acid Value Not more than 4.Iodine Value Not more than 3.Free Glycerin Not more than 1.0%.Lead Not more than 1 mg/kg.1-Monoglycerides Content Not less than 12.0% and notmore than 18.0%.Residue on Ignition Not more than 0.1%.Saponification Value Not less than 145 and not morethan 165.
TESTS
Acid Value Suspend about 10 g of sample, accuratelyweighed, in a flask containing 50 mL of a 1:1 mixture ofalcohol and ether that has been neutralized to phenolphthaleinwith 0.1 N sodium hydroxide. Connect the flask with a suitablecondenser, and while frequently shaking, warm for about 10min. Add 1 mL of phenolphthalein TS, and titrate with 0.1N sodium hydroxide until the solution remains faintly pinkafter shaking for 30 s. Calculate the acid value by the formula
56.1V × N/W,
in which V is the volume, in milliliters, of the 0.1 N sodiumhydroxide solution; N is the normality of the sodium hydroxidesolution; and W is the weight, in grams, of sample taken.Iodine Value Determine as directed under Iodine Value,Appendix VII.
FCC V Monographs / Glyceryl-Lacto Esters of Fatty Acids / 203
Free Glycerin Determine as directed under Free Glycerinor Propylene Glycol, Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.1-Monoglycerides Content Determine as directed under 1-Monoglycerides, Appendix VII, using about 1 g of a samplethat has been melted at a temperature not higher than 80°,stirred, and accurately weighed.
Note: If the sample titration is less than 0.8 volumesof the blank titration, discard and repeat, using a smallerweight of sample.
Calculate the percentage of 1-monoglycerides, as glycerylmonobehenate, by the formula
20.73N × (B × S)/W,
in which 20.73 is one-twentieth of the molecular weight ofglyceryl monobehenate; N is the normality of the sodiumthiosulfate; B and S are the volumes, in milliliters, of 0.1 Nsodium thiosulfate consumed by the blank and the sample,respectively; and W is the weight, in grams, of sample taken.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 5-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.
Packaging and Storage Store in tight containers at a tem-perature not higher than 35°.
Glyceryl-Lacto Esters of Fatty Acids
Lactated Mono-Diglycerides; Lactic and Fatty Acid Estersof Glycerol
INS: 472b
DESCRIPTION
Glyceryl-Lacto Esters of Fatty Acids occur as a waxy solidthat varies in consistency from soft to hard. They are a mixtureof partial lactic and fatty acid esters of glycerin. They aredispersible in hot water and are moderately soluble in hotisopropanol, in xylene, and in cottonseed oil.
Function Emulsifier; stabilizer.
REQUIREMENTS
Identification Transfer 1 mL of the Sample Solution re-maining at the end of the test for Total Lactic Acid (below)into a 25-mL glass-stoppered test tube, add 0.1 mL of cupricsulfate solution (1 g of CuSO4·5H2O in 25 mL of water) and6 mL of sulfuric acid, and mix. Stopper loosely, heat in aboiling water bath for 5 min, and then cool in an ice bath for 5
min. Remove from the ice bath, add 0.1 mL of p-phenylphenolsolution (75 mg dissolved in 5 mL of 1 N sodium hydroxide),and mix. Allow to stand at room temperature for 1 min, thenheat in a boiling water bath for 1 min. A deep, blue-violetcolor indicates the presence of lactic acid.Lead Not more than 0.5 mg/kg.Residue on Ignition Not more than 0.1%.Unsaponifiable Matter Not more than 2.0%.
The following specifications should conform to the representa-tions of the vendor: Acid Value, Free Glycerin, 1-Monoglycer-ide Content, Total Lactic Acid, and Water.
TESTS
Acid Value Determine as directed in Method II under AcidValue, Appendix VII.Free Glycerin Determine as directed under Free Glycerinor Propylene Glycol, Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.1-Monoglyceride Content Determine as directed under 1-Monoglycerides, Appendix VII.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Total Lactic Acid Transfer an accurately weighed portionof melted sample, equivalent to between 140 and 170 mg oflactic acid, into a 250-mL Erlenmeyer flask. Pipet 20 mL of0.5 N alcoholic potassium hydroxide into the flask, connectan air condenser at least 65 cm long, and reflux for 30 min.Run a blank determination (see General Provisions) usingthe same volume of 0.5 N alcoholic potassium hydroxide. Add20 mL of water to each flask, then disconnect the condensers,evaporate to a volume of 20 mL, and cool to about 40°. Addmethyl red TS to each flask, and titrate the blank with 0.5 Nhydrochloric acid. While swirling the sample flask, add ex-actly the same volume of 0.5 N hydrochloric acid as with theblank. Add 50 mL of hexane to each flask. Swirl the sampleflask vigorously to dissolve the fatty acids, then quantitativelytransfer the contents of each flask into separate 250-mL sepa-rators, and shake for 30 s. Collect the aqueous phases in 300-mL Erlenmeyer flasks, wash the hexane solutions with 50mL of water, and combine the wash solutions with the originalaqueous phases in the Erlenmeyer flasks, discarding the hex-ane solutions. Titrate with 0.1 N potassium hydroxide, usingphenolphthalein TS as the indicator, to a pink color that per-sists for at least 30 s. Calculate the percent of total lactic acidby the formula
[9.008(S – B)(N)]/W,
in which 9.008 is the equivalence factor for lactic acid; (S –B) is the difference, in milliliters, between the volumes of 0.1N potassium hydroxide required for the sample and for theblank, respectively; N is the exact normality of the potassiumhydroxide solution; and W is the weight, in grams, of thesample taken.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.
204 / Glyceryl Monooleate / Monographs FCC V
Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Glyceryl MonooleateMonoolein; 1,2,3-Propanetriol
C H
H2C
H2C
HO
OH
O C (CH2)7 CH CH (CH2)7CH3
O
C21H40O4 Formula wt 356.54
INS: 471 CAS: [25496-72-4]
FEMA: 2526
DESCRIPTION
Glyceryl Monooleate occurs as a clear liquid at room tempera-ture. It has a mild, fatty taste. It is prepared by esterifyingglycerin with food-grade oleic acid in the presence of a suitablecatalyst such as aluminum oxide. It also occurs in many animaland vegetable fats such as tallow and cocoa butter. It is solublein hot alcohol and in chloroform; very slightly soluble in coldalcohol, in ether, and in petroleum ether; and insoluble inwater. It melts at around 15°. It may also contain tri- anddiesters.
Function Emulsifier; flavoring agent.
REQUIREMENTS
Identification Glyceryl Monooleate exhibits the followingtypical composition profile of fatty acids determined as di-rected under Fatty Acid Composition, Appendix VII:
Fatty Acid: ≤12 12:0 14:0 16:0 16:1Weight % (Range): 0 0 <4 1–5 <9Fatty Acid: 18:0 18:1 18:2 ≥20Weight % (Range): <3.0 ≥82 3–7 <1.5
Assay Not less than 35.0% monoglycerides, calculated onthe anhydrous basis.Acid Value Not more than 6.Free Glycerin Not more than 6.0%.Hydroxyl Value Between 300 and 330.Iodine Value Between 58 and 80.Lead Not more than 1 mg/kg.Residue on Ignition Not more than 0.1%.Saponification Value Between 160 and 176.Water Not more than 1.0%.
TESTS
AssayPropionating Reagent Mix 10 mL of pyridine and 20 mL
of propionic anhydride.Internal Standard Solution Transfer about 400 mg of hex-
adecyl hexadecanoate, accurately weighed, into a 100-mLvolumetric flask, dilute with chloroform to volume, and mix.
Standard Preparation Transfer about 50 mg of USP Mon-oglycerides Reference Standard, accurately weighed, into a25-mL flask, add 5 mL of Internal Standard Solution by pipet,and mix. When solution is complete, immerse the flask in awater bath maintained at a temperature between 45° and 50°,and volatilize the chloroform with the aid of a stream ofnitrogen. Add 3.0 mL of Propionating Reagent, and heat ona hot plate at 75° for 30 min. Evaporate the reagents with theaid of a stream of nitrogen and gentle steam heat. Add 15mL of chloroform, and swirl to dissolve the residue.
Assay Preparation Transfer about 50 mg of sample, accu-rately weighed, into a 25-mL conical flask, and proceed asdirected for Standard Preparation, beginning with ‘‘add 5mL of Internal Standard Solution. . . .’’
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flame-ionization detector, and containing a 2.4-m × 4-mm (id) boro-silicate glass column, or equivalent, packed with 2% liquidphase, 5% phenyl methyl silicone on 80- to 100-mesh support(Supelcoport, or equivalent). Maintain the column isother-mally at a temperature between 270° and 280°, and the injec-tion port and detector block at about 310°. Use helium as thecarrier gas at a flow rate of about 70 mL/min.
System Suitability Chromatograph 6 to 10 injections ofthe Standard Preparation as directed under Procedure. Theresolution factor, R, between the peaks for the derivatizedglyceryl hexadecanoate and glyceryl octadecanoate is not lessthan 2.0, and the relative standard deviation of the ratio ofthe peak area of the derivatized glyceryl cis-9-octadecanoateto that of the hexadecyl hexadecanoate is not more than 2.0%.
Procedure Inject a suitable portion of the Standard Prepa-ration into the gas chromatograph, and record the chromato-gram. Measure the areas under the peaks, and record thevalues of the sum of the areas under the derivatized monoglyc-eride peaks and of the area under the hexadecyl hexadecanoatepeak as AS and AD, respectively. Calculate the response factor,F, taken by the formula
(AS/AD)(WD/WS),
in which WD and WS are the weights, in milligrams, of hexade-cyl hexadecanoate and USP Monoglycerides Reference Stan-dard, respectively, in the Standard Preparation. Similarlyinject a suitable portion of the Assay Preparation, and recordthe chromatogram. Measure the areas under the peaks, andrecord the values of the sum of the areas under the derivatizedmonoglyceride peaks and of the area under the hexadecylhexadecanoate peak as aU and aD, respectively. Calculate thequantity, in milligrams, of monoglycerides in the amount ofsample taken by the formula
(WD/F)(aU/aD).
FCC V Monographs / Glyceryl Monostearate / 205
Acid Value Determine as directed under Acid Value, Ap-pendix VII.Free Glycerin
Propionating Reagent Mix 10 mL of pyridine with 20mL of propionic anhydride.
Internal Standard Solution Dissolve a suitable quantityof tributyrin, accurately weighed, in chloroform and dilutequantitatively with chloroform to obtain a solution having aconcentration of about 0.2 mg/mL.
Standard Preparation Transfer about 15 mg of glycerinand about 50 mg of tributyrin, both accurately weighed, intoa glass-stoppered, 25-mL conical flask, add 3 mL of Propio-nating Reagent, and heat at 75° for 30 min. Volatilize thereagents with the aid of a stream of nitrogen at room tempera-ture, add about 12 mL of chloroform, and mix. Dilute about1 mL of this mixture with chloroform to about 20 mL, and mix.
Test Preparation Transfer about 50 mg of sample, accu-rately weighed, into a glass-stoppered, 25-mL conical flask;add 5 mL of Internal Standard Solution by pipet; and mix todissolve. Immerse the flask in a water bath maintained at atemperature between 45° and 50°, and volatilize the chloro-form with the aid of a stream of nitrogen. Add 3 mL ofPropionating Reagent, and heat at 75° for 30 min. Volatilizethe reagents with the aid of a stream of nitrogen at roomtemperature, add about 5 mL of chloroform, and mix.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flame-ionization detector and containing a 2.4-m × 4-mm borosilicateglass column, or equivalent, packed with 2% liquid phaseconsisting of a high-molecular-weight compound of polyeth-ylene glycol and a diepoxide (Carbowax 20 M, or equivalent)on an 80- to 100-mesh siliceous earth support (ChromosorbW AW DMCS, or equivalent). Maintain the column isother-mally at a temperature between 190° and 200° and the injec-tion port and detector block at about 300° and 310°, respec-tively. Use helium as the carrier gas at a flow rate of about70 mL/min.
System Suitability Chromatograph 6 to 10 injections ofthe Standard Preparation as directed under Procedure. Theresolution factor, R, between the peaks for the derivatizedglycerin and tributyrin is not less than 4.0, and the relativestandard deviation of the ratio of their peak areas is not morethan 2.0%.
Procedure Inject a suitable portion of the Standard Prepa-ration into the gas chromatograph, or equivalent, and recordthe chromatogram. Measure the areas under the peaks andrecord the values of the areas under the tripropionin andtributyrin peaks as AS and AD, respectively. Calculate theresponse factor, F, taken by the formula
(AD/AS)(WS/WD),
in which WS and WD are the weights, in milligrams, of glycerinand tributyrin, respectively, in the Standard Preparation. Sim-ilarly inject a suitable portion of the Test Preparation, andrecord the chromatogram. Measure the areas under the peaksand record the values of the areas under the tripropionin andtributyrin peaks as aU and aD, respectively. Calculate thepercentage of glycerin by the formula
100F(aU/aD)(wD/wU),
in which wD is the weight, in milligrams, of tributyrin in 5mL of Internal Standard Solution, and wU is the weight, inmilligrams, of Glyceryl Monooleate in the Test Preparation.Hydroxyl Value Determine as directed in Method II underHydroxyl Value, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 5-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB, using 0.5 g of sample and 20 mL of a 1:1methanol:chloroform mixture.
Packaging and Storage Store in tight, light-resistant con-tainers.
Glyceryl MonostearateMonostearin; 1,2,3-Propanetriol Octadecanoate
CAS: [31566-31-1]
DESCRIPTION
Glyceryl Monostearate occurs as a white, waxlike solid, asflakes, or as beads. It is a mixture of Glyceryl Monostearateand glyceryl monopalmitate. It may contain a suitable antioxi-dant. It is soluble in hot organic solvents such as acetone,alcohol, and ether and in mineral or fixed oils. It is dispersiblein hot water with the aid of soap or suitable surfactants.
Function Emulsifier.
REQUIREMENTS
Identification Heat the sample with 3 parts water to 2° to5° above its melting point. An irreversible gel forms whenthe sample is held at this temperature.Assay Not less than 90.0% monoglycerides of saturatedfatty acids.Acid Value Not more than 6.Free Glycerin Not more than 1.2%.Hydroxyl Value Between 300 and 330.Iodine Value Not more than 3.Lead Not more than 1 mg/kg.Melting Range Not below 65°.Residue on Ignition Not more than 0.1%.Saponification Value Not less than 150 and not morethan 165.
206 / Glyceryl Monostearate / Monographs FCC V
TESTS
AssayPropionating Reagent Mix 10 mL of pyridine and 20 mL
of propionic anhydride.Internal Standard Solution Transfer about 400 mg of hex-
adecyl hexadecanoate, accurately weighed, into a 100-mLvolumetric flask, dissolve in chloroform, dilute with chloro-form to volume, and mix.
Standard Preparation Transfer about 50 mg of USP Mon-oglycerides Reference Standard, accurately weighed, to a 25-mL conical flask, add 5 mL of Internal Standard Solution bypipet, and mix. When solution is complete, immerse the flaskin a water bath maintained at a temperature between 45° and50°, and volatilize the chloroform with the aid of a stream ofnitrogen. Add 3.0 mL of Propionating Reagent, and heat theflask on a hot plate at 75° for 30 min. Evaporate the reagentswith the aid of a stream of nitrogen and gentle steam heat.Add 15 mL of chloroform, and swirl to dissolve the residue.
Assay Preparation Transfer about 50 mg of sample, accu-rately weighed, into a 25-mL conical flask, and proceed asdirected for Standard Preparation, beginning with ‘‘add 5mL of Internal Standard Solution. . . .’’
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flame-ionization detector and containing a 2.4-m × 4-mm borosilicateglass column, or equivalent, packed with 2% liquid phase 5%phenyl methyl silicone (SE 52, or equivalent) on an 80- to100-mesh siliceous earth support (Diatoport S, or equivalent).Maintain the column isothermally at a temperature between270° and 280°, and the injection port and detector block atabout 310°. Use helium as the carrier gas at a flow rate ofabout 70 mL/min.
System Suitability Chromatograph 6 to 10 injections ofthe Standard Preparation as directed under Procedure. Theresolution factor, R, between the peaks for the derivatizedglyceryl hexadecanoate and glyceryl octadecanoate is not lessthan 2.0, and the relative standard deviation of the ratio ofthe peak area of the derivatized glyceryl octadecanoate to thatof the hexadecyl hexadecanoate is not more than 2.0%.
Procedure Inject a suitable portion of the Standard Prepa-ration into the gas chromatograph, or equivalent, and recordthe chromatogram. Measure the areas under the peaks, andrecord the values of the sum of the areas under the derivatizedmonoglyceride peaks and of the area under the hexadecylhexadecanoate peak as AS and AD, respectively. Calculate theresponse factor, F, taken by the formula
(AS/AD)(WD/WS),
in which WD and WS are the weights, in milligrams, of hexade-cyl hexadecanoate and USP Monoglycerides Reference Stan-dard, respectively, in the Standard Preparation. Similarly,inject a suitable portion of the Assay Preparation and recordthe chromatogram. Measure the areas under the peaks, andrecord the values of the sum of the areas under the derivatizedmonoglyceride peaks and of the area under the hexadecylhexadecanoate peak as aU and aD, respectively. Calculate thequantity, in milligrams, of monoglycerides in the amount ofsample taken by the formula
(WD/F)(aU/aD).
Acid Value Determine as directed under Acid Value, Ap-pendix VII.Free Glycerin
Propionating Reagent Mix 10 mL of pyridine with 20mL of propionic anhydride.
Internal Standard Solution Dissolve a suitable quantityof tributyrin, accurately weighed, in chloroform, and dilutequantitatively with chloroform to obtain a solution with aconcentration of about 0.2 mg/mL.
Standard Preparation Transfer about 15 mg of glycerinand about 50 mg of tributyrin, both accurately weighed, intoa glass-stoppered, 25-mL conical flask, add 3 mL of Propio-nating Reagent, and heat at 75° for 30 min. Volatilize thereagents with the aid of a stream of nitrogen at room tempera-ture, add about 12 mL of chloroform, and mix. Dilute about1 mL of this mixture with chloroform to about 20 mL, and mix.
Test Preparation Transfer about 50 mg of sample, accu-rately weighed, into a glass-stoppered, 25-mL conical flask,add 5 mL of Internal Standard Solution by pipet, and mix todissolve. Immerse the flask in a water bath maintained at atemperature between 45° and 50°, and volatilize the chloro-form with the aid of a stream of nitrogen. Add 3 mL ofPropionating Reagent, and heat at 75° for 30 min. Volatilizethe reagents with the aid of a stream of nitrogen at roomtemperature, add about 5 mL of chloroform, and mix.
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a flame-ionization detector and containing a 2.4-m × 4-mm borosilicateglass column, or equivalent, packed with 2% liquid phaseconsisting of a high-molecular-weight compound of polyeth-ylene glycol and a diepoxide (Carbowax 20 M, or equivalent)on an 80- to 100-mesh siliceous earth support (ChromosorbW AW DMCS, or equivalent). Maintain the column isother-mally at a temperature between 190° and 200° and the injec-tion port and detector block at about 300° and 310°, respec-tively. Use helium as the carrier gas at a flow rate of about70 mL/min.
System Suitability Chromatograph 6 to 10 injections ofthe Standard Preparation as directed under Procedure. Theresolution factor, R, between the peaks for the derivatizedglycerin and tributyrin is not less than 4.0, and the relativestandard deviation of the ratio of their peak areas is not morethan 2.0%.
Procedure Inject a suitable portion of the Standard Prepa-ration into the gas chromatograph, and record the chromato-gram. Measure the areas under the peaks, and record thevalues of the areas under the tripropionin and tributyrin peaksas AS and AD, respectively. Calculate the response factor, F,taken by the formula
(AD/AS)(WS/WD),
in which WS and WD are the weights, in milligrams, of glycerinand tributyrin, respectively, in the Standard Preparation. Sim-ilarly, inject a suitable portion of the Test Preparation, andrecord the chromatogram. Measure the areas under the peaks,and record the values of the areas under the tripropionin and
FCC V Monographs / Glyceryl Palmitostearate / 207
tributyrin peaks as aU and aD, respectively. Calculate thepercentage of glycerin by the formula
100F(aU/aD)(wD/wU),
in which wD is the weight, in milligrams, of tributyrin in 5mL of Internal Standard Solution, and wU is the weight, inmilligrams, of sample in the Test Preparation.Hydroxyl Value Determine as directed in Method II underHydroxyl Value, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Melting Range Determine as directed under Melting Range,Appendix VII.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 5-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.
Packaging and Storage Store in tight, light-resistant con-tainers.
Glyceryl Palmitostearate
DESCRIPTION
Glyceryl Palmitostearate occurs as a fine powder or waxysolid. It is a mixture of fatty acid glycerides, primarily glycerylesters of palmitic and stearic acids. The waxy solid melts atabout 55°. Glyceryl Palmitostearate is soluble in chloroform,but practically insoluble in water and in alcohol.
Function Emulsifier
REQUIREMENTS
Identification Transfer about 100 mg of sample into a small,conical flask fitted with a suitable reflux condenser. Transfer50 mg each of USP Palmitic Acid Reference Standard andUSP Stearic Acid Reference Standard into a similar flask toserve as the Standard Solution. Treat the contents of each flaskas follows: Add 5.0 mL of a solution prepared by dissolving 14g of boron trifluoride in methanol to make 100 mL [commer-cial reagent, 14% w/v, may be used (Applied Science, orequivalent)]. Swirl to mix, and reflux for 15 min. Cool, transferthe reaction mixture with the aid of 10 mL of chromatographic-grade hexane to a 60-mL separator, and add 10 mL of waterand 10 mL of saturated sodium chloride solution. Shake, allowthe mixture to separate, then drain and discard the lower,aqueous layer. Pass the hexane layer through 6 g of anhydroussodium sulfate into a suitable flask.
Concomitantly introduce a 1-�L to 2-�L portion of thefiltered hexane solution from each of the two flasks into a gas
chromatograph, or equivalent, and obtain the chromatograms.Use a gas chromatograph, or equivalent, equipped with aflame-ionization detector and a 1.5-m × 3-mm (id) columnpacked with 15% diethylene glycol succinate polyester onflux-calcined, acid-washed siliceous earth. Maintain the col-umn at 165°. Set the temperatures of the inlet port and thedetector to 210°. Use helium as the carrier gas. The retentiontimes of the main peaks of methyl palmitate and methyl stear-ate obtained in the Sample Solution chromatogram correspondto those of the main peaks obtained from the Standard Solu-tion. The ratio of the response of the main peaks to the sumof all the responses is between 0.42 and 0.55 for methylpalmitate, and between 0.43 and 0.55 for methyl stearate.Acid Value Not more than 6.Free Glycerin Not more than 1%.Iodine Value Not more than 3.Lead Not more than 1 mg/kg.1-Monoglycerides Content Not more than 18.0%.Residue on Ignition Not more than 0.1%.Saponification Value Between 170 and 200.
TESTS
Acid Value Transfer about 10 g of sample, accuratelyweighed, into a flask, and add 50 mL of a 1:1 ethanol:diethylether mixture that has been neutralized to phenolphthaleinwith 0.1 N sodium hydroxide. Connect the flask to a suitablecondenser, and warm it slowly while frequently shaking it.Add 1 mL of phenolphthalein TS, and titrate with 0.1 Nsodium hydroxide until the solution remains faintly pink aftershaking it for 30 s. Calculate the acid value by the formula
56.11V × (N/W),
in which V is the volume, in milliliters, of the 0.1 N sodiumhydroxide solution used; N is the normality of the sodiumhydroxide solution; and W is the weight, in grams, of thesample taken.Free Glycerin Determine as directed under Free Glycerinor Propylene Glycol, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method,under Lead Limit Test, Appendix IIIB.1-Monoglycerides Content Determine as directed under 1-Monoglycerides, Appendix VII, using about 1 g of sample,accurately weighed, that has been melted at a temperature nothigher than 80° and mixed.
Calculate the percentage of 1-monoglycerides as a normal-ized content of monopalmitin and monostearin by the formula
[17.2N × (B − S)]/W,
in which 17.2 is one-twentieth of the formula weight of glyc-eryl monopalmitostearate; N is the normality of the sodiumthiosulfate solution; B and S are the volumes, in milliliters,of 0.1 N sodium thiosulfate solution consumed by the blankand by the sample, respectively; and W is the weight, in grams,of the sample taken.
208 / Glyceryl Tristearate / Monographs FCC V
Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 5-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.
Packaging and Storage Store in tight containers at a tem-perature no higher than 25°.
Glyceryl TristearateTristearin; Stearin; Octadecanoic Acid; 1,2,3-PropaneTristearoyl Ester
C57H110O6 Formula wt 891.49
CAS: [555-43-1]
DESCRIPTION
Glyceryl Tristearate occurs as a white, microfine, crystallinepowder. It is prepared by reacting glycerin with stearic acidin the presence of a suitable catalyst such as aluminum oxide.It is also found in many animal and vegetable fats such astallow and cocoa butter. It is soluble in hot alcohol and inchloroform; very slightly soluble in cold alcohol, in ether,and in petroleum ether; but insoluble in water.
Function Crystallization accelerator; lubricant; surface-fin-ishing agent.
REQUIREMENTS
Identification Glyceryl Tristearate exhibits the followingtypical composition profile of fatty acids determined as di-rected under Fatty Acid Composition, Appendix VII:
Fatty Acid: ≤12 12:0 14:0 16:0 16:1Weight % (Range): 0.0–0.3 0.0–0.5 0.0–1.0 0.0–0.1 0.0–0.1Fatty Acid: 18:0 18:1 18:2 ≥20Weight % (Range): >95 0.0–0.5 0.0–0.5 0.0–0.5
Acid Value Not more than 1.0.Free Glycerin Not more than 0.5%.Hydroxyl Value Not more than 5.0.Iodine Number Not more than 1.0.Lead Not more than 1 mg/kg.Melting Range Between 69° and 73°.Residue on Ignition Not more than 0.1%.Saponification Value Between 186 and 192.Unsaponifiable Matter Not more than 0.5%.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VII.Free Glycerin Determine as directed under Free Glycerinand Propylene Glycol, Appendix VII.
Hydroxyl Value Determine as directed in Method II underHydroxyl Value, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Melting Range Determine as directed in Procedure forClass II under Melting Range or Temperature, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 5-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.
Packaging and Storage Store in tight, light-resistant con-tainers.
GlycineAminoacetic Acid; Glycocoll
H2NCH2COOH
C2H5NO2 Formula wt 75.07
INS: 640 CAS: [56-40-6]
DESCRIPTION
Glycine occurs as a white, crystalline powder. One gramdissolves in about 4 mL of water. It is very slightly solublein alcohol and in ether. Its solution is acid to litmus.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC2H5NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.2%.Residue on Ignition Not more than 0.1%.
TESTS
Assay Transfer about 175 mg of sample, previously driedat 105° for 3 h and accurately weighed, into a 250-mL flask.Dissolve the sample in 50 mL of glacial acetic acid, add 2drops of crystal violet TS, and titrate with 0.1 N perchloricacid to a blue-green endpoint.
View IR
FCC V Monographs / Grape Skin Extract / 209
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions) andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 7.507 mg of C2H5NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in well-closed containers.
Grapefruit Oil, ColdpressedGrapefruit Oil, Expressed; Oil of Shaddock
CAS: [8016-20-4]
DESCRIPTION
Grapefruit Oil, Coldpressed, occurs as a yellow, sometimesred, liquid that often shows a flocculent separation of waxymaterial. It is the oil obtained by expression from the freshpeel of the grapefruit Citrus paradisi Macfayden (Citrus decu-mana L.) (Fam. Rutaceae). It is soluble in most fixed oilsand in mineral oil, often with opalescence or cloudiness. Itis slightly soluble in propylene glycol and insoluble in glyc-erin. It may contain a suitable antioxidant.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown under Infrared Spectra, usingthe same test conditions as specified therein.Angular Rotation Between +91° and +96°.Refractive Index Between 1.475 and 1.478 at 20°.Residue on Evaporation Between 5.0% and 10.0%.Specific Gravity Between 0.848 and 0.856.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Residue on Evaporation Determine as directed under Resi-due on Evaporation, Appendix VI, heating a sample for 5 h.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Grape Skin ExtractEnocianina
INS: 163(ii) CAS: [11029-12-2]
DESCRIPTION
Grape Skin Extract occurs as a red to purple powder or liquidconcentrate. It is prepared by aqueous extraction of grapemarc remaining from the pressing of grapes to obtain juice.Extraction is effected with water containing sulfur dioxide.During the steeping process, sulfur dioxide is added, and thesugar content is reduced by fermentation; further concentra-tion removes most of the alcohol. The primary color compo-nents are anthocyanins such as the glucosides of malvidin,peonidin, petunidin, delphinidin, or cyanidin. Other compo-nents naturally present are sugars, tartrates, malates, tannins,and minerals. The powder may contain an added carrier suchas maltodextrin, modified starch, or gum. In acid solution,Grape Skin Extract is red; in neutral to alkaline solution, itis unstable and violet to blue.
Function Color.
REQUIREMENTS
Identification Transfer 1 g of sample and 1 g of potassiummetabisulfite to a 100-mL volumetric flask, dissolve in about50 mL of pH 3.0 Citrate–Citric Acid Buffer (see Assay, below),and dilute to volume with the same buffer. The red colorcaused by anthocyanins is bleached.Assay Not less than 90% of the color strength as representedby the vendor.Arsenic Not more than 1 mg/kg.Lead Not more than 5 mg/kg.
TESTS
AssaypH 3.0 Citrate–Citric Acid Buffer Add, dropwise, 0.1 M
sodium citrate to 0.1 M citric acid until a pH of 3.0 is reached,as determined by a glass electrode.
Procedure Transfer about 0.2 g of sample, accuratelyweighed, to a 100-mL volumetric flask, dissolve it in about25 mL of pH 3.0 Citrate–Citric Acid Buffer, and dilute tovolume with the same buffer. Remove any undissolved mate-rial by filtration or centrifugation. Adjust the pH to 3.0, anddetermine the absorbance of the clarified solution at the maxi-mum, near 525 nm, in a cell with a 1-cm pathlength. The
View IR
210 / Guar Gum / Monographs FCC V
color strength, expressed as the absorbance of a 1% solutionin a cell of 1-cm pathlength, is calculated as
A525/S,
in which A525 is the absorbance at 525 nm and S is the weight,in grams, of sample.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.
Packaging and Storage Store liquid Grape Skin Extractwith aseptic packaging or in high-density polyethylene con-tainers at 4° to 14°. Store powdered Grape Skin Extract infiber drums at room temperature.
Guar GumINS: 412 CAS: [9000-30-0]
DESCRIPTION
Guar Gum occurs as a white to yellow-white powder. It is agum obtained from the ground endosperms of Cyamopsistetragonolobus (L.) Taub (Fam. Leguminosae) (synonym Cy-amopsis psoraloides [Lam.] D.C.). It consists chiefly of ahigh-molecular-weight polysaccharide composed of galactoseand mannose units and may be described chemically as agalactomannan. It is dispersible in either hot or cold water,forming a sol, having a pH between 5.4 and 7.0, that may beconverted to a gel by the addition of small amounts of sodiumborate.
Function Stabilizer; thickener; emulsifier.
REQUIREMENTS
IdentificationA. Transfer a 2-g sample into a 400-mL beaker, moisten
it thoroughly with about 4 mL of isopropyl alcohol, add, withvigorous stirring, 200 mL of cold water, and continue to stiruntil the gum is completely and uniformly dispersed. Anopalescent, viscous dispersion forms.
B. Transfer 100 mL of the dispersion prepared in Identifica-tion Test A into another 400-mL beaker, heat the mixture ina boiling water bath for about 10 min, and then cool to roomtemperature. No appreciable increase in viscosity develops.Acid-Insoluble Matter Not more than 7.0%.Ash (Total) Not more than 1.5%.Galactomannans Not less than 70.0%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 15.0%.Protein Not more than 10.0%.Starch Passes test.
TESTS
Acid-Insoluble Matter Transfer 1.5 g of sample, accuratelyweighed, into a 250-mL beaker containing 150 mL of waterand 1.5 mL of sulfuric acid. Cover the beaker with a watchglass, and heat the mixture on a steam bath for 6 h, rubbingdown the wall of the beaker frequently with a rubber-tippedstirring rod and replacing any water lost by evaporation. Atthe end of the 6-h heating period, add about 500 mg of asuitable filter aid, previously dried for 3 h at 105° and accu-rately weighed, and filter through a tared, sintered-glass filtercrucible. Wash the residue several times with hot water, drythe crucible and its contents at 105° for 3 h, cool in a desicca-tor, and weigh. The difference between the weight of the filteraid and that of the residue is the weight of Acid-InsolubleMatter.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Galactomannans The difference between 100 and the sumof the percent Acid-Insoluble Matter, Total Ash, Loss on Dry-ing, and Protein represents the percent Galactomannans.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 5 h.Protein Determine as directed in Method I under NitrogenDetermination, Appendix IIIC, using about 3.5 g of sample,accurately weighed, transferred into a 500-mL Kjeldahl flask.The percentage of nitrogen determined, multiplied by 6.25,gives the percentage of protein in the sample.Starch Add a few drops of iodine TS to a 1:10 aqueoussolution. No blue color appears.
Packaging and Storage Store in well-closed containers.
Gum ArabicAcacia
INS: 414 CAS: [9000-01-5]
DESCRIPTION
Gum Arabic occurs as a dried, gummy exudation obtainedfrom the stems and branches of Acacia senegal (L.) Willdenowor of related species of Acacia (Fam. Leguminosae). Theunground product occurs as white or yellow-white, spheroidaltears of varying size or in angular fragments. It is also availablecommercially as white to yellow-white flakes, granules, orpowder. One gram dissolves in 2 mL of water, forming asolution that flows readily and is acid to litmus. It is insolublein alcohol.
FCC V Monographs / Gum Ghatti / 211
Function Stabilizer; emulsifier.
REQUIREMENTS
Identification Add 0.2 mL of diluted lead subacetate TS to10 mL of a cold 1:50 aqueous solution. A flocculent or curdy,white precipitate forms immediately.Arsenic Not more than 3 mg/kg.Ash (Acid-Insoluble) Not more than 0.5%.Ash (Total) Not more than 4.0%.Insoluble Matter Not more than 1.0%.Lead Not more than 5 mg/kg.Loss on Drying Not more than 15.0%.Starch or Dextrin Passes test.Tannin-Bearing Gums Passes test.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Ash (Acid-Insoluble) Determine as directed under Ash (AcidInsoluble), Appendix IIC.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Insoluble Matter Dissolve 5 g of sample in about 100 mLof water contained in a 250-mL Erlenmeyer flask, add 10 mLof 2.7 N hydrochloric acid, and boil gently for 15 min. Filterthe hot solution by suction through a tared filtering crucible,wash the residue thoroughly with hot water, dry at 105° for2 h, and weigh.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in thecontrol.Loss on Drying (Note: Powder unground samples suffi-ciently to pass through a No. 40 sieve, and mix well beforeweighing and drying.) Determine as directed under Losson Drying, Appendix IIC, drying a sample at 105° for 5 h.Starch or Dextrin Boil a 1:50 aqueous solution, cool,and add a few drops of iodine TS. No blue or red colorappears.Tannin-Bearing Gums Add about 0.1 mL of ferric chlo-ride TS to 10 mL of a 1:50 aqueous solution. No blackcoloration or precipitate forms.
Packaging and Storage Store in well-closed containers.
Gum GhattiIndian Gum
CAS: [9000-28-6]
DESCRIPTION
Gum Ghatti occurs as colorless or light to dark tan tears. Itis also available as a gray to red-gray powder. It is the dried
gummy exudate from the stems of Anogeissus latifolia Wall(Fam. Combretaceae). It is a complex, water-soluble, acidicpolysaccharide composed of the calcium and magnesium saltsof L-arabinose, D-galactose, D-mannose, D-xylose, and D-glucuronic acids in the approximate molar ratio of 10:6:2:1:2.It is slightly soluble in water, but it is insoluble in 90% alcohol.
Function Emulsifier.
REQUIREMENTS
Labeling Indicate the viscosity in centipoises.Identification
Lead Acetate Solution (Caution: Use gloves and gogglesto avoid contact with skin and eyes. Use an effective fume-removal device or other respiratory protection.) Activate 50to 60 g of lead (II) oxide by heating it for 2.5 to 3 h in afurnace at 650° to 670° (cooled product should have a lemoncolor). Boil 80 g of lead acetate trihydrate and 40 g of thefreshly activated lead (II) oxide with 250 g of water in a 500-mL Erlenmeyer flask provided with a reflux condenser for45 min. Cool, filter off any residue, and dilute with recentlyboiled water to a density of 1.25 at 20°. Add 4 mL of waterto 1 mL of the lead acetate solution, and filter.
Procedure Add 0.2 mL of the Lead Acetate Solution to5 mL of a cold 1:100 aqueous solution. A slight precipitate orclear solution results in which an opaque flocculent precipitateforms on the addition of 1 mL of 3 N ammonium hydroxide.Arsenic Not more than 3 mg/kg.Ash (Acid-Insoluble) Not more than 1.75%.Ash (Total) Not more than 6.0%.Insoluble Matter Not more than 1.0%.Lead Not more than 5 mg/kg.Loss on Drying Not more than 14.0%.Viscosity A 5% solution exhibits a viscosity, measured incentipoises, within the range stated on the label.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Ash (Acid-Insoluble) Determine as directed under Ash(Acid-Insoluble), Appendix IIC.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Insoluble Matter Dissolve 5 g of sample, accuratelyweighed, in about 100 mL of water contained in a 250-mLErlenmeyer flask, add 10 mL of 2.7 N hydrochloric acid, andboil gently for 15 min. Filter the hot solution, using suctionthrough a tared filtering crucible, wash thoroughly with hotwater, dry at 105° for 2 h, and weigh.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in thecontrol.Loss on Drying Determine as directed under Loss onDrying, Appendix IIC, drying a ground sample at 105° for5 h. Powder unground samples to pass through a No. 40sieve, and mix well before weighing.
212 / Gum Guaiac / Monographs FCC V
Viscosity Determine as directed under Viscosity of Cellu-lose Gum, Appendix IIB, at 75°, using spindle No. 2 at60 rpm.
Packaging and Storage Store in well-closed containers.
Gum GuaiacGuaiac Resin
INS: 314 CAS: [9000-29-7]
DESCRIPTION
Gum Guaiac occurs as irregular masses enclosing fragmentsof vegetable tissues; as large, nearly homogeneous masses;and occasionally, as more-or-less rounded or ovoid tears. Itis externally brown-black to dusky brown, acquiring a greencolor on long exposure to air, the fractured surface having aglassy luster, the thin pieces being transparent and varyingin color from brown to yellow-orange. The powder is moderateyellow-brown, becoming olive brown on exposure to air. Itis the resin of the wood of Guajacum officinale L. or Guajacumsanctum L. (Fam. Zygophyllaceae). Gum Guaiac dissolvesincompletely, but readily, in alcohol, in ether, in chloroform,and in solutions of alkalies. It is slightly soluble in carbondisulfide.
Function Antioxidant.
REQUIREMENTS
IdentificationA. Add 1 drop of ferric chloride TS to 5 mL of a 1:100
alcoholic solution. A blue color appears that gradually changesto green, finally becoming green-yellow.
B. A mixture of 5 mL of a 1:100 alcoholic solution and 5mL of water becomes blue when shaken with 20 mg of leadperoxide. Filter the solution, and boil a portion of the filtrate.The color disappears but may be restored by adding leadperoxide and shaking the filtrate. Add a few drops of 2.7 Nhydrochloric acid to a second portion of the filtrate. The colorimmediately disappears.Alcohol-Insoluble Residue Not more than 15.0%.Ash (Acid-Insoluble) Not more than 2.0%.Ash (Total) Not more than 5.0%.Lead Not more than 2 mg/kg.Melting Range Between 85° and 90°.Rosin Passes test.
TESTS
Alcohol-Insoluble Residue Place 2 g of sample, finely pow-dered and accurately weighed, in a dry, tared extraction thim-ble, and extract it with alcohol in a suitable continuous extrac-tion apparatus for 3 h or until completely extracted. Dry the
insoluble residue remaining in the thimble for 4 h at 105°,and weigh.Ash (Acid-Insoluble) Determine as directed under Ash(Acid-Insoluble), Appendix IIC.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 4 �g of lead (Pb) ion in the control.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Rosin A 1:10 solution of sample in petroleum ether is color-less, and when mixed and shaken with an equal quantity ofa fresh, 1:200 aqueous solution of cupric acetate, is not moregreen than a similar solution of cupric acetate in petroleumether.
Packaging and Storage Store in well-closed containers.
Helium
He Formula wt 4.00
INS: 939 CAS: [7440-59-7]
DESCRIPTION
Helium occurs as a colorless gas that is not combustible anddoes not support combustion. It is very slightly soluble inwater. One liter of the gas weighs about 180 mg at 0° and760 mm Hg.
Function Processing aid.
REQUIREMENTS
Note: Reduce the sample gas cylinder pressure with aregulator. Measure the sample gas with a gas volumemeter downstream from the detector tube to minimizecontamination of or change to the gas samples.
The detector tube called for in one test is describedunder Solutions and Indicators.
IdentificationA. Insert a burning wood splinter into an inverted test tube
filled with sample gas. The flame is extinguished.
Note: Use caution.
B. A small balloon filled with sample gas shows buoyancy.Assay Not less than 99.0% of He, by volume.Air Not more than 1.0%, by volume.Carbon Monoxide Not more than 10 ppm, by volume.Odor Passes test.
TESTS
Assay (See Chromatography, Appendix IIA.) Use a gaschromatograph equipped with a thermal-conductivity detector
FCC V Monographs / Hexanes / 213
and a 6-m × 4-mm (id) column, or equivalent, packed withporous polymer beads (PoraPak Q, or equivalent), which per-mit complete separation of nitrogen and oxygen from Helium,although nitrogen and oxygen may not be separated from eachother. Maintain the column at 60°. Use industrial-grade helium(99.99%) as the carrier gas. Introduce a gas sample into thegas-sampling valve. Select the operating conditions of the gaschromatograph, or equivalent, so that the peak signal of anair–helium certified standard (a mixture of 1.0% air in indus-trial-grade helium is available from most suppliers) corre-sponds to not less than 70% of the full-scale reading. Thepeak response produced by the sample gas exhibits a retentiontime corresponding to that produced by the air–helium stan-dard and, when compared with the peak response of thatstandard, indicates not more than 1.0% air, by volume, andnot less than 99.0% of He, by volume.Air Determine as directed under Assay (above).Carbon Monoxide Pass 1050 � 50 mL of sample gasthrough a carbon monoxide detector tube at the rate specifiedfor the tube. The indicator change corresponds to not morethan 10 ppm, by volume.Odor Carefully open the sample gas cylinder valve to pro-duce a moderate flow of sample gas. Do not direct the gasstream toward the face, but deflect a portion of the streamtoward the nose. No appreciable odor is discernible.
Packaging and Storage Store in appropriate gas cylinders.
Heptylparabenn-Heptyl-p-hydroxybenzoate
HO COO(CH2)6CH3
C14H20O3 Formula wt 236.31
CAS: [1085-12-7]
DESCRIPTION
Heptylparaben occurs as small, colorless crystals or as a white,crystalline powder. It is very slightly soluble in water, but isfreely soluble in alcohol and in ether.
Function Preservative; antimicrobial agent.
REQUIREMENTS
Identification Dissolve 500 mg of sample in 10 mL of 1N sodium hydroxide, boil for 30 min, allow the solution toevaporate to a volume of about 5 mL, and cool. Acidify thesolution with 2 N sulfuric acid, collect the crystals on a filter,wash several times with small portions of water, and dry ina desiccator over silica gel. The p-hydroxybenzoic acid so
obtained melts between 212° and 217° (see Melting Rangeor Temperature, Appendix IIB).Assay Not less than 99.0% and not more than 100.5% ofC14H20O3, calculated on the dried basis.Acidity Passes test.Lead Not more than 2 mg/kg.Loss on Drying Not more than 0.5%.Melting Range Between 48° and 51°.Residue on Ignition Not more than 0.05%.
TESTS
Assay Transfer 3.5 g of sample, accurately weighed, into aflask, add 40.0 mL of 1 N sodium hydroxide, and rinse thesides of the flask with water. Cover with a watch glass, boilgently for 1 h, cool, and titrate the excess sodium hydroxidewith 1 N sulfuric acid to pH 6.5. Perform a blank determination(see General Provisions) with the same quantities of the samereagents in the same manner, and make any necessary correc-tion. Each milliliter of 1 N sodium hydroxide is equivalentto 236.3 mg of C14H2OO3, calculated on the dried basis.Acidity Mix 750 mg of sample with 15 mL of water, heatat 80° for 1 min, cool, and filter. The filtrate is acid or neutralto litmus. Add 0.2 mL of 0.1 N sodium hydroxide and 2 dropsof methyl red TS to 10 mL of the filtrate. The solution isyellow, without even a light cast of pink.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample in a desiccator over silicagel for 5 h.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in tight containers.
Hexanes
Mixed Paraffinic Hydrocarbons
C6H14 Formula wt 86.18
CAS: [110-54-3]
DESCRIPTION
Hexanes occur as a clear, colorless, flammable liquid. It iscomposed predominantly of C6, with some C5 and C7, isomericparaffins. The relative proportion of isomers varies with theproducer and the production lot. It is soluble in alcohol, inacetone, and in ether and is insoluble in water.
214 / 4-Hexylresorcinol / Monographs FCC V
Function Extraction solvent.
REQUIREMENTS
Benzene Not more than 0.05%.Color (APHA) Not more than 10.Distillation Range Between 56° and 71°.Lead Not more than 1 mg/kg.Nonvolatile Residue Not more than 10 mg/kg.Specific Gravity Between 0.655 and 0.675.Sulfur Not more than 5 mg/kg.
TESTS
Benzene Determine as directed under Benzene, AppendixIIIC.Color (APHA) Dilute 2.0 mL of platinum–cobalt stock solu-tion (APHA No. 500) with water in a 100-mL volumetricflask. Compare this solution (APHA No. 10) with 100 mLof sample in 100-mL Nessler tubes, viewed vertically over awhite background.Distillation Range Determine as directed under DistillationRange, Appendix IIB.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Nonvolatile Residue Evaporate 150 mL (about 100 g) ofsample to dryness in a tared dish on a steam bath. Dry theresidue at 105° for 30 min, cool, and weigh.Specific Gravity Determine by any reliable method (seeGeneral Provisions).Sulfur Determine as directed under Sulfur, Appendix IIIC.
Packaging and Storage Store in tight containers, protectedfrom fire.
4-HexylresorcinolHexylresorcinol; 4-Hexyl-1,3-benzenediol
CH3
OH
HO
C12H18O2 Formula wt 194.27
CAS: [136-77-6]
DESCRIPTION
4-Hexylresorcinol occurs as a white powder. It is very slightlysoluble in water and freely soluble in ether and in acetone.
Caution: 4-Hexylresorcinol is irritating to the oral mu-cosa and respiratory tract and to the skin, and its solutionin alcohol has vesicant properties.
Function Color stabilizer; enzymatic browning inhibitor.
REQUIREMENTS
IdentificationA. Add 1 mL of nitric acid to 1 mL of a saturated solution
of sample. A light red color appears.B. Add 1 mL of bromine TS to 1 mL of a saturated solution
of the sample. A yellow flocculent precipitate forms. Add 2mL of 6 N ammonium hydroxide, and the precipitate dissolves,producing a yellow solution.
C. The infrared absorption spectrum of a potassium bromidedispersion of the sample exhibits maxima only at the samewavelengths as those of a typical spectrum as shown in thesection on Infrared Spectra, using the same test conditionsas specified therein.Assay Not less than 98.0% and not more than 100.5% ofC12H18O2 after drying.Acidity Passes test.Lead Not more than 2 mg/kg.Melting Range Between 62° and 67°.Mercury Not more than 3 mg/kg.Nickel Not more than 2 mg/kg.Residue on Ignition Not more than 0.1%.Resorcinol and Other Phenols Negative by test.
TESTS
Assay Dissolve 70 to 100 mg of sample, previously driedover silica gel for 4 h and accurately weighed, in 10 mL ofmethanol in a 250-mL iodine flask. Add 30.0 mL of 0.1 Nbromine, then quickly add 5 mL of hydrochloric acid, andinsert the stopper in the flask immediately. Cool the flaskunder running water to room temperature, shake vigorouslyfor 5 min, then set aside for 5 min. Add 6 mL of potassiumiodide TS around the stopper, cautiously loosen the stopper,again insert the stopper tightly, and swirl gently. Add 1 mLof chloroform, and titrate the liberated iodine with 0.1 Nsodium thiosulfate, adding 3 mL of starch TS as the endpointis approached. Perform a blank determination (see GeneralProvisions), and make any necessary correction. Calculate themilligrams of C12H18O2 in the sample taken by the formula
4.857 × (B – S),
in which 4.857 is the milliequivalent factor, B is the numberof milliliters of 0.1 N sodium thiosulfate required for theblank, and S is the number of milliliters of 0.1 N sodiumthiosulfate required for the sample.Acidity Dissolve 250 mg of sample in 500 mL of water,add a few drops of methyl red TS, and titrate with 0.02 Nsodium hydroxide. Not more than 1.0 mL is required forneutralization (0.05%).Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 2-g sample.
View IR
FCC V Monographs / High-Fructose Corn Syrup / 215
Melting Range Determine as directed in Procedure forClass I under Melting Range or Temperature, Appendix IIB.Mercury (Note: Select all reagents for this test to have aslow a content of mercury as practicable, and store all reagentsolutions in containers of borosilicate glass. Specially cleanall glassware used in this test by soaking it in warm 8 N nitricacid for 30 min and rinsing with water. Keep flasks for thisdetermination separate from other flasks, and use them onlyfor mercury determinations.)
Standard Solution Transfer 34.0 mg of mercuric chlorideto a 250-mL volumetric flask. Add 1 drop of hydrochloricacid, and dissolve in and dilute to volume with water. Transfer1.0 mL of this solution to a 100-mL volumetric flask, add 1drop of hydrochloric acid, and dilute with water to volume.Transfer 1.0 mL of this solution to a 500-mL volumetric flask,add 1 drop of hydrochloric acid, and dilute with water tovolume.
Test Solution Transfer 134 mg of sample to a 250-mLbeaker, and cautiously add 10 mL of 11 N nitric acid and 10mL of 18 N sulfuric acid. Digest with the aid of heat ina well-ventilated hood until brown fumes cease to evolve.Cautiously add an additional 10 mL of 11 N nitric acid, andcontinue heating until no more fumes evolve. Cool, transferto a 200-mL volumetric flask, and dilute to volume with water.
Procedure Transfer 100 mL of Standard Solution to a300-mL mercury analysis reaction vessel, add 2 drops of a1:20 potassium permanganate solution, and mix (the solutionshould be purple; add additional permanganate solution, drop-wise, if necessary). Add 5 mL of 11 N nitric acid, stir, andallow to stand for not less than 15 s. Add 5 mL of 18 Nsulfuric acid, stir, and allow to stand for not less than 45 s.Add 5 mL of a 3:200 hydroxylamine hydrochloride solution,stir, and allow to stand until the solution turns light yellowor colorless. Add 5 mL of a 1:10 stannous chloride solution,immediately insert the aerator connected to an air pump, anddetermine the maximum absorbance of the treated StandardSolution at the mercury resonance line of 253.65 nm, with asuitable atomic absorption spectrophotometer equipped witha mercury hollow-cathode lamp and an absorption cell thatpermits the flameless detection of mercury.
Note: Disregard the presence of insoluble matter in thissolution; mix before use.
In a closed system with a circulating air pump, connect acalcium chloride drying tube and an aerator inserted in a 300-mL reaction vessel so that air passed through the treatedpreparation contained in the reaction vessel evaporates anymetallic mercury present. In a similar manner, treat 100 mLof the Test Solution and 100 mL of water (reagent blank),and determine the maximum absorbances at the same wave-length. The absorbance of the solution from the Test Solutiondoes not exceed that of the solution from the Standard So-lution.
Note: Check the zero setting of the instrument fre-quently.
NickelStandard Nickel Solution Transfer 40.1 mg of nickel chlo-
ride hexahydrate, accurately weighed, into a 1-L volumetric
flask, dilute with water to volume, and mix. Transfer 10.0mL of this solution to a 100-mL volumetric flask, dilute tovolume, and mix. Each milliliter of this solution contains 1�g of nickel ion.
Dissolve 2 g of sample in methanol to yield 20 mL. Add3 mL of bromine TS and 2 mL of a 1:5 citric acid solution,and mix. Add 10 mL of 6 N ammonium hydroxide and 1 mLof a 1:100 dimethylglyoxime:ethanol solution. Mix, dilutewith water to 50 mL, and allow to stand for 5 min. Any colorthe solution produces is not more intense than that of a solutioncontaining 4 mL of Standard Nickel Solution and treated inthe same manner.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Resorcinol and Other Phenols Shake about 1 g of samplewith 50 mL of water for a few minutes, filter, and add 3 dropsof ferric chloride TS to the filtrate. No red or blue colorappears.
Packaging and Storage Store in tight, light-resistant con-tainers.
High-Fructose Corn Syrup
DESCRIPTION
High-Fructose Corn Syrup (HFCS) occurs as a water whiteto light yellow, somewhat viscous liquid that darkens at hightemperatures. It is a saccharide mixture prepared as a clear,aqueous solution from high-dextrose-equivalent corn starchhydrolysate by the partial enzymatic conversion of glucose(dextrose) to fructose, using an insoluble glucose isomerasepreparation that complies with 21 CFR 184.1372 and that hasbeen obtained from a pure culture fermentation that producesno antibiotics. It is miscible in all proportions with water.
Function Nutritive sweetener.
REQUIREMENTS
Labeling Indicate the color range and presence of sulfurdioxide if the residual concentration is greater than 10 mg/kg.Identification Add a few drops of a 1:10 aqueous solutionto 5 mL of hot alkaline cupric tartrate TS. A copious redprecipitate of cuprous oxide forms.Assay 42% HFCS: Not less than 97.0% total saccharides,expressed as a percentage of solids, of which not less than42.0% consists of fructose, not less than 92.0% consists ofmonosaccharides, and not more than 8.0% consists of othersaccharides. 55% HFCS: Not less than 95.0% total saccha-rides, expressed as a percentage of solids, of which not lessthan 55.0% consists of fructose, not less than 95.0% consistsof monosaccharides, and not more than 5.0% consists of othersaccharides.
216 / High-Fructose Corn Syrup / Monographs FCC V
Arsenic Not more than 1 mg/kg.Color Within the range specified by the vendor.Lead Not more than 0.1 mg/kg.Residue on Ignition Not more than 0.05%.Sulfur Dioxide Not more than 0.003%.Total Solids 42% HFCS: Not less than 70.5%; 55% HFCS:Not less than 76.5%.
TESTS
AssayApparatus (See Chromatography, Appendix IIA.) Use a
suitable high-performance liquid chromatography system suchas described in Standard Analytical Methods of the CornRefiners Association, equipped with a 22- to 31-cm stainless-steel column, or equivalent, a strip-chart recorder, and a differ-ential refractometer detector maintained at 45° � 0.005°.
Stationary Phase Use prepacked macroreticular polysty-rene sulfonate divinylbenzene cation-exchange resin (2% to8% cross-linked, 8- to 25-�m particle size), preferably in thecalcium or silver form. Examples of acceptable resins are Bio-Rad Aminex HPX-87C, or equivalent, for separating DP1–DP4 saccharides, and Aminex HPX-42C and HPX-42A, orequivalent, for separating DP1–DP7 saccharides. Maintain thecolumn at 85° during operation.
Mobile Phase Use degassed, purified water passedthrough a 0.22-�m filter before use; maintain the water at85° during operation of the chromatograph.
Standardization Prepare a Standard Solution containinga total of about 10% solids, using sugars of known purity (e.g.,USP Fructose Reference Standard; USP Dextrose ReferenceStandard, or NIST Standard Reference Material; maltose, Ald-rich Chemical Company; or equivalent) that approximates,on the dry basis, the composition of the sample to be analyzed.Dissolve each standard sugar, accurately weighed, in 20 mLof purified water contained in a 50-mL beaker. Heat on asteam bath until all sugars are dissolved, then cool, and transferto a 100-mL volumetric flask. Dilute to volume with waterand mix. Freeze the solution if it is to be reused.
If a corn syrup or maltodextrin is used to supply a DP4+
fraction, take care to include all saccharides in the standardcomposition calculation.
Compute the dry-basis concentration, in percent, of eachindividual component in the Standard Solution by the formula
(WC/WI) × 100,
in which WC is the weight of the sugar of interest and Wi
is the sum of the weights of all sugar components. Standardizeby injecting 10 to 20 �L (about 1.0 to 2.0 mg of solids) ofthe standard sugar solution into the chromatograph. Integratethe peaks and normalize. Sum the individual DP4+ responsesfrom the normalized printout to obtain the total DP4+ normal-ized response. Calculate the response factors as follows (seeChromatography, Appendix IIA):
RI = (known concentration, dry basis %)/(measured concen-tration, normalized %),
in which RI is the response factor for component i.
Compute the response factor for each component relativeto glucose (R′I) using the following equation:
R′I = RI/RG′,
in which RG is the response factor for glucose. The R′I forDP4+ should be programmed as a default value (if automatedequipment is used) and used to compute the concentration ofhigher saccharides.
Sample Analysis Determine the solids content (below) ofthe sample, and dilute to approximately 10% solids with water.Inject a volume (10 to 50 �L) appropriate for the specificsolids content into the chromatograph.
Calculation Calculate the concentration of each compo-nent as follows:
CI = (AI × RI × 100)/(ANRN),
in which AI is the area recorded for that component and ANRN
is the sum of the product of the areas (A) and response factors(R) for all components detected.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds and 1 mL of Standard Arsenic Solution(1 �g As).Color
Apparatus Use a suitable variable-wavelength spectro-photometer capable of measuring percent transmittancethroughout the visible spectrum and designed to permit theuse of sample and reference cells with pathlengths of 2 to 4 cm.The transmittance of all paired cells should agree within 0.5%.
Standard Solution Dissolve 0.10 g of reagent-grade potas-sium dichromate (K2Cr2O7) in 1 L of water, and mix thor-oughly.
Procedure Using water in the sample and reference cellsof 2-cm pathlength, normalize the percent transmittance scaleof the spectrophotometer to 100%. Leave the reference cellin place and replace the water in the sample cell with theStandard Solution. Determine the wavelength at which thesolution exhibits exactly 54.5% transmittance. This wave-length is defined as �c, the corrected 450-nm wavelength.Remove the 2-cm cells from the spectrophotometer, and withwater in the sample and reference cells of 4-cm pathlength,adjust the percent transmittance scale to 100% with the spec-trophotometer set at �c. Leave the reference cell in place, andreplace the water in the sample cell with sample. Measurethe percent transmittance (T450). Remove the sample cell, setthe wavelength at 600 nm, replace the sample with water,and adjust the percent transmittance scale to 100%. Determinethe percent transmittance at 600 nm (T600) with the samesample in the sample cell. Calculate the Color of the sampletaken using the following formula:
(log T600 – log T450)/4,
in which T600 is the percent transmittance at 600 nm and T450
is the percent transmittance at 450 nm.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 5-g sample.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIIB, igniting a 10-g sample.
FCC V Monographs / L-Histidine Monohydrochloride / 217
Sulfur Dioxide Determine as directed under Sulfur DioxideDetermination, Appendix X, using a 50-g sample.Total Solids Determine the refractive index of a sample at20° or 45°, and use the tables in High-Fructose Corn SyrupSolids, under Total Solids, Appendix X, to obtain the percentTotal Solids.
Packaging and Storage Store in tight containers.
L-HistidineL-�-Amino-4(or 5)-imidazolepropionic Acid
N NH
CH2CCOOH
H NH2
C6H9N3O2 Formula wt 155.16
CAS: [71-00-1]
DESCRIPTION
L-Histidine occurs as white crystals or as a crystalline powder.It is soluble in water, very slightly soluble in alcohol, andinsoluble in ether. It melts with decomposition between about277° and 288°.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H9N3O2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.2%.Optical (Specific) Rotation [�]D
20°: Between +11.5° and+13.5°, calculated on the dried basis; or [�]D
25°: Between+12.0° and +14.0°, calculated on the dried basis.Residue on Ignition Not more than 0.2%.
TESTS
Assay Dissolve about 150 mg of sample, previously driedat 105° for 3 h and accurately weighed, in 3 mL of formicacid and 50 mL of glacial acetic acid, and titrate with 0.1 Nperchloric acid, determining the endpoint potentiometrically.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 15.52 mg of C6H9N3O2.
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 11 g of previously dried sample in sufficient 6 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
L-Histidine MonohydrochlorideL-�-Amino-4(or 5)-imidazolepropionic AcidMonohydrochloride
N NH
CH2CCOOH
H NH2·HCl
C6H9N3O2·HCl·H2O Formula wt 209.63
CAS: monohydrate [5934-29-2]
DESCRIPTION
L-Histidine Monohydrochloride occurs as white crystals or asa crystalline powder. It is soluble in water, and insoluble inalcohol and in ether. It melts with decomposition at about250° (after drying).
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H9N3O2·HCl·H2O, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Optical (Specific) Rotation [�]D
20°: Between +8.5° and+10.5°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 100 mg of sample, previously driedat 105° for 3 h and accurately weighed, in 3 mL of formic
View IR
View IR
218 / Hops Oil / Monographs FCC V
acid, add exactly 15.0 mL of 0.1 N perchloric acid, and heaton a water bath for 30 min.
Caution: Handle perchloric acid in an appropriatefume hood.
After cooling, add 45 mL of glacial acetic acid, and titratethe excess perchloric acid with 0.1 N sodium acetate, determin-ing the endpoint potentiometrically. Perform a blank determi-nation (see General Provisions), and make any necessarycorrection. Each milliliter of 0.1 N perchloric acid is equivalentto 10.48 mg of C6H9N3O2·HCl·H2O.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in thecontrol.Loss on Drying Determine as directed under Loss onDrying, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 11 g of previously dried sample in sufficient 6N hydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed, light-resistantcontainers.
Hops Oil
CAS: [8007-04-3]
DESCRIPTION
Hops Oil occurs as a light yellow to green-yellow liquid witha characteristic, aromatic odor. Age darkens the color, andthe oil tends to become viscous. It is the volatile oil obtainedby steam distillation of the freshly dried membranous conesof the female plants of Humulus lupulus L. or Humulus ameri-canus Nutt. (Fam. Moraceae). It is soluble in most fixedoils and, sometimes with opalescence, in mineral oil. It ispractically insoluble in glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 11.0.Angular Rotation Between −2° and +2°5′.Refractive Index Between 1.470 and 1.494 at 20°.Saponification Value Between 14 and 69.
Solubility in Alcohol Passes test.Specific Gravity Between 0.825 and 0.926.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI, using about 5 g of sample, accurately weighed.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed in Saponifica-tion Value under Esters, Appendix VI, using about 5 g ofsample, accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample usually isnot soluble in 95% alcohol. Older oils are less soluble thanfresh oils.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Hydrochloric Acid
HCl Formula wt 36.46
INS: 507 CAS: [7647-01-0]
DESCRIPTION
Hydrochloric Acid occurs as a clear, colorless or slightlyyellow, corrosive liquid. It is a water solution of hydrogenchloride of varied concentrations. It is miscible with waterand with alcohol. Concentrations of Hydrochloric Acid areexpressed in percent by weight or may be expressed in degreesBaumé (°Bé) from which percents of Hydrochloric Acid andspecific gravities may readily be derived (see HydrochloricAcid Table, Appendix IIC). The usually available concentra-tions are 18°, 20°, 22°, and 23°Bé. Concentrations above13°Bé (19.6%) fume in moist air, lose hydrogen chloride,and create a corrosive atmosphere. Because of these character-istics, observe suitable precautions during sampling and analy-sis to prevent losses.
Note: Hydrochloric Acid is produced by various meth-ods that might impart trace amounts of organic com-pounds as impurities. The manufacturer, vendor, or useris responsible for identifying the specific organic com-pounds that are present and for meeting the Require-ments for organic compounds (below). Methods areprovided for their determination under Tests. Inapplying the procedures, use any necessary standards
View IR
FCC V Monographs / Hydrochloric Acid / 219
to quantitate the organic compounds present in eachspecific product.
The variety of organic impurities that might conceiv-ably be found in Hydrochloric Acid is such that it isimpossible to provide a comprehensive and accuratelist here. Therefore, the manufacturer, vendor, or useris responsible for establishing the suitability of suchHydrochloric Acid for its intended application in foodsor food processing in accordance with the provision ofTrace Impurities (see General Provisions).
Function Acidifier.
REQUIREMENTS
Labeling Indicate the content, by weight, of HydrochloricAcid (HCl). Alternatively, indicate the range of HydrochloricAcid content, the range of degrees Baumé, and/or the specificgravity range.Identification A sample gives positive tests for Chloride,Appendix IIIA.Assay Not less than 97.0% and not more than 103.0% ofthe labeled amount of HCl, or within the range specified onthe label.Color Passes test.Degrees Baumé Within the range shown on the label orclaimed by the vendor.Iron Not more than 5 mg/kg.Lead Not more than 1 mg/kg.Nonvolatile Residue Not more than 0.5%.Organic Compounds
Total Organic Compounds (Non-Fluorine-Containing)Not more than 5 mg/kg, including
Benzene Not more than 0.05 mg/kg.Fluorinated Organic Compounds (total) Not more than
0.0025%.Oxidizing Substances (as Cl2) Not more than 0.003%.Reducing Substances (as SO3) Not more than 0.007%.Specific Gravity Within the range specified or implied bythe vendor.Sulfate Not more than 0.5%.
TESTS
Assay Accurately tare a 125-mL glass-stoppered Erlen-meyer flask containing 35.0 mL of 1 N sodium hydroxide.Without the use of vacuum, partially fill a 10-mL serologicalpipet from near the bottom of a flask containing the sample,remove any acid adhering to the outside, and discard the firstmilliliter flowing from the pipet. Hold the tip of the pipetjust above the surface of the sodium hydroxide solution, andtransfer between 2.5 and 3 mL of the sample into the flask,leaving at least 1 mL in the pipet. Stopper the flask, gentlyswirl to mix the contents, and accurately weigh to obtain thesample weight. Add methyl orange TS, and titrate the excesssodium hydroxide with 1 N Hydrochloric Acid. Each milliliterof 1 N sodium hydroxide is equivalent to 36.46 mg of HCl.
Color A sample shows no more color than does MatchingFluid A under Readily Carbonizable Substances, AppendixIIB.Degrees Baumé Transfer about 200 mL of sample, pre-viously cooled to a temperature below 15° into a 250-mLhydrometer cylinder. Insert a suitable Baumé hydrometergraduated at 0.1 °Bé intervals, adjust the temperature to 15.6°,and note the reading at the bottom of the meniscus.Iron Dilute 4.3 mL (5 g) of sample to 40 mL with water,and add about 40 mg of ammonium persulfate and 10 mL ofammonium thiocyanate TS. Any red color produced does notexceed that produced by 2.5 mL of Iron Standard Solution(25 �g Fe) (see Solutions and Indicators) in an equal volumeof solution containing the same quantities of ACS Reagent-Grade Hydrochloric Acid and the reagents used in the test.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Nonvolatile Residue Transfer 1 g of sample into a taredglass dish, evaporate to dryness on a steam bath, and thendry at 110° for 1 h, cool in a desiccator, and weigh. Theweight of the residue does not exceed 5 mg.Organic Compounds (Note: Use either of the methods pre-sented below for analysis of all listed elements, except forbenzene, which requires the Vapor Partitioning Method.)
VAPOR PARTITIONING METHOD
This method is suitable for the determination of extractableorganic compounds at 0.05 to 100 mg/kg but is most appro-priate for organic compounds with a vapor pressure greaterthan 10 mm Hg at 25°.
Preparation of Standard Solutions Prepare a standard so-lution of each of the organic compounds to be quantitated inHydrochloric Acid (known to be free of interfering impurities)at approximate concentrations of 5 mg/kg, or within �50%of the concentrations in the samples to be analyzed.
Place a stirring bar in a 1-L volumetric flask equipped witha ground-glass stopper, and tare the combination. Fill the flaskwith reagent-grade Hydrochloric Acid so that no air space ispresent when the flask is stoppered, and determine the weightof the Hydrochloric Acid. Calculate the volume (V), in microli-ters, of each organic component to be added using the equation
V = (C × W)/(D × 1000),
in which C is the desired concentration, in milligrams perkilogram, of the organic compound; W is weight, in grams,of the Hydrochloric Acid; D is the density, in milligrams permicroliter, of the organic compound; and 1000 is a conversionfactor with the units of grams per kilogram. Add the calculatedamount of each component to the Hydrochloric Acid with asyringe (ensure that the syringe tip is under the solution sur-face), stopper the flask, and stir the solution for at least 2 husing a magnetic stirrer.
Calibration Treat the standards in the same way as de-scribed for the sample under Procedure (below). Determinea blank for each lot of reagent-grade Hydrochloric Acid, andcalculate a response factor (R) by dividing the concentration(C), in milligrams per kilogram, for each component by the
220 / Hydrochloric Acid / Monographs FCC V
peak area (A) for that component (subtract any area obtainedfrom the blank sample):
R = C/(A – area of blank).
Gaseous compounds present special problems in the prepara-tion of standards. Therefore, to determine response factorsfor gaseous compounds use the following Method of MultipleExtractions. Dilute a sample of Hydrochloric Acid known tocontain the gaseous compound of interest with an equal vol-ume of water. Draw 20 mL of this solution into a 50-mLglass syringe; then draw 20 mL of air into the syringe, capwith a rubber septum, and place the syringe on a shaker for5 min. Withdraw 1 mL of the vapor through the septum, andinject it into the chromatograph. Expel the vapor phase fromthe 50-mL syringe, draw in another 20 mL of air, repeat theextraction, and inject another 1-mL vapor sample into thechromatograph. Carry out the extraction and analysis on thesame sample of acid six times. For each impurity, plot thearea (AN) determined for extraction (n) against the differencebetween AN and the area determined for extraction (N + 1);that is, plot AN against (AN – AN+1). The slope of this line isthe extraction efficiency (E) for that impurity into the air.
Inject 1 mL of a 0.1% (by volume) standard gas sampleof each impurity in air into the chromatograph, and determinethe absolute factor (FA), in grams, per peak area (A) by thefollowing formula:
FA = (M × 4.0816 × 10−8)/A,
in which M is the molecular weight of the compound.The concentration (C), in milligrams per kilogram, of the
component in the original sample is calculated by the formula
C = (A × FA × 1.6949 × 106)/E,
in which A is the peak area corresponding to the compound(as above), FA is the absolute factor, and E is extractionefficiency. The response factor is then calculated as
R = C/A.
Procedure (See Chromatography, Appendix IIA.) Use agas chromatograph equipped with a flame-ionization detectorand a 4-m × 2-mm (id) stainless-steel column, or equivalent,packed with 15%, by weight, methyl trifluoropropyl silicone(DCFS 1265, or QF-1, or OV-210, or SP-2401) stationaryphase on 80- to 100-mesh Gas Chrom R, or the equivalent.Condition a newly packed column at 120° and with a 30-mL/min helium flow for at least 2 h (preferably overnight) beforeit is attached to the detector. For analysis, maintain the columnisothermally at 105°; the injection port and detector at 250°;the carrier gas flow rate at 11 mL/min; with fuel gas flowsoptimized for the gas chromatograph and detector in use.Change the experimental conditions as necessary for optimalresolution and sensitivity. The signal-to-noise ratio should beat least 10:1.
Dilute a 10-mL sample with an equal volume of water.Draw this solution into a 50-mL glass syringe. Then draw 20mL of air into the syringe, cap with a rubber septum, andplace the syringe on a shaker for 5 min. Draw 1 mL of the vaporthrough the septum, and inject it into the gas chromatograph.
Approximate elution times, in minutes, for some specific or-ganic compounds are as follows:
Methane and acetylene .....................................................1.70Methyl chloride .................................................................2.21Vinyl chloride ...................................................................2.291,1,1-Trichlorofluoromethane ...........................................2.62Ethyl chloride.................................................................. 2.90Vinylidene chloride......................................................... 3.20Methylene chloride ......................................................... 3.64Chloroform ...................................................................... 4.491,1-Dichloroethane .......................................................... 4.53Carbon tetrachloride........................................................ 4.861,1,1-Trichloroethane ...................................................... 5.50Benzene ........................................................................... 6.00Trichloroethylene ............................................................ 6.22Ethylene dichloride ......................................................... 6.61Propylene dichloride ....................................................... 8.41Perchloroethylene............................................................ 9.73
Alternative columns may be required to resolve some combi-nations of components. Methyl chloride and vinyl chlorideare resolved by a 3.7-m × 3-mm (id) squalane column, orequivalent, at 45° and a helium flow of 10 mL/min. Chloro-form and 1,1-dichloroethane are resolved by a 4-m × 3-mm(id) DC 550R column, or equivalent, at 110° and a heliumflow of 12 mL/min.
Calculation Calculate the concentration (C) in milligramsper kilogram of each compound by multiplying its correspond-ing peak area (A) by the appropriate response factor (R) deter-mined in the Calibration protocol:
C = R × A.
Precision The relative standard deviation at 5 mg/kgshould not exceed 15% for five analyses.
SOLVENT EXTRACTION METHOD
This method is suitable for the determination of extractableorganic compounds at 0.3 to 100 mg/kg, but is most appro-priate for organic compounds with vapor pressures less than10 mm Hg at 25°.
Preparation of Standards Prepare the Standard Solutionas described under the Vapor Partitioning Method.
Calibration Extract a sample of the Standard Solution asdirected under Procedure (below) and inject it into the gaschromatograph, or equivalent. Determine a blank for each lotof reagent-grade Hydrochloric Acid and perchloroethylene byextracting the Hydrochloric Acid in the same way as forthe standard. Calculate a response factor (R) by dividing theconcentration (C), in milligrams per kilogram, for each com-ponent by the peak area (A) for that component (subtract anyarea obtained from the blank sample):
R = C/(A – area of blank).
Procedure The conditions for the gas chromatograph, orequivalent, are the same as for the Vapor Partitioning Method,except that the column temperature is set at 120°, and thecarrier-gas flow is set to 21 mL/min. Accurately transfer 90mL of sample and 10 mL of perchloroethylene (free of interfer-
FCC V Monographs / Hydrogenated Starch Hydrolysate / 221
ing impurities) into a narrow-mouth, 4-oz bottle. Place thebottle in a mechanical shaker for 30 min. Separate the twophases (perchloroethylene on the bottom) and inject 3 �L ofthe perchloroethylene extract into the gas chromatograph, orequivalent. Approximate elution times, in minutes, for somechlorinated organic compounds are as follows:
Vinylidene chloride......................................................... 2.94Methylene chloride ......................................................... 3.27Chloroform ...................................................................... 3.83Carbon tetrachloride........................................................ 4.071,1,1-Trichloroethane ...................................................... 4.50Trichloroethylene ............................................................ 4.97Ethylene dichloride ......................................................... 5.26Propylene dichloride ....................................................... 6.36Perchloroethylene............................................................ 6.951,1,1,2-Tetrachloroethane.............................................. 10.121,1,2,2-Tetrachloroethane.............................................. 13.70Pentachloroethane ...........................................................16.19
To determine perchloroethylene and higher-boiling impuri-ties, substitute methylene chloride (free of interfering impuri-ties) for perchloroethylene in the extraction step. For higher-boiling impurities such as monochlorobenzene and the threedichlorobenzenes, use a 2.74-m × 2.1-mm (id) stainless-steelcolumn packed with 10% carbowax 20M/2% KOH on 80- to100-mesh chromasorb W (acid washed), set at 150° and witha nitrogen flow of 35 mL/min.
Calculation Calculate the concentration (C), in milli-grams per kilogram, of each compound by multiplying thecorresponding peak area (A) (subtract any area obtained froma blank sample) by the appropriate response factor (R) deter-mined in the Calibration protocol:
C = R × (A – area of blank).
Precision The relative standard deviation at 5 mg/kgshould not exceed 15% for five analyses.Oxidizing Substances (as Cl2) Transfer 1 mL of sampleinto a 30-mL test tube, dilute to 20 mL with freshly boiledand cooled water, and add 1 mL of potassium iodide TS and1 mL of starch TS. Stopper the test tube, and mix thoroughly.Any blue color does not exceed that produced in a controlconsisting of 1.0 mL of 0.001 N iodine in an equal volumeof water containing the same quantities of the same reagentsand 1 mL of ACS Reagent-Grade Hydrochloric Acid.Reducing Substances (as SO3) Transfer 1 mL of ACS Re-agent-Grade Hydrochloric Acid into a 30-mL test tube, diluteto 20 mL with recently boiled and cooled water, and add 1mL of potassium iodide TS, 1 mL of starch TS, and 2.0 mLof 0.001 N iodine. Stopper the test tube, and mix thoroughly.The blue color that appears does not disappear when 1 mLof sample is added.Specific Gravity Determine at 15.6° with a hydrometer, orcalculate it from the degrees Baumé observed in the DegreesBaumé Test.Sulfate Dilute 1 g of sample to 100.0 mL with water, transfer5.0 mL of this dilution into a 50-mL tall-form Nessler tube,and dilute to 20 mL with water. Add a drop of phenolphthaleinTS, neutralize the solution with 6 N ammonium hydroxide,
and then add 1 mL of 2.7 N Hydrochloric Acid. Add 3 mL ofbarium chloride TS to the resulting clear solution, previouslyfiltered, if necessary, dilute to 50 mL with water, and mix.Prepare a control consisting of 1 mL of ACS Reagent-GradeHydrochloric Acid and 250 �g of sulfate (SO4) and the samequantities of the reagents used for the sample. Any turbidityshown in the sample does not exceed that shown in the control.
Packaging and Storage Store in tight containers.
Hydrogenated Starch Hydrolysate
Polyglucitol
CH2OHOH
CH2OH
HO
OH
OH
O
CH2OH
HO
OH
OHO
CH2OHOH
CH2OH
OH
OH
O
CH2OH
OH
OHO
CH2OHOH
CH2OH
OH
OHO
O
CH2OH
HO
OH
OHn
Sorbitol Maltitol
Hydrogenated Polysaccharides
C6H14O6 Formula wt, Sorbitol 182.17C12H24O11 Formula wt, Maltitol 344.31C12H24O11 plus Formula wt, Dextrose Monomer 162.14C6H10O5 for eachadditional glucosemoiety in the chain
CAS: [68425-17-2]
DESCRIPTION
Hydrogenated Starch Hydrolysate occurs as a concentrated,aqueous solution or spray-dried or dried powder. It is a mixtureof sorbitol, maltitol, maltitriol, and hydrogenated polysaccha-rides containing greater than three D-glucopyranosyl unitsjoined by �-1,4-linkages and terminated with a D-glucityl unit.It is soluble in water.
Function Humectant; texturizing agent; stabilizer; thick-ener; crystal modification agent.
222 / Hydrogen Peroxide / Monographs FCC V
REQUIREMENTS
Assay Passes test.Chloride Not more than 50 mg/kg, on the dry basis.Lead Not more than 1 mg/kg.Loss on Drying Dry Samples: Not more than 15%; LiquidSamples: Not more than 50%.Nickel Not more than 2 mg/kg.Reducing Sugars Not more than 1%, on the dry basis.Residue on Ignition Not more than 0.1%.
TESTS
AssayMobile Phase Use degassed, deionized water.
Standard Solutions Dissolve accurately weighed quanti-ties of USP Sorbitol, USP Dextrose, USP Maltitol, and high-purity maltose monohydrate (Sigma, or equivalent, and pre-viously dried at 105° to constant weight) in sufficient MobilePhase to obtain solutions having concentrations of 0.1% (w/v) for each.
Assay Solution Transfer 0.1 g of sample, accuratelyweighed, into a 100-mL volumetric flask, and dilute to volumewith deionized water. Transfer approximately 10 mL of thissolution into a separate container, and add approximately 0.2g of an MB-1 mixed-bed resin. Shake this mixture for 30 s,and filter through a 0.45-micron nylon disc filter.
Procedure Use a liquid chromatograph equipped with adifferential refractive index detector and a 20-cm × 10-mm (id)column (Phenomenex ‘‘Rezex’’ 4% silver oligosaccharide, orequivalent). Maintain the column at 80° and the Mobile Phaseflow rate at 0.3 mL/min.
Separately inject about 50-�L portions of the Assay Solutionand Standard Solutions into the chromatograph, record thechromatograms, and measure the responses for the majorpeaks. The elution order for the standards is maltose, maltitol,dextrose, and sorbitol. The differential refractive index detec-tor should show similar response factors.
Calculate the response factors of the standards by dividingtheir concentrations by their peak areas. Calculate the concen-tration of each component in the sample by multiplying theindividual peak areas corresponding to each component bythe appropriate response factor calculated for its standard,giving the concentration in w/w percent. A comparison ofsorbitol and dextrose peak areas shows that at least 95% ofthe sum of these peak areas is sorbitol. A similar comparisonof maltitol and maltose peak areas shows that at least 95%is maltitol. Neither sorbitol nor maltitol fractions comprisemore than 50% of the sample.Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB. Anyturbidity produced by a 10.0-g sample does not exceed thatshown in a control containing 5 mL of the standard solution.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 5-g sample.Loss on Drying Transfer 5 g of dry sample or 1 g of liquidsample, accurately weighed, into an oven, and dry at 105°
for 4 h. Place the sample in a cool desiccator until it hascooled to room temperature, and weigh.Nickel Determine as directed under Nickel Limit Test, Ap-pendix IIIB, using a 20-g sample.Reducing Sugars
Cupri–Citric Solution Transfer 25 g of copper sulfate, 50g of citric acid, and 144 g of anhydrous sodium carbonateinto a 1000-mL beaker, and dissolve in and dilute to volumewith deionized water.
Caution: Add the anhydrous sodium carbonate slowly.
Procedure Dissolve 1.0 g of sample in 6 mL of deionizedwater with the aid of gentle heat, if necessary. Cool, and add20-mL of Cupri–Citric Solution and a few glass beads. Heatso that boiling begins after 4 min, and continue boiling for3 min. Cool rapidly, and add 100 mL of a 2.4% (v/v) solutionof glacial acetic acid and 20.0 mL of 0.025 M iodine. Whileshaking continuously, add 25 mL of a 6:94 (v/v) mixtureof hydrochloric acid:deionized water. After the precipitatedissolves, titrate the excess iodine with 0.05 M sodium thiosul-fate, using 1 mL of starch solution as the indicator, addedtowards the end of the titration. Not less than 12.8 mL (1%)of 0.05 M sodium thiosulfate is required.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC.
Packaging and Storage Store in well-closed containers.
Hydrogen Peroxide
H2O2 Formula wt 34.01
CAS: [7722-84-1]
DESCRIPTION
Hydrogen Peroxide occurs as a clear, colorless liquid. Thegrades of Hydrogen Peroxide suitable for food use usuallyhave a concentration between 30% and 50%. It is misciblewith water.
Note: Although Hydrogen Peroxide undergoes exother-mic decomposition in the presence of dirt and otherforeign materials, it is safe and stable under recom-mended conditions of handling and storage. Informationon safe handling and use may be obtained from thesupplier.
Function Bleaching, oxidizing agent; starch modifier; anti-microbial agent.
REQUIREMENTS
Identification Shake 1 mL of sample with 10 mL of watercontaining 1 drop of 2 N sulfuric acid, and add 2 mL of ether.The subsequent addition of a drop of potassium dichromate
FCC V Monographs / Hydrogen Peroxide / 223
TS produces an evanescent blue color in the water layer thatupon agitation and standing passes into the ether layer.Assay Not less than the labeled concentration or within therange stated on the label.Acidity (as H2SO4) Not more than 0.03%.Iron Not more than 0.5 mg/kg.Lead Not more than 4 mg/kg.Phosphate Not more than 0.005%.Residue on Evaporation Not more than 0.006%.Tin Not more than 10 mg/kg.
TESTS
Assay Transfer a volume of sample, equivalent to about 300mg of H2O2 and accurately weighed, into a 100-mL volumetricflask, dilute to volume with water, and mix thoroughly. Add25 mL of 2 N sulfuric acid to a 20.0-mL portion of thissolution, and titrate with 0.1 N potassium permanganate. Eachmilliliter of 0.1 N potassium permanganate is equivalent to1.701 mg of H2O2.Acidity (as H2SO4) Dilute 9 mL (10 g) of sample in 90 mLof carbon dioxide-free water, add methyl red TS, and titratewith 0.02 N sodium hydroxide. The volume of sodium hydrox-ide solution should not be more than 3 mL greater than thevolume required for a blank test on 90 mL of the water usedfor dilution.Iron Evaporate 18 mL (20 g) of sample to dryness with 10mg of sodium chloride on a steam bath, dissolve the residuein 2 mL of hydrochloric acid, and dilute to 50 mL with water.Add about 40 mg of ammonium persulfate crystals and 10mL of ammonium thiocyanate TS, and mix. Any red or pinkcolor does not exceed that produced by 1.0 mL of Iron Stan-dard Solution (10 �g Fe) in an equal volume of solutioncontaining the quantities of the reagents used in the test.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, with the following modifications: (1) Prepare only oneDiluted Standard Lead Solution by transferring 40 mL ofLead Nitrate Stock Solution into a 1000-mL volumetric flaskand diluting to volume with water to obtain a solution con-taining 4 �g/mL of lead (Pb) ion; (2) Replace the first para-graph under Sample Preparation with the following: Transfer10 g of sample, accurately weighed, into an evaporation dish;(3) Under Procedure, determine the absorbances of the SampleSolution and Diluted Standard Lead Solution only⎯the ab-sorbance of the Sample Solution is less than or equal to thatof the Diluted Standard Lead Solution.Phosphate Evaporate 400 mg of sample to dryness on asteam bath. Dissolve the residue in 25 mL of approximately0.5 N sulfuric acid, add 1 mL of ammonium molybdate solu-tion [500 mg of (NH4)6Mo7O24·4H2O in each 10 mL of water]and 1 mL of p-methylaminophenol sulfate TS, and allow itto stand for 2 h. Any blue color does not exceed that producedby 2.0 mL of Phosphate Standard Solution (20 �g PO4)(see Solutions and Indictors) in an equal volume of solutioncontaining the quantities of the reagents used in the test.Residue on Evaporation Evaporate 25 g of sample to dry-ness in a tared porcelain or silica dish on a steam bath, and
continue drying to constant weight at 105°. The weight ofthe residue does not exceed 1.5 mg.Tin
Aluminum Chloride Solution Dissolve 8.93 g of alumi-num chloride (AlCl3·6H2O) in sufficient water to make1000 mL.
Gelatin Solution On the day of use, dissolve 100 mg ofgelatin in 50 mL of boiled water that has been cooled tobetween 50° and 60°.
Tin Stock Solution Dissolve 250.0 mg of lead-free tin foilin 10 to 15 mL of hydrochloric acid, and dilute to 250.0 mLwith 1:2 hydrochloric acid.
Standard Solution On the day of use, transfer 5.0 mL ofTin Stock Solution into a 100-mL volumetric flask, dilute tovolume with water, and mix. Transfer 2.0 mL of this solution(100 �g Sn) into a 250-mL Erlenmeyer flask, and add 15 mLof water, 5 mL of nitric acid, and 2 mL of sulfuric acid. Placea small, stemless funnel in the mouth of the flask, and heatuntil strong fumes of sulfuric acid evolve. Cool, add 5 mLof water, evaporate again to strong fumes, and cool. Repeatthe addition of water and heating to strong fumes, then add15 mL of water, heat to boiling, and cool. Dilute to about 35mL with water, add 1 drop of methyl red TS and 2.0 mL ofthe Aluminum Chloride Solution, and mix. Make the solutionjust alkaline by adding, dropwise, ammonium hydroxide andstirring gently, then add 0.1 mL in excess.
Caution: To avoid dissolving the aluminum hydroxideprecipitate, do not add more ammonium hydroxide than0.1 mL in excess.
Centrifuge for about 15 min at 4000 rpm, and then decantthe supernatant liquid as completely as possible without dis-turbing the precipitate. Dissolve the precipitate in 5 mL of1:2 hydrochloric acid, add 1.0 mL of the Gelatin Solution,and dilute to 20.0 mL with a saturated solution of aluminumchloride.
Sample Solution Transfer 9 mL (10 g) of sample into a250-mL Erlenmeyer flask, and add 15 mL of water, 5 mL ofnitric acid, and 2 mL of sulfuric acid. Mix, and heat gently ona hot plate to initiate and maintain a vigorous decomposition.When decomposition is complete, place a small, stemlessfunnel in the mouth of the flask, and continue as directed forthe Standard Solution, beginning with ‘‘and heat until strongfumes of sulfuric acid evolve.’’
Procedure Rinse a polarographic cell or other vessel witha portion of the Standard Solution, then add a suitable volumeto the cell, immerse it in a constant-temperature bath main-tained at 35° � 0.2°, and deaerate by bubbling oxygen-freenitrogen or hydrogen through the solution for at least 10 min.Insert the dropping mercury electrode of a suitable polaro-graph, and record the polarogram from −0.2 to −0.7 V ata sensitivity of 0.0003 �A/mm, using a saturated calomelreference electrode. In the same manner, record a polarogramof a portion of the Sample Solution at the same current sensitiv-ity. The height of the wave produced by the Sample Solutionis not greater than that produced by the Standard Solution atthe same half-wave potential.
224 / Hydroxylated Lecithin / Monographs FCC V
Packaging and Storage Store in a cool place in containerswith a vent in the stopper.
Hydroxylated Lecithin
CAS: [8029-76-3]
DESCRIPTION
Hydroxylated Lecithin occurs as a light yellow substance thatmay vary in consistency from fluid to plastic, depending onthe content of free fatty acid and oil and on whether it containsdiluents. It is derived from a complex mixture of acetone-insoluble phosphatides from soybean and other plant lecithins,consisting chiefly of phosphatidyl choline, phosphatidyl etha-nolamine, and phosphatidyl inositol as well as other minorphospholipids and glycolipids mixed with varying amountsof triglycerides, fatty acids, sterols, and carbohydrates. Themixture is treated with hydrogen peroxide, benzoyl peroxide,lactic acid, and sodium hydroxide or with hydrogen peroxide,acetic acid, and sodium hydroxide to produce a hydroxylatedproduct having an iodine value approximately 10% lower thanthat of the starting material. It is partially soluble in waterbut hydrates readily to form emulsions; it is more dispersibleand hydrates more readily than crude lecithin.
Function Emulsifier; clouding agent.
REQUIREMENTS
Acetone-Insoluble Matter (as phosphatides) Not less than50.0%.Acid Value Not more than 70.Hexane-Insoluble Matter Not more than 0.3%.Iodine Value Between 85 and 95.Lead Not more than 1 mg/kg.Peroxide Value Not more than 100.Water Not more than 1.5%.
TESTS
Acetone-Insoluble Matter (as phosphatides)Purification of Phosphatides Dissolve 10 g of sample in
20 mL of petroleum ether, add 50 mL of acetone to thesolution, chill, and decant. Dry the solids under flowing nitro-gen under a hood. Dissolve 5 g of the solids in 10 mL ofpetroleum ether, and add 25 mL of acetone to the solution.Transfer approximately equal portions of the precipitate toeach of two 40-mL centrifuge tubes, using additional portionsof acetone to facilitate the transfer. Stir thoroughly, dilute to40 mL with acetone, stir again, chill for 15 min in an icebath, stir again, and then centrifuge for 5 min. Decant theacetone, crush the solids with a stirring rod, refill the tubewith acetone, stir, chill, centrifuge, and decant as before.The solids after the second centrifugation require no further
purification and may be used for preparing the Phosphatide–Acetone Solution. Five grams of the purified phosphatides arerequired to saturate about 16 L of acetone.
Phosphatide–Acetone Solution Add a quantity of purifiedphosphatides to sufficient acetone, previously cooled to about5°, to form a saturated solution, and maintain the mixture atthis temperature for 2 h, shaking it vigorously at 15-minintervals. Decant the solution through a rapid filter paper,avoiding the transfer of any undissolved solids to the paperand conducting the filtration under refrigerated conditions(not above 5°).
Procedure If the sample is plastic or semisolid, soften aportion of it by warming it in a water bath at a temperaturenot exceeding 60°, and then mix it thoroughly. Transfer about2 g of a well-mixed sample, accurately weighed, into a 40-mL centrifuge tube, previously tared with a glass stirring rod,and add 15 mL of Phosphatide–Acetone Solution from a buret.Warm the mixture in a water bath until the sample melts, butavoid evaporation of the acetone. Stir until the sample iscompletely disintegrated and dispersed, transfer the tube intoan ice bath, chill for 5 min, remove from the ice bath, andadd about 10 mL of Phosphatide–Acetone Solution, previouslychilled for 5 min in an ice bath. Stir the mixture to completedispersion of the sample, dilute to 40 mL with chilled (5°)Phosphatide–Acetone Solution, stir to complete dispersion ofthe sample, and return the tube and contents to the ice bathfor 15 min. Subsequently stir again while still in the ice bath,remove the stirring rod, and centrifuge the mixture immedi-ately for 5 min. Decant the supernatant liquid from the centri-fuge tube; crush the centrifuged solids with the stirring rod;refill the tube to the 40-mL mark with chilled (5°) Phospha-tide–Acetone Solution; and repeat the chilling, stirring, centrif-ugation, and decantation procedure. After the second centrifu-gation and decantation of the supernatant acetone, again crushthe solids with the stirring rod, and place the tube and itscontents in a horizontal position at room temperature untilthe excess acetone has evaporated. Mix the residue again, drythe centrifuge tube and its contents at 105° for 45 min in aforced-draft oven, cool, and weigh. Calculate the percentageof acetone-insoluble matter by the formula
(100R/S) – B,
in which R is the weight, in grams, of residue, S is the weight,in grams, of the sample taken, and B is the percentage ofhexane-insoluble matter determined as directed under Hexane-Insoluble Matter (below).Acid Value If the sample is plastic or semisolid, soften aportion by warming it in a water bath at a temperature notexceeding 60°, and then mix it thoroughly. Transfer about 2g of a well-mixed sample, accurately weighed, into a 250-mL Erlenmeyer flask, and dissolve it in 50 mL of petroleumether. Add 50 mL of ethanol, previously neutralized to phenol-phthalein with 0.1 N sodium hydroxide, to this solution, andmix well. Using phenolphthalein TS as the indicator, titratewith 0.1 N sodium hydroxide to a pink endpoint that persistsfor 5 s. Calculate the Acid Value by the formula
5.6 × A/W,
FCC V Monographs / Hydroxypropyl Methylcellulose / 225
in which A is the volume, in milliliters, of 0.1 N sodiumhydroxide consumed, and W is the weight, in grams, of thesample taken.Hexane-Insoluble Matter Determine as directed underHexane-Insoluble Matter, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the FlameAtomic Absorption Spectrophotometric Method under LeadLimit Test, Appendix IIIB, using a 10-g sample.Peroxide Value Transfer about 10 g of sample, accuratelyweighed, into a suitable container, add 30 mL of a 3:2 solutionof glacial acetic acid:chloroform, and mix. Add 1 mL of asaturated solution of potassium iodide, mix, and allow to standfor 10 min. Add 100 mL of water, begin titrating with 0.05N sodium thiosulfate, adding starch TS as the endpoint isapproached, and continue the titration until the blue starchcolor has just disappeared. Perform a blank determination(see General Provisions), and make any necessary correction.Calculate the peroxide value, as milliequivalents of peroxideper kilogram of sample, by the formula
[S × N × 1000]/W,
in which S is the net volume, in milliliters, of sodium thiosul-fate solution required for the sample; N is the exact normalityof the sodium thiosulfate solution; and W is the weight, ingrams, of the sample taken.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Hydroxypropyl CelluloseModified Cellulose
INS: 463 CAS: [9004-64-2]
DESCRIPTION
Hydroxypropyl Cellulose occurs as a white powder. It is acellulose ether containing hydroxypropyl substitution. It maycontain a suitable anticaking agent. It is soluble in water andin certain organic solvents.
Function Emulsifier; film coating; protective colloid; stabi-lizer; suspending agent; thickener.
REQUIREMENTS
IdentificationA. Shake a 0.1% solution of sample. A layer of foam
appears (distinction from cellulose gum).B. Add 5 mL of a 5.0% solution of copper sulfate (or
aluminum sulfate) to 5 mL of a 0.5% solution of sample. Noprecipitate forms (distinction from cellulose gum).
Assay Not more than 80.5% of hydroxypropoxyl groups(⎯OCH2CHOHCH3) after drying, equivalent to not morethan 4.6 hydroxypropyl groups per anhydroglucose unit.Lead Not more than 3 mg/kg.Loss on Drying Not more than 5.0%.pH of a 1% Solution Between 5.0 and 8.0.Residue on Ignition Not more than 0.5%.Viscosity of a 10% Solution Not less than 145 centipoises.
TESTS
Assay Determine as directed under Hydroxypropoxyl Deter-mination, Appendix IIIC, using 85 mg of sample, previouslydried at 105° for 3 h and accurately weighed.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds but with a 2-g sample, and 6 �g of lead(Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.pH of a 1% Solution Determine as directed under pH Deter-mination, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Viscosity of a 10% Solution Determine as directed underViscosity of Cellulose Gum, Appendix IIB, using an accuratelyweighed sample, equivalent to 40 g of Hydroxypropyl Cellu-lose on the dried basis, in a tared sample container.
Packaging and Storage Store in well-closed containers.
Hydroxypropyl MethylcellulosePropylene Glycol Ether of Methylcellulose; ModifiedCellulose, HPMC
O
H OR
CH2OR
OR
H
H
H
HO
n
in whichR = H or CH3 or CH2CHOHCH3
INS: 464 CAS: [9004-65-3]
DESCRIPTION
Hydroxypropyl Methylcellulose occurs as a white to off-white, fibrous powder or as granules. It is the propylene glycol
226 / Hydroxypropyl Methylcellulose / Monographs FCC V
ether of methylcellulose in which both the hydroxypropyl andthe methyl groups are attached to the anhydroglucose rings ofcellulose by ether linkages. Several product types are availablethat are defined by varying combinations of methoxyl andhydroxypropoxyl content. It is soluble in water and in certainorganic solvent systems. Aqueous solutions are surface active,form films upon drying, and undergo a reversible transforma-tion from sol to gel upon heating and cooling, respectively.
Function Thickening agent; stabilizer; emulsifier.
REQUIREMENTS
IdentificationA. Add 1 g of sample to 100 mL of water. It swells and
disperses to form a clear to opalescent, mucilaginous solution,depending on the intrinsic viscosity, which is stable in thepresence of most electrolytes.
B. Add 1 g of sample to 100 mL of boiling water, and stirthe mixture. A slurry forms that when cooled to 20°, dissolvesto form a clear or opalescent, mucilaginous solution.
C. Pour a few milliliters of the solution prepared for Identifi-cation Test B onto a glass plate, and allow the water toevaporate. A thin, self-sustaining film forms.Assay for Hydroxypropoxyl Groups Between a minimumof 3.0% and a maximum of 12.0% of hydroxypropoxyl groups(⎯ OCH2CHOHCH3), within the range claimed by the vendorfor any product type.Assay for Methoxyl Groups Between a minimum of 19.0%and a maximum of 30.0% of methoxyl groups (⎯OCH3),within the range claimed by the vendor for any product type.Lead Not more than 3 mg/kg.Loss on Drying Not more than 5.0%.Residue on Ignition Not more than 1.5% for products withviscosities of 50 centipoises or above; not more than 3.0%for products with viscosities below 50 centipoises.Viscosity The viscosity of an aqueous solution containing2 g of sample per 100 g of solution is not less than 80.0%and not more than 120.0% of that stated on the label forviscosity types of 100 centipoises or less, and not less than75.0% and not more than 140.0% of that stated on the labelfor viscosity types higher than 100 centipoises.
TESTS
Assay (Caution: Perform all steps involving hydriodic acidcarefully in a well-ventilated hood. Use goggles, acid-resistantgloves, and other appropriate safety equipment. Be extremelycareful when handling the hot vials because they are underpressure. In the event of hydriodic acid exposure, wash withcopious amounts of water and seek medical attention at once.)
Internal Standard Solution Transfer about 2.5 g of tolu-ene, accurately weighed, into a 1000-mL volumetric flaskcontaining 10 mL of o-xylene, dilute with o-xylene to volume,and mix.
Standard Preparation Transfer about 135 mg of adipicacid into a suitable serum vial, add 4.0 mL of hydriodic acidfollowed by 4.0 mL of the Internal Standard Solution, andclose the vial securely with a septum stopper. Accurately
weigh the vial and its contents, add 30 �L of isopropyl iodidewith a syringe through the septum, reweigh, and calculate theweight of isopropyl iodide added. Similarly, add 90 �L ofmethyl iodide, and calculate the weight added. Shake well,and allow the layers to separate.
Assay Preparation Transfer about 0.065 g of sample, ac-curately weighed, into a 5-mL vial equipped with a pressure-tight septum closure, add an amount of adipic acid equal tothe weight of the sample, and pipet 2 mL of the InternalStandard Solution into the vial. Cautiously pipet 2 mL ofhydriodic acid into the mixture, immediately secure the clo-sure, and accurately weigh. Shake the vial for 30 s, heat at150° for 20 min, remove from the heat, shake again, usingextreme caution, and heat at 150° for 40 min. Allow the vialto cool for about 45 min, and weigh. If the weight loss isgreater than 10 mg, discard the mixture and prepare anotherAssay Preparation.
Chromatographic System (See Chromatography, Appen-dix IIA) Use a gas chromatograph equipped with a thermalconductivity detector and a 1.8-m × 4-mm glass column, orequivalent, packed with 10% methylsilicone oil (UCW 982or equivalent) on 100- to 120-mesh flux-calcined chromato-graphic siliceous earth (Chromosorb WHP, or equivalent).Maintain the column at 100° and the injection port and detectorat 200°. Use helium as the carrier gas with a flow rate of 20mL/min.
Calibration Inject about 2 �L of the upper layer of theStandard Preparation into the chromatograph, and recordthe chromatogram. The retention times for methyl iodide,isopropyl iodide, toluene, and o-xylene are approximately 3,5, 7, and 13 min, respectively. Calculate the relative responsefactor, F, of equal weights of toluene and methyl iodide bythe formula
Q/A,
in which Q is the quantity ratio of methyl iodide to toluenein the Standard Preparation, and A is the peak area ratio ofmethyl iodide to toluene obtained from the Standard Prepara-tion. Similarly, calculate the relative response factor, F′, ofequal weights of toluene and isopropyl iodide by the formula
Q′/A′,
in which Q′ is the quantity ratio of isopropyl iodide to toluenein the Standard Preparation, and A′ is the peak area ratioof isopropyl iodide to toluene obtained from the StandardPreparation.
Procedure Inject about 2 �L of the upper layer of theAssay Preparation into the chromatograph, and record thechromatogram. Calculate the percentage of methoxyl groups(⎯OCH3) in the sample by the formula
2 × (31/142) × F × a × (W/w),
in which 31/142 is the ratio of the formula weights of methoxylto methyl iodide; a is the ratio of the area of the methyl iodidepeak to that of the toluene peak obtained from the AssayPreparation; W is the weight, in grams, of toluene in theInternal Standard Solution; and w is the weight, in grams, ofsample taken. Similarly, calculate the percentage of hydroxy-
FCC V Monographs / Indigotine / 227
propoxyl groups (⎯OCH2CHOHCH3) in the sample by theformula
2 × (75/170) × F′ × a′ × (W/w),
in which a′ is the ratio of the area of the isopropyl iodidepeak to that of the toluene peak obtained from the AssayPreparation.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution containing 2 g of sampleprepared as directed for organic compounds, and 6 �g of leadion (Pb) in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 3-g sample at 105° for 2 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.Viscosity Accurately weigh a sample, equivalent to 2 g ofsolids on the dried basis, transfer to a wide-mouth 250-mLcentrifuge bottle, and add 98 g of water previously heated tobetween 80° and 90°. Stir with a mechanical stirrer for 10min, then place the bottle in an ice bath until solution iscomplete, adjust the weight of the solution with water to 100g if necessary, and centrifuge it to expel any entrapped air.Adjust the temperature of the solution to 20° � 0.1°, anddetermine the viscosity as directed under Viscosity of Methyl-cellulose, Appendix IIB.
Packaging and Storage Store in well-closed containers.
Indigotine1
CI Food Blue 1; Indigotine Disulfonate; Indigo Carmine;CI 73015; Class: Indigoid
NaO3S C
NH
C
O
SO3NaC
NH
O
C
C16H8N2O8S2Na2 Formula wt 466.36
INS: 132 CAS: [860-22-0]
DESCRIPTION
Indigotine occurs as a blue-brown to red-brown powder orgranules. It is principally the disodium salt of 2-(1,3-dihydro-
1To be used or sold for use to color food that is marketed in the UnitedStates, this color additive must be from a batch that has been certifiedby the U.S. Food and Drug Administration (FDA). If it is not from anFDA-certified batch, it is not a permitted color additive for food use inthe United States, even if it is compositionally equivalent. The nameFD&C Blue No. 2 can be applied only to FDA-certified batches of thiscolor additive. Indigotine is a common name given to the uncertifiedcolorant. See the monograph entitled FD&C Blue No. 2 for directionsfor producing an FDA-certified batch.
3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid. It dissolves in water to give a solutionthat is blue at neutrality, blue-violet in acid, and green toyellow-green in base. When dissolved in concentrated sulfuricacid, it yields a blue-violet solution that turns blue whendiluted with water. It is insoluble in ethanol.
Function Color.
REQUIREMENTS
Identification A freshly prepared aqueous solution con-taining 20 mg of sample per liter exhibits absorbance intensi-ties (A) and wavelength maxima as follows: at pH 7, A = 0.82at 610 nm; at pH 1, A = 0.81 at 610 nm; and at pH 13, A =0.2 at 610 nm, and A = 0.31 at 442 nm.Assay Not less than 85.0% total coloring matter.Arsenic Not more than 3 mg/kg.Ether Extracts (combined) Not more than 0.2%.Lead Not more than 10 mg/kg.Loss on Drying (Volatile Matter) at 135°, Chlorides, andSulfates (as sodium salts) Not more than 15.0% in combi-nation.Mercury Not more than 1 mg/kg.Subsidiary and Isomeric Colors
Disodium Salt of 2-(1,3-Dihydro-3-oxo-7-sulfo-2H-indole-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic Acid Notmore than 18.0%.
Sodium Salt of 2-(1,3-Dihydro-3-oxo-2H-indole-2-yli-dene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic Acid Notmore than 2.0%.Uncombined Intermediates and Products of Side Reac-tions
Isatin-5-sulfonic Acid Not more than 0.4%.5-Sulfoanthranilic Acid Not more than 0.2%.
Water-Insoluble Matter Not more than 0.4%.
TESTS
Assay Determine the total color strength as the weight per-cent of the sample using Methods I and II in Total Colorunder Colors, Appendix IIIC. Express the Total Color as theaverage of the two results.
Method I (Sample Preparation) Transfer 175 to 225 mgof sample, accurately weighed, into a 1-L volumetric flask;dissolve in and dilute to volume with water. The absorptivity(a) for Indigotine is 0.0478 mg/L/cm at 610 nm.
Method II (Sample Preparation) Transfer approximately0.3 g of sample, accurately weighed, into the titration flask.The stoichiometric factor (FS) for Indigotine is 4.29.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Chloride Determine as directed in Sodium Chloride underColors, Appendix IIIC.Ether Extracts Determine as directed in Ether Extractsunder Colors, Appendix IIIC.
228 / Inositol / Monographs FCC V
Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 10 �g of lead (Pb) ion in the control.Loss on Drying (Volatile Matter) at 135° Determine asdirected in Loss on Drying (Volatile Matter) under Colors,Appendix IIIC.Mercury Determine as directed in Mercury under Colors,Appendix IIIC.Subsidiary and Isomeric Colors
Apparatus Use a 40- × 2.5-cm (id) glass column packedwith Celite (Johns Mansville No. 595, or equivalent) preparedas described under Procedure. Dissolve 20 g of hydroxyl-amine hydrochloride in 500 mL of water, place the solutioninto a 2-L separatory funnel, and add 450 mL of butanol,450 mL of chloroform, 300 mL of water, and 100 mL ofhydrochloric acid. Agitate the mixture well, periodically vent-ing the funnel. After settling, separate and store the bottomlayer (organic), the Mobile Phase, and the top layer (aqueous),the Stationary Phase.
Sample Solution Dissolve approximately 100 mg of sam-ple, accurately weighed, in 100 mL of Stationary Phase; warmon a steam bath, if necessary, to dissolve the sample.
Procedure Slurry 12 g of Celite with 7 mL of StationaryPhase, and pour the slurry into the column. Mix 5 mL ofSample Solution with 10 g of Celite, and pour the mixtureinto the column over the slurry, ensuring that the sample isquantitatively transferred to the column.
Elute the column with the Mobile Phase. Collect the mono-sulfonated derivative, the first band eluting, in a 25-mL gradu-ated cylinder, and note the volume. Collect the next band,the isomeric (unsulfonated) derivative, in a similar manner.
Mix each aliquot collected with an equal volume of hexane,and transfer to a separatory funnel. Extract this mixture withthree 15-mL aliquots of water; combine the water extracts, andcalculate the percent concentration (P) of the monosulfonatedderivative (a = 0.0513 mg/L/cm at 615 nm) and the isomericderivative (a = 0.0478 mg/L/cm at 610 nm) by the equation
P = (A × V)/(a × W × 10),
in which A is the absorbance; V is the volume of extract; ais the absorptivity, in milligrams per liter per centimeter; andW is the weight, in milligrams, of the sample.Sulfate Determine as directed in Sodium Sulfate under Col-ors, Appendix IIIC.Uncombined Intermediates and Products of Side Reac-tions Determine as directed for Method I in UncombinedIntermediates and Products of Side Reactions under Colors,Appendix IIIC. Calculate the concentration of isatin-5-sul-fonic acid using an absorptivity of 0.089 mg/L/cm at 245 nm.Water-Insoluble Matter Determine as directed in Water-Insoluble Matter under Colors, Appendix IIIC.
Packaging and Storage Store in well-closed containers.
Inositol1,2,3,5/4,6-Cyclohexanehexol; i-Inositol; meso-Inositol;myo-Inositol
H
OH OH
OH
OH H
H H
HO H
OHH
C6H12O6 Formula wt 180.16
CAS: [87-89-8]
DESCRIPTION
Inositol occurs as fine, white crystals or as a white, crystallinepowder. Its solutions are neutral to litmus. It is opticallyinactive. It is stable in air. One gram is soluble in 6 mL ofwater. It is slightly soluble in alcohol, and is insoluble inether and in chloroform.
Function Nutrient.
REQUIREMENTS
IdentificationA. Add 6 mL of nitric acid to 1 mL of a 1:50 aqueous
solution in a porcelain evaporating dish, and evaporate todryness on a water bath. Dissolve the residue in 1 mL ofwater, add 0.5 mL of a 1:10 aqueous solution of strontiumacetate, and again evaporate to dryness on a steam bath. Aviolet color appears.
B. The inositol hexaacetate obtained in the Assay meltsbetween 212° and 216° (see Melting Range or Temperature,Appendix IIB).Assay Not less than 97.0% of C6H12O6 after drying.Calcium Passes test.Chloride Not more than 0.005%.Lead Not more than 4 mg/kg.Loss on Drying Not more than 0.5%.Melting Range Between 224° and 227°.Residue on Ignition Not more than 0.1%.Sulfate Not more than 0.006%.
TESTS
Assay Transfer about 200 mg of sample, previously driedat 105° for 4 h and accurately weighed, to a 250-mL beaker,add 5 mL of a 1:50 mixture of 2 N sulfuric acid:acetic anhy-dride, and cover the beaker with a watch glass. Heat on asteam bath for 20 min, then chill in an ice bath, and add 100mL of water. Boil for 20 min, allow to cool, and transferquantitatively, with the aid of a little water, to a 250-mLseparator. Extract the solution with six successive 30-, 25-,
FCC V Monographs / Iron, Carbonyl / 229
20-, 15-, 10-, and 10-mL portions of chloroform, using thesolvent to rinse the original flask. Collect the chloroformextracts in a second 250-mL separator, and wash the combinedextracts with 10 mL of water. Transfer the chloroform extractsthrough a funnel containing a pledget of cotton into a 150-mL tared Soxhlet flask. Wash the separator and funnel with10 mL of chloroform, and add to the combined extracts.Evaporate to dryness on a steam bath, dry in an oven at 105°for 1 h, cool in a desiccator, and weigh. The weight of theinositol hexaacetate obtained, multiplied by 0.4167, representsthe equivalent of C6H12O6.Calcium Add 1 mL of ammonium oxalate TS to 10 mL ofa 1:10 aqueous solution of sample. The solution remains clearfor at least 1 min.Chloride Determine as directed in Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB. Anyturbidity produced by a 400-mg sample does not exceed thatproduced in a control containing 20 �g of chloride (Cl) ion.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 4 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.Sulfate Determine as directed in Sulfate Limit Test, underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 5-g sample does not exceed that produced ina control containing 300 �g of sulfate (SO4).
Packaging and Storage Store in well-closed containers.
Invert SugarInvert Sugar Syrup
CAS: [8013-17-0]
DESCRIPTION
Invert Sugar occurs as a hygroscopic liquid. It is a mixtureof glucose and fructose that results from the hydrolysis ofsucrose. Invert Sugar is marketed as Invert Sugar Syrup andcontains dextrose (glucose), fructose, and sucrose in variousamounts as represented by the manufacturer. It is very solublein water, in glycerin, and in glycols and is very sparinglysoluble in acetone and in ethanol.
Function Nutritive sweetener.
REQUIREMENTS
Labeling Indicate the percentages of sucrose and InvertSugar.
Identification (See Chromatography, Appendix IIA.) Pre-pare a 10% solution of sample in purified water and inject7.5 �L of the solution into a high-performance liquid chroma-tograph equipped with a differential refractometer detectorand a column packed with a cation exchange resin maintainedat 85°. Use purified water as the liquid phase, at a flowrate of 0.7 mL/min. The chromatogram of the sample givesappropriate elution times for fructose, glucose, and sucrosewhen compared with a standard solution containing 1 g ofeach saccharide in 100 mL of water.Assay Not less than 90.0% and not more than 110.0% ofthe labeled amount of sucrose and of Invert Sugar.Lead Not more than 0.5 mg/kg.pH Not less than 3.0 and not more than 5.5.Residue on Ignition Not more than 0.2%.Total Solids As represented by the vendor.Total Sugars Not less than 99.5% of the total solids content.
TESTS
Assay Determine as directed in Invert Sugar under TotalSolids, Appendix X.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 5-g sample.pH Determine as directed under pH Determination, Appen-dix IIB.Residue on Ignition Determine as directed in Method IIunder Residue on Ignition, Appendix IIC.Total Solids Determine as directed in Invert Sugar underTotal Solids, Appendix X, using the table provided.Total Sugars Calculate the percent Total Sugars (TS) bythe equation
TS = PI + PS,
in which PI is the percentage of Invert Sugar and PS is thepercentage of sucrose as determined under Assay (above).
Packaging and Storage Store in tight containers.
Iron, CarbonylFe At wt 55.85
CAS: [37220-42-1]
DESCRIPTION
Iron, Carbonyl, occurs as a dark gray powder. It is elementaliron produced by the decomposition of iron pentacarbonyl.When viewed under a microscope having a magnifying powerof 500 diameters or greater, it appears as spheres built upwith concentric shells. It is stable in dry air.
Function Nutrient.
230 / Iron, Electrolytic / Monographs FCC V
REQUIREMENTS
Identification A sample dissolves in dilute mineral acidswith the evolution of hydrogen and the formation of solutionsof the corresponding salts, which give positive tests for Fer-rous Salts (Iron), Appendix IIIA.Assay Not less than 98.0% of Fe.Acid-Insoluble Substances Not more than 0.2%.Arsenic Not more than 3 mg/kg.Lead Not more than 4 mg/kg.Mercury Not more than 2 mg/kg.Sieve Analysis Not less than 100% passes through a 200-mesh sieve; not less than 95% passes through a 325-meshsieve.
TESTS
Assay Determine as directed in the monograph for Iron,Reduced.Acid-Insoluble Substances Determine as directed in themonograph for Iron, Electrolytic.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using the following solution: Dissolve 1.0 gof sample in 25 mL of 2 N sulfuric acid, heat on a steam bathuntil the evolution of hydrogen ceases, cool, and dilute to 35mL with water.Lead (Note: When preparing all aqueous solutions and rins-ing glassware before use, employ water that has been passedthrough a strong-acid, strong-base, mixed-bed ion-exchangeresin before use. Select all reagents to have as low a contentof lead as practicable, and store all reagent solutions in con-tainers of borosilicate glass. Clean glassware before use bysoaking in warm 8 N nitric acid for 30 min and by rinsingwith deionized water.)
Ascorbic Acid–Sodium Iodide Solution Transfer 20 g ofascorbic acid and 38.5 g of sodium iodide to a 200-mL volu-metric flask, dissolve in and dilute to volume with water,and mix.
Trioctylphosphine Oxide Solution (Caution: This solu-tion causes irritation. Avoid contact with eyes, skin, and cloth-ing. Take special precautions in disposing of unused portionsof solutions to which this reagent is added.) Transfer 5.0g of trioctylphosphine oxide to a 100-mL volumetric flask.Dissolve in and dilute to volume with 4-methyl-2-pentanone,and mix.
Lead Nitrate Stock Solution (100 �g/mL) Transfer 159.8mg of reagent-grade lead nitrate [Pb(NO3)2] into a 1000-mLvolumetric flask, dissolve it in 100 mL of water containing1 mL of nitric acid, and dilute to volume with water.
Standard Preparation and Blank Preparation Transfer5.0 mL of Lead Nitrate Stock Solution into a 100-mL volumet-ric flask, dilute to volume with water, and mix. Transfer 2.0mL of the resulting solution into a 50-mL beaker. Add 8 mLof hydrochloric acid and 2 mL of nitric acid to this beaker,and to a separate 50-mL beaker (blank). Place a ribbed watchglass over each beaker, and evaporate to dryness on a steambath. Add 10 mL of 9 N hydrochloric acid to each beaker,and transfer the resulting solutions, with the aid of about 10mL of water, to separate 50-mL volumetric flasks. Add 20
mL of Ascorbic Acid–Sodium Iodide Solution and 5.0 mL ofTrioctylphosphine Oxide Solution to each flask, shake for 30s, and allow to separate. Add water to bring the organic solventlayer into the neck of each flask, shake again, and allow thelayers to separate. The organic solvent layers are the BlankPreparation and the Standard Preparation, and they contain0.0 and 2.0 �g of lead per milliliter, respectively.
Test Preparation Transfer 1.0 g of sample to a 50-mLbeaker, and cover it with a ribbed watch glass. Slowly add 8mL of hydrochloric acid and 2 mL of nitric acid, keeping thebeaker covered as much as possible. After the initial reactionsubsides, evaporate to dryness on a steam bath, cool, anddissolve the residue in 10 mL of 9 N hydrochloric acid,warming if necessary to effect solution. Cool, and transferthe resultant solution, with the aid of about 10 mL of water,into a 50-mL volumetric flask. Add 20 mL of Ascorbic Acid–Sodium Iodide Solution and 5 mL of Trioctylphosphine OxideSolution, shake for 30 s, and allow the layers to separate. Addwater to bring the organic solvent layer into the neck of theflask, shake again, and allow the layers to separate. The or-ganic solvent layer is the Test Preparation.
Procedure Concomitantly determine the absorbance ofthe Blank Preparation, the Standard Preparation, and theTest Preparation at the lead emission line at 283.3 nm, witha suitable atomic absorption spectrophotometer equipped witha lead hollow-cathode lamp and an air–acetylene flame, using4-methyl-2-pentanone to set the instrument to zero. In a suit-able analysis, the absorbance of the Blank Preparation is notgreater than 20% of the difference between the absorbanceof the Standard Preparation and the absorbance of the BlankPreparation. The absorbance of the Test Preparation doesnot exceed that of the Standard Preparation.Mercury Determine as directed in the monograph for Iron,Reduced, but use 2 g of sample and 40 mL of Sodium CitrateSolution in preparing the Sample Solution, and prepare theDiluted Standard Mercury Solution as follows: Transfer 4.0mL of Mercury Stock Solution into a 250-mL volumetric flask,dilute to volume with 1 N hydrochloric acid, and mix (1 mL =4 �g Hg). Modify the first sentence of the Procedure to read:‘‘Prepare a control by treating 1.0 mL of Diluted StandardMercury Solution (4 �g Hg) in the same manner. . . .’’Sieve Analysis Determine as directed under Sieve Analysisof Granular Metal Powders, Appendix IIC.
Packaging and Storage Store in well-closed containers.
Iron, ElectrolyticFe At wt 55.85
CAS: [7439-89-6]
DESCRIPTION
Iron, Electrolytic, occurs as an amorphous, lusterless, gray-black powder. It is elemental iron obtained by electrode posi-tion. It is stable in dry air.
FCC V Monographs / Iron, Reduced / 231
Function Nutrient.
REQUIREMENTS
Identification A sample dissolves in dilute mineral acidswith the evolution of hydrogen and the formation of solutionsof the corresponding salts, which give positive tests for Fer-rous Salts (Iron), Appendix IIIA.Assay Not less than 97.0% of Fe.Acid-Insoluble Substances Not more than 0.2%.Arsenic Not more than 3 mg/kg.Lead Not more than 4 mg/kg.Mercury Not more than 2 mg/kg.Sieve Analysis Not less than 100% passes through a 100-mesh sieve; not less than 95% passes through a 325-meshsieve.
TESTS
Assay Determine as directed in the monograph for Iron,Reduced.Acid-Insoluble Substances Dissolve 1 g of sample in 25mL of 2 N sulfuric acid, and heat on a steam bath until theevolution of hydrogen ceases. Filter through a tared filtercrucible, wash with water until free from sulfate, dry at 105°for 1 h, cool, and weigh.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using the following solution: Dissolve 1 g ofsample in 25 mL of 2 N sulfuric acid, heat on a steam bathuntil the evolution of hydrogen ceases, cool, and dilute to 35mL with water.Lead Determine as directed in the monograph for Iron,Carbonyl.Mercury Determine as directed in the monograph for Iron,Reduced, but use 2 g of sample and 40 mL of Sodium CitrateSolution in preparing the Sample Solution, and prepare theDiluted Standard Mercury Solution as follows: Transfer 4.0mL of Mercury Stock Solution into a 250-mL volumetric flask,dilute to volume with 1 N hydrochloric acid, and mix (1 mL =4 �g of Hg). Modify the first sentence of the Procedure toread: ‘‘Prepare a control by treating 1.0 mL of Diluted Stan-dard Mercury Solution (4 �g Hg) in the same manner. . . .’’Sieve Analysis Determine as directed under Sieve Analysisof Granular Metal Powders, Appendix IIC.
Packaging and Storage Store in well-closed containers.
Iron, Reduced
Fe At wt 55.85
CAS: [7439-89-6]
DESCRIPTION
Iron, Reduced, occurs as a gray-black powder. It is elementaliron obtained by a chemical process. It is lusterless or has
not more than a slight luster. When viewed under a microscopehaving a magnifying power of 100 diameters, it appears asan amorphous powder, free from particles having a crystallinestructure. It is stable in dry air.
Function Nutrient.
REQUIREMENTS
Identification A sample dissolves in dilute mineral acidswith the evolution of hydrogen and the formation of solutionsof the corresponding salts, which give positive tests for Fer-rous Salts (Iron), Appendix IIIA.Assay Not less than 96.0% of Fe.Acid-Insoluble Substances Not more than 1.25%.Arsenic Not more than 8 mg/kg.Lead Not more than 10 mg/kg.Mercury Not more than 5 mg/kg.Sieve Analysis Not less than 100% passes through a 100-mesh sieve.
TESTS
Assay Transfer about 200 mg of sample, accuratelyweighed, into a 300-mL Erlenmeyer flask, add 50 mL of 2N sulfuric acid, and close the flask with a stopper containinga Bunsen valve (made by inserting a glass tube connected toa short piece of rubber tubing with a slit on the side and aglass rod inserted in the other end and arranged so that gasescan escape but air cannot enter). Heat on a steam bath untilthe iron is dissolved, cool the solution, dilute it with 50 mLof recently boiled and cooled water, add 2 drops of orthophen-anthroline TS, and titrate with 0.1 N ceric sulfate until thered color changes to a weak blue. Each milliliter of 0.1 Nceric sulfate is equivalent to 5.585 mg of Fe.Acid-Insoluble Substances Dissolve 1.0 g of sample in 25mL of 2 N sulfuric acid, and heat on a steam bath until theevolution of hydrogen ceases. Filter through a tared filtercrucible, wash with water until free from sulfate, and dry at105° for 1 h. The weight of the residue does not exceed12.5 mg.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using the following solution: Dissolve 1.0 gof sample in 25 mL of 2 N sulfuric acid, heat on a steam bathuntil the evolution of hydrogen ceases, cool, and dilute to 35mL with water.Lead Determine as directed in the monograph for Iron,Carbonyl.Mercury
Dithizone Stock Solution Dissolve 30 mg of dithizone in1000 mL of chloroform, add 5 mL of alcohol, and mix. Storein a refrigerator in a dark bottle. Prepare fresh each month.
Dithizone Extraction Solution On the day of use, dilute30 mL of Dithizone Stock Solution to 100 mL with chloroform.
Hydroxylamine Hydrochloride Solution Dissolve 20 g ofhydroxylamine hydrochloride in sufficient water to makeabout 65 mL, transfer the solution into a separator, add a fewdrops of thymol blue TS, and then add ammonium hydroxideuntil a yellow color appears. Add 10 mL of 1:25 sodium
232 / Isobutane / Monographs FCC V
diethyldithiocarbamate solution, mix, and allow to stand for5 min. Extract the solution with successive 10- to 15-mLportions of chloroform until a 5-mL test portion of the chloro-form extract does not develop a yellow color when shakenwith a dilute cupric sulfate solution. Add 2.7 N hydrochloricacid until the extracted solution is pink, adding one or twomore drops of thymol blue TS, if necessary, then dilute to100 mL with water, and mix.
Mercury Stock Solution Transfer 33.8 mg of mercuricchloride, accurately weighed, into a 100-mL volumetric flask,dissolve in 1 N hydrochloric acid, dilute to volume with theacid, and mix. Each milliliter contains the equivalent of 250mg of mercury.
Diluted Standard Mercury Solution Transfer 2.0 mL ofMercury Stock Solution into a 100-mL volumetric flask, diluteto volume with 1 N hydrochloric acid, and mix. Each millilitercontains the equivalent of 5 mg of mercury.
Sodium Citrate Solution Dissolve 250 g of sodium citratedihydrate in 1000 mL of water.
Sample Solution Transfer 1 g of sample into a 250-mLbeaker, add 20 mL of 1:2 nitric acid, and digest on a steambath for about 45 min. Add 5 mL of 1:3 hydrochloric acid,and continue heating on the steam bath until the sample isdissolved. Cool to room temperature, and filter, if necessary,through a medium-porosity filter paper. Wash the paper witha few milliliters of water, add 20 mL of Sodium CitrateSolution and 1 mL of Hydroxylamine Hydrochloride Solutionto the filtrate, and adjust the pH to 1.8 with ammonium hy-droxide.
Procedure (Note: Because mercuric dithizonate is lightsensitive, this procedure should be performed in subduedlight.) Prepare a control by treating 1.0 mL of Diluted StandardMercury Solution (5 mg Hg) in the same manner and withthe same reagents as directed for the preparation of the SampleSolution. Transfer the control and the Sample Solution intoseparate 250-mL separators, and treat both solutions as fol-lows: Extract with 5 mL of Dithizone Extraction Solution,shaking the mixtures vigorously for 1 min. Drain carefully,collecting the chloroform in another separator. If the chloro-form does not show a pronounced green color caused byexcess reagent, add another 5 mL of the extraction solution,shake again, and drain into the separator. Continue the extrac-tion with 5-mL portions, if necessary, collecting each succes-sive extract in the second separator, until the final chloroformlayer contains dithizone in marked excess. Add 15 mL of 1:3hydrochloric acid, to the combined chloroform extracts, shakethe mixture vigorously for 1 min, and discard the chloroform.Extract with 2 mL of chloroform, drain carefully, and discardthe chloroform. Add 1 mL of 0.05 M disodium EDTA and 2mL of 6 N acetic acid to the aqueous layer. Slowly add 5 mLof 6 N ammonium hydroxide, and cool the separator. Transferthe solution into a 150-mL beaker, adjust the pH to 1.8 with6 N ammonium hydroxide or 1:10 nitric acid, using a pHmeter, and return the solution to the separator. Add 5.0 mLof Dithizone Extraction Solution, and shake vigorously for 1min. Allow the layers to separate, insert a plug of cotton intothe stem of the separator, and collect the dithizone extract ina test tube. Determine the absorbance of each solution in 1-
cm cells at 490 nm with a suitable spectrophotometer, usingchloroform as the blank. The absorbance of the Sample Solu-tion does not exceed that of the control.Sieve Analysis Determine as directed under Sieve Analysisof Granular Metal Powders, Appendix IIC.
Packaging and Storage Store in well-closed containers.
Isobutane
CH3CH(CH3)2
C4H10 Formula wt 58.12
CAS: [75-28-5]
DESCRIPTION
Isobutane occurs as a colorless, flammable gas. Its boilingtemperature is about −11°.
Function Propellant; aerating agent.
REQUIREMENTS
Caution: Isobutane is highly flammable and explosive.Perform sampling and analytical operations in a well-ventilated fume hood.
Identification
A. The infrared absorption spectrum exhibits maxima,among others, at about the following wavelengths, in �m:3.4 (vs), 6.8 (s), 7.2 (m), and 10.9 (m).
B. The vapor pressure of a test sample, obtained as directedin the Sampling Procedure (below) and determined at 21° bymeans of a suitable pressure gauge, is approximately 1600mm Hg (17 psi).Assay Not less than 95.0% of C4H10.Acidity of Residue Passes test.High-Boiling Residue Not more than 5 mg/kg.Sulfur Compounds Passes test.Water Not more than 10 mg/kg.
TESTS
Sampling Procedure Use a stainless steel sampling cylin-der equipped with a stainless steel valve and having a capacityof not less than 200 mL and a pressure rating of 240 psi ormore. Dry the cylinder with the valve open at 110° for 2 h,and evacuate the hot cylinder to less than 1 mm Hg. Closethe valve, and cool and weigh the cylinder. Tightly connectone end of a charging line to the Isobutane container, andloosely connect the other end to the sampling cylinder. Care-fully open the Isobutane container, and allow the Isobu-
FCC V Monographs / Isobutylene–Isoprene Copolymer / 233
tane to flush out the charging line through the loose connec-tion. Avoid excessive flushing that causes moisture to freezein the charging line and connections. Tighten the fitting onthe sampling cylinder, and open the sampling cylinder valve,allowing the Isobutane to flow into the evacuated cylinder.Continue sampling until the desired amount of sample isobtained, then close the Isobutane container valve, and finally,close the sampling cylinder valve.
Caution: Do not overload the sampling cylinder.
Weigh the charged sampling cylinder again, and calculate thesample weight.
Assay
Chromatographic System (See Chromatography, Appen-dix IIA.) Use a gas chromatograph equipped with a thermal-conductivity detector and a 6-m × 3-mm (id) aluminum col-umn, or equivalent, packed with 10 weight percent tetraethy-lene glycol dimethyl ether liquid phase, on a support ofcrushed firebrick (GasChrom R, or equivalent), which hasbeen calcined or burned with a clay binder above 900° andsilanized, or equivalent. Use helium as the carrier gas at arate of 50 mL/min. Maintain the column at 33°.
System Suitability The peak responses obtained for Isobu-tane in the chromatograms from duplicate determinationsagree within 1%.
Procedure Connect one Isobutane cylinder to the chroma-tograph through a suitable sampling valve and a flow controlvalve downstream from the sampling valve. Flush the liquidsample through the sampling valve, taking care to avoid trap-ping gas or air in the valve. Inject a suitable volume, typically2 �L, of Isobutane into the chromatograph, and record thechromatogram.
Calculation Calculate the purity of the sample using thefollowing formula:
(100 × B)/(x + y + z + . . .),
in which B is the sample response and x, y, z,. . . representthe sum of all the responses in the chromatogram.Acidity of Residue Add 10 mL of water to the residueobtained in High-Boiling Residue (below), mix by swirlingfor about 30 s, add 2 drops of methyl orange TS, insert thestopper in the tube, and shake vigorously. No pink or redcolor appears in the aqueous layer.High-Boiling Residue Prepare a cooling coil from coppertubing [about 6.1 m × 6 mm (od)] to fit into a suitable vacuum-jacketed flask. Immerse the cooling coil in a mixture of dryice and acetone in a vacuum-jacketed flask, and connect oneend of the tubing to a sampling cylinder (see Sampling Proce-dure, above). Carefully open the sampling cylinder valve,flush the cooling coil with about 50 mL of the liquified Isobu-tane, and discard this portion of liquid. Continue deliveringliquid from the cooling coil, and collect it in a previouslychilled 1000-mL sedimentation cone until the cone is filledto the 1000-mL mark (approximately 600 g). Allow the liquidto evaporate, using a warm water bath maintained at about40° to reduce evaporating time. When all of the liquid hasevaporated, rinse the sedimentation cone with two 50-mL
portions of pentane, and combine the rinsings in a tared, 150-mL evaporating dish. Transfer 100 mL of the pentane solventto a second tared, 150-mL evaporating dish, place both evapo-rating dishes on a water bath, evaporate to dryness, and heatthe dishes in an oven at 100° for 60 min. Cool the dishes ina desiccator, and weigh. Repeat the heating for 15-min periodsuntil successive weighings are within 0.1 mg. The weight ofthe residue obtained from the sample is the difference betweenthe weights of the residues in the two evaporating dishes.Calculate the milligrams per kilogram of high-boiling residuebased on a sample weight of 600 g.Sulfur Compounds Carefully open the container valve toproduce a moderate flow of gas. Do not direct the gas streamtoward the face, but deflect a portion of the stream towardthe nose. The gas is free from the characteristic odor of sulfurcompounds.Water Determine as directed under Water Determination,Appendix IIB, using the following modifications: (a) Providethe closed-system titrating vessel with an opening, and passthrough it a coarse-porosity gas dispersion tube connected toa sampling cylinder. (b) Dilute the reagent with anhydrousmethanol to give a water equivalence factor of between 0.2and 1.0 mg/mL; age this diluted solution for at least 16 hbefore standardization. (c) Obtain a 100-g sample as directedin the Sampling Procedure (above), and introduce the sampleinto the titration vessel through the gas dispersion tube at arate of about 100 mL of gas per minute; if necessary, heatthe sampling cylinder gently to maintain this flow rate.
Packaging and Storage Store in tight cylinders protectedfrom excessive heat.
Isobutylene–Isoprene Copolymer
Butyl Rubber
CAS: [9010-85-9]
DESCRIPTION
Isobutylene–Isoprene Copolymer is a synthetic copolymercontaining from 0.5 to 3.0 molar percent of isoprene, theremainder consisting of isobutylene. It is prepared by copoly-merization of isobutylene and isoprene in methyl chloridesolution, using aluminum chloride as the catalyst. After com-pletion of polymerization, the rubber particles are treated withhot water containing a suitable food-grade deagglomeratingagent, such as stearic acid. Finally, the coagulum is dried toremove residual volatiles.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple dissolved in hot toluene and evaporated on a potassium
View IR
234 / DL-Isoleucine / Monographs FCC V
bromide plate exhibits relative maxima at the same wave-lengths as those of a typical spectrum as shown in the sectionon Infrared Spectra, using the test conditions as specifiedtherein.Lead Not more than 3 mg/kg.Total Unsaturation Not more than 3.0 molar percent, asisoprene.
TESTS
Lead Determine as directed under Sample Solution for LeadLimit Test, Appendix IV.Total Unsaturation Determine as directed under Total Un-saturation, Appendix IV.
Packaging and Storage Store in well-closed containers.
DL-IsoleucineDL-2-Amino-3-methylvaleric Acid
OHH3C
H3C H
H NH2
O
C6H13NO2 Formula wt 131.17
CAS: [443-79-8]
DESCRIPTION
DL-Isoleucine occurs as a white, crystalline powder. It is solu-ble in water, and practically insoluble in alcohol and in ether.It melts with decomposition at about 292°. The pH of a 1:100aqueous solution is between 5.5 and 7.0. It is optically inactive.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H13NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 250 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 N
perchloric acid to the first appearance of a pure green coloror until the blue color disappears completely.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.12 mg of C6H13NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers.
L-IsoleucineL-2-Amino-3-methylvaleric Acid
OHH3C
H3C H
H NH2
O
C6H13NO2 Formula wt 131.17
CAS: [73-32-5]
DESCRIPTION
L-Isoleucine occurs as crystalline leaflets or as a white, crystal-line powder. It is soluble in 25 parts of water, slightly solublein hot alcohol, and soluble in diluted mineral acids and inalkaline solutions. It sublimes at between 168° and 170°, andmelts with decomposition at about 284°. The pH of a 1:100aqueous solution is between 5.5 and 7.0.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H13NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Optical (Specific) Rotation [�]D
20°: Between +38.6° and+41.5°, calculated on the dried basis; or [�]D
25°: Between+38.2° and +41.1°, calculated on the dried basis.Residue on Ignition Not more than 0.2%.
View IRView IR
FCC V Monographs / Juniper Berries Oil / 235
TESTS
Assay Dissolve about 250 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid, add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to the first appearance of a pure green coloror until the blue color disappears completely.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.12 mg of C6H13NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 4 g of previously dried sample in sufficient 6 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers.
Isopropyl Alcohol2-Propanol; Isopropanol
CH3CHOHCH3
C3H8O Formula wt 60.10
CAS: [67-63-0]
DESCRIPTION
Isopropyl Alcohol occurs as a clear, colorless, flammableliquid. It is miscible with water, with ethyl alcohol, with ether,and with many other organic solvents. Its refractive index at20° is about 1.377.
Function Extraction solvent.
REQUIREMENTS
Identification The refractive index at 20°, determined asdirected under Refractive Index, Appendix IIB, is about 1.377.Assay Not less than 99.7% of C3H8O, by weight.Acidity (as acetic acid) Not more than 10 mg/kg.Distillation Range Within a range of 1°, including 82.3°.Lead Not more than 1 mg/kg.Nonvolatile Residue Not more than 10 mg/kg.Solubility in Water Passes test.
Specific Gravity Not more than 0.7840.Substances Reducing Permanganate Passes test.Water Not more than 0.2%.
TESTS
Assay (See Chromatography, Appendix IIA.) Determine thecontent of propan-2-ol and volatile impurities using a suitablegas chromatograph equipped with flame-ionization detectorand a 1.8-m × 6-mm (id) steel column, or equivalent, packedwith 10% P.E.G. 400 on 60- to 80-mesh Chromosorb W (orequivalent). Maintain the column at 90°, and set both theinjection port temperature and the detector temperature to150°. Use helium as the carrier gas, with a flow rate of 45mL/min. Inject between 1-�L and 5-�L samples, and fromthe chromatograms so obtained, determine the content of eachconstituent by the method of area normalization.Acidity (as acetic acid) Add 2 drops of phenolphthalein TSto 100 mL of water, add 0.01 N sodium hydroxide to the firstpink color that persists for at least 30 s, then add 50 mL(about 39 g) of sample, and mix. Not more than 0.7 mL of0.01 N sodium hydroxide is required to restore the pink color.Distillation Range Determine as directed under DistillationRange, Appendix IIB.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Nonvolatile Residue Evaporate 125 mL (about 100 g) ofsample to dryness in a tared dish on a steam bath, dry theresidue at 105° for 30 min, cool, and weigh.Solubility in Water Mix 10 mL of sample with 40 mL ofwater. After 1 h, the solution is as clear as an equal volumeof water.Specific Gravity The specific gravity of a sample, deter-mined by any reliable method (see General Provisions), isnot greater than 0.7840 at 25°/25° (equivalent to 0.7870 at20°/20°).Substances Reducing Permanganate Transfer 50 mL ofsample into a 50-mL glass-stoppered cylinder, add 0.25 mLof 0.1 N potassium permanganate, mix, and allow to standfor 10 min: The pink color is not entirely discharged.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers, remotefrom fire.
Juniper Berries Oil
CAS: [8012-91-7]
DESCRIPTION
Juniper Berries Oil occurs as a colorless, faintly green oryellow liquid with a characteristic odor and an aromatic, bitter
View IR
236 / Kaolin / Monographs FCC V
taste. It is the volatile oil obtained by steam distillation fromthe dried ripe fruit of the plant Juniperus communis L. var.erecta Pursh (Fam. Cupressaceae). It is soluble in most fixedoils and in mineral oil. It is insoluble in glycerin and inpropylene glycol. The oil tends to polymerize during longstorage.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation Between −15° and 0°.Refractive Index Between 1.474 and 1.484 at 20°.Specific Gravity Between 0.854 and 0.879.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Kaolin
China Clay
CAS: [1332-58-7]
DESCRIPTION
Kaolin occurs as a fine, white to yellow-white or gray powderthat becomes darker when moistened. It is a purified clayconsisting mainly of alumina, silica, and water. It is insolublein water, in alcohol, in dilute acids, and in alkali solutions.
Function Anticaking agent.
REQUIREMENTS
Identification Mix 1 g of sample with 10 mL of water and5 mL of sulfuric acid in a porcelain dish, and evaporate untilthe water is removed. Continue heating until dense, whitefumes of sulfur trioxide evolve, then cool, and cautiously add20 mL of water. Boil for a few minutes, and filter. A gray
residue of silica remains on the filter. Add 6 N ammoniumhydroxide to a portion of the filtrate. A gelatinous, whiteprecipitate of aluminum hydroxide forms that is insoluble inan excess of 6 N ammonium hydroxide.Acid-Soluble Substances Not more than 2.0%.Arsenic Not more than 3 mg/kg.Carbonate Passes test.Iron Passes test.Lead Not more than 10 mg/kg.Loss on Ignition Not more than 15.0%.Sulfide Passes test.
TESTS
Acid-Soluble Substances Mix 1 g of sample with 20 mLof 2.7 N hydrochloric acid for 15 min, and filter. Evaporate10 mL of the filtrate to dryness in a tared dish, ignite gently,cool, and weigh the residue (R). Calculate the percent Acid-Soluble Substances by the formula
(2R × 100)/w,
in which w is the sample weight.
Sample Solution for the Determination of Arsenicand Lead Transfer 10.0 g of sample into a 250-mLflask, and add 50 mL of 0.5 N hydrochloric acid. Attacha reflux condenser to the flask, heat on a steam bathfor 30 min, cool, and let the undissolved material settle.Decant the supernatant liquid through Whatman No. 3filter paper, or equivalent, into a 100-mL volumetricflask, retaining as much as possible of the insolublematerial in the beaker. Wash the slurry and beakerwith three 10-mL portions of hot water, decanting eachwashing through the filter into the flask. Finally, washthe filter paper with 15 mL of hot water, cool the filtrateto room temperature, dilute to volume with water,and mix.
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using 10 mL of the Sample Solution (above).Carbonate Mix 1 g of sample with 10 mL of water, cool,and keep the mixture cool while adding 5 mL of sulfuric acid.No effervescence occurs during the addition of the acid.Iron Mix 2 g of sample with 10 mL of water in a mortar,and add 500 mg of sodium salicylate. No more than a lightred tint appears.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using 10 mL of the Sample Solution (above), and10 �g of lead (Pb) ion in the control.Loss on Ignition Ignite 2 g of sample, accurately weighed,in a tared crucible at 575° � 25° to constant weight, cool,and weigh.Sulfide Add 1 g of sample to 25 mL of water in a 250-mL flask, then add 15 mL of 2.7 N hydrochloric acid, andimmediately cover the top of the flask with filter paper moist-ened with lead acetate TS. Heat to boiling, and boil for severalminutes. The paper does not show any brown coloration.
Packaging and Storage Store in well-closed containers.
FCC V Monographs / Kelp / 237
Karaya Gum
Sterculia Gum
INS: 416 CAS: [9000-36-6]
DESCRIPTION
Karaya Gum occurs in tears of variable size or in broken,irregular pieces having a somewhat crystalline appearance.In this form, it is a pale yellow to pink-brown, translucentsubstance that is compact and homogeneous with a dull luster.It is sometimes admixed with a few darker fragments andoccasional pieces of bark. In the powdered form, it is lightto pink-gray. It is a dried, gummy exudation from Sterculiaurens Roxburgh and other species of Sterculia (Fam. Sterculi-aceae), or from Cochlospermum gossypium A. P. De Condolleor other species of Cochlospermum Kunth (Fam. Bixaceae).Karaya Gum is insoluble in alcohol, but it swells in water toform a gel.
Function Stabilizer; thickener; emulsifier.
REQUIREMENTS
Identification
A. Add 2 g of sample to 50 mL of water. The sample swellsto form a stiff, granular, slightly opalescent mucilage.
B. Add a few drops of Millon’s Reagent to a 1:100 aqueoussolution. A white, curdy precipitate forms.
C. The sample swells in 60% alcohol.Ash (Acid-Insoluble) Not more than 1.0%.Insoluble Matter Not more than 3.0%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 20.0%.Starch Passes test.Viscosity of a 1% Solution Not less than the minimum orwithin the range claimed by the vendor.
TESTS
Ash (Acid-Insoluble) Determine as directed under Ash(Acid-Insoluble), Appendix IIC.Insoluble Matter Transfer about 5 g of sample, accuratelyweighed, into a 250-mL Erlenmeyer flask, add a 1:1 mixtureof 2.7 N hydrochloric acid:water, cover the flask with a watchglass, and boil the solution gently until it loses its viscosity.Filter the solution through a tared filtering crucible, wash theresidue with water until the washings are free from acid, dryat 105° for 1 h, and weigh.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 4 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, using an unground sample that has beenpowdered until it passes through a No. 40 sieve. Mix wellbefore weighing, and dry at 105° for 5 h.
Starch Add a few drops of iodine TS to a 1:10 aqueoussolution. No blue color appears.Viscosity Transfer a 4-g sample, finely powdered, into thecontainer of a stirring apparatus equipped with blades capableof being adjusted to about 1000 rpm. Add 10 mL of alcoholto the sample, swirl to wet it uniformly, and then add 390mL of water, avoiding the formation of lumps. Stir the mixturefor 7 min, pour the resulting dispersion into a 500-mL bottle,insert a stopper, and allow to stand for about 12 h in a waterbath at 25°. Determine the apparent viscosity at this tempera-ture with a model LVF Brookfield, or equivalent, viscometer(see Viscosity of Cellulose Gum, Appendix IIB) using a suit-able spindle, speed, and factor.
Packaging and Storage Store in well-closed containers.
Kelp
DESCRIPTION
Kelp occurs as a dark green to olive brown, dry substance.It is the dehydrated seaweed obtained from the class Phaeo-phyceae (brown algae) of the genera Macrocystis (includingM. pyrifera and related species) and Laminaria (including L.digitata, L. cloustoni, and L. saccharina). The seaweed maybe chopped to provide coarse particles and/or it may be groundto provide a fine powder.
Function Nutrient (source of iodine).
REQUIREMENTS
Arsenic Not more than 1 mg/kg.Ash (Total) Not more than 45.0%.Iodine Content Between 0.1% and 0.5%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 13.0%.
TESTS
ArsenicDistillation-Reducing Solution Dissolve 36 g of ACS low-
arsenic ferrous chloride (FeCl2·4H2O) in 500 mL of 6.6 Nhydrochloric acid. Prepare fresh on the day of use.
Apparatus Refer to the figure Special Apparatus for theDetermination of Inorganic Arsenic (Arsenic Limit Test, Ap-pendix IIIB). Have all parts available for assembly during theProcedure.
Procedure Accurately weigh 2.00 g of sample that haspreviously been ground to pass through a 60-mesh screen,and transfer it to the distillation flask (A). Add 50 mL ofDistillation-Reducing Solution, connect the flask to the re-ceiver chamber (B), complete the assembly of the apparatus,and begin circulating tap water through the condenser (C).Half-fill the lower two bulbs of the splash head (D) with water.
238 / Konjac Flour / Monographs FCC V
Maneuver the stopcock to cause the contents of the receiverchamber to drain into the distillation flask, heat the flask untilthe temperature above the solution reaches 106° to 108°, andcontinue refluxing at this temperature for 45 min. Close thestopcock, continue heating at 108° to 110°, and collect 30 to33 mL of distillate in the receiver chamber. Remove theheating source and allow the temperature to drop to about 80°.
Drain the distillate from the receiver chamber into a 250-mL beaker that is contained in an ice-water bath. Close thestopcock, and add a second 50-mL portion of the Distillation-Reducing Solution through the thermometer opening to thedistillation flask. Replace the thermometer, increase the tem-perature to 108° to 110°, and collect a second 30- to 33-mLportion of distillate in the receiver chamber.
Drain the second distillate into the beaker containing thefirst portion, and continue cooling in the ice-water bath untilthe combined distillate cools to room temperature. Removethe splash head, and wash its contents into the beaker. Also,wash down the insides of the condenser and receiver chamberwith water, collecting the washings in the beaker. Filter thebeaker contents through a Whatman No. 40, or equivalent,filter paper, collecting the filtrate in a 300-mL Erlenmeyerflask having a 24/40 standard-taper joint, to be used later asan arsine generator flask. Wash the filter three times withwater so that the final volume of filtrate measures 200 mL.
Refer to the Arsenic Limit Test, Appendix IIIB. Add 2 mLof potassium iodide TS and 0.5 mL of Stannous ChlorideSolution to the Erlenmeyer flask, and continue as directed inthe Procedure, beginning with, ‘‘Allow the mixture to standfor 30 min at room temperature . . .’’ but use 6.0 mL, ratherthan 3.0 mL, of Standard Arsenic Solution in the preparationof the standard.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Iodine Content Transfer about 2 g of sample, accuratelyweighed, into a large porcelain crucible, and mix thoroughlywith 10 g of potassium carbonate. Place the sample in a mufflefurnace, starting with low heat, and then ignite at 500° to600° for 20 min or until combustion is complete. Dissolvethe ash in about 200 mL of boiling water, filter, and washthe filter paper with two 15-mL portions of boiling water,adding the washings to the filtrate. Cool to room temperature,neutralize to methyl red TS with approximately 20 mL of85% phosphoric acid diluted with 20 mL of water, and thenadd 5 mL in excess. Cool the reaction mixture on an ice bath,and add bromine TS (about 5 mL) until a permanent yellowcolor appears. Gently boil the solution to remove all freebromine, adding water if necessary to maintain a volume of200 mL or more. Boil for an additional 5 min after the brominecolor has completely disappeared. Add a few milligrams ofsalicylic acid, stir, and cool to about 20°. Add 1 mL of thediluted phosphoric acid solution and 5 mL of potassium iodideTS, and titrate immediately with 0.01 N sodium thiosulfate,using starch TS as the indicator. Each milliliter of 0.01 Nsodium thiosulfate is equivalent to 211.5 �g of iodine (I).Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.
Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 4 h.
Packaging and Storage Store in well-closed containers.
Konjac Flour
Konjac; Konnyaku; Konjac Gum; Yam Flour
CAS: [37220-17-0]
DESCRIPTION
Konjac Flour occurs as a cream to light tan powder. It is ahydrocolloidal polysaccharide obtained from the tubers ofvarious species of Amorphophallus. Konjac Flour is a high-molecular-weight, nonionic glucomannan primarily con-sisting of mannose and glucose at a respective molar ratio ofapproximately 1.6:1.0. It is a slightly branched polysaccharideconnected by �-1,4 linkages and has an average molecularweight of 200 to 2000 kDa. Acetyl groups along the glucoman-nan backbone contribute to solubility properties and are lo-cated, on average, every 9 to 19 sugar units. Konjac Flour isdispersible in hot or cold water and forms a highly viscoussolution with a pH between 4.0 and 7.0. Solubility is increasedby heat and mechanical agitation. Addition of mild alkali tothe solution results in the formation of a heat-stable gel thatresists melting, even under extended heating conditions.
Function Gelling agent; thickener; film former; stabilizer.
REQUIREMENTS
Identification
A. Microscopic Test Stain about 0.1 g of sample with0.01% methylene blue powder in 50% isopropyl alcohol, andobserve microscopically. The sample should have flattenedelliptical particles that are generally 100 to 500 �m in lengthalong the long axis. Unground Konjac Flour is clearly distin-guished from other hydrocolloids by the presence of saclikecells that contain glucomannan. The surface of these cellshas a reticulated structure. Particles of Konjac Flour are alsobirefringent under polarized light. These visual characteristicsmay remain even if the sample is finely ground, but they areless pronounced.
B. Gel Test At room temperature, add 5 mL of a 4%sodium borate solution to a 1% solution of sample in a testtube, and shake vigorously. A gel forms. (Konjac Flour solu-tions gel in the presence of sodium borate, similar in reactionto that of galactomannans such as guar gum and locustbean gum.)
C. Heat-Stable Gel Test Prepare a 2% solution of sampleby heating it in a boiling water bath for 30 min with continuousagitation and then cooling the solution to room temperature.
FCC V Monographs / Lactic Acid / 239
For each gram of sample used to prepare the 2% solution,add 1 mL of 10% potassium carbonate solution to the fullyhydrated sample at ambient temperature. Heat the mixture ina water bath to 85°, and hold quiescently for 2 h withoutagitation. The sample forms a thermally stable gel under theseconditions. (Related hydrocolloids such as guar gum and lo-cust bean gum do not form thermally stable gels and arenegative by this test.)Arsenic Not more than 3 mg/kg.Ash (Total) Not more than 5.0%.Carbohydrate (Total) Not less than 75.0%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 15.0%.Protein Not more than 8.0%.
TESTS
Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Carbohydrate (Total) The remainder, after subtractingfrom 100% the sum of the percentages of Ash (Total), Losson Drying, and Protein, represents the percentage of carbohy-drates (glucomannans) in the sample.Lead Determine as directed for Flame Atomic AbsorptionSpectrophotometric Method under the Lead Limit Test, Ap-pendix IIIB, using a 5-g sample and the Diluted StandardLead Solutions specified for a 1 mg/kg Lead Limit.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 5 h.Protein Determine as directed under Nitrogen Determina-tion, Appendix IIIC, using about 3.5 g of sample, accuratelyweighed, transferred into a 500-mL Kjeldahl flask. Determinethe percent of protein by the formula
%N × 5.7,
in which N is nitrogen and 5.7 is the conversion factor toprotein.
Packaging and Storage Store cool and dry in a closedcontainer away from direct heat and sunlight.
Labdanum Oil
CAS: [8016-26-0]
DESCRIPTION
Labdanum Oil occurs as a golden yellow, viscous liquid witha powerful, balsamic odor, which on dilution, is reminiscentof ambergris. It turns dark brown on standing. It is the volatileoil obtained by steam distillation from crude labdanum gumextracted from the perennial shrub Cistus ladaniferus L. (Fam.
Cistaceae). It is soluble in most fixed oils and in mineral oil,but it is insoluble in glycerin and in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Between 18 and 86.Angular Rotation Between +0°15’ and +7°.Ester Value Between 31 and 86.Refractive Index Between 1.492 and 1.507 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.905 and 0.993.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed in Ester Value underEsters, Appendix VI, using about 1 g of sample, accuratelyweighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 0.5 mL of 90% alcohol, but the solution usually becomesopalescent or turbid on further dilution.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Lactic Acid
�-Hydroxypropionic Acid; 2-Hydroxypropionic Acid
C3H6O3 Formula wt 90.08
INS: 270 CAS: L(+)-Lactic Acid [79-33-4]CAS: DL-Lactic Acid [598-82-3]
DESCRIPTION
Lactic Acid occurs as a colorless or yellow, syrupy liquidconsisting of a mixture of lactic acid (C3H6O3) and lactic acidlactate (C6H10O5). It is obtained by the lactic fermentation of
View IR
240 / Lactic Acid / Monographs FCC V
sugars or is prepared synthetically. It is usually available insolutions containing the equivalent of from 50% to 90% lacticacid. It is hygroscopic, and when concentrated by boiling, theacid condenses to form lactic acid lactate, 2-(lactoyloxy)pro-panoic acid, that on dilution and heating, hydrolyzes to LacticAcid. It is miscible with water and with alcohol.
Function Acidifier.
REQUIREMENTS
Labeling Indicate the concentration of Lactic Acid.Identification A sample gives positive tests for Lactate,Appendix IIIA.Assay Not less than 95.0% and not more than 105.0% ofthe labeled concentration of C3H6O3.Chloride Not more than 0.1%.Citric, Oxalic, Phosphoric, or Tartaric Acid Passes test.Cyanide Not more than 5 mg/kg.Iron Not more than 10 mg/kg.Lead Not more than 0.5 mg/kg.Residue on Ignition Not more than 0.1%.Sugars Passes test.Sulfate Not more than 0.25%.
TESTS
Assay Transfer an accurately weighed quantity of sample,equivalent to 3 g of Lactic Acid, into a 250-mL flask, add50.0 mL of 1 N sodium hydroxide, mix, and boil for 20 min.Add phenolphthalein TS, titrate the excess alkali in the hotsolution with 1 N sulfuric acid, perform a blank determination(see General Provisions), and make any necessary correction.Each milliliter of 1 N sodium hydroxide is equivalent to 90.08mg of C3H6O3.Chloride Determine as directed in the Chloride Limit Testunder Chloride and Sulfate Limit Tests, Appendix IIIB, dilut-ing 20 g of sample to 1000 mL with water and mixing thor-oughly. Any turbidity a 1.0-mL (20 mg of Lactic Acid) portionof this solution produces does not exceed that shown in acontrol containing 20 �g of chloride (Cl) ion.Citric, Oxalic, Phosphoric, or Tartaric Acid Dilute 1 gof sample to 10 mL with water, add 40 mL of calcium hydrox-ide TS, and boil for 2 min. The solution does not becometurbid.Cyanide
p-Phenylenediamine–Pyridine Mixed Reagent Dissolve200 mg of p-phenylenediamine hydrochloride in 100 mL ofwater, warming to effect solution. Cool, allow the solids tosettle, and use the supernatant liquid to make the mixed re-agent. Dissolve 128 mL of pyridine in 365 mL of water, add10 mL of hydrochloric acid, and mix. To prepare the mixedreagent, mix 30 mL of the p-phenylenediamine solution withall of the pyridine solution, and allow to stand for 24 h beforeusing. The mixed reagent is stable for about 3 weeks whenstored in an amber bottle.
Sample Solution Transfer an accurately weighed quantityof sample, equivalent to 20.0 g of Lactic Acid, into a 100-mL volumetric flask, dilute to volume with water, and mix.
Cyanide Standard Solution Dissolve 0.25 g of potassiumcyanide in 100 mL of 0.1 N sodium hydroxide. Transfer a 1-mL aliquot into a 100-mL volumetric flask, dilute to volumewith 0.1 N sodium hydroxide, and mix. Each milliliter of thissolution contains 10 �g of cyanide (CN).
Procedure Pipet a 10-mL aliquot of the Sample Solutioninto a 50-mL beaker. Pipet 1.0 mL of the Cyanide StandardSolution into a second 50-mL beaker, and add 10 mL of water.Place the beakers in an ice bath, and adjust the pH to between9 and 10 with 20% sodium hydroxide, stirring slowly andadding the reagent slowly to avoid overheating. Allow thesolutions to stand for 3 min, and then slowly add 10% phos-phoric acid to a pH between 5 and 6. Transfer the solutionsinto 100-mL separators containing 25 mL of cold water, andrinse the beakers and pH meter electrodes with a few millilitersof cold water, collecting the washings in the respective separa-tors. Add 2 mL of bromine TS, stopper, and mix. Add 2 mLof 2% sodium arsenite solution, stopper, and mix. Add 10mL of n-butanol to the clear solutions, stopper, and mix.Finally, add 5 mL of p-Phenylenediamine–Pyridine MixedReagent, mix, and allow to stand for 15 min. Remove anddiscard the aqueous phases, and filter the alcohol phases into10-mm cells. The absorbance of the Sample Solution, deter-mined at 480 nm with a suitable spectrophotometer, is nogreater than that of the Cyanide Standard Solution.Iron Add 2 mL of 1:20 hydrochloric acid to the ash obtainedin the test for Residue on Ignition (below), and evaporate todryness on a steam bath. Dissolve the residue in 1 mL ofhydrochloric acid, dilute to 40 mL with water, and add about40 mg of ammonium persulfate crystals and 10 mL of ammo-nium thiocyanate TS. Any red or pink color does not exceedthat produced by 2.0 mL of Iron Standard Solution (20 �gFe) in an equal volume of solution containing the quantitiesof reagents used in the test.Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample. Save theash for the test for Iron (above).Sugars Add 5 drops of sample to 10 mL of hot alkalinecupric tartrate TS. No red precipitate forms.Sulfate Determine as directed in the Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB, diluting 10g of sample to 100 mL with water, and mixing thoroughly.Any turbidity produced by a 1.6-mL (160 mg of Lactic Acid)portion of this solution does not exceed that shown in a controlcontaining 400 �g of sulfate (SO4) ion.
Packaging and Storage Store in tight containers.
FCC V Monographs / Lactylated Fatty Acid Esters of Glycerol and Propylene Glycol / 241
Lactose4-O-�-Galactopyranosyl-D-glucose
O
CH2OH
OH
OH
HO
O
CH2OH
OH
OH
O
CH
(α-Lactose)
C12H22O11 Formula wt, anhydrous 342.30C12H22O11·H2O Formula wt, monohydrate 360.32
CAS: anhydrous [63-42-3]CAS: monohydrate [5989-81-1]
DESCRIPTION
Lactose occurs as a white to creamy white, crystalline powder.It is normally obtained from whey. It may be anhydrous,contain one molecule of water of hydration, or contain amixture of both forms if it has been prepared by a spray-drying process. It is soluble in water, very slightly soluble inalcohol, and insoluble in chloroform and in ether.
Function Nutritive sweetener; processing aid; humectant(anhydrous form); texturizer.
REQUIREMENTS
Labeling Indicate whether it is anhydrous or the monohy-drate or a mixture of both forms if it has been prepared bya spray-drying process.Identification Add 5 mL of 1 N sodium hydroxide to 5 mLof a hot, saturated solution of sample, and gently warm themixture. The liquid turns yellow and, finally, brown-red. Coolto room temperature, and add a few drops of alkaline cuprictartrate TS. A red precipitate of cuprous oxide forms.Assay Not less than 98.0% and not more than 100.5% ofC12H22O11, calculated on the dried basis.Arsenic Not more than 0.5 mg/kg.Lead Not more than 0.5 mg/kg.Loss on Drying Monohydrate and spray-dried mixture: Notless than 4.5% and not more than 5.5%; Anhydrous: Not morethan 1.0%.pH Not less than 4.5 and not more than 7.5, in a 10%aqueous solution.Residue on Ignition Not more than 0.3%.
TESTS
Assay Determine as directed under Lactose, Appendix X.Transfer about 2 g of sample, accurately weighed, to a 100-mL volumetric flask. Add 10 mL of fructose internal standardsolution, dilute to volume with water, and mix. Perform theanalysis within 24 h.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 4 g of sample in 35 mLof water, and 2.0 mL of Standard Arsenic Solution in thecontrol (2 �g As).Lead Determine as directed for Method I in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 5-g sample.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a 2-g sample at 120° for 16 h.pH
Sample Preparation Transfer 10 g of sample, accuratelyweighed, into a clean, dry 100-mL Erlenmeyer flask, and add90 mL of recently boiled water set at 25°. Shake until theparticles are evenly suspended and the mixture is free oflumps. Heat the sample to boiling, and shake frequently toaid dissolution. Let the suspension stand for 10 min, decantthe supernate into the hydrogen-ion vessel, and quickly coolto 25°.
Procedure Determine as directed under pH Determina-tion, Appendix IIB, using pH 4.01 and 9.18 buffer solutionsto standardize the pH meter.Residue on Ignition Determine as directed in Method Iunder Residue on Ignition, Appendix IIC, igniting a 2-gsample.
Packaging and Storage Store in well-closed containersprotected from humidity.
Lactylated Fatty Acid Esters of Glyceroland Propylene Glycol
Propylene Glycol Lactostearate
INS: 478
DESCRIPTION
Lactylated Fatty Acid Esters of Glycerol and Propylene Glycoloccur as a substance that varies in consistency from a softsolid to a hard, waxy solid. They are a mixture of partiallactic and fatty acid esters of propylene glycol and glycerinproduced by the lactylation of a product obtained by reactingedible fats or oils with propylene glycol. They are dispersiblein hot water, and are moderately soluble in hot isopropanol,in chloroform, and in soybean oil.
Function Emulsifier; stabilizer; whipping agent; plasticizer.
242 / Lactylated Fatty Acid Esters of Glycerol and Propylene Glycol / Monographs FCC V
REQUIREMENTS
Identification Place about 150 mg of melted sample into a16- × 125-mm tube equipped with a screw cap having a Teflonliner, and add 4 mL of absolute methanol, 4 drops of a 25%sodium methoxide solution in absolute methanol, and a boilingchip. Cap the tube, reflux for 15 min, and cool to roomtemperature. Extract as follows: Add 8 drops of a 15% potas-sium acid sulfate solution, 4 mL of water, and 4 mL of n-hexane; cap the tube; shake for 1 min; and centrifuge for 30to 60 s. Decant and discard the n-hexane layer, and repeatthe extraction with three additional 4-mL portions of n-hexane,discarding each extract. Transfer the aqueous alcoholic phasefrom the tube into a 50-mL round-bottom, glass-stopperedflask; place the flask in a water bath at 50° to 55°; andevaporate to near dryness (about 0.5 mL of residue) in a rotaryfilm evaporator under full water aspirator vacuum.
Caution: Do not heat above 55°.
Remove the flask from the evaporator, add 1 mL of a 1:1solution of 0.5 N hydrochloric acid:methanol, swirl for severalminutes, and decant the clear solution into a small flask. Injecta portion of this solution into a suitable gas chromatograph,or equivalent, and obtain the chromatogram. (See Chromatog-raphy, Appendix IIA.) Use a gas chromatograph equippedwith a flame-ionization detector and a 1.8-m × 3-mm (id)column packed with 80- to 100-mesh Porapak Q (ethylvinyl-benzene–divinylbenzene polymer porous beads), or equiva-lent. Maintain the column at 175° to 210°, heated at a rateof 4°/min, and held at 210° until the glycerin is eluted. Setthe inlet port temperature to 310° and the detector to 385°.Use helium as the carrier gas, with a flow rate of 50 mL/min.Use a recorder with a range of 0 to 1 mV and a 1-s full-scale deflection at a chart speed of 6.5 mm/min. From thechromatogram so obtained, identify the peaks by their relativepositions on the chart. The major peaks, representing propyl-ene glycol, methyl lactate, lactic acid, and glycerin, in theorder listed, may be identified with suitable reference sub-stances. Major peaks may also be identified by their relativeretention times using a suitable internal standard.Acid Value Not more than 12.0.Lead Not more than 2 mg/kg.Water-Insoluble Combined Lactic Acid Between 14.0%and 18.0%.
The following specifications should conform to the representa-tions of the vendor: Free Glycerin, Free Lactic Acid, 1-Mono-glyceride Content, Total Lactic Acid, and Water.
TESTS
Acid Value Determine as directed in Method II under AcidValue, Appendix VII.Free Glycerin Determine as directed under Free Glycerinor Propylene Glycol, Appendix VII.Free Lactic Acid Transfer about 15 g of sample, accuratelyweighed, into a beaker, dissolve it in about 75 mL of benzene,and transfer the solution into a 500-mL glass-stoppered gradu-ate. Wash the beaker with about 125 mL of benzene in dividedportions, adding the washings to the graduate. Add 200 mL
of water to the graduate, and shake vigorously for 1 min.After 125 mL or more of the aqueous phase has separated,pipet 100.0 mL of the aqueous phase into an Erlenmeyerflask, add 1 mL of phenolphthalein TS, and titrate with 0.5N sodium hydroxide to the first appearance of a slight pinkcolor. Calculate the percentage of free lactic acid in the sampleby the formula
[45.04 × V × N]/(0.5 × W),
in which 45.04 is the equivalence factor for lactic acid; V isthe volume, in milliliters, of 0.5 N sodium hydroxide required;N is the exact normality of the sodium hydroxide solution;and W is the weight, in grams, of the sample.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.1-Monoglyceride Content Determine as directed under 1-Monoglycerides, Appendix VII.Total Lactic Acid Transfer about 3 g of sample, accuratelyweighed, into a 250-mL glass-stoppered flask, pipet 50.0 mLof 0.7 N alcoholic potassium hydroxide into the flask, attachan air condenser, and boil gently on a steam bath for 30 minor until the sample is completely saponified. Remove the flaskfrom the steam bath, immediately remove the air condenser,and allow the solution to cool until it begins to gel. Add 75.0mL of 0.5 N hydrochloric acid, mix, and transfer the solutioninto a 500-mL separator, washing the flask with two 15-mLportions of water and adding them to the solution in theseparator. Cool to 35° or lower, and extract with 100 mL ofdiethyl ether. Transfer the aqueous layer into a second 500-mLseparator, and wash the ether layer with two 20-mL portions ofwater, adding the wash water to the original aqueous phasein the second separator. Retain the ether solution. Extract theaqueous phase with a second 100-mL portion of diethyl ether,and transfer the aqueous phase into a 500-mL Erlenmeyerflask. Combine and wash the ether extracts with five 20-mLportions of water, and add the wash water to the flask. Add1 mL of phenolphthalein TS to the combined aqueous phasesin the Erlenmeyer flask, and titrate with 0.5 N sodium hydrox-ide to the first appearance of a slight pink color. Performa blank determination (see General Provisions), make anynecessary correction, and calculate the percent of total lacticacid in the sample taken by the formula
[45.04 × (S − B) × N]/W,
in which 45.04 is the equivalence factor for lactic acid; S −B represents the difference, in milliliters, between the volumesof 0.5 N sodium hydroxide required for the sample and forthe blank, respectively; N is the exact normality of the sodiumhydroxide solution; and W is the weight, in grams, of thesample.Water Determine as directed under Water Determination,Appendix IIB.Water-Insoluble Combined Lactic Acid Transfer about 3g of sample, accurately weighed, into a 250-mL separatorwith the aid of 100 mL of benzene, and wash with three 30-mL portions of water, discarding the washings. Transfer thebenzene layer into a 250-mL glass-stoppered Erlenmeyerflask, wash the separator with a few milliliters of benzene,
FCC V Monographs / Lactylic Esters of Fatty Acids / 243
and completely evaporate the combined benzene solution todryness. Pipet 50.0 mL of 0.7 N alcoholic potassium hydroxideinto the flask, attach an air condenser, boil gently on a steambath for 30 min or until the sample is completely saponified,and remove the flask from the steam bath. Immediately re-move the air condenser, and allow the solution to cool untilit begins to jell. Add 75.0 mL of 0.5 N hydrochloric acid, mix,and transfer the solution into a 500-mL separator, washing theflask with two 15-mL portions of water. Cool to 35° or lower,and extract with 100 mL of diethyl ether. Transfer the waterlayer to a second 500-mL separator, and wash the diethylether with two 20-mL portions of water, adding the washwater to the original aqueous phase in the second separator.Retain the ether solution. Extract the aqueous phase with asecond 100-mL portion of diethyl ether, and transfer the aque-ous phase to a 500-mL Erlenmeyer flask. Combine and washthe ether extracts with five 20-mL portions of water, and addthe wash water to the flask. Add 1 mL of phenolphthaleinTS to the combined aqueous phases in the flask, and titratewith 0.5 N sodium hydroxide to the first appearance of aslight pink color. Perform a blank determination (see GeneralProvisions), make any necessary correction, and calculate thepercent of water-insoluble combined lactic acid in the sampletaken by the formula
[45.04(S − B)(N)]/W,
in which 45.04 is the equivalence factor for lactic acid; S −B represents the difference, in milliliters, between the volumesof 0.5 N sodium hydroxide required for the sample and forthe blank, respectively; N is the exact normality of the sodiumhydroxide solution; and W is the weight, in grams, of thesample.
Packaging and Storage Store in well-closed containers.
Lactylic Esters of Fatty Acids
DESCRIPTION
Lactylic Esters of Fatty Acids occur as liquids to hard, waxysolids. They are mixed fatty acid esters of lactic acid and itspolymers, with minor quantities of free lactic acid, polylacticacid, and fatty acids. They are dispersible in hot water andare soluble in organic solvents and in vegetable oils.
Function Emulsifier; surface-active agent.
REQUIREMENTS
IdentificationA. Take 1 mL of the solution obtained in the test for
Total Lactic Acid (below) after titrating with 0.1 N potassiumhydroxide, and transfer it into a 25-mL glass-stoppered testtube. Add 0.1 mL of cupric sulfate solution (1 g of Cu-
SO4·5H2O in 25 mL of water) and 6 mL of sulfuric acid, andmix. Stopper loosely, heat in a boiling water bath for 5 min,then cool in an ice bath for 5 min, and remove from the bath.Add 0.1 mL of p-phenylphenol TS, mix, allow to stand atroom temperature for 1 min, and then heat in a boiling waterbath for 1 min. A deep, blue-violet color indicates the presenceof lactic acid.
B. Apparatus (See Chromatography, Appendix IIA.) As-semble a suitable apparatus for ascending thin-layer chroma-tography. Prepare a slurry of chromatographic silica gel con-taining about 13% of calcium sulfate (1 g to each 2 mL ofwater) as the binder, apply a uniformly thin layer to glassplates of convenient size, dry in air for 10 min, and activateby drying at 100° for 1 h. Store the cool plates in a clean,dry place until ready for use.
Sample Solution Transfer 1 g of sample into a 10-mLvolumetric flask and dissolve in and dilute to volume withhexane.
Stearic Acid Solution Transfer 250 mg of stearic acid intoanother 10-mL volumetric flask, and dissolve in and diluteto volume with hexane.
Procedure Spot 2 �L of the Sample Solution and 1 �Lof the Stearic Acid Solution approximately 1.5 cm from thebottom of the plate, allow the spots to dry, and then placethe plate in a suitable chromatographic chamber containinga 4:4:92 (v/v) mixture of acetone:glacial acetic acid:hexane.Develop by ascending chromatography until the solvent fronttravels 15 cm beyond the sample spot. Remove the plate fromthe chamber, dry thoroughly in air, and spray evenly witha saturated solution of chromium trioxide in sulfuric acid.Immediately place the sprayed plate on a hot plate in a hood,heat it to about 200°, char until white fumes of sulfur trioxidecease to evolve, and cool to room temperature. The spots fromthe Sample Solution are located according to the following Rf
values: Stearic Acid: 1.00; Fatty Acid: 1.00; Acylated Mono-lactic Acid: 0.84; Acetylated Dilactic Acid: 0.76; AceylatedTrilactic Acid: 0.68; and Tetralactic Acid: 0.62.Lead Not more than 2 mg/kg.
The following specifications should conform to the representa-tions of the vendor: Acid Value, Acylated Monolactic Acid,Acylated Polylactic Acid, Free Fatty Acids, Total Lactic Acid,Saponification Value, and Water.
TESTS
Assay for Acylated Monolactic Acid, Acylated PolylacticAcid, and Free Fatty Acids Transfer about 100 mg of sam-ple into a small, conical flask fitted with a suitable refluxcondenser. Add 5.0 mL of a solution prepared by dissolving14 g of boron trifluoride in methanol to make 100 mL (acommercial reagent, 14% w/v, may be used; Applied Science,or equivalent). Swirl to mix, and reflux for 15 min. Cool,transfer the reaction mixture with the aid of 10 mL of chro-matographic-grade hexane to a 60-mL separator, and add 10mL of water and 10 mL of saturated sodium chloride solution.Shake, allow the mixture to separate, then drain and discardthe lower, aqueous layer. Pass the hexane layer through 6 gof anhydrous sodium sulfate into a suitable flask. Inject 0.5
244 / Lactylic Esters of Fatty Acids / Monographs FCC V
to 2.0 �L of the hexane solution obtained into a suitablegas chromatographic apparatus, or equivalent, using a 10-�Lcapacity Hamilton fixed needle, or equivalent. (See Chroma-tography, Appendix IIA.) Use a gas chromatograph equippedwith a flame-ionization detector and a 1.2-m × 6.3-mm (id)column packed with 20% SE-30 or SE-52, or equivalentgrades of silicone rubber gums, on Chromosorb P or W orDiatoport S, or equivalent grades of diatomaceous material.Maintain the column between 150° and 310°, heated at a rateof 4°/min. Set the inlet port temperature to 335° and thedetector to 315°. Use helium as the carrier gas at a flow rateof about 54 mL/min throughout the determination. Use arecorder that has an attenuator switch, a range of 0- to 1-mV,and a 1-s full-scale deflection at a chart speed of 12.7 mm/min. Adjust the sample size so that the major peak is notattenuated more than ×8. From the chromatogram so obtained,identify the peaks by their relative position on the chart. Theesters, appearing in the order of increasing number of carbonatoms in the fatty acid and in order of increasing length ofthe polymer, are eluted as follows:
T myristateT palmitateT stearateT palmitoyl lactylate (2-palmitoyloxypropionate)T stearoyl lactylate (2-stearoyloxypropionate)T palmitoyl lactoyl lactylateT stearoyl lactoyl lactylateT palmitoyl dilactoyl lactylateT stearoyl dilactoyl lactylateT palmitoyl trilactoyl lactylateT stearoyl trilactoyl lactylateT palmitoyl tetralactoyl lactylate
Other esters may be determined by interpolation of a conven-tional carbon number-retention plot.
Determine the composition of the sample, using the areanormalization method, by the formula
%i = 100Ai/∑(Ai + . . . + An),
in which i represents the component of interest, Ai is theequalized area for the component of interest, and ∑(Ai + . . .+ An) is the sum of the equalized areas.
If free and polylactic acids are present, as determined below,the results should be corrected by multiplying %i by [(100 −% free and polylactic acid)/100].Acid Value Determine as directed in Method II under AcidValue, Appendix VII.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIB, using a 10-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII.Total Free and Polylactic Acids Transfer about 500 mgof sample, previously melted and accurately weighed, into a50-mL glass-stoppered separator with the aid of 15 mL ofhexane, and add 10 mL of water. Invert the separator tentimes, and allow it to stand until the layers have separated.Filter the aqueous layer through a plug of glass wool into a125-mL flask, wash the hexane with two 10-mL portions of
water, and combine the aqueous layers. Add 5.0 mL of 0.1N sodium hydroxide to the flask, and then heat the flask ona steam bath for 15 min under a nitrogen atmosphere. Titratewith 0.1 N hydrochloric acid, using phenolphthalein TS asthe indicator, to the disappearance of the pink color. Conducta blank determination (see General Provisions), using 30 mLof water and 5.0 mL of 0.1 N sodium hydroxide, and makeany necessary correction. Calculate the percent of free andpolylactic acids in the sample by the formula
[(B − S) × 9.008]/W,
in which B − S represents the difference, in milliliters, betweenthe volumes of 0.1 N hydrochloric acid required for the blankand for the sample, respectively; 9.008 is an equivalence factorfor the lactic acid; and W is the weight, in grams, of thesample.Total Lactic Acid Transfer an accurately weighed portionof melted sample, equivalent to between 140 mg and 170 mgof lactic acid, into a 250-mL Erlenmeyer flask. Pipet 20 mLof 0.5 N alcoholic potassium hydroxide into the flask, connectan air condenser, at least 65 cm long, and reflux for 30 min.Run a blank determination (see General Provisions) usingthe same volume of 0.5 N alcoholic potassium hydroxide. Add20 mL of water to each flask, then disconnect the condensers,evaporate to a volume of about 20 mL, and cool to about 40°.Add methyl red TS to each flask, and titrate the blank with0.5 N hydrochloric acid. While swirling the sample flask, addexactly the same volume of 0.5 N hydrochloric acid. Add 50mL of hexane to each flask. Swirl the sample flask vigorouslyto dissolve the fatty acids, then quantitatively transfer thecontents of each flask into separate 250-mL separators, andshake for 30 s. Collect the aqueous phases in 300-mL Erlen-meyer flasks, wash the hexane solutions with 50 mL of water,and combine the wash solution with the original aqueousphases in the Erlenmeyer flasks, discarding the hexane solu-tion. Titrate with 0.1 N potassium hydroxide, using phenol-phthalein TS as the indicator, to a pink color that persists forat least 30 s. Calculate the percent of total lactic acid by theformula
[9.008(S − B)(N)]/W,
in which 9.008 is the equivalence factor for lactic acid; (S −B) is the difference, in milliliters, between the volumes of 0.1N potassium hydroxide required for the sample and for theblank, respectively; N is the exact normality of the potassiumhydroxide solution; and W is the weight, in grams, of thesample.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight, plastic-lined contain-ers in a cool, dry place.
FCC V Monographs / Lard (Unhydrogenated) / 245
Lanolin, Anhydrous
Wool Fat
INS: 913 CAS: [8006-54-0]
DESCRIPTION
Lanolin, Anhydrous, occurs as a purified, yellow-white, semi-solid, fatlike substance. It is extracted from the wool of sheep.It is insoluble in water, but mixes with about twice its weightof water without separation. It is soluble in chloroform andin ether.
Function Masticatory substance in chewing gum base.
REQUIREMENTS
Acid Value Not more than 1.12.Iodine Value Between 18 and 36.Lead Not more than 3 mg/kg.Loss on Heating Not more than 0.5%.Melting Range Between 36° and 42°.
TESTS
Acid Value Determine as directed for Method I under AcidValue, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Proceed as directed under Sample Solution for LeadLimit Test, Appendix IV.Loss on Heating Heat a 5-g sample on a steam bath, withfrequent stirring, to constant weight.Melting Range Determine as directed under Melting Rangeor Temperature, Appendix IIB.
Packaging and Storage Store in well-closed containers,preferably at a temperature not exceeding 30°.
Lard (Unhydrogenated)
DESCRIPTION
Lard (Unhydrogenated) is an off white fat obtained by dryor wet (steam) rendering of fresh fatty porcine tissues (cuttingsand trimmings) shortly after slaughtering. Rendered Lard maybe bleached, or bleached and deodorized. It is soft to semisolidat 27° and melts completely at 42°.
Rendered, Bleached, and Bleached-Deodorized lards areoff white semisolids at 21° to 27°. Bleached, and Bleached-Deodorized lards, which are pale yellow and clear at 54°,
differ from Rendered Lard, which is pale yellow, clear tohazy, and may contain extraneous matter.
Function Coating agent; texturizer.
SPECIFIC REQUIREMENTS
Rendered Bleached Bleached-Lard Lard Deodorized Lard
Color (AOCS- Not more Not more Not more thanWesson) than 3.0 red than 1.5 red 1.5 red
Free Fatty Acids Not more Not more Not more than(as oleic acid) than 1.0% than 1.0% 0.1%
Hexane- Not more Not more Not more thanInsoluble Matter than 0.1% than 0.05% 0.05%
Iodine Value Between 46 Between 46 Between 46 andand 70 and 70 70
Unsaponifiable Not more Not more Not more thanMatter than 1.5% than 1.5% 1.5%
Water Not more Not more Not more thanthan 0.5% than 0.1% 0.1%
GENERAL REQUIREMENTS
Identification Unhydrogenated Lard exhibits the followingcomposition profile of fatty acids, determined as directedunder Fatty Acid Composition, Appendix VII:
Fatty Acid: <14:0 14:0 14:1 15:0 16:0Weight % (Range): <0.5 0.5–2.5 0.2 <0.1 20–32Fatty Acid: 16:1 17:0 17:1 18:0 18:1Weight % (Range): 1.7–5 <1.0 <0.7 5.0–24 35–62Fatty Acid: 18:2 18:3 20:0 20:1Weight % (Range): 3.0–16 <2.0 <1.0 <1.0
Lead Not more than 0.1 mg/kg.Peroxide Value Not more than 10 meq/kg.
TESTS
Color (AOCS-Wesson) Determine as directed under Color(AOCS-Wesson), Appendix VII.Free Fatty Acids (as oleic acid) Determine as directed underFree Fatty Acids, Appendix VII, using the following equiva-lence factor (e) in the formula given in the procedure:
Free fatty acids as oleic acid, e = 28.2.
Hexane-Insoluble Matter If the sample is plastic or semi-solid, soften a portion by warming it at a temperature notexceeding 60°, and then mix it thoroughly. Transfer 100 g ofwell-mixed sample into a 1500-mL wide-mouth Erlenmeyerflask, add 1000 mL of solvent hexane, and shake until thesample is dissolved. Filter the resulting solution through a600-mL Corning ‘‘C’’ porosity, or equivalent, filtering funnelthat previously has been dried at 105° for 1 h, cooled in adesiccator, and weighed. Wash the flask with two successive250-mL portions of solvent hexane, and pass the washingsthrough the filter. Dry the funnel at 105° for 1 h, cool to room
246 / Laurel Leaf Oil / Monographs FCC V
temperature in a desiccator, and weigh. From the gain inweight of the funnel, calculate the percentage of the hexane-insoluble matter in the sample.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB, using a 3-g sample.Peroxide Value Accurately weigh about 10 g of sample,add 30 mL of a 3:2 mixture of glacial acetic acid:chloroform,and mix. Add 1 mL of a saturated solution of potassiumiodide, mix the solution for 1 min, add 100 mL of water, andimmediately begin titrating with 0.05 N sodium thiosulfate,adding starch TS as the endpoint is approached, and continuethe titration until the blue starch color has just disappeared.Perform a blank determination (see General Provisions), andmake any necessary correction. Calculate the peroxide value,as milliequivalents of peroxide per kilogram of sample, bythe formula
[S × N × 1000]/W,
in which S is the net volume, in milliliters, of sodium thiosul-fate solution required for the sample; N is the exact normalityof the sodium thiosulfate solution; and W is the weight, ingrams, of the sample taken.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB; however, in the Procedure, use 50 mL ofchloroform in place of 35 to 40 mL of methanol to dissolvethe sample.
Packaging and Storage Store in well-closed containers.
Laurel Leaf Oil
Bay Leaf Oil
CAS: [8006-78-8]
DESCRIPTION
Laurel Leaf Oil occurs as a light yellow to yellow liquidwith an aromatic, spicy odor. It is the oil obtained by steamdistillation from the leaves of Laurus nobilis L. (Fam. Lau-raceae). It is soluble in most fixed oils, and it is solublewith cloudiness in mineral oil and in propylene glycol. It isinsoluble in glycerin.
Note: The oil from Laurus nobilis L. should not beconfused with that of the West Indian bay tree or theCalifornia bay laurel.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as those
of a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Acid Value Not more than 3.0.Angular Rotation Between −10° and −19°.Refractive Index Between 1.465 and 1.470 at 20°.Saponification Value Between 15 and 45.Saponification Value after Acetylation Between 36 and85.Solubility in Alcohol Passes test.Specific Gravity Between 0.905 and 0.929.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed in Saponifica-tion Value under Esters, Appendix VI, using about 5 g ofsample, accurately weighed.Saponification Value after Acetylation Proceed as di-rected under Total Alcohols, Appendix VI, using about 2.5 gof acetylated oil, accurately weighed. Calculate the saponifica-tion value by the formula
28.05 × A/B,
in which A is the number of milliliters of 0.5 N alcoholicpotassium hydroxide consumed in the titration and B is theweight, in grams, of the acetylated oil.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 1 mL of 80% alcohol and remains in solution upon dilutionto 10 mL.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Lauric AcidDodecanoic Acid
CH3(CH2)10COOH
C12H24O2 Formula wt 200.32
CAS: [143-07-7]
DESCRIPTION
Lauric Acid occurs as a white or faintly yellow, somewhatglossy, crystalline solid or powder. It is obtained from coconut
View IR
FCC V Monographs / Lavender Oil / 247
oil and other plant fats. It is practically insoluble in water,but is soluble in alcohol, in chloroform, and in ether.
Function Component in the manufacture of other food-grade additives; defoaming agent.
REQUIREMENTSAcid Value Between 252 and 287.Iodine Value Not more than 3.0.Lead Not more than 0.1 mg/kg.Residue on Ignition Not more than 0.1%.Saponification Value Between 253 and 287.Titer (Solidification Point) Between 26° and 44°.Unsaponifiable Matter Not more than 0.3%.Water Not more than 0.2%.
TESTS
Acid Value Determine as directed in Method I under AcidValue, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed for Method II in the AtomicAbsorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test, Appendix IIIB.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 10-g sample.Saponification Value Determine as directed under Saponi-fication Value, Appendix VII, using about 3 g of sample,accurately weighed.Titer (Solidification Point) Determine as directed under So-lidification Point, Appendix IIB.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Lavandin Oil, Abrial Type
CAS: [8022-15-9]
DESCRIPTION
Lavandin Oil, Abrial Type, occurs as a pale yellow to yellowliquid with a slight, camphoraceous odor that is strongly sug-gestive of lavender. It is obtained by steam distillation of thefresh flowering tops of a hybrid, Lavandula abrialis unofficial(Fam. Labiatae), of true lavender, Lavandula officinalis, orof spike lavender, Lavandula latifolia. It is soluble in most
fixed oils and in propylene glycol. It is soluble with opales-cence in mineral oil, but it is relatively insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 28.0% and not more than 35.0% ofesters, calculated as linalyl acetate (C12H20O2).Angular Rotation Between −2° and −5°.Refractive Index Between 1.460 and 1.464 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.885 and 0.893.
TESTS
Assay Determine as directed in Ester Determination underEsters, Appendix VI, using about 3 g of sample, accuratelyweighed, and 98.15 as the equivalence factor (e) in the calcu-lation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 2 mL of 70% alcohol. A slight opalescence sometimesdevelops on further dilution.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Lavender Oil
CAS: [8000-28-0]
FEMA: 2622
DESCRIPTION
Lavender Oil occurs as a colorless or yellow liquid with thecharacteristic odor and taste of lavender flowers. It is thevolatile oil obtained by steam distillation from the fresh flow-ering tops of Lavandula officinalis Chaix ex Villars (Lavan-
View IR
View IR
248 / Lecithin / Monographs FCC V
dula vera De Candolle) (Fam. Labiatae). It is soluble in alcoholand in most vegetable oils, but is insoluble in propylene glycol.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 35.0% of esters, calculated as linalylacetate (C12H20O2).Alcohol Passes test.Angular Rotation Between −3° and −10°.Refractive Index Between 1.459 and 1.470 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.875 and 0.888.
TESTS
Assay Determine as directed for Ester Determination underEsters, Appendix VI, using about 5 g of sample, accuratelyweighed, and 98.15 as the equivalence factor (e) in the calcu-lation.Alcohol Transfer 5 mL of sample into a narrow, graduated,glass-stoppered, 10-mL cylinder; add 5 mL of water; andshake. The volume of the oil does not diminish.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 4 mL of 70% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers.
Lecithin
INS: 322 CAS: [8002-43-5]
DESCRIPTION
Lecithin, both natural and refined grades, occurs as asubstance varying in consistency from plastic to fluid de-pending on free fatty acid and oil content and on thepresence or absence of other diluents. Its color varies fromlight yellow to brown, depending on the source, on cropvariations, and on whether it is bleached or unbleached.Lecithin is obtained from soybeans and other plant sources.
It is a complex mixture of acetone-insoluble phosphatidesthat consists chiefly of phosphatidyl choline, phosphatidylethanolamine, and phosphatidyl inositol combined with vari-ous amounts of other substances such as triglycerides, fattyacids, and carbohydrates. Refined grades of Lecithin maycontain any of these components in varying proportionsand combinations depending on the type of fractionationused. In its oil-free form, the preponderance of triglyceridesand fatty acids is removed and the product contains 90%or more of phosphatides representing all or certain fractionsof the total phosphatide complex. Edible diluents, such ascocoa butter and vegetable oils, often replace soybean oilto improve functional and flavor characteristics. Lecithin isonly partially soluble in water, but it readily hydrates toform emulsions. The oil-free phosphatides are soluble infatty acids, but they are practically insoluble in fixed oils.When all phosphatide fractions are present, Lecithin ispartially soluble in alcohol and practically insoluble inacetone.
Function Antioxidant; emulsifier.
REQUIREMENTS
Acetone-Insoluble Matter (as phosphatides) Not less than50.0%.Acid Value Not more than 36.Hexane-Insoluble Matter Not more than 0.3%.Lead Not more than 1 mg/kg.Peroxide Value Not more than 100.Water Not more than 1.5%.
TESTS
Acetone-Insoluble Matter (as phosphatides)Purification of Phosphatides Dissolve 10 g of sample in
20 mL of petroleum ether, add 50 mL of acetone to thesolution, chill, and decant. Dry the solids under flowing nitro-gen under a hood. Dissolve 5 g of the solids in 10 mL ofpetroleum ether, and add 25 mL of acetone to the solution.Transfer approximately equal portions of the precipitate toeach of two 40-mL centrifuge tubes, using additional portionsof acetone to facilitate the transfer. Stir thoroughly, dilute to40 mL with acetone, stir again, chill for 15 min in an icebath, stir again, and then centrifuge for 5 min. Decant theacetone, crush the solids with a stirring rod, refill the tubewith acetone, stir, chill, centrifuge, and decant as before.The solids after the second centrifugation require no furtherpurification and may be used for preparing the Phosphatide–Acetone Solution. Five grams of the purified phosphatides arerequired to saturate about 16 L of acetone.
Phosphatide–Acetone Solution Add a quantity of purifiedphosphatides to sufficient acetone, previously cooled to about5°, to form a saturated solution, and maintain the mixture atthis temperature for 2 h, shaking it vigorously at 15-minintervals. Decant the solution through a rapid filter paper,avoiding the transfer of any undissolved solids to the paperand conducting the filtration under refrigerated conditions(not above 5°).
FCC V Monographs / Lemongrass Oil / 249
Procedure If the sample is plastic or semisolid, soften aportion of it by warming it in a water bath at a temperaturenot exceeding 60°, and then mix it thoroughly. Transfer about2 g of a well-mixed sample, accurately weighed, into a 40-mL centrifuge tube, previously tared with a glass stirring rod,and add 15 mL of Phosphatide–Acetone Solution from a buret.Warm the mixture in a water bath until the sample melts, butavoid evaporation of the acetone. Stir until the sample iscompletely disintegrated and dispersed, transfer the tube intoan ice bath, chill for 5 min, remove from the ice bath, andadd about 10 mL of Phosphatide–Acetone Solution, previouslychilled for 5 min in an ice bath. Stir the mixture to completedispersion of the sample, dilute to 40 mL with chilled (5°)Phosphatide–Acetone Solution, stir to complete dispersion ofthe sample, and return the tube and contents to the ice bathfor 15 min. Subsequently stir again while still in the ice bath,remove the stirring rod, and centrifuge the mixture immedi-ately for 5 min. Decant the supernatant liquid from the centri-fuge tube; crush the centrifuged solids with the stirring rod;refill the tube to the 40-mL mark with chilled (5°) Phospha-tide–Acetone Solution; and repeat the chilling, stirring, centri-fugation, and decantation procedure. After the second centrifu-gation and decantation of the supernatant acetone, again crushthe solids with the stirring rod, and place the tube and itscontents in a horizontal position at room temperature untilthe excess acetone has evaporated. Mix the residue again, drythe centrifuge tube and its contents at 105° for 45 min in aforced-draft oven, cool, and weigh. Calculate the percentageof acetone-insoluble substances by the formula
(100R/S) − B,
in which R is the weight, in grams, of residue; S is the weight,in grams, of the sample taken; and B is the percentage ofhexane-insoluble matter determined as directed under Hexane-Insoluble Matter (below).Acid Value If the sample is plastic or semisolid, soften aportion by warming it in a water bath at a temperature notexceeding 60°, and then mix it thoroughly. Transfer about 2g of a well-mixed sample, accurately weighed, into a 250-mL Erlenmeyer flask, and dissolve it in 50 mL of petroleumether. Add 50 mL of ethanol, previously neutralized to phenol-phthalein with 0.1 N sodium hydroxide, to this solution, andmix well. Using phenolphthalein TS as the indicator, titratewith 0.1 N sodium hydroxide to a pink endpoint that persistsfor 5 s. Calculate the Acid Value by the formula
5.6 × A/W,
in which A is the volume, in milliliters, of 0.1 N sodiumhydroxide consumed, and W is the weight, in grams, of thesample taken.Hexane-Insoluble Matter If the sample is plastic or semi-solid, soften a portion by warming it at a temperature notexceeding 60°, and then mix it thoroughly. Transfer 10 g ofwell-mixed sample into a 250-mL wide-mouth Erlenmeyerflask, add 100 mL of solvent hexane, and shake until thesample is dissolved. Filter the resulting solution through a
30-mL Corning ‘‘C’’ porosity, or equivalent, filtering funnelthat previously has been dried at 105° for 1 h, cooled in adesiccator, and weighed. Wash the flask with two successive25-mL portions of solvent hexane, and pass the washingsthrough the filter. Dry the funnel at 105° for 1 h, cool to roomtemperature in a desiccator, and weigh. From the gain inweight of the funnel, calculate the percentage of the hexane-insoluble matter in the sample.Lead Determine as directed for Method II in the FlameAtomic Absorption Spectrophotometric Method under LeadLimit Test, Appendix IIIB, using a 10-g sample.Peroxide Value Transfer about 10 g of sample, accuratelyweighed, into a suitable container, add 30 mL of a 3:2 solutionof glacial acetic acid:chloroform, and mix. Add 1 mL of asaturated solution of potassium iodide, mix, and allow to standfor 10 min. Add 100 mL of water, begin titrating with 0.05N sodium thiosulfate, adding starch TS as the endpoint isapproached, and continue the titration until the blue starchcolor has just disappeared. Perform a blank determination(see General Provisions), and make any necessary correction.Calculate the peroxide value, as milliequivalents of peroxideper kilograms of sample, by the formula
[S × N × 1000]/W,
in which S is the net volume, in milliliters, of sodium thiosul-fate solution required for the sample; N is the exact normalityof the sodium thiosulfate solution; and W is the weight, ingrams, of the sample taken.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in well-closed containers.
Lemongrass OilCAS: [8007-02-1]
FEMA: 2624
DESCRIPTION
Lemongrass Oil is a volatile oil prepared by steam distillationof freshly cut and partially dried cymbopogon grasses indige-nous to tropical and subtropical areas. Two types of Lem-ongrass Oil are commercially available. The East Indian type,also known as Cochin, Native, and British Indian LemongrassOil, usually occurs as a dark yellow to light brown-red liquidwith a pronounced heavy lemon odor. The West Indian type,also known as Madagascar, Guatemala, or other country oforigin Lemongrass Oil, occurs as a light yellow to light brownliquid with a lemon odor of a lighter character than the EastIndian type oil. Lemongrass Oils are soluble in mineral oil,freely soluble in propylene glycol, but practically insoluble
View IR
250 / Lemon Oil, Coldpressed / Monographs FCC V
in water and in glycerin. The East Indian type dissolves readilyin alcohol, but the West Indian type yields cloudy solutions.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate whether it is the East Indian or WestIndian type.Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown under Infrared Spectra usingthe same test conditions as specified therein.Assay Not less than 75.0%, by volume, of aldehydes, calcu-lated as citral (C10H16O).Angular Rotation Between −10° and 0°.Refractive Index Between 1.483 and 1.489.Solubility in Alcohol Passes test.Specific Gravity East Indian Type: Between 0.894 and0.904; West Indian Type: Between 0.869 and 0.894.
TESTS
Assay Mix 50.0 mL of sample with 500 mg of tartaric acid,shake for 5 min, and filter. Dry the filtered oil over anhydroussodium sulfate, and then pipet 10.0 mL of the clear, treatedoil into a 150-mL cassia flask. Add 75 mL of a 30% solutionof sodium bisulfite, stopper the flask, and shake until a semi-solid to solid sodium bisulfite addition product has formed.Allow the mixture to stand at room temperature for 5 min,then loosen the stopper, and immerse the flask in a water bathheated to between 85° and 90°. Maintain the water bath at thistemperature, shaking the flask occasionally, until the additionproduct dissolves, and then continue heating and intermittentlyshaking for another 30 min. When the liquids have separatedcompletely, add enough 30% sodium bisulfite solution to raisethe lower level of the oily layer within the graduated portionof the flask’s neck. Calculate the percentage, by volume, ofthe citral by the formula
100 − (V × 10),
in which V is the number of milliliters of separated oil in thegraduated neck of the cassia flask.Angular Rotation Determine as directed under Optical Ro-tation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. East Indian Type: One milliliterdissolves in 3 mL of 70% alcohol, usually with slight turbidity;West Indian Type: Yields a cloudy solution with 70%, 80%,90%, and 95% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in full, tight containers.Avoid exposure to excessive heat.
Lemon Oil, Coldpressed
Lemon Oil, ExpressedCAS: [8008-56-8]
FEMA: 2625
DESCRIPTION
Lemon Oil, Coldpressed, occurs as a pale to deep yellow orgreen-yellow liquid with the characteristic odor and taste ofthe outer part of fresh lemon peel. It is the volatile oil obtainedby expression, without the aid of heat, from the fresh peel ofthe fruit of Citrus limon L. Burmann filius (Fam. Rutaceae)with or without the previous separation of the pulp and thepeel. It is miscible with dehydrated alcohol and with glacialacetic acid. It may contain a suitable antioxidant.
Note: Do not use Lemon Oil that has a terebinthine odor.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate whether it is the California type or theItalian type.Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay California Type: Not less than 2.2% and not morethan 3.8% of aldehydes, calculated as citral (C10H16O); ItalianType: Not less than 3.0% and not more than 5.5% of aldehydes,calculated as citral (C10H16O).Angular Rotation Between +57° and +65.6°.Refractive Index Between 1.473 and 1.476 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.849 and 0.855.Ultraviolet Absorbance California Type: Not less than 0.2;Italian Type: Not less than 0.49.
TESTS
Assay Determine as directed in the Hydroxylamine/Tert-Butyl Alcohol Method under Aldehydes and Ketones, Appen-dix VI, using about 5 mL of sample, accurately weighed.Allow the mixture to stand for 15 min, occasionally shaking,before titrating, and use 76.12 as the equivalence factor (e)in the calculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of 95% alcohol, sometimes with a slight haze.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
View IR
FCC V Monographs / Lemon Oil, Distilled / 251
Ultraviolet Absorbance Determine as directed under Ultra-violet Absorbance of Citrus Oils, Appendix VI, using about250 mg of sample, accurately weighed. The maximum ab-sorbance occurs at 315 � 3 nm.
Packaging and Storage Store in full, tight containers.Avoid exposure to excessive heat.
Lemon Oil, Desert Type, Coldpressed
Lemon Oil Arizona
DESCRIPTION
Lemon Oil, Desert Type, Coldpressed, occurs as a pale todeep yellow or green-yellow liquid with the characteristicodor and taste of the outer part of fresh lemon peel. It is thevolatile oil obtained by expression, without the aid of heat,from the fresh peel of the fruit of Citrus limon L. Burmannfilius (Fam. Rutaceae), with or without the previous separationof the pulp and peel. It is miscible with dehydrated alcohol andwith glacial acetic acid. It may contain a suitable antioxidant.
Note: Do not use if it has a terebinthine odor.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 1.7% of aldehydes, calculated as citral(C10H16O).Angular Rotation Between +67° and +78°.Refractive Index Between 1.473 and 1.476.Solubility in Alcohol Passes test.Specific Gravity Between 0.846 and 0.851.Ultraviolet Absorbance Not less than 0.20.
TESTS
Assay Determine as directed in the Hydroxylamine/Tert-Butyl Alcohol Method under Aldehydes and Ketones, Appen-dix VI, using about 5 mL of sample, accurately weighed.Allow the mixture to stand for 15 min, shaking occasionally,before titrating, and use 76.12 as the equivalence factor (e)in the calculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.
Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 3 mL of alcohol, sometimes with a slight haze.Specific Gravity Determine by any reliable method (seeGeneral Provisions).Ultraviolet Absorbance Determine as directed under Ultra-violet Absorbance of Citrus Oils, Appendix VI, using about250 mg of sample, accurately weighed. The maximum ab-sorbance occurs at 315 � 3 nm.
Packaging and Storage Store in full, tight containers.Avoid exposure to excessive heat.
Lemon Oil, Distilled
DESCRIPTION
Lemon Oil, Distilled, occurs as a colorless to pale yellowliquid with the characteristic odor of fresh lemon peel. It isthe volatile oil obtained by distillation from the fresh peel orjuice of the fruit of Citrus limon L. Burmann filius (Fam.Rutaceae), with or without the previous separation of thejuice, pulp, and peel. It is soluble in most fixed oils, in mineraloil, and in alcohol (with haze). It is insoluble in glycerin andin propylene glycol. It may contain a suitable antioxidant.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseshown in a typical spectrum as shown in the section on Infra-red Spectra, using the same test conditions as specified therein.Aldehydes Between 1.0% and 3.5% of aldehydes, calcu-lated as citral (C10H16O).Angular Rotation Between +55° and +75°.Refractive Index Between 1.470 and 1.475 at 20°.Specific Gravity Between 0.842 and 0.856.Ultraviolet Absorbance Not more than 0.01.
TESTS
Aldehydes Determine as directed in the Hydroxylamine/Tert-Butyl Alcohol Method under Aldehydes and Ketones,Appendix VI, using about 5 mL of sample, accuratelyweighed, and 76.12 as the equivalence factor (e) in the calcula-tion. Allow the mixture to stand at room temperature for 1 hbefore titrating.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.
View IR
View IR
252 / DL-Leucine / Monographs FCC V
Specific Gravity Determine by any reliable method (seeGeneral Provisions).Ultraviolet Absorbance Determine as directed under Ultra-violet Absorbance of Citrus Oils, Appendix VI, using about250 mg of sample, accurately weighed. The maximum ab-sorbance occurs at 315 � 5 nm.
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers.
DL-LeucineDL-2-Amino-4-methylvaleric Acid
OHH3C
H3C H NH2
O
C6H13NO2 Formula wt 131.17
CAS: [328-39-2]
DESCRIPTION
DL-Leucine occurs as small, white crystals or as a crystallinepowder. It is freely soluble in water, slightly soluble in alcohol,and insoluble in ether. It melts with decomposition at about290°. The pH of a 1:100 aqueous solution is between 5.5 and7.0. It is optically inactive.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H13NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.3%.Residue on Ignition Not more than 0.1%.
TESTS
Assay Dissolve about 400 mg of sample, accuratelyweighed, in 3 mL of formic acid and 50 mL of glacial aceticacid. Add 2 drops of crystal violet TS, and titrate with 0.1 Nperchloric acid to the first appearance of a pure-green coloror until the blue color disappears completely.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.12 mg of C6H13NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers.
L-LeucineL-2-Amino-4-methylvaleric Acid
OHH3C
H3C H NH2
O
C6H13NO2 Formula wt 131.17
INS: 641 CAS: [61-90-5]
DESCRIPTION
L-Leucine occurs as small, white, lustrous plates, or as a white,crystalline powder. One gram dissolves in about 40 mL ofwater and in about 100 mL of glacial acetic acid. It is sparinglysoluble in alcohol, and soluble in dilute hydrochloric acid andin solutions of alkali hydroxides and carbonates.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H13NO2, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 0.2%.Optical (Specific) Rotation [�]D
20°: Between +14.5° and+16.5°, calculated on the dried basis; or [�]D
25°: Between+14.8° and +16.8°, calculated on the dried basis.Residue on Ignition Not more than 0.1%.
TESTS
Assay Transfer about 400 mg of sample, previously driedat 105° for 3 h and accurately weighed, into a 250-mL flask.
View IR
View IR
FCC V Monographs / Lime Oil, Distilled / 253
Dissolve the sample in 3 mL of formic acid and about 50 mLof glacial acetic acid, add 2 drops of crystal violet TS, andtitrate with 0.1 N perchloric acid to a blue-green endpoint.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination (see General Provisions), andmake any necessary correction. Each milliliter of 0.1 N per-chloric acid is equivalent to 13.12 mg of C6H13NO2.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 4 g of sample in sufficient 6 N hydrochloric acidto make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 1-g sample.
Packaging and Storage Store in well-closed containers.
Lime Oil, Coldpressed
Lime Oil, ExpressedCAS: [8008-26-2]
FEMA: 2631
DESCRIPTION
Lime Oil, Coldpressed, occurs as a yellow to brown-green togreen liquid that often shows a waxy separation and has afresh lime-peel odor. It is the volatile oil obtained by expres-sion from the fresh peel or crushed whole fruit of Citrusaurantifolia Swingle (Mexican type) or Citrus latifolia (Tahi-tian type) (Fam. Rutaceae). It is soluble in most fixed oilsand in mineral oil, but is insoluble in glycerin and in propyleneglycol. It may contain a suitable antioxidant.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate whether it is the Mexican or Tahitiantype.Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Mexican Type: Not less than 4.5% and not more than8.5% of aldehydes (as citral); Tahitian Type: Not less than
3.2% and not more than 7.5% of aldehydes (as citral).Angular Rotation Mexican Type: Between +35° and +41°;Tahitian Type: Between +38° and +53°.Refractive Index Mexican Type: Between 1.482 and 1.486;Tahitian Type: Between 1.476 and 1.486.Residue on Evaporation Mexican Type: Between 10.0%and 14.5%; Tahitian Type: Between 5.0% and 12.0%.Specific Gravity Mexican Type: Between 0.872 and 0.881;Tahitian Type: Between 0.858 and 0.876.Ultraviolet Absorbance Mexican Type: Not less than 0.45;Tahitian Type: Not less than 0.24.
TESTS
Assay Determine as directed in the Hydroxylamine/Tert-Butyl Alcohol Method under Aldehydes and Ketones, Appen-dix VI, using about 5 mL of sample, accurately weighed.Allow the mixture to stand for 1 h, occasionally shaking,before titrating, and use 76.12 as the equivalence factor (e)in the calculation.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, using an Abbé or other refractometer of equal or greateraccuracy.Residue on Evaporation Determine as directed under Resi-due on Evaporation, Appendix VI, using a 3-g sample andheating for 6 h.Specific Gravity Determine by any reliable method (seeGeneral Provisions).Ultraviolet Absorbance Determine as directed under Ultra-violet Absorbance of Citrus Oils, Appendix VI, using about 20mg of sample, accurately weighed. The maximum absorbanceoccurs at 315 � 3 nm.
Packaging and Storage Store in full, tight containers.Avoid exposure to excessive heat.
Lime Oil, Distilled
DESCRIPTION
Lime Oil, Distilled, occurs as a colorless to green-yellowliquid with a mild citrus, floral odor. It is the volatile oilobtained by distillation from the juice or the whole crushedfruit of Citrus aurantifolia Swingle (Fam. Rutaceae). It issoluble in most fixed oils and in mineral oil, but it is insolublein glycerin and in propylene glycol. It may contain a suitableantioxidant.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as
View IR
View IR
254 / Limestone, Ground / Monographs FCC V
those of a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Aldehydes Between 0.5% and 2.5% of aldehydes, calcu-lated as citral (C10H16O).Angular Rotation Between +34° and +47°.Refractive Index Between 1.474 and 1.477 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.855 and 0.863.
TESTS
Aldehydes Determine as directed in the Hydroxylamine/Tert-Butyl Alcohol Method under Aldehydes and Ketones,Appendix VI, using about 5 g of sample, accurately weighed,and 76.12 as the equivalence factor (e) in the calculation.Allow the mixture to stand at room temperature for 15 minbefore titrating.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, using an Abbé or other refractometer of equal or greateraccuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 5 mL of 90% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Limestone, Ground
DESCRIPTION
Limestone, Ground, is produced as a fine, white to off white,microcrystalline powder mainly consisting of calcium carbon-ate. It is obtained by crushing, grinding, and classifying natu-rally occurring limestone benefited by flotation and/or airclassification. It is stable in air. It is practically insoluble inwater and in alcohol. The presence of any ammonium salt orcarbon dioxide increases its solubility in water, but the pres-ence of any alkali hydroxide reduces its solubility.
Function Texturizing and release agent and modifier forchewing gum base and chewing gum.
REQUIREMENTS
Identification A sample dissolves with effervescence in 1N acetic acid, in 2.7 N hydrochloric acid, and in 1.7 N nitricacid, and the resulting solutions, after boiling, give positivetests for Calcium, Appendix IIIA.
Assay Not less than 94.0% and not more than 100.5% ofCaCO3 after drying.Acid-Insoluble Substances Not more than 2.5%.Arsenic Not more than 3 mg/kg.Fluoride Not more than 0.005%.Lead Not more than 3 mg/kg.Loss on Drying Not more than 2.0%.Magnesium and Alkali Salts Not more than 3.5%.
TESTS
Assay Transfer about 200 mg of sample, previously driedat 200° for 4 h and accurately weighed, into a 400-mL beaker,add 10 mL of water, and swirl to form a slurry. Cover thebeaker with a watch glass, and introduce 2 mL of 2.7 Nhydrochloric acid from a pipet inserted between the lip of thebeaker and the edge of the watch glass. Swirl the contents ofthe beaker to dissolve the sample. Wash down the sides ofthe beaker, the outer surface of the pipet, and the watchglass, and dilute to about 100 mL with water. While stirring,preferably with a magnetic stirrer, add about 30 mL of 0.05M disodium EDTA from a 50-mL buret, add 15 mL of 1 Npotassium hydroxide and 300 mg of hydroxy naphthol blueindicator, and continue the titration to a blue endpoint. Eachmilliliter of 0.05 M disodium EDTA is equivalent to 5.004mg of CaCO3.Acid-Insoluble Substances Suspend 5 g of sample in 25mL of water, agitate while cautiously adding 25 mL of 1:2hydrochloric acid, and add water to make a volume of about200 mL. Heat the solution to boiling, cover, digest on a steambath for 1 h, cool, and filter. Wash the precipitate with wateruntil the last washing shows no chloride with silver nitrateTS, and then ignite it. The weight of the residue does notexceed 125 mg.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a solution of 1 g of sample in 10 mLof 2.7 N hydrochloric acid.Fluoride Determine as directed in Method III under Fluo-ride Limit Test, Appendix IIIB.Lead
Sample Solution Cautiously dissolve 5 g of sample in 25mL of 1:2 hydrochloric acid, and evaporate to dryness on asteam bath. Dissolve the residue in about 15 mL of water,and dilute to 25 mL (1 mL = 200 mg).
Procedure Determine as directed under Lead Limit Test,Appendix IIIB, using a 5-mL portion of the Sample Solution,and 3 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 200° for 4 h.Magnesium and Alkali Salts Mix 1 g of sample with 40mL of water, carefully add 5 mL of hydrochloric acid, mix,and boil for 1 min. Rapidly add 40 mL of oxalic acid TS,and stir vigorously until precipitation is well established. Im-mediately add 2 drops of methyl red TS, then add 6 N ammo-nium hydroxide, dropwise, until the mixture is just alkaline,and cool. Transfer the mixture to a 100-mL cylinder, diluteto 100 mL with water, and let it stand for 4 h or overnight.Decant the clear, supernatant liquid through a dry filter paper,and place 50 mL of the clear filtrate in a platinum dish. Add
FCC V Monographs / Linoleic Acid / 255
0.5 mL of sulfuric acid, and evaporate the mixture on a steambath to a small volume. Carefully evaporate the remainingliquid to dryness over a free flame, and continue heating untilthe ammonium salts have been completely decomposed andvolatilized. Finally, ignite the residue to constant weight. Theweight of the residue does not exceed 17.5 mg.
Packaging and Storage Store in well-closed containers.
Linaloe Wood OilCAS: [8006-86-8]
DESCRIPTION
Linaloe Wood Oil occurs as a colorless to yellow liquid witha pleasant, flowery odor. It is the volatile oil obtained bysteam distillation from the wood of Bursera delpechianaPoiss. (Fam. Burseraceae) and other Bursera species. It issoluble in most fixed oils and in propylene glycol. It is solublein mineral oil, but it becomes opalescent or turbid on dilution.It is insoluble in glycerin.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 85.0% of alcohols, calculated as linalool(C10H18O).Acid Value Not more than 3.0.Angular Rotation Between −5° and −13°.Ester Value Between 40 and 75.Refractive Index Between 1.459 and 1.463 at 20°.Solubility in Alcohol Passes test.Specific Gravity Between 0.876 and 0.883.
TESTS
Assay Determine as directed under Linalool Determination,Appendix VI, using about 1.5 g of acetylated oil, accuratelyweighed, for the saponification.Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Ester Value Determine as directed in Ester Value underEsters, Appendix VI, using about 2.5 g of sample, accuratelyweighed.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.
Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 5 mL of 60% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Linoleic Acid(Z),(Z)-9,12-Octadecadienoic Acid
CH3(CH2)4CH CHCH2CH CH(CH2)7COOH
C18H32O2 Formula wt 280.45
CAS: [60-33-3]
DESCRIPTION
Linoleic Acid occurs as a colorless to pale yellow, oily liquidthat is easily oxidized by air. It is an essential fatty acidand the major constituent of many vegetable oils, includingcottonseed, soybean, peanut, corn, sunflower seed, safflower,poppy seed, and linseed. Its specific gravity is about 0.901,and its refractive index is about 1.469. It has a boiling pointranging from 225° to 230° and a melting point around −5°.One milliliter dissolves in 10 mL of petroleum ether. It isfreely soluble in ether; soluble in absolute alcohol and inchloroform; and miscible with dimethylformamide, fat sol-vents, and oils. It is insoluble in water.
Function Flavoring adjuvant; nutrient.
REQUIREMENTS
Identification Linoleic Acid exhibits the following compo-sition profile of fatty acids determined as directed under FattyAcid Composition, Appendix VII.
Fatty Acid: 14:0 16:0 18:0 18:1 18:2 18:3Weight % (Range): <1.0 3–5 <1.0 <25.0 >60.0 <9.0
Assay Not less than 60.0% of fatty acid C18:2, equivalentto C18H32O2, calculated on the anhydrous basis.Acid Value Between 196 and 202.Iodine Value Between 145 and 160.Lead Not more than 2 mg/kg.Residue on Ignition Not more than 0.01%.Unsaponifiable Matter Not more than 2.0%.Water Not more than 0.5%.
TESTS
Assay Determine as directed under Fatty Acid Composition,Appendix VII.
View IR
256 / Locust (Carob) Bean Gum / Monographs FCC V
Acid Value Determine as directed in Method I under AcidValue, Appendix VII.Iodine Value Determine as directed under Iodine Value,Appendix VII.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 5-g sample.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 10-g sample.Unsaponifiable Matter Determine as directed under Unsa-ponifiable Matter, Appendix VII, using a 10-g sample.Water Determine as directed under Water Determination,Appendix IIB.
Packaging and Storage Store in tight containers.
Locust (Carob) Bean GumLocust Bean Gum; Carob Bean Gum
INS: 410 CAS: [9000-40-2]
DESCRIPTION
Locust (Carob) Bean Gum occurs as a white to yellow-whitepowder. It is obtained from the ground endosperms of Cerato-nia siliqua (L.) Taub. (Fam. Leguminosae). It consists chieflyof a high-molecular-weight hydrocolloidal polysaccharide,composed of galactose and mannose units combined throughglycosidic linkages, which may be described chemically as agalactomannan. It is dispersible in either hot or cold water,forming a sol having a pH between 5.4 and 7.0, which maybe converted to a gel by the addition of small amounts ofsodium borate.
Function Stabilizer; thickener.
REQUIREMENTS
IdentificationA. Transfer a 2-g sample into a 400-mL beaker, moisten
it with about 4 mL of isopropyl alcohol, while vigorouslystirring add 200 mL of cold water, and continue stirring untilthe gum is uniformly dispersed. An opalescent, slightly vis-cous solution forms. Save this solution for IdentificationTest B.
B. Transfer 100 mL of the solution prepared in Identifica-tion Test A into another 400-mL beaker, heat the mixture ina boiling water bath for about 10 min, and then cool toroom temperature. The viscosity of the solution increasesappreciably (distinction from guar gum).Acid-Insoluble Matter Not more than 4.0%.Arsenic Not more than 3 mg/kg.Ash (Total) Not more than 1.2%.Galactomannans Not less than 75.0%.Lead Not more than 5 mg/kg.
Loss on Drying Not more than 14.0%.Protein Not more than 7.0%.Starch Passes test.
TESTS
Acid-Insoluble Matter Transfer 1.5 g of sample, accuratelyweighed, into a 250-mL beaker containing 150 mL of waterand 1.5 mL of sulfuric acid. Cover the beaker with a watchglass, and heat the mixture on a steam bath for 6 h, rubbingdown the wall of the beaker frequently with a rubber-tippedstirring rod and replacing any water lost by evaporation. Sub-sequently add about 500 mg of a suitable filter aid, previouslydried for 3 h at 105° and accurately weighed, and filter througha tared, sintered-glass filter crucible. Wash the residue severaltimes with hot water, dry the crucible and its contents at 105°for 3 h, cool in a desiccator, and weigh. The difference betweenthe weight of the filter aid and that of the residue is the weightof the Acid-Insoluble Matter.Arsenic Determine as directed under Arsenic Limit Test,Appendix IIIB, using a Sample Solution prepared as directedfor organic compounds.Ash (Total) Determine as directed under Ash (Total), Ap-pendix IIC.Galactomannans Add the percentages of Acid-InsolubleMatter, Total Ash, Loss on Drying, and Protein, and subtractthe total from 100%. The difference represents the percentageof galactomannans in the sample.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 5 h.Protein Determine as directed under Nitrogen Determina-tion, Appendix IIIC, using about 3.5 g of sample, accuratelyweighed, transferred into a 500-mL Kjeldahl flask. The per-cent of nitrogen determined, multiplied by 6.25, gives thepercent of protein in the sample.Starch Add a few drops of iodine TS to a 1:10 aqueoussolution of the sample. No blue color appears.
Packaging and Storage Store in well-closed containers.
Lovage OilCAS: [8016-31-7]
DESCRIPTION
Lovage Oil occurs as a yellow-green-brown to deep brown liq-uid with a strong, characteristic aromatic odor and taste. It isthe volatile oil obtained by steam distillation of the fresh rootof the plant Levisticum officinale L. Koch syn. Angelica levisti-cum, Baillon (Fam. Umbelliferae). It is soluble in most fixedoils and slightly soluble, with opalescence, in mineral oil, butit is relatively insoluble in glycerin and in propylene glycol.
View IR
FCC V Monographs / Mace Oil / 257
Note: This oil becomes darker and more viscous underthe influence of air and light.
Function Flavoring agent.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima (that may vary in intensity) atthe same wavelengths as those of a typical spectrum as shownin the section on Infrared Spectra, using the same test condi-tions as specified therein.Acid Value Between 2.0 and 16.0.Angular Rotation Between −1° and +5°.Refractive Index Between 1.536 and 1.554 at 20°.Saponification Value Between 238 and 258.Solubility in Alcohol Passes test.Specific Gravity Between 1.030 and 1.057.
TESTS
Acid Value Determine as directed under Acid Value, Ap-pendix VI.Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Saponification Value Determine as directed in Saponifica-tion Value under Esters, Appendix VI, using 1.5 g of sample,accurately weighed.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 4 mL of 95% ethanol, sometimes with slight turbidity. Theage of the oil has an adverse effect upon solubility.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
L-Lysine Monohydrochloride2,6-Diaminohexanoic Acid Hydrochloride
NH2(CH2)4CCOOH·HCl
H NH2
C6H14N2O2·HCl Formula wt 182.65
CAS: [657-27-2]
DESCRIPTION
L-Lysine Monohydrochloride occurs as a white or nearlywhite, free-flowing, crystalline powder. It is freely soluble in
water, but it is almost insoluble in alcohol and in ether. Itmelts at about 260° with decomposition.
Function Nutrient.
REQUIREMENTS
Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Assay Not less than 98.5% and not more than 101.5% ofC6H14N2O2·HCl, calculated on the dried basis.Lead Not more than 5 mg/kg.Loss on Drying Not more than 1.0%.Optical (Specific) Rotation [�]D
20°: Between +20.3° and+21.5°, calculated on the dried basis; or [�]D
25°: Between+20.4° and +21.4°, calculated on the dried basis.Residue on Ignition Not more than 0.2%.
TESTS
Assay Dissolve about 100 mg of sample, previously driedat 105° for 3 h and accurately weighed, in 2 mL of formicacid, add exactly 15.0 mL of 0.1 N perchloric acid, and heaton a water bath for 30 min.
Caution: Handle perchloric acid in an appropriatefume hood.
After cooling, add 45 mL of glacial acetic acid, and titratethe excess perchloric acid with 0.1 N sodium acetate, determin-ing the endpoint potentiometrically. Perform a blank determi-nation (see General Provisions), and make any necessarycorrection. Each milliliter of 0.1 N perchloric acid is equivalentto 9.133 mg of C6H14N2O2·HCl.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a Sample Solution prepared as directed fororganic compounds, and 5 �g of lead (Pb) ion in the control.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 3 h.Optical (Specific) Rotation Determine as directed underOptical (Specific) Rotation, Appendix IIB, using a solutioncontaining 8 g of a previously dried sample in sufficient 6 Nhydrochloric acid to make 100 mL.Residue on Ignition Determine as directed under Residueon Ignition, Appendix IIC, igniting a 2-g sample.
Packaging and Storage Store in well-closed containers.
Mace OilCAS: [8007-12-3]
DESCRIPTION
Mace Oil occurs as a colorless to pale yellow liquid with thecharacteristic odor and taste of nutmeg. It is the volatile oil
View IRView IR
258 / Magnesium Carbonate / Monographs FCC V
obtained by steam distillation from the ground, dried arillodeof the ripe seed of Myristica fragrans Houtt. (Fam. Myristica-ceae). Two types of oil, the East Indian and the West Indian,are commercially available. It is soluble in most fixed oilsand in mineral oil, but it is insoluble in glycerin and in propyl-ene glycol.
Function Flavoring agent.
REQUIREMENTS
Labeling Indicate whether it is the East Indian or WestIndian type.Identification The infrared absorption spectrum of the sam-ple exhibits relative maxima at the same wavelengths as thoseof a typical spectrum as shown in the section on InfraredSpectra, using the same test conditions as specified therein.Angular Rotation East Indian Type: Between +2° and+30°; West Indian Type: Between +20° and +45°.Refractive Index East Indian Type: Between 1.474 and1.488; West Indian Type: Between 1.469 and 1.480 at 20°.Solubility in Alcohol Passes test.Specific Gravity East Indian Type: Between 0.880 and0.930; West Indian Type: Between 0.854 and 0.880.
TESTS
Angular Rotation Determine as directed under Optical(Specific) Rotation, Appendix IIB, using a 100-mm tube.Refractive Index Determine as directed under RefractiveIndex, Appendix IIB, using an Abbé or other refractometerof equal or greater accuracy.Solubility in Alcohol Determine as directed under Solubilityin Alcohol, Appendix VI. One milliliter of sample dissolvesin 4 mL of 90% alcohol.Specific Gravity Determine by any reliable method (seeGeneral Provisions).
Packaging and Storage Store in a cool place protectedfrom light in full, tight containers that are made from steelor aluminum and that are suitably lined.
Magnesium CarbonateMgCO3 Formula wt, anhydrous 84.314MgCO3·Mg(OH)2·5H2O Formula wt, basic 485.65MgCO3·H2O Formula wt, monohydrate 102.33
INS: 504(i) CAS: anhydrous [546-93-0]CAS: basic [39409-82-0]
CAS: monohydrate [23389-33-5]
DESCRIPTION
Magnesium Carbonate occurs as light, white, friable masses,or as a bulky, white powder. It is a basic hydrated magnesium
carbonate or a normal hydrated magnesium carbonate. It isstable in air. It is practically insoluble in water, to which,however, it imparts a slightly alkaline reaction. It is insolublein alcohol, but dissolves, with effervescence, in dilute acids.
Function pH control; drying agent; color-retention agent;anticaking agent; carrier.
REQUIREMENTS
Identification When treated with 2.7 N hydrochloric acid, asample dissolves with effervescence, and the resulting solutiongives positive tests for Magnesium, Appendix IIIA.Assay The equivalent of not less than 40.0% and not morethan 43.5% of MgO.Acid-Insoluble Substances Not more than 0.05%.Calcium Oxide Not more than 0.6%.Lead Not more than 2 mg/kg.Soluble Salts Not more than 1%.
TESTS
Assay Dissolve about 1 g of sample, accurately weighed,in 30.0 mL of 1 N sulfuric acid, add methyl orange TS, andtitrate the excess acid with 1 N sodium hydroxide. From thevolume of 1 N sulfuric acid consumed, deduct the volume of1 N sulfuric acid corresponding to the content of calciumoxide in the weight of the sample taken for the assay. Thedifference is the volume of 1 N sulfuric acid equivalent tothe magnesium oxide present. Each milliliter of 1 N sulfuricacid is equivalent to 20.16 mg of MgO and to 28.04 mg of CaO.Acid-Insoluble Substances Mix 5.0 g of sample with 75mL of water; while agitating, add hydrochloric acid in smallportions until no more of the sample dissolves; and boil for5 min. If an insoluble residue remains, filter through a suitabletared porous bottom porcelain crucible, wash well with wateruntil the last washing is free from chloride, ignite at 800° �25° for 45 min, cool, and weigh.
Note: Avoid exposing the crucible to sudden tempera-ture changes.
Calcium Oxide Dissolve about 1 g of sample, accuratelyweighed, in a mixture of 3 mL of sulfuric acid and 22 mLof water. Add 50 mL of alcohol, and allow the mixture tostand overnight. If crystals of magnesium sulfate separate,warm the mixture to about 50° to dissolve them. Filter througha suitable tared porous bottom porcelain crucible, previouslywashed with 2 N sulfuric acid, water, and alcohol. Wash thecrystals on the porous disk several times with a 2:1 (v/v)mixture of alcohol:2 N sulfuric acid. Ignite the crucible andcontents at 450° � 25° to constant weight. The weight ofcalcium sulfate so obtained, multiplied by 0.4119, gives theequivalent of calcium oxide in the sample taken for the test.
Note: Avoid exposing the crucible to sudden tempera-ture changes.
Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Soluble Salts Mix 2.0 g of sample with 100 mL of a 1:1(v/v) mixture of n-propyl alcohol:water. Heat the mixture to the
FCC V Monographs / Magnesium Gluconate / 259
boiling point with constant stirring, cool to room temperature,add water to make 100 mL, and filter. Evaporate 50 mL of thefiltrate on a steam bath to dryness, and dry in an oven at 105°for 1 h. The weight of the residue does not exceed 10 mg.
Packaging and Storage Store in well-closed containers.
Magnesium ChlorideMgCl2·6H2O Formula wt 203.30
INS: 511 CAS: [7791-18-6]
DESCRIPTION
Magnesium Chloride occurs as colorless flakes or crystals. Itcontains six molecules of water of hydration. It is hygroscopic,very soluble in water, and freely soluble in alcohol.
Function Color-retention agent; firming agent.
REQUIREMENTS
Identification A 1:10 aqueous solution gives positive testsfor Magnesium and for Chloride, Appendix IIIA.Assay Not less than 99.0% and not more than 105.0% ofMgCl2·6H2O.Ammonium Not more than 0.005%.Lead Not more than 4 mg/kg.Sulfate Not more than 0.03%.
TESTS
Assay Dissolve about 450 mg of sample, accuratelyweighed, in 25 mL of water, add 5 mL of ammonia–ammo-nium chloride buffer TS and 0.1 mL of eriochrome black TS,and titrate with 0.05 M disodium EDTA until the solution turnsblue. Each milliliter of 0.05 M disodium EDTA is equivalent to10.16 mg of MgCl2·6H2O.Ammonium Dissolve 1 g of sample in 90 mL of water, andslowly add 10 mL of a freshly boiled and cooled solution of1:10 sodium hydroxide. Allow the mixture to settle, thendecant 20 mL of the supernatant liquid into a color comparisontube, dilute to 50 mL with water, and add 2 mL of Nessler’sreagent. Any color does not exceed that produced by 10 �gof ammonium (NH4) ion in 48 mL of water and 2 mL of thesodium hydroxide solution.Lead Determine as directed under Lead Limit Test, Appen-dix IIIB, using a solution of 1 g of sample, accurately weighed,in 10 mL of water; and 4 �g of lead (Pb) ion in the control.Sulfate Determine as directed in the Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 1-g sample does not exceed that shown in acontrol containing 300 �g of sulfate (SO4).
Packaging and Storage Store in tight containers.
Magnesium Gluconate
[CH2OH(CHOH)4COO]2Mg
C12H22MgO14 Formula wt, anhydrous 414.60C12H22MgO14·2H2O Formula wt, dihydrate 450.63
INS: 580 CAS: anhydrous [3632-91-5]CAS: dihydrate [59625-89-7]
DESCRIPTION
Magnesium Gluconate occurs as a white to off white powderor granulate. It is anhydrous, the dihydrate, or a mixture ofboth. It is very soluble in water and is sparingly soluble inalcohol. It is insoluble in ether.
Function Nutrient.
REQUIREMENTS
IdentificationA. A 1:20 aqueous solution gives positive tests for Magne-
sium, Appendix IIIA.B. Dissolve a quantity of sample in water, heating in a water
bath at 60° if necessary, to obtain a Test Solution containing10 mg/mL. Similarly, prepare a Standard Solution of USPPotassium Gluconate Reference Standard in water, dilutingto 10 mg/mL. Apply separate 5-�L portions of the Test Solu-tion and the Standard Solution on a suitable thin-layer chro-matographic plate (see Chromatography, Appendix IIA)coated with a 0.25-mm layer of chromatographic silica gel,and allow to dry. Develop the chromatogram in a solventsystem consisting of a mixture of alcohol, water, ammoniumhydroxide, and ethyl acetate (50:30:10:10) until the solventfront has moved about three-fourths of the length of the plate.Remove the plate from the chamber, and dry it at 110° for20 min. Allow to cool, and spray with a spray reagent preparedas follows: Dissolve 2.5 g of ammonium molybdate in about50 mL of 2 N sulfuric acid in a 100-mL volumetric flask,add 1.0 g of ceric sulfate, swirl to dissolve, dilute with 2 Nsulfuric acid to volume, and mix. After spraying, heat theplate at 110° for about 10 min. The principal spot obtainedfrom the Test Solution corresponds in color, size, and Rf valueto that obtained from the Standard Solution.Assay Not less than 98.0% and not more than 102.0% ofC12H22MgO14, calculated on the anhydrous basis.Chloride Not more than 0.05%.Lead Not more than 2 mg/kg.Reducing Substances Not more than 1.0%.Sulfate Not more than 0.05%.Water Between 3.0% and 12.0%.
TESTS
Assay Dissolve about 800 mg of sample, accuratelyweighed, in 20 mL of water, add 5 mL of ammonia–ammo-nium chloride buffer TS and 0.1 mL of eriochrome black TS,
260 / Magnesium Hydroxide / Monographs FCC V
and titrate with 0.05 M disodium EDTA to a blue endpoint.Each milliliter of 0.05 M disodium EDTA is equivalent to20.73 mg of C12H22MgO14.Chloride Determine as directed in the Chloride Limit Test,under Chloride and Sulfate Limit Tests, Appendix IIIB, dis-solving 1 g of sample in 100 mL of water. Any turbidityproduced by a 10-mL portion of this solution does not exceedthat shown in a control containing 50 �g of chloride (Cl) ion.Lead Determine as directed in the Flame Atomic AbsorptionSpectrophotometric Method under Lead Limit Test, AppendixIIIB, using a 10-g sample.Reducing Substances Transfer about 1 g of sample, accu-rately weighed, into a 250-mL Erlenmeyer flask, dissolve itin 10 mL of water, add 25 mL of alkaline cupric citrate TS,and cover the flask with a small beaker. Boil gently for exactly5 min and cool rapidly to room temperature. Add 25 mL ofa 1:10 acetic acid solution, 10.0 mL of 0.1 N iodine, 10 mLof 2.7 N hydrochloric acid, and 3 mL of starch TS, and titratewith 0.1 N sodium thiosulfate to the disappearance of theblue color. Calculate the weight, in milligrams, of reducingsubstances (as D-glucose) by the formula
27(V1N1 − V2N2),
in which 27 is an empirically determined equivalence factorfor D-glucose; V1 and N1 are the volume and normality, respec-tively, of the iodine solution; and V2 and N2 are the volumeand normality, respectively, of the sodium thiosulfate solution.Sulfate Determine as directed for Sulfate Limit Test underChloride and Sulfate Limit Tests, Appendix IIIB. Any turbidityproduced by a 200-mg sample does not exceed that shownin a control containing 100 �g of sulfate (SO4) ion.Water Determine as directed for Method 1b in the KarlFischer Titrimetric Method under Water Determination, Ap-pendix IIB. Allow 30 min for the sample to dissolve, performa blank determination (see General Provisions), and makeany necessary correction.
Packaging and Storage Store in well-closed containers.
Magnesium Hydroxide
Mg(OH)2 Formula wt 58.32
INS: 528 CAS: [1309-42-8]
DESCRIPTION
Magnesium Hydroxide occurs as a white, bulky powder. Itis soluble in dilute acids but practically insoluble in waterand in alcohol.
Function pH control; drying agent; color-retention agent.
REQUIREMENTS
Identification A 1:20 aqueous solution in 2.7 N hydrochlo-ric acid gives positive tests for Magnesium, Appendix IIIA.
Assay Not less than 95.0% and not more than 100.5% ofMg(OH)2 after drying.Alkalies (Free) and Soluble Salts Passes test.Calcium Oxide Not more than 1%.Lead Not more than 2 mg/kg.Loss on Drying Not more than 2.0%.Loss on Ignition Between 30.0% and 33.0%.
TESTS
Assay Transfer about 400 mg of sample, previously driedat 105° for 2 h and accurately weighed, into an Erlenmeyerflask. Add 25.0 mL of 1 N sulfuric acid, and after solutionis complete, add methyl red TS, and titrate the excess acidwith 1 N sodium hydroxide. From the volume of 1 N sulfuricacid consumed, deduct the volume of 1 N sulfuric acid corre-sponding to the content of calcium oxide in the sample takenfor the assay. The difference is the volume of 1 N sulfuricacid equivalent to the Mg(OH)2 in the sample taken. Eachmilliliter of 1 N sulfuric acid is equivalent to 29.16 mg ofMg(OH)2 and to 28.04 mg of CaO.Alkalies (Free) and Soluble Salts Boil 2 g of sample with100 mL of water for 5 min in a covered beaker, then filterwhile hot. Titrate 50 mL of the cooled filtrate with 0.1 Nsulfuric acid, using methyl red TS as the indicator. Not morethan 2 mL of the acid is consumed. Evaporate 25 mL of thefiltrate to dryness, and dry at 105° for 3 h. Not more than 10mg of residue remains.Calcium Oxide Dissolve about 500 mg of sample, accu-rately weighed, in a mixture of 3 mL of sulfuric acid and 22mL of water. Add 50 mL of alcohol, and allow the mixtureto stand overnight. If crystals of magnesium sulfate separate,warm the mixture to about 50° to dissolve them. Filter througha suitable tared, porous-bottom porcelain crucible, previouslywashed with 2 N sulfuric acid, water, and alcohol. Wash thecrystals on the porous disk several times with a mixture of 2volumes of alcohol and 1 volume of 2 N sulfuric acid. Ignitethe crucible and contents at 450° � 25° to constant weight.The weight of calcium sulfate thus obtained, multiplied by0.4119, gives the equivalent of calcium oxide in the sampletaken for the test.
Note: Avoid exposing the crucible to sudden tempera-ture changes.
Lead Determine as directed in the APDC Extraction Methodunder Lead Limit Test, Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing, Appendix IIC, drying a sample at 105° for 2 h.Loss on Ignition Transfer about 500 mg of sample, accu-rately weighed, into a tared platinum crucible, and ignite, in-creasing the heat gradually, to constant weight at 800° � 25°.
Packaging and Storage Store in tight containers.
Next Page
3/Flavor Chemicals
Contents
Specifications for Flavor Chemicals . . . 517Test Methods for Flavor Chemicals . . . 630Gas Chromatographic (GC) Assay of Flavor Chemicals . . . 635
515
FCC V Flavor Chemicals / 517
SPECIFICATIONS FOR FLAVOR CHEMICALS
Specifications for flavoring agents other than the essential oilsare presented in tabular form rather than as separate monographs.Specifications for all such ingredients from the Food ChemicalsCodex, Fourth Edition, together with a number of new specifica-tions, are provided on the following pages of this section. Follow-ing the tabular specifications are the Test Methods for FlavorChemicals (M-1 through M-17) and the Gas ChromatographicAssay of Flavor Chemicals used to determine the assay valuesand various other physico-chemical properties of the flavors. Theinfrared spectra, used for identification and comparison purposes,are provided in the section entitled Infrared Spectra.
Explanatory Notes to Tabular SpecificationsThe Food Chemicals Codex, Fifth Edition, uses the nomenclatureconvention (Z) and (E) to specify the structure of acyclic doublebonds in organic chemicals, specifically ‘‘cis’’ and ‘‘trans.’’
The FCC name of the substance is followed, where available,by the number assigned to the substance by the Flavor and ExtractManufacturers Association (FEMA) and by its synonym(s). Theexplanatory notes to the specifications in this section applythroughout the tabular series and are as follows:
Note 1 (Odor) Odor terms are general descriptors and do notindicate the source of the material.
Note 2 (Solubility) Approximate solubilities (see General Pro-visions) are indicated by the following abbreviations: vs = verysoluble; s = soluble; ss = slightly soluble; vss = very slightlysoluble; m = miscible; ins = insoluble or practically insoluble.Other abbreviations are as follows: alc = alcohol; gly = glyc-erin; org = organic; prop = propylene (as in propylene glycol);veg = vegetable (as in vegetable oil); vol = volatile.
Note 3 (B.P.) Boiling points (B.P.) are expressed in °C. Theyare approximate values given for information only and not asrequirements.
Note 4 (Solubility in Alcohol) Determine the solubility in alco-hol at 25° as directed in the general method, Appendix VI,Essential Oils and Flavors.
Note 5 (I.D.) The notation ‘‘IR’’ in the identification (I.D.)column indicates that an infrared absorption spectrum is pro-vided for the particular substance in the section entitled Infra-red Spectra. Where the IR requirement is specified, the infraredabsorption spectrum of the sample shall exhibit maxima at thesame wavelengths as those shown in the respective spectrum,using the test conditions as specified therein.
Note 6 (Assay) Assay requirements are specified as minimumvalues (unless a range of assay values is given) and are statedin weight percent unless otherwise indicated. References toassay methods are indicated by citations in parentheses, e.g.,‘‘(M-1a),’’ to methods provided under Test Methods for FlavorChemicals.
Note 7 (A.V.) Unless otherwise indicated, determine the acidvalue (A.V.) as directed in M-16, using phenolphthalein TSas the indicator unless another indicator is specified for anindividual substance. Where Method II is specified, determinethe acid value as directed in the general method, AppendixVII, Fats and Related Substances.
Note 8 (Ref. Index) Refractive index (Ref. Index) determina-tions are made at 20° unless another temperature is specified,according to the general method, Appendix II, Physical Testsand Determinations.
Note 9 (Sp. Gr.) Specific gravity (Sp. Gr.) determinations aremade at 25° unless another temperature is specified by anyreliable method (see General Provisions).
Note 10 (Other Requirements) Numerical limits for other re-quirements are specified as maximum values unless otherwiseindicated (max = maximum; NLT = not lower than or not lessthan, as appropriate). Test methods are indicated by citationsin parentheses, which refer either to methods given in thesection that follows this tabular section or to general methodsgiven in Appendix VI, Essential Oils and Flavors.
518 / Acetaldehyde / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Acetaldehyde flammable, colorless liq/ m—alc, org solvents,
FEMA No. 2003 pungent, ethereal water/44.05/C2H4O/
CH3CHOAcetic Aldehyde; Ethanal 21°
Acetaldehyde Diethyl Acetal colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2002 ethereal, fruity oils; mL 95%
Acetal ss—water/ ethanol
102°H3C HC
O
O
C2H5
C2H5
118.17/C6H14O2/
Acetanisole colorless to pale yel s—most fixed oils, 1 g in 5 mL
FEMA No. 2005 fused solid/hawthorn prop glycol; 50% alc
4-Acetylanisole; ins—gly/
p-Methoxyacetophenone 153° (26 mm Hg)CH3O C
O
CH3
150.18/C9H10O2/
Acetoin Monomer Monomer Monomer
FEMA No. 2008
Acetyl Methyl Carbinol; colorless to pale yel liq/ m—alc, prop glycol,
Dimethylketol; 3-Hydroxy-2- buttery water;88.11/C4H8O2/
CH3CH(OH)COCH3butanone ins—veg oils/
148°
Dimer Dimer Dimer
white to pale yel powder/ s—hot prop glyc;
odorless ss—weak alkali;
ins—most solvents
176.21/C8H16O4/
HO C
H3C
C
C
CH3
C
OH
HO
H3C CH3
OH
HO C
CH3
C
O C H
C
CH3
H O
CH3 CH3
OH
(a)
(b)
Acetophenone practically colorless liq vs—most fixed oils, 1 mL in 5
FEMA No. 2009 above 20°/ prop glycol; mL 50% alc
Acetylbenzene; Methyl Phenyl very sweet, pungent s—alc, chloroform,
Ketone ether;
ss—water;
120.15/C8H8O/
C CH3
O
ins—gly/
202°
3-Acetyl-2,5-dimethyl Furan yel liq/ s—alc, most fixed
FEMA No. 3391 powerful, slightly oils, prop glycol;
2,5-Dimethyl-3-acetylfuran roasted, nutty ss—water/
83° (11 mm Hg)
138.17/C8H10O2/
OH3C CH3
COCH3
Complete Table
FCC V Flavor Chemicals / 3-Acetyl-2,5-dimethyl Furan / 519
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 99.0% of C2H4O 5.0 0.804–0.811 Residue on Evap.—0.006% (M-16)
(M-2b) (0°/20°)
IR 97.0% of C6H14O2 1.379–1.384 0.821–0.827
(M-1b)
IR 98.0% of C9H10O2 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Lead—10 mg/kg (M-9)
Monomer Monomer Monomer Monomer
IR 96.0% of C4H8O2 1.417–1.422 0.995–1.019
(M-1b)
Dimer Dimer
96.0% of C4H8O2
(M-1b)
IR 98.0% of C8H8O 1.533–1.535 1.025–1.028 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Solidification Pt.—NLT 19° (Appendix IIB)
IR 99.0% of C8H10O2 1.484–1.492 1.027–1.048
(M-1a)
520 / 2-Acetylpyrazine / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
2-Acetylpyrazine colorless to pale yel 1 g in 20 mL
FEMA No. 3126 cryst/ 95% alc
Methyl Pyrazinyl Ketone popcorn
122.13/C6H6N2O/
N
NCH3
O
3-Acetylpyridine colorless to yel liq/ s—acids, alc, ether,
FEMA No. 3424 sweet, nutty, popcorn water/
Methyl Pyridyl Ketone 230°
121.14/C7H7NO/
N
COCH3
2-Acetylpyrrole white to pale brown fine ins—prop glycol, veg 1 g in 6 mL
FEMA No. 3202 cryst/ oils, water/ ethanol
Methyl 2-Pyrrolyl Ketone bready 220°
109.13/C6H7NO/
N
H
COCH3
2-Acetyl Thiazole colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3328 popcorn oils; mL 95%
ins—water/ ethanol
89° at 12 mm
91° (1 mm Hg)
127.17/C5H5NOS/
S
N
CH3
O
Allyl Cyclohexanepropionate colorless liq/ m—alc, chloroform, 1 mL in 4
FEMA No. 2026 pineapple ether; mL 80% alc
Allyl-3-cyclohexanepropionate ins—gly, water
196.29/C12H20O2/
CH2CH2COOCH2CH CH2
Allyl Heptanoate colorless to pale yel liq/ 210° 1 mL in 1
FEMA No. 2031 sweet, pineapple mL 95% alc170.25/C10H18O2/
CH3(CH2)5COOC3H5Allyl Heptoate
Allyl Hexanoate colorless to light yel liq/ m—alc, most fixed 1 mL in 6
FEMA No. 2032 strong, pineapple oils; mL 70% alc156.22/C9H16O2/
CH3(CH2)4COOCH2CH CH2Allyl Caproate ins—prop glycol,
water/
185°
Allyl �-Ionone colorless to yel liq/ s—alc; 1 mL in 1
FEMA No. 2033 fruity, woody ins—water/ mL 90% alc
Allyl Ionone 265° gives clear
soln
232.37/C16H24O/
CH2CH2CH
O
CH2
Complete Table
FCC V Flavor Chemicals / Allyl �-Ionone / 521
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 99.0% of C6H6N2O Melting Range—between 75° and 78°
(M-1a) (Appendix IIB)
IR 98.0% of C7H7NO 1.530–1.540 1.100–1.115 Water—0.5% (Appendix IIB, KF)
(M-1a)
98.0% of C6H7NO Melting Range—between 88° and 92°
(M-1a) (Appendix IIB)
Residue on Ignit.—0.3% (Appendix IIC)
IR 98.0% of C5H5NOS 1.542–1.552 1.219–1.226
(M-1b)
IR 98.0% of C12H20O2 5.0 1.457–1.462 0.945–0.950 Allyl Alcohol—NMT 0.1% (M-1b)
(M-1b)
IR 97.0% of C10H18O2 1.0 1.426–1.430 0.880–0.885 Allyl Alcohol—NMT 0.1% (M-1b)
(M-1b)
IR 98.0% of C9H16O2 1.0 1.422–1.426 0.884–0.890 Allyl Alcohol—NMT 0.1% (M-1b)
(M-1b)
IR 88.0% of C16H24O 1.502–1.507 0.926–0.932 Allyl Alcohol—NMT 0.1% (M-1b)
(M-1b)
522 / Allyl Isothiocyanate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Allyl Isothiocyanate colorless to pale yel, m—alc, carbon
FEMA No. 2034 strongly refractive liq/ disulfide, ether/99.16/C4H5NS/
CH2 CH CH2 N C Sirritating, acrid taste, 150°
mustard (caution:
lachrymator)
Allyl Isovalerate colorless to pale yel liq/ 155° 1 mL in 1
FEMA No. 2045 fruity, apple mL 95% alc142.20/C8H14O2/
(CH3)2CHCH2CO2CH2CH CH2Allyl Isopentanoate
Allyl Phenoxy Acetate colorless to pale yel liq/ ss—prop glycol; 1 mL in 1
FEMA No. 2038 honey, pineapple vss—water; mL 95%
ins—veg oils/ ethanol
265°
192.21/C11H12O3/
O CH2 C O CH2
O
CH CH2
Allyl Propionate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2040 ethereal, fruity oils; mL 95%
ins—water/ ethanolH2C CH CH2 O C C2H5
O
114.15/C6H10O2/
124°
1-Amyl Alcohol colorless to pale yel liq/ s—prop glycol, veg
FEMA No. 2056 fusel, winey oils; water/CH3(CH2)4OH
88.15/C5H12O/
1-Pentanol 136°
Amyl Butyrate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2059 fruity, banana oils; water/ mL 95%CH3CH2CH2COOCH2(CH2)3CH3
158.23/C9H18O2/
1-Pentyl Butyrate 184°–188° ethanol
�-Amylcinnamaldehyde yel liq/ s—most fixed oils; 1 mL in 5
FEMA No. 2061 strong, floral, jasmine on ins—gly, prop glycol/ mL 80% alc
Amylcinnamaldehyde dilution, spicy 285°
202.30/C14H18O/
CH C CHO
(CH2)4CH3
Amyl Cinnamate colorless to pale yel liq/ s—most fixed oils; 1 mL in 7
FEMA No. 2063 faint, balsamic, cocoa ss—prop glycol; mL 80% alc
Isoamyl Cinnamate; Isoamyl ins—gly/ may be
3-Phenyl Propenate 310° opalescent
218.28/C14H18O2/
CH CHCOO(CH2)4CH3
Amyl Formate colorless to pale yel liq/ m—alc/
FEMA No. 2068 fruity 128°–130°
1-Pentyl Formate
116.16/C6H12O2/
CH3(CH2)4OCH
O
Complete Table
FCC V Flavor Chemicals / Amyl Formate / 523
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 93.0% of C4H5NS 1.527–1.531 1.013–1.020 Allyl Alcohol—NMT 0.1% (M-1b)
(M-1a) Distillation Range—between 148° and 154°
(Appendix IIB)
Phenols—passes test (M-17)
IR 98.0% of C8H14O2 1.0 1.413–1.418 0.879–0.884 Allyl Alcohol—NMT 0.1% (M-1b)
(one isomer)
(M-1b)
IR 97.0% of C11H12O3 1.0 1.513–1.518 1.100–1.105
(M-1b)
IR 97.0% of C6H10O2 2.0 1.408–1.413 0.912–0.917
(M-1b)
98.0% of C5H12O 1.407–1.412 0.810–0.816
(M-1b)
98.0% of C9H18O2 1.0 1.409–1.414 0.863–0.866
(sum of isomers)
(M-1b)
IR 97.0% of C14H18O 5.0 1.554–1.559 0.963–0.968 Chlorinated Cmpds.—passes test (Appendix
(sum of two isomers; VI)
90% main isomer)
(M-1b)
IR 96.0% of C14H18O2 1.0 1.535–1.539 0.992–0.997
(sum of n-, 2-methyl
butyl, and 3-methyl butyl
isomers)
(M-1b)
92.0% of C6H12O2 5.0 add ice to 1.396–1.402 0.881–0.887
(sum of n-, 2-methyl soln
butyl, and 3-methyl butyl
isomers)
(M-1b)
524 / Amyl Heptanoate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Amyl Heptanoate colorless to pale yel liq/ 245° 1 mL in 1
FEMA No. 2073 fruity mL 95% alc200.32/C12H24O2/
CH3(CH2)5COO(CH2)4CH3Pentyl Heptanoate
Amyl Octanoate colorless liq/ s—alc, most fixed 1 mL in 7
FEMA No. 2079 fruity oils; mL 80% alc214.35/C13H26O2/
CH3(CH2)6COOC5H11Amyl Caprylate; Isoamyl ss—prop glycol; remains clear
Caprylate; Isoamyl Octanoate ins—gly, water/ to 10 mL
260°
Amyl Propionate colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2082 fruity, apricot-pineapple oils; mL 70% alc144.21/C8H16O2/
CH3CH2COOC5H11Isoamyl Propionate ins—gly, prop glycol,
water/
160°
Anethole colorless to faintly yel liq ss—water; 1 mL in 2
FEMA No. 2086 at or above 23°; sweet m—chloroform, mL alc
trans-Anethole; Isoestragole; taste/ ether/
p-Propenylanisole anise 234°
148.20/C10H12O/
CH3O CH CHCH3
Anisole colorless liq/ s—alc, ether;
FEMA No. 2097 phenolic, anise ins—water/
Methylphenyl Ether 154°
108.14/C7H8O/
OCH3
Anisyl Acetate colorless to slightly yel s—alc, most fixed 1 mL in 6
FEMA No. 2098 liq/ oils; mL 60% alc
p-Methoxybenzyl Acetate floral, fruity, balsamic ins—gly, prop glycol/ remains in
235° soln to 10
180.20/C10H12O3/
CH3O CH2OOCCH3
mL
Anisyl Alcohol colorless to slightly yel s—most fixed oils; 1 mL in 1
FEMA No. 2099 liq/ ss—gly/ mL 50% alc
Anisic Alcohol; p- floral 259° remains in
Methoxybenzyl Alcohol soln to 10
138.17/C8H10O2/
CH3O CH2OH
mL
Anisyl Formate colorless to pale yel liq/ 100° 1 mL in 1
FEMA No. 2101 sweet, floral, tonka mL 95% alc
p-Methoxybenzyl Formate
166.18/C9H10O3/
CH3O CH2 O CH
O
Complete Table
FCC V Flavor Chemicals / Anisyl Formate / 525
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
93.0% of C12H24O2 1.0 1.422–1.426 0.859–0.863
(sum of n-, 2-methyl
butyl, and 3-methyl butyl
isomers)
(M-1a)
IR 98.0% of C13H26O2 1.0 1.425–1.429 0.855–0.861
(sum of n-, 2-methyl
butyl, and 3-methyl butyl
isomers)
(M-1b)
IR 98.0% of C8H16O2 1.0 1.405–1.409 0.866–0.871
(sum of n-, 2-methyl
butyl, and 3-methyl butyl
isomers)
(M-1b)
IR 99.0% of C10H12O 1.557–1.562 0.983–0.988 Angular Rotation—between −0.15° and +0.15°
(M-1b) (Appendix IIB, 100-mm tube)
Distillation Range—between 231° and 237°
(Appendix IIB)
Phenols—passes test (M-17)
Solidification Pt.—NLT 20° (Appendix IIB)
IR 97.0% of C7H8O 1.515–1.518 0.990–0.993 Distillation Range—within a 2° range
(M-1b) (Appendix IIB)
Phenols—passes test (M-17)
IR 97.0% of C10H12O3 1.0 1.511–1.516 1.104–1.111
(M-1b)
IR 97.0% of C8H10O2 1.0 1.542–1.547 1.110–1.115 Aldehydes—1.0% as anisaldehyde (M-1b)
(M-1b) Solidification Pt.—min 23.5° (Appendix IIB)
IR 90.0% of C9H10O3 3.0 1.521–1.525 1.138–1.142
(M-1b)
526 / Benzaldehyde / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Benzaldehyde colorless liq, burning ss—water;
FEMA No. 2127 taste/ m—alc, ether, most
bitter almond oil fixed oils, vol oils/
178°
106.12/C7H6O/
CHO
Benzaldehyde Glyceryl Acetal colorless to pale yel liq/ 185° 1 mL in 1
FEMA No. 2129 mild almond mL 95% alc
Mixture of 1,2- and 1,3-
Benzaldehyde Cyclic Acetals of
Glycerin
180.20/C10H12O3/
O
OOH
(a)
(b)
O
O OH
1,2-Benzodihydropyrone colorless to pale yel liq/ 272° 1 mL in 1
FEMA No. 2381 coconut mL 95% alc
Dihydrocoumarin
148.16/C9H8O2/
O O
Benzophenone white rhombic cryst or s—most fixed oils; 1 g in 10 mL
FEMA No. 2134 flaky solid/ ss—prop glycol; 80% alc
Benzoylbenzene; Diphenyl delicate, persistent, rose ins—gly/
Ketone 305°
182.22/C13H10O/
C
O
Benzyl Acetate colorless liq/ s—alc, most fixed 1 mL in 5
FEMA No. 2135 sweet, floral, fruity oils, prop glycol; mL 60% alc
ins—gly, water/
214°
150.18/C9H10O2/
CH2OOCCH3
Benzyl Alcohol colorless liq with a sharp m—alc, chloroform,
FEMA No. 2137 burning taste/ ether, 1 mL in 30 mL
Phenyl Carbinol faint, aromatic water/
206° (decomp)
108.14/C7H8O/
CH2OH
Benzyl Benzoate colorless, oily liq/ m—alc, chloroform,
FEMA No. 2138 slight, aromatic ether;
ins—gly, water/
323°
212.25/C14H12O2/
COOCH2
Benzyl Butyrate colorless liq/ s—alc, most fixed 1 mL in 2
FEMA No. 2140 floral, fruity, plum oils; mL 80% alc
Benzyl n-Butyrate ins—gly, prop glycol,
water/
178.23/C11H14O2/
CH2OOCCH2CH2CH3
239°
Complete Table
FCC V Flavor Chemicals / Benzyl Butyrate / 527
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C7H6O 1.544–1.547 1.041–1.046 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Hydrocyanic Acid—passes test (M-8)
IR 95.0% of C10H12O3 2.0 1.535–1.541 1.181–1.191
(sum of four isomers;
each isomer between 5%
and 40%)
(M-1a)
IR 99.0% of C9H8O2 1.555–1.559 1.186–1.192 Solidification Pt.—NLT 22° (Appendix IIB)
(M-1b)
IR 98.0% of C13H10O Chlorinated Cmpds.—passes test (Appendix
(M-1a) VI)
Lead—10 mg/kg (M-9)
Solidification Pt.—NLT 47° (Appendix IIB)
IR 98.0% of C9H10O2 1.0 (phenol 1.501–1.504 1.052–1.056 Chlorinated Cmpds.—passes test (Appendix
(M-1b) red TS) VI)
IR 99.0% of C7H8O 1.539–1.541 1.042–1.047 Aldehydes—0.2% (M-1b)
(M-1a) Chlorinated Cmpds.—passes test (Appendix
VI)
Distillation Range—NLT 95% between 202.5°
and 206.5° (Appendix IIB)
IR 99.0% of C14H12O2 1.0 1.568–1.570 1.116–1.120 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Solidification Pt.—NLT 18° (Appendix IIB)
IR 98.0% of C11H14O2 1.0 1.492–1.496 1.006–1.009
(M-1b)
528 / Benzyl Cinnamate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Benzyl Cinnamate white to pale yel solid/ s—most fixed oils; 1 g in 8 mL
FEMA No. 2142 sweet, balsamic ins—gly, prop glycol/ 90% alc
195° (5 mm Hg)
238.29/C16H14O2/
CH CHCOOCH2
Benzyl Formate colorless to pale yel liq/ 203° 1 mL in 1
FEMA No. 2145 sweet, balsamic, floral mL 95% alc136.15/C8H8O2/
CH2 O CH
O
Benzyl Isobutyrate colorless liq/ s—alc, most fixed 1 mL in 6
FEMA No. 2141 floral, fruity, jasmine oils; mL 70% alc
Benzyl 2-Methyl Propionate ss—prop glycol;
ins—gly/
178.23/C11H14O2/
CH2OOCCH(CH3)2
229°
Benzyl Isovalerate colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2152 fruity, herbaceous, apple oils; mL 80% alc
Benzyl 3-Methyl Butyrate ss—prop glycol; remains in
ins—gly, water/ soln on
192.26/C12H16O2/
CH2OOCCH2CH(CH3)2
246° dilution
Benzyl Phenylacetate colorless liq/ m—alc, chloroform, 1 mL in 3
FEMA No. 2149 sweet, floral, honey ether/ mL 90% alc
undertone 317° gives clear
soln
226.27/C15H14O2/
CH2COOCH2
Benzyl Propionate colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2150 sweet, floral, fruity oils; mL 70% alc
Benzyl Propanoate ss—prop glycol; remains clear
ins—gly, water/ to 10 mL
164.20/C10H12O2/
CH2OOCCH2CH3
222°
Benzyl Salicylate almost colorless liq/ s—most fixed oils; 1 mL in 5
FEMA No. 2151 faint, sweet ins—gly, prop glycol/ mL 95% alc
300°
228.25/C14H12O3/
OH
COOCH2
Borneol white to off-white cryst/ ss—prop glycol; 1 g in 2 mL
FEMA No. 2157 piney, camphoraceous vss—water; 95% ethanol
ins—veg oils/
210°CH3
154.25/C10H18O/
H3C CH3
H
OH
Complete Table
FCC V Flavor Chemicals / Borneol / 529
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C16H14O2 1.0 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Solidification Pt.—between 33.0° and 35.0°
(Appendix IIB)
IR 95.0% of C8H8O2 3.0 1.508–1.515 1.082–1.092
(M-1b)
IR 97.0% of C11H14O2 1.0 1.488–1.492 1.000–1.005
(M-1b)
IR 98.0% of C12H16O2 1.0 1.486–1.490 0.983–0.989
(one isomer)
(M-1b)
IR 98.0% of C15H14O2 1.0 1.553–1.558 1.095–1.099
(M-1b)
IR 98.0% of C10H12O2 1.0 1.496–1.500 1.028–1.032
(M-1b)
IR 98.0% of C14H12O3 1.0 (phenol 1.573–1.582 1.176–1.180 Solidification Pt.—NLT 23.5° (Appendix IIB)
(M-1b) red TS)
IR 97.0% of C10H18O Melting Point—202° min.
(M-1b)
530 / Bornyl Acetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Bornyl Acetate colorless liq, semicryst s—alc, most fixed 1 mL in 3
FEMA No. 2159 mass, or white cryst oils; mL 70% alc
L-Bornyl Acetate solid/ ss—water; remains in
sweet, herbaceous, piney ins—gly, prop glycol/ soln to 10
226° mL
196.29/C12H20O2/
H3C C CH3
CH CH2H2C
H2C C CHOOCCH3
CH3
2-Butanone colorless, mobile liq/ m—alc, ether, most
FEMA No. 2170 ethereal, nauseating fixed oils, 1 mL in 472.11/C4H8O/
CH3COCH2CH3Methyl Ethyl Ketone mL water/
78.6°–80°
Butan-3-one-2-yl Butanoate white to slightly yel liq/ s—alc, prop glycol,
FEMA No. 3332 sweet, red berry character most fixed oils;
ins—water
158.20/C8H14O3/
O
O
O
Butyl Acetate colorless, mobile liq/ m—alc, ether, prop
FEMA No. 2174 strong, fruity glycol, 1 mL in 145
116.16/C6H12O2/
CH3COO(CH2)3CH3n-Butyl Acetate mL water/
126°
Butyl Alcohol colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2178 vinous in 15 mL water/74.12/C4H10O/
CH3(CH2)2CH2OH1-Butanol 117.7°
Butyl Butyrate colorless liq/ ss—prop glycol,
FEMA No. 2186 fruity, pineapple on water, 1 mL in 3 mL144.21/C8H16O2/
CH3CH2CH2COOC4H9n-Butyl n-Butyrate dilution 70% alc;
m—alc, ether, most
veg oils/
165°
Butyl Butyryllactate colorless liq/ s—prop glycol; 1 mL in 3
FEMA No. 2190 mild, buttery, cream m—alc, most fixed mL 70% alc
Butyl Ester; Butyrate; oils;
Butyryllactic Acid; Lactic Acid ins—water
216.28/C11H20O4/
CH3CHCOOC4H9
CH3CH2CH2COO
Complete Table
FCC V Flavor Chemicals / Butyl Butyryllactate / 531
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C12H20O2 1.0 1.462–1.466 0.981–0.985 Angular Rotation—between −39.5° and −45.0°
(M-1b) (Appendix IIB, 100-mm tube)
Solidification Pt.—NLT 25° (Appendix IIB)
IR 99.5% of C4H8O 2.0 (M-15) 1.375–1.384 0.801–0.803 Distillation Range—within 1.5° (Appendix IIB)
(M-1b) Water—0.2% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 98.0% of C8H14O3 1.408–1.429 0.972–0.992
(M-1a)
IR 98.0% of C6H12O2 2.0 (M-15) 1.393–1.396 0.876–0.880 Distillation Range—between 120° and 128°
(M-1b) (Appendix IIB)
IR 99.5% of C4H10O 2.0 (M-15) 1.397–1.402 0.807–0.809 Butyl Ether—0.15% (M-1b)
(M-1b) Distillation Range—max. 1.5° between
beginning and end (Appendix IIB)
IR 98.0% of C8H16O2 1.0 1.405–1.407 0.867–0.871
(M-1b)
IR 95.0% of C11H20O4 1.0 1.420–1.423 0.970–0.974
(M-1b)
532 / 2-sec-Butyl Cyclohexanone / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
2-sec-Butyl Cyclohexanone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3261 camphoraceous oils; mL 95%
Freskomenthe ins—water/ ethanol
76° at 8 mm
154.25/C10H18O/
O
Butyl Isobutyrate colorless liq/ m—alc, ether, most 1 mL in 7
FEMA No. 2188 fresh, fruity, apple- fixed oils; mL 60% alc144.21/C8H16O2/
(CH3)2CHCOOC4H9 pineapple ins—gly, prop glycol,
water/
166°
Butyl Isovalerate colorless to pale yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2218 fruity oils; mL 95% alc158.24/C9H18O2/
(CH3)2CHCH2COOC4H9 ins—prop glycol,
water/
175°
Butyl 2-Methyl Butyrate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3393 fruity oils; mL 95%
ins—water/ ethanol
173° at 730 mm
158.24/C9H18O2/
C4H9 O C
O
CH
CH3
C2H5
Butyl Phenylacetate colorless to pale yel liq/ 260° 1 mL in 1
FEMA No. 2209 honey, rose mL 95% alc196.26/C12H16O2/
O
O
Butyl Stearate colorless, waxy solid/ s—alc, most fixed 1 mL in 6
FEMA No. 2214 odorless to faintly fatty oils; mL 95% alc340.59/C22H44O2/
CH3(CH2)16COO(CH2)3CH3Butyl Octadecanoate ins—prop glycol,
water/
223°
Butyraldehyde colorless, mobile liq/ s—1 mL in 15 mL
FEMA No. 2219 pungent, nutty water;72.11/C4H8O/
CH3(CH2)2CHOButyl Aldehyde m—alc, ether/
74.8°
Butyric Acid colorless liq/ m—alc, most fixed
FEMA No. 2221 strong, rancid, buttery oils, prop glycol88.11/C4H8O2/
CH3(CH2)2COOH water/
164°
Complete Table
FCC V Flavor Chemicals / Butyric Acid / 533
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C10H18O 1.456–1.462 0.910–0.915
(sum of two isomers)
(M-1b)
IR 97.0% of C8H16O2 1.0 1.401–1.404 0.859–0.864
(one isomer)
(M-1b)
IR 97.0% of C9H18O2 1.0 1.407–1.411 0.856–0.859
(one isomer)
(M-1b)
IR 98.0% of C9H18O2 1.0 1.407–1.413 0.858–0.863
(M-1b)
IR 98.0% of C12H16O2 1.0 1.488–1.492 0.990–0.997
(M-1a)
Melting Range—between 17° and 21°
(Appendix IIB)
Iodine Value—1 max (Appendix VII)
Saponification Value—between 165 and 180
(Appendix VI)
IR 98.0% of C4H8O 5.0 (methyl 1.381–1.387 0.797–0.802 Distillation Range—between 72° and 80° (first
(M-2c) red TS) 95%, Appendix IIB)
para-Butyraldehyde—2.5% (M-1b)
Water—0.5% (Appendix IIB, KF)
IR 99.0% of C4H8O2 1.397–1.399 0.953–0.957 Reducing Subs.—passes test (M-14)
(M-3a)
534 / �-Butyrolactone / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
�-Butyrolactone colorless to slightly yel s—water;
FEMA No. 3291 liq/ m—alc/
faint, sweet, caramel 204°
86.09/C4H6O2/
O O
Camphene colorless cryst mass/ s—alc;
FEMA No. 2229 camphoraceous-oily m—most fixed oils;
ins—water/
159°
136.24/C10H16/
CH2
CH3
CH3
d-Camphor white to gray translucent s—alc; 1 mL in 1
FEMA No. 2230 cryst or fused mass/ ins—most fixed oils, mL 95% alc
minty, ethereal prop glycol, water/
204°
152.24/C10H16O/
CH3
H3C
H3C
Carvacrol colorless to pale yel liq/ s—alc, ether; 1 mL in 4
FEMA No. 2245 pungent, spicy, thymol ins—water/ mL 60% alc
238° gives clear
soln
150.22/C10H14O/
CH3
OHHC
H3C
H3C
l-Carveol colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2247 spearminty oils; mL 95% alc
p-Mentha-6,8-dien-2-ol ins—water/
226°–227° (751 mm
Hg)
152.24/C10H16O/
OH
d-Carvone colorless to light yel liq/ s—prop glycol, most 1 mL in 5
FEMA No. 2249 caraway fixed oils; mL 60% alc
dextro-Carvone; d-1-Methyl-4- m—alc;
isopropenyl-6-cyclohexen-2-one ins—gly/
230°
150.22/C10H14O/
CH3
CH3C CH2
O
l-Carvone colorless to pale s—prop glycol, most 1 mL in 2
FEMA No. 2249 strawberry colored liq/ fixed oils; mL 70% alc
levo-Carvone; l-1-Methyl-4- spearminty m—alc;
isopropenyl-6-cyclohexen-2-one ins—gly/
231°
150.22/C10H14O/
CH3
CH3C CH2
O
Complete Table
FCC V Flavor Chemicals / l-Carvone / 535
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C4H6O2 1.430–1.440 1.120–1.130
(M-1a)
80.0% of C10H16 Solidification Pt.—40° (Appendix IIB)
(M-1a)
IR Melting Range—between 174° and 179°
(Appendix IIB)
Angular Rotation—between +41° and +43°
(Appendix IIB)
IR 98.0% of C10H14O 1.521–1.526 0.974–0.980 Angular Rotation—between −117° and −130°
(M-1b) (Appendix IIB)
IR 96.0% of C10H14O 1.493–1.497 0.947–0.953 Angular Rotation—between −117° and −130°
(M-1b) (Appendix IIB)
IR 95.0% of C10H16O 1.496–1.499 0.955–0.960 Angular Rotation—between +50° and +60°
(M-1b) (Appendix IIB, 100-mm tube)
IR 97.0% of C10H14O 1.495–1.502 0.956–0.960 Angular Rotation—between −57° and −62°
(M-1b) (Appendix IIB, 100-mm tube)
536 / l-Carvyl Acetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
l-Carvyl Acetate colorless to pale yel liq/ s—alc/
FEMA No. 2250 spearminty 77°–79° (0.1 mm
p-Mentha-6,8-dien-2-yl Acetate Hg)
194.27/C12H18O2/
CH3
O C
O
CH3
H3C CH2
�-Caryophyllene colorless to slightly yel, s—alc, ether; 1 mL in 6
FEMA No. 2252 oily liq/ ins—water/ mL 95% alc
woody, spicy 256° gives clear
soln
204.36/C15H24/
CH3
CH2
H3C
CH3
Cinnamaldehyde yel, strongly refractive m—alc, chloroform, 1 mL in 5
FEMA No. 2286 liq/ ether, fixed and vol mL 60% alc
Cinnamal; Cinnamic Aldehyde cinnamon, burning oils, 1 g in 700 mL
aromatic taste water/
132.16/C9H8O/
CH CHCHO
248°
Cinnamic Acid white cryst scales/ s—acetic acid, 1 g in 7 mL
FEMA No. 2288 honey-floral acetone, benzene, 95% alc
3-Phenylpropenoic Acid most fixed oils, 1 g
in 2000 mL water/
148.16/C9H8O2/
CH CHCOOH
300°
Cinnamyl Acetate colorless to slightly yel m—alc, chloroform, 1 mL in 5
FEMA No. 2293 liq/ ether, most fixed oils; mL 70% alc
sweet, balsamic, floral ins—gly, water/
264°
176.22/C11H12O2/
CH CHCH2OOCCH3
Cinnamyl Alcohol white to slightly yel cryst s—most fixed oils, 1 g in 1 mL
FEMA No. 2294 solid/ prop glycol; 70% alc
Cinnamic Alcohol balsamic ins—gly/ remains in
258° soln to 10
134.18/C9H10O/
CH CHCH2OH
mL
Cinnamyl Butyrate colorless to pale yel liq/ 300° 1 mL in 1
FEMA No. 2296 fruity, balsamic mL 95% alc204.27/C13H16O2/
CH CHCH2OOC(CH2)2CH3
Cinnamyl Cinnamate mixture of (Z) and (E) 370° 1 mL in 1
FEMA No. 2298 isomers; low-melting mL 95% alc
solid
264.32/C18H16O2/
CH CHCOOCH2CH CH
Complete Table
FCC V Flavor Chemicals / Cinnamyl Cinnamate / 537
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C12H18O2 1.0 1.473–1.479 0.964–0.970 Angular Rotation—between −90° and −120°
(M-1b) (Appendix IIB)
IR 80.0% of C15H24 1.498–1.504 0.897–0.910 Angular Rotation—between −5° and −10°
(M-1a) (Appendix IIB, 100-mm tube)
Phenols—3.0% (M-1b)
IR 98.0% of C9H8O 10.0 1.619–1.623 1.046–1.050 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 99.0% of C9H8O2 (after Melting Range—NLT 130° (Appendix IIB)
drying) Residue on Ignit.—0.05% (Appendix IIC)
(M-3b)
IR 98.0% of C11H12O2 1.0 1.539–1.543 1.050–1.054
(M-1b)
IR 98.0% of C9H10O Aldehydes—1.5% (M-1b)
(M-1b) Chlorinated Cmpds.—passes test (Appendix
VI)
Solidification Pt.—NLT 31° (Appendix IIB)
IR 96.0% of C13H16O2 1.0 1.525–1.530 1.010–1.015
(M-1b)
IR 95.0% of C18H16O2 2.0
(M-1b)
538 / Cinnamyl Formate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Cinnamyl Formate colorless to slightly yel m—alc, chloroform, 1 mL in 2
FEMA No. 2299 liq/ ether, most fixed oils; mL 80% alc
green, herbaceous, ins—water/ gives clear
balsamic 250° soln
162.19/C10H10O2/
CH CHCH2OOCH
Cinnamyl Isobutyrate colorless to pale yel liq/ 254° 1 mL in 1
FEMA No. 2297 sweet, balsamic, fruity mL 95% alc
204.27/C13H16O2/
CHCH2OOCHCH3
CH3
CH
Cinnamyl Isovalerate colorless to slightly yel m—alc, chloroform, 1 mL in 1
FEMA No. 2302 liq/ most fixed oils, ether; mL 90% alc
spicy, floral, fruity ins—gly, prop glycol,
water/
218.30/C14H18O2/
CH CHCH2OOCCH2CHCH3
CH3313°
Cinnamyl Propionate colorless to pale yel liq/ m—alc, chloroform,
FEMA No. 2301 spicy, fruity, balsamic ether, most fixed oils;
ins—gly, prop glycol,
water/
190.24/C12H14O2/
CH CHCH2OOCCH2CH3
289°
Citral pale yel liq/ s—most fixed oils, 1 mL in 7
FEMA No. 2303 strong, lemon min oil, prop glycol; mL 70% alc
Mixture of Geranial [(E)-3,7- ins—gly/
dimethyl-2,6-octadien-1-al] and 228°
Neral [the (Z) isomer]
152.24/C10H16O/
CO
H
CH
O(a) Geranial
(b) Neral
Citronellal colorless to slightly yel s—alc, most fixed 1 mL in 5
FEMA No. 2307 liq/ oils; mL 70% alc
3,7-Dimethyl-6-octen-1-al intense lemon-citronella- ss—prop glycol; remains clear
rose ins—gly, water/ on dilution
206°
154.25/C10H18O/
CH
O
Citronellol colorless, oily liq/ s—most fixed oils, 1 mL in 2
FEMA No. 2309 rosy prop glycol; mL 70% alc
3,7-Dimethyl-6-octen-1-ol ss—water; remains in
ins—gly/ soln to 10
225° mL
156.27/C10H20O/
CH2OH
Citronellyl Acetate colorless liq/ s—alc, most fixed 1 mL in 9
FEMA No. 2311 fruity oils; mL 70% alc
3,7-Dimethyl-6-octen-1-yl ins—gly, prop glycol,
Acetate water/
229°
198.31/C12H22O2/
CH2OCO
CH3
Complete Table
FCC V Flavor Chemicals / Citronellyl Acetate / 539
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 92.0% of C10H10O2 3.0 1.550–1.556 1.077–1.082 Cinnamyl Alcohol—8.0% (M-1a)
(M-1a)
IR 96.0% of C13H16O2 3.0 1.523–1.528 1.006–1.009
(M-1b)
IR 95.0% of C14H18O2 3.0 1.518–1.524 0.991–0.996
(one major isomer)
(M-1b)
IR 98.0% of C12H14O2 3.0 1.532–1.537 1.029–1.035
(one isomer)
(M-1b)
IR 96.0% of C10H16O 1.486–1.490 0.885–0.891
(sum of neral and
geranial)
(M-1b)
IR 85.0% of aldehydes as 3.0 1.446–1.456 0.850–0.860 Angular Rotation—between −1° and +11°
C10H18O (Appendix IIB, 100-mm tube)
(one isomer)
(M-1b)
IR 90.0% of total alcohols 1.454–1.462 0.850–0.860 Aldehydes—1.0% as citronellal (M-2d; 5 g/
as C10H20O 66.08)
(Appendix VI; 1.2 g/ Esters—1.0% as citronellyl acetate (Appendix
78.13) VI; 5 g/99.15)
IR 92.0% of total esters as 1.0 1.440–1.450 0.883–0.893
C12H22O2
(Appendix VI; 1.4 g/
99.15)
540 / Citronellyl Butyrate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Citronellyl Butyrate colorless liq/ m—alc, ether, most 1 mL in 6
FEMA No. 2312 strong, fruity-rosy fixed oils, mL 80% alc
3,7-Dimethyl-6-octen-1-yl chloroform; gives clear
Butyrate ins—water/ soln
245°
226.36/C14H26O2/
CH2OCO
CH2CH2CH3
Citronellyl Formate colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2314 strong, fruity, floral oils; mL 80% alc
3,7-Dimethyl-6-octen-1-yl ss—prop glycol; remains in
Formate ins—gly, water/ soln to 10
235° mL
184.28/C11H20O2/
CH2OCO
H
Citronellyl Isobutyrate colorless liq/ m—alc, chloroform, 1 mL in 6
FEMA No. 2313 fruity-rosy ether, most fixed oils; mL 80% alc
3,7-Dimethyl-6-octen-1-yl ins—water/ gives clear
Isobutyrate 249° soln
226.36/C14H26O2/
CH2OCO
CH(CH3)2
Citronellyl Propionate colorless liq/ m—alc, most fixed 1 mL in 4
FEMA No. 2316 fruity-rosy oils; mL 80% alc
Citronellyl Propanoate; 3,7- ins—water/ gives clear
Dimethyl-6-octen-1-yl 242° soln
Propionate
212.33/C13H24O2/
CH2OCO
CH2CH3
p-Cresyl Acetate colorless liq/ s—most fixed oils, 1 mL in 2
FEMA No. 3073 strong, floral prop glycol; mL 70% alc
p-Methylphenyl Acetate; ins—gly/
p-Tolyl Acetate 212°
150.18/C9H10O2/
H3C OOCCH3
Cuminic Aldehyde colorless to pale yel liq/ s—alc, ether; 1 mL in 4
FEMA No. 2341 strong, pungent, cumin ins—water/ mL 70% alc
Cuminal; Cuminaldehyde; oil 236°
p-Cuminic Aldehyde;
p-Isopropylbenzaldehyde
148.20/C10H12O/
CH3CH CHO
CH3
Cyclamen Aldehyde colorless to pale yel liq/ s—most fixed oils; 1 mL in 3
FEMA No. 2743 strong, floral ins—gly, prop glycol/ mL 80% alc
2-Methyl-3-(p- 270°
isopropylphenyl)propionaldehyde
190.29/C13H18O/
CH CH2
CH3
CH CHO
CH3
CH3
Cyclohexyl Acetate colorless to pale yel liq/ s—alc/
FEMA No. 2349 green, fruity 174°142.20/C8H14O2/
OCOCH3
Complete Table
FCC V Flavor Chemicals / Cyclohexyl Acetate / 541
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 90.0% of total esters as 1.0 1.444–1.448 0.873–0.883
C14H26O2
(Appendix VI; 1.5 g/
113.2)
IR 86.0% of total esters as 3.0 1.443–1.452 0.890–0.903
C11H20O2
(Appendix VI; 1.0 g/
92.14)
IR 92.0% of total esters as 1.0 1.440–1.448 0.870–0.880
C14H26O2
(Appendix VI; 1.5 g/
113.2)
IR 90.0% of total esters as 1.0 1.443–1.449 0.877–0.886
C13H24O2
(Appendix VI; 1.2 g/
95.12)
IR 98.0% of C9H10O2 1.0 (phenol 1.499–1.502 1.044–1.050 Free Cresol—1.0% (M-17)
(one isomer) red TS)
(M-1b)
IR 95.0% of C10H12O 5.0 1.528–1.534 0.975–0.980 Chlorinated Cmpds.—passes test (Appendix
(M-2a) VI)
IR 90.0% of C13H18O 5.0 1.503–1.508 0.946–0.952
(sum of two isomers;
major isomer 85.0%
min)
(M-1b)
98.0% of C8H14O2 1.0 1.436–1.441 0.966–0.970
(M-1b)
542 / p-Cymene / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
p-Cymene colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2356 kerosene ins—water, prop mL 95% alc
glycol/
177°
134.22/C10H14/
CH(CH3)2
CH3
(E),(E)-2,4-Decadienal yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3135 powerful, oily, chicken oils; mL 95%
trans,trans-2,4-Decadienal fat ins—water/ ethanol
104° (7 mm Hg)
152.24/C10H16O/
C C
CH3(CH2)4
H
H
C CH CHO
H
�-Decalactone colorless liq/ vs—alc, prop glycol, 1 mL in 1
FEMA No. 2361 coconut-fruity, buttery on veg oils; mL 95%
dilution ins—water/ ethanol
281°
170.25/C10H18O2/
OCH3(CH2)4 O
�-Decalactone colorless to pale yel liq/ s—prop glycol; veg 1 mL in 1
FEMA No. 2360 fruity, peach oils; mL 95% alc
4-Hydroxydecanoic Acid ins—water/
170.25/C10H18O2/
CH3(CH2)5CHCH2CH2C O
OLactone 281°
Decanal colorless to light yel liq/ m—alc, most fixed
FEMA No. 2362 fatty, floral-orange on oils, prop glycol156.27/C10H20O/
CH3(CH2)8CHOAldehyde C-10; Capraldehyde dilution (may be turbid);
ins—gly, water/
209°
(E)-2-Decenal slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2366 orange, wax oils; mL 95%
trans-2-Decenal ins—water/ ethanol
229°
154.25/C10H18O/
C CCHOH
CH3(CH2)6 H
(Z)-4-Decenal colorless to slightly yel s—alc, most fixed 1 mL in 1
FEMA No. 3264 liq/ oils; mL 95%
cis-4-Decenal orange, fatty ins—water/ ethanol
78°–80° (10 mm Hg)
154.25/C10H18O/
C CH
CH3(CH2)4
H
(CH2)2CHO
Decyl Alcohol colorless liq/ s—alc, ether, min oil, 1 mL in 3
FEMA No. 2365 floral, waxy, fruity prop glycol, most mL 60% alc
158.28/C10H22O/
CH3(CH2)8CH2OHAlcohol C-10; 1-Decanol fixed oils;
ins—gly, water/
233°
Complete Table
FCC V Flavor Chemicals / Decyl Alcohol / 543
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C10H14 1.489–1.491 0.853–0.855
(M-1a)
IR 89.0% of C10H16O 1.514–1.519 0.866–0.876
(sum of two isomers)
(M-1a)
IR 98.0% of C10H18O2 5.0 (Appendix 1.454–1.459 0.964–0.971
(M-1b) VII; A.V.
Method II)
IR 95.0% of C10H18O2 1.0 1.447–1.451 0.949–0.954
(M-1a)
IR 92.0% of C10H20O 10.0 1.426–1.430 0.823–0.832
(M-1b)
IR 92.0% of C10H18O 1.452–1.457 0.836–0.846
(one isomer)
(M-1a)
IR 90.0% of C10H18O 1.442–1.447 0.843–0.850
(M-1a)
IR 98.0% of C10H22O 1.0 1.435–1.439 0.826–0.831 Solidification Pt.—NLT 5° (Appendix IIB)
(M-1b)
544 / Diacetyl / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Diacetyl yel to yel-green liq/ s—gly, water;
FEMA No. 2370 powerful, buttery in very m—alc, most fixed
2,3-Butanedione; dilute soln oils, prop glycol/
86.09/C4H6O2/
CH3 C C
O
CH3
O
Dimethyldiketone; 88°
Dimethylglyoxal
Dibenzyl Ether colorless to pale yel liq/ 298° 1 mL in 1
FEMA No. 2371 earthy mL 95% alc198.26/C14H14O/
O
1,2-Di[(1′-ethoxy)ethoxy]propane colorless to pale yel liq/
FEMA No. 3534 odorless when pure
CH2 O CH O CH2CH3
CH3
CH O CH O CH2CH3
CH3 CH3
220.31/C11H24O4/
Diethyl Malonate colorless liq/ s—most fixed oils, 1 mL in 1.5
FEMA No. 2375 slight, fruity prop glycol; mL 60% alc
Ethyl Malonate; Malonic Ester ss—alc, water;
ins—gly, min oil/
200°
160.17/C7H12O4/
CH2
COOCH2CH3
COOCH2CH3
Diethyl Sebacate colorless to slightly yel m—alc, ether, other
FEMA No. 2376 liq/ org solvents, most258.36/C14H26O4/
C2H5OOC(CH2)8COOC2H5Ethyl Sebacate faint, winy, fruity fixed oils;
ins—water/
302°
Diethyl Succinate colorless, mobile liq/ s—1 mL in 50 mL
FEMA No. 2377 faint, winy, ethereal water;174.20/C8H14O4/
C2H5OOCCH2CH2COOC2H5Ethyl Succinate m—alc, ether, most
fixed oils/
217°
Dihydrocarveol almost colorless, oily liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2379 spearmint oils; mL 95%
ins—water/ ethanol
225°
154.25/C10H18O/
H3C CH2
OH
CH3
Complete Table
FCC V Flavor Chemicals / Dihydrocarveol / 545
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C4H6O2 1.393–1.397 0.979–0.985 Solidification Pt.—between –2.0° and –4.0°
(M-1b) (Appendix IIB)
IR 98.0% of C14H14O 1.557–1.565 1.039–1.044 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
97.0% of C11H24O4 0.1 1.409–1.413 0.915–0.925
(M-1b)
IR 98.0% of C7H12O4 1.0 1.413–1.416 1.053–1.056
(M-1b)
IR 98.0% of C14H26O4 1.0 1.435–1.438 0.960–0.965
(M-1b)
IR 99.0% of C8H14O4 2.0 1.036–1.040 1.419–1.423 Diethyl Maleate—0.03% (M-1b)
(M-1a) Water—0.05% (Appendix IIB, KF)
96.0% of C10H18O 1.477–1.481 0.921–0.926
(sum of two isomers)
(M-1a)
546 / d-Dihydrocarvone / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
d-Dihydrocarvone almost colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3565 herbaceous, spearmint oils; mL 95% alc
d-2-Methyl-5-(1-methylethenyl)- ins—water/
cyclohexanone 222°
154.24/C10H16O/
CH3
O
H3C CH2
2,6-Dimethoxy Phenol white to brown cryst/ ss—prop glyc, veg 1 g in 2 mL
FEMA No. 3137 smoky oils; 95% ethanol
ins—water/
262°
154.17/C8H10O3/
CH3O
OH
OCH3
Dimethyl Anthranilate pale yel liq with pale s—most fixed oils; 1 mL in 3
FEMA No. 2718 blue fluorescence/ ss—prop glycol; mL 80% alc
Methyl N-Methyl Anthranilate grape ins—gly, water/ remains in
256° soln to 10
165.19/C9H11NO2/
NHCH3
COOCH3
mL
Dimethyl Benzyl Carbinol white cryst solid; may s—min oil, most 1 mL in 3
FEMA No. 2393 exist in supercooled form fixed oils, prop mL 50% alc
�,�-Dimethylphenethyl Alcohol as colorless to pale yel glycol; remains in
liq/ ins—gly soln to 10
150.22/C10H14O/
CH2COH
CH3
CH3 floral mL
Dimethyl Benzyl Carbinyl colorless liq; solidifies at s—most fixed oils; 1 mL in 4
Acetate room temp/ ss—prop glycol; mL 70% alc
FEMA No. 2392 floral, fruity ins—water/
�,�-Dimethylphenethyl Acetate 250°
192.26/C12H16O2/
CH2COOCCH3
CH3
CH3
Dimethyl Benzyl Carbinyl almost colorless liq/ s—alc, most fixed 1 mL in 1
Butyrate prune oils; mL 95%
FEMA No. 2394 ins—prop glycol, ethanol
�,�-Dimethylphenethyl Butyrate water/CH2COOC(CH2)2CH3
CH3
CH3
220.31/C14H20O2/
237°–255°
3,4-Dimethyl 1,2- pale yel to orange cryst/ ss—prop glycol; 1 g in 3 mL
Cyclopentandione maple ins—veg oils, water/ 95% ethanol
FEMA No. 3268 142°
126.16/C7H10O2/
O
O
CH3H3C
Complete Table
FCC V Flavor Chemicals / 3,4-Dimethyl 1,2-Cyclopentandione / 547
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 92.0% of C10H16O 1.470–1.474 0.923–0.928 Angular Rotation—between +14.0° and +22°
(sum of two isomers) (Appendix IIB)
(M-1a)
IR 98.0% of C8H10O3 Melting Range—between 53.0° and 56.0°
(M-1b) (Appendix IIB)
98.0–101.3% of total 1.577–1.583 1.124–1.132 Solidification Pt.—NLT 14° (Appendix IIB)
esters as C9H11NO2
(Appendix VI; 1.1 g/
82.60)
IR 97.0% of C10H14O 1.0 1.514–1.517 (20°, 0.972–0.977 Chlorinated Cmpds.—passes test (Appendix
(M-1b) as supercooled VI)
liq) Solidification Pt.—NLT 22° (Appendix IIB)
IR 98.0% of C12H16O2 1.0 1.490–1.495 0.995–1.002 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Solidification Pt.—NLT 28° (Appendix IIB)
IR 95.0% of C14H20O2 1.484–1.489 0.960–0.981
(M-1b)
IR 95.0% of C7H10O2 Melting Range—between 64.0° and 72.0°
(M-1b) (Appendix IIB)
548 / 2,6-Dimethyl-5-heptenal / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
2,6-Dimethyl-5-heptenal pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2389 melon ss—prop glycol; mL 95% alc
ins—water/
140.23/C9H16O/
CH3C
CH3
CH(CH2)2CHCHO
CH3
116°–124° (100 mm
Hg)
3,7-Dimethyl-1-octanol colorless liq/ s—most fixed oils, 1 mL in 3
FEMA No. 2391 sweet, rose prop glycol; mL 70% alc
Dimethyl Octanol; ins—gly/
Tetrahydrogeraniol 213°
158.28/C10H22O/
CH2OH
2,3-Dimethylpyrazine colorless to slightly yel m—org solvents, 1 mL in 1
FEMA No. 3271 liq/ water/ mL 95%
nutty, cocoa 156° ethanol
108.14/C6H8N2/
N
N CH3
CH3
2,5-Dimethylpyrazine colorless to slightly yel m—water, org
FEMA No. 3272 liq/ solvents/
earthy, potato 155°
108.14/C6H8N2/
N
N
CH3
H3C
2,6-Dimethylpyrazine white to yel, lumpy cryst/ s—water, org
FEMA No. 3273 nutty, coffee solvents/
155°
108.14/C6H8N2/
N
N
CH3H3C
2,5-Dimethylpyrrole colorless to yellow, oily vs—alc, ether;
liq/ vss—water/
burnt 165°
95.14/C6H9N/
N CH3H3C
H
Dimethyl Succinate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2396 mild, fruity oils; mL 95% alc
ins—water/
146.14/C6H10O4/
CH3OC COCH3(CH2)2
O O
196°
Dimethyl Sulfide colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2746 disagreeable, intense oils; mL 95% alc
62.14/C2H6S/
CH3SCH3Methyl Sulfide; Thiobismethane boiled cabbage ins—water/
37°
Complete Table
FCC V Flavor Chemicals / Dimethyl Sulfide / 549
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 85.0% of C9H16O 5.0 1.442–1.447 0.848–0.854
(M-2d; 1 g/14.01)
IR 90.0% of total alcohols 1.0 1.435–1.445 0.826–0.842
as C10H22O (Appendix
VI; 1.2 g/79.15)
IR 95.0% of C6H8N2 1.506–1.509 1.000–1.022 Distillation Range—between 152° and 157°
(M-1a) (Appendix IIB)
Solidification Pt.—11° to 13° (Appendix IIB)
Tri- and Tetrapyrazines—5% (by GC assay)
Water—0.5% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 99.0% of C6H8N2 1.497–1.501 0.980–1.000 Solidification Pt.—between 12° and 17°
(M-1a) (Appendix IIB)
Water—0.5% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 98.0% of C6H8N2 0.965 (50°) Melting Range—between 35° and 40°
(M-1a) (Appendix IIB)
Residue on Ignit.—0.1% (Appendix IIC)
Water—0.5% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 98.0% of C6H9N 1.503–1.506 0.932–0.942 Water—0.5% (Appendix IIB, KF; use freshly
(M-1a) dist. pyridine as solvent)
98.0% of C6H10O4 1.0 1.418–1.421 1.114–1.118
(M-1b)
IR 99.0% of C2H6S 1.431–1.441 0.842–0.847
(M-1a)
550 / Diphenyl Ether / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Diphenyl Ether colorless to white to pale s—veg oils; 1 g in 2 mL
FEMA No. 3667 yel liq/ vss—water/ 95% ethanol
Diphenyl Oxide rose 259°
170.21/C12H10O/
O
�-Dodecalactone colorless to yel liq/ vs—alc, prop glycol, 1 mL in 1
FEMA No. 2401 coconut-fruity, buttery on veg oils; mL 95%
dilution ins—water/ ethanol
140°–141° (1 mm
198.31/C12H22O2/
O OCH3(CH2)6Hg)
�-Dodecalactone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2400 fruity, peach, pear oils; mL 95% alc
4-Hydroxydodecanoic Acid ins—water/
198.31/C12H22O2/
CH3(CH2)6CH(CH2)3C O
Lactone 131° (1.5 mm Hg)
(E)-2-Dodecen-1-al slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2402 fatty, citrus oils; mL 95%
trans-2-Dodecen-1-al ins—water/ ethanol
272°
182.31/C12H22O/
C CCH3(CH2)8
H
H
CHO
Estragole colorless to light yel liq/ s—alc; 1 mL in 6
FEMA No. 2411 anise ins—water/ mL 80% alc
p-Allylanisole; Methyl Chavicol 216° gives clear
solnCH3O CH2CH CH2
148.20/C10H12O/
Ethone white to pale yel cryst/ 1 g in 7 mL
FEMA No. 2673 nutty, maple 95% alc
1-(p-Methoxyphenyl)-1-penten-
3-one
190.24/C12H14O2/
CH3O CH CHCOCH2CH3
Ethyl Acetate colorless liq; vol at low m—alc, ether, gly,
FEMA No. 2414 temp; flammable/ most fixed oils, volCH3COOC2H5
88.11/C4H8O2/
fragrant, acetous, ethereal oils, 1 mL in 10 mL
water/
54°
Ethyl Acetoacetate colorless to very light m—alc, ether, ethyl
FEMA No. 2415 yel, mobile liq/ acetate, 1 mL in 12
Acetoacetic Ester; Ethyl 3- fruity mL water/
Oxybutanoate 181°
130.14/C6H10O3/
CH3C CHCOOC2H5
OH
CH3COCH2COOC2H5
Ethyl Acrylate colorless, mobile liq; m—alc, ether, 1 mL
FEMA No. 2418 lachrymator/ in 50 mL water/100.12/C5H8O2/
CH2 CHCOOC2H5 intense, harsh, fruity 99°
Complete Table
FCC V Flavor Chemicals / Ethyl Acrylate / 551
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 99.0% of C12H10O freezes 1.070–1.074 Melting Range—between 26.0° and 30.0°
(M-1b)
IR 98.0% of C12H22O2 8.0 (Appendix 1.458–1.461 0.942–0.950 Saponification Value—between 278 and 286
(sum of two isomers; � VII; A.V. (Appendix VI, 1-g sample)
isomer 95.0% min) Method II)
(M-1a)
97.0% of C12H22O2 1.0 1.451–1.456 0.933–0.938
(M-1a)
IR 93.0% of C12H22O 1.454–1.460 0.839–0.849
(M-1a)
IR 95.0% of C10H12O 1.519–1.524 0.960–0.968
(M-1a)
IR 98.0% of C12H14O2 Solidification Pt.—min 59.0° (Appendix IIB)
(M-1b)
IR 99.0% of C4H8O2 5.0 1.370–1.375 0.894–0.898 Distillation Range—between 76° and 77.5°
(M-1b) (bromocresol (Appendix IIB)
purple TS) Methyl Cmpds.—passes test (M-10)
Readily Carb. Subs.—passes test (M-12)
Residue on Evap.—0.02% (M-16, 10-g
sample, 105°)
IR 97.5% of C6H10O3 5.0 1.418–1.421 1.022–1.027
(M-1b) (bromocresol
purple TS)
IR 99.5% of C5H8O2 5.0 0.916–0.919 Antioxidants—0.022% (M-6)
(M-1b) Water—0.05% (Appendix IIB, KF)
552 / Ethyl p-Anisate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Ethyl p-Anisate colorless to slightly yel s—alc, chloroform, 1 mL in 7
FEMA No. 2420 liq/ ether; mL 60% alc
Ethyl p-Methoxybenzoate light, fruity, anise ins—water/ gives clear
270° soln
180.20/C10H12O3/
CH3O COOC2H5
Ethyl Anthranilate colorless to amber- s—alc, most fixed 1 mL in 2
FEMA No. 2421 colored liq/ oils, prop glycol/ mL 70% alc
Ethyl o-Aminobenzoate floral, orange blossom 267°
165.19/C9H11NO2/
NH2
COOC2H5
Ethyl Benzoate colorless liq/ s—alc, most fixed 1 mL in 6
FEMA No. 2422 heavy, floral, fruity oils, prop glycol; mL 60% alc
ins—gly, water/
212°
150.18/C9H10O2/
COOC2H5
Ethyl Benzoyl Acetate colorless to light yel liq/ 265°
FEMA No. 2423 whiskey
COCH2CO2CH2CH3
192.21/C11H12O3/
Ethyl-(E)-2-butenoate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3486 sweet, ethereal oils; mL 95% alc114.14/C6H10O2/
CH3 CH CH COO CH2 CH3Ethyl-trans-2-butenoate; Ethyl ins—water/
Crotonate 136°
2-Ethylbutyraldehyde colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2426 pungent in 50 mL water/
117°
100.16/C6H12O/
C2H5
CH3CH2CHCHO
Ethyl Butyrate colorless liq/ s—most fixed oils, 1 mL in 3
FEMA No. 2427 banana-pineapple prop glycol; mL 60% alc116.16/C6H12O2/
CH3CH2CH2COOC2H5 ins—gly/
121°
2-Ethylbutyric Acid colorless liq/ m—alc, ether, 1 mL
FEMA No. 2429 mildly rancid in 65 mL water/
99° (18 mm Hg)
116.16/C6H12O2/
C2H5
CHCOOHCH3CH2
Ethyl Cinnamate colorless, oily liq/ m—alc, ether, most 1 mL in 5
FEMA No. 2430 faint, cinnamon fixed oils; mL 70% alc
Ethyl 3-Phenylpropenate ins—gly, water/
272°
176.22/C11H12O2/
CH CHCOOC2H5
Complete Table
FCC V Flavor Chemicals / Ethyl Cinnamate / 553
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C10H12O3 1.0 1.522–1.526 1.101–1.104
(M-1b)
IR 96.0% of total esters as 1.0 1.563–1.566 1.115–1.120 Solidification Pt.—NLT 13° (Appendix IIB)
C9H11NO2
(Appendix VI; 1.5 g/
82.6)
IR 98.0% of C9H10O2 1.0 1.502–1.506 1.043–1.046 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
88.0% of C11H12O3 2.0 1.528–1.533 1.107–1.120
(M-1b)
IR 97.0% of C6H10O2 5.0 1.422–1.427 0.913–0.920
(M-1b)
95.0% of C6H12O 2.0 1.398–1.404 0.808–0.814 Distillation Range—NLT 95% between 100°
(M-1b) and 120° (Appendix IIB)
IR 98.0% of C6H12O2 1.0 1.391–1.394 0.870–0.877
(M-1b)
IR 98.0% of C6H12O2 1.408–1.418 0.917–0.922 Distillation Range—between 190° and 200°
(M-3b) (Appendix IIB)
Water—0.2% (Appendix IIB, KF)
IR 98.0% of C11H12O2 1.0 1.558–1.561 1.045–1.051
(M-1b)
554 / Ethyl Decanoate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Ethyl Decanoate colorless liq/ s—most fixed oils; 1 mL in 4
FEMA No. 2432 oily, brandy ins—gly, prop glycol/ mL 80% alc200.32/C12H24O2/
CH3(CH2)8COOC2H5Ethyl Caprate 243°
2-Ethyl-3,5(6)-dimethylpyrazine colorless to slightly yel s—prop glycol, veg
FEMA No. 3149 liq/ oils/
roasted cocoa 180°–181°
136.20/C8H12N2/
N
N CH2CH3
CH3
H3C
Ethylene Brassylate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 3543 sweet, musky ins—prop glycol, mL 95% alc
water/
138°–142° (1 mm
Hg)
270.37/C15H26O4/
CH2
(CH2)9
CH2
C O CH2
C O CH2
O
O
2-Ethyl Fenchol pale yel liq/ s—alc, prop glycol,
FEMA No. 3491 sharp, camphoraceous, most fixed oils;
earthy ins—water/
105° (15 mm Hg)
182.31/C12H22O/
OH
CH3
CH3
CH2CH3
CH3
Ethyl Formate colorless liq/ s—most fixed oils, 1 mL in 5
FEMA No. 2434 sharp, rum prop glycol, water mL 50% alc74.08/C3H6O2/
HCOOC2H5 (decomp);
ss—min oil;
ins—gly/
54°
4-Ethyl Guaiacol colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2436 warm, spicy, medicinal oils; mL 95% alc
4-Hydroxy-3- ins—water/
methylethylbenzene 235°
152.19/C9H12O2/
CH2CH3
OH
OCH3
Ethyl Heptanoate colorless liq/ ss—prop glycol; 1 mL in 3
FEMA No. 2437 winy-brandy m—alc, chloroform, mL 70% alc158.24/C9H18O2/
CH3(CH2)5COOC2H5Ethyl Heptoate most fixed oils;
ins—gly/
189° (72% water
azeotrope, 98.5°)
Complete Table
FCC V Flavor Chemicals / Ethyl Heptanoate / 555
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C12H24O2 1.0 1.424–1.427 0.862–0.867
(M-1b)
IR 95.0% of C8H12N2 1.500–1.503 0.950–0.970 Water—0.1% (Appendix IIB, KF; use freshly
(M-1a) dist. pyridine as solvent)
IR 95.0% of C15H26O4 1.0 1.468–1.473 1.040–1.045
(M-1a)
IR 95.0% of C12H22O 1.470–1.491 0.946–0.967
(M-1a)
IR 95.0% of C3H6O2 1.359–1.363 0.916–0.921 Acidity—0.2% (M-5)
(M-1b)
IR 98.0% of C9H12O2 1.525–1.530 1.061–1.064
(M-1a)
IR 98.0% of C9H18O2 1.0 1.411–1.415 0.867–0.872
(M-1b)
556 / Ethyl Hexanoate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Ethyl Hexanoate colorless liq/ s—most fixed oils; 1 mL in 2
FEMA No. 2439 winy ss—prop glycol; mL 70% alc144.21/C8H16O2/
CH3(CH2)4COOC2H5Ethyl Caproate; Ethyl Capronate ins—gly/
166°
2-Ethyl Hexanol colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3151 green oils; mL 95%130.23/C8H18O/
CH3(CH2)3CH(C2H5)CH2OH2-Ethyl-1-hexanol ins—water/ ethanol
183°
5-Ethyl 3-Hydroxy 4-Methyl pale yel to yel liq/ s—prop glycol, veg 1 mL in 2
2(5H)-Furanone maple oils; mL 95%
FEMA No. 3153 ss—water/ ethanol
Maple Furanone 83° (0.5 mm Hg)
O O
OH
142.15/C7H10O3/
Ethyl Isobutyrate colorless liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2428 fruity oils; mL 95% alc116.16/C6H12O2/
(CH3)2CHCOOC2H5 ins—water/
112°–113°
Ethyl Isovalerate colorless liq/ s—prop glycol, 1 mL
FEMA No. 2463 strong, fruity, vinous, in 350 mL water;130.19/C7H14O2/
(CH3)2CHCH2COOC2H5Ethyl 3-Methylbutyrate apple on dilution m—alc, most fixed
oils/
135°
Ethyl Lactate colorless liq/ vs—alc, ether,
FEMA No. 2440 cheesy chloroform, water/118.13/C5H10O3/
CH3CHOHCOOC2H5Ethyl 2-Hydroxypropionate 154°
Ethyl Laurate colorless, oily liq/ m—alc, chloroform, 1 mL in 9
FEMA No. 2441 fruity-floral ether; mL 80% alc228.38/C14H28O2/
CH3(CH2)10COOC2H5Ethyl Dodecanoate ins—water/ gives clear
269° soln
Ethyl Levulinate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2442 fruity, apple, green oils; mL 95% alc144.17/C7H12O3/
CH3COCH2CH2COOC2H5 ins—water/
93°–94° (18 mm Hg)
Ethyl 2-Methylbutyrate colorless liq/ s—alc, prop glycol; 1 mL in 1
FEMA No. 2443 strong, green-fruity, apple vss—water; mL 95%
m—most fixed oils/ ethanol
130.19/C7H14O2/
CH3CH2OOCCHCH2CH3
CH3 133°
Complete Table
FCC V Flavor Chemicals / Ethyl 2-Methylbutyrate / 557
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C8H16O2 1.0 1.406–1.409 0.867–0.871
(M-1b)
97.0% of C8H18O 1.429–1.434 0.830–0.834
(M-1b)
IR 95% of C7H10O3 1.488–1.493
(M-1b)
IR 98.0% of C6H12O2 1.0 1.385–1.391 0.862–0.868
(M-1b)
IR 98.0% of C7H14O2 2.0 1.395–1.399 0.862–0.866
(one isomer)
(M-1b)
IR 98.0% of C5H10O3 1.0 1.410–1.420 1.029–1.032
(M-1b)
IR 98.0% of C14H28O2 1.0 1.430–1.434 0.858–0.863
(M-1b)
IR 98.0% of C7H12O3 2.0 1.420–1.425 1.007–1.013
(M-1b)
95.0% of C7H14O2 2.0 1.393–1.400 0.863–0.870
(one isomer)
(M-1b)
558 / Ethyl 2-Methylpentanoate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Ethyl 2-Methylpentanoate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 3488 fruity ins—prop glycol, mL 95%144.21/C8H16O2/
CH3CH2CH2CH(CH3)CO2CH2CH3 water/ ethanol
153°
Ethyl Methylphenylglycidate colorless to pale yel liq/ s—most fixed oils, 1 mL in 3
FEMA No. 2444 strong, fruity, strawberry prop glycol; mL 70% alc
Aldehyde C-16; Strawberry ins—gly/
Aldehyde 272°–275°
206.24/C12H14O3/
C CHCOOCH2CH3
CH3
O
2-Ethyl-3-methylpyrazine colorless to slightly yel s—prop glycol, veg 1 mL in 1
FEMA No. 3155 liq/ oils, water/ mL 95%
strong, raw potato 57° (10 mm Hg) ethanol
122.17/C7H10N2/
N
N CH2CH3
CH3
Ethyl 3-Methylthiopropionate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3343 onion, fruity, sweet oils; mL 95%148.23/C6H12O2S/
CH3SCH2CH2COOCH2CH3 ins—water/ ethanol
89°–91° (15 mm Hg)
Ethyl Myristate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2445 waxy ins—prop glycol, mL 95% alc256.43/C16H32O2/
CH3(CH2)12COOC2H5 water/
178°–180° (12 mm
Hg)
Ethyl Nonanoate colorless liq/ m—alc, prop glycol; 1 mL in 10
FEMA No. 2447 fatty, fruity, cognac ins—water/ mL 70% alc186.29/C11H22O2/
CH3(CH2)7COOC2H5Ethyl Pelargonate 229°
Ethyl Octanoate colorless liq/ s—most fixed oils; 1 mL in 4
FEMA No. 2449 winy-brandy, fruity-floral ss—prop glycol; mL 70% alc172.27/C10H20O2/
CH3(CH2)6COOC2H5Ethyl Caprylate; Ethyl Octoate ins—gly, water/
209°
Ethyl Oleate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2450 floral ins—prop glycol, mL 95%310.52/C20H38O2/
CH3(CH2)7CH CH(CH2)7COOCH2CH3Ethyl 9-Octadecenoate water/ ethanol
205°–208°
Ethyl Oxyhydrate (so-called) colorless liq/ m—alc, gly, prop
FEMA No. 2996 sharp rum glycol;
Rum Ether, So-Called ins—veg oils, water
Complete Table
FCC V Flavor Chemicals / Ethyl Oxyhydrate (so-called) / 559
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C8H16O2 1.0 1.401–1.404 0.859–0.865
(M-1b)
IR 98.0% of C12H14O3 2.0 1.504–1.513 1.086–1.096
(sum of two isomers)
(M-1b)
IR 98.0% of C7H10N2 1.502–1.505 0.978–0.988 Water—0.1% (Appendix IIB, KF; use freshly
(M-1a) dist. pyridine as solvent)
IR 99.0% of C6H12O2S 1.457–1.463 1.030–1.035
(M-1b)
IR 98.0% of C16H32O2 1.0 1.434–1.438 0.857–0.862
(M-1b)
IR 98.0% of C11H22O2 3.0 1.420–1.424 0.863–0.867
(M-1b)
IR 98.0% of C10H20O2 1.0 1.416–1.420 0.863–0.867
(M-1b)
1.0 1.448–1.453 0.868–0.873 Saponification Value—between 175 and 190
(Appendix VI)
Alcohol Content—min 14.0% by vol, at 15.56°
(M-4)
Ester Value—min 25 (Appendix VI, 1- to 3-g
sample)
560 / Ethyl Phenylacetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Ethyl Phenylacetate colorless or nearly s—alc, most fixed 1 mL in 3
FEMA No. 2452 colorless liq/ oils; mL 70% alc
sweet, honey ins—gly, prop glycol,
water/
164.20/C10H12O2/
CH2COOC2H5
228°
Ethyl Phenylglycidate colorless to slightly yel s—alc, chloroform, 1 mL in 6
FEMA No. 2454 liq/ ether; mL 70% alc,
strong, strawberry ins—water/ and in 1 mL
96° (0.5 mm Hg) 80% alc
192.21/C11H12O3/
C CHCOOCH2CH3
H
O gives clear
solns
Ethyl Propionate colorless liq/ s—most fixed oils, 1
FEMA No. 2456 fruity, rum, ethereal mL in 42 mL water;102.13/C5H10O2/
CH3CH2COOC2H5 m—alc, prop glycol/
99°
3-Ethyl Pyridine colorless to yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3394 tobacco oils; mL 95%
ins—water/ ethanol
166°N
107.16/C7H9N/
Ethyl Salicylate colorless liq/ s—alc, acetic acid, 1 mL in 4
FEMA No. 2458 wintergreen most fixed oils; mL 80% alc
ss—gly, water/ gives clear
234° soln
166.18/C9H10O3/
OH
COOC2H5
Ethyl 10-Undecenoate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2461 waxy, coconut ins—prop glycol, mL 95%212.33/C13H24O2/
H2C CH(CH2)8CO2C2H5 water/ ethanol
258°–259°
Ethyl Valerate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2462 fruity ss—prop glycol; mL 95%130.19/C7H14O2/
CH3(CH2)3COOCH2CH3Ethyl n-Pentanoate ins—water/ ethanol
145°
Ethyl Vanillin fine white or slightly yel s—alc, chloroform, 1 g in 5 mL
FEMA No. 2464 cryst; affected by strong ether, prop glycol 95% ethanol
3-Ethoxy-4- light/ solns of alkali
hydroxybenzaldehyde strong, vanilla hydroxides, 1 g in
100 mL water at 50°
166.18/C9H10O3/
CHO
OH
OC2H5
Complete Table
FCC V Flavor Chemicals / Ethyl Vanillin / 561
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C10H12O2 1.0 1.496–1.500 1.027–1.032 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 98.0% of C11H12O3 1.516–1.521 1.120–1.125
(Appendix VI; Ester
Determination; 1.4 g/
96.11)
IR 97.0% of C5H10O2 2.0 1.383–1.385 0.886–0.889
(M-1b)
IR 97.0% of C7H9N 1.500–1.505 0.951–0.957
(M-1b)
IR 99.0% of C9H10O3 1.0 (phenol 1.520–1.525 1.126–1.130
(M-1b) red TS)
98.0% of C13H24O2 1.0 1.436–1.440 0.877–0.879
(M-1b)
98.0% of C7H14O2 1.0 1.399–1.404 0.870–0.875
(M-1b)
IR 98.0% of C9H10O3 Melting Range—76° to 78° (Appendix IIB, dry
(M-1b) over P2O5/4 h)
Loss on Drying—0.5% (Appendix IIC,
P2O5/4 h)
Residue on Ignit.—0.05% (Appendix IIC, 2-g
sample)
562 / Eucalyptol / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Eucalyptol colorless liq/ s—alc, most fixed 1 mL in 5
FEMA No. 2465 camphoraceous; pungent, oils, gly, prop glycol/ mL 60% alc
1,8-Cineol; 1,8 Epoxy-p- cooling taste 176°
menthane; 1:8 Oxido-p-
menthane
154.25/C10H18O/
CH3
H3C CH3
O
Eugenol colorless to pale yel liq/ ss—water; 1 mL in 2
FEMA No. 2467 pungent, spicy taste; m—alc, chloroform, mL 70% alc
4-Allylguaiacol; 4-Allyl-2- darkens and thickens on ether, most fixed oils/
methoxyphenol; Eugenic Acid air exposure/ 256°
strong clove
164.20/C10H12O2/
OH
OCH3
CH2CH CH2
Eugenyl Acetate fused solid, melts at s—alc, ether; 1 mL in 5
FEMA No. 2469 warm room temp to a ins—water/ mL 70% alc
Aceteugenol; Acetyl Eugenol; 4- pale yel liq/ 282°
Allyl-2-methoxy-phenyl Acetate; mild, clove
Eugenol Acetate
206.24/C12H14O3/
CH2CH
OCH3
OOCCH3
CH2
Farnesol slightly yel liq/ ins—water/ 1 mL in 1
FEMA No. 2478 mild, oily 263° mL 95%
3,7,11-Trimethyl-2,6,10- ethanol
dodecatrien-1-ol
222.37/C15H26O/
OHH3C
CH3 CH3 CH3
d-Fenchone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2479 camphoraceous oils; mL 95%
ins—water/ ethanol
192°
CH3
152.24/C10H16O/
CH3
CH3
O
Fenchyl Alcohol white to pale yel cryst/ s—veg oils; 1 g in 1 mL
FEMA No. 2480 camphoraceous vss—water/ 95% ethanol
201°CH3
154.25/C10H18O/
CH3
CH3
OH
Furfural colorless to yel oily liq, s—veg oils; 1 mL in 1
FEMA No. 2489 turns red-brown on long ss—prop glycol, mL 95%
2-Furaldehyde; Pyromucic storage/ water/ ethanol
Aldehyde sweet, bready 162°
96.09/C5H4O2/
OCHO
Complete Table
FCC V Flavor Chemicals / Furfural / 563
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.5% of C10H18O 1.455–1.460 0.921–0.924 Angular Rotation—between 0.5° and +0.5°
(M1-a) (Appendix IIB, 100-mm tube)
IR 98.0% of C10H12O2 1.540–1.542 1.064–1.070 Hydrocarbons—passes test (M-7)
(M-1b)
IR 98.0% of C12H14O3 1.0 (phenol 1.514–1.522 1.077–1.082 Solidification Pt.—NLT 25° (Appendix IIB)
(M-1b) red TS) (as supercooled (as melted,
liq) supercooled liq)
IR 96.0% of C15H26O 1.487–1.492 0.884–0.891
(sum of four isomers)
(M-1a)
IR 97.0% of C10H16O 1.460–1.467 0.940–0.948 Angular Rotation—between −68° and −46°
(M-1b)
IR 97.0% of C10H18O Melting Point—between 35.0° and 40.0°
(M-1b)
IR 96.0% of C5H4O2 1.0 1.522–1.528 1.154–1.158
(M-1b)
564 / Furfuryl Alcohol / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Furfuryl Alcohol pale yel to brown liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2491 caramel oils, water/ mL 95%
169° ethanol
98.10/C5H6O2/
O CH2OH
Furfuryl Mercaptan yel to brown liq/ s—veg oils; 1 mL in 1
FEMA No. 2493 coffee ss—prop glycol mL 95%
ins—water/ ethanol
155°O CH2
SH
114.16/C5H6OS/
2-Furyl Methyl Ketone yel to brown liq/ ss—prop glycol, veg 1 mL in 2
FEMA No. 3163 coffee oils; mL 95%
vss—water/ ethanol
67° at 10 mmOCH3
O
110.11/C6H6O2/
Fusel Oil, Refined colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2497 winy, whiskey oils; mL 95% alc
ins—water/
128°–130°
Geraniol colorless liq/ s—most fixed oils, 1 mL in 3
FEMA No. 2507 rose prop glycol; mL 70% alc
trans-3,7-Dimethyl-2,6-octadien- ss—water; remains in
1-ol; E-3,7-Dimethyl-2,6- ins—gly/ soln to 10
octadien-1-ol 230° mL
CH2OH
154.25/C10H18O/
Geranyl Acetate colorless liq/ s—alc, most fixed 1 mL in 9
FEMA No. 2509 floral oils; mL 70 % alc
3,7-Dimethyl-2,6-octadien-1-yl ss—prop glycol;
Acetate ins—gly, water/
245°
CH2OC
196.29/C12H20O2/
O
CH3
Geranyl Benzoate slightly yel liq/ m—alc, chloroform; 1 mL in 4
FEMA No. 2511 floral, resembling ylang- ins—water/ mL 90% alc
3,7-Dimethyl-2,6-octadien-1-yl ylang oil 305° gives clear
Benzoate solnCH2OC
258.36/C17H22O2/
O
Geranyl Butyrate colorless to pale yel liq/ s—alc, most fixed 1 mL in 6
FEMA No. 2512 fruity, rose oils; mL 80% alc
3,7-Dimethyl-2,6-octadien-1-yl ins—gly, prop glycol,
Butyrate water/
253°
CH2OC
224.34/C14H24O2/
O
CH2CH2CH3
Complete Table
FCC V Flavor Chemicals / Geranyl Butyrate / 565
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C5H6O2 1.481–1.490 1.126–1.136
(M-1b)
IR 95.0% of C5H6OS 1.529–1.534 1.124–1.135
(M1-b)
IR 97.0% of C10H6O2 1.505–1.510 1.102–1.107
(M-1b)
IR 95.0% of 2- and 3- 1.405–1.410 0.807–0.813 Angular Rotation—between −0.5° and −2.0°
methyl butanol (Appendix IIB)
(M-1a)
IR 88.0% of total alcohols 1.469–1.478 0.870–0.885 Aldehydes—1.0% as citronellal (M-2d; 5 g/
as C10H18O 77.13)
(Appendix VI; 1.2 g/ Esters—1.0% as geranyl acetate (Appendix VI;
77.13) 5 g/98.15)
IR 90.0% of total esters as 1.458–1.464 0.900–0.914
C12H20O2
(Appendix VI; 1.0 g/
98.15)
IR 95.0% of total esters as 1.0 1.516–1.521 0.983–0.989
C17H22O2
(Appendix VI; Ester
Determination; 1.5 g/
129.2)
IR 92.0% of total esters as 1.0 1.455–1.462 0.889–0.904
C14H24O2
(Appendix VI; 1.0 g/
112.2)
566 / Geranyl Formate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Geranyl Formate colorless to pale yel liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2514 fresh, leafy, rose oils; mL 80% alc
3,7-Dimethyl-2,6-octadien-1-yl ins—gly, prop glycol,
Formate water/
216°
CH2OC
182.26/C11H18O2/
O
H
Geranyl Isovalerate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2518 rose oils; mL 95%
ins—water/ ethanol
279°CH2
238.37/C15H26O2/
O C
O
CH2 CH
CH3
CH3
Geranyl Phenylacetate yel liq/ m—alc, chloroform, 1 mL in 4
FEMA No. 2516 honey-rose ether; mL 90% alc
3,7-Dimethyl-2,6-octadien-1-yl ins—water/ gives clear
Phenylacetate 278° solnCH2OC
272.39/C18H24O2/
O
CH2
Geranyl Propionate colorless liq/ s—alc, most fixed 1 mL in 4
FEMA No. 2517 rosy, fruity oils; mL 80% alc
3,7-Dimethyl-2,6-octadien-1-yl ins—gly, prop glycol,
Propionate water/
253°
CH2OC
210.32/C13H22O2/
O
CH2CH3
Glyceryl Tripropanoate colorless to pale yel liq/ 175°–176° (20 mm
FEMA No. 3286 odorless with a bitter Hg)
Tripropionin taste
260.29/C12H20O6/
COCCH2CH3
O
COCCH2CH3
O
COCCH2CH3
OH
H
H
H
H
(E),(E)-2,4-Heptadienal slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3164 fatty, green oils; mL 95%
trans,trans-2,4-Heptadienal ins—water/ ethanol
100° (35 mm Hg)
110.16/C7H10O/
C C
CH3CH2
H
H
C CH CHO
H
�-Heptalactone colorless, slightly oily s—prop glycol;
FEMA No. 2539 liq/ m—alc, most fixed
coconut, sweet, malty, oils;
caramel ins—water/
128.17/C7H12O2/
OCH3(CH2)2 O
61° (2 mm Hg)
Complete Table
FCC V Flavor Chemicals / �-Heptalactone / 567
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 85.0% of total esters as 3.0 (Appendix 1.457–1.466 0.906–0.920
C11H18O2 VI)
(Appendix VI; 1.0 g/
91.13)
IR 95.0% of C15H26O2 1.0 1.452–1.462 0.881–0.894
(sum of neryl and
geranyl isomers)
(M-1b)
IR 97.0% of total esters as 2.0 1.506–1.511 0.971–0.978
C18H24O2
(Appendix VI; 1.6 g/
136.2)
IR 92.0% of total esters as 1.0 1.456–1.464 0.896–0.913
C13H22O2
(Appendix VI; 1.6 g/
105.2)
97.1% of C12H20O6 2.0 1.431–1.435 1.078–1.082
(M-1b)
IR 92.0% of C7H10O 1.531–1.537 0.878–0.888
(sum of isomers)
(M-1a)
IR 98.0% of C7H12O2 1.439–1.445 0.989–0.998
(M-1a)
568 / Heptanal / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Heptanal colorless to slightly yel ss—water; 1 mL in 2
FEMA No. 2540 liq/ m—alc, ether, most mL 70% alc114.19/C7H14O/
CH3(CH2)5CHOAldehyde C-7; Heptaldehyde penetrating, oily fixed oils/ gives clear
153° soln
2,3-Heptandione yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2543 butter, cheese oils; mL 95%
Acetyl Valeryl ins—water/ ethanol
64° (18 mm Hg)
O
O
128.17/C7H12O2/
2-Heptanone colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2544 fruity, spicy in 250 mL water/114.19/C7H14O/
CH3CO(CH2)4CH3Methyl Amyl Ketone 151°
3-Heptanone colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2545 fruity, green, fatty in 70 mL water/114.19/C7H14O/
CH3(CH2)3COCH2CH3Ethyl Butyl Ketone 149°
(Z)-4-Hepten-1-al slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3289 fatty, green oils; mL 95%
cis-4-Hepten-1-al ins—water/ ethanol
60° (25 mm Hg)
112.17/C7H12O/
C CH
CH3CH2
H
(CH2)2CHO
Heptyl Alcohol colorless liq/ ss—water; 1 mL in 2
FEMA No. 2548 fatty, winy m—alc, ether, most mL 60% alc116.20/C7H16O/
CH3(CH2)5CH2OHEnanthic Alcohol fixed oils/ gives clear
175° soln
�-Hexalactone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2556 herbaceous, sweet oils; mL 95% alc
4-Hydroxyhexanoic Acid ins—water/
114.14/C6H10O2/
CH3CH2CHCH2CH2C O
OLactone 220°
Hexanal almost colorless liq/ vss—water;
FEMA No. 2557 fatty-green, grassy m—alc, prop glycol,100.16/C6H12O/
CH3(CH2)4CHOCaproic Aldehyde; Hexaldehyde; most fixed oils/
Aldehyde C-6 131°
Hexanoic Acid colorless to very pale yel, m—alc, most fixed
FEMA No. 2559 oily liq/ oils, ether, 1 mL in116.16/C6H12O2/
CH3(CH2)4COOHCaproic Acid cheesy, sweat 250 mL water/
223°
Complete Table
FCC V Flavor Chemicals / Hexanoic Acid / 569
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 92.0% of C7H14O 10.0 1.412–1.420 0.815–0.820
(M-1b)
IR 97.0% of C7H12O2 1.411–1.418 0.916–0.923
(M-1b)
IR 95.0% of C7H14O 2.0 1.405–1.411 0.811–0.816 Distillation Range—between 147° and 154°
(M-1b) (Appendix IIB)
Residue on Evap.—5 mg/100 mL (M-16, 100-
mL sample)
Water—0.3% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 97.0% of C7H14O 2.0 1.404–1.411 0.813–0.818 Distillation Range—between 143° and 151°
(M-1b) (Appendix IIB)
Water—0.3% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 98.0% of C7H12O 1.432–1.436 0.843–0.855
[sum of two isomers;
(Z)-4-isomer 93.0% min]
(M-1a)
IR 97.0% of C7H16O 1.0 1.423–1.427 0.820–0.824 Aldehydes—1.0% heptanal (M-1b)
(M-1b)
IR 98.0% of C6H10O2 1.0 1.437–1.442 1.020–1.025
(M-1a)
97.0% of C6H12O 10.0 1.402–1.407 0.808–0.817
(M-1a)
IR 98.0% of C6H12O2 1.415–1.418 0.923–0.928 Solidification Pt.—NLT −4.5° (Appendix IIB)
(M-3a)
570 / (E)-2-Hexen-1-al / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
(E)-2-Hexen-1-al pale yel liq/ s—alc, prop glycol, 1 mL in 1
FEMA No. 2560 strong, fruity-green, most fixed oils; mL 95%
trans-2-Hexen-1-al vegetable vss—water/ ethanol
47° (17 mm Hg)
98.14/C6H10O/
C CCHOH
CH3(CH2)2 H
(E)-2-Hexen-1-ol almost colorless liq/ s—alc, prop glycol,
FEMA No. 2562 strong, fruity-green most fixed oils;
trans-2-Hexen-1-ol vss—water/
158°
100.16/C6H12O/
C C
H
CH3(CH2)2
CH2OH
H
(Z)-3-Hexenol colorless liq/ s—alc, prop glycol,
FEMA No. 2563 powerful, grassy-green most fixed oils;
cis-3-Hexen-1-ol vss—water/
156°
100.16/C6H12O/
C C
CH3CH2
H
(CH2)2OH
H
(E)-2-Hexenyl Acetate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2564 green note ss—prop glycol; mL 95% alc
trans-2-Hexen-1-yl Acetate ins—water/
166°
142.20/C8H14O2/
C C
H
CH3COCH2
(CH2)2CH3
H
O
(Z)-3-Hexenyl Acetate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3171 powerful green note oils; mL 95% alc
cis-3-Hexen-1-yl Acetate ins—water/
198°
142.20/C8H14O2/
H3C O(CH2)2
C C
H
CH2CH3
H
O
(Z)-3-Hexenyl Butyrate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3402 green, fruity oils; mL 95%
ins—water/ ethanol
96° (20 mm Hg)
170.25/C10H18O2/
CO
(CH2)2
C C
H
CH2CH3
H
O
CH3(CH2)2
(Z)-3-Hexenyl Formate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3353 green oils; mL 95%
ins—water/ ethanol
72° (40 mm Hg)
128.17/C7H12O2/
H O(CH2)2
C C
H
CH2CH3
H
O
(Z)-3-Hexenyl Isovalerate colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3498 sweet, apple oils; prop glycol, mL 95%
cis-3-Hexen-1-yl Isovalerate water/ ethanol
199°
184.28/C11H20O2/
C C(CH2)2OOCCH2CH(CH3)2CH3CH2
H H
Complete Table
FCC V Flavor Chemicals / (Z)-3-Hexenyl Isovalerate / 571
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
92.0% of C6H10O 1.445–1.449 0.841–0.850
(M-1a)
IR 95.0% of C6H12O 1.436–1.441 0.839–0.844
(M-1a)
IR 98.0% of C6H12O [sum 1.436–1.443 0.846–0.850
of (Z)- and (E)-isomers;
min 92% (Z)]
(M-1a)
IR 98.0% of C8H14O2 [sum 1.425–1.430 0.890–0.897
of (Z)- and (E)-isomers;
min 90% (E)]
(M-1a)
IR 98.0% of C8H14O2 [sum 1.0 1.425–1.429 0.896–0.901
of (Z)- and (E)-isomers;
min 92% (Z)]
(M-1a)
IR 97.0% of C10H18O2 1.0 1.427–1.435 0.880–0.887
(one isomer)
(M-1b)
IR 95.0% of C7H12O2 5.0 1.424–1.430 0.907–0.915
(M-1b) (add ice to
soln)
95.0% of C11H20O2 2.0 1.429–1.435 0.872–0.877
[sum of two isomers;
(Z)-isomer 92.0% min]
(M-1a)
572 / (Z)-3-Hexenyl 2-Methylbutyrate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
(Z)-3-Hexenyl 2-Methylbutyrate almost colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3497 powerful, fruity, unripe oils; mL 95%
cis-3-Hexenyl 2-Methylbutyrate apples ins—water/ ethanol
105° (25 mm Hg)
184.28/C11H20O2/
C CH
CH3CH2
H
(CH2)2OOCCHCH2CH3
CH3
Hexyl Acetate colorless liq/ s—veg oils; 1 mL in 1
FEMA No. 2565 fruity ss—prop glycol; mL 95% alc144.21/C8H16O2/
CH3(CH2)5OOCCH3 ins—water/
168°–170°
Hexyl Alcohol colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2567 mild, sweet, green in 175 mL water/102.18/C6H14O/
CH3(CH2)4CH2OH1-Hexanol; Alcohol C-6 157°
Hexyl-2-butenoate colorless liq/ s—alc, most fixed
FEMA No. 3354 fruity oils;170.25/C10H18O2/
CH3(CH2)5OOCCH CHCH3 ins—water, prop
glycol
Hexyl Butyrate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2568 fruity ss—prop glycol; mL 95%172.27/C10H20O2/
CH3(CH2)5OOC(CH2)2CH3 ins—water/ ethanol
205°
�-Hexylcinnamaldehyde pale yel liq/ s—most fixed oils; 1 mL in 1
FEMA No. 2569 jasmine ins—gly, prop glycol/ mL 90% alc
174° (15 mm Hg)
216.32/C15H20O/
CH C(CH2)5CH3
CHO
Hexyl Hexanoate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2572 fruity oils; mL 95%200.32/C12H24O2/
CH3(CH2)5OOC(CH2)4CH3 ins—water/ ethanol
245°
Hexyl Isovalerate colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3500 pungent, fruity oils; mL 95% alc186.29/C11H22O2/
(CH3)2CHCH2COOCH2(CH2)4CH3 ins—water/
215°
Hexyl 2-Methylbutyrate colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3499 strong, fresh-green, fruity oils; mL 95%
ins—water/ ethanol
217°–219°
186.29/C11H22O2/
CH3
CH3(CH2)5OOCCHCH2CH3
Complete Table
FCC V Flavor Chemicals / Hexyl 2-Methylbutyrate / 573
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
95.0% of C11H20O2 2.0 1.430–1.434 0.876–0.880
[sum of two isomers;
(Z)-isomer 92.0% min]
(M-1a)
IR 98.0% of C8H16O2 1.0 1.407–1.411 0.868–0.872
(M-1b)
IR 97.0% of C6H14O 2.0 1.415–1.420 0.816–0.821
(M-1b)
IR 95.0% of C10H18O2 1.428–1.449 0.880–0.900
(M-1a)
IR 98.0% of C10H20O2 1.0 1.414–1.420 0.860–0.866
(M-1b)
IR 95.0% of C15H20O 5.0 1.548–1.552 0.953–0.959 Chlorinated Cmpds.—passes test (Appendix
(sum of two isomers) VI)
(M-1b)
IR 98.0% of C12H24O2 1.0 1.421–1.427 0.857–0.863
(M-1b)
95.0% of C11H22O2 2.0 1.417–1.421 0.853–0.857
(sum of two isomers;
isovalerate isomer 92.0%
min)
(M-1b)
95.0% of C11H22O2 2.0 1.416–1.421 0.854–0.859
(one isomer)
(M-1a)
574 / Hydroxycitronellal / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Hydroxycitronellal colorless liq/ s—most fixed oils, 1 mL in 1
FEMA No. 2583 sweet, floral, lily prop glycol; mL 50% alc
7-Hydroxy-3,7-dimethyl Octanal ins—gly/
241°
172.27/C10H20O2/
CH
OHO
Hydroxycitronellal Dimethyl colorless liq/ s—most fixed oils, 1 mL in 2
Acetal floral prop glycol; mL 50% alc
FEMA No. 2585 ins—gly/
7-Hydroxy-3,7-dimethyl Octanal: 252°
Acetal
218.34/C12H26O3/
CHOCH3
OCH3
HO
4-Hydroxy-2,5-dimethyl-3(2H)- white to pale yel solid/ 1 g in 1 mL
furanone fruity, caramel, burnt 95% alc
FEMA No. 3174 sugar
O
O OH
CH3H3C
128.13/C6H8O3/
6-Hydroxy-3,7-dimethyloctanoic colorless, low-melting vs—-water;
Acid Lactone solid/ s—alc
FEMA No. 3355 maple syrup or brown
sugar
170.25/C10H18O2/
O
H3C
O
H3C CH3
4-(p-Hydroxyphenyl)-2-butanone white solid/ ins—prop glycol, veg 1 g in 2 mL
FEMA No. 2588 raspberry oils, water 95% alc164.20/C10H12O2/
HO
O
Indole white, lustrous, flaky, s—alc, most fixed 1 g in 3 mL
FEMA No. 2593 cryst solid/ oils, prop glycol; 70% alc
unpleasant odor in high ins—gly/
conc., free of fecal 253°–254°
quality; floral on dilution
117.15/C8H7N/
HN
�-Ionone colorless to pale yel liq/ s—alc, most fixed 1 mL in 10
FEMA No. 2594 warm, woody, violet- oils, prop glycol; mL 60% alc
4-(2,6,6-Trimethyl-2- floral ins—gly, water/
cyclohexenyl)-3-butene-2-one 237°
192.30/C13H20O2/
O
Complete Table
FCC V Flavor Chemicals / �-Ionone / 575
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C10H20O2 5.0 1.447–1.450 0.918–0.923
(M-1b)
IR 95.0% of C12H26O3 1.0 1.441–1.444 0.925–0.930 Free Hydroxy Citronellal—3.0% (M-1b)
(M-1b)
IR 98.0% of C6H8O3 in a
suitable solvent
(M-1a)
90.0% of C10H18O2 1.457–1.461 0.966–0.973
(M-1a)
IR 98.0% of C10H12O2 Melting Range—between 82° and 84°
(M-1b) (Appendix IIB)
IR 99.0% of C8H7N Solidification Pt.—NLT 51° (Appendix IIB,
(M-1a) dry over H2SO4)
IR 85.0% of C13H20O2 1.497–1.502 0.927–0.933
(97.0% sum of �-, �-,
�-, and �-isomers)
(M-1b)
576 / �-Ionone / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
�-Ionone colorless to pale straw- s—alc, most fixed 1 mL in 1
FEMA No. 2595 colored liq/ oils, prop glycol; mL 95%
4-(2,6,6-Trimethyl-1- warm, woody, dry ins—gly, water/ ethanol
cyclohexenyl)-3-butene-2-one 239°
192.30/C13H20O/
O
Isoamyl Acetate colorless liq/ ss—water; 1 mL in 3
FEMA No. 2055 fruity, pear, banana m—alc, ether, ethyl mL 60% alc130.19/C7H14O2/
CH3COOCH2CH2CH(CH3)2Amyl Acetate; �-Methyl Butyl acetate, most fixed gives clear
Acetate oils; soln
ins—gly, prop glycol/
145°
Isoamyl Alcohol colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2057 winy oils; mL 95%88.15/C5H12O/
(CH3)2CHCH2CH2OH ins—water/ ethanol
130°
Isoamyl Benzoate colorless to pale yel liq/ 261° (746 mm Hg) 1 mL in 1
FEMA No. 2058 pungent fruit mL 95% alc196.26/C12H16O2/
CH2OOCCH2CH(CH3)2
Isoamyl Butyrate colorless liq/ s—alc, most fixed 1 mL in 4
FEMA No. 2060 fruity oils; mL 70% alc158.24/C9H18O2/
CH3(CH2)2COOCH2CH2CH(CH3)2Amyl Butyrate ins—gly, prop glycol,
water/
179°
Isoamyl Formate colorless liq/ s—alc, most fixed 1 mL in 4
FEMA No. 2069 plum oils, prop glycol; mL 60% alc116.16/C6H12O2/
HCOOCH2CH2CH(CH3)2Amyl Formate ss—water; remains in
ins—gly/ soln to 10
124° mL
Isoamyl Hexanoate colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2075 fruity oils; mL 80% alc186.29/C11H22O2/
CH3(CH2)4COOCH2CH2CH(CH3)2Amyl Hexanoate; Isoamyl ins—gly, prop glycol, gives clear
Caproate; Pentyl Hexanoate water/ soln
222°
Complete Table
FCC V Flavor Chemicals / Isoamyl Hexanoate / 577
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C13H20O 1.517–1.522 0.940–0.947
(M-1b)
IR 95.0% of C7H14O2 1.0 1.400–1.404 0.868–0.878
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
98.0% of C5H12O 1.405–1.410 0.807–0.813
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
IR 98.0% of C12H16O2 1.0 1.492–1.496 0.986–0.992
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1a)
IR 98.0% of C9H18O2 1.0 1.409–1.414 0.861–0.866
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
IR 92.0% of C6H12O2 3.0 1.396–1.400 0.881–0.889
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
IR 98.0% of C11H22O2 1.0 1.418–1.422 0.858–0.863
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
578 / Isoamyl Isobutyrate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Isoamyl Isobutyrate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3507 fruity oils; mL 95%
ins—water/ ethanol
169°
158.24/C9H18O2/
HC CH2 CH2OOCCH
CH3
CH3
CH3
CH3
Isoamyl Isovalerate colorless liq/ ss—prop glycol 1 mL in 6
FEMA No. 2085 fruity, apple m—alc, most fixed mL 70% alc172.27/C10H20O2/
(CH3)2CHCH2COOCH2CH2CH(CH3)2Amyl Valerate; oils;
Amyl Isovalerate ins—water/
192°
Isoamyl Phenyl Acetate colorless to pale yel liq/ 268° 1 mL in 1
FEMA No. 2081 chocolate, honey mL 95% alc206.29/C13H18O2/
CH2COOCH2CH2CH(CH3)2
Isoamyl Salicylate colorless liq/ m—alc, chloroform, 1 mL in 3
FEMA No. 2084 floral ether, most fixed oils; mL 90% alc
Amyl Salicylate ins—gly, prop glycol, remains in
water/ soln on
277° dilution
208.26/C12H16O3/
COOCH2CH2CH(CH3)2
OH
Isoborneol white cryst solid/ ss—prop glycol; 1 g in 1 mL
FEMA No. 2158 piney, camphoraceous ins—veg oils/ 95% alc
214°
154.25/C10H18O/
OH
Isobornyl Acetate colorless liq when fresh, s—alc, most fixed 1 mL in 3
FEMA No. 2160 yellows upon storage/ oils; mL 70% alc
camphoraceous, piney, ss—prop glycol;
balsamic ins—gly, water/
227°
196.29/C12H20O2/
OC
CH3
O
Isobutyl Acetate colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2175 fruity, banana on dilution oils, prop glycol, 1 mL 95%116.16/C6H12O2/
CH3COOCH2CH(CH3)2 mL in 180 mL water/ ethanol
116°
Isobutyl Alcohol colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2179 penetrating, winy in 140 mL water/74.12/C4H10O/
(CH3)2CHCH2OH 108°
Complete Table
FCC V Flavor Chemicals / Isobutyl Alcohol / 579
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C9H18O2 1.0 1.404–1.410 0.853–0.859
(M-1b)
98.0% of C10H20O2 2.0 1.411–1.414 0.851–0.857
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
IR 98.0% of C13H18O2 1.0 1.485–1.490 0.975–0.981
(sum of 2-methyl butyl,
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
IR 98.0% of C12H16O3 1.0 (phenol 1.505–1.509 1.047–1.053
(sum of 2-methyl butyl, red TS)
3-methyl butyl, and n-
pentyl isomers)
(M-1b)
IR Melting Range—between 212° and 214°
(Appendix IIB)
IR 97.0% of C12H20O2 1.0 1.462–1.465 0.979–0.984 Angular Rotation—between −4° and 0°
(M-1b) (Appendix IIB, 100-mm tube)
IR 90.0% of C6H12O2 1.0 1.389–1.392 0.862–0.871
(M-1b)
IR 98.0% of C4H10O 2.0 1.392–1.397 0.799–0.801
(M-1a)
580 / Isobutyl-2-butenoate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Isobutyl-2-butenoate colorless liq/ s—alc, prop glycol, 1 mL in 1
FEMA No. 3432 powerful, fruity most fixed oils; mL 95%142.20/C8H14O2/
(CH3)2CHCH2OOCCH CHCH3 ss—water/ ethanol
71°
Isobutyl Butyrate colorless liq/ s—alc, most fixed 1 mL in 8
FEMA No. 2187 sweet, fruity, apple, oils; mL 60% alc144.21/C8H16O2/
C3H7COOCH2CH(CH3)22-Methyl Propanyl Butyrate pineapple ss—water;
ins—gly/
157°
Isobutyl Cinnamate colorless liq/ m—alc, chloroform, 1 mL in 3
FEMA No. 2193 sweet, fruity, balsamic ether, most fixed oils; mL 80% alc
ins—water/ gives clear
271° soln
204.27/C13H16O2/
CH CHCOOCH2CHCH3
CH3
Isobutyl Formate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2197 fruity oils; mL 95%
ins—water/ ethanol
98°
102.13/C5H10O2/
HC CH2 OOCH
CH3
CH3
Isobutyl Hexanoate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2202 fruity oils; mL 95%
ins—water/ ethanol
203°
172.27/C10H20O2/
HC CH2 OOC(CH2)4CH3
CH3
CH3
Isobutyl Phenylacetate colorless liq/ s—alc, most fixed 1 mL in 2
FEMA No. 2210 rose, honey oils; mL 80% alc
ins—gly, prop glycol, remains in
water/ soln to 10
192.26/C12H16O2/
CH2COOCH2CHCH3
CH3 247° mL
Isobutyl Salicylate colorless liq/ s—most fixed oils; 1 mL in 9
FEMA No. 2213 orchid ins—gly, prop glycol/ mL 80% alc
260° remains in
soln to 10
mL
194.23/C11H14O3/
OH
COOCH2CHCH3
CH3
Isobutyraldehyde colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2220 sharp, pungent in 125 mL water/72.11/C4H8O/
(CH3)2CHCHO 64°
Isobutyric Acid colorless liq/ m—alc, most fixed
FEMA No. 2222 strong, penetrating odor oils, gly, prop glycol;88.11/C4H8O2/
(CH3)2CHCOOH2-Methyl Propanoic Acid; of rancid butter ins—water/
Isopropylformic Acid 155°
Complete Table
FCC V Flavor Chemicals / Isobutyric Acid / 581
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C8H14O2 1.426–1.430 0.880–0.900
(M-1a)
IR 98.0% of C8H16O2 1.0 1.402–1.405 0.858–0.863
(M-1b)
IR 98.0% of C13H16O2 1.0 1.539–1.541 1.001–1.004
(sum of two isomers)
(M-1b)
IR 94.0% of C5H10O2 2.0, add ice to
(M-1b) solution
IR 98.0% of C10H20O2 1.0 1.411–1.417 0.853–0.859
(M-1b)
IR 98.0% of C12H16O2 1.0 1.486–1.488 0.984–0.988
(M-1b)
IR 98.0% of C11H14O3 1.0 (phenol 1.507–1.510 1.062–1.066
(M-1b) red TS)
IR 98.0% of C4H8O 5.0 (methyl 0.783–0.788
(M-1b) red TS)
IR 99.0–101.1% of C4H8O2 1.392–1.395 0.944–0.948 Reducing Subs.—passes test (M-14)
(M-3a)
582 / Isoeugenol / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Isoeugenol pale yel, viscous liq/ s—most fixed oils, 1 mL in 5
FEMA No. 2468 floral, carnation ether; mL 50% alc
2-Methoxy-4-propenylphenol ins—gly/
266°
164.20/C10H12O2/
CH3O
HO CH CHCH3
Isoeugenyl Acetate white cryst/ s—alc, most fixed 1 g in 27 mL
FEMA No. 2470 spicy, clove oils, chloroform; 95% alc
2-Methoxy-4-propenyl Phenyl ins—water gives clear
Acetate soln
206.24/C12H14O3/
OOCCH3
OCH3
CH CHCH3
Isopropyl Acetate colorless, mobile liq/ m—alc, ether, most
FEMA No. 2926 ethereal fixed oils, 1 g in 72102.13/C5H10O2/
CH3COOCH(CH3)2 mL water/
88°
Isopulegol colorless liq/ 91° (12 mm Hg) 1 mL in 4
FEMA No. 2962 harsh, camphoraceous, mL 60% alc
p-Menth-4-en-3-ol mint, with rose leaf and gives clear
geranium background soln
154.25/C10H18O/
CH3
OH
CH3C CH2
Isovaleric Acid colorless to pale yel liq/ s—alc, chloroform,
FEMA No. 3102 disagreeable, rancid, ether, water/102.13/C5H10O2/
(CH3)2CHCH2COOHIsopropylacetic Acid cheese 175°
Lauryl Alcohol colorless liq above 21°/ s—most fixed oils, 1 mL in 3
FEMA No. 2617 fatty prop glycol; mL 70% alc186.34/C12H26O/
CH3(CH2)10CH2OH1-Dodecanol; Alcohol C-12 ins—gly, water/ remains clear
259° to 10 mL
Lauryl Aldehyde colorless to light yel liq s—alc, most fixed
FEMA No. 2615 (may solidify at room oils, prop glycol184.32/C12H24O/
CH3(CH2)10CHOAldehyde C-12; Dodecanal temp)/ (may be turbid);
fatty ins—gly, water/
249°
Levulinic Acid yel to brown liq; may 245° 1 mL in 1
FEMA No. 2627 congeal/ mL 95% alc116.12/C5H8O3/
CH3COCH2CH2COOH smoky, caramel
Complete Table
FCC V Flavor Chemicals / Levulinic Acid / 583
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 99.0% of C10H12O2 1.572–1.577 1.079–1.085 Solidification Pt.—NLT 12° (Appendix IIB)
(sum of two isomers)
(M-1b)
IR 98.0% of C12H14O3 2.0 (phenol Solidification Pt.—NLT 76° (Appendix IIB)
(sum of two isomers; red TS)
main isomer 95.0% min)
(M-1b)
IR 99.0% of C5H10O2 2.0 0.866–0.869
(M-1b)
IR 95.0% of total alcohols 1.0 1.470–1.475 0.904–0.913 Aldehydes—1.0% as citronellal (M-2d; 10 g/
as C10H18O 77.13)
(Appendix VI; 1.2 g/ Angular Rotation—between 0° and −7°
77.12 with 2-h reflux) (Appendix IIB, 100-mm tube)
IR 99.0% of C5H10O2 1.401–1.405 0.923–0.928
(M-3a)
IR 97.0% of C12H26O 1.0 1.440–1.444 0.830–0.836 Solidification Pt.—NLT 21° (Appendix IIB)
(M-1b)
IR 92.0% of C12H24O 10.0 1.433–1.439 0.826–0.836
(M-1b)
IR 97.0% of C5H8O3 1.440–1.445 1.136–1.142 Solidification Pt.—min 27° (Appendix IIB)
(M-3a)
584 / d-Limonene / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
d-Limonene colorless liq/ ss—gly;
FEMA No. 2633 mildly citrus, free from m—alc, most fixed
d-p-Mentha-1,8-diene; Cinene camphoraceous and oils;
terpene notes ins—prop glycol,
water/
177°
136.24/C10H16/
CH3
CH2C CH3
l-Limonene colorless liq/ m—alc, most fixed
l-p-Mentha-1,8-diene refreshing, light, clean oils;
ins—water/
177°
136.24/C10H16/
CH3
CH2C CH3
Linalool colorless liq/ s—most fixed oils, 1 mL in 4
FEMA No. 2635 pleasant, floral prop glycol; mL 60% alc
3,7-Dimethyl-1,6-octadien-3-ol ins—gly/
198°OH
154.25/C10H18O/
Linalool Oxide colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3746 floral oils; mL 95%
ins—water/ ethanol
188°
170.25/C10H18O2/
O C
CH3
CH3
OH
Linalyl Acetate colorless liq/ ss—prop glycol; 1 mL in 5
FEMA No. 2636 floral, fruity m—alc, most fixed mL 70% alc
3,7-Dimethyl-1,6-octadien-3-yl oils;
Acetate ins—gly, water/
220°OC
196.29/C12H20O2/
O
CH3
Linalyl Benzoate yel to brown-yel liq/ s—alc, chloroform, 1 mL in 1
FEMA No. 2638 tuberose ether; mL 90% alc
3,7-Dimethyl-1,6-octadien-3-yl ins—water/ gives clear
Benzoate 263° soln
O
258.36/C17H22O2/
CO
Complete Table
FCC V Flavor Chemicals / Linalyl Benzoate / 585
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 93.0% of C10H16 1.471–1.474 0.838–0.843 Angular Rotation—between +96° and +104°
(M-1a) (Appendix IIB, 100-mm tube)
Peroxide Value—5.0 (M-11)
95.0% of C10H16 1.469–1.473 0.837–0.841 Angular Rotation—between −90° and −61°
(M-1a) (Appendix IIB, 100-mm tube)
Peroxide Value—5.0 (M-11)
IR 92.0% of C10H18O 1.461–1.465 0.858–0.867 Esters—0.5% as linalylacetate (Appendix VI;
(M-1b) 10 g/98.15)
IR 98.0% of C10H18O2 1.449–1.455 0.940–0.947
(sum of cis and trans
isomers)
(M-1b)
IR 90.0% of total esters as 1.0 1.449–1.457 0.895–0.914
C12H20O2
(M-1b)
IR 75.0% of C17H22O2 5.0 1.505–1.520 0.980–0.999
(M-1b)
586 / Linalyl Formate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Linalyl Formate colorless liq/ s—alc, most fixed 1 mL in 6
FEMA No. 2642 fresh, citrus, green, oils; mL 70% alc
3,7-Dimethyl-1,6-octadien-3-yl herbaceous, bergamot ss—prop glycol,
Formate water;
ins—gly/OC
182.26/C11H18O2/
O
H202°
Linalyl Isobutyrate colorless to slightly yel m—alc, chloroform, 1 mL in 3
FEMA No. 2640 liq/ ether; mL 80% alc
3,7-Dimethyl-6-octadien-3-yl sweet, fresh, rosy ins—water/ gives clear
Isobutyrate 230° solnOC
224.34/C14H24O2/
O
CH(CH3)CH3
Linalyl Propionate colorless or almost s—alc, most fixed 1 mL in 2
FEMA No. 2645 colorless liq/ oils; mL 80% alc
3,7-Dimethyl-6-octadien-3-yl fresh, floral, sweet, ss—prop glycol;
Propionate fruity, pear ins—gly/
226°OC
210.32/C13H22O2/
O
CH2CH3
Maltol Isobutyrate colorless to yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3462 strawberry oils; mL 95%
ins—water/ ethanol
176° (7 mm Hg)
O
O
OC
CCH3
O
CH3
196.20/C10H12O4/
H3C
Menthol colorless, hexagonal vs—alc, vol oils; 1 mL in 1
FEMA No. 2665 crysts, usually like ss—water/ mL 95%
3-p-Menthanol [NOTE: l- needles; fused masses or 212° ethanol
Menthol is natural or synthetic, cryst powder/
dl-Menthol is synthetic] peppermint
156.27/C10H20O/
H3C
OH
CH
CH3
CH3
l-Menthone almost colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2667 mint oils; mL 95%
l-p-Menthan-3-one vss—water/ ethanol
207°
154.25/C10H18O/
CH3
O
H3C CH3
Complete Table
FCC V Flavor Chemicals / l-Menthone / 587
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 90.0% of C11H18O2 3.0 1.453–1.458 0.910–0.918
(M-1b)
IR 95.0% of C14H24O2 1.0 1.446–1.451 0.882–0.888
(M-1b)
IR 92.0% of C13H22O2 1.0 1.449–1.454 0.893–0.902
(M-1b)
IR 96.0% of C10H12O4 10.0 1.493–1.501 1.140–1.153
(M-1b)
IR Melting Range (l-menthol)—41° to 44°
(Appendix IIB)
Nonvol. Res.—0.05% (M-16)
Readily Ox. Subs. (dl-menthol)—pass (M-13)
Specific Rotation (l-menthol)— between −45°
and −51° (Appendix IIB)
Specific Rotation (dl-menthol)—between −2°
and +2° (Appendix IIB)
96.0% of C10H18O 1.0 1.448–1.453 0.888–0.895 Angular Rotation—min −20° (Appendix IIB,
(sum of two isomers) 100-mm tube)
(M-1b)
588 / dl-Menthyl Acetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
dl-Menthyl Acetate colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 2668 mild, minty oils, prop glycol; mL 95%
dl-p-Menthan-3-yl Acetate ss—gly, water/ ethanol
228°–229°
198.31/C12H22O2/
CH3
OOCCH3
H3C CH3
l-Menthyl Acetate colorless liq/ s—alc, prop glycol,
FEMA No. 2668 mild, minty most fixed oils;
l-p-Menthan-3-yl Acetate ss—water/
229°–230°
198.31/C12H22O2/
CH3
OOCCH3
H3C CH3
2-Mercaptopropionic Acid colorless to pale yel liq/ m—water, alc, ether, 1 mL in 1
FEMA No. 3180 roasted, meaty acetone/ mL 95% alc106.16/C3H6O2S/
CH3CH(SH)COOH 117°
p-Methoxybenzaldehyde colorless to slightly yel s—prop glycol; 1 mL in 3
FEMA No. 2670 liq/ m—alc, ether, most mL 60% alc
Anisic Aldehyde; p- hawthorn fixed oils; gives clear
Anisaldehyde ins—alc, water/ soln
248°
136.15/C8H8O2/
CHO
OCH3
2-Methoxy 3- (or 5- or 6-) colorless to pale yel liq/ s—veg oils; water/ 1 mL in 1
Isopropyl Pyrazine bell pepper, raw potato 120°–125° (20 mm mL 95%
FEMA No. 3358 Hg) ethanol
N
N OCH3
CHH3C
CH3
152.20/C8H12N2O/
2-Methoxy-3(5)-methylpyrazine colorless liq/ s—org solvents, 1 mL in 1
FEMA No. 3183 roasted, hazelnut water mL 95%
ethanol
124.14/C6H8N2O/
N
N OCH3
CH3
4-p-Methoxyphenyl-2-butanone colorless to pale yel liq/ 277° 1 mL in 1
FEMA No. 2672 sweet, floral, fruity mL 95% alc
Anisyl Acetone
178.23/C11H14O2/
CH2CH2COCH3
OCH3
Complete Table
FCC V Flavor Chemicals / 4-p-Methoxyphenyl-2-butanone / 589
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
97.0% of C12H22O2 2.0 1.443–1.450 0.921–0.926
(sum of two isomers)
(M-1b)
98.0% of C12H22O2 2.0 1.443–1.447 0.921–0.926 Angular Rotation—between −70° and −69°
(one major isomer) (Appendix IIB, 100-mm tube)
(M-1b)
IR 98.0% of C3H6O2S 1.479–1.484 1.192–1.200
(M-3a)
IR 97.5% of C8H8O2 6.0 1.571–1.574 1.119–1.123 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 97.0% of C8H12N2O 1.492–1.499 1.010–1.022
(sum of three isomers)
(M-1b)
IR 99.0% of C6H8N2O 1.506–1.510 1.070–1.090
(sum of two isomers)
(M-1b)
IR 98.0% of C11H14O2 1.517–1.521 1.042–1.048
(M-1a)
590 / 2-Methoxypyrazine / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
2-Methoxypyrazine colorless to yellow liq/ s—alc;
FEMA No. 3302 nutty, cocoa ins—water/
61° (29 mm Hg)
110.12/C5H6N2O/
N
N OCH3
Methyl Acetate colorless liq/ 57.5° 1 mL in 1
FEMA No. 2676 ethereal, fruity mL 95% alc74.08/C3H6O2/
CH3COOCH3
4-Methyl Acetophenone colorless or nearly s—most fixed oils, 1 mL in 10
FEMA No. 2677 colorless liq/ prop glycol; mL 50% alc
Methyl p-Tolyl Ketone fruity-floral, resembling ins—gly/
acetophenone 226°
134.18/C9H10O/
H3C CCH3
O
p-Methyl Anisole colorless liq/ s—most fixed oils; 1 mL in 3
FEMA No. 2681 ylang-ylang ins—gly, prop glycol/ mL 80% alc
p-Cresyl Methyl Ether; 174° remains in
Methyl p-Cresol soln on
122.17/C8H10O/
H3C OCH3
dilution
Methyl Anthranilate colorless to pale yel liq s—most fixed oils, 1 mL in 5
FEMA No. 2682 with blue fluorescence/ prop glycol; mL 60% alc
grape ins—gly/ remains in
256° soln to 10
mL
151.16/C8H9NO2/
COOCH3
NH2
Methyl Benzoate colorless liq/ s—alc, most fixed 1 mL in 4
FEMA No. 2683 deep, pungent, floral oils, prop glycol; mL 60% alc
ins—gly/
198°
136.15/C8H8O2/
COOCH3
Methylbenzyl Acetate colorless liq/ s—most fixed oils; 1 mL in 2
FEMA No. 3072 sweet, nutty ss—prop glycol; mL 70% alc
o-Tolyl Acetate ins—gly remains clear
on dilution
164.20/C10H12O2/
CH2OOCCH3
CH3
�-Methylbenzyl Alcohol colorless liq above room vs—gly; 1 mL in 3
FEMA No. 2685 temp/ s—most fixed oils, mL 50% alc
Methyl Phenylcarbinol; mild, hyacinth prop glycol/
�-Phenethyl Alcohol 204°
122.17/C8H10O/
CHCH3
OH
2-Methyl Butanal colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2691 chocolate oils; mL 95%86.13/C5H10O/
CH3CH2CH(CH3)CHO ins—water/ ethanol
93°
Complete Table
FCC V Flavor Chemicals / 2-Methyl Butanal / 591
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 99.0% of C5H6N2O 1.508–1.511 1.110–1.140
(M-1b) (20°)
IR 98.0% of C3H6O2 1.0 1.358–1.363 0.927–0.932
(M-1b)
IR 95.0% of C9H10O 1.530–1.535 0.996–1.004 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 98.5% of C8H10O 1.510–1.513 0.966–0.970 Cresol—0.5% (M-1b)
(M-1a)
IR 98.0% of total esters as 1.581–1.585 1.161–1.169 Solidification Pt.—NLT 23.8° (Appendix IIB)
C8H9NO2 (as supercooled
(Appendix VI; 1.0 g/ liq)
75.59)
IR 98.0% of C8H8O2 1.0 1.514–1.518 1.082–1.088 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 98.0% of C10H12O2 1.0 1.501–1.504 1.030–1.035 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 99.0% of C8H10O 1.525–1.529 1.009–1.014 Ketones—1.0% as acetophenone (M-2d; 10.0 g/
(M-1b) 60.07)
Solidification Pt.—NLT 19° (Appendix IIB)
97.0% of C5H10O 10.0 1.388–1.393 0.799–0.804
(M-1b)
592 / 3-Methyl Butanal / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
3-Methyl Butanal colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2692 chocolate oils; mL 95%86.13/C5H10O/
(CH3)2CHCH2CHOIsovaleraldehyde ins—water/ ethanol
93°
2-Methylbutyl Acetate colorless to pale yel liq/ 138°
FEMA No. 3644 banana130.18/C7H14O2/
CH3CH2CH(CH3)CH2OOCCH3
2-Methylbutyl Isovalerate colorless liq/ s—alc, most fixed
FEMA No. 3506 herbaceous, fruity oils;
2-Methylbutyl-3-methylbutanoate ins—water/
191°–195°
172.27/C10H20O2/
CH3CH2CHCH2OOCCH2CH(CH3)2
CH3
Methyl Butyrate colorless liq/ 102° 1 mL in 1
FEMA No. 2693 fruity mL 95%102.13/C5H10O2/
CH3(CH2)2COOCH3 ethanol
2-Methylbutyric Acid colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2695 fruity oils; mL 95% alc
ins—water/
176°
102.13/C5H10O2/
CH3CH2CHCOOH
CH3
�-Methylcinnamaldehyde yel liq/ s—most fixed oils, 1 mL in 3
FEMA No. 2697 cinnamon prop glycol; mL 70% alc
ins—gly/ remains clear
148° (27 mm Hg) on dilution
146.19/C10H10O/
CH CCHO
CH3
Methyl Cinnamate white to slightly yel cryst s—alc, most fixed 1 g in 4 mL
FEMA No. 2698 mass/ oils, gly, prop glycol; 80% alc
fruity, balsamic ins—water/
260°
162.19/C10H10O2/
CH CHCOOCH3
6-Methylcoumarin white cryst solid/ ins—prop glycol, veg 1 g in 20 mL
FEMA No. 2699 coconut oils, water/ 95% alc
303° (725 mm Hg)
160.17/C10H8O2/
O
H3C
O
Methyl Cyclopentenolone white, cryst powder/ s—alc, prop glycol; 1 g in 5 mL
FEMA No. 2700 nutty, maple-licorice ss—most fixed oils, 90% alc
3-Methylcyclopentane-1,2-dione aroma in dilute soln 1 g in 72 mL water
112.13/C6H8O2/
CH3
O
O
Complete Table
FCC V Flavor Chemicals / Methyl Cyclopentenolone / 593
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C5H10O 10.0 1.388–1.391 0.795–0.802
(M-1b)
97.0% of C7H14O2 1.0 1.399–1.404 0.872–0.877
(M-1b)
98.0% of C10H20O2 2.0 1.413–1.416 0.852–0.857
(M-1a)
98.0% of C5H10O2 1.0 1.386–1.390 0.892–0.897
(M-1b)
IR 98.0% of C5H10O2 1.404–1.408 0.932–0.936
(M-3a)
IR 97.0% of C10H10O 5.0 1.602–1.607 1.035–1.039
(one major isomer)
(M-1b)
IR 98.0% of C10H10O2 2.0 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Lead—10 mg/kg (M-9)
IR 99.0% of C10H8O2 Melting Range—between 73° and 76°
(M-1a) (Appendix IIB)
IR Melting Range—between 104° and 108°
(Appendix IIB)
594 / 5H-5-Methyl-6,7-dihydrocyclopenta[b]pyrazine / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
5H-5-Methyl-6,7- yel to brown liq/ s—prop glycol, veg 1 mL in 1
dihydrocyclopenta[b]pyrazine peanut oils; mL 95%
FEMA No. 3306 ss—water/ ethanol
200°
N
N
134.18/C8H10N2/
Methyl Eugenol colorless to pale yel liq/ s—most fixed oils; 1 mL in 2
FEMA No. 2475 delicate, clove-carnation ins—gly, prop glycol/ mL 70% alc
Eugenyl Methyl Ether; 1,2- 249° remains clear
Dimethoxy-4-allylbenzene to 10 mL
178.23/C11H14O2/
OCH3
OCH3
CH2CH CH2
5-Methyl Furfural yel to brown liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2702 nutty, caramel oils; mL 95%
ins—water/ ethanol
187°
110.11/C6H6O2/
OH3C C H
O
Methyl Furoate pale yel to brown liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2703 fruity oils; mL 95%
ins—water/ ethanol
181°
126.11/C6H6O3/
O C
O
OCH3
6-Methyl-5-hepten-2-one slightly yel liq/ m—alc, most fixed 1 mL in 2
FEMA No. 2707 sharp, citrus-lemongrass oils, ether; mL 70% alc
Methyl Heptenone ins—water/ gives clear
73° (18 mm Hg) soln
126.20/C8H14O/
CH3C CHCH2CH2COCH3
CH3
Methyl Hexanoate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2708 fruity oils; mL 95%130.19/C7H14O2/
CH3 OOC(CH2)4CH3 ins—water ethanol
151°
Methyl Hexyl Ketone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2802 apple oils; mL 95% alc128.21/C8H16O/
CH3(CH2)5COCH32-Octanone ins—water/
175°
Methyl Ionones clear to pale yel to yel 232°–270° 1 mL in 1
Mixture of �-, �-, �- or �-iso, liq/ mL 95%
and �-isomers woody, orris ethanol
206.3/C14H22O/
O
(α-iso)
Complete Table
FCC V Flavor Chemicals / Methyl Ionones / 595
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
98.0% of C8H10N2 1.525–1.535 1.048–1.059
(M-1b)
IR 98.0% of C11H14O2 1.532–1.536 1.032–1.036 Eugenol—1.0% (M-1b)
(one major isomer)
(M-1b)
IR 97.0% of C6H6O2 5.0 1.525–1.535 1.095–1.110
(M-1b)
IR 98.0% of C6H6O3 5.0 1.483–1.500 1.174–1.180
(M-1b)
IR 98.0% of C8H14O 1.438–1.442 0.846–0.851
(M-1b)
IR 98.0% of C7H14O2 2.0 1.402–1.408 0.880–0.886
(M-1b)
IR 95.0% of C8H16O 1.0 1.414–1.418 0.813–0.818
(M-1a)
88.0% of C14H22O 5.0 1.497–1.507 0.925–0.934
(sum of four isomers)
(M-1b)
596 / Methyl Isobutyrate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Methyl Isobutyrate colorless liq/ 90° 1 mL in 1
FEMA No. 2694 fruity mL 95% alc102.13/C5H10O2/
CH3COOCH(CH3)2
Methyl Isoeugenol colorless to pale yel liq/ s—most fixed oils; 1 mL in 2
FEMA No. 2476 delicate, clove-carnation ins—gly, prop glycol/ mL 70% alc
4-Allyl-1,2-dimethoxy Benzene; 270° remains in
Isoeugenyl Methyl Ether; soln to 10
4-Propenyl Veratrole mL
178.23/C11H14O2/
CH3O
CH3O
CH CHCH3
5-Methyl-2-isopropyl-2-hexenal slightly yel liq/ s—alc, most fixed
FEMA No. 3406 herbaceous, woody, oils;
Isodihydrolavandulal fruity, chocolate ins—water, prop
glycol/
154.25/C10H18O/
CH3
CHCH2CHCH3 CCHO
CH(CH3)2 73° (10 mm Hg)
Methyl Isovalerate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2753 apple oils; mL 95%
ins—water/ ethanol
114°
116.16/C6H12O2/
CH3 OOC CH2 CH
CH3
CH3
Methyl 2-Methylbutyrate almost colorless liq/ s—alc, most fixed
FEMA No. 2719 sweet, fruity, apple oils;
Methyl 2-Methylbutanoate ins—water/
115°
116.16/C6H12O2/
CH3OOCCHCH2CH3
CH3
Methyl-3-methylthiopropionate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2720 onion oils; mL 95%134.19/C5H10O2S/
CH3SCH2CH2CO2CH3 ins—water/ ethanol
74°–75°
(18 mm Hg)
Methyl �-Naphthyl Ketone white or nearly white s—most fixed oils; 1 g in 5 mL
FEMA No. 2723 cryst solid/ ss—prop glycol; 95% alc
2-Acetonaphthone orange blossom ins—gly/
300°
170.21/C12H10O/
C
O
CH3
Methyl 2-Octynoate colorless to slightly yel s—most fixed oils; 1 mL in 5
FEMA No. 2729 liq/ ss—prop glycol; mL 70% alc154.21/C9H14O2/
CH3(CH2)5 CCOOCH3Methyl Heptine Carbonate powerful, unpleasant, ins—gly/
violet when diluted 217°
2-Methylpentanoic Acid colorless to pale yel liq/ 196°–197° 1 mL in 1
FEMA No. 2754 caramel, pungent mL 95% alc116.16/C6H12O2/
CH3
CH3CH2CH2CHCOOH
Complete Table
FCC V Flavor Chemicals / 2-Methylpentanoic Acid / 597
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C5H10O2 1.0 1.382–1.386 0.884–0.888
(M-1b)
IR 85.0% of C11H14O2 1.566–1.569 1.047–1.053 Isoeugenol—1.0% (M-1b)
(one isomer)
(M-1a)
IR 90.0% of C10H18O 1.448–1.454 0.842–0.850
(sum of isomers)
(M-1a)
IR 95.0% of C6H12O2 1.0 1.390–1.396 0.878–0.884
(M-1b)
92.0% of C6H12O2 2.0 1.393–1.397 0.879–0.883
(M-1b)
IR 97.0% of C5H10O2S 1.0 1.462–1.468 1.069–1.078
(M-1a)
IR 99.0% of C12H10O Solidification Pt.—NLT 53° (Appendix IIB)
(M-1b)
IR 96.0% of C9H14O2 1.0 1.446–1.449 0.919–0.924 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 98.0% of C6H12O2 1.411–1.416 0.916–0.923
(M-3a)
598 / 4-Methylpentanoic Acid / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
4-Methylpentanoic Acid colorless to pale yel liq/ 199°–201° 1 mL in 1
FEMA No. 3463 sour, penetrating mL 95% alc116.16/C6H12O2/
CH3
CH3CHCH2CH2COOH
4-Methyl-2-pentanone colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2731 fruity, ethereal in 50 mL water/100.16/C6H12O/
CH3COCH2CH(CH3)2Methyl Isobutyl Ketone 117°
2-Methyl-2-pentenoic Acid colorless to pale yel liq 123° (30 mm Hg) 1 mL in 1
FEMA No. 3195 (high-purity material may mL 95% alc
solidify at room temp,
with a melting point
114.14/C6H10O2/
CH3CH2CH CCOOH
CH3
range of 24°–26°)
Methyl Phenylacetate colorless or nearly s—alc, most fixed 1 mL in 6
FEMA No. 2733 colorless liq/ oils; mL 60% alc
honey, jasmine ins—gly, prop glycol,
water/
150.18/C9H10O2/
CH2COOCH3
215°
Methyl Phenylcarbinyl Acetate colorless liq/ s—most fixed oils, 1 mL in 7
FEMA No. 2684 gardenia gly; mL 60% alc
�-Phenyl Ethyl Acetate ins—water/
214°
164.20/C10H12O2/
CHCH3
OCOCH3
5-Methyl 2-Phenyl 2-Hexenal colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3199 cocoa oils; mL 95%
ins—water/ ethanol
89° (26 mm Hg)H
O
188.27/C13H16O/
2-Methyl Propyl 3-Methyl colorless to pale yel liq/ m—alc/
Butyrate fruity 170°158.24/C9H18O2/
(CH3)2CHCH2COOCH2CH(CH3)2FEMA No. 3369
Isobutyl Isovalerate
2-Methylpyrazine colorless to slightly yel m—water, alc,
FEMA No. 3309 liq/ acetone, most fixed
nutty, cocoa oils/
137°
94.12/C5H6N2/
N
N
CH3
Complete Table
FCC V Flavor Chemicals / 2-Methylpyrazine / 599
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C6H12O2 1.412–1.417 0.919–0.926
(M-3a)
99.0% of C6H12O 2.0 1.392–1.397 0.796–0.799 Distillation Range—between 114° and 117°
(M-2d) (M-15) (Appendix IIB)
Water—0.1% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 98.0% of C6H10O2 1.455–1.465 0.978–0.985
(M-3a)
IR 98.0% of C9H10O2 1.0 1.503–1.509 1.061–1.067 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 97.0% of C10H12O2 2.0 1.493–1.497 1.023–1.026 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
92.0% of C13H16O 4.0 1.529–1.536 0.963–0.979
[sum of (E)- and (Z)-
isomers]
(M-1b)
98.0% of C9H18O2 1.0 1.404–1.408 0.850–0.854
(M-1b)
IR 99.0% of C5H6N2 1.504–1.506 1.010–1.030 Water—0.5% (Appendix IIB, KF; use freshly
(M-1a) dist. pyridine as solvent)
600 / Methyl Salicylate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Methyl Salicylate colorless, to yel liq/ s—alc, glacial acetic 1 mL in 7
FEMA No. 2745 wintergreen acid; mL 70% alc
ss—water/ may be
222° (decomp) slightly
152.15/C8H8O3/
COOCH3
OH cloudy
4-Methyl-5-thiazole Ethanol colorless to pale yel liq; 135° (7 mm Hg)
FEMA No. 3204 may darken upon aging/
Sulfurol meaty
143.20/C9H9NOS/
N
S
H3C
HOCH2H2C
Methyl Thiobutyrate 118.20/C5H10OS/ colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3310 CH3—SOC(CH2)2CH3 pungent oils; mL 95%
ins—water/ ethanol
143°
3-Methylthiopropionaldehyde colorless to pale yel liq/ 165°–166° 1 mL in 1
FEMA No. 2747 meaty potato mL 95% alc
Methional
104.17/C4H8OS/
O
CH3S(CH2)2CH
2-Methylundecanal colorless to slightly yel s—most fixed oils,
FEMA No. 2749 liq/ alc, prop glycol (may
Aldehyde C-12 MNA; Methyl n- fatty be turbid);
Nonyl Acetaldehyde ins—gly/
184.32/C12H24O/
CH3(CH2)8CHCHO
CH3
171°
Methyl Valerate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2752 fruity oils; mL 95%116.16/C6H12O2/
CH3 OOC(CH2)3CH3 ins—water/ ethanol
128°
Myrcene colorless to pale yel liq/ s—alc, most fixed
FEMA No. 2762 sweet, balsamic oils;
7-Methyl-3-methylene-1,6- ins—water/
octadiene 167°
CH2
136.24/C10H16/
CH2
CH3C CH3
Myristaldehyde colorless to pale yel liq/ ins—ethanol, prop
FEMA No. 2763 fatty, orris glycol, veg oils,212.38/C14H28O/
CH3(CH2)12CHOTetradecanal water/
260°
Complete Table
FCC V Flavor Chemicals / Myristaldehyde / 601
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C8H8O3 1.0 (phenol 1.535–1.538 1.180–1.185
(M-1b) red TS)
IR 98.0% of C9H9NOS 1.548–1.552 1.196–1.210
(M-1b)
IR 97.0% of C5H10OS 3.0 1.461–1.467 0.964–0.970
(M-1b)
IR 98.0% of C4H8OS 1.484–1.493 1.038–1.048
(M-1a)
IR 94.0% of C12H24O 10.0 1.431–1.436 0.822–0.830
(M-1b)
IR 98.0% of C6H12O2 1.0 1.395–1.401 0.885–0.891
(M-1b)
90.0% of C10H16 1.466–1.471 0.789–0.793 Peroxide Value—50.0 (M-11)
(M-1a)
85.0% of C14H28O 5.0 1.438–1.445 0.825–0.830
(M-2a)
602 / Myristyl Alcohol / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Myristyl Alcohol colorless to white, waxy, s—ether;
1-Tetradecanol; Tetradecyl solid flakes/ ss—alc;214.38/C14H30O/
CH3(CH2)12CH2OHAlcohol waxy ins—water/
289°
�-Naphthyl Ethyl Ether white to pale yel cryst/ ss—prop glycol, veg 1 mL in 5
FEMA No. 2768 floral oils; mL 95%
Nerolin II; Nerolin Bromelia ins—water/ ethanol
282°
172.23/C12H12O/
O C2H5
Nerol colorless liq/ m—alc, chloroform, 1 mL in 9
FEMA No. 2770 fresh, sweet, rose ether; mL 50% alc
cis-3,7-Dimethyl-2,6-octadien- ins—water/ gives clear
1-ol 227° soln
154.25/C10H18O/
CH2OH
Nerolidol colorless to straw-colored s—most fixed oils, 1 mL in 4
FEMA No. 2772 liq/ prop glycol; mL 70% alc
3,7,11-Trimethyl-1,6,10- faint, floral, rose, apple ins—gly/
dodecatrien-3-ol 276°
222.37/C15H26O/
OH
Neryl Acetate colorless to pale yel liq/ s—veg oils; 1 mL in 1
FEMA No. 2773 sweet, floral ss—prop glycol; mL 95% alc
cis-3,7-Dimethyl-2,6-octadien-1- ins—water/
yl Acetate 134° (25 mm Hg)
196.29/C12H20O2/
CH2COOCH3
CH3
H3C CH3
(E),(E)-2,4-Nonadienal slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3212 strong, fatty, floral oils; mL 95%
trans,trans-2,4-Nonadienal ins—water/ ethanol
97° (10 mm Hg)
138.21/C9H14O/
C CH
C
CH3(CH2)3
H CH
H
CHO
(E),(Z)-2,6-Nonadienal slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3377 powerful, violet, oils; mL 95%
trans,cis-2,6-Nonadienal cucumber ins—water/ ethanol
94° (18 mm Hg)
138.21/C9H14O/
C CH H
CH3CH2 CH2CH2
C CH CHO
H
Complete Table
FCC V Flavor Chemicals / (E),(Z)-2,6-Nonadienal / 603
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
98.0% of C14H30O 1.0 Melting Range—between 38° and 41°
(M-1b) (Appendix IIB)
Iodine Value—3.0 max. (Appendix VII)
Saponification Value—1.0 max. (Appendix VI)
IR 97.0% of C12H12O Melting Point—NLT 30.0° (Appendix IIB)
(M-1b)
IR 95.0% of total alcohols 1.467–1.478 0.875–0.880
as C10H18O
(Appendix VI; 1.2 g/
77.13)
IR 97.0% of C15H26O 1.478–1.483 0.870–0.880 Angular Rotation (Natural)—between +11° and
(sum of two isomers) +14° (Appendix IIB, 100-mm tube)
(M-1b) Esters—0.5% as nerolidyl acetate (Appendix
VI; 10 g/132.7)
IR 96.0% of C12H20O2; 1.0 1.458–1.464 0.905–0.914
[predominantly (Z)-
isomer by M-1a]
(M-1b)
IR 89.0% of C9H14O 1.517–1.523 0.865–0.880
(one major isomer)
(M-1a)
IR 96.0% of C9H14O 1.470–1.475 0.850–0.870
(sum of two isomers;
90.0% major isomer)
(M-1a)
604 / (E),(Z)-2,6-Nonadienol / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
(E),(Z)-2,6-Nonadienol white to yel liq/ ins—water/ 1 mL in 1
FEMA No. 2780 powerful, green, 196° mL 95%
trans,cis-2,6-Nonadienol vegetable ethanol
140.22/C9H16O/
C CH H
CH3CH2 CH2CH2
C CH CH2OH
H
�-Nonalactone colorless to pale yel liq/ s—prop glycol; veg 1 mL in 1
FEMA No. 3356 coconut oils; mL 95% alc
5-Hydroxynonanoic Acid, ins—water/
Lactone 250°OCH3(CH2)3 O
156.22/C9H16O2/
�-Nonalactone colorless to slightly yel s—alc, most fixed 1 mL in 5
FEMA No. 2781 liq/ oils, prop glycol; mL 60% alc
Aldehyde C-18, So-Called coconut ins—water/
156.22/C9H16O2/
OCH3(CH2)4 O121°–122° (6 mm
Hg)
Nonanal colorless to light yel liq/ s—alc, most fixed
FEMA No. 2782 fatty; citrus-rose on oils, prop glycol;142.24/C9H18O/
CH3(CH2)7CHOAldehyde C-9; Pelargonic dilution ins—gly/
Aldehyde 93° (23 mm Hg)
Nonanoic Acid colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2784 fatty oils; mL ethanol158.24/C9H18O2/
CH3(CH2)7COOHins—water/
254°
2-Nonanone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2785 fruity, floral, fatty, oils; mL 95% alc142.24/C9H18O/
CH3CO(CH2)6CH3Methyl Heptyl Ketone herbaceous ins—water/
195°
(E)-2-Nonenal white to slightly yel liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3213 fatty, violet oils; mL 95%
trans-2-Nonenal ins—water/ ethanol
88° (12 mm Hg)
140.22/C9H16O/
C CH
CHO
CH3(CH2)5
H
(E)-2-Nonen-1-ol white liq/ ins—water/ 1 mL in 1
FEMA No. 3379 fatty, violet 105° (12 mm Hg) mL 95%
trans-2-Nonenol ethanol
142.24/C9H18O/
C CH
CH2OH
CH3(CH2)5
H
(Z)-6-Nonen-1-ol white to slightly yel liq/ ins—water/ 1 mL in 1
FEMA No. 3465 powerful, melon 115° (20 mm Hg) mL 95%
cis-6-Nonen-1-ol ethanol
142.24/C9H18O/
C CH
(CH2)4CH2OH
H
CH3CH2
Complete Table
FCC V Flavor Chemicals / (Z)-6-Nonen-1-ol / 605
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 92.0% of C9H16O 1.464–1.471 0.860–0.880
(one major isomer)
(M-1a)
IR 98.0% of C9H16O2 1.454–1.459 0.980–0.986
(M-1a)
IR 98.0% of C9H16O2 2.0 1.446–1.450 0.958–0.966
(M-1b)
IR 92.0% of C9H18O 10.0 1.422–1.429 0.820–0.830
(M-1b)
98.0% of C9H18O2 1.431–1.435 0.901–0.906
(M-3a)
IR 97.0% of C9H18O 1.418–1.423 0.817–0.823
(M-1a)
IR 92.0% of C9H16O 1.450–1.460 0.840–0.850
(one major isomer)
(M-1a)
IR 96.0% of C9H18O 1.444–1.452 0.830–0.850
(one major isomer)
(M-1a)
IR 95.0% of C9H18O 1.446–1.452 0.850–0.870
(one major isomer)
(M-1a)
606 / Nonyl Acetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Nonyl Acetate colorless liq/ s—alc, ether; 1 mL in 6
FEMA No. 2788 floral, fruity ins—water/ mL 70% alc186.29/C11H22O2/
CH3COO(CH2)8CH3 212° gives clear
soln
Nonyl Alcohol colorless liq/ m—alc, chloroform, 1 mL in 3
FEMA No. 2789 rose-citrus ether; mL 60% alc144.26/C9H20O/
CH3(CH2)7CH2OH1-Nonanol; Alcohol C-9 ins—water/ gives clear
213° soln
�-Octalactone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3214 coconut oils; mL 95%
5-Hydroxyoctanoic Acid Lactone ins—water/ ethanol
234°
142.20/C8H14O2/
O OCH3CH2CH2
�-Octalactone colorless to slightly yel s—alc;
FEMA No. 2796 liq/ ss—water/
sweet, coconut, fruity 234°
142.20/C8H14O2/
OCH3(CH2)3 O
Octanal colorless to light yel liq/ s—alc, most fixed
FEMA No. 2797 fatty-orange oils, prop glycol;128.21/C8H16O/
CH3(CH2)6CHOAldehyde C-8; Caprylic ins—gly/
Aldehyde 171°
3-Octanol colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3581 strong, oily-nutty, oils; mL 95%
herbaceous ins—water/ ethanol
174°
130.23/C8H18O/
OH
CH3(CH2)4CHCH2CH3
(E)-2-Octen-1-al slightly yel liq/ s—alc, most fixed
FEMA No. 3215 fatty, green oils;
trans-2-Octen-1-al ss—water/
84° (19 mm Hg)
126.20/C8H14O/
C CH
CHO
CH3(CH2)4
H
1-Octen-3-ol colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2805 mushroom, herbaceous oils; mL 95% alc
Amyl Vinyl Carbinol ins—water/
128.21/C8H16O/
CH3(CH2)4CHCH CH2
OH 175°
(Z)-3-Octen-1-ol white to slightly yel liq/ ins—water/ 1 mL in 1
FEMA No. 3467 musty, mushroom 174° mL 95%
cis-3-Octen-1-ol ethanol
128.21/C8H16O/
C CH
(CH2)2OH
H
CH3(CH2)3
Complete Table
FCC V Flavor Chemicals / (Z)-3-Octen-1-ol / 607
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C11H22O2 1.0 1.422–1.426 0.864–0.868
(M-1b)
IR 97.0% of C9H20O 1.0 1.431–1.435 0.824–0.830
(M-1b)
IR 98.0% of C8H14O2 1.452–1.458 0.995–1.000
(M-1a)
IR 95.0% of C8H14O2 8.0 1.443–1.447 0.970–0.980
(M-1a)
IR 92.0% of C8H16O 10.0 1.417–1.425 0.810–0.830
(M-1b)
97.0% of C8H18O 1.425–1.429 0.817–0.824
(M-1a)
IR 92.0% of C8H14O 1.450–1.455 0.830–0.850
[as (E)-isomer]
(M-1a)
IR 97.0% of C8H16O 1.434–1.442 0.831–0.839
(M-1a)
IR 95.0% of C8H16O 1.440–1.446 0.830–0.850
[as (Z)-isomer]
(M-1a)
608 / 1-Octen-3-yl Acetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
1-Octen-3-yl Acetate almost colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3582 metallic, mushroom oils; mL 95%
ins—water, prop ethanol
glycol/
170.25/C10H18O2/
CH3(CH2)3CH2CHOOCCH3
CH CH2
80° (15 mm Hg)
1-Octen-3-yl Butyrate almost colorless liq/ s—alc, most fixed 1 mL in 1
FEMA No. 3612 metallic, mushroom oils; mL 95%
ss—prop glycol; ethanol
ins—water/
198.31/C12H22O2/
CH3(CH2)3CH2CHOOC(CH2)2CH3
CH CH2
225°–229°
Octyl Acetate colorless liq/ m—alc, most fixed 1 mL in 4
FEMA No. 2806 fruity, orange, jasmine oils, org solvents; mL 70% alc172.27/C10H20O2/
CH3COO(CH2)7CH3 ins—water/ gives clear
208° soln
3-Octyl Acetate colorless liq/ s—alc, prop glycol,
FEMA No. 3583 rosy-minty most fixed oils;
ss—water/
187°
172.27/C10H20O2/
CH3(CH2)3CH2CHOOCCH3
CH2CH3
Octyl Alcohol colorless liq/ s—most fixed oils, 1 mL in 5
FEMA No. 2800 sharp fatty-citrus prop glycol; mL 50% alc130.23/C8H14O/
CH3(CH2)6CH2OHAlcohol C-8; 1-Octanol; Capryl ins—gly/
Alcohol 195°
Octyl Formate colorless liq/ s—most fixed oils, 1 mL in 5
FEMA No. 2809 fruity min oil, prop glycol; mL 70% alc158.24/C9H18O2/
HCOO(CH2)7CH3 ins—gly/ remains in
200° soln to 10
mL
Octyl Isobutyrate colorless to pale yel liq/ 245° 1 mL in 1
FEMA No. 2808 refreshing, herbaceous mL 95% alc200.32/C12H24O2/
CH3(CH2)7OOCCH(CH3)2Octyl 2-Methylpropanoate
�-Pentadecalactone white to tan or blue-gray s—veg oils; 1 g in 1 mL
FEMA No. 2840 cryst/ ins—prop glycol/ 95% alc
Cyclopentadecanolide; musky 137° (2 mm Hg)
Exaltolide; Thibetolide
240.38/C15H28O2/
(CH2)14C O
O
2,3-Pentanedione yel to yel-green liq/ m—alc, prop glycol, 1 mL in 3
FEMA No. 2841 penetrating, buttery on most fixed oils; mL 50% alc
Acetyl Propionyl dilution ins—gly, water/
108°
100.12/C5H8O2/
CH3CH2C CCH3
O O
Complete Table
FCC V Flavor Chemicals / 2,3-Pentanedione / 609
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C10H18O2 1.418–1.428 0.865–0.886
(M-1b)
IR 95.0% of C12H22O2 1.423–1.433 0.859–0.880
(M-1b)
IR 98.0% of C10H20O2 1.0 1.418–1.421 0.865–0.868
(M-1b)
98.0% of C10H20O2 2.0 1.414–1.419 0.856–0.860
(M-1b)
IR 98.0% of C8H18O 1.0 1.428–1.431 0.822–0.830
(M-1b)
IR 96.0% of C9H18O2 1.0 1.418–1.420 0.869–0.874
(M-1b)
98.0% of C12H24O2 1.0 1.420–1.425 0.853–0.858
(M-1a)
IR 99.0% of C15H28O2 Solidification Pt.—NLT 35° (Appendix IIB)
(M-1b)
IR 93.0% of C5H8O2 1.402–1.406 0.952–0.962
(M-1b)
610 / 2-Pentanone / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
2-Pentanone colorless, mobile liq/ m—alc, ether, 1 mL
FEMA No. 2842 fruity, ethereal in 25 mL water/86.13/C5H10O/
CH3COCH2CH2CH3Methyl Propyl Ketone 102°
�-Phellandrene colorless to slightly yel s—alc; 1 mL in 1
FEMA No. 2856 liq/ ins—water mL 95% alc
p-Mentha-1,5-diene herbaceous; minty gives clear
background soln
136.24/C10H16/
CH3
CH(CH3)2
Phenethyl Acetate colorless liq/ s—alc, most fixed 1 mL in 2
FEMA No. 2857 sweet, rosy, honey oils, prop glycol; mL 70% alc
2-Phenethyl Acetate ins—gly, water/ remains clear
232° to 10 mL
164.20/C10H12O2/
CH2CH2OOCCH3
Phenethyl Alcohol colorless liq/ s—most fixed oils, 1 mL in 2
FEMA No. 2858 rose water, prop glycol/ mL 50% alc
2-Phenylethyl Alcohol 219° remains clear
to 10 mL
122.17/C8H10O/
CH2CH2OH
Phenethyl Isobutyrate colorless to slightly yel s—alc, most fixed 1 mL in 3
FEMA No. 2862 liq/ oils; mL 80% alc
fruity, rosy ins—water/ gives clear
230° soln
192.26/C12H16O2/
CH2CH2OOCCHCH3
CH3
Phenethyl Isovalerate colorless to slightly yel s—alc, most fixed 1 mL in 3
FEMA No. 2871 liq/ oils; mL 80% alc
fruity, rosy ins—water/ gives clear
263° soln
206.28/C13H18O2/
CH2CH2OOCCH2CHCH3
CH3
2-Phenethyl 2-Methylbutyrate colorless liq/ s—alc, most fixed
FEMA No. 3632 floral-fruity oils;
ins—water
206.28/C13H18O2/
CH2CH2OOCCHCH2CH3
CH3
Phenethyl Phenylacetate colorless to slightly yel s—alc; 1 mL in 4
FEMA No. 2866 liq above 26°/ ins—water/ mL 90% alc
rosy, hyacinth 325° gives clear
soln
240.30/C16H16O2/
CH2CH2OOCCH2
Phenethyl Salicylate white cryst/ s—alc; 1 g in 20 mL
FEMA No. 2868 balsamic ins—water/ 95% alc
370° gives clear
soln
242.27/C15H14O3/
CH2CH2OOC
OH
Complete Table
FCC V Flavor Chemicals / Phenethyl Salicylate / 611
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C5H10O 2.0 1.387–1.392 0.801–0.806
(M-1b)
IR 1.471–1.477 0.835–0.865 Angular Rotation—between −80° and −120°
(Appendix IIB, 100-mm tube)
IR 98.0% of C10H12O2 1.0 1.497–1.501 1.030–1.034
(M-1b)
IR 99.0% of C8H10O 1.531–1.534 1.017–1.020 Chlorinated Cmpds.—passes test (Appendix
(one isomer) VI)
(M-1a)
IR 98.0% of C12H16O2 1.0 1.486–1.490 0.987–0.990
(M-1b)
IR 98.0% of C13H18O2 1.0 1.484–1.486 0.973–0.976
(one major isomer)
(M-1b)
95.0% of C13H18O2 2.0 1.484–1.488 0.973–0.977
(M-1b)
IR 98.0% of C16H16O2 1.0 1.548–1.552 1.079–1.082 Solidification Pt.—NLT 26° (Appendix IIB)
(M-1b) (may solidify)
IR 98.0% of C15H14O3 1.0 (phenol Solidification Pt.—NLT 41° (Appendix IIB)
(M-1b) red TS)
612 / Phenoxyethyl Isobutyrate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Phenoxyethyl Isobutyrate colorless liq/ m—alc, chloroform, 1 mL in 3
FEMA No. 2873 honey, rose ether; mL 70% alc
ins—water/ gives clear
125°–127° (4 mm soln
208.26/C12H16O3/
OCH2CH2OOCCHCH3
CH3Hg)
Phenylacetaldehyde colorless to slightly yel, s—most fixed oils, 1 mL in 2
FEMA No. 2874 oily liq; becomes more prop glycol; mL 80% alc
�-Toluic Aldehyde viscous on aging/ ins—gly/
harsh; hyacinth on 195°
120.15/C8H8O/
CH2CHO
dilution
Phenylacetaldehyde Dimethyl colorless liq/ s—most fixed oils, 1 mL in 2
Acetal green, spicy, floral prop glycol; mL 70% alc
FEMA No. 2876 ins—gly/ remains clear
219° to 10 mL
166.22/C10H14O2/
CH2CH
OCH3
OCH3
Phenylacetic Acid glistening white cryst s—most fixed oils,
FEMA No. 2878 solid/ gly;
�-Toluic Acid persistent, disagreeable, ss—water/
suggestive of geranium 265°
136.15/C8H8O2/
CH2COOH
leaf and rose when
diluted
Phenylethyl Anthranilate colorless to pale yel cryst s—alc/
FEMA No. 2859 mass/ 324°
neroli, grape undertone
241.29/C15H15NO2/
CH2CH2OC O
NH2
Phenylethyl Butyrate colorless to pale yel liq/ 238° 1 mL in 1
FEMA No. 2861 green, hay mL 95% alc192.26/C12H16O2/
CH2CH2OOCCH2CH2CH3
Phenyl Ethyl Cinnamate white to pale yel cryst/ s—prop glycol, veg 1 mL in 1
FEMA No. 2863 floral oils; mL 95%
ins—water ethanol
252.31/C17H16O2/
OC
O
Complete Table
FCC V Flavor Chemicals / Phenyl Ethyl Cinnamate / 613
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C12H16O3 1.0 1.492–1.495 1.044–1.048
(M-1b)
IR 90.0% of C8H8O 5.0 1.525–1.545 1.025–1.045
(M-1b)
IR 95.0% of C10H14O2 1.0 1.493–1.496 1.000–1.006 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
Free Phenyl Acetaldehyde—1.0% (M-1b)
IR 99.0% of C8H8O2 (after Melting Range—between 76° and 78°
drying) (Appendix IIB, Class Ia)
(M-3b) Lead—10 mg/kg (M-9)
IR 98.0% of C15H15NO2 1.0 Solidification Pt.—NLT 40° (Appendix IIB)
(M-1b)
IR 98.0% of C12H16O2 1.0 1.487–1.492 0.991–0.995
(M-1b)
IR 99.0% of C17H16O2 1.0 Melting Point—NLT 54.0° (Appendix IIB)
(M-1b)
614 / Phenyl Ethyl Propionate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Phenyl Ethyl Propionate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2867 rose oils; mL 95%
ins—water/ ethanol
245°
178.23/C11H14O2/
OC
O
C2H5
3-Phenyl-1-propanol colorless, slightly viscous s—most fixed oils, 1 mL in 1
FEMA No. 2885 liq/ prop glycol; mL 70% alc
Phenylpropyl Alcohol; sweet, hyacinth- ins—gly/
Hydrocinnamyl Alcohol mignonette 236°
136.19/C9H12O/
CH2CH2CH2OH
2-Phenylpropionaldehyde water-white to pale yel s—most fixed oils;
FEMA No. 2886 liq/ ss—prop glycol;
Hydratropic Aldehyde; �-Methyl floral ins—gly/
Phenylacetaldehyde 222°
134.18/C9H10O/
CHCHO
CH3
3-Phenylpropionaldehyde colorless to slightly yel m—alc, ether; 1 mL in 7
FEMA No. 2887 liq/ ins—water/ mL 60% alc
Hydrocinnamaldehyde; strong, pungent, floral, 97°–98° (12 mm Hg) remains clear
Phenylpropyl Aldehyde hyacinth on dilution
134.18/C9H10O/
CH2CH2CHO
2-Phenylpropionaldehyde colorless to slightly yel s—alc, ether; 1 mL in 7
Dimethyl Acetal liq/ ins—water/ mL 60% alc,
FEMA No. 2888 mushroom 241° and in 3 mL
Hydratropic Aldehyde Dimethyl 70% alc
Acetal gives clear
180.25/C11H16O2/
CH
CH3
CH
OCH3
OCH3solns
3-Phenylpropyl Acetate colorless liq/ s—alc; 1 mL in 3
FEMA No. 2890 spicy, floral ins—water/ mL 70% alc
244° gives clear
soln
178.23/C11H14O2/
CH2CH2CH2OOCCH3
�-Pinene colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 2902 fresh, piney oils; mL 95%
2,6,6- ins—water/ ethanol
Trimethylbicyclo(3.1.1)hept-2- 155°
ene; 2-Pinene; l-�-Pinene
136.24/C10H16/
CH3
H3C
CH3
Complete Table
FCC V Flavor Chemicals / �-Pinene / 615
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 98.0% of C11H14O2 1.0 1.491–1.497 1.009–1.015
(M-1b)
IR 98.0% of C9H12O 1.524–1.528 0.998–1.002 Free 3-Phenyl(M-1b) Propionaldehyde—0.5% (M-1b)
IR 95.0% of C9H10O 5.0 1.515–1.520 0.998–1.006
(M-1b)
IR 90.0% of aldehydes 10.0 1.520–1.532 1.010–1.020 Chlorinated Cmpds.—passes test (Appendix
(M-1b) VI)
IR 95.0% of C11H16O2 1.492–1.497 0.989–0.994 Free 2-Phenylpropionaldehyde—3.0% (M-1b)
(M-1b)
IR 98.0% of C11H14O2 1.0 1.494–1.497 1.012–1.015
(M-1b)
97.0% of C10H16 1.464–1.468 0.855–0.860 Angular Rotation—between −20° and −50°
(M-1a) (Appendix IIB)
616 / �-Pinene / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
�-Pinene colorless liq/ s—most fixed oils; 1 mL in 3
FEMA No. 2903 resinous-piney ins—water, prop mL 95%
6,6-Dimethyl-2- glycol/ ethanol
methylenebicyclo[3.1.1]heptane 165°
136.24/C10H16/
CH3
H3C
CH2
Piperidine colorless to pale yel liq/ 106°
FEMA No. 2908 ammoniacal, fishy,
Hexahydropyridine nauseating
85.15/C5H11N/
NH
Piperonal white cryst substance/ vs—alc; 1 g in 4 mL
FEMA No. 2911 floral, heliotrope, free s—most fixed oils, 70% alc
3,4-(Methylenedioxy)- from safrole by-odor prop glycol;
benzaldehyde; Heliotropine; ins—gly, water/O
O
150.13/C8H6O3/
CHO
Piperonyl Aldehyde 264°
Propenylguaethol white cryst powder/ s—veg oils; 1 g in 15 mL
FEMA No. 2922 vanilla ins—water, 1 g in 20 95% ethanol
1-Ethoxy-2-hydroxy-4- mL 95% alc
propenylbenzene
178.23/C11H14O2/
OC2H5
OH
HC CHCH3
Propionaldehyde colorless, mobile liq/ m—alc, ether, water/
FEMA No. 2923 sharp, pungent 49°58.08/C3H6O/
CH3CH2CHO
Propyl Acetate colorless liq/ 102° 1 mL in 1
FEMA No. 2925 ethereal mL 95% alc102.13/C5H10O2/
CH3CH2CH2OOCCH3n-Propyl Acetate
Propyl Alcohol colorless liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2928 ethereal oils; mL 95% alc60.09/C3H8O/
CH3CH2CH2OHn-Propanol m—water/
97°
p-Propyl Anisole colorless to pale yel liq/ s—most fixed oils; 1 mL in 5
FEMA No. 2930 anise, with sassafras ins—gly, prop glycol/ mL 80% alc
Dihydroanethole background 215° remains in
soln on
150.22/C10H14O/
CH3O CH2CH2CH3
dilution
Complete Table
FCC V Flavor Chemicals / p-Propyl Anisole / 617
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
97.0% of C10H16 1.477–1.481 0.867–0.871 Angular Rotation—between −20° and −50°
(M-1a) (Appendix IIB)
IR 98.0% of C5H11N 1.450–1.454 0.858–0.862
(M-1a)
IR 99.0% of C8H6O3 Lead—10 mg/kg (M-9)
(M-1b) Solidification Pt.—NLT 35° (Appendix IIB)
IR 99.0% of C11H14O2 Melting Range—between 85° and 88°
(M-1a) (Appendix IIB)
Residue on Ignit.—0.1% (Appendix IIC, 2-g
sample)
IR 97.0% of C3H6O 5.0 (M-15) 0.800–0.805 Distillation Range—between 46° and 50° (first
(M-2c) 97%, Appendix IIB)
Water—2.5% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 97.0% of C5H10O2 1.0 1.382–1.387 0.880–0.886
(M-1b)
IR 99.0% of C3H8O 1.383–1.388 0.800–0.805
(M-1b)
IR 99.0% of C10H14O 1.502–1.506 0.940–0.943
(M-1a)
618 / Propyl Formate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Propyl Formate colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 2943 ethereal oils; mL 95%
ins—water/ ethanol
80°
88.11/C4H8O2/
H3C CH2 CH2 O C H
O
Propyl Mercaptan colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3521 onion oils; mL 95%
ins—water/ ethanol
67°
76.16/C3H8S/
H3C
H2C
CH2
S H
Propyl Propionate colorless to pale yel liq/ 123° 1 mL in 1
FEMA No. 2958 fruity mL 95% alc116.16/C6H12O2/
CH3CH2CH2OOCCH2CH3n-Propyl Propionate
Pyrrole colorless to yel liq, s—alc, most fixed
FEMA No. 3386 darkens on aging/ oils;
nutty, sweet, warm, ss—water/
ethereal 130° (decomp)
67.09/C4H5N/
NH
Rhodinol colorless liq/ s—most fixed oils, 1 mL in 1.2[see Citronellol, Geraniol, and Nerol]FEMA No. 2980 pronounced rose prop glycol; mL 70% alc
ins—gly/
68°–70° (1.8 mm
Hg)
Rhodinyl Acetate colorless to slightly yel s—alc, most fixed 1 mL in 2
FEMA No. 2981 liq/ oils; mL 80% alc[see Citronellyl Acetate and Geranyl Acetate]
light, fresh, rose ins—gly, prop glycol, remains in
water/ soln to 10
237° mL
Rhodinyl Formate colorless to slightly yel s—alc, most fixed 1 mL in 2[see Citronellyl Formate]FEMA No. 2984 liq/ oils; mL 80% alc
leafy, rose ins—gly, prop glycol, gives clear
water/ soln
220°
Salicylaldehyde colorless to yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3004 phenolic oils; mL 95%
ins—water/ ethanol
197°OH
O H
122.12/C7H6O2/
Complete Table
FCC V Flavor Chemicals / Salicylaldehyde / 619
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of C4H8O2 5.0 (add ice to 1.374–1.380 0.898–0.904
(M-1b) soln)
IR 97.0% of C3H8S 1.436–1.442 0.838–0.844
(M-1b)
IR 98.0% of C6H12O2 1.0 1.391–1.396 0.873–0.879
(M-1b)
IR 98.0% of C4H5N 1.507–1.510 0.950–0.980 Distillation Range—between 125° and 130°
(M-1a) (20°) (Appendix IIB)
Water—0.5% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 82.0% of total alcohols 1.463–1.473 0.860–0.880 Angular Rotation—between −4° and −9°
as C10H20O (Appendix IIB, 100-mm tube)
(Appendix VI; 1.2 g/ Esters—1.0% as citronellyl acetate (Appendix
78.14) VI; 5 g/99.15)
IR 87.0% of total esters as 1.0 1.450–1.458 0.895–0.908 Angular Rotation—between −2° and −6°
C12H22O2 (Appendix IIB, 100-mm tube)
(Appendix VI; 1.3 g/
99.15)
IR 85.0% of total esters as 2.0 1.453–1.458 0.901–0.908
C11H20O2
(Appendix VI; 1.3 g/
92.14)
IR 97.0% of C7H6O2 10.0 1.570–1.576 1.159–1.170
(M-1b) (use phenol
red indicator)
620 / Santalol / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Santalol colorless to slightly yel, vs—alc, most fixed 1 mL in 4
FEMA No. 3006 viscous liq/ oils, prop glycol; mL 70% alc
[Mixture of �- and �-isomers] sandalwood ins—gly, water/ gives clear
302° soln
220.35/C15H24O/
H2CHC C
CH2
CH
HCHC
CH3
CH2CH2CH
CH3
CCH2OH
CH3
Santalyl Acetate colorless to slightly yel s—alc; 1 mL in 9
FEMA No. 3007 liq/ ins—water/ mL 80% alc[mixture of α- and β- isomers from acetylation of Santalol]
sandalwood 315° gives clear
soln
�-Terpinene colorless liq/ s—alc, most fixed 1 mL in 2
FEMA No. 3558 lemon oils; mL 95%
1-Methyl-4-(1-methylethyl)-1,3- ins—water/ ethanol
cyclohexadiene 173°
136.24/C10H16/
CH3
H3C CH3
�-Terpinene colorless liq/ s—alc, most fixed 1 mL in 3
FEMA No. 3559 herbaceous, citrus oils; mL 95%
1-Methyl-4-(1-methylethyl)-1,4- ins—water/ ethanol
cyclohexadiene 182°
136.24/C10H16/
CH3
H3C CH3
Terpinen-4-ol colorless to pale yel liq/ s—alc/ 1 mL in 1
FEMA No. 2248 piney 88° (6 mm Hg) mL 95%
4-Carvomenthenol ethanol
154.25/C10H18O/
OH
�-Terpineol colorless, viscous liq s—prop glycol, veg 1 mL in 2
FEMA No. 3045 (high-purity material may oils; mL 70% alc,
p-Menth-1-en-8-ol solidify)/ ss—gly, water/ 4 mL 60%
lilac 217° alc, 8 mL
50% alc
154.25/C10H18O/
OH
Complete Table
FCC V Flavor Chemicals / �-Terpineol / 621
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 95.0% of total alcohols 1.505–1.509 0.965–0.975 Angular Rotation—between −11° and −19°
as C15H24O (Appendix IIB, 100-mm tube)
(Appendix VI; 1.6 g/
110.18)
IR 95.0% of total esters as 1.0 1.488–1.491 0.980–0.986
C17H26O2
(Appendix VI; 1.6 g/
131.2)
89.0% of C10H16 1.475–1.480 0.833–0.838
(M-1a)
95.0% of C10H16 1.473–1.477 0.841–0.845
(M-1a)
92.0% of C10H18O 1.476–1.480 0.928–0.934
(M-1b)
IR 96.0% of C10H18O 1.482–1.485 0.930–0.936
[sum of �-, (E)-�-, (Z)-
�-, �-, terpinen-4-ol, and
terpinen-1-ol isomers]
(M-1a)
622 / Terpinyl Acetate / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Terpinyl Acetate colorless liq/ s—alc, most fixed 1 mL in 5
FEMA No. 3047 sweet, refreshing, oils, min oil, prop mL 70% alc
Menthen-1-yl-8 Acetate herbaceous glycol; remains in
ss—gly; soln to 10
ins—water/ mL
220°
196.29/C12H20O2/
OC
CH3
O
Terpinyl Propionate colorless to slightly yel s—gly; 1 mL in 2
FEMA No. 3053 liq/ ss—prop glycol; mL 80% alc
Menthen-1-yl-8 Propionate sweet, floral, herbaceous, m—alc, chloroform, gives clear
lavender ether, most fixed oils; soln
ins—water/
240°
210.32/C13H22O2/
OC
CH2CH3
O
�-Tetradecalactone colorless to pale yel liq/ s—prop glycol, veg 1 mL in 1
FEMA No. 3590 fruity oils; mL 95%
ins—water/ ethanol
130° (5 mm Hg)
226.36/C14H26O2/
O OCH3(CH2)8
Tetrahydrofurfuryl Alcohol colorless liq/ 178°
FEMA No. 3056 mild, warm, oily, caramel102.13/C5H10O2/
OCH2OH
Tetrahydrolinalool colorless liq/ s—alc, most fixed
FEMA No. 3060 distinct floral oils;
3,7-Dimethyl-3-octanol ins—water/
71° (6 mm Hg)
158.28/C10H22O/
OH
2,3,5,6-Tetramethylpyrazine white cryst or powder/ s—alc, prop glycol,
FEMA No. 3237 fermented soybeans most fixed oils;
ss—water/
190°
136.20/C8H12N2/
N
N
H3C CH3
CH3H3C
Thymol white cryst/ s—water, prop 1 g in 1 mL
FEMA No. 3066 phenol glycol, veg oils/ 95% alc
232°
150.22/C10H14O/
CH3
OH
H3C CH3
Complete Table
FCC V Flavor Chemicals / Thymol / 623
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C12H20O2 1.464–1.467 0.953–0.962
(sum of �-, (E)-, �-, (Z)-
�-, �-, terpinen-4-ol, and
terpinen-1-ol isomers)
(M-1b)
IR 95.0% of C13H22O2 1.0 1.462–1.468 0.947–0.952
(sum of �-, (E)-, �-, (Z)-
�-, �-, terpinen-4-ol, and
terpinen-1-ol isomers)
(M-1b)
IR 98.0% of C14H26O2 5.0 1.459–1.465 0.931–0.937
(M-1b)
IR 99.0% of C5H10O2 1.452–1.453 1.050–1.052
(M-1a)
95.0% of C10H22O 1.431–1.435 0.823–0.829
(M-1a)
IR 95.0% of C8H12N2 Melting Range—between 85° and 90°
(M-1a) (Appendix IIB)
Water—0.2% (Appendix IIB, KF; use freshly
dist. pyridine as solvent)
IR 99.0% of C10H14O Melting Range—between 49° and 51°
(M-1a) (Appendix IIB)
624 / Tolualdehyde, Mixed Isomers / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Tolualdehyde, Mixed Isomers colorless liq/ 198° 1 mL in 1
FEMA No. 3068 cherry mL 95% alc
Tolyl Aldehyde, mixed isomers;
Methylbenzaldehyde
120.15/C8H8O/
CHO
CH3
p-Tolualdehyde colorless liq/ 83°–85° (11 mm Hg) 1 mL in 1
FEMA No. 3068 cherry mL 95% alc
p-Tolyl Aldehyde; p-
Methylbenzaldehyde
120.15/C8H8O/
CHO
CH3
p-Tolyl Isobutyrate colorless liq/ s—alc; 1 mL in 7
FEMA No. 3075 fruity ins—water/ mL 70% alc
p-Cresyl Isobutyrate 237° gives clear
soln
178.23/C11H14O2/
H3C OOCCHCH3
CH3
Tributyrin colorless, somewhat oily s—alc, chloroform,
FEMA No. 2223 liq/ ether;
Glyceryl Tributyrate; Butyrin almost odorless, slightly ins—water/
fatty 308°
302.37/C15H26O6/
H COCOC3H7
COCOC3H7H
COCOC3H7H
H
H
2-Tridecanone white to pale yel solid/ s—prop glycol, veg 1 g in 1 mL
FEMA No. 3388 herbal oils; 95% ethanol
ins—water/
134° (10 mm Hg)
198.35/C13H26O/
CH3C
(CH2)10CH3
O
2-Tridecenal white or slightly yellow s—alc, most fixed 1 mL in 1
FEMA No. 3082 liq/ oils; mL 95%196.33/C13H24O/
CH3(CH2)9CH CHCHOoily, citrus ins—water ethanol
Trimethylamine gas/ 2.9°
FEMA No. 3241 pungent, fishy,
ammoniacal
59.11/C3H9N/
CH3
NH3C CH3
3,5,5-Trimethyl Hexanal colorless to pale yel liq/ 67° (2.5 mm Hg)
FEMA No. 3524 melon, green142.24/C9H18O/
(CH3)3CCH2CH(CH3)CH2CHO
Complete Table
FCC V Flavor Chemicals / 3,5,5-Trimethyl Hexanal / 625
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 94.0% of C8H8O 5.0 1.540–1.548 1.019–1.029
(sum of three isomers)
(M-1b)
97.0% of C8H8O 5.0 1.542–1.548 1.012–1.018
(M-1b)
IR 95.0% of C11H14O2 1.0 1.485–1.489 0.990–0.996
(M-1b)
IR 99.0% of C15H26O6 5.0 1.431–1.441 1.034–1.037
(M-1b)
IR 95.0% of C13H26O Melting Point—NLT 27.0° (Appendix IIB)
(M-1b)
IR 92.0% of C13H24O 1.455–1.460 0.842–0.862
(M-1a)
98.0% of C3H9N in a
suitable solvent
(M-1a)
97.0% 5.0 1.419–1.424 0.817–0.823
(M-1b)
626 / 2,4,5-Trimethyl �-3-Oxazoline / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
2,4,5-Trimethyl �-3-Oxazoline yel orange liq/ s—alc, prop glycol,
FEMA No. 3525 powerful, musty, slight water;
green, wood, nut ins—most fixed oils
113.16/C6H11NO/
O
NH3C
H3C CH3
2,3,5-Trimethylpyrazine colorless to slightly yel s—org solvents, 1 mL in 1
FEMA No. 3244 liq/ water/ mL 95%
baked potato, peanut 171° ethanol
122.17/C7H10N2/
N
NH3C
CH3
CH3
�-Undecalactone colorless to pale yel liq/ 152°–155° (10.5 mm 1 mL in 1
FEMA No. 3294 creamy, peach Hg) mL 95% alc
5-Hydroxyundecanoic Acid
Lactone
184.28/C11H20O2/
O
O
H3C(CH2)4H2C
�-Undecalactone colorless to slightly yel s—alc, most fixed 1 mL in 5
FEMA No. 3091 liq/ oils, prop glycol; mL 60% alc
Aldehyde C-14 Pure, So-Called; fruity, peach ins—gly, water/
Peach Aldehyde 297°
184.28/C11H20O2/
CH3(CH2)6CHCH2CH2
O C O
Undecanal colorless to slightly yel s—most fixed oils,
FEMA No. 3092 liq/ prop glycol;170.30/C11H22O/
CH3(CH2)9CHOAldehyde C-11 Undecyclic; sweet, fatty, floral ins—gly, water/
n-Undecyl Aldehyde 223°
2-Undecanone colorless to pale yel liq/ 231°–232° 1 mL in 1
FEMA No. 3093 citrus, fatty, rue mL 95% alc170.30/C11H22O/
CH3CO(CH2)9CH3Methyl Nonyl Ketone
1,3,5-Undecatriene clear, colorless to pale 88° 1 Torr 1 mL in 25
FEMA No. 3795 yel liq/ mL 95% alc150.26/C11H18/
oily, waxy, peppery
10-Undecenal colorless to light yel liq/ s—most fixed oils,
FEMA No. 3095 fatty; rose on dilution prop glycol;168.28/C11H20O/
H2C CH(CH2)8CHOAldehyde C-11 Undecylenic; ins—gly, water/
Undecen-10-al 235°
(E)-2-Undecenol white to slightly yel liq/ ins—water 1 mL in 1
oily, sweet, floral mL 95%170.30/C11H22O/
CH3(CH2)7CH CHCH2OHethanol
Undecyl Alcohol colorless liq/ s—most fixed oils;
FEMA No. 3097 fatty-floral ins—water/172.31/C11H24O/
CH3(CH2)9CH2OHAlcohol C-11 146° (30 mm Hg)
Complete Table
FCC V Flavor Chemicals / Undecyl Alcohol / 627
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 94.0% of C6H11NO 1.414–1.435 0.911–0.932
(M-1a)
IR 98.0% of C7H10N2 1.503–1.507 0.970–0.980 Water—0.2% (Appendix IIB, KF; use freshly
(M-1a) dist. pyridine as solvent)
IR 98.0% of C11H20O2 1.457–1.461 0.956–0.961
(sum of two isomers;
� isomer 96.0% min)
(M-1a)
IR 98.0% of C11H20O2 5.0 1.448–1.453 0.942–0.945
(sum of two isomers;
� isomer 96.0% min)
(M-1b)
IR 92.0% of C11H22O 10.0 1.430–1.435 0.825–0.832
(M-1b)
IR 96.0% of C11H22O 5.0 1.428–1.432 0.822–0.826
(M-1a)
IR 90% of C11H18 1.508–1.517 0.787–0.793
(sum of isomers 90%
min)
(M-1b)
IR 90.0% of C11H20O 6.0 1.441–1.447 0.840–0.850
(M-1b)
IR 92.0% of C11H22O 1.448–1.453 0.840–0.846
(M-1a)
IR 97.0% of C11H24O 1.437–1.443 0.820–0.840
(M-1a)
628 / Valeraldehyde / Flavor Chemicals FCC V
General Information and Description
Name of Substance/ Formula Wt/Formula/ Physical Form/ Solubility2/ Solubility
Synonyms Structure Odor1 B.P.3 in Alcohol4
Valeraldehyde colorless to pale yel liq/ 103° 1 mL in 1
FEMA No. 3098 chocolate mL 95% alc86.13/C5H10O/
CH3CH2CH2CH2CHO
Valeric Acid colorless to pale yel, m—alc, ether, 1 mL
FEMA No. 3101 mobile liq/ in 40 mL water/102.13/C5H10O2/
CH3(CH2)3COOHPentanoic Acid unpleasant, penetrating, 186°
rancid
�-Valerolactone colorless to slightly yel m—alc, most fixed
FEMA No. 3103 liq/ oils, water/
warm, sweet, herbaceous 207°
100.12/C5H8O2/
OH3C O
Vanillin fine, white to slightly yel s—alc, chloroform,
FEMA No. 3107 cryst, usually needles/ ether, 1 g in 100 mL
4-Hydroxy-3- odor and taste of vanilla water at 25°, in 20
methoxybenzaldehyde mL gly, in 20 mL
water at 80°
152.15/C8H8O3/
CHO
OCH3
OH
Veratraldehyde white to tan or blue-gray 281° 1 g in 1 mL
FEMA No. 3109 flakes or solid/ 95% alc
Methyl Vanillin; Veratryl sweet, vanilla
Aldehyde; 3,4-
Dimethoxybenzaldehyde
166.18/C9H10O3/
H3CO
OCH3
CHO
Zingerone yel to yel-brown liq (may 290°
FEMA No. 3124 solidify at room temp)/
spicy
194.23/C11H14O3/
HO
CH3O
CH2CH2COCH3
Complete Table
FCC V Flavor Chemicals / Zingerone / 629
Requirements
I.D. Assay A.V. Ref.
Test5 Min. %6 Max.7 Index8 Sp. Gr.9 Other Requirements10
IR 97.0% of C5H10O 5.0 1.390–1.395 0.805–0.809
(M-2a)
IR 99.0% of C5H10O2 1.405–1.412 0.935–0.940
(M-3b)
IR 95.0% of C5H8O2 1.431–1.434 1.047–1.054
(M-1b)
IR 97.0% of C8H8O3 (on Loss on Drying—0.5% (Appendix IIC, silica
dried basis) gel/4 h)
(M-1b) Melting Range—between 81° and 83°
(Appendix IIB)
Residue on Ignit.—0.05% (Appendix IIC, 2-g
sample)
95.0% of C9H10O3 Solidification Pt.—NLT 40° (Appendix IIB)
(M1-b)
IR 95.0% of C11H14O3 1.538–1.545 1.136–1.140
(M-1b)
630 / Flavor Chemicals FCC V
TEST METHODS FOR FLAVOR CHEMICALS
This section provides the test methods by which certain flavorchemicals listed in the preceding tabular section are to beanalyzed.
M-1 ASSAY BY GASCHROMATOGRAPHY
M-1a General Method, Polar Column
Proceed as directed under GC Assay of Flavor Chemicals.The composition of the polar column and the conditions ofanalysis may be varied at the discretion of the analyst, pro-vided that such changes would result in equal or improvedseparations and/or quantification as would be obtained by useof the particular column material and test conditions specifiedtherein.
M-1b General Method, Nonpolar Column
Proceed as directed under GC Assay of Flavor Chemicals.The composition of the nonpolar column and the conditionsof analysis may be varied at the discretion of the analyst,provided that such changes would result in equal or improvedseparations and/or quantification as would be obtained by useof the particular column material and test conditions specifiedtherein.
M-2 ASSAYS FOR CERTAINALDEHYDES AND KETONES
M-2a Aldehydes—Hydroxylamine tert-Butyl AlcoholMethod
Hydroxylamine Solution Dissolve 45 g of reagent-gradehydroxylamine hydrochloride in 130 mL of water, add 850mL of tert-butyl alcohol, mix, and using a pH meter, neutralizeto a pH of 3.0 to 3.5 with sodium hydroxide.
Caution: Do not heat the solution.
Procedure Transfer an accurately weighed quantity of sam-ple, as specified below, into a 250-mL glass-stoppered flask.Add 50 mL of the Hydroxylamine Solution, mix thoroughly,and allow to stand at room temperature for the time specified.Titrate with 0.5 N sodium hydroxide to the same pH as thatof the Hydroxylamine Solution used. Calculate the percentaldehyde or ketone by the equation
AK = (S)(100e)/W,
in which AK is the percent aldehyde or ketone; S is the number
of milliliters of 0.5 N sodium hydroxide consumed in thetitration of the sample; e is the equivalence factor given below;and W is the weight, in milligrams, of the sample taken.
Sample ReactionWeight Time 1 mL of 0.5 N NaOH
Substance (g) (min) Equivalent to
Cuminic Aldehyde 1 60 74.11 mg of C10H12OMyristaldehyde 1.5 60 106.18 mg of C14H28OValeraldehyde 1 60 43.07 mg of C5H10O
M-2b Procedure Requiring the Use of Sealed GlassVials or Ampules
Transfer 65 mL of 0.5 N hydroxylamine hydrochloride and50.0 mL of 0.5 N triethanolamine into a suitable heat-resistantpressure bottle provided with a tight closure that can be fas-tened securely. Replace the air in the bottle by passing agentle stream of nitrogen for 2 min through a glass tubepositioned so that the end is just above the surface of theliquid. Add the quantity of sample specified below, containedin a sealed glass ampule, to the mixture in the pressure bottle.Introduce several pieces of 8-mm glass rod, cap the bottle,and shake vigorously to break the ampule. Allow the bottleto stand at room temperature for the time specified, swirlingoccasionally. Cool, if necessary, and uncap the bottle cau-tiously to prevent any loss of the contents. Titrate with 0.5N sulfuric acid to pH 3.4, using a suitable pH meter. Performa residual blank titration (see General Provisions). Each milli-liter of 0.5 N sulfuric acid is equivalent to the amount specifiedbelow.
Sample ReactionWeight Time 1 mL of 0.5 N H2SO4
Substance (mg) (min) Equivalent to
Acetaldehyde 600 30 22.03 mg of C2H4O
M-2c Aldehydes—Hydroxylamine Method
Hydroxylamine Hydrochloride Solution Dissolve 50 g ofhydroxylamine hydrochloride (preferably reagent grade orfreshly recrystallized before using) in 90 mL of water, anddilute to 1000 mL with aldehyde-free alcohol. Adjust thesolution to a pH of 3.4 with 0.5 N alcoholic potassium hy-droxide.
Procedure Transfer an accurately weighed quantity of sam-ple, as specified in the table below, into a 125-mL Erlenmeyerflask. Add 30 mL of Hydroxylamine Hydrochloride Solution,mix thoroughly, and allow to stand at room temperature for thetime specified below. Titrate with 0.5 N alcoholic potassium
FCC V Flavor Chemicals / 631
hydroxide to a green-yellow endpoint that matches the colorof 30 mL of Hydroxylamine Hydrochloride Solution in a 125-mL flask when the same volume of bromophenol blue TShas been added to each flask, or preferably, using a suitablepH meter, titrate to a pH of 3.4. Calculate the percent aldehyde(A) by the equation
A = (S – b)(100e)/W,
in which S is the number of milliliters of 0.5 N alcoholicpotassium hydroxide consumed in the titration of the sample;b is the number of milliliters of 0.5 N alcoholic potassiumhydroxide consumed in the titration of the blank; e is theequivalence factor given below; and W is the weight, in milli-grams, of the sample taken.
Sample ReactionWeight Time 1 mL of 0.5 N KOH
Substance (mg) (min) Equivalent to
Butyraldehyde 900 60 36.06 mg of C4H8OIsobutyraldehyde 900 60 36.06 mg of C4H8OPropionaldehyde 750 30 29.04 mg of C3H6O
M-2d Ketones—Hydroxylamine Method
Hydroxylamine Solution Dissolve 20 g of hydroxylaminehydrochloride (reagent grade or, preferably, freshly crystal-lized) in 40 mL of water, and dilute to 400 mL with alcohol.While stirring, add 300 mL of 0.5 N alcoholic potassiumhydroxide, and filter. Use this solution within 2 days.
Procedure Transfer an accurately weighed quantity of sam-ple, as specified below, into a 250-mL glass-stoppered flask.Add 75.0 mL of Hydroxylamine Solution to this flask and toa similar flask for a residual blank titration (see GeneralProvisions). Attach the flask to a suitable condenser, refluxthe mixture for the time specified, and then cool to roomtemperature. Titrate both flasks with 0.5 N hydrochloric acidto the same green-yellow endpoint using bromophenol blueTS as the indicator or, preferably, using a pH meter, to a pHof 3.4. (If the indicator is used, the endpoint color must bethe same as that produced when the blank is titrated to a pHof 3.4.) Calculate the percent ketone by the equation
K = (b – S)(100e)/W,
in which K is the percent ketone; b is the number of millilitersof 0.5 N hydrochloric acid consumed in the residual blanktitration; S is the number of milliliters of 0.5 N hydrochloricacid consumed in the titration of the sample; e is the equiva-lence factor given below; and W is the weight, in milligrams,of the sample taken.
Sample ReactionWeight Time 1 mL of 0.5 N HCl
Substance (mg) (min) Equivalent to
4-Methyl-2-pentanone 1200 60 50.08 mg of C6H12O
M-3 ASSAY BY TITRIMETRICPROCEDURES
M-3a Direct Aqueous Acid Base Titrations
Transfer an accurately weighed amount of sample, as specifiedbelow, into a 250-mL Erlenmeyer flask containing 75 to 100mL of water, add phenolphthalein TS, and titrate with 0.5 Nsodium hydroxide to the first pink color that persists for 15 s.Each milliliter of 0.5 N sodium hydroxide is equivalent to theamount of substance as specified below.
SampleWeight 1 mL of 0.5 N NaOH
Substance (g) Equivalent to
Butyric Acid 1.5 44.06 mg of C4H8O2
Hexanoic Acid 2.0 58.08 mg of C6H12O2
Isobutyric Acid 1.5 44.06 mg of C4H8O2
Isovaleric Acid 1.5 51.07 mg of C5H10O2
Levulinic Acid 1.0 58.08 mg of C5H8O3
2-Mercaptopropionic Acid 1.0 53.08 mg of C3H6O2S2-Methyl-2-pentenoic Acid 2.0 57.02 mg of C6H10O2
2-Methylbutyric Acid 1.0 51.06 mg of C5H10O2
4-Methylpentanoic Acid 2.0 58.05 mg of C6H12O2
2-Methylpentanoic Acid 2.0 58.05 mg of C6H12O2
Nonanoic Acid 1.0 79.12 mg of C9H18O2
M-3b Direct Aqueous Alcoholic Acid Base Titrations
Dissolve 1 g of sample, accurately weighed, in 50% ethanol/water that previously has been neutralized to phenolphthaleinTS with 0.1 N sodium hydroxide. Titrate with 0.5 N sodiumhydroxide to a pink color. Each milliliter of titrant is equivalentto the amount of substance specified below.
Conditions for Direct Aqueous Alcoholic Acid Base Titrations
1 mL of 0.5 N NaOHSubstance Equivalent to
Cinnamic Acid (dried indesiccator 3 h over silica gel) 74.08 mg of C9H8O2
2-Ethylbutyric Acid 58.08 mg of C6H12O2
Phenylacetic Acid (dried 3 hover H2SO4) 68.08 mg of C8H8O2
Valeric Acid 51.07 mg of C5H10O2
M-4 ALCOHOL CONTENT OF ETHYLOXYHYDRATE
Mix 25.0 mL of sample with an equal volume of water in aseparator, saturate with sodium chloride, and extract withthree 25-mL portions of solvent hexane. Extract the combinedsolvent hexane extracts with three 10-mL portions of a satu-rated solution of sodium chloride, and then discard the solvent
632 / Flavor Chemicals FCC V
hexane solutions. Combine the saline solutions in a suitabledistillation flask, and distill, collecting 25 mL of distillate.The specific gravity of the distillate is not greater than 0.9814,indicating an alcohol content of not less than 14.0% byvolume.
M-5 ACIDITY DETERMINATION BYIODOMETRIC METHOD
Ethyl Formate (Acidity as Formic Acid) Transfer about 5g of sample, accurately weighed, into a glass-stoppered flaskcontaining a solution of 500 mg of potassium iodate and 2 gof potassium iodide in 50 mL of water. Titrate the liberatediodine with 0.1 N sodium thiosulfate, using starch TS asthe indicator. Each milliliter of 0.1 N sodium thiosulfate isequivalent to 4.603 mg of CH2O2.
M-6 LIMIT TEST FOR ANTIOXIDANTSIN ETHYL ACRYLATE
Preliminary Examination of the Sample Wash a 25-mLportion of the sample with 25 mL of a 1:10 solution of sodiumhydroxide. Any yellow or brown coloration in the extractindicates the presence of hydroquinone, in which case bothof the procedures below (A and B) must be followed to deter-mine the antioxidant content. If the sodium hydroxide extractremains colorless, the first procedure (A) need not be run,and the antioxidant content is determined by the second proce-dure (B) alone.
A. Determination of Hydroquinone
Carbonyl-Free Methanol Add 5 g of 2,4-dinitrophenylhy-drazine to 500 mL of anhydrous methanol, heat the mixtureunder a reflux condenser for 2 h, and then recover the methanolby distillation. Store the carbonyl-free methanol in tight con-tainers.
2,4-Dinitrophenylhydrazine Solution Dissolve 100 mg of2,4-dinitrophenylhydrazine in 50 mL of Carbonyl-Free Meth-anol, add 4 mL of hydrochloric acid, and dilute to 100 mLwith water.
Sodium Carbonate Solution Dissolve 530 mg of sodiumcarbonate in sufficient water to make 100 mL.
Pyridine–Diethanolamine Solution Mix 5 mL of dietha-nolamine with 500 mL of freshly distilled pyridine.
Calibration Curve Transfer 25 mg of hydroquinone, accu-rately weighed, into a 100-mL volumetric flask, add sufficientbutyl acetate to volume, and mix thoroughly (250 �g/mL).Prepare a series of standards by transferring 1.0-, 2.0-, 3.0-,4.0-, and 6.0-mL portions of this solution into separate 50-mL volumetric flasks, and diluting each aliquot to 50.0 mL
with butyl acetate. One milliliter of each of these standardscontains 5, 10, 15, 20, and 30 �g, respectively, of hydroqui-none. Transfer 1.0 mL of each solution into separate 25-mLglass-stoppered graduates, and continue as directed in theProcedure, beginning with ‘‘. . . add 2.0 mL of water. . . .’’Plot a calibration curve of absorbance versus micrograms ofhydroquinone. Fifteen micrograms of hydroquinone shouldbe equivalent to approximately 0.30 units of absorbance, andthe curve should intersect the origin.
Procedure Using a hypodermic syringe, transfer 0.2 mLof sample, accurately weighed, into a 25-mL glass-stopperedgraduate, add 2.0 mL of water, stopper the graduate, and mixthe contents well without allowing contact between the liquidand the stopper. Add 0.5 mL of Sodium Carbonate Solutionto the mixture, and immediately shake gently for 5 s, avoidingcontact between the solution and the stopper. Immediatelyadd 1.0 mL of a 15% (v/v) solution of sulfuric acid, shakeas previously directed, and add 1 mL of 2,4-Dinitrophenylhy-drazine Solution. Stopper the graduate and place it in a waterbath, maintained at a temperature between 70° and 72°, for1 h. Shake samples three times during the heating period.Cool the graduate to room temperature, dilute the contents to15 mL with water, add 5.8 mL of benzene, stopper, shakevigorously, and then allow the phases to separate. Using asuitable pipet, transfer 2.0 mL of the benzene layer into a testtube, add 10.0 mL of Pyridine–Diethanolamine Solution, andmix. Transfer a portion of this solution into a 2-cm cell, anddetermine the absorbance at 620 nm with a suitable spectro-photometer, using as a blank 1.0 mL of butyl acetate treatedin the same manner as the sample except that 5.0 mL ofbenzene is used for the extraction instead of 5.8 mL. Fromthe previously prepared Calibration Curve, read the micro-grams of hydroquinone and/or benzoquinone correspondingto the absorbance of the solution from the sample, and recordthis value as w. Calculate the milligrams per kilogram ofhydroquinone (mg/kg HQ) in the sample by the formula
1000w/W,
in which W is the weight, in milligrams, of the sample taken.
B. Determination of Hydroquinone Monomethyl Ether
Antioxidant-Free Ethyl Acrylate Wash a suitable volumeof the sample with three separate, similar-sized volumes ofa 1:10 sodium hydroxide solution. After the last washing, adda small amount of sodium chloride, if necessary, to removeany turbidity that may be present.
Calibration Curve Transfer 25.0 mg of hydroquinonemonomethyl ether, accurately weighed, into a 100-mL volu-metric flask, add Antioxidant-Free Ethyl Acrylate to volume,and shake to effect complete solution (250 �g/mL). Preparea series of standards by transferring 1.0-, 5.0-, 10.0-, and 20.0-mL portions of this solution into separate 25-mL volumetricflasks, diluting each to volume with Antioxidant-Free EthylAcrylate, and mixing. One milliliter of each of the standardscontains 10, 50, 100, and 200 �g, respectively, of hydroqui-none monomethyl ether. Transfer 5.0 mL of each solutioninto separate 50-mL volumetric flasks, dilute each to volumewith isooctane, and mix. Determine the absorbance of each
FCC V Flavor Chemicals / 633
solution in a 1-cm silica cell at 292 nm with a suitable spectro-photometer, using a 1:10 dilution of Antioxidant-Free EthylAcrylate as the blank. Plot a calibration curve of absorbanceversus micrograms of hydroquinone monomethyl ether. Thecurve should be linear and should intersect the origin.
Procedure Transfer 5.0 mL of sample, accuratelyweighed, into a 50-mL volumetric flask, dilute to volumewith isooctane, and mix. Determine the absorbance of thissolution in a 1-cm silica cell at 292 nm with a suitable spectro-photometer, using a 1:10 dilution of Antioxidant-Free EthylAcrylate in isooctane as the blank. From the previously pre-pared Calibration Curve read the micrograms of hydroquinonemonomethyl ether corresponding to the absorbance of thesample solution, and record this value as w. Calculate themilligrams per kilogram of hydroquinone monomethyl ether(mg/kg HMME) in the sample by the formula
w/W,
in which W is the weight, in grams, of the sample taken.
Note: If the first sodium hydroxide extract obtainedunder Preliminary Examination of the Sample (or underAntioxidant-Free Ethyl Acrylate) showed a yellow col-oration, the true mg/kg HMME is obtained by sub-tracting the mg/kg HQ, obtained under section A, fromthe apparent mg/kg HMME.
M-7 LIMIT TEST FORHYDROCARBONS IN EUGENOL
Dissolve 1 mL of sample in 20 mL of 0.5 N sodium hydroxidecontained in a stoppered 50-mL tube, add 18 mL of water,and mix. A clear mixture results immediately, but it maybecome turbid when exposed to air.
M-8 LIMIT TEST FOR HYDROCYANICACID IN BENZALDEHYDE
Shake 0.5 mL of sample with 5 mL of water, add 0.5 mL of1 N sodium hydroxide and 0.1 mL of ferrous sulfate TS, andwarm the mixture gently. Upon the addition of a slight excessof hydrochloric acid, no green-blue color or blue precipitateevolves within 15 min.
M-9 LIMIT TEST FOR LEADA Sample Solution containing a 1-g sample and prepared asdirected for organic compounds meets the requirements ofthe Lead Limit Test, Appendix IIIB, using 10 �g of lead (Pb)ion in the control.
M-10 LIMIT TEST FOR METHYLCOMPOUNDS IN ETHYLACETATE
Transfer 20 mL of sample into a 500-mL separator, add asolution of 20 g of sodium hydroxide in 50 mL of water,stopper the separator, and wrap it securely in a towel forprotection against the heat of the reaction. Shake the mixturevigorously for about 5 min, cautiously opening the stopcockfrom time to time to permit the escape of air. Continue shakingthe mixture vigorously until a homogeneous liquid results,then distill, and collect about 25 mL of the distillate. Add 1drop of dilute phosphoric acid (1:20) and 1 drop of a 1:20solution of potassium permanganate to 1 drop of the distillate.Mix, allow to stand for 1 min, and add, dropwise, a 1:20solution of sodium bisulfite until the color disappears. If abrown color remains, add 1 drop of the dilute phosphoricacid. Add to the colorless solution 5 mL of a freshly prepared1:2000 solution of chromotropic acid in 75% sulfuric acid,and heat on a steam bath for 10 min at 60°. No violet colorappears.
M-11 LIMIT TEST FOR PEROXIDEVALUE
Add 10 mL of sample to 50 mL of a 3:2 (v/v) mixture ofglacial acetic acid and chloroform. Add 1 mL of a saturatedsolution of potassium iodide to this solution, allow to standfor exactly 1 min with gentle shaking, and then introduce 100mL of water and a few drops of starch TS. Titrate immediatelywith 0.1 N sodium thiosulfate. Each milliliter of 0.1 N sodiumthiosulfate, multiplied by 5, equals the peroxide value, ex-pressed in millimoles of peroxide per liter of the sample.
M-12 LIMIT TEST FOR READILYCARBONIZABLE SUBSTANCESIN ETHYL ACETATE
Carefully pour 2 mL of sample onto 10 mL of 95% sulfuricacid to form separate layers. No discoloration appears within15 min.
M-13 LIMIT TEST FOR READILYOXIDIZABLE SUBSTANCES INdl-MENTHOL
Transfer 500 mg of dl-menthol into a clean, dry test tube,and add 10 mL of potassium permanganate solution (preparedby diluting 3 mL of 0.1 N potassium permanganate to 100
634 / Flavor Chemicals FCC V
mL with water). Place the test tube in a beaker of watermaintained between 45° and 50°. At 30-s intervals, quicklyremove the test tube from the bath and shake. The color ofpotassium permanganate is still apparent after 5 min.
M-14 LIMIT TEST FOR REDUCINGSUBSTANCES
Dilute 2 mL of sample in a glass-stoppered flask with 50 mLof water and 5 mL of sulfuric acid, shaking the flask duringthe addition. While the solution is still warm, titrate with 0.1N potassium permanganate. Not more than 1 mL is requiredto produce a pink color that persists for 30 min.
M-15 ACID VALUEDissolve about 10 g of sample, accurately weighed, in 50 mLof alcohol, previously neutralized to phenolphthalein with 0.1N sodium hydroxide. (Add 50 g of ice when testing cinnamylformate, citronellyl formate, geranyl formate, isoamyl for-mate, or linalyl formate.) Add 1 mL of phenolphthalein TS,and titrate with 0.1 N sodium hydroxide until the solutionremains faintly pink after shaking for 10 s, unless otherwisedirected in the individual monograph. Calculate the acid value(AV) by the equation
AV = (5.61 × S)/W,
in which S is the number of milliliters of 0.1 N sodiumhydroxide consumed in the titration of the sample, and W isthe weight, in grams, of the sample.
When phenol red TS is specified as the indicator in theindividual monograph, proceed as directed above, and titratewith 0.1 N sodium hydroxide to the appearance of the firstendpoint, a yellow-orange color.
M-16 RESIDUE ON EVAPORATIONTransfer the quantity of sample specified in the monograph,accurately weighed, into a suitable evaporating dish that has
previously been heated on a steam bath, cooled to room tem-perature in a desiccator, and accurately weighed. Weigh thesample in the dish. Heat the evaporating dish containing thesample on the steam bath for 1 h. Cool the dish and its contentsto room temperature in a desiccator, and accurately weigh.Calculate the residue as percent of the sample used.
M-17 QUALITATIVE TEST FORPHENOLS USING FERRICCHLORIDE
Allyl Isothiocyanate Dilute 1 mL of the sample with 5 mLof alcohol, and add 1 drop of ferric chloride TS. A blue colordoes not immediately appear.
Anethole Shake 1 mL of sample with 20 mL of water, andallow the liquids to separate. Filter the water layer through afilter paper previously moistened with water, and add 3 dropsof ferric chloride TS to 10 mL of the filtrate. No purple colorappears.
Anisole Shake 1 mL of sample with about 20 mL of water,allow the layers to separate, collect the water layer in a testtube, and add a few drops of ferric chloride TS. No green,blue, or purple color appears.
Cresyl Acetate (Test for Free Cresol)
Ferric Chloride Solution Add 1.5 g of anhydrous ferricchloride to 850 mL of chloroform contained in a 2-L beaker.Add 100 mL of ethylene glycol monobutyl ether. When theferric chloride has dissolved, add 50 mL of pyridine, mix,and filter through a Büchner funnel.
Procedure Transfer 5 mL of sample into a 15-mm testtube, and add 10 mL of the Ferric Chloride Solution. Thecolor of the solution is not a darker green than is a solutionof 5 mL of a 1% solution of cresol in cresol-free methyl p-cresol mixed with 10 mL of the Ferric Chloride Solution.
FCC V Flavor Chemicals / 635
GAS CHROMATOGRAPHIC (GC) ASSAY OF FLAVOR CHEMICALS
This procedure applies both to the assay of flavor chemicalsand to the quantitation of minor components in flavor chemi-cals. Analysts following this procedure and performing thetest should obtain sufficient resolution of major and eventrace components of a mixture to calculate accurately theconcentration of the desired component; should be familiarwith the general principles, usual techniques, and instrumentalvariables normally met in gas chromatographic analysis; andshould pay particular attention to the following:
1. Stability of baseline, return to baseline before and aftereach peak of interest, and minimum use of recorder attenu-ation.
2. Any incompatibility between a sensitive sample compo-nent and column support, liquid substrate, or constructionmaterial.
3. The response to different components of the same ordifferent detectors. Because sizable errors may be encounteredin correlating area percent directly to weight percent, analystsmust know the methods for calculating response factors.
4. Where limits for minor components are specified inthe column entitled Other Requirements in the above tabularspecifications for flavor chemicals, analysts should use au-thentic materials to confirm the retention times of minor com-ponents. Determine the quantity of components following theinstructions below under Calculations and Methods.
I GC CONDITIONS FOR ANALYSISColumn: Open tubular capillary column of fused silica 30m × 0.25 to 0.53 mm (id), or equivalent.
Stationary phase:1. For a nonpolar column (or equivalent): methyl sili-
cone gum, or equivalent (preferably a bonded and cross-linkeddimethyl polysiloxane);
2. For a polar column (or equivalent): polyethylene gly-col, or equivalent (preferably a bonded and cross-linked poly-ethylene glycol);
3. The stationary phase coating should have a thickness of0.25 to 3 �m.
Carrier gas: Helium flowing at a linear velocity of 20 to 40cm/s.
Sample size: 0.1 to 1.0 �L.
Split ratio: [for 0.25-mm to 0.35-mm (id) columns only]50:1 to 200:1, typically, making sure that no one componentexceeds the capacity of the column. Peak fronting is indicativeof an overloaded column.
Inlet temperature: 225° to 275°.
Detector temperature: 250° to 300°.
Detectors: Use a thermal conductivity detector or a flameionization detector or a mass spectrometer, operating all asrecommended by the manufacturer.
Oven program: 50° to 240°, increasing the temperature by5°/min; and holding at 240° for 5 min.
Analysts may also use any GC conditions providing separa-tions equal to (or better than) those obtained with the abovemethod, but in the case of a dispute, the above methodmust stand.
II CALCULATIONS AND METHODSA. Peak area integration with total area detected normalized
to 100%, using electronic integrators: Use an electronic peakintegrator in accordance with the manufacturer’s recommen-dations, ensuring that the integration parameters permit properintegration of the peaks of a variety of shapes and magnitudesand do not interpret baseline shifts and noise spikes as areacontributed by the sample. Use internal or external standardsas needed to confirm that the total GC peak area correspondsto 100% of the components present in the sample.
B. Results obtained as described above are based on theassumption that the entire sample has eluted and the peaksof all of the components have been included in the calculation.They will be incorrect if any part of the sample does not eluteor if not all of the peaks are measured. In such cases, and inall methods described above, the internal standard methodmay be used to determine percentages based on the totalsample. For this method, measurements are required of thepeaks of the component(s) being assayed and of the internalstandard.
An accurately weighed or pipetted mixture of the internalstandard and the sample is prepared and chromatographed,the area ratio(s) of the component(s) to the standard is com-puted, and the percentage(s) of the component(s) is calculated.
If this calculation is to be applied, the substance used asthe standard should be one that meets the following criteria:
a. Its detector response is similar to that of the component(s)to be determined. In general, the more nearly the chemicalstructure of the component resembles that of the standard, thecloser the response will be.
b. Its retention time is close to, but not well resolved from,that of the component(s).
c. The internal standard is never a natural component foundin the sample.
The weight ratio of the internal standard to the sampleshould be such that the internal standard and the componentsought produce approximately equal peaks. This is, of course,not possible if several components of interest are at differentlevels of concentration.
636 / Flavor Chemicals FCC V
If the internal standard method is applied properly, it maybe assumed that the ratio of the weight of component to theweight of internal standard is exactly proportional to the peakarea ratio, and under these conditions no correction factor isneeded. The sample is first run by itself to determine whetherthe internal standard would mask any component by peaksuperposition. If there is no interference, a mixture is preparedof the sample and of the internal standard in the specifiedweight ratio, and the percentages of the internal standard andof the sample in the mixture are calculated. The mixture ischromatographed, and the areas of the component peak andthe internal standard peak are calculated by one of the methodsdescribed above.
The calculations are as follows:
1a. % Component in Mixture / % Internal Standard inMixture = Component Area / Internal Standard Area
or
1b. % Component in Mixture = % Internal Standard inMixture × (Component Area / Internal Standard Area)
2. % Component in Sample = (% Component in Mixture× 100) / % Sample in Mixture
If calibration is necessary, mixtures should be prepared of theinternal standard and component, either of 100% or of knownpurity. The number of mixtures and the weight ratios to beused depend on the component being analyzed. Usually, aminimum of three mixtures will be required. The weight ratioof one is chosen so that the heights of component and standardare equal. The ratios of the other two may be two-thirds andfour-thirds of this value. Each mixture should be chromato-graphed at least three times, and the areas calculated. Thefactor for each chromatograph should be calculated as speci-fied below, and the averages taken for each mixture. Anoverall average factor is calculated from them. The calibrationshould be performed periodically.
1. Factor = [(Weight Component × % Purity) / (Weight ofInternal Standard × % Purity)] × [Internal Standard Area /Component Area]
2. % Component in Sample Mixture = (Component Area× Factor × % Internal Standard in Sample Mixture) / InternalStandard Area
3. % Component in Sample = (% Component in SampleMixture × 100) / % Sample in Sample Mixture
III GC SYSTEM SUITABILITY TESTSAMPLE
The GC system suitability test sample consists of an equal-weight mixture of FCC-quality acetophenone, benzyl alcohol,benzyl acetate, linalool, and hydroxycitronellal.
Using the test sample described below, periodically test theperformance of and resolution provided by the gas chromato-graph employed. The test sample must display results compa-rable in quantitative composition, peak shape, and elutionorder to those specified herein. The quantitative compositionshould not deviate from the results listed below by more than�10%. Analyze the GC test sample using the GC Conditionsfor Analysis given above.
Order of Elution Normalized % Area (FID)
Component in TestSample Nonpolar Polar Nonpolar Polar
Benzyl Alcohol 1 4 22.0 21.3Acetophenone 2 2 21.1 21.4Linalool 3 1 20.8 21.0Benzyl Acetate 4 3 18.6 19.1Hydroxycitronellal 5 5 16.7 16.7
4/Infrared Spectra
INTRODUCTION
The infrared absorption spectra contained in this section areprovided in conjunction with the requirements for Identifica-tion as specified for a number of substances in this edition
ORGANIZATION
The spectra are arranged in alphabetical order.
SAMPLE PREPARATION
Most of the substances for which spectra are provided areliquids at or near room temperature. Unless otherwise noted
637
in the caption for an individual spectrum, the spectra foressential oils and flavor chemicals were obtained on the neatliquids contained in fixed-volume sodium chloride cells orbetween salt plates. For solids, the sample was prepared as apotassium bromide pellet or a mineral oil (Nujol or equivalent)dispersion, as indicated in the individual spectrum captions.The remaining substances were prepared as directed underIdentification in the individual monographs.
638 / Table of Contents / Infrared Spectra FCC V
Table of Contents for Infrared Spectra
Acetaldehyde 642Acetaldehyde Diethyl Acetal 642Acetanisole 642Acetoin 643Acetophenone 6433-Acetyl-2,5-dimethyl Furan 643N-Acetyl-L-methionine 6442-Acetylpyrazine 6443-Acetylpyridine 6442-Acetyl Thiazole 645DL-Alanine 645L-Alanine 645Allyl Cyclohexanepropionate 646Allyl Heptanoate 646Allyl Hexanoate 646Allyl �-Ionone 647Allyl Isothiocyanate 647Allyl Isovalerate 647Allyl Phenoxy Acetate 648Allyl Propionate 648Almond Oil, Bitter, FFPA 648Ambrette Seed Oil 649�-Amylcinnamaldehyde 649Amyl Cinnamate 649Amyl Octanoate 650Amyl Propionate 650Amyris Oil, West Indian Type 650Anethole 651Angelica Root Oil 651Angelica Seed Oil 651Anise Oil 652Anisole 652Anisyl Acetate 652Anisyl Alcohol 653Anisyl Formate 653L-Arginine 653L-Arginine
Monohydrochloride 654L-Asparagine 654Aspartame-Acesulfame Salt 654DL-Aspartic Acid 655L-Aspartic Acid 655Balsam Peru Oil 655Basil Oil, Comoros Type 656Basil Oil, European Type 656Bay Oil 656Benzaldehyde 657Benzaldehyde Glyceryl Acetal 6571,2-Benzodihydropyrone 657Benzophenone 658Benzyl Acetate 658Benzyl Alcohol 658Benzyl Benzoate 659Benzyl Butyrate 659
Benzyl Cinnamate 659Benzyl Formate 660Benzyl Isobutyrate 660Benzyl Isovalerate 660Benzyl Phenylacetate 661Benzyl Propionate 661Benzyl Salicylate 661Bergamot Oil, Coldpressed 662Birch Tar Oil, Rectified 662Black Pepper Oil 662Bois de Rose Oil 663Borneol 663Bornyl Acetate 663Butadiene-Styrene 50/50 Rubber
(Solid) 664Butadiene-Styrene 75/25 Rubber
(Emulsion-PolymerizedLatex) 664
Butadiene-Styrene 75/25 Rubber(Emulsion-PolymerizedSolid) 664
Butadiene-Styrene 50/50 Rubber(Latex) 665
2-Butanone 665Butan-3-one-2-yl Butanoate 665Butyl Acetate 666Butyl Alcohol 666Butyl Butyrate 666Butyl Butyryllactate 6672-sec-Butyl Cyclohexanone 667Butyl 667Butyl Isovalerate 668Butyl 2-Methyl Butyrate 668Butyl Phenylacetate 668Butyraldehyde 669Butyric Acid 669�-Butyrolactone 669Caffeine 670d-Camphor 670Cananga Oil 670Candelilla Wax 671Caraway Oil 671Cardamom Oil 671Carrot Seed Oil 672Carvacrol 672l-Carveol 672d-Carvone 673l-Carvone 673l-Carvyl Acetate 673�-Caryophyllene 674Cascarilla Oil 674Cassia Oil 674Cedar Leaf Oil 675Celery Seed Oil 675
Chamomile Oil, English Type 675Chamomile Oil, German Type 676Cinnamaldehyde 676Cinnamic Acid 676Cinnamon Bark Oil, Ceylon
Type 677Cinnamon Leaf Oil 677Cinnamyl Acetate 677Cinnamyl Alcohol 678Cinnamyl Butyrate 678Cinnamyl Cinnamate 678Cinnamyl Formate 679Cinnamyl Isobutyrate 679Cinnamyl Is sovalerate 679Cinnamyl Propionate 680Citral 680Citronellal 680Citronellol 681Citronellyl Acetate 681Citronellyl Butyrate 681Citronellyl Formate 682Citronellyl Isobutyrate 682Citronellyl Propionate 682Clary Oil 683Clove Leaf Oil 683Clove Oil 683Clove Stem Oil 684Cognac Oil, Green 684Copaiba Oil 684Coriander Oil 685Costus Root Oil 685p-Cresyl Acetate 685Cubeb Oil 686Cuminic Aldehyde 686Cumin Oil 686Cyclamen Aldehyde 687�-Cyclodextrin 687p-Cymene 687L-Cysteine
Monohydrochloride 688L-Cystine 688(E),(E)-2,4-Decadienal 688�-Decalactone 689�-Decalactone 689Decanal 689Decanoic Acid 690(E)-2-Decenal 690(Z)-4-Decenal 690Decyl Alcohol 691Diacetyl 691Dibenzyl Ether 691Diethyl Malonate 692Diethyl Sebacate 692Diethyl Succinate 692
FCC V Infrared Spectra / Table of Contents / 639
d-Dihydrocarvone 693Dill Seed Oil, European Type 693Dill Seed Oil, Indian Type 693Dillweed Oil, American Type 6942,6-Dimethoxy Phenol 694Dimethyl Benzyl Carbinol 694Dimethyl Benzyl Carbinyl
Acetate 695Dimethyl Benzyl Carbinyl
Butyrate 6953,4-Dimethyl 1,2-
Cyclopentandione 695Dimethyl Dicarbonate 6962,6-Dimethyl-5-heptenal 6963,7-Dimethyl-1-octanol 6962,3-Dimethylpyrazine 6972,5-Dimethylpyrazine 6972,6-Dimethylpyrazine 6972,5-Dimethylpyrrole 698Dimethyl Sulfide 698Diphenyl Ether 698�-Dodecalactone 699(E)-2-Dodecen-1-al 699Estragole 699Ethone 700Ethyl Acetate . 700Ethyl Acetoacetate 700Ethyl Acrylate 701Ethyl p-Anisate 701Ethyl Anthranilate 701Ethyl Benzoate 702Ethyl-(E)-2-butenoate 7022-Ethylbutyraldehyde 702Ethyl Butyrate 7032-Ethylbutyric Acid 703Ethyl Cinnamate 703Ethyl Decanoate 7042-Ethyl-3,5(6)-
dimethylpyrazine 704Ethylene Brassylate 7042-Ethyl Fenchol 705Ethyl Formate 7054-Ethyl Guaiacol 705Ethyl Heptanoate 706Ethyl Hexanoate 7065-Ethyl 3-Hydroxy 4-Methyl
2(5H)-Furanone 706Ethyl Isobutyrate 707Ethyl Isovalerate 707Ethyl Lactate 707Ethyl Laurate . 708Ethyl Levulinate 708Ethyl 2-Methylpentanoate 708Ethyl Methylphenylglycidate 7092-Ethyl-3-methylpyrazine 709Ethyl 3-Methylthiopropionate 709Ethyl Myristate 710Ethyl Nonanoate 710
Ethyl Octanoate 710Ethyl Phenylacetate 711Ethyl Phenylglycidate 711Ethyl Propionate 7113-Ethyl Pyridine 712Ethyl Salicylate 712Ethyl Vanillin 712Eucalyptol 713Eucalyptus Oil 713Eugenol 713Eugenyl Acetate 714Farnesol 714d-Fenchone 714Fenchyl Alcohol 715Fennel Oil 715Fir Needle Oil, Canadian
Type 715Fir Needle Oil, Siberian Type 716Furfural 716Furfuryl Alcohol 716Furfuryl Mercaptan 7172-Furyl Methyl Ketone 717Fusel Oil, Refined 717Garlic Oil 718Geraniol 718Geranium Oil, Algerian Type 718Geranyl Acetate 719Geranyl Benzoate 719Geranyl Butyrate 719Geranyl Formate 720Geranyl Isovalerate 720Geranyl Phenylacetate 720Geranyl Propionate 721Ginger Oil 721L-Glutamic Acid 721L-Glutamic Acid
Hydrochloride 722L-Glutamine 722Glycerol Ester of Gum Rosin 722Glycerol Ester of Partially
Dimerized Rosin 723Glycerol Ester of Partially
Hydrogenated Gum Rosin 723Glycerol Ester of Partially
Hydrogenated Wood Rosin 723Glycerol Ester of Polymerized
Rosin 724Glycerol Ester of Tall Oil
Rosin 724Glycerol Ester of Wood Rosin 724Glycine 725Grapefruit Oil, Coldpressed 725(E),(E)-2,4-Heptadienal 725�-Heptalactone 726Heptanal 7262,3-Heptandione 7262-Heptanone 7273-Heptanone 727
(Z)-4-Hepten-1-al 727Heptyl Alcohol 728�-Hexalactone 728Hexanoic Acid 728(E)-2-Hexen-1-ol 729(Z)-3-Hexenol 729(E)-2-Hexenyl Acetate 729(Z)-3-Hexenyl Acetate 730(Z)-3-Hexenyl Butyrate 730(Z)-3-Hexenyl Formate 730Hexyl Acetate 731Hexyl Alcohol 731Hexyl-2-butenoate 731Hexyl Butyrate 732�-Hexylcinnamaldehyde 732Hexyl Hexanoate 7324-Hexylresorcinol 733L-Histidine 733L-Histidine
Monohydrochloride 733Hops Oil 734Hydroxycitronellal 734Hydroxycitronellal Dimethyl
Acetal 7344-Hydroxy-2,5-dimethyl-3(2H)-
furanone 7354-(p-Hydroxyphenyl)-2-
butanone 735Indole 735�-Ionone 736�-Ionone 736Isoamyl Acetate 736Isoamyl Benzoate 737Isoamyl Butyrate 737Isoamyl Formate 737Isoamyl Hexanoate 738Isoamyl Isobutyrate 738Isoamyl Phenyl Acetate 738Isoamyl Salicylate 739Isoborneol 739Isobornyl Acetate 739Isobutyl Acetate 740Isobutyl Alcohol 740Isobutyl-2-butenoate 740Isobutyl Butyrate 741Isobutyl Cinnamate 741Isobutylene-Isoprene
Copolymer 741Isobutyl Formate 742Isobutyl Hexanoate 742Isobutyl Phenylacetate 742Isobutyl Salicylate 743Isobutyraldehyde 743Isobutyric Acid 743Isoeugenol 744Isoeugenyl Acetate 744DL-Isoleucine 744L-Isoleucine 745
640 / Table of Contents / Infrared Spectra FCC V
Isopropyl Acetate 745Isopulegol 745Isovaleric Acid 746Juniper Berries Oil 746Labdanum Oil 746Laurel Leaf Oil 747Lauryl Alcohol 747Lauryl Aldehyde 747Lavandin Oil, Abrial Type 748Lavender Oil 748Lemongrass Oil 748Lemon Oil, Coldpressed 749Lemon Oil, Desert Type,
Coldpressed 749Lemon Oil, Distilled, Brazil
Type 749DL-Leucine 750L-Leucine 750Levulinic Acid 750Lime Oil, Coldpressed, Mexican
Type 751Lime Oil, Coldpressed, Tahitian
Type 751Lime Oil, Distilled 751d-Limonene 752Linaloe Wood Oil 752Linalool 752Linalool Oxide 753Linalyl Acetate 753Linalyl Benzoate 753Linalyl Formate 754Linalyl Isobutyrate 754Linalyl Propionate 754Lovage Oil 755L-Lysine Monohydrochloride 755Mace Oil 755Maltol Isobutyrate 756Mandarin Oil, Coldpressed 756Marjoram Oil, Spanish Type 756Marjoram Oil, Sweet 757Mentha Arvensis Oil, Partially
Dementholized 757Menthol 7572-Mercaptopropionic Acid 758DL-Methionine 758L-Methionine 758p-Methoxybenzaldehyde 7592-Methoxy 3- (or 5- or 6-)
Isopropyl Pyrazine 7592-Methoxy-3(5)-
methylpyrazine 7594-p-Methoxyphenyl-2-
butanone 7602-Methoxypyrazine 760Methyl Acetate 7604-Methyl Acetophenone 761p-Methyl Anisole 761Methyl Anthranilate 761
Methyl Benzoate 762�-Methylbenzyl Acetate 762�-Methylbenzyl Alcohol 7623-Methyl Butanal 7632-Methylbutyric Acid 763�-Methylcinnamaldehyde 763Methyl Cinnamate 7646-Methylcoumarin 764Methyl Cyclopentenolone 764Methyl Ester of Rosin, Partially
Hydrogenated 765Methyl Eugenol 7655-Methyl Furfural 765Methyl Furoate 7666-Methyl-5-hepten-2-one 766Methyl Hexanoate 766Methyl Hexyl Ketone 767Methyl Isobutyrate 767Methyl Isoeugenol 7675-Methyl-2-isopropyl-2-
hexenal 768Methyl Isovalerate 768Methyl-3-
methylthiopropionate 768Methyl �-Naphthyl Ketone 769Methyl 2-Octynoate 7692-Methylpentanoic Acid 7694-Methylpentanoic Acid 7702-Methyl-2-pentenoic Acid 770Methyl Phenylacetate 770Methyl Phenylcarbinyl
Acetate 7715-Methyl 2-Phenyl 2-Hexenal 7712-Methylpyrazine 771Methyl Salicylate 7724-Methyl-5-thiazole Ethanol 772Methyl Thiobutyrate 7723-Methylthiopropionaldehyde 7732-Methylundecanal 773Methyl Valerate 773Monoammonium L-Glutamate 774Monopotassium L-Glutamate 774Monosodium L-Glutamate 774Morpholine 775Myrrh Oil 775�-Naphthyl Ethyl Ether 775Nerol 776Nerolidol 776Neryl Acetate 776(E),(E)-2,4-Nonadienal 777(E),(Z)-2,6-Nonadienal 777(E),(Z)-2,6-Nonadienol 777�-Nonalactone 778�-Nonalactone 778Nonanal 7782-Nonanone 779(E)-2-Nonenal 779(E)-2-Nonen-1-ol 779
(Z)-6-Nonen-1-ol 780Nonyl Acetate 780Nonyl Alcohol 780Nutmeg Oil 781�-Octalactone 781�-Octalactone 781Octanal 782(E)-2-Octen-1-al 7821-Octen-3-ol 782(Z)-3-Octen-1-ol 7831-Octen-3-yl Acetate 7831-Octen-3-yl Butyrate 783Octyl Acetate 784Octyl Alcohol 784Octyl Formate 784Olibanum Oil 785Onion Oil 785Orange Oil, Bitter,
Coldpressed 785Orange Oil, Coldpressed 786Orange Oil, Distilled 786Origanum Oil, Spanish Type 786Orris Root Oil 787Palmarosa Oil 787Paraffin, Synthetic 787Parsley Herb Oil 788Parsley Seed Oil 788Pennyroyal Oil 788�-Pentadecalactone 789Pentaerythritol Ester of Partially
Hydrogenated Wood Rosin 789Pentaerythritol Ester of Wood
Rosin 7892,3-Pentanedione 7902-Pentanone 790Peppermint Oil 790Petitgrain Oil, Paraguay Type 791Petroleum Wax (Refined) 791Petroleum Wax
(Microcrystalline) 791Petroleum Wax, Synthetic 792�-Phellandrene 792Phenethyl Acetate 792Phenethyl Alcohol 793Phenethyl Isobutyrate 793Phenethyl Isovalerate 793Phenethyl Phenylacetate 794Phenethyl Salicylate 794Phenoxyethyl Isobutyrate 794Phenylacetaldehyde 795Phenylacetaldehyde Dimethyl
Acetal 795Phenylacetic Acid 795DL-Phenylalanine 796L-Phenylalanine 796Phenylethyl Anthranilate 796Phenylethyl Butyrate 797Phenyl Ethyl Cinnamate 797
FCC V Infrared Spectra / Table of Contents / 641
Phenyl Ethyl Propionate 7973-Phenyl-1-propanol 7982-Phenylpropionaldehyde 7983-Phenylpropionaldehyde 7982-Phenylpropionaldehyde Dimethyl
Acetal 7993-Phenylpropyl Acetate 799Pimenta Leaf Oil 799Pimenta Oil 800Pine Needle Oil, Dwarf 800Pine Needle Oil, Scotch Type 800Piperidine 801Piperonal 801Polyethylene 801Polyisobutylene 802Polyvinyl Acetate 802L-Proline 802Propenylguaethol 803Propionaldehyde 803Propyl Acetate 803Propyl Alcohol 804p-Propyl Anisole 804Propyl Formate 804Propyl Mercaptan 805Propyl Propionate 805Pyrrole 805Rhodinol 806Rhodinyl Acetate 806Rhodinyl Formate 806
Rice Bran Wax 807Rosemary Oil 807Rose Oil 807Rue Oil 808Sage Oil, Dalmatian Type 808Sage Oil, Spanish Type 808Salatrim 809Salicylaldehyde 809Sandalwood Oil, East Indian
Type 809Santalol 810Santalyl Acetate 810Savory Oil (Summer Variety) 810DL-Serine 811L-Serine 811Spearmint Oil 811Spike Lavender Oil 812Sucrose Acetate Isobutyrate 812Tangerine Oil, Coldpressed 812Tarragon Oil 813�-Terpineol 813Terpinyl Acetate 813Terpinyl Propionate 814�-Tetradecalactone 814Tetrahydrofurfuryl Alcohol 8142,3,5,6-Tetramethylpyrazine 815L-Threonine 815Thyme Oil 815Thymol 816
Tolualdehyde (MixedIsomers) 816
p-Tolyl Isobutyrate 816Triacetin 817Tributyrin 8172-Tridecanone 8172-Tridecenal 8182,4,5-Trimethyl �-3-Oxazoline 8182,3,5-Trimethylpyrazine 818DL-Tryptophan 819L-Tryptophan 819L-Tyrosine 819�-Undecalactone 820�-Undecalactone 820Undecanal 8202-Undecanone 8211,3,5-Undecatriene 82110-Undecenal 821(E)-2-Undecenol 822Undecyl Alcohol 822Valeraldehyde 822Valeric Acid 823�-Valerolactone 823L-Valine 823Vanillin 824Vegetable Oil Phytosterol
Esters 824Wintergreen Oil 824Zingerone 825
642 / Acetaldehyde / Infrared Spectra FCC V
Acetaldehyde
TR
AN
SM
ITT
AN
CE
(%)
Acetaldehyde Diethyl Acetal
TR
AN
SM
ITT
AN
CE
(%)
Acetanisole
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / 3-Acetyl-2,5-dimethyl Furan / 643
Acetoin
TR
AN
SM
ITT
AN
CE
(%)
Acetophenone
TR
AN
SM
ITT
AN
CE
(%)
3-Acetyl-2,5-dimethyl Furan
TR
AN
SM
ITT
AN
CE
(%)
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644 / N-Acetyl-L-methionine / Infrared Spectra FCC V
N-Acetyl-L-methionine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
2-Acetylpyrazine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
3-Acetylpyridine
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / L-Alanine / 645
2-Acetyl Thiazole
TR
AN
SM
ITT
AN
CE
(%)
DL-Alanine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
L-Alanine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
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646 / Allyl Cyclohexanepropionate / Infrared Spectra FCC V
Allyl Cyclohexanepropionate
TR
AN
SM
ITT
AN
CE
(%)
Allyl Heptanoate
TR
AN
SM
ITT
AN
CE
(%)
Allyl Hexanoate
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Allyl Isovalerate / 647
Allyl �-Ionone
TR
AN
SM
ITT
AN
CE
(%)
Allyl Isothiocyanate
TR
AN
SM
ITT
AN
CE
(%)
Allyl Isovalerate
TR
AN
SM
ITT
AN
CE
(%)
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648 / Allyl Phenoxy Acetate / Infrared Spectra FCC V
Allyl Phenoxy Acetate
TR
AN
SM
ITT
AN
CE
(%)
Allyl Propionate
TR
AN
SM
ITT
AN
CE
(%)
Almond Oil, Bitter, FFPA
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Amyl Cinnamate / 649
Ambrette Seed Oil
TR
AN
SM
ITT
AN
CE
(%)
�-Amylcinnamaldehyde
TR
AN
SM
ITT
AN
CE
(%)
Amyl Cinnamate
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
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650 / Amyl Octanoate / Infrared Spectra FCC V
Amyl Octanoate
TR
AN
SM
ITT
AN
CE
(%)
Amyl Propionate
TR
AN
SM
ITT
AN
CE
(%)
Amyris Oil, West Indian Type
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Angelica Seed Oil / 651
Anethole
TR
AN
SM
ITT
AN
CE
(%)
Angelica Root Oil
TR
AN
SM
ITT
AN
CE
(%)
Angelica Seed Oil
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
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652 / Anise Oil / Infrared Spectra FCC V
Anise Oil
TR
AN
SM
ITT
AN
CE
(%)
Anisole
TR
AN
SM
ITT
AN
CE
(%)
Anisyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / L-Arginine / 653
Anisyl Alcohol
TR
AN
SM
ITT
AN
CE
(%)
Anisyl Formate
TR
AN
SM
ITT
AN
CE
(%)
L-Arginine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
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654 / L-Arginine Monohydrochloride / Infrared Spectra FCC V
L-Arginine Monohydrochloride (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
L-Asparagine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
Aspartame-Acesulfame Salt (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
View Monograph
FCC V Infrared Spectra / Balsam Peru Oil / 655
DL-Aspartic Acid (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
L-Aspartic Acid (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
Balsam Peru Oil
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
View Monograph
656 / Basil Oil, Comoros Type / Infrared Spectra FCC V
Basil Oil, Comoros Type
TR
AN
SM
ITT
AN
CE
(%)
Basil Oil, European Type
TR
AN
SM
ITT
AN
CE
(%)
Bay Oil
TR
AN
SM
ITT
AN
CE
(%)
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View Monograph
View Monograph
FCC V Infrared Spectra / 1,2-Benzodihydropyrone / 657
Benzaldehyde
TR
AN
SM
ITT
AN
CE
(%)
Benzaldehyde Glyceryl Acetal
TR
AN
SM
ITT
AN
CE
(%)
1,2-Benzodihydropyrone
TR
AN
SM
ITT
AN
CE
(%)
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658 / Benzophenone / Infrared Spectra FCC V
Benzophenone
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Alcohol
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Benzyl Cinnamate / 659
Benzyl Benzoate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Butyrate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Cinnamate
TR
AN
SM
ITT
AN
CE
(%)
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660 / Benzyl Formate / Infrared Spectra FCC V
Benzyl Formate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Isobutyrate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Isovalerate
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Benzyl Salicylate / 661
Benzyl Phenylacetate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Propionate
TR
AN
SM
ITT
AN
CE
(%)
Benzyl Salicylate
TR
AN
SM
ITT
AN
CE
(%)
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662 / Bergamot Oil, Coldpressed / Infrared Spectra FCC V
Bergamot Oil, Coldpressed
TR
AN
SM
ITT
AN
CE
(%)
Birch Tar Oil, Rectified
TR
AN
SM
ITT
AN
CE
(%)
Black Pepper Oil
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
View Monograph
FCC V Infrared Spectra / Bornyl Acetate / 663
Bois de Rose Oil
TR
AN
SM
ITT
AN
CE
(%)
Borneol
TR
AN
SM
ITT
AN
CE
(%)
Bornyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
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664 / Butadiene-Styrene 50/50 Rubber / Infrared Spectra FCC V
Butadiene-Styrene 50/50 Rubber (Solid)
TR
AN
SM
ITT
AN
CE
(%)
Butadiene-Styrene 75/25 Rubber (Emulsion-Polymerized Latex)
TR
AN
SM
ITT
AN
CE
(%)
Butadiene-Styrene 75/25 Rubber (Emulsion-Polymerized Solid)
TR
AN
SM
ITT
AN
CE
(%)
FCC V Infrared Spectra / Butan-3-one-2-yl Butanoate / 665
Butadiene-Styrene 50/50 Rubber (Latex)
TR
AN
SM
ITT
AN
CE
(%)
2-Butanone
TR
AN
SM
ITT
AN
CE
(%)
Butan-3-one-2-yl Butanoate
TR
AN
SM
ITT
AN
CE
(%)
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666 / Butyl Acetate / Infrared Spectra FCC V
Butyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
Butyl Alcohol
TR
AN
SM
ITT
AN
CE
(%)
Butyl Butyrate
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Butyl Isobutyrate / 667
Butyl Butyryllactate
TR
AN
SM
ITT
AN
CE
(%)
2-sec-Butyl Cyclohexanone
TR
AN
SM
ITT
AN
CE
(%)
Butyl Isobutyrate
TR
AN
SM
ITT
AN
CE
(%)
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668 / Butyl Isovalerate / Infrared Spectra FCC V
Butyl Isovalerate
TR
AN
SM
ITT
AN
CE
(%)
Butyl 2-Methyl Butyrate
TR
AN
SM
ITT
AN
CE
(%)
Butyl Phenylacetate
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / �-Butyrolactone / 669
Butyraldehyde
TR
AN
SM
ITT
AN
CE
(%)
Butyric Acid
TR
AN
SM
ITT
AN
CE
(%)
�-Butyrolactone
TR
AN
SM
ITT
AN
CE
(%)
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670 / Caffeine / Infrared Spectra FCC V
Caffeine (Mineral Oil Mull)
TR
AN
SM
ITT
AN
CE
(%)
d-Camphor
TR
AN
SM
ITT
AN
CE
(%)
Cananga Oil
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Cardamom Oil / 671
Candelilla Wax
TR
AN
SM
ITT
AN
CE
(%)
Caraway Oil
TR
AN
SM
ITT
AN
CE
(%)
Cardamom Oil
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
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672 / Carrot Seed Oil / Infrared Spectra FCC V
Carrot Seed Oil
TR
AN
SM
ITT
AN
CE
(%)
Carvacrol
TR
AN
SM
ITT
AN
CE
(%)
l-Carveol
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
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FCC V Infrared Spectra / l-Carvyl Acetate / 673
d-Carvone
TR
AN
SM
ITT
AN
CE
(%)
l-Carvone
TR
AN
SM
ITT
AN
CE
(%)
l-Carvyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
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674 / �-Caryophyllene / Infrared Spectra FCC V
�-Caryophyllene
TR
AN
SM
ITT
AN
CE
(%)
Cascarilla Oil
TR
AN
SM
ITT
AN
CE
(%)
Cassia Oil
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Chamomile Oil, English Type / 675
Cedar Leaf Oil
TR
AN
SM
ITT
AN
CE
(%)
Celery Seed Oil
TR
AN
SM
ITT
AN
CE
(%)
Chamomile Oil, English Type
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
View Monograph
676 / Chamomile Oil, German Type / Infrared Spectra FCC V
Chamomile Oil, German Type
TR
AN
SM
ITT
AN
CE
(%)
Cinnamaldehyde
TR
AN
SM
ITT
AN
CE
(%)
Cinnamic Acid
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
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FCC V Infrared Spectra / Cinnamyl Acetate / 677
Cinnamon Bark Oil, Ceylon Type
TR
AN
SM
ITT
AN
CE
(%)
Cinnamon Leaf Oil
TR
AN
SM
ITT
AN
CE
(%)
Cinnamyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
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678 / Cinnamyl Alcohol / Infrared Spectra FCC V
Cinnamyl Alcohol
TR
AN
SM
ITT
AN
CE
(%)
Cinnamyl Butyrate
TR
AN
SM
ITT
AN
CE
(%)
Cinnamyl Cinnamate
TR
AN
SM
ITT
AN
CE
(%)
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FCC V Infrared Spectra / Cinnamyl Isovalerate / 679
Cinnamyl Formate
TR
AN
SM
ITT
AN
CE
(%)
Cinnamyl Isobutyrate
TR
AN
SM
ITT
AN
CE
(%)
Cinnamyl Isovalerate
TR
AN
SM
ITT
AN
CE
(%)
View Flavor Chemical
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680 / Cinnamyl Propionate / Infrared Spectra FCC V
Cinnamyl Propionate
TR
AN
SM
ITT
AN
CE
(%)
Citral
TR
AN
SM
ITT
AN
CE
(%)
Citronellal
TR
AN
SM
ITT
AN
CE
(%)
View Flavor Chemical
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FCC V Infrared Spectra / Citronellyl Butyrate / 681
Citronellol
TR
AN
SM
ITT
AN
CE
(%)
Citronellyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
Citronellyl Butyrate
TR
AN
SM
ITT
AN
CE
(%)
View Flavor Chemical
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682 / Citronellyl Formate / Infrared Spectra FCC V
Citronellyl Formate
TR
AN
SM
ITT
AN
CE
(%)
Citronellyl Isobutyrate
TR
AN
SM
ITT
AN
CE
(%)
Citronellyl Propionate
TR
AN
SM
ITT
AN
CE
(%)
View Flavor Chemical
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FCC V Infrared Spectra / Clove Oil / 683
Clary Oil
TR
AN
SM
ITT
AN
CE
(%)
Clove Leaf Oil
TR
AN
SM
ITT
AN
CE
(%)
Clove Oil
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
View Monograph
684 / Clove Stem Oil / Infrared Spectra FCC V
Clove Stem Oil
TR
AN
SM
ITT
AN
CE
(%)
Cognac Oil, Green
TR
AN
SM
ITT
AN
CE
(%)
Copaiba Oil
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
View Monograph
View Monograph
FCC V Infrared Spectra / p-Cresyl Acetate / 685
Coriander Oil
TR
AN
SM
ITT
AN
CE
(%)
Costus Root Oil
TR
AN
SM
ITT
AN
CE
(%)
p-Cresyl Acetate
TR
AN
SM
ITT
AN
CE
(%)
View Monograph
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686 / Cubeb Oil / Infrared Spectra FCC V
Cubeb Oil
TR
AN
SM
ITT
AN
CE
(%)
Cuminic Aldehyde
TR
AN
SM
ITT
AN
CE
(%)
Cumin Oil
TR
AN
SM
ITT
AN
CE
(%)
Next Page
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5/General Tests and Assays
Contents
APPENDIX I:APPARATUS FOR TESTS AND ASSAYS . . . 831
Oxygen Flask Combustion . . . 831Thermometers . . . 831Volumetric Apparatus . . . 832Weights and Balances . . . 833
APPENDIX II:PHYSICAL TESTS ANDDETERMINATIONS . . . 834A. Chromatography . . . 834
Column Chromatography . . . 834Thin-Layer Chromatography . . . 835Gas Chromatography . . . 836High-Performance Liquid
Chromatography . . . 838B. Physicochemical Properties . . . 841
Distillation Range . . . 841Melting Range or Temperature . . . 842Optical (Specific) Rotation . . . 844pH Determination . . . 844Readily Carbonizable Substances . . . 845Refractive Index . . . 846Solidification Point . . . 846Viscosity . . . 848
Viscosity of Dimethylpolysiloxane . . . 848Viscosity of Methylcellulose . . . 849Viscosity of Cellulose Gum . . . 850
Water Determination . . . 851Method I: Karl Fischer Titrimetric
Method . . . 851
827
Method II: Toluene DistillationMethod . . . 853
C. Others . . . 854Ash (Acid-Insoluble) . . . 854Ash (Total) . . . 854Hydrochloric Acid Table . . . 854Loss on Drying . . . 855Oil Content of Synthetic Paraffin . . . 855Residue on Ignition (Sulfated Ash) . . . 857Sieve Analysis of Granular Metal
Powders . . . 858Sulfuric Acid Table . . . 858
APPENDIX III:CHEMICAL TESTS ANDDETERMINATIONS . . . 859A. Identification Tests . . . 859B. Limit Tests . . . 861
Arsenic Limit Test . . . 861Cadmium Limit Test . . . 863Chloride and Sulfate Limit Tests . . . 863
Chloride Limit Test . . . 863Sulfate Limit Test . . . 863
1,4-Dioxane Limit Test . . . 863Fluoride Limit Test . . . 864
Method I: Thorium Nitrate ColorimetricMethod . . . 864
Method II: Ion-Selective Electrode MethodA . . . 865
Method III: Ion-Selective Electrode MethodB . . . 865
828 / Contents / General Tests and Assays FCC V
Method IV: Ion-Selective Electrode MethodC . . . 866
Method V . . . 866Lead Limit Test . . . 867
Dithizone Method . . . 867Flame Atomic Absorption Spectrophotometric
Method . . . 868Atomic Absorption Spectrophotometric
Graphite Furnace Method . . . 869Method I . . . 869Method II . . . 870
APDC Extraction Method . . . 871Manganese Limit Test . . . 872Mercury Limit Test . . . 872
Method I . . . 872Method II . . . 873
Nickel Limit Test . . . 874Method I . . . 874Method II . . . 874
Phosphorus Limit Test . . . 874Selenium Limit Test . . . 875
Method I . . . 875Method II . . . 876
C. Others . . . 876Alginates Assay . . . 876�-Amino Nitrogen (AN) Determination . . . 877Ammonia Nitrogen (NH3-N)
Determination . . . 877Benzene (in Paraffinic Hydrocarbon
Solvents) . . . 878Colors . . . 880
Chromium . . . 880Ether Extracts . . . 880Leuco Base . . . 881Mercury . . . 881Sodium Chloride . . . 882Sodium Sulfate . . . 882Total Color . . . 882Uncombined Intermediates and Products of
Side Reactions . . . 884Glutamic Acid . . . 886Hydroxypropoxyl Determination . . . 887Methoxyl Determination . . . 887Nitrogen Determination (Kjeldahl
Method) . . . 888Method I . . . 888Method II . . . 889
Sulfur (by Oxidative Microcoulometry) . . . 889
APPENDIX IV:CHEWING GUM BASE POLYMERS . . . 892
Bound Styrene . . . 892Molecular Weight . . . 892
Polyethylene . . . 892Polyisobutylene (Flory Method) . . . 893Polyvinyl Acetate . . . 893
Quinones . . . 893Residual Styrene . . . 894Sample Solution for Arsenic Limit Test . . . 895
Sample Solution for Lead Limit Test . . . 895Total Unsaturation . . . 895
APPENDIX V:ENZYME ASSAYS . . . 896
Acid Phosphatase Activity . . . 898Aminopeptidase (Leucine) Activity . . . 899�-Amylase Activity (Nonbacterial) . . . 900�-Amylase Activity (Bacterial) . . . 901Catalase Activity . . . 902Cellulase Activity . . . 902Chymotrypsin Activity . . . 904Diastase Activity (Diastatic Power) . . . 904�-Galactosidase Activity . . . 905�-Glucanase Activity . . . 906Glucoamylase Activity (Amyloglucosidase
Activity) . . . 907Glucose Isomerase Activity . . . 908Glucose Oxidase Activity . . . 909Hemicellulase Activity . . . 909Invertase Activity . . . 911Lactase (Neutral) (�-Galactosidase) Activity . . . 911Lactase (Acid) (�-Galactosidase) Activity . . . 913Lipase Activity . . . 914Lipase (Microbial) Activity for Medium- and Long-
Chain Fatty Acids . . . 914Lysozyme Activity . . . 915Maltogenic Amylase Activity . . . 916Milk-Clotting Activity . . . 917Pancreatin Activity . . . 917Pepsin Activity . . . 920Phospholipase A2 Activity . . . 920Phytase Activity . . . 921Plant Proteolytic Activity . . . 922Proteolytic Activity, Bacterial (PC) . . . 923Proteolytic Activity, Fungal (HUT) . . . 924Proteolytic Activity, Fungal (SAP) . . . 925Pullulanase Activity . . . 926Transglutaminase Activity . . . 927Trypsin Activity . . . 928
APPENDIX VI:ESSENTIAL OILS AND FLAVORS . . . 929
Acetals . . . 929Acid Value . . . 929Aldehydes . . . 929Aldehydes and Ketones . . . 929
Hydroxylamine Method . . . 929Hydroxylamine tert-Butyl Alcohol
Method . . . 930Neutral Sulfite Method . . . 930
Chlorinated Compounds . . . 930Esters . . . 930Linalool Determination . . . 931Percentage of Cineole . . . 931Phenols . . . 931Phenols, Free . . . 932Residue on Evaporation . . . 932Solubility in Alcohol . . . 932
FCC V General Tests and Assays / Contents / 829
Total Alcohols . . . 932Ultraviolet Absorbance of Citrus Oils . . . 932Volatile Oil Content . . . 933
APPENDIX VII:FATS AND RELATED SUBSTANCES . . . 934
Acetyl Value . . . 934Acid Value . . . 934
Method I: Commercial Fatty Acids . . . 934Method II: Animal Fats and Vegetable and
Marine Oils . . . 934Chlorophyll . . . 934Cold Test . . . 935Color (AOCS-Wesson) . . . 935Fatty Acid Composition . . . 935Free Fatty Acids . . . 936Free Glycerin or Propylene Glycol . . . 936Hexane-Insoluble Matter . . . 936Hydroxyl Value . . . 936
Method I . . . 937Method II . . . 937
Iodine Value . . . 937Melting Range . . . 9381-Monoglycerides . . . 938Total Monoglycerides . . . 938Oxyethylene Determination . . . 939Peroxide Value . . . 940Reichert-Meissl Value . . . 940Saponification Value . . . 941Soap . . . 942Specific Gravity . . . 942Stability (Active Oxygen Method) . . . 942Unsaponifiable Matter . . . 942Volatile Acidity . . . 943
APPENDIX VIII:OLEORESINS . . . 944
Color Value . . . 944Curcumin Content . . . 944Piperine Content . . . 944
Residual Solvent . . . 945Scoville Heat Units . . . 946Volatile Oil Content . . . 946
APPENDIX IX:ROSINS AND RELATED SUBSTANCES . . . 947
Acid Number . . . 947Softening Point . . . 947
Drop Method . . . 947Ring-and-Ball Method . . . 948
Viscosity . . . 950
APPENDIX X:CARBOHYDRATES (STARCHES, SUGARS,AND RELATED SUBSTANCES) . . . 951
Acetyl Groups . . . 951Crude Fat . . . 951Invert Sugar . . . 951Lactose . . . 952Propylene Chlorohydrin . . . 953Reducing Sugars Assay . . . 954Sulfur Dioxide Determination . . . 955Total Solids . . . 957
Glucose Syrup (Corn Syrup) . . . 957High-Fructose Corn Syrup Solids . . . 958Liquid Fructose . . . 958Maltodextrin . . . 959Invert Sugar . . . 960Sucrose . . . 960
SOLUTIONS AND INDICATORS . . . 962Colorimetric Solutions . . . 962Standard Buffer Solutions . . . 962Standard Solutions for the Preparation of Controls
and Standards . . . 963Test Solutions (TS) and Other Reagents . . . 963Volumetric Solutions . . . 970Indicators . . . 975Indicator Papers and Test Papers . . . 976Detector Tubes . . . 977
830 / List of Figures / General Tests and Assays FCC V
List of Figures
1 Chromatographic Separation of Two Substances. . . 840
2 Asymmetrical Chromatographic Peak . . . 8413 Apparatus for Determination of Solidification
Point . . . 8474 Stirrer for Solidification Point Determination
. . . 8475 Ubbelohde Viscometer for Dimethylpolysiloxane
. . . 8496 Methylcellulose Viscometers . . . 8497 Agitator for Viscosity of Cellulose Gum . . . 8508 Moisture Distillation Apparatus . . . 8549 Assembly for Checking Pore Diameter of Filter
Sticks . . . 85610 Filtration Assembly for Determination of Oil
Content . . . 85611 General Apparatus for Arsenic Test . . . 86112 Modified Bethge Apparatus for the Distillation
of Arsenic Tribromide . . . 86113 Special Apparatus for the Distillation of Arsenic
Trichloride . . . 86114 Special Apparatus for the Determination of
Inorganic Arsenic . . . 86215 Closed-System Vacuum Distillation Apparatus
for 1,4-Dioxane . . . 86416 Aeration Apparatus for Mercury Limit Test
. . . 87217 Apparatus for Alginates Assay . . . 87618 Typical Chromatogram for the Determination of
Benzene in Hexanes Using Column No. 5. . . 878
19 Illustration of A/B Ratio . . . 87820 Illustration of A/B Ratio for a Small Component
Peak on the Tail of a Large Peak . . . 87821 Upward Displacement-Type Liquid–Liquid
Extractor with Sintered-Glass Diffuser . . . 88022 (a) Schematic Diagram of Apparatus for
Photometric Mercury Vapor M Method; (b)Quartz Combustion Tube with Boat and CopperOxide Packing; (c) Schematic Diagram of TrapUsed to Contain Ascarite, Dehydrite, andAluminum Oxide . . . 881
23 Titanous Chloride Titration Apparatus . . . 88324 Allura Red–Top Trace: Eluant Monitored at 254
nm; Bottom Trace: Eluant Monitored at 375 to385 nm . . . 885
25 Tartrazine–Top Trace: Eluant Monitored at 254nm; Bottom Trace: Eluant Monitored at 375 to385 nm . . . 885
26 Sunset Yellow–Top Trace: Eluant Monitored at254 nm; Bottom Trace: Eluant Monitored at 375to 385 nm . . . 886
27 Apparatus for Hydroxypropoxyl Determination. . . 887
28 Chart for Converting Percentage of Substitution,by Weight, of Hydroxypropoxyl Groups toMolecular Substitution per Glucose Unit . . . 888
29 Distillation Apparatus for MethoxylDetermination . . . 888
30 Microcoulometric Titrating System for theDetermination of Sulfur in Hexanes . . . 890
31 Raney Nickel Reduction Apparatus . . . 89132 Column Assembly for Assay of Immobilized
Glucose Isomerase . . . 90833 Typical Spectrogram of Lemon Oil . . . 93334 Apparatus for Determination of Volatile Oil
Content . . . 93335 Apparatus for Oxyethylene Distribution . . . 94036 Reichert-Meissl Distillation Apparatus . . . 94137 Modified Hortvet-Sellier Distillation Apparatus
. . . 94338 Clevenger Traps . . . 94539 Apparatus for Drop Softening Point
Determination . . . 94740 Shouldered Ring, Ring Holder, Ball-Centering
Guide, and Assembly of Apparatus ShowingTwo Rings . . . 949
41 Assembly of Apparatus Showing Stirrer andSingle-Shouldered Ring . . . 949
42 Butt-Type Extractor for Crude Fat Determination. . . 951
43 The Optimized Monier-Williams Apparatus. . . 955
44 Diagram of Bubbler . . . 956
FCC V General Tests and Assays / Appendix I / 831
APPENDIX I: APPARATUS FOR TESTS AND ASSAYS
OXYGEN FLASK COMBUSTION
Apparatus The apparatus consists of a heavy-walled,deeply lipped or cupped, conical flask of a volume suitable forthe complete combustion of the sample in which the particularelement is being determined (e.g., see Selenium Limit Test,Appendix IIIB). The flask is fitted with a ground-glass stopperto which is fused a sample carrier consisting of heavy-gaugeplatinum wire and a piece of welded platinum gauze measuringabout 1.5 × 2 cm. A suitable apparatus may be obtained asCatalog Nos. 6513-C20 (500-mL capacity) and 6513-C30(1000-mL capacity) from the Arthur H. Thomas Co., P.O. Box779, Philadelphia, PA 19105. Equivalent apparatus availablefrom other sources, or other suitable apparatus embodyingthe principles described herein, may also be used.
Procedure (Caution: Analysts should wear safety glassesand should use a suitable safety shield between themselvesand the apparatus. Further safety measures should be observedas necessary to ensure maximum protection of the analysts.Furthermore, the flask must be scrupulously clean and freefrom even traces of organic solvents. Samples containingwater of hydration or more than 1% of moisture should bedried at 140° for 2 h before combustion, unless otherwisedirected.) Accurately weigh the amount of sample specifiedin the monograph or general test. Solids should be weighedon a 4-cm square piece of halide-free paper, which should befolded around the sample. Liquid samples not exceeding 0.2mL in volume should be weighed in tared cellulose acetatecapsules [available as Catalog Nos. 6513-C80 (100 capsules)and 6513-C82 (1000 capsules) from the Arthur H. ThomasCo.]; gelatin capsules are satisfactory for liquid samples ex-ceeding 0.2 mL in volume.
Note: Gelatin capsules may contain significant amountsof combined halide or sulfur, in which case a blankdetermination should be made as necessary.
Place the sample, together with a filter paper fuse-strip, inthe platinum gauze sample holder. Place the absorbing liquid,as specified in the individual monograph or general test, intothe flask, moisten the joint of the stopper with water, andflush the air from the flask with a stream of rapidly flowingoxygen, swirling the liquid to facilitate its taking up oxygen.
Note: Saturation of the liquid with oxygen is essentialfor successful performance of this procedure.
Ignite the fuse-strip by suitable means. If the strip is ignitedoutside the flask, immediately plunge the sample holder intothe flask, invert the flask so that the absorption solution makesa seal around the stopper, and hold the stopper firmly in place.If the ignition is carried out in a closed system, the inversionof the flask may be omitted. After combustion is complete,
shake the flask vigorously, and allow it to stand for not lessthan 10 min with intermittent shaking. Continue as directedin the individual monograph or general test chapter.
THERMOMETERS
Thermometers suitable for Food Chemicals Codex use con-form to the specifications of the American Society for Testingand Materials, ASTM Standards E 1, and are standardized inaccordance with ASTM Method E 77.
The thermometers are of the mercury in glass type, andthe column above the liquid is filled with nitrogen. Theymay be standardized for ‘‘total immersion’’ or for ‘‘partialimmersion’’ and should be used as near as practicable underthe same condition of immersion.
Total immersion means standardization with the thermome-ter immersed to the top of the mercury column, with theremainder of the stem and the upper expansion chamber ex-posed to the ambient temperature. Partial immersion meansstandardization with the thermometer immersed to the indi-cated immersion line etched on the front of the thermometer,with the remainder of the stem exposed to the ambient temper-ature. If used under any other condition of immersion, anemergent-stem correction is necessary to obtain correct tem-perature readings.
Thermometer Specifications
Range SubdivisionsASTM ImmersionNo. E1 °C °F °C °F (mm)
For General Use
1 C –20 to +150 — 1 — 761 F — 0 to 302 — 2 762 C –5 to +300 — 1 — 762 F — 20 to 580 — 2 763 C –5 to +400 — 1 — 763 F — 20 to 760 — 2 76
For Determination of Softening Point
15 C –2 to +80 — 0.2 — total15 F — 30 to 180 — 0.5 total16 C 30 to 200 — 0.5 — total16 F — 85 to 392 — 1 total
For Determination of Kinematic Viscosity
44 F — 66.5 to 71.5 — 0.1 total45 F — 74.5 to 79.5 — 0.1 total28 F — 97.5 to 102.5 — 0.1 total46 F — 119.5 to 124.5 — 0.1 total29 F — 127.5 to 132.5 — 0.1 total47 F — 137.5 to 142.5 — 0.1 total48 F — 177.5 to 182.5 — 0.1 total30 F — 207.5 to 212.5 — 0.1 total
832 / Appendix I / General Tests and Assays FCC V
Thermometer Specifications (continued)
Range SubdivisionsASTM ImmersionNo. E1 °C °F °C °F (mm)
For Determination of Distillation Range
37 C –2 to +52 — 0.2 — 10038 C 24 to 78 — 0.2 — 10039 C 48 to 102 — 0.2 — 10040 C 72 to 126 — 0.2 — 10041 C 98 to 152 — 0.2 — 100102 C 123 to 177 — 0.2 — 100103 C 148 to 202 — 0.2 — 100104 C 173 to 227 — 0.2 — 100105 C 198 to 252 — 0.2 — 100106 C 223 to 277 — 0.2 — 100107 C 248 to 302 — 0.2 — 100
For Determination of Solidification Point
89 C –20 to +10 — 0.1 — 7690 C 0 to 30 — 0.1 — 7691 C 20 to 50 — 0.1 — 7692 C 40 to 70 — 0.1 — 7693 C 60 to 90 — 0.1 — 7694 C 80 to 110 — 0.1 — 7695 C 100 to 130 — 0.1 — 7696 C 120 to 150 — 0.1 — 76100 C 145 to 205 — 0.2 — 76101 C 195 to 305 — 0.5 — 76
For Special Use
14 Ca 38 to 82 — 0.1 — 7938 Cb –2 to +68 — 0.2 — 4518 Cc 34 to 42 — 0.1 — total18 Fc — 94 to 108 — 0.2 total22 Cc 95 to 103 — 0.1 — total22 Fc — 204 to 218 — 0.2 total23 Cd 18 to 28 — 0.2 — 9024 Cd 30 to 54 — 0.2 — 9054 Fe — 68 to 213 — 0.5 total71 Ff — –35 to +70 — 1 76
aFor determination of melting range of Class III solids.bFor determination of the titer of fatty acids.cFor determination of Saybolt viscosity.dFor determination of Engler viscosity.eFor determination of congealing point.fFor determination of oil in wax.
In selecting a thermometer, careful consideration shouldbe given to the conditions under which it is to be used. Thepreceding table lists several ASTM thermometers, togetherwith their usual conditions of use, which may be required inFood Chemicals Codex tests. Complete specifications forthese thermometers are given in ‘‘ASTM Standards on Ther-mometers.’’
VOLUMETRIC APPARATUS
Most of the volumetric apparatus available in the UnitedStates is calibrated at 20°, although the temperatures generallyprevailing in laboratories more nearly approach 25°, which
is the temperature specified generally for tests and assays.This discrepancy is inconsequential provided the room tem-perature is reasonably constant and the apparatus has beencalibrated accurately prior to and under the conditions of itsintended use.
Before use, all volumetric ware must be cleaned in such amanner that when rinsed with water, no droplet of water canbe seen on the inside walls. Many kinds of ‘‘degreasing’’solutions are available, and the user should consult the manu-facturer’s literature for the system of choice.
Use To attain the degree of precision required in manyassays involving volumetric measurements and directing thata quantity be ‘‘accurately measured,’’ the apparatus must bechosen and used with exceptional care. Where less than 10mL of titrant is to be measured, a 10-mL buret or microburetgenerally is required.
The design of volumetric apparatus is an important factorin ensuring accuracy. For example, the length of the graduatedportions of graduated cylinders should be not less than fivetimes the inside diameter, and the tips of burets should permitan outflow of not more than 0.5 mL/s.
Pipets and burets must be allowed to drain properly in use.Usually, transfer pipets for dilute aqueous solutions shoulddrain for the time specified by the manufacturer before thetip is touched to the wall of the vessel. Buret volumes shouldnot be read immediately upon delivery of the titrant. A suitablelength of time should elapse to allow the titrant retained onthe walls to drain down. A time interval of 5 to 10 s is usuallysufficient.
Standards of Accuracy The capacity tolerances for volu-metric flasks, transfer pipets, and burets are those acceptedby the National Institute of Standards and Technology (ClassA),1 as indicated in the accompanying tables. Use Class Avolumetric apparatus unless otherwise specified in the individ-ual monograph. For plastic volumetric apparatus, the acceptedcapacity tolerances are Class B.2
Volumetric Flasks
Designated Volume (mL)
10 25 50 100 250 500 1000
Limit of error (mL) 0.02 0.03 0.05 0.08 0.12 0.15 0.30Limit of error (%) 0.20 0.12 0.10 0.08 0.05 0.03 0.03
Transfer Pipets
Designated Volume (mL)
1 2 5 10 25 50 100
Limit of error (mL) 0.006 0.006 0.01 0.02 0.03 0.05 0.08Limit of error (%) 0.6 0.30 0.20 0.20 0.12 0.10 0.08
1 See ‘‘Testing of Glass Volumetric Apparatus,’’ NBS Circ. 602, April1, 1959. Apparatus meeting the specifications of NB SIR 74–461 (‘‘TheCalibration of Small Volumetric Laboratory Glassware’’), as well as ofANSI/ASTM E 694–79 (‘‘Specifications for Volumetric Ware’’), is alsoacceptable.
2 See ASTM E 288, Fed. Spec. NNN-F-289, and ISO Standard 284.
FCC V General Tests and Assays / Appendix I / 833
Burets
Designated Volume (mL)
10 (‘‘micro’’ type) 25 50
Subdivisions (mL) 0.02 0.10 0.10Limit of error (mL) 0.02 0.03 0.05
The capacity tolerances for measuring (i.e., ‘‘graduated’’)pipets of up to and including 10-mL capacity are somewhatlarger than those for the corresponding sizes of transfer pipets,namely, 0.01, 0.02, and 0.03 mL for the 2-, 5-, and 10-mLsizes, respectively.
Transfer and measuring pipets calibrated ‘‘to deliver’’should be drained in a vertical position and then touched againstthe wall of the receiving vessel to drain the tips. Volume read-ings on burets should be estimated to the nearest 0.01 mL for25- and 50-mL burets, and to the nearest 0.005 mL for 5- and10-mL burets. Pipets calibrated ‘‘to contain’’ may be called forin special cases, generally for measuring viscous fluids. In suchcases, the pipet should be washed clean, after draining, and thewashings added to the measured portion.
WEIGHTS AND BALANCES
Food Chemicals Codex tests and assays are designed for usewith three types of analytical balances, known as micro-,semimicro-, and macro-.
By custom, microbalances weigh objects with a sensitivitydown to the microgram range (or lower); semimicrobalancesdown to the 0.01-mg range; and analytical macrobalancesdown to the 0.1-mg range.
Tolerances The analytical weights meet the tolerances ofthe American National Standard ANSI/ASTM E617, ‘‘Labo-ratory Weights and Precision Mass Standards.’’ This standardis incorporated by reference and should be consulted for fulldescriptions and information on the tolerances and construc-tion of weights.3 Where quantities of 25 mg or less are to be‘‘accurately weighed,’’ any applicable corrections for weightsshould be used.
3 Copies of ASTM Standard E 617-81 (Reapproved 1985) may beobtained from the American Society for Testing and Materials, 1916Race Street, Philadelphia, PA 19103.
Class 1.1 weights are used for calibration of low-capacity,high-sensitivity balances. They are available in various de-nominations from 1 to 500 mg. The tolerance for any denomi-nation in this class is 5 �g. They are recommended for calibra-tion of balances using optical or electrical methods foraccurately weighing quantities below 20 mg.
Class 1 weights are designated as high-precision standardsfor calibration. They may be used for accurately weighingquantities below 20 mg.
Class 2 weights are used as working standards for calibra-tion, built-in weights for analytical balances, and laboratoryweights for routine analytical work.
Class 3 and Class 4 weights are used with moderate-preci-sion laboratory balances.
Use Where substances are to be ‘‘accurately weighed’’ inan assay or a test, the weighing is to be performed in suchmanner as to limit the error to 0.1% or less. For example, aquantity of 50 mg is to be weighed to the nearest 0.05 mg;a quantity of 0.1 g is to be weighed to the nearest 0.1 mg;and a quantity of 10 g is to be weighed to the nearest 10 mg.
Calibration All precision balances and weights should becalibrated periodically (preferably at least once a year) and arecord kept of the calibration date and results. The user mayhave a set of weights calibrated by the nearest Departmentof Weights and Measurements (or its equivalent). This isusually done for little or no charge. Alternatively, an indepen-dent, outside company may be retained for the purpose ofperforming such calibrations.
Buoyancy Effect When a weighing is to be performed withan accuracy of 0.1% or better, the buoyancy effect should notbe neglected. The equation to be used in correcting for thiseffect is
MV = MA[1 + 0.0012(1/DO + DW)],
in which MV is the mass in vacuum; MA is the mass in air;0.0012 is the density of air; DO is the density of the weighedobject; and DW is the density of the calibrated weights.
834 / Appendix II / General Tests and Assays FCC V
APPENDIX II: PHYSICAL TESTS AND DETERMINATIONS
A. CHROMATOGRAPHY
Note: Chromatographic separations may also be charac-terized according to the type of instrumentations orapparatus used. The types of chromatography that maybe used in the Food Chemicals Codex are column, thin-layer, gas, and high-pressure or high-performance liquidchromatography.
The Committee on Food Chemicals Codex recog-nizes that the field of chromatography continues toadvance. Accordingly, the use of equivalent or im-proved systems is acceptable with appropriate vali-dation.
For the purposes of the Food Chemicals Codex, chromatogra-phy is defined as an analytical technique whereby a mixtureof chemicals may be separated by virtue of their differentialaffinities for two immiscible phases. One of these, the station-ary phase, consists of a fixed bed of small particles with alarge surface area, while the other, the mobile phase, is a gasor liquid that moves constantly through, or over the surfaceof, the fixed phase. Chromatographic systems achieve theirability to separate mixtures by selectively retarding the pas-sage of some compounds through the stationary phase whilepermitting others to move more freely. Therefore, the chroma-togram may be evaluated qualitatively by determining the Rf,or retardation factor, for each of the eluted substances. TheRf is a measure of that fraction of its total elution time that anycompound spends in the mobile phase. Because this fraction isdirectly related to the fraction of the total amount of the solutethat is in the mobile phase, the Rf can be expressed as
Rf = VmCm/(VmCm + VsCs),
in which Vm and Vs are the volumes of the mobile and station-ary phase, respectively, and Cm and Cs are the concentrationsof the solute in either phase at any time. This can be simpli-fied to
Rf = Vm/(Vm + KVs),
in which K = Cs/Cm and is an equilibrium constant that indi-cates this differential affinity of the solute for the phases.Alternatively, a new constant, k, the capacity factor, may beintroduced, giving another form of the expression:
Rf = 1/(1 + k),
in which k = KVs/Vm. The capacity factor, k, which is normallyconstant for small samples, is a parameter that expresses theability of a particular chromatographic system to interact witha solute. The larger the k value, the more the sample is retarded.
Both the retardation factor and the capacity factor may beused for qualitative identification of a solute or for developingstrategies for improving separation. In terms of parameterseasily obtainable from the chromatogram, the Rf is defined
as the ratio of the distance traveled by the solute band to thedistance traveled by the mobile solvent in a particular time.The capacity factor, k, can be evaluated by the expression
k = (tr − to)/to,
in which tr, the retention time, is the elapsed time from thestart of the chromatogram to the elution maximum of thesolute, and to is the retention time of a solute that is notretained by the chromatographic system.
Retardation of the solutes by the stationary phase may beachieved by one or a combination of mechanisms. Certainsubstances, such as alumina or silica gel, interact with thesolutes primarily by adsorption, either physical adsorption,in which the binding forces are weak and easily reversible,or chemisorption, in which strong bonding to the surfacecan occur. Another important mechanism of retardation ispartition, which occurs when the solute dissolves in the sta-tionary phase, usually a liquid coated as a thin layer on thesurface of an inert particle or chemically bonded to it. If theliquid phase is a polar substance (e.g., polyethylene glycol)and the mobile phase is nonpolar, the process is termed nor-mal-phase chromatography. When the stationary phase is non-polar (e.g., octadecylsilane) and the mobile phase is polar,the process is reversed-phase chromatography. For the separa-tion of mixtures of ionic species, insoluble polymers calledion exchangers are used as the stationary phase. Ions of thesolutes contained in the mobile phase are adsorbed onto thesurface of the ion exchanger while at the same time displacingan electrically equivalent amount of less strongly bound ionsto maintain the electroneutrality of both phases. The chromato-graphic separation of mixtures of large molecules such asproteins may be accomplished by a mechanism called sizeexclusion chromatography. The stationary phases used arehighly cross-linked polymers that have imbibed a sufficientamount of solvent to form a gel. The separation is based onthe physical size of the solvated solutes; those that are toolarge to fit within the interstices of the gel are eluted rapidly,while the smaller molecules permeate into the pores of thegel and are eluted later. In any chromatographic separation,more than one of the above mechanisms may be occurringsimultaneously.
Chromatographic separations may also be characterizedaccording to the type of instrumentation or apparatus used.The types of chromatography that may be used in the FCCare column, thin-layer, gas, and high-performance liquid chro-matography.
COLUMN CHROMATOGRAPHY
Apparatus The equipment needed for column chromatogra-phy is not elaborate, consisting only of a cylindrical glass or
FCC V General Tests and Assays / Appendix II / 835
Teflon tube that has a restricted outflow orifice. The dimen-sions of the tube are not critical and may vary from 10 to 40mm in inside diameter and from 100 to 600 mm in length.For a given separation, greater efficiency may be obtainedwith a long narrow column, but the resultant flow rate willbe lower. A fritted-glass disk may be seated in the end of thetube to act as a support for the packing material. The columnis fitted at the end with a stopcock or other flow-restrictiondevice to control the rate of delivery of the eluant.
Procedure The stationary phase is introduced into the col-umn either as a dry powder or as a slurry in the mobile phase.Because a homogeneous bed free of void spaces is necessaryto achieve maximum separation efficiency, the packing mate-rial is introduced in small portions and allowed to settle beforefurther additions are made. Settling may be accomplished byallowing the mobile phase to flow through the bed, by tappingor vibrating the column if a dry powder is used, or by com-pressing each added portion using a tamping rod. The rodcan be a solid glass, plastic, or metal cylinder whose diameteris slightly smaller than that of the column, or it can be athinner rod onto the end of which has been attached a diskof suitable diameter. Ion-exchange resins and exclusion poly-mers are never packed as dry powders because after introduc-tion of the mobile phase, they will swell and create sufficientpressure to shatter the column. When the packing has beencompleted, the sample is introduced onto the top of the col-umn. If the sample is soluble, it is dissolved in a minimumamount of the mobile phase, pipetted onto the column, andallowed to percolate into the top of the bed. If it is not solubleor if the volume of solution is too large, it may be mixedwith a small amount of the column packing. This material isthen transferred to the chromatographic tube to form the topof the bed.
The chromatogram is then developed by adding the mobilephase to the column in small portions and allowing it topercolate through the packed bed either by gravity or underthe influence of pressure or vacuum. Development of thechromatogram takes place by selective retardation of the com-ponents of the mixture as a result of their interaction with thestationary phase. In column chromatography, the stationaryphase may act by adsorption, partition, ion exchange, exclu-sion of the solutes, or a combination of these effects.
When the development is complete, the components of thesample mixture may be detected and isolated by either of twoprocedures. The entire column may be extruded carefully fromthe tube, and if the compounds are colored or fluorescentunder ultraviolet light, the appropriate segments may be cutfrom the column using a razor blade. If the components arecolorless, they may be visualized by painting or spraying athin longitudinal section of the surface of the chromatogramwith color-developing reagents. The chemical may then beseparated from the stationary phase by extraction with a strongsolvent such as methanol and subsequently quantitated bysuitable methods.
In the second procedure, the mobile phase may be allowedto flow through the column until the components of the mix-ture successively appear in the effluent. This eluate may becollected in fractions and the mobile phase evaporated if
desired. The chemicals present in each fraction may then bedetermined by suitable analytical techniques.
THIN-LAYER CHROMATOGRAPHY
In thin-layer chromatography (TLC), the stationary phase isa uniform layer of a finely divided powder that has beencoated on the surface of a glass or plastic sheet and that isheld in place by a binder. The capacity of the system isdependent on the thickness of the layer, which may rangefrom 0.1 to 2.0 mm. The thinner layers are used primarilyfor analytical separations, while the thicker layers, becauseof their greater sample-handling ability, are useful for prepara-tive work.
Substances that are used as coatings in TLC include silicagel, alumina, cellulose, and reversed-phase packings. Separa-tions occur because of adsorption of the solutes from themobile phase onto the surface of the thin layer. However,adsorption of water from the air or solvent components fromthe mobile phase can give rise to partition or liquid−liquidchromatography. Specially coated plates are available thatpermit ion-exchange or reversed-phase separations.
Apparatus Acceptable apparatus and materials for thin-layer chromatography consist of the following:
Glass Plates Flat glass plates of uniform thicknessthroughout their areas. The most common sizes are 20, 10,and 5 cm × 20 cm. (Aluminum plates also are commonly used.)
Aligning Tray An aligning tray or other suitable flat sur-face is used to align and hold plates during application of theadsorbent.
Adsorbent The adsorbent may consist of finely dividedadsorbent materials for chromatography. It can be applieddirectly to the glass plate, or it can be bonded to the plate bymeans of plaster of Paris or with starch paste. Pretreatedchromatographic plates are available commercially.
Spreader A suitable spreading device that, when movedover the glass plate, applies a uniform layer of adsorbent ofthe desired thickness over the entire surface of the plate.
Storage Rack A rack of convenient size to hold the pre-pared plates during drying and transportation.
Developing Chamber A glass chamber that can accommo-date one or more plates and can be properly closed and sealed.It is fitted with a plate-support rack that can support the plateswhen the lid of the chamber is in place.
Note: Preformed TLC plates available commerciallymay also be used.
Procedure Clean the plates scrupulously, as by immersionin a chromic acid cleansing mixture, rinse them with copiousquantities of water until the water runs off the plates withoutleaving any visible water or oily spots, and dry.
Arrange the plate or plates on the aligning tray, and securethem so that they will not slip during the application of theadsorbent. Mix an appropriate quantity of adsorbent and liq-
836 / Appendix II / General Tests and Assays FCC V
uid, usually water, which when shaken for 30 s gives a smoothslurry that will spread evenly with the aid of a spreader.Transfer the slurry to the spreader, and apply the coating atonce before the binder begins to harden. Move the spreadersmoothly over the plates from one end of the tray to the other.Remove the spreader, and wipe away excess slurry. Allowthe plates to set for 10 min, and then place them in the storagerack and dry at 105° for 30 min or as directed in the individualmonograph. Store the finished plates in a desiccator.
Equilibrate the atmosphere in the Developing Chamber byplacing in it a volume of the mobile phase in excess of thatrequired for complete development of the chromatogram,cover the chamber with its lid, and allow it to stand for atleast 30 min.
Apply the Sample Solution and the Standard Solution atpoints about 1.5 cm apart and about 2 cm from the loweredge of the plate (the lower edge is the first part over whichthe spreader moves in the application of the adsorbent layer),and allow to dry. A template will aid in determining the spotpoints and the 10- to 15-cm distance through which the solventfront should move.
Arrange the plate on the supporting rack (sample spotson the bottom), and introduce the rack into the developingchamber. The solvent in the chamber must be deep enoughto reach the lower edge of the adsorbent, but must not touchthe spot points. Seal the cover in place, and maintain thesystem until the solvent ascends to a point 10 to 15 cm abovethe initial spots; this usually requires 15 min to 1 h. Removethe plates, and dry them in air. Measure and record the distanceof each spot from the point of origin. If so directed, spraythe spots with the reagent specified, observe, and comparethe sample with the standard chromatogram.
Detection and Identification Detection and identificationof solute bands is done by methods essentially the same asthose described in Column Chromatography. However, inTLC an additional method called fluorescence quenching isalso used. In this procedure, an inorganic phosphor is mixedwith the adsorbent before it is coated on the plate. When thedeveloped chromatogram is irradiated with ultraviolet light,the surface of the plate fluoresces with a characteristic color,except in those places where ultraviolet-absorbing solutes aresituated. These quench the fluorescence and are detectable asdark spots.
Detection with an ultraviolet light source suitable for obser-vations with short (254-nm) and long (360-nm) ultravioletwavelengths may be called for in some cases.
Quantitative Analysis Two methods are available if quanti-tation of the solute is necessary. In the first, the bands aredetected and their positions marked. Those areas of adsorbentcontaining the compounds of interest are scraped from thesurface of the plate into a centrifuge tube. The chemicals areextracted from the adsorbent with the aid of a suitable strongsolvent, the suspension is centrifuged, and the supernatantlayer is subjected to appropriate methods of quantitativeanalysis.
The second method involves the use of a scanning densitom-eter. This is a spectrophotometric device that directs a beam
of monochromatic radiation across the surface of the plate.After interaction with the solutes in the adsorbent layer, theradiation is detected as transmitted or reflected light and arecording of light intensity versus distance traveled is pro-duced. The concentration of a particular species is proportionalto the area under its peak and can be determined accuratelyby comparison with standards.
GAS CHROMATOGRAPHY
The distinguishing features of gas chromatography are a gas-eous mobile phase and a solid or immobilized liquid stationaryphase. Liquid stationary phases are available in packed orcapillary columns. In the packed columns, the liquid phaseis deposited on a finely divided, inert solid support, such asdiatomaceous earth or porous polymer, which is packed intoa column that typically has a 2- to 4-mm id and is 1 to 3 mlong. In capillary columns, which contain no particles, theliquid phase is deposited on the inner surface of the fusedsilica column and may be chemically bonded to it. In gas−solidchromatography, the solid phase is an active adsorbent, such asalumina, silica, or carbon, packed into a column. Polyaromaticporous resins, which are sometimes used in packed columns,are not coated with a liquid phase.
When a volatile compound is introduced into the carriergas and carried into the column, it is partitioned betweenthe gas and stationary phases by a dynamic countercurrentdistribution process. The compound is carried down the col-umn by the carrier gas, retarded to a greater or lesser extentby sorption and desorption in the stationary phase. The elutionof the compound is characterized by the partition ratio, k, adimensionless quantity also called the capacity factor. It isequivalent to the ratio of the time required for the compoundto flow through the column (the retention time) to the retentiontime of a nonretarded compound. The value of the capacityfactor depends on the chemical nature of the compound; thenature, amount, and surface area of the liquid phase; and thecolumn temperature. Under a specified set of experimentalconditions, a characteristic capacity factor exists for everycompound. Separation by gas chromatography occurs only ifthe compounds concerned have different capacity factors.
Apparatus A gas chromatograph consists of a carrier gassource, an injection port, column, detector, and recordingdevice. The injection port, column, and detector are carefullytemperature controlled. The typical carrier gas is helium ornitrogen, depending on the column and detector in use. The gasis supplied from a high-pressure cylinder and passes throughsuitable pressure-reducing valves to the injection port andcolumn. Compounds to be chromatographed, either in solutionor as gases, are injected into the gas stream at the injectionport. Depending on the configuration of the apparatus, thetest mixture may be injected directly into the column or bevaporized in the injection port and mixed into the flowingcarrier gas before entering the column.
FCC V General Tests and Assays / Appendix II / 837
Once in the column, compounds in the test mixture areseparated by virtue of differences in their capacity factors,which in turn depend on their vapor pressure and degree ofinteraction with the stationary phase. The capacity factor,which governs resolution and retention times of componentsof the test mixture, is also temperature dependent. The useof temperature-programmable column ovens takes advantageof this dependence to achieve efficient separation of com-pounds differing widely in vapor pressure.
As resolved compounds emerge from the column, they passthrough a detector, which responds to the amount of eachcompound present. The type of detector to be used dependson the nature of the compounds to be analyzed, and is specifiedin the individual monograph. Detectors are heated above themaximum column operating temperature to prevent condensa-tion of the eluting compounds.
Detector output is recorded as a function of time, producinga chromatogram, which consists of a series of peaks on a timeaxis. Each peak represents a compound in the vaporized testmixture, although some peaks may overlap. The elution timeis characteristic of the individual compounds (qualitative anal-ysis), and the peak area is a function of the amount present(quantitative analysis).
Injectors Sample injection devices range from simple sy-ringes to fully programmable automatic injectors. The amountof sample that can be injected into a capillary column withoutoverloading is small compared with the amount that can beinjected into a packed column, and may be less than thesmallest amount that can be manipulated satisfactorily bysyringe. Capillary columns are therefore used with injectorsable to split samples into two fractions, a small one that entersthe column and a large one that goes to waste (split injector).Such injectors may also be used in a splitless mode for analysesof trace or minor components.
Purge and trap injectors are equipped with a sparging deviceby which volatile compounds in solution are carried into alow-temperature trap. When sparging is complete, trappedcompounds are thermally desorbed into the carrier gas byrapid heating of the temperature-programmable trap.
Headspace injectors are equipped with a thermostaticallycontrolled sample-heating chamber. Solid or liquid samplesin tightly closed containers are heated in the chamber for afixed period of time, allowing the volatile components in thesample to reach an equilibrium between the nongaseous phaseand the gaseous or headspace phase.
After this equilibrium has been established, the injectorautomatically introduces a fixed amount of the headspace inthe sample container into the gas chromatograph.
Columns Capillary columns, which are usually made offused silica, have a 0.2- to 0.53-mm id and are 5 to 30 mlong. The liquid or stationary phase is 0.1 to 1.0 �m thick,although nonpolar stationary phases may be up to 5 �m thick.
Packed columns, made of glass or metal, are 1 to 3 m long,with a 2- to 4-mm id. Those used for analysis typically haveliquid phase loadings of about 5% (w/w) on a solid support.
Supports for analysis of polar compounds on low-capacity,low-polarity liquid phase columns must be inert to avoid peaktailing. The reactivity of support materials can be reduced by
silanizing before coating with liquid phase. Acid-washed,flux-calcined diatomaceous earth is often used for drug analy-sis. Support materials are available in various mesh sizes,with 80- to 100-mesh and 100- to 120-mesh being morecommonly used with 2- to 4-mm columns. Because of theabsence of a solid support, capillary compounds are muchmore inert than packed columns.
Retention time and the peak efficiency depend on the carriergas flow rate; retention time is also directly proportional tocolumn length, while resolution is proportional to the squareroot of the column length. For packed columns, the carriergas flow rate is usually expressed in milliliters per minute atatmospheric pressure and room temperature. It is measuredat the detector outlet with a soap film flow meter while thecolumn is at operating temperature. Unless otherwise specifiedin the individual monograph, flow rates for packed columnsare 60 to 75 mL/min for 4-mm id columns and ~30 mL/minfor 2-mm id columns.
For capillary columns, linear flow velocity is often used in-stead of flow rate. This is conveniently determined from thelength of the column and the retention time of a dilute methanesample, provided a flame-ionization detector is in use. Typicallinear velocities are 20 to 60 cm/s for helium. At high operatingtemperatures there is sufficient vapor pressure to result in agradual loss of liquid phase, a process called ‘‘bleeding.’’
Detectors Flame-ionization detectors are used for mostanalyses, with lesser use made of thermal conductivity, elec-tron-capture, nitrogen−phosphorus, and mass spectrometricdetectors. For quantitative analyses, detectors must have awide linear dynamic range: the response must be directlyproportional to the amount of compound present in the detectorover a wide range of concentrations. Flame-ionization detec-tors have a wide linear range (~106) and are sensitive toorganic compounds. Unless otherwise specified in individualmonographs, flame-ionization detectors with either helium ornitrogen carrier gas are to be used for packed columns, andhelium is used for capillary columns.
The thermal conductivity detector detects changes in thethermal conductivity of the gas stream as solutes are eluted.Although its linear dynamic range is smaller than that of theflame-ionization detector, it is quite rugged and occasionallyused with packed columns, especially for compounds that donot respond to flame-ionization detectors.
The alkali flame-ionization detector, sometimes called anNP or nitrogen−phosphorus detector, contains a thermionicsource, such as an alkali-metal salt or a glass element con-taining rubidium or other metal, that results in the efficientionization of organic nitrogen and phosphorus compounds. Itis a selective detector that shows little response to hydro-carbons.
The electron-capture detector contains a radioactive source(usually 63Ni) of ionizing radiation. It exhibits an extremelyhigh response to compounds containing halogens and nitrogroups but little response to hydrocarbons. The sensitivityincreases with the number and atomic weight of the halogenatoms.
Data Collection Devices Modern data stations receive thedetector output, calculate peak areas, and print chromato-
838 / Appendix II / General Tests and Assays FCC V
grams, complete with run parameters and peak data. Chro-matographic data may be stored and reprocessed, with integra-tion and other calculation variables being changed as required.Data stations are used also to program the chromatograph,controlling most operational variables and providing for longperiods of unattended operation.
Data can also be collected for manual measurement onsimple recorders or on integrators whose capabilities rangefrom those providing a printout of peak areas to those provid-ing chromatograms with peak areas and peak heights calcu-lated and data stored for possible reprocessing.
Procedure Capillary columns must be tested to ensure thatthey comply with the manufacturers’ specifications beforethey are used. These tests consist of the following injections:a dilute methane sample to determine the linear flow velocity;a mixture of alkanes (e.g., C14, C15, and C16) to determineresolution; and a polarity test mixture to check for active siteson the column. The latter mixture may include a methyl ester,an unsaturated compound, a phenol, an aromatic amine, a diol,a free carboxylic acid, and a polycyclic aromatic compound,depending on the samples to be analyzed.
Packed columns must be conditioned before use until thebaseline and other characteristics are stable. This may be doneby operation at a temperature above that called for by themethod or by repeated injections of the compound or mixtureto be chromatographed. A suitable test for support inertnessshould be done. Very polar molecules (like free fatty acids)may require a derivatization step.
Before any column is used for assay purposes, a calibrationcurve should be constructed to verify that the instrumentalresponse is linear over the required range and that the curvepasses through the origin. If the compound to be analyzed isadsorbed within the system, the calibration curve will intersectthe abscissa at a nonzero value. This may result in error,particularly for compounds at low concentrations determinedby a procedure based on a single reference point. At highconcentrations, the liquid phase may be overloaded, leadingto loss of peak height and symmetry.
Assays require quantitative comparison of one chromato-gram with another. A major source of error is irreproducibilityin the amount of sample injected, notably when manual injec-tions are made with a syringe. The effects of variability can beminimized by addition of an internal standard, a noninterferingcompound present at the same concentration as in the sampleand standard solutions. The ratio of peak response of theanalyte to that of the internal standard is compared fromone chromatogram to another. Where the internal standard ischemically similar to the substance being determined, there isalso compensation for minor variations in column and detectorcharacteristics. In some cases, the internal standard may becarried through the sample preparation procedure before gaschromatography to control other quantitative aspects of theassay. Automatic injectors greatly improve the reproducibilityof sample injections and reduce the need for internal standards.
Many monographs require that system suitability require-ments be met before samples are analyzed, see System Suit-ability below.
HIGH-PERFORMANCE LIQUIDCHROMATOGRAPHY
High-performance liquid chromatography (HPLC) is a separa-tion technique based on a solid stationary phase and a liquidmobile phase. Separations are achieved by partition, adsorp-tion, exclusion, or ion-exchange processes, depending on thetype of stationary phase used. HPLC has distinct advantagesover gas chromatography for the analysis of nonvolatile or-ganic compounds. Compounds to be analyzed are dissolved ina liquid, and most separations take place at room temperature.
As in gas chromatography, the elution time of a compoundcan be described by the capacity factor, k, which depends onthe chemical nature of the composition and flow rate of themobile phase, and the composition and surface area of thestationary phase. Column length is an important determinantof resolution. Only compounds having different capacity fac-tors can be separated by HPLC.
Apparatus A liquid chromatograph consists of one, two,or more reservoirs containing the mobile phase, a pump toforce the mobile phase through the system at high pressure,an injector to introduce the sample into the mobile phase, a chro-matographic column, a detector, and a data collection devicesuch as a computer, integrator, or recorder. Short, 3-, 5-, 10-,and 25-cm, small-bore columns containing densely packed par-ticles of stationary phase provide for the rapid exchange of com-pounds between the mobile and stationary phases. In additionto receiving and reporting detector output, computers are usedto control chromatographic settings and operations, thus pro-viding for long periods of unattended operation.
Pumping Systems HPLC pumping systems deliver me-tered amounts of mobile phase from the solvent reservoirs tothe column through high-pressure tubing and fittings. Modernsystems consist of one or more computer-controlled meteringpumps that can be programmed to vary the ratio of mobilephase components, as is required for gradient chromatography,or to mix isocratic mobile phases (i.e., mobile phases havinga fixed ratio of solvents). However, the proportion of ingredi-ents in premixed isocratic mobile phases can be more accu-rately controlled than in those delivered by most pumpingsystems. Operating pressures up to 5000 psi with deliveryrates up to about 10 mL/min are typical. Pumps used forquantitative analysis should be constructed of materials inertto corrosive mobile phase components and be capable ofdelivering the mobile phase at a constant rate with minimalfluctuations over extended periods of time.
Injectors After dissolution in mobile phase or other suit-able solution, compounds to be chromatographed are injectedinto the mobile phase, either manually by syringe or loopinjectors, or automatically by autosamplers. The latter consistof a carousel or rack to hold sample vials with tops that havea pierceable septum or stopper and an injection device totransfer sample from the vials to a calibrated, fixed-volumeloop from which it is loaded into the chromatograph. Someautosamplers can be programmed to control sample volume,the number of injections and loop rinse cycles, the intervalbetween injections, and other operating variables.
FCC V General Tests and Assays / Appendix II / 839
Some valve systems incorporate a calibrated sample loopthat is filled with test solution for transfer to the column inthe mobile phase. In other systems, test solution is transferredto a cavity by syringe and then switched into the mobile phase.
Columns For most analyses, separation is achieved bypartition of compounds in the test solution between the mobileand stationary phases. Systems consisting of polar stationaryphases and nonpolar mobile phases are described as normalphase, while the opposite arrangement, polar mobile phasesand nonpolar stationary phases, is called reversed-phase chro-matography. Partition chromatography is almost always usedfor hydrocarbon-soluble compounds of a molecular weightthat is less than 1000. The affinity of a compound for thestationary phase, and thus its retention time on the column,is controlled by making the mobile phase more or less polar.Mobile phase polarity can be varied by the addition of asecond, and sometimes a third or even a fourth, component.
Stationary phases for modern, reversed-phase liquid chro-matography typically consist of an organic phase chemicallybound to silica or other materials. Particles are usually 3, 5,or 10 �m in diameter, but sizes may range up to 50 �mfor preparative columns. Small particles thinly coated withorganic phase allow fast mass transfer and, hence, rapid trans-fer of compounds between the stationary and mobile phases.Column polarity depends on the polarity of the bound func-tional groups, which range from relatively nonpolar octadecylsilane to very polar nitrile groups.
Columns used for analytical separations usually have inter-nal diameters of 2 to 4.6 mm; larger diameter columns areused for preparative chromatography. Columns may be heatedto give more efficient separations, but only rarely are theyused at temperatures above 60° because of potential stationaryphase degradation or mobile phase volatility. Unless otherwisespecified in the individual monograph, columns are used atan ambient temperature.
Ion-exchange chromatography is used to separate water-soluble, ionizable compounds of molecular weights that areless than 2000. The stationary phases are usually syntheticorganic resins; cation-exchange resins contain negativelycharged active sites and are used to separate basic substancessuch as amines; while anion-exchange resins have positivelycharged active sites for separation of compounds with nega-tively charged groups such as phosphate, sulfonate, or carbox-ylate groups. Water-soluble ionic or ionizable compounds areattracted to the resins, and differences in affinity bring aboutthe chromatographic separation. The pH of the mobile phase,temperature, ion type, ionic concentration, and organic modifi-ers affect the equilibrium, and these variables can be adjustedto obtain the desired degree of separation.
In size-exclusion chromatography, columns are packed witha porous stationary phase. Molecules of the compounds beingchromatographed are filtered according to size. Those toolarge to enter the pores pass unretained through the column(total exclusion). Smaller molecules enter the pores and areincreasingly retained as molecular size decreases. These col-umns are typically used to remove high molecular weightmatrices or to characterize the molecular weight distributionof a polymer.
Detectors Many compendial HPLC methods require theuse of spectrophotometric detectors. Such a detector consistsof a flow-through cell mounted at the end of the column. Abeam of ultraviolet radiation passes through the flow cell andinto the detector. As compounds elute from the column, theypass through the cell and absorb the radiation, resulting inmeasurable energy level changes.
Fixed, variable, and photodiode array (PDA) detectors arewidely available. Fixed wavelength detectors operate at asingle wavelength, typically 254 nm, emitted by a low-pres-sure mercury lamp. Variable wavelength detectors contain acontinuous source, such as a deuterium or high-pressure xenonlamp, and a monochromator or an interference filter to gener-ate monochromatic radiation at a wavelength selected by theoperator. Modern variable wavelength detectors can be pro-grammed to change wavelength while an analysis is in progress.Multi-wavelength detectors measure absorbance at two or morewavelengths simultaneously. In diode array multi-wavelengthdetectors, continuous radiation is passed through the samplecell, then resolved into its constituent wavelengths, which areindividually detected by the photodiode array. These detectorsacquire absorbance data over the entire UV-visible range, thusproviding the analyst with chromatograms at multiple, se-lectable wavelengths and spectra of the eluting peaks. Diodearray detectors usually have lower signal-to-noise ratios thanfixed orvariable wavelength detectors, and thus are less suitablefor analysis of compounds present at low concentrations.
Differential refractometer detectors measure the differencebetween the refractive index of the mobile phase alone andthat of the mobile phase containing chromatographed com-pounds as it emerges from the column. Refractive index detec-tors are used to detect non-UV absorbing compounds, butthey are less sensitive than UV detectors. They are sensitiveto small changes in solvent composition, flow rate, and tem-perature, so that a reference column may be required to obtaina satisfactory baseline.
Fluorometric detectors are sensitive to compounds that areinherently fluorescent or that can be converted to fluorescentderivatives either by chemical transformation of the compoundor by coupling with fluorescent reagents at specific functionalgroups. If derivatization is required, it can be done beforechromatographic separation or, alternatively, the reagent canbe introduced into the mobile phase just before its enteringthe detector.
Potentiometric, voltammetric, or polarographic electro-chemical detectors are useful for the quantitation of speciesthat can be oxidized or reduced at a working electrode. Thesedetectors are selective, sensitive, and reliable, but requireconducting mobile phases free of dissolved oxygen and reduc-ible metal ions. A pulseless pump must be used, and caremust be taken to ensure that the pH, ionic strength, and temper-ature of the mobile phase remain constant. Working electrodesare prone to contamination by reaction products with conse-quent variable responses.
Electrochemical detectors with carbon-paste electrodes maybe used advantageously to measure nanogram quantities ofeasily oxidized compounds, notably phenols and catechols.
840 / Appendix II / General Tests and Assays FCC V
Data Collection Devices Modern data stations receive andstore detector output and print out chromatograms completewith peak heights, peak areas, sample identification, andmethod variables. They are also used to program the liquidchromatograph, controlling most variables and providing forlong periods of unattended operation.
Data also may be collected on simple recorders for manualmeasurement or on stand-alone integrators, which range incomplexity, from those providing a printout of peak areas tothose providing a printout of peak areas and peak heightscalculated and data stored for possible subsequent re-processing.
Procedure The mobile phase composition significantly in-fluences chromatographic performance and the resolution ofcompounds in the mixture being chromatographed. Composi-tion has a much greater effect than temperature on the capacityfactor, k.
In partition chromatography, the partition coefficient, andhence the separation, can be changed by addition of anothercomponent to the mobile phase. In ion-exchange chromatogra-phy, pH and ionic strength as well as changes in the composi-tion of the mobile phase affect capacity factors. The techniqueof continuously increasing mobile phase strength during thechromatographic run is called gradient elution or solvent pro-gramming. It is sometimes used to chromatograph complexmixtures of components differing greatly in their capacityfactors. Detectors that are sensitive to change in solvent com-position, such as the differential refractometer, are more diffi-cult to use with the gradient elution technique.
For accurate quantitative work, high-purity, ‘‘HPLC-grade’’ solvents and reagents must be used. The detector musthave a broad linear dynamic range, and compounds to bemeasured must be resolved from any interfering substances.The linear dynamic range of a compound is the range overwhich the detector signal response is directly proportional tothe amount of the compound. For maximum flexibility inquantitative work, this range should be about three orders ofmagnitude. HPLC systems are calibrated by plotting peakresponses in comparison with known concentrations of a refer-ence standard, using either an external or an internal standard-ization procedure.
Reliable quantitative results are obtained by external cali-bration if automatic injectors or autosamplers are used. Thismethod involves direct comparison of the peak responsesobtained by separately chromatographing the test and refer-ence standard solutions. If syringe injection, which is irrepro-ducible at the high pressures involved, must be used, betterquantitative results are obtained by the internal calibrationprocedure where a known amount of a noninterfering com-pound, the internal standard, is added to the test and referencestandard solutions, and the ratios of peak responses of theanalyte and internal standard are compared.
Because of normal variations in equipment, supplies, andtechniques, a system suitability test is required to ensure thata given operating system may be generally applicable. Themain features of System Suitability tests are described below.
For information on the interpretation of results, see thesection Interpretation of Chromatograms.
FIGURE 1 Chromatographic Separation of Two Substances.
Interpretation of Chromatograms Fig. 1 represents a typi-cal chromatographic separation of two substances, 1 and 2,in which tR(1) and tR(2) are the respective retention times; h,h/2, and Wh/2 are the height, the half-height, and the width athalf-height, respectively, for peak 1; and W1 and W2 are therespective widths of peaks 1 and 2 at the baseline. Air peaksare a feature of gas chromatograms and correspond to thesolvent front in liquid chromatography.
Chromatographic retention times are characteristic of thecompounds they represent but are not unique. Coincidenceof retention times of a test and a reference substance can beused as a feature in construction of an identity profile but isinsufficient on its own to establish identity. Absolute retentiontimes of a given compound vary from one chromatogram tothe next. Comparisons are normally made in terms of relativeretention, which is calculated by the equation
� = (tR(2) − tR(0))/(tR(1) − tO),
in which tR(2) and tR(1) are the retention times, measured fromthe point of injection, of the test and reference substances,respectively, determined under identical experimental condi-tions on the same column, and tO is the retention time of anonretained substance, such as methane in this case, of gaschromatography.
In this and the following expressions, the correspondingretention volumes or linear separations on the chromatogram,both of which are directly proportional to retention time, maybe substituted in the equations. Where the value of tO is small,Rr may be estimated from the retention times measured fromthe point of injection (tR(2)/tR(1)).
The number of theoretical plates, N, is a measure of columnefficiency. For Gaussian peaks, it is calculated by the equa-tions
N = 16(tR/W)2 or N = 5.54(tR/W½)2,
in which tR is the retention time of the substance and W isthe width of the peak at its base, obtained by extrapolatingthe relatively straight sides of the peak to the baseline. W1/2
is the peak width at half-height, obtained directly by electronicintegrators. The value of N depends on the substance beingchromatographed as well as the operating conditions such asmobile phase or carrier gas flow rates and temperature, thequality of the packing, the uniformity of the packing withinthe column, and for capillary columns, the thickness of thestationary phase film and the internal diameter and length ofthe column.
The separation of two components in a mixture, the resolu-tion, R, is determined by the equation
R = 2(tR(2) − tR(1))/(W2 + W1),
FCC V General Tests and Assays / Appendix II / 841
in which tR(2) and tR(1) are the retention times of the twocomponents, and W2 and W1 are the corresponding widths atthe bases of the peaks obtained by extrapolating the relativelystraight sides of the peaks to the baseline.
Peak areas and peak heights are usually proportional to thequantity of compound eluting. These are commonly measuredby electronic integrators but may be determined by more classi-cal approaches. Peak areas are generally used but may be lessaccurate ifpeak interference occurs. Formanual measurements,the chart should be run faster than usual, or a comparator shouldbe used to measure the width at half-height and the width at thebase of the peak, to minimize error in these measurements. Foraccurate quantitative work, the components to be measuredshould be separated from anyinterfering components. Peak tail-ing and fronting and the measurement of peaks on solvent tailsare to be avoided (see Fig. 2). The relative standard deviationis expressed by the equation
SR (%) = (100/X) {[�N
i−1(Xi − X)2]/(N − 1)}½,
in which SR is the relative standard deviation in percent, X isthe mean of the set of N measurements, and Xi is an individualmeasurement. When an internal standard is used, the measure-ment Xi usually refers to the measurement of relative area, As,
Xi = As = ar/ai,
in which ar is the area of the peak corresponding to thestandard substance and ai is the area of the peak correspondingto the internal standard. When peak heights are used, themeasurement Xi refers to the measurement of relativeheights, Hs,
Xi = Hs = hr/hi,
in which hr is the height of the peak corresponding to thestandard substance and hi is the height of the peak correspond-ing to the internal standard.
System Suitability Such tests are an integral part of gasand liquid chromatographic methods. They are used to verifythat the resolution and reproducibility of the chromatographicsystem are adequate for the analysis to be done. The tests arebased on the concept that the equipment, electronics, analyticaloperations, and samples to be analyzed constitute an integralsystem that can be evaluated as such.
The resolution, R, is a function of column efficiency, N,and is specified to ensure that closely eluting compounds areresolved from each other, to establish the general resolving
FIGURE 2 Asymmetrical Chromatographic Peak.
power of the system, and to ensure that internal standards areresolved from the analyte. Column efficiency may be specifiedalso as a system suitability requirement, especially if there isonly one peak of interest in the chromatogram; however,it is a less reliable means to ensure resolution than directmeasurement. Column efficiency is a measure of peak sharp-ness, which is important for the detection of trace components.
Replicate injections of a standard preparation used in theassay or other standard solution are compared to ascertainwhether requirements for precision are met. Unless otherwisespecified in the individual monograph, data from five replicateinjections of the analyte are used to calculate the relativestandard deviation if the requirement is 2.0% or less; datafrom six replicate injections are used if the relative standarddeviation requirement is more than 2.0%.
The tailing factor, T, a measure of peak symmetry, is unityfor perfectly symmetrical peaks, and its value increases astailing becomes more pronounced. In some cases, values lessthan unity may be observed. As peak asymmetry increases,integration, and hence precision, becomes less reliable. Thecalculation is expressed by the equation
tailing factor = T = W0.05/2f.
These tests are performed by collecting data from replicateinjections of standard or other solutions as specified in theindividual monograph. The specification of definitive parame-ters in a monograph does not preclude the use of other suitableoperating conditions (see Procedures under Tests and Assaysin General Provisions). Adjustments of operating conditionsto meet system suitability requirements may be necessary.
Unless otherwise directed in the monograph, system suit-ability parameters are determined from the analyte peak.
To ascertain the effectiveness of the final operating system,it should be subjected to a suitability test before use and duringtesting whenever there is a significant change in equipment orin a critical reagent or when a malfunction is suspected.
B. PHYSICOCHEMICAL PROPERTIES
DISTILLATION RANGE
Scope This method is to be used for determining the distilla-tion range of pure or nearly pure compounds or mixtureshaving a relatively narrow distillation range of about 40° orless. The result so determined is an indication of purity, notnecessarily of identity. Products having a distillation rangeof greater than 40° may be determined by this method if awide-range thermometer, such as ASTM E1, 1C, 2C, or 3C,is specified in the individual monograph.
842 / Appendix II / General Tests and Assays FCC V
DefinitionsDistillation Range The difference between the tempera-
ture observed at the start of a distillation and that observedat which a specified volume has distilled, or at which the drypoint is reached.
Initial Boiling Point The temperature indicated by thedistillation thermometer at the instant the first drop of conden-sate leaves the end of the condenser tube.
Dry Point The temperature indicated at the instant the lastdrop of liquidevaporates from the lowest point in the distillationflask, disregarding any liquid on the side of the flask.
ApparatusDistillation Flask A 200-mL round-bottom distilling flask
of heat-resistant glass is preferred when sufficient sample (inexcess of 100 mL) is available for the test. If a sample ofless than 100 mL must be used, a smaller flask having acapacity of at least double the volume of the liquid taken maybe employed. The 200-mL flask has a total length of 17 to19 cm, and the inside diameter of the neck is 20 to 22 mm.Attached about midway on the neck, approximately 12 cmfrom the bottom of the flask, is a side arm 10 to 12.7 cmlong and 5 mm in internal diameter, which forms an angleof 70° to 75° with the lower portion of the neck.
Condenser Use a straight glass condenser of heat-resistanttubing, 56 to 60 cm long and equipped with a water jacketso that about 40 cm of the tubing is in contact with the coolingmedium. The lower end of the condenser may be bent toprovide a delivery tube or it may be connected to a bentadapter that serves as the delivery tube.
Note: All-glass apparatus with standard-taper groundjoints may be used alternatively if the assembly em-ployed provides results equal to those obtained withthe flask and condenser described above.
Receiver The receiver is a 100-mL cylinder that is gradua-ted in 1-mL subdivisions and calibrated ‘‘to contain.’’ It isused for measuring the sample as well as for receiving thedistillate.
Thermometer An accurately standardized partial-immer-sion thermometer having the smallest practical subdivisions(not greater than 0.2°) is recommended to avoid the necessityfor an emergent stem correction. Suitable thermometers areavailable as the ASTM E1 Series 37C through 41C, and 102Cthrough 107C, or as the MCA types R-1 through R-4 (seeThermometers, Appendix I).
Source of Heat A Bunsen burner is the preferred source ofheat. An electric heater may be used, however, if it is shown togive results comparable to those obtained with the gas burner.
Shield The entire burner and flask assembly should beprotected from external air currents. Any efficient shield maybe employed for this purpose.
Flask Support A heat-resistant board, 5 to 7 mm in thick-ness and having a 10-cm circular hole, is placed on a suitablering or platform support and fitted loosely inside the shieldto ensure that hot gases from the source of heat do not comein contact with the sides or neck of the flask. A second 5- to7-mm thick heat-resistant board, 14- to 16-cm square and
provided with a 30- to 40-mm circular hole, is placed on topof the first board. This board is used to hold the 200-mLdistillation flask, which should be fitted firmly on the boardso that direct heat is applied to the flask only through theopening in the board.
Procedure (Note: For materials boiling below 50°, cool theliquid to below 10° before sampling, receive the distillate ina water bath cooled to below 10°, and use water cooled tobelow 10° in the condenser.) Measure 100 � 0.5 mL of theliquid in the 100-mL graduate, and transfer the sample, to-gether with an efficient antibumping device, into the distillingflask. Do not use a funnel in the transfer or allow any of thesample to enter the side arm of the flask. Place the flask onthe heat-resistant boards, which are supported on a ring orplatform, and position the shield for the flask and burner.Connect the flask and condenser, place the graduate underthe outlet of the condenser tube, and insert the thermometer.The thermometer should be located in the center of the neckso that the top of the contraction chamber (or bulb, if 37C or38C is used) is level with the bottom of the outlet to the sidearm. Regulate the heating so that the first drop of liquid iscollected within 5 to 10 min. Read the thermometer at theinstant the first drop of distillate falls from the end of thecondenser tube, and record as the initial boiling point. Con-tinue the distillation at the rate of 4 or 5 mL of distillate perminute, noting the temperature as soon as the last drop ofliquid evaporates from the bottom of the flask (dry point) orwhen the specified percentage has distilled over. Correct theobserved temperature readings for any variation in the baro-metric pressure from the normal (760 mm) by allowing 0.1°for each 2.7 mm of variation, adding the correction if thepressure is lower, or subtracting if higher, than 760 mm.
When a total-immersion thermometer is used, correct forthe temperature of the emergent stem by the formula
0.00015 × N(T − t),
in which N represents the number of degrees of emergentstem from the bottom of the stopper, T represents the observedtemperatures of the distillation, and t represents the tempera-ture registered by an auxiliary thermometer, the bulb of whichis placed midway of the emergent stem, adding the correctionto the observed readings of the main thermometer.
MELTING RANGE OR TEMPERATURE
For purposes of the FCC, the melting range or temperatureof a solid is defined as those points of temperature withinwhich or the point at which the solid coalesces and is com-pletely melted when determined as directed below. Any appa-ratus or method capable of equal accuracy may be used. Theaccuracy should be checked frequently by the use of one ormore of the six USP Melting Point Reference Standards,preferably the one that melts nearest the melting temperatureof the compound to be tested.
FCC V General Tests and Assays / Appendix II / 843
Five procedures for the determination of melting range ortemperature are given herein, varying in accordance with thenature of the substance. When no class is designated in themonograph, use the procedure for Class I.
The procedure known as the mixed melting point determina-tion, whereby the melting range of a solid under test is com-pared with that of an intimate mixture of equal parts of thesolid and an authentic specimen of it, may be used as aconfirmatory identification test. Agreement of the observa-tions on the original and the mixture usually constitutes reli-able evidence of chemical identity.
Apparatus The melting range apparatus consists of a glasscontainer for a bath of colorless fluid, a suitable stirring device,an accurate thermometer (see Appendix I), and a controlledsource of heat. The bath fluid is selected consistent with thetemperature required, but light paraffin is used generally,and certain liquid silicones are well adapted to the highertemperature ranges. The fluid is deep enough to permit immer-sion of the thermometer to its specified immersion depth sothat the bulb is still about 2 cm above the bottom of the bath.The heat may be supplied electrically or by an open flame.The capillary tube is about 10 cm long, with an internaldiameter of 0.8 to 1.2 mm, and with walls 0.2 to 0.3 mm thick.
The thermometer is preferably one that conforms to thespecifications provided under Thermometers, Appendix I, se-lected for the desired accuracy and range of temperature.
Procedure for Class I Reduce the sample to a very finepowder, and unless otherwise directed, render it anhydrouswhen it contains water of hydration by drying it at the tempera-ture specified in the monograph, or when the substance con-tains no water of hydration, dry it over a suitable desiccantfor 16 to 24 h.
Charge a capillary glass tube, one end of which is sealed,with a sufficient amount of the dry powder to form a column inthe bottom of the tube 2.5 to 3.5 mm high when packed downas closely as possible by moderate tapping on a solid surface.
Heat the bath until a temperature approximately 30° belowthe expected melting point is reached, attach the capillarytube to the thermometer, and adjust its height so that thematerial in the capillary is level with the thermometer bulb.Return the thermometer to the bath, continue the heating, withconstant stirring, at a rate of rise of approximately 3°/minuntil a temperature 3° below the expected melting point isattained, then carefully regulate the rate to about 1° to 2°/min until melting is complete.
The temperature at which the column of the sample isobserved to collapse definitely against the side of the tube atany point is defined as the beginning of melting, and thetemperature at which the sample becomes liquid throughoutis defined as the end of melting. The two temperatures fallwithin the limits of the melting range.
Procedure for Class Ia Prepare the sample and charge thecapillary glass tube as directed for Class I. Heat the bath untila temperature 10° � 1° below the expected melting range isreached, then introduce the charged tube, and heat at a rate
of rise of 3° � 0.5°/min until melting is complete. Recordthe melting range as for Class I.
Procedure for Class Ib Place the sample in a closed con-tainer, and cool to 10° or lower for at least 2 h. Without previouspowdering, charge the cooled material into the capillary tubeas directed for Class I, immediately place the charged tube ina vacuum desiccator, and dry at a pressure not exceeding 20mm Hg for 3 h. Immediately upon removal from the desiccator,fire-seal the open end of the tube. As soon as is practicable,proceed with the determination of the melting range as follows:Heat the bath until a temperature of 10° � 1° below the expectedmelting range is reached, then introduce the charged tube, andheat at a rate of rise of 3° � 0.5°/min until melting is complete.Record the melting range as directed in Class I.
If the particle size of the material is too large for thecapillary, precool the sample as directed above, then with aslittle pressure as possible, gently crush the particles to fit thecapillary, and immediately charge the tube.
Procedure for Class II Carefully melt the material to betested at as low a temperature as possible, and draw it into acapillary tube that is left open at both ends to a depth of about10 mm. Cool the charged tube at 10°, or lower, for 24 h, orin contact with ice for at least 2 h. Then attach the tube tothe thermometer by means of a rubber band, adjust it in awater bath so that the upper edge of the material is 10 mmbelow the water level, and heat as directed for Class I, exceptwithin 5° of the expected melting temperature, regulate therate of rise of temperature to 0.5° to 1.0°/min. The temperatureat which the material is observed to rise in the capillary tubeis the melting temperature.
Procedure for Class III Melt a quantity of the substanceslowly, while stirring, until it reaches a temperature of 90°to 92°. Remove the source of heat, and allow the moltensubstance to cool to a temperature of 8° to 10° above theexpected melting point. Chill the bulb of an ASTM 14Cthermometer (see Appendix I) to 5°, wipe it dry, and whileit is still cold, dip it into the molten substance so that approxi-mately the lower half of the bulb is submerged. Withdraw itimmediately, and hold it vertically away from the heat untilthe wax surface dulls, then dip it for 5 min into a water bathhaving a temperature not higher than 16°.
Fix the thermometer securely in a test tube so that the lowerpoint is 15 mm above the bottom of the test tube. Suspendthe test tube in a water bath adjusted to about 16°, and raisethe temperature of the bath at the rate of 2°/min to 30°, thenchange to a rate of 1°/min, and note the temperature at whichthe first drop of melted substance leaves the thermometer.Repeat the determination twice on a freshly melted portionof the sample. If the variation of three determinations is lessthan 1°, take the average of the three as the melting point. Ifthe variation of three determinations is greater than 1°, maketwo additional determinations and take the average of the five.
844 / Appendix II / General Tests and Assays FCC V
OPTICAL (SPECIFIC) ROTATION
Many chemicals in a pure state or in solution are opticallyactive in the sense that they cause incident polarized light toemerge in a plane forming a measurable angle with the planeof the incident light. When this effect is large enough forprecise measurement, it may serve as the basis for an assayor an identity test. In this connection, the optical rotation isexpressed in degrees, as either angular rotation (observed)or specific rotation (calculated with reference to the specificconcentration of 1 g of solute in 1 mL of solution, measuredunder stated conditions).
Specific rotation of a liquid substance usually is expressedby the equation [�]x
t = a/ld, and for solutions of solid sub-stances, expressed by the equation [�]x
t = 100a/lpd =100a/lc, in which a is the corrected observed rotation, indegrees, at temperature t; x is the wavelength of the lightused; l is the length of the polarimeter cell, in dm; d is thespecific gravity of the liquid or solution at the temperatureof observation; p is the concentration of the solution expressedas the number of grams of substance in 100 g of solution;and c is the concentration of the solution expressed as thenumber of grams of substance in 100 mL of solution. Theconcentrations p and c should be calculated on the dried oranhydrous basis, unless otherwise specified. Spectral linesmost frequently employed are the D line of sodium (doubletat 589.0 and 589.6 nm) and the yellow-green line of mercuryat 546.1 nm. The specific gravity and the rotatory power varyappreciably with the temperature.
The accuracy and precision of optical rotatory measure-ments will be increased if they are carried out with due regardfor the following general considerations.
Supplement the source of illumination with a filtering sys-tem capable of transmitting light of a sufficiently monochro-matic nature. Precision polarimeters generally are designedto accommodate interchangeable disks to isolate the D linefrom sodium light or the 546.1-nm line from the mercuryspectrum. With polarimeters not thus designed, cells con-taining suitably colored liquids may be employed as filters(see also A. Weissberger and B. W. Rossiter, Techniques ofChemistry, Vol. I: Physical Methods of Chemistry, Part 3,Wiley-Interscience, New York, 1972).
Pay special attention to temperature control of the solutionand of the polarimeter. Make accurate and reproducible obser-vations to the extent that differences between replicates, orbetween observed and true values of rotation (the latter valuehaving been established by calibration of the polarimeter scalewith suitable standards), calculated in terms of either specificrotation or angular rotation, whichever is appropriate, do notexceed one-fourth of the range given in the individual mono-graph for the rotation of the article being tested. Generally,a polarimeter accurate to 0.05° of angular rotation, and capableof being read with the same precision, suffices for FCC pur-poses; in some cases, a polarimeter accurate to 0.01°, or less,of angular rotation, and read with comparable precision, maybe required.
Fill polarimeter tubes in such a way as to avoid creating orleaving air bubbles, which interfere with the passage of the
beam of light. Interference from bubbles is minimized withtubes in which the bore is expanded at one end. However, tubesof uniform bore, such as semimicro- or micro-tubes, requirecare for proper filling. At the time of filling, the tubes and theliquid or solution should be at a temperature not higher than thatspecified for the determination to guard against the formation ofa bubble upon cooling and contraction of the contents.
In closing tubes having removable end plates fitted withgaskets and caps, the latter should be tightened only enoughto ensure a leak-proof seal between the end plate and thebody of the tube. Excessive pressure on the end plate mayset up strains that result in interference with the measurements.In determining the specific rotation of a substance of lowrotatory power, loosen the caps and tighten them again be-tween successive readings in the measurement of both therotation and the zero point. Differences arising from end platestrain thus generally will be revealed and appropriate adjust-ments to eliminate the cause may be made.
Procedure In the case of a solid, dissolve the substance in asuitable solvent, reserving a separate portion of the latter for ablank determination. Make at least five readings of the rotationof the solution, or of the substance itself if liquid, at 25° or thetemperature specified in the individual monograph. Replace thesolution with the reserved portion of the solvent (or, in the caseof a liquid, use the empty tube), make the same number of read-ings, and use the average as the zero point value. Subtract thezero point value from the average observed rotation if the twofigures are of the same sign, or add if opposite in sign, to obtainthe corrected observed rotation.
Calculation Calculate the specific rotation of a liquid sub-stance, or of a solid in solution, by application of one of thefollowing formulas:
(I) for liquid substances,
[�]xt = a/ld,
(II) for solutions of solids,
[�]xt = 100a/lpd = 100a/lc,
in which a is the corrected observed rotation, in degrees, attemperature t; x is the wavelength of the light used; l is thelength, in dm, of the polarimeter cell; d is the specific gravityof the liquid or solution at the temperature of observation; pis the concentration of the solution expressed as the numberof grams of substance in 100 g of solution; and c is theconcentration of the solution expressed as the number of gramsof substance in 100 mL of solution. The concentrations p andc should be calculated on the dried or anhydrous basis, unlessotherwise specified.
pH DETERMINATION
Principle The definition of pH is the negative log of thehydrogen ion concentration in moles per liter of aqueous
FCC V General Tests and Assays / Appendix II / 845
solutions. Measure pH potentiometrically by using a pH meteror colorimetrically by using pH indicator paper.
Scope This method is suitable to determine the pH of aque-ous solutions. While pH meters, calibrated with aqueous solu-tions, are sometimes used to make measurements in semia-queous solutions or in nonaqueous polar solutions, the valueobtained is the apparent pH value only and should not becompared with the pH of aqueous solutions. For nonpolarsolutions, pH has no meaning, and pH electrodes may bedamaged by direct contact with these solutions. Referencesto the pH of nonpolar solutions or liquids usually indicate thepH of a water extract of the nonpolar liquid or the apparentpH of a mixture of the nonpolar liquid in a polar liquid suchas alcohol or alcohol–water mixtures.
Procedure [Potentiometric Method (pH Meter)]Calibration Select two standard buffers to bracket, if pos-
sible, the anticipated pH of the unknown substances. Thesecommercially available standards and the sample should be atthe same temperature, within two degrees. Set the temperaturecompensator of the pH meter to the temperature of the samplesand standards. Follow the manufacturer’s instructions for set-ting temperature compensation and for adjusting the outputduring calibration. Rinse the electrodes with distilled or deion-ized water, and blot them dry with clean, absorbent laboratorytissue. Place the electrode(s) in the first standard buffer solu-tion, and adjust the standardization control so that the pHreading matches the stated pH of the standard buffer. Repeatthis procedure with fresh portions of the first buffer solutionuntil two successive readings are within � 0.02 pH units withno further adjustment. Rinse the electrodes, blot them dry,and place them in a portion of the second standard buffersolution. Following the manufacturer’s instructions, adjust theslope control (not the standardization control) until the outputdisplays the pH of the second standard buffer.
Repeat the sequence of standardization with both buffersuntil pH readings are within � 0.02 pH units for both bufferswithout adjustments to either the slope or standardizationcontrols. The pH of the unknown may then be measured,using either a pH electrode in combination with a referenceelectrode or a single combination electrode. Select electrodesmade of chemically resistant glass when measuring samplesof either low or high pH.
pH Indicator Paper Test papers impregnated with acid–base indicators, although less accurate than pH meters, offera convenient way to determine the pH of an aqueous solution.They may be purchased in rolls or strips covering all or partof the pH range; papers covering a narrow part of the pHrange can be sensitive to differences of 0.2 pH units. Sometest papers comprise a plastic strip with small squares of testpaper attached. The different squares are sensitive to differentpH ranges. When using this type of test paper, wet all of thesquares with the test sample to ensure a correct pH reading.
Test paper can contaminate the sample being tested; there-fore, do not dip it into the sample. Either use a clean glassrod to remove a drop of the test solution and place it on thetest paper, or transfer a small amount of the sample to a smallcontainer, dip the test paper into this portion, and compare
the developed color with the color comparison chart providedwith the test paper to determine the pH of the sample.
READILY CARBONIZABLESUBSTANCES
ReagentsSulfuric Acid, 95% Add a quantity of sulfuric acid of
known concentration to sufficient water to adjust the finalconcentration to between 94.5% and 95.5% of H2SO4. Becausethe acid concentration may change upon standing or uponintermittent use, check the concentration frequently and eitheradjust solutions assaying more than 95.5% or less than 94.5%by adding either diluted or fuming sulfuric acid, as required,or discard them.
Cobaltous Chloride CS Dissolve about 65 g of cobaltouschloride (CoCl2·6H2O) in enough of a mixture of 25 mL ofhydrochloric acid and 975 mL of water to make 1000 mL.Pipet 5 mL of this solution into a 250-mL iodine flask, add5 mL hydrogen peroxide TS (3%) and 15 mL of a 1:5 solutionof sodium hydroxide, boil for 10 min, cool, and add 2 g ofpotassium iodide and 20 mL of 1:4 sulfuric acid. When theprecipitate has dissolved, titrate the liberated iodine with 0.1N sodium thiosulfate. The titration is sensitive to air oxidationand should be blanketed with carbon dioxide. Each milliliterof 0.1 N sodium thiosulfate is equivalent to 23.79 mg ofCoCl2·6H2O. Adjust the final volume of the solution by addingenough of the mixture of hydrochloric acid and water so thateach milliliter contains 59.5 mg of CoCl2·6H2O.
Cupric Sulfate CS Dissolve about 65 g of cupric sulfate(CuSO4·5H2O) in enough of a mixture of 25 mL of hydrochlo-ric acid and 975 mL of water to make 1000 mL. Pipet 10 mLof this solution into a 250-mL iodine flask; add 40 mL ofwater, 4 mL of acetic acid, and 3 g of potassium iodide; andtitrate the liberated iodine with 0.1 N sodium thiosulfate,adding starch TS as the indicator. Each milliliter of 0.1 Nsodium thiosulfate is equivalent to 24.97 mg of CuSO4·5H2O.Adjust the final volume of the solution by adding enough of themixture of hydrochloric acid and water so that each millilitercontains 62.4 mg of CuSO4·5H2O.
Ferric Chloride CS Dissolve about 55 g of ferric chloride(FeCl3·6H2O) in enough of a mixture of 25 mL of hydrochloricacid and 975 mL of water to make 1000 mL. Pipet 10 mLof this solution into a 250-mL iodine flask; add 15 mL ofwater, 5 mL of hydrochloric acid, and 3 g of potassium iodide;and allow the mixture to stand for 15 min. Dilute with 100mL of water, and titrate the liberated iodine with 0.1 N sodiumthiosulfate, adding starch TS as the indicator. Perform a blankdetermination with the same quantities of the same reagentsand in the same manner, and make any necessary correction.Each milliliter of 0.1 N sodium thiosulfate is equivalent to27.03 mg of FeCl3·6H2O. Adjust the final volume of thesolution by adding the mixture of hydrochloric acid and waterso that each milliliter contains 45.0 mg of FeCl3·6H2O.
846 / Appendix II / General Tests and Assays FCC V
Platinum–Cobalt CS Transfer 1.246 g of potassium chlo-roplatinate (K2PtCl6) and 1.00 g of crystallized cobaltouschloride (CoCl2·6H2O) into a 1000-mL volumetric flask, dis-solve in about 200 mL of water and 100 mL of hydrochloricacid, dilute to volume with water, and mix. This solution hasa color of 500 APHA units.
Note: Use this solution only when specified in an indi-vidual monograph.
Procedure Unless otherwise directed, add the specifiedquantity of the substance, finely powdered if in solid form,in small portions to the comparison container, which is madeof colorless glass resistant to the action of sulfuric acid andcontains the specified volume of 95% Sulfuric Acid.
Stir the mixture with a glass rod until solution is complete,allow the solution to stand for 15 min, unless otherwise di-rected, and compare the color of the solution with that of thespecified matching fluid in a comparison container that alsois of colorless glass and has the same internal and cross-section dimensions, viewing the fluids transversely against abackground of white porcelain or white glass.
When heat is directed to effect solution of the substancein the 95% Sulfuric Acid, mix the sample and the acid in atest tube, heat as directed, cool, and transfer the solution tothe comparison container for matching.
Matching Fluids For purposes of comparison, a series of20 matching fluids, each designated by a letter of the alphabet,is provided, the composition of each being as indicated in theaccompanying table. To prepare the matching fluid specified,pipet the prescribed volumes of the colorimetric test solutions(CS) and water into one of the matching containers, and mixthe solutions in the container.
Matching Fluidsa
Parts of Parts of Parts ofMatching Cobaltous Ferric Cupric Parts ofFluid Chloride CS Chloride CS Sulfate CS Water
A 0.1 0.4 0.1 4.4B 0.3 0.9 0.3 8.5C 0.1 0.6 0.1 4.2D 0.3 0.6 0.4 3.7E 0.4 1.2 0.3 3.1F 0.3 1.2 0.0 3.5G 0.5 1.2 0.2 3.1H 0.2 1.5 0.0 3.3I 0.4 2.2 0.1 2.3J 0.4 3.5 0.1 1.0K 0.5 4.5 0.0 0.0L 0.8 3.8 0.1 0.3M 0.1 2.0 0.1 2.8N 0.0 4.9 0.1 0.0O 0.1 4.8 0.1 0.0P 0.2 0.4 0.1 4.3Q 0.2 0.3 0.1 4.4
Matching Fluidsa (continued)
Parts of Parts of Parts ofMatching Cobaltous Ferric Cupric Parts ofFluid Chloride CS Chloride CS Sulfate CS Water
R 0.3 0.4 0.2 4.1S 0.2 0.1 0.0 4.7T 0.5 0.5 0.4 3.6
aSolutions A–D, very light brown-yellow.Solutions E–L, yellow through red-yellow.Solutions M–O, green-yellow.Solutions P–T, light pink.
REFRACTIVE INDEX
The refractive index of a transparent substance is the ratio ofthe velocity of light in air to its velocity in that material underlike conditions. It is equal to the ratio of the sine of the angleof incidence made by a ray in air to the sine of the angle ofrefraction made by the ray in the material being tested. Therefractive index values specified in this Codex are for theD line of sodium (589 nm) unless otherwise specified. Thedetermination should be made at the temperature specified inthe individual monograph, or at 25° if no temperature isspecified. This physical constant is used as a means for identi-fication of, and detection of impurities in, volatile oils andother liquid substances. The Abbé refractometer, or otherrefractometers of equal or greater accuracy, may be employedat the discretion of the operator.
SOLIDIFICATION POINT
Scope This method is designed to determine the solidifica-tion point of food-grade chemicals having appreciable heatsof fusion. It is applicable to chemicals having solidificationpoints between −20° and +150°. Necessary modifications willbe noted in individual monographs.
Definition Solidification Point is an empirical constant de-fined as the temperature at which the liquid phase of a sub-stance is in approximate equilibrium with a relatively smallportion of the solid phase. It is measured by noting the maxi-mum temperature reached during a controlled cooling cycleafter the appearance of a solid phase.
The solidification point is distinguished from the freezingpoint in that the latter term applies to the temperature ofequilibrium between the solid and liquid state of pure com-pounds.
Some chemical compounds have more than one temperatureat which there may be an equilibrium between the solid and
FCC V General Tests and Assays / Appendix II / 847
liquid state depending on the crystal form of the solid that ispresent.
Apparatus The apparatus illustrated in Figs. 3 and 4 con-sists of the components described in the following paragraphs.
Thermometer A thermometer having a range not ex-ceeding 30°, graduated in 0.1° divisions, and calibrated for76-mm immersion should be employed. A satisfactory seriesof thermometers, covering a range from −20° to +150°, isavailable as ASTM-E1 89C through 96C (see Thermometers,Appendix I). A thermometer should be chosen such that thesolidification point is not obscured by the cork stopper of thesample container.
Sample Container Use a standard glass 25- × 150-mmtest tube with a lip, fitted with a two-hole cork stopper tohold the thermometer in place and to allow adequate stirringwith a stirrer.
Air Jacket For the air jacket, use a standard glass 38- ×200-mm test tube with a lip and fitted with a cork or rubberstopper bored with a hole into which the sample containercan easily be inserted up to the lip.
Cooling Bath Use a 2000-mL beaker or a similar, suitablecontainer as a cooling bath. Fill it with an appropriate coolingmedium such as glycerin, mineral oil, water, water and ice,or alcohol–dry ice.
Stirrer The stirrer (Fig. 4) consists of a 1-mm in diameter(B & S gauge 18), corrosion-resistant wire bent into a seriesof three loops about 25 mm apart. It should be made so thatit will move freely in the space between the thermometer andthe inner wall of the sample container. The shaft of the stirrershould be of a convenient length designed to pass looselythrough a hole in the cork holding the thermometer. Stirringmay be hand operated or mechanically activated at 20 to 30strokes/min.
Assembly Assemble the apparatus in such a way that thecooling bath can be heated or cooled to control the desiredtemperature ranges. Clamp the air jacket so that it is heldrigidly just below the lip, and immerse it in the cooling bathto a depth of 160 mm.
FIGURE 3 Apparatus for Determination of Solidification Point.
FIGURE 4 Stirrer for Solidification Point Determination.
Sample Preparation The solidification point of chemicalsis usually determined as they are received. Some may behygroscopic, however, and will require special drying. If thisis necessary, it will be noted in the individual monographs.
Products that are normally solid at room temperature mustbe carefully melted at a temperature about 10° above theexpected solidification point. Care should be observed to avoidheating in such a way as to decompose or distill any portionof a sample.
Procedure Adjust the temperature of the cooling bath toabout 5° below the expected solidification point. Fit the ther-mometer and stirrer with a cork stopper so that the thermome-ter is centered and the bulb is about 20 mm from the bottomof the sample container. Transfer a sufficient amount of thesample, previously melted if necessary, into the sample con-tainer to fill it to a depth of about 90 mm when in the moltenstate. Place the thermometer and stirrer in the sample con-tainer, and adjust the thermometer so that the immersion linewill be at the surface of the liquid and so that the end of thebulb is 20 � 4 mm from the bottom of the sample container.When the temperature of the sample is about 5° above theexpected solidification point, place the assembled sample tubein the air jacket.
Allow the sample to cool while stirring, at the rate of 20to 30 strokes/min, in such a manner that the stirrer does nottouch the thermometer. Stir the sample continuously duringthe remainder of the test.
The temperature at first will gradually fall, then will becomeconstant as crystallization starts and continues under equilib-rium conditions, and finally will start to drop again. Somechemicals may supercool slightly below (0.5°) the solidifica-tion point; as crystallization begins, the temperature will riseand remain constant as equilibrium conditions are established.Other products may cool more than 0.5° and cause deviationfrom the normal pattern of temperature change. If the tempera-ture rise exceeds 0.5° after the initial crystallization begins,repeat the test, and seed the melted compound with smallcrystals of the sample at 0.5° intervals as the temperatureapproaches the expected solidification point. Crystals for seed-
848 / Appendix II / General Tests and Assays FCC V
ing may be obtained by freezing a small sample in a test tubedirectly in the cooling bath. It is preferable that seed of thestable phase be used from a previous determination.
Observe and record the temperature readings at regularintervals until the temperature rises from a minimum, dueto supercooling, to a maximum and then finally drops. Themaximum temperature reading is the solidification point.Readings 10 s apart should be taken to establish that thetemperature is at the maximum level and should continueuntil the drop in temperature is established.
VISCOSITY
Viscosity is a fluid’s measured internal resistance to flow.Thick, slow-moving fluids have higher viscosities than thin,free-flowing fluids. The basic unit of measure for viscosityis the poise or Pascal second, Pa·s, in SI units. The relationshipbetween poise and Pa·s is 1 poise = 0.1 Pa·s. Since commonlyencountered viscosities are often fractions of 1 poise, viscosi-ties are commonly expressed as centipoises (one centipoise =0.01 poise). Poise or centipoise is the unit of measure forabsolute viscosity. Kinematic viscosity also is commonly usedand is determined by dividing the absolute viscosity of thetest liquid by the density of the test liquid at the same tempera-ture as the viscosity measurement and is expressed as stokesor centistokes (poise/density = stokes). The specified tempera-ture is important: viscosity varies greatly with temperature,generally decreasing with increasing temperature.
Absolute viscosity can be determined directly if accuratedimensions of the measuring instruments are known. It iscommon practice to calibrate an instrument with a fluid ofknown viscosity and to determine the unknown viscosity ofanother fluid by comparison with that of the known viscosity.
Many substances, such as gums, have a variable viscosity,and most of them are less resistant to flow at higher flow(more correctly, shear) rates. In such cases, select a given setof conditions for measurement, and consider the measurementobtained to be an apparent viscosity. Since a change in theconditions of measurement would yield a different value forthe apparent viscosity of such substances, the operator mustclosely adhere to the instrument dimensions and conditionsfor measurement.
Measuring Viscosity Several common methods are avail-able for measuring viscosity. Two very common ones are theuse of capillary tubes such as Ubbelohde, Ostwald, or Cannon-Fenske viscometer tubes and the use of a rotating spindlesuch as the Brookfield viscometer.
Determine the viscosity in capillary tubes by measuringthe amount of time it takes for a given volume of liquid toflow through a calibrated capillary tube. Calibrate the capillarytube by using liquids of known viscosity. The calibration maybe supplied with the viscometer tube when purchased alongwith specific instructions for its use. Many types of capillaryviscometer tubes are available, and exact procedures will vary
with the type of tube chosen. Examples of procedures are inthe following sections: Viscosity of Dimethylpolysiloxane andViscosity of Methylcellulose. In general, calibrate capillaryviscometers by filling the viscometers per the manufacturer’sinstructions and allowing the filled tube to equilibrate to thegiven temperature in a constant-temperature bath. Draw theliquid to the top graduation line, and measure the time, inseconds, it takes for the liquid to flow from the upper mark tothe lower mark in the capillary tube. Calculate the viscometerconstant, k, by the equation
k = v/dt,
in which v is the known viscosity, in centipoises, of thestandard liquid; d is the density, at the specified temperature,of the liquid; and t is the time, in seconds, for the liquid topass from the upper mark to the lower mark. It is not necessaryto recalibrate the tube unless changes or repairs are made toit. To measure viscosity, introduce the unknown liquid intothe viscometer tube in the same way as the calibration standardwas introduced, and measure the time, in seconds, it takesfor the liquid to flow from the upper mark to the lower mark.Calculate viscosity by the equation
v = kdt,
in which v is the viscosity to be determined, k is the viscometerconstant, and d is the density of the liquid being measured.
Using rotational viscometers provides a particularly rapidand convenient method for determining viscosity. They em-ploy a rotating spindle or cup immersed in the liquid, andthey measure the resistance of the liquid to the rotation ofthe spindle or cup. A wide range of viscosities can be measuredwith one instrument by using spindles or cups of different sizesand by rotating them at different speeds. The manufacturersupplies the calibration of viscosity versus the spindle sizeand speed, which can be checked by using fluids of knownviscosity. Take a measurement by allowing the sample tocome to the desired temperature in a constant-temperaturebath and immersing the spindle or cup to the depth specifiedby the manufacturer. Allow the spindle or cup to rotate untila constant reading is obtained. Multiply the reading by a factorsupplied by the manufacturer for a given spindle or cup andgiven rotational speed to obtain the viscosity. The exact proce-dures will vary with the particular instrument. An exampleis given in the section on Viscosity of Cellulose Gum.
Another method to determine viscosity uses the falling-ballviscometer. Determine viscosity by noting the time it takesfor a ball to fall through the distance between two marks ona tube filled with the unknown liquid (the tube is generallyin a constant-temperature bath). Use balls of different weightsto measure a wide range of viscosities. Calculate the viscosityby using manufacturer-supplied constants for the ball used.These instruments can be quite precise for Newtonian liquids,that is, liquids that do not have viscosities that vary with flow(more correctly, shear) rate.
Three specific methods are described below:
Viscosity of Dimethylpolysiloxane
Apparatus The Ubbelohde suspended level viscometer,shown in Fig. 5, is preferred to determine the viscosity of
FCC V General Tests and Assays / Appendix II / 849
FIGURE 5 Ubbelohde Viscometer for Dimethylpolysiloxane(all dimensions are in mm).
dimethylpolysiloxane. Alternatively, a Cannon-Ubbelohdeviscometer may be used.
Select a viscometer having a minimum flow time of at least200 s. Use a No. 3 size Ubbelohde, or a No. 400 size Cannon-Ubbelohde, viscometer for the range of 300 to 600 centistokes.The viscometer should be fitted with holders that satisfy thedimensional positions of the separate tubes as shown in thediagram and that hold the viscometer vertically. Filling linesin bulb A indicate the minimum and maximum volumes ofliquid to be used for convenient operation. The volume ofbulb B is approximately 5 mL.
Calibration of the Viscometer Determine the viscosityconstant, C, for each viscometer by using an oil of knownviscosity.1 Charge the viscometer by tilting the instrumentabout 30 degrees from the vertical, with bulb A below thecapillary, and then introduce enough of the sample into tubel to bring the level up to the lower filling line. The levelshould not be above the upper filling line when the viscometeris returned to the vertical position and the sample has drainedfrom tube l. Charge the viscometer in such a manner that theU-tube at the bottom fills completely without trapping air.
After the viscometer has been in a constant-temperaturebath (25° � 0.2°) long enough for the sample to reach tempera-ture equilibrium, place a finger over tube 3, and apply suctionto tube 2 until the liquid reaches the center of bulb C. Removesuction from tube 2, then remove the finger from tube 3, andplace it over tube 2 until the sample drops away from thelower end of the capillary. Remove the finger from tube 2,
1Oils of known viscosities may be obtained from the Cannon InstrumentCo., P.O. Box 812, State College, PA 16801. For determining the viscosityof dimethylpolysiloxane, choose an oil with a viscosity as close as possibleto that of the type of sample to be tested.
and measure the time, to the nearest 0.1 s, required for themeniscus to pass from the first timing mark (T1) to the sec-ond (T2).
Calculate the viscometer constant, C, by the equation
C = cs/t1,
in which cs is the viscosity, in centistokes, and t1 is the effluxtime, in seconds, for the standard liquid.
Determination of the Viscosity of DimethylpolysiloxaneCharge the viscometer with the sample in the same manneras described for the calibration procedure; determine the effluxtime, t2; and calculate the viscosity of the dimethylpolysilox-ane by the formula
V = C × t2.
Viscosity of Methylcellulose
Apparatus Viscometers used to determine the viscosity ofmethylcellulose and some related compounds are illustratedin Fig. 6 and consist of three parts: a large filling tube, A; anorifice tube, B; and an air vent to the reservoir, C.
There are two basic types of methylcellulose viscometers—one for cellulose derivatives of a range between 1500 and4000 centipoises, and the other for less viscous ones. Each typeof viscometer is modified slightly for the different viscosities.
Calibration of the Viscometer Determine the viscometerconstant, K, for each viscometer by using an oil of known
FIGURE 6 Methylcellulose Viscometers.
850 / Appendix II / General Tests and Assays FCC V
viscosity.2 Place an excess of the liquid that is to be tested(adjusted to 20° � 0.1°) in the filling tube, A, and transfer itto the orifice tube, B, by gentle suction, taking care to keepthe liquid free from air bubbles by closing the air vent tube,C. Adjust the column of liquid in tube B so it is even withthe top graduation line. Open both tubes B and C to permit theliquid to flow into the reservoir against atmospheric pressure.
Note: Failure to open air vent tube C before determiningthe viscosity will yield false values.
Record the time, in seconds, for the liquid to flow from theupper mark to the lower mark in tube B.
Calculate the viscometer constant, K, from the equation
K = V/dt,
in which V is the viscosity, in centipoises, of the liquid; K isthe viscometer constant; d is the specific gravity of the liquidtested at 20°/20°; and t is the time, in seconds, for the liquidto pass from the upper to the lower mark.
For the calibration, all values in the equation are knownor can be determined except K, which must be solved. If atube is repaired, it must be recalibrated to avoid obtainingsignificant changes in the value of K.
Determination of the Viscosity of Methylcellulose Preparea 2% solution of methylcellulose or other cellulose derivative,by weight, as directed in the monograph. Place the solutionin the proper viscometer and determine the time, t, requiredfor the solution to flow from the upper mark to the lower markin orifice tube B. Separately determine the specific gravity, d,at 20°/20°. Viscosity, V = Kdt.
Viscosity of Cellulose Gum
Apparatus Use a Brookfield Model LV series viscometer,analog or digital, or equivalent type viscometer for the deter-mination of viscosity of aqueous solutions of cellulose gumwithin the range of 25 to 10,000 centipoises at 25°. Rotationalviscometers of this type have spindles for use in determiningthe viscosity of different viscosity types of cellulose gum. Thespindles and speeds for determining viscosity within differentranges are tabulated below.
Viscometer Spindles Required for Given Speeds
Viscosity Range Spindle Speed Scale Factor(centipoises) No. (rpm)
10–100 1 60 100 1100–200 1 30 100 2200–1000 2 30 100 101000–4000 3 30 100 404000–10,000 4 30 100 200
2Oils of known viscosities may be obtained from the Cannon InstrumentCo., P.O. Box 812, State College, PA 16801. For determining the viscosityof methylcellulose, choose an oil that has a viscosity as close as possibleto that of the type of sample to be tested.
Mechanical Stirrer Use an agitator essentially as shownin Fig. 7 that can be attached to a variable-speed motor capableof operating at 900 � 100 rpm under varying load conditions.
Note: The agitator may be fabricated from stainlesssteel or glass as shown in Fig. 7. Where this procedureis specified for viscosity measurements by reference inother monographs, equivalent three-blade agitators maybe used. Agitators are commercially available from Div-tech Equipment Company, Cincinnati, Ohio, or fromHercules, Inc., Wilmington, Delaware.
Sample Container Use a glass jar about 152 mm deephaving an od of approximately 64 mm and a capacity of about340 g.
Water Bath Use a water bath capable of maintaining aconstant temperature. Set the temperature to 25°, and maintainit within �0.2°.
Thermometer Use an ASTM Saybolt Viscosity Thermom-eter having a range from 19° to 27° and conforming to therequirements for Thermometer 17C as described in ASTMSpecification E1.
Sample Preparation Accurately weigh an amount of sam-ple equivalent to 4.8 g of cellulose gum on the dried basis,and record the actual quantity required, in grams, as S. Transferan accurately measured volume of water equivalent to 240− S g into the sample container. Position the stirrer in thesample container, allowing minimal clearance between thestirrer and the bottom of the container. Begin stirring, andslowly add the sample. Adjust the stirring speed to approxi-mately 900 � 100 rpm. Mix for exactly 2 h. Do not allow
FIGURE 7 Agitator for Viscosity of Cellulose Gum.
FCC V General Tests and Assays / Appendix II / 851
the stirring speed to exceed 1200 rpm. Remove the stirrer,cap the sample container, and transfer the sample containerinto a constant-temperature water bath, maintained at 25° �0.2°, for 1 h. Check the sample temperature with a thermome-ter at the end of 1 h to ensure that the test temperature hasbeen reached.
Procedure Remove the sample container from the waterbath, shake vigorously for 10 s, and measure the viscositywith the Brookfield viscometer, using the proper spindle andspeed indicated in the accompanying table. Be sure to usethe viscometer guard, and allow the spindle to rotate for 3min before taking the reading. Calculate the viscosity, incentipoises, by multiplying the reading observed by the appro-priate factor from the table.
WATER DETERMINATION
Method I (Karl Fischer Titrimetric Method)
Determine the water by Method Ia, unless otherwise specifiedin the individual monograph.
Method Ia (Direct Titration)
Principle The titrimetric determination of water is basedon the quantitative reaction of water with an anhydrous solu-tion of sulfur dioxide and iodine in the presence of a bufferthat reacts with hydrogen ions.
In the original titrimetric solution, known as Karl FischerReagent, the sulfur dioxide and iodine are dissolved in pyri-dine and methanol. Pyridine-free reagents are more commonlyused now. The test specimen may be titrated with the KarlFischer Reagent directly, or the analysis may be carried outby a residual titration procedure. The stoichiometry of thereaction is not exact, and the reproducibility of the determina-tion depends on such factors as the relative concentrations ofthe Karl Fischer Reagent ingredients, the nature of the inertsolvent used to dissolve the test specimen, the apparent pHof the final mixture, and the technique used in the particulardetermination. Therefore, an empirically standardized tech-nique is used to achieve the desired accuracy. Precision in themethod is governed largely by the extent to which atmosphericmoisture is excluded from the system. The titration of wateris usually carried out with the use of anhydrous methanol asthe solvent for the test specimen; however, other suitablesolvents may be used for special or unusual test specimens.
Substances that may interfere with the test results are ferricion, chlorine, and similar oxidizing agents, as well as signifi-cant amounts of strong acids or bases, phosgene, or anythingthat will reduce iodide to iodine, poison the reagent, and showthe sample to be bone dry when water may be present (falsenegative). 8-Hydroxyquinoline may be added to the vessel toeliminate interference from ferric ion. Chlorine interferencecan be eliminated with SO2 or unsaturated hydrocarbon. Ex-cess pyridine or other amines may be added to the vessel toeliminate the interference of strong acids. Excess acetic acid
or other carboxylic acid can be added to reduce the interferenceof strong bases. Aldehydes and ketones may react with thesolution, showing the sample to be wet while the detectornever reaches an endpoint (false positive).
Apparatus Any apparatus may be used that provides foradequate exclusion of atmospheric moisture and for determi-nation of the endpoint. In the case of a colorless solution thatis titrated directly, the endpoint may be observed visually asa change in color from canary yellow to amber. The reverseis observed in the case of a test specimen that is titratedresidually. More commonly, however, the endpoint is deter-mined electrometrically with an apparatus employing a simpleelectrical circuit that serves to impress about 200 mV ofapplied potential between a pair of platinum electrodes (about5 mm2 in area and about 2.5 cm apart) immersed in thesolution to be titrated. At the endpoint of the titration, a slightexcess of the reagent increases the flow of current to between50 and 150 microamperes for 30 s to 30 min, depending onthe solution being titrated. The time is shortest for substancesthat dissolve in the reagent. The longer times are required forsolid materials that do not readily go into solution in the KarlFischer Reagent. With some automatic titrators, the abruptchange in current or potential at the endpoint serves to closea solenoid-operated valve that controls the buret deliveringthe titrant. A commercially available apparatus generally com-prises a closed system consisting of one or two automaticburets and a tightly covered titration vessel fitted with thenecessary electrodes and a magnetic stirrer. The air in thesystem is kept dry with a suitable desiccant such as phosphoruspentoxide, and the titration vessel may be purged by meansof a stream of dry nitrogen or a current of dry air.
Reagent The Karl Fischer Reagent may be prepared asfollows: Add 125 g of iodine to a solution containing 670mL of methanol and 170 mL of pyridine, and cool. Place 100mL of pyridine in a 250-mL graduated cylinder, and keepingthe pyridine cold in an ice bath, pass in dry sulfur dioxideuntil the volume reaches 200 mL. Slowly add this solution,with shaking, to the cooled iodine mixture. Shake to dissolvethe iodine, transfer the solution to the apparatus, and allowthe solution to stand overnight before standardizing. One milli-liter of this solution, when freshly prepared, is equivalent toapproximately 5 mg of water, but it deteriorates gradually;therefore, standardize it within 1 h before use, or daily incontinual use. Protect the solution from light while in use.Store any bulk stock of the solution in a suitably sealed,glass-stoppered container, fully protected from light and underrefrigeration.
A commercially available, stabilized solution of a KarlFischer-type reagent may be used. Commercially availablereagents containing solvents or bases other than pyridine and/or alcohols other than methanol also may be used. These maybe single solutions or reagents formed in situ by combiningthe components of the reagents present in two discrete solu-tions. The diluted Karl Fischer Reagent called for in somemonographs should be diluted as directed by the manufacturer.Either methanol, or another suitable solvent such as ethyleneglycol monomethyl ether, may be used as the diluent.
852 / Appendix II / General Tests and Assays FCC V
Test Preparation Unless otherwise specified in the individ-ual monograph, use an accurately weighed or measuredamount of the specimen under test estimated to contain 10 to250 mg of water.
Where the monograph specifies that the specimen undertest is hygroscopic, accurately weigh a sample of the specimeninto a suitable container. Use a dry syringe to inject an appro-priate volume of methanol, or other suitable solvent, accu-rately measured, into the container and shake to dissolve thespecimen. Dry the syringe, and use it to remove the solutionfrom the container and transfer it to a titration vessel preparedas directed under Procedure. Repeat the procedure with asecond portion of methanol, or other suitable solvent, accu-rately measured; add this washing to the titration vessel; andimmediately titrate. Determine the water content, in milli-grams, of a portion of solvent of the same total volume asthat used to dissolve the specimen and to wash the containerand syringe, as directed under Standardization of Water Solu-tion for Residual Titrations, and subtract this value from thewater content, in milligrams, obtained in the titration of thespecimen under test.
Standardization of the Reagent Place enough methanolor other suitable solvent in the titration vessel to cover theelectrodes, and add sufficient Karl Fischer Reagent to givethe characteristic color or 100 � 50 microamperes of directcurrent at about 200 mV of applied potential. Pure methanolcan make the detector overly sensitive, particularly at lowppm levels of water, causing it to deflect to dryness and slowlyrecover with each addition of reagent. This slows down thetitration and may allow the system to actually pick up ambientmoisture during the resulting long titration. Adding chloro-form or a similar nonconducting solvent will retard this sensi-tivity and can improve the analysis.
For determination of trace amounts of water (less than 1%),quickly add 25 �L (25 mg) of pure water, using a 25- or 50-�L syringe, and titrate to the endpoint. The water equivalencefactor F, in milligrams of water per milliliter of reagent, isgiven by the formula
25/V,
in which V is the volume, in milliliters, of the Karl FischerReagent consumed in the second titration.
For the precise determination of significant amounts ofwater (more than 1%), quickly add between 25 and 250 mg(25 to 250 �L) of pure water, accurately weighed by differencefrom a weighing pipet or from a precalibrated syringe ormicropipet, the amount of water used being governed bythe reagent strength and the buret size, as referred to underVolumetric Apparatus. Titrate to the endpoint. Calculate thewater equivalence factor, F, in milligrams of water per millili-ter of reagent by the formula
W/V,
in which W is the weight, in milligrams, of the water, and Vis the volume, in milliliters, of the Karl Fischer Reagentrequired.
Procedure Unless otherwise specified, transfer 35 to 40 mLof methanol or other suitable solvent to the titration vessel,
and titrate with the Karl Fischer Reagent to the electrometricor visual endpoint to consume any moisture that may bepresent. (Disregard the volume consumed because it does notenter into the calculations.) Quickly add the Test Preparation,mix, and again titrate with the Karl Fischer Reagent to theelectrometric or visual endpoint. Calculate the water contentof the specimen, in milligrams, by the formula
SF,
in which S is the volume, in milliliters, of the Karl FischerReagent consumed in the second titration, and F is the waterequivalence factor of the Karl Fischer Reagent.
Method 1b (Residual Titration)
Principle See the information in the section entitled Princi-ple under Method Ia. In the residual titration, add excess KarlFischer Reagent to the test specimen, allow sufficient timefor the reaction to reach completion, and titrate the uncon-sumed Karl Fischer Reagent with a standard solution of waterin a solvent such as methanol. The residual titration procedureis generally applicable and avoids the difficulties that may beencountered in the direct titration of substances from whichthe bound water is released slowly.
Apparatus, Reagent, and Test Preparation Use those inMethod Ia.
Standardization of Water Solution for Residual Titra-tion Prepare a Water Solution by diluting 2 mL of purewater to 1000 mL with methanol or another suitable solvent.Standardize this solution by titrating 25.0 mL with the KarlFischer Reagent, previously standardized as directed underStandardization of the Reagent. Calculate the water content,in milligrams per milliliter, of the Water Solution with theformula
VF/25,
in which V is the volume of the Karl Fischer Reagent con-sumed, and F is the water equivalence factor of the KarlFischer Reagent. Determine the water content of the WaterSolution weekly, and standardize the Karl Fischer Reagentagainst it periodically as needed. Store the Water Solution ina tightly capped container.
Procedure Where the individual monograph specifies thewater content is to be determined by Method Ib, transfer 35to 40 mL of methanol or other suitable solvent into the titrationvessel, and titrate with the Karl Fischer Reagent to the electro-metric or visual endpoint. Quickly add the Test Preparation,mix, and add an accurately measured excess of the KarlFischer Reagent. Allow sufficient time for the reaction toreach completion, and titrate the unconsumed Karl FischerReagent with standardized Water Solution to the electrometricor visual endpoint. Calculate the water content of the speci-men, in milligrams, with the formula
F(X′ − XR),
in which F is the water equivalence factor of the Karl FischerReagent; X′ is the volume, in milliliters, of the Karl Fischer
FCC V General Tests and Assays / Appendix II / 853
Reagent added after introduction of the specimen; X is thevolume, in milliliters, of standardized Water Solution requiredto neutralize the unconsumed Karl Fischer Reagent; and Ris the ratio V/25 (milliliters of Karl Fischer Reagent/millilitersof Water Solution), determined from the Standardization ofWater Solution for Residual Titration.
Method Ic (Coulometric Titration)
Principle Use the Karl Fischer reaction in the coulometricdetermination of water. In this determination, iodine is notadded in the form of a volumetric solution, but is producedin an iodide-containing solution by anodic oxidation. Thereaction cell usually consists of a large anode compartmentand a small cathode compartment that are separated by adiaphragm. Other suitable types of reaction cells (e.g., withoutdiaphragms) may be used. Each compartment has a platinumelectrode that conducts current through the cell. Iodine, whichis produced at the anode electrode, immediately reacts withthe water present in the compartment. When all the water hasbeen consumed, an excess of iodine occurs, which can bedetected potentiometrically, thus indicating the endpoint. Pre-electrolysis, which can take several hours, eliminates moisturefrom the system. Therefore, changing the Karl Fischer Re-agent after each determination is not practical. Individualdeterminations may be carried out in succession in the samereagent solution. A requirement for this method is that eachcomponent of the test specimen be compatible with the othercomponents and that no side reactions take place. Samplesmay be transferred into the vessel as solids or as solutionsby means of injection through a septum. Gases can be intro-duced into the cell by means of a suitable gas inlet tube. Forthe water determination of solids, another common techniqueis to dissolve the solid in a suitable solvent and then inject aportion of this solution into the cell. In the case of insolublesolids, water may be extracted using suitable solvents, andthen the extracts injected into the coulometric cell. Alterna-tively, an evaporation technique may be used in which thesample is heated in a tube and the water is evaporated andcarried into the cell by means of a stream of dry, inert gas.Precision in the method is predominantly governed by theextent to which atmospheric moisture is excluded from thesystem. Control of the system may be monitored by measuringthe amount of baseline drift. The titration of water in solidtest specimens is usually carried out with the use of anhydrousmethanol as the solvent. Other suitable solvents may be usedfor special or unusual test specimens. This method is particu-larly suited to chemically inert substances such as hydrocar-bons, alcohols, and ethers. In comparison with the volumetricKarl Fischer titration, coulometry is a micro-method. Themethod uses extremely small amounts of current. It is predom-inantly used for substances with a very low water content(0.1% to 0.0001%).
Apparatus Any commercially available apparatus con-sisting of an absolutely tight system fitted with the necessaryelectrodes and a magnetic stirrer is appropriate. The instru-ment’s microprocessor controls the analytical procedure anddisplays the results. Calibration of the instrument is not neces-
sary as the current consumed can be measured absolutely.Proper operation of the instrument can be confirmed by in-jecting 1 �L of water into the vessel. The instrument shouldread 1000 �g of water on reaching the endpoint.
Reagent See Reagent under Method Ia.
Test Preparation Using a dry syringe, inject an appropriatevolume of test specimen estimated to contain 0.5 to 5 mg ofwater, accurately measured, into the anolyte solution. Thesample may also be introduced as a solid, accurately weighed,into the anolyte solution. Perform coulometric titration, anddetermine the water content of the specimen under test.
Alternatively, when the specimen is a suitable solid, dis-solve an appropriate quantity, accurately weighed, in anhy-drous methanol or another suitable solvent, and inject a suit-able portion into the anolyte solution.
When the specimen is an insoluble solid, extract the waterby using a suitable anhydrous solvent from which an appro-priate quantity, accurately weighed, may be injected into theanolyte solution. Alternatively use an evaporation technique.
Procedure Quickly inject the Test Preparation, or transferthe solid sample, into the anolyte, mix, and perform the coulo-metric titration to the electrometric endpoint. Read the watercontent of the Test Preparation directly from the instrument’sdisplay, and calculate the percent that is present in the sub-stance.
Method II (Toluene Distillation Method)
Principle This method determines water by distillation ofa sample with an immiscible solvent, usually toluene.
Apparatus Use a glass distillation apparatus (see Fig. 8)provided with 24/40 ground-glass connections. The compo-nents consist of a 500-mL short-neck, round-bottom flaskconnected by means of a trap to a 400-mm water-cooledcondenser. The lower tip of the condenser should be about 7mm above the surface of the liquid in the trap after distillationconditions have been established (see Procedure).
The trap should be constructed of well-annealed glass, thereceiving end of which is graduated to contain 5 mL andsubdivided into 0.1-mL divisions, with each 1-mL line num-bered from 5 mL beginning at the top. Calibrate the receiverby adding 1 mL of water, accurately measured, to 100 mLof toluene contained in the distillation flask. Conduct thedistillation, and calculate the volume of water obtained asdirected in the Procedure. Add another milliliter of water tothe cooled apparatus, and repeat the distillation. Continue inthis manner until five 1-mL portions of water have been added.The error at any indicated capacity should not exceed 0.05mL. The source of heat is either an oil bath or an electricheater provided with a suitable means of temperature control.The distillation may be better controlled by insulating the tubeleading from the flask to the receiver. It is also advantageous toprotect the flask from drafts. Clean the entire apparatus withpotassium dichromate−sulfuric acid cleaning solution, rinsethoroughly, and dry completely before using.
854 / Appendix II / General Tests and Assays FCC V
FIGURE 8 Moisture Distillation Apparatus.
Procedure Place in the previously cleaned and dried flaska quantity of the substance, weighed accurately to the nearest0.01 g, that is expected to yield from 1.5 to 4 mL of water.If the substance is of a pastelike consistency, weigh it in aboat of metal foil that will pass through the neck of the flask.If the substance is likely to cause bumping, take suitableprecautions to prevent it. Transfer about 200 mL of ACSreagent-grade toluene into the flask, and swirl to mix it withthe sample. Assemble the apparatus, fill the receiver withtoluene by pouring it through the condenser until it begins tooverflow into the flask, and insert a loose cotton plug in thetop of the condenser. Heat the flask so that the distillationrate will be about 200 drops/min, and continue distilling untilthe volume of water in the trap remains constant for 5 min.Discontinue the heating, use a copper or nichrome wire spiralto dislodge any drops of water that may be adhering to theinside of the condenser tube or receiver, and wash down withabout 5 mL of toluene. Disconnect the receiver, immerse itin water at 25° for at least 15 min or until the toluene layeris clear, and then read the volume of water. Conduct a blankdetermination using the same volume of toluene as used whendistilling the sample mixture, and make any necessary correc-tion (see General Provisions).
C. OTHERS
ASH (Acid-Insoluble)
Boil the ash obtained as directed under Ash (Total), below,with 25 mL of 2.7 N hydrochloric acid for 5 min, collect theinsoluble matter on a tared, porous-bottom porcelain filtercrucible or ashless filter, wash it with hot water, ignite toconstant weight at 675° � 25°, and weigh. Calculate thepercent acid-insoluble ash from the weight of the sampletaken.
Note: Avoid exposing the crucible to sudden tempera-ture changes.
ASH (Total)
Unless otherwise directed, accurately weigh about 3 g of thesample in a tared crucible, ignite it at a low temperature (about550°), not to exceed a very dull redness, until it is free fromcarbon, cool it in a desiccator, and weigh. If a carbon-freeash is not obtained, wet the charred mass with hot water,collect the insoluble residue on an ashless filter paper, andignite the residue and filter paper until the ash is white ornearly so. Finally, add the filtrate, evaporate it to dryness,and heat the whole to a dull redness. If a carbon-free ash isstill not obtained, cool the crucible, add 15 mL of ethanol,break up the ash with a glass rod, then burn off the ethanol,again heat the whole to a dull redness, cool it in a desiccator,and weigh.
HYDROCHLORIC ACID TABLE
Percent Percent°Bé Sp. Gr. HCl °Bé Sp. Gr. HCl
1.00 1.0069 1.40 7.25 1.0526 10.552.00 1.0140 2.82 7.50 1.0545 10.943.00 1.0211 4.25 7.75 1.0564 11.324.00 1.0284 5.69 8.00 1.0584 11.715.00 1.0357 7.15 8.25 1.0603 12.095.25 1.0375 7.52 8.50 1.0623 12.485.50 1.0394 7.89 8.75 1.0642 12.875.75 1.0413 8.26 9.00 1.0662 13.266.00 1.0432 8.64 9.25 1.0681 13.656.25 1.0450 9.02 9.50 1.0701 14.046.50 1.0469 9.40 9.75 1.0721 14.436.75 1.0488 9.78 10.00 1.0741 14.837.00 1.0507 10.17 10.25 1.0761 15.22
FCC V General Tests and Assays / Appendix II / 855
Percent Percent°Bé Sp. Gr. HCl °Bé Sp. Gr. HCl
10.50 1.0781 15.62 21.7 1.1760 34.6410.75 1.0801 16.01 21.8 1.1770 34.8311.00 1.0821 16.41 21.9 1.1779 35.0211.25 1.0841 16.81 22.0 1.1789 35.2111.50 1.0861 17.21 22.1 1.1798 35.4011.75 1.0881 17.61 22.2 1.1808 35.5912.00 1.0902 18.01 22.3 1.1817 35.7812.25 1.0922 18.41 22.4 1.1827 35.9712.50 1.0943 18.82 22.5 1.1836 36.1612.75 1.0964 19.22 22.6 1.1846 36.3513.00 1.0985 19.63 22.7 1.1856 36.5413.25 1.1006 20.04 22.8 1.1866 36.7313.50 1.1027 20.44 22.9 1.1875 36.9313.75 1.1048 20.86 23.0 1.1885 37.1419.2 1.1526 30.00 23.1 1.1895 37.3619.3 1.1535 30.18 23.2 1.1904 37.5819.4 1.1544 30.35 23.3 1.1914 37.8019.5 1.1554 30.53 23.4 1.1924 38.0319.6 1.1563 30.71 23.5 1.1934 38.2619.7 1.1572 30.90 23.6 1.1944 38.4919.8 1.1581 31.08 23.7 1.1953 38.7219.9 1.1590 31.27 23.8 1.1963 38.9520.0 1.1600 31.45 23.9 1.1973 39.1820.1 1.1609 31.64 24.0 1.1983 39.4120.2 1.1619 31.82 24.1 1.1993 39.6420.3 1.1628 32.01 24.2 1.2003 39.8620.4 1.1637 32.19 24.3 1.2013 40.0920.5 1.1647 32.38 24.4 1.2023 40.3220.6 1.1656 32.56 24.5 1.2033 40.5520.7 1.1666 32.75 24.6 1.2043 40.7820.8 1.1675 32.93 24.7 1.2053 41.0120.9 1.1684 33.12 24.8 1.2063 41.2421.0 1.1694 33.31 24.9 1.2073 41.4821.1 1.1703 33.50 25.0 1.2083 41.7221.2 1.1713 33.69 25.1 1.2093 41.9921.3 1.1722 33.88 25.2 1.2103 42.3021.4 1.1732 34.07 25.3 1.2114 42.6421.5 1.1741 34.26 25.4 1.2124 43.0121.6 1.1751 34.45 25.5 1.2134 43.40
Source: Courtesy of the Manufacturing Chemists’ Association of theUnited States.
Specific gravity determinations were made at 60°F, comparedwith water at 60°F.
From the specific gravities, the corresponding degreesBaumé were calculated by the following formula:
degrees Baumé = 145 − (145/sp. gr.).
Baumé hydrometers for use with this table must be gradua-ted by the above formula, which should always be printed onthe scale.
Allowance for Temperature10° to 15°Bé: 1/40 °Bé or 0.0002 sp. gr. for 1°F15° to 22°Bé: 1/30 °Bé or 0.0003 sp. gr. for 1°F22° to 25°Bé: 1/28 °Bé or 0.00035 sp. gr. for 1°F
LOSS ON DRYING
This procedure is used to determine the amount of volatile mat-ter expelled under the conditions specified in the monograph.Because the volatile matter may include material other than ad-sorbed moisture, this test is designed for compounds in whichthe loss on drying may not definitely be attributable to wateralone. For substances appearing to contain water as the onlyvolatile constituent, the Direct (Karl Fischer) TitrationMethod, provided under Water, Appendix IIB, is usually appro-priate.
Procedure Unless otherwisedirected in themonograph,con-duct the determination on 1 to 2 g of the substance, previouslymixed and accurately weighed. If the sample is in the form oflarge crystals, reduce the particle size to about 2 mm, quicklycrushing the sample to avoid absorption or loss of moisture.Tare a glass-stoppered, shallow weighing bottle that has beendried for 30 min under the same conditions to be used in thedetermination. Transfer the sample to the bottle, replace thecover, and weigh the bottle and its contents. By gentle sidewaysshaking, distribute the sample as evenly as possible to a depthof about 5 mm for most substances and not over 10 mm in thecase of bulky materials. Place the loaded bottle in the dryingchamber, removing the stopper and leaving it also in the cham-ber, and dry at the temperature and for the length of time speci-fied in the monograph. Upon opening the chamber, close thebottle promptly and allow it to come to room temperature, pref-erably in a desiccator, before weighing.
Where drying in vacuum is specified in the monograph,use a pressure as low as that obtainable by an aspirating waterpump (not higher than 20 mm Hg).
If the test substance melts at a temperature lower than thatspecified for the determination, preheat the bottle and itscontents for 1 to 2 h at a temperature 5° to 10° below themelting range, then continue drying at the specified tempera-ture for the determination. When drying the sample in a desic-cator, ensure that the desiccant is kept fully effective byreplacing it frequently.
OIL CONTENT OF SYNTHETICPARAFFIN
ApparatusFilter Stick Use either a 10-mm diameter sintered-glass
filter stick of 10- to 15-�m maximum pore diameter, or afilter stick made of stainless steel and having a 0.5-in. diskof 10- to 15-�m maximum pore diameter. Determine confor-mance with the pore diameter specified as follows: Cleansintered-glass filter sticks by soaking in hydrochloric acid, orstainless steel sticks by soaking in nitric acid, wash with water,rinse with acetone, and dry in air followed by drying in anoven at 105° for 30 min.
856 / Appendix II / General Tests and Assays FCC V
FIGURE 9 Assembly for Checking Pore Diameter of FilterSticks.
Thoroughly wet the clean filter stick by soaking in water,and then connect it with an apparatus (see Fig. 9) consistingof a mercury-filled manometer, readable to 0.5 mm; a cleanand filtered air supply; a drying bulb filled with silica gel;and a needle-valve type air pressure regulator. Apply pressureslowly from the air source, and immerse the filter just belowthe surface of water contained in a beaker.
Note: If a head of liquid is noted above the surface ofthe filter after it is inserted into the water, the backpressure thus produced should be subtracted from theobserved pressure when the pore diameter is calculatedas directed below.
Increase the air pressure to 10 mm below the acceptablepressure limit, and then increase the pressure at a slow, uni-form rate of about 3 mm Hg per minute until the first bubblepasses through the filter. This can be conveniently observedby placing the beaker over a mirror. Read the manometerwhen the first bubble passes off the underside of the filter.Calculate the pore diameter, in micrometers, by the formula
2180/p,
in which p is the observed pressure, in millimeters, correctedfor any back pressure as mentioned above.
Filtration Assembly Connect the Filter Stick with an airpressure inlet tube and delivery nozzle and ground-glass jointto fit a 25- × 170-mm test tube as shown in Fig. 10. If astainless steel Filter Stick is used, make the connection to thetest tube by means of a cork.
Cooling Bath Use a suitable insulated box having 1-in.holes in the center to accommodate any desired number oftest tubes. The bath may be filled with a suitable mediumsuch as kerosene and may be cooled by circulating a refrigerantthrough coils, or by using solid carbon dioxide, to produce atemperature of 30° � 2°F.
Air Pressure Regulator Use a suitable pressure-reductionvalve, or other suitable regulator, that will supply air to theFiltration Assembly at the volume and pressure required togive an even flow of filtrate (see Procedure). Connect the
regulator with rubber tubing to the end of the Filter Stick inthe Filtration Assembly.
Thermometer Use an ASTM Oil in Wax Thermometerhaving the range of −35° to +70°F and conforming to therequirements for an ASTM 71F thermometer (see Thermome-ters, Appendix I).
Weighing Bottles Use glass-stoppered conical bottles hav-ing a capacity of 15 mL. The bottles are used as evaporatingflasks in the Procedure.
Evaporation Assembly The assembly consists of an evap-orating cabinet capable of maintaining a temperature of 95°� 2°F around the evaporation flasks, and air jets (4 � 0.2mm id) for delivering a stream of clean, dry air verticallydownward into the flasks. In the Procedure below, supporteach jet so that the tip is 15 � 5 mm above the surface of theliquid at the start of the evaporation. Supply the air (purified bypassage through a tube of 1-cm bore packed loosely to aheight of 20 cm with absorbent cotton) at the rate of 2 to 3L/min per jet. The cleanliness of the air should be checkedperiodically to ensure that not more than 0.1 mg of residueis obtained when 4 mL of methyl ethyl ketone is evaporatedas directed in the Procedure.
Wire Stirrer Use a 250-mm length of stiff iron or ni-chrome wire of about No. 20 B & S gauge. Form a 10-mmdiameter loop at each end, and bend the loop at the bottomend so that the plane of the loop is perpendicular to the lengthof the wire.
Sample Selection If the sample weighs about 1 kg or less,obtain a representative portion by melting the entire sample
FIGURE 10 Filtration Assembly for Determination of OilContent.
FCC V General Tests and Assays / Appendix II / 857
and stirring thoroughly. For samples heavier than about 1kg, exercise special care to ensure that a truly representativeportion is obtained, noting that the oil may not be distributeduniformly throughout the sample and that mechanical opera-tions may have expressed some of the oil.
Procedure Melt a representative portion of the sample ina beaker, using a water bath or oven maintained at 160° to210°F. As soon as the sample is completely melted, thoroughlymix it by stirring. Preheat a dropper pipet, provided with arubber bulb and calibrated to deliver 1 � 0.05 g of moltensample, and withdraw a 1-g portion of the sample as soon aspossible after it has melted. Hold the pipet in a vertical posi-tion, and carefully transfer its contents into a clean, dry testtube previously weighed to the nearest milligram. Evenly coatthe bottom of the tube by swirling, allow the tube to cool,and weigh to the nearest milligram. Calculate the sampleweight, in grams, and record it as B (see Calculation). Pipet15 mL of methyl ethyl ketone (ASTM Specification D 740,or equivalent) into the tube, and immerse the tube up to thetop of the liquid in a hot water or steam bath. Stir with anup-and-down motion with the wire stirrer, and continue heat-ing and stirring until a homogeneous solution is obtained,exercising care to avoid loss of solvent by prolonged boiling.
Note: If it appears that a clear solution will not beobtained, stir until any undissolved material is welldispersed so as to produce a slightly cloudy solution.
After the sample solution is prepared, plunge the test tubeinto an 800-mL beaker of ice water, and continue to stir untilthe contents are cold. Remove the stirrer, then remove thetest tube from the bath, dry the outside of the tube with acloth, and weigh to the nearest 100 mg. Calculate the weight,in grams, of solvent in the test tube, and record it as C (seeCalculation). Place the tube in the cooling bath, maintainedat −30° � 2°F, and stir continuously with the thermometeruntil the temperature reaches −25° � 0.5°F, maintaining theslurry at a uniform consistency and taking precautions toprevent the sample from setting up on the walls of the tubeor forming crystals.
Place the filter stick in a test tube and cool at −30° � 2°Fin the cooling bath for a minimum of 10 min. Immerse thecooled filter stick in the sample, then connect the filtrationassembly, seating the ground-glass joint of the filter so as tomake an airtight seal. Placed an unstoppered weighing bottle,previously weighed together with the glass stopper to thenearest 0.1 mg, under the delivery nozzle of the filtrationassembly.
Note: Suitable precautions and proper analytical tech-nique should be applied to ensure the accuracy of theweight of the bottle. Before determining its weight, thebottle and its stopper should have been cleaned anddried, then rinsed with methyl ethyl ketone, wiped dryon the outside, dried in the evaporation assembly forabout 5 min, and cooled. Then allow it to stand forabout 10 min near the balance before weighing.
Apply air pressure to the filtration assembly, immediatelycollect about 4 mL of filtrate in the weighing bottle, and
release the air pressure to permit the liquid to drain backslowly from the delivery nozzle. Stopper the bottle, andweigh it to the nearest 10 mg without waiting for it tocome to room temperature. Remove the stopper, transferthe bottle to the evaporation assembly maintained at 95°� 2°F, and place it under an air jet centered inside theneck, with the tip 15 � 5 mm above the surface of theliquid. After the solvent has evaporated (usually less than30 min), stopper the bottle, and allow it to stand near thebalance for about 10 min before it is weighed to the nearest0.1 mg. Repeat the evaporation procedure for 5-min periodsuntil the loss between successive weighings is not morethan 0.2 mg. Determine the weight of the oil residue, ingrams, by subtracting the weight of the empty stopperedbottle from the weight of the stoppered bottle plus the oilresidue after the evaporation procedure, and record theresults as A (see Calculation). Determine the weight ofsolvent evaporated, in grams, by subtracting the weight ofthe bottle plus oil residue from the weight of the bottleplus filtrate, and record the result as D (see Calculation).
Calculation Calculate the percent, by weight, of oil in thesample by the formula
(100 AC/BD) − 0.15,
in which 0.15 is a factor to correct for solubility of the samplein the solvent at −25°F.
RESIDUE ON IGNITION (Sulfated Ash)
Method I (for Solids)Transfer the quantity of the sample directed in the individualmonograph onto a tared 50- to 100-mL platinum dish or othersuitable container, and add sufficient 2 N sulfuric acid tomoisten the entire sample. Heat gently, using a hot plate, anArgand burner, or an infrared heat lamp, until the sample isdry and thoroughly charred, then continue heating until all ofthe sample has been volatilized or nearly all of the carbonhas been oxidized, and cool. Moisten the residue with 0.1 mLof sulfuric acid, and heat in the same manner until the remain-der of the sample and any excess sulfuric acid have beenvolatilized. To promote volitilization of sulfuric acid, add afew pieces of ammonium carbonate just before completingignition. Finally, ignite to constant weight in a muffle furnaceat 800° � 25° for 15 min, or longer if necessary to completeignition, cool in a desiccator, and weigh.
Method II (for Liquids)Unless otherwise directed, transfer the required weight of thesample onto a tared 75- to 100-mL platinum dish. Heat gently,using an Argand or Meker burner, until the sample ignites,then allow the sample to burn until it self-extinguishes. Cool,then wet the residue with 2 mL of concentrated sulfuric acid,and heat the sample over a low flame until dry. Ignite to
858 / Appendix II / General Tests and Assays FCC V
constant weight in a muffle furnace at 800° � 25° for 30min, or longer if necessary for complete ignition, cool in adesiccator, and weigh.
SIEVE ANALYSIS OF GRANULARMETAL POWDERS(Based on ASTM Designation: B 214)
Apparatus
Sieves Use a set of standard sieves, ranging from 80-mesh to 325-mesh, conforming to the specifications in ASTMDesignation: E 11 (Sieves for Testing Purposes).
Sieve Shaker Use a mechanically operated sieve shakerthat imparts to the set of sieves a horizontal rotary motion ofbetween 270 and 300 rotations/min and a tapping action ofbetween 140 and 160 taps/min. The sieve shaker is fitted witha plug to receive the impact of the tapping device. The entireapparatus is rigidly mounted—bolted to a solid foundation,preferably of concrete. Preferably a time switch is providedto ensure the accuracy of test duration.
Procedure Assemble the sieves in consecutive order byopening size, with the coarsest sieve (80-mesh) at the top,and place a solid-collecting pan below the bottom sieve (325-mesh). Place 100.0 g of the test sample, W, on the top sieve,and close the sieve with a solid cover. Securely fasten theassembly to the sieve shaker, and operate the shaker for 15min. Remove the most coarse sieve from the nest, gently tapits contents to one side, and pour the contents onto a tared,glazed paper. Using a soft brush, transfer onto the next finersieve any material adhering to the bottom of the sieve andframe. Place the sieve just removed upside down on the papercontaining the retained portion, and tap the sieve. Accuratelyweigh the paper and its contents, and record the net weightof the fraction, F, obtained. Repeat this process for each sievein the nest and for the portion of the sample that has beencollected in the bottom pan. Record the total of the fractionsretained on the sieves as T and that portion collected in thepan as t. The combined total, S, of T + t is the amount of thesample, W, recovered in the test. Calculate the percent recov-ery by the formula
S/W × 100.
If the percent recovery is less than 99.0%, check the conditionof the sieves and for possible errors in weighing, and repeatthe test. If the percent recovery is not less than 99.0%, calculatethe percent retained on each sieve by the formula
F/W × 100.
Calculate the percent through the smallest mesh sieve fromthe portion collected in the pan by the formula
[(100 − t)/W] × 100.
SULFURIC ACID TABLE
Percent Percent°Bé Sp. Gr. H2SO4 °Bé Sp. Gr. H2SO4
0 1.0000 0.00 36 1.3303 42.631 1.0069 1.02 37 1.3426 43.992 1.0140 2.08 38 1.3551 45.353 1.0211 3.13 39 1.3679 46.724 1.0284 4.21 40 1.3810 48.105 1.0357 5.28 41 1.3942 49.476 1.0432 6.37 42 1.4078 50.877 1.0507 7.45 43 1.4216 52.268 1.0584 8.55 44 1.4356 53.669 1.0662 9.66 45 1.4500 55.0710 1.0741 10.77 46 1.4646 56.4811 1.0821 11.89 47 1.4796 57.9012 1.0902 13.01 48 1.4948 59.3213 1.0985 14.13 49 1.5104 60.7514 1.1069 15.25 50 1.5263 62.1815 1.1154 16.38 51 1.5426 63.6616 1.1240 17.53 52 1.5591 65.1317 1.1328 18.71 53 1.5761 66.6318 1.1417 19.89 54 1.5934 68.1319 1.1508 21.07 55 1.6111 69.6520 1.1600 22.25 56 1.6292 71.1721 1.1694 23.43 57 1.6477 72.7522 1.1789 24.61 58 1.6667 74.3623 1.1885 25.81 59 1.6860 75.9924 1.1983 27.03 60 1.7059 77.6725 1.2083 28.28 61 1.7262 79.4326 1.2185 29.53 62 1.7470 81.3027 1.2288 30.79 63 1.7683 83.3428 1.2393 32.05 64 1.7901 85.6629 1.2500 33.33 64.25 1.7957 86.3330 1.2609 34.63 64.50 1.8012 87.0431 1.2719 35.93 64.75 1.8068 87.8132 1.2832 37.26 65 1.8125 88.6533 1.2946 38.58 65.25 1.8182 89.5534 1.3063 39.92 65.50 1.8239 90.6035 1.3182 41.27 66 1.8354 93.19
Source: Courtesy of the Manufacturing Chemists’ Association of theUnited States.
Specific gravity determinations were made at 60°F, comparedwith water at 60°F. The values given above for aqueoussulfuric acid solutions were adopted as standard in 1904 bythe Manufacturing Chemists’ Association of the United States.
From the specific gravities, the corresponding degreesBaumé were calculated by the following equation:
°Baumé = 145 − (145/sp. gr.).
Baumé hydrometers for use with this table must be gradua-ted by the above formula, which should always be printedon the scale. Acids stronger than 66°Bé should have theirpercentage compositions determined by chemical analysis.
FCC V General Tests and Assays / Appendix III / 859
APPENDIX III: CHEMICAL TESTS AND DETERMINATIONS
A. IDENTIFICATION TESTS
The identification tests described in Section A of this Appendixare frequently referred to in the Food Chemicals Codex forthe presumptive identification of FCC-grade chemicals takenfrom labeled containers. These tests are not intended to beapplied to mixtures unless so specified.
AcetateAcetic acid or acetates, when warmed with sulfuric acid andalcohol, form ethyl acetate, recognizable by its characteristicodor. With neutral solutions of acetates, ferric chloride TSproduces a deep red color that is destroyed by the additionof a mineral acid.
AluminumSolutions of aluminum salts yield with 6 N ammonia a white,gelatinous precipitate that is insoluble in an excess of the 6N ammonia. The same precipitate is produced by 1 N sodiumhydroxide, but it dissolves in an excess of this reagent.
AmmoniumAmmonium salts are decomposed by 1 N sodium hydroxidewith the evolution of ammonia, recognizable by its alkalineeffect on moistened red litmus paper. The decomposition isaccelerated by warming.
BenzoateNeutral solutions of benzoates yield a salmon coloredprecipitate with ferric chloride TS. From moderately concen-trated solutions of benzoate, 2 N sulfuric acid precipitates freebenzoic acid, which is readily soluble in ether.
BicarbonateSee Carbonate.
BisulfiteSee Sulfite.
BromideFree bromine is liberated from solutions of bromides uponthe dropwise addition of chlorine TS. When shaken withchloroform, the bromine dissolves, coloring the chloroformred to red-brown. A yellow-white precipitate, which is insolu-ble in nitric acid and slightly soluble in 6 N ammonia, isproduced when solutions of bromides are treated with silvernitrate TS.
CalciumInsoluble oxalate salts are formed when solutions of calciumsalts are treated in the following manner: Using 2 drops ofmethyl red TS as the indicator, neutralize a 1:20 solution ofa calcium salt with 6 N ammonia, then add 2.7 N hydrochloric
acid, dropwise, until the solution is acid. A white precipitateof calcium oxalate forms upon the addition of ammoniumoxalate TS. This precipitate is insoluble in acetic acid butdissolves in hydrochloric acid.
Calcium salts moistened with hydrochloric acid impart atransient yellow-red color to a nonluminous flame.
CarbonateCarbonates and bicarbonates effervesce with acids, yieldinga colorless gas that produces a white precipitate immediatelywhen passed into calcium hydroxide TS. Cold solutions ofsoluble carbonates are colored red by phenolphthalein TS,whereas solutions of bicarbonates remain unchanged or areslightly changed.
ChlorideSolutions of chlorides yield with silver nitrate TS a white,curdy precipitate that is insoluble in nitric acid but soluble ina slight excess of 6 N ammonia.
CitrateTo 15 mL of pyridine add a few milligrams of a citrate salt,dissolved or suspended in 1 mL of water, and shake. Add 5mL of acetic anhydride to this mixture, and shake. A lightred color appears.
CobaltSolutions of cobalt salts (1:20) in 2.7 N hydrochloric acidyield a red precipitate when heated on a steam bath with anequal volume of a hot, freshly prepared 1:10 solution of 1-nitroso-2-naphthol in 9 N acetic acid. Solutions of cobaltsalts yield a yellow precipitate when saturated with potassiumchloride and treated with potassium nitrite and acetic acid.
CopperWhen solutions of cupric compounds are acidified withhydrochloric acid, a red film of metallic copper is depositedon a bright untarnished surface of metallic iron. An excessof 6 N ammonia, added to a solution of a cupric salt, producesfirst a blue precipitate and then a deep blue colored solution.Solutions of cupric salts yield with potassium ferrocyanideTS a red-brown precipitate, insoluble in diluted acids.
HypophosphiteHypophosphites evolve spontaneously flammable phosphinewhen strongly heated. Solutions of hypophosphites yield awhite precipitate with mercuric chloride TS. This precipitatebecomes gray when an excess of hypophosphite is present.Hypophosphite solutions, acidified with sulfuric acid andwarmed with copper sulfate TS, yield a red precipitate.
IodideSolutions of iodides, upon the dropwise addition of chlorineTS, liberate iodine, which colors the solution yellow to red.Chloroform is colored violet when shaken with this solution.
860 / Appendix III / General Tests and Assays FCC V
The iodine thus liberated gives a blue color with starch TS.In solutions of iodides, silver nitrate TS produces a yellow,curdy precipitate that is insoluble in nitric acid and in 6 Nammonia.
IronSolutions of ferrous and ferric compounds yield a blackprecipitate with ammonium sulfide TS. This precipitate isdissolved by cold 2.7 N hydrochloric acid with the evolutionof hydrogen sulfide.
Ferric Salts Potassium ferrocyanide TS (10%) producesa dark blue precipitate in acid solutions of ferric salts. Withan excess of 1 N sodium hydroxide, a red-brown precipitateis formed. Solutions of ferric salts produce with ammoniumthiocyanate TS (1.0 N) a deep red color that is not destroyedby diluted mineral acids.
Ferrous Salts Potassium ferricyanide TS (10%) producesa dark blue precipitate in solutions of ferrous salts. This precip-itate, which is insoluble in dilute hydrochloric acid, is decom-posed by 1 N sodium hydroxide. Solutions of ferrous saltsyield with 1 N sodium hydroxide a green-white precipitate,the color rapidly changing to green and then to brown whenshaken.
LactateWhen solutions of lactates are acidified with sulfuric acid,potassium permanganate TS (0.1 N) is added, and the mixtureis heated, acetaldehyde is evolved. This can be detected byallowing the vapor to come into contact with a filter paperthat has been moistened with a freshly prepared mixture ofequal volumes of 20% aqueous morpholine and sodiumnitroferricyanide TS. A blue color is produced.
MagnesiumSolutions of magnesium salts in the presence of ammoniumchloride yield no precipitate with ammonium carbonate TS,but a white crystalline precipitate, which is insoluble in 6 Nammonium hydroxide, is formed on the subsequent additionof sodium phosphate TS (6%).
ManganeseSolutions of manganous salts yield with ammonium sulfideTS a salmon colored precipitate that dissolves in acetic acid.
NitrateWhen a solution of a nitrate is mixed with an equal volumeof sulfuric acid, the mixture cooled, and a solution of ferroussulfate superimposed, a brown color is produced at thejunction of the two liquids. Brown-red fumes are evolvedwhen a nitrate is heated with sulfuric acid and metallic copper.Nitrates do not decolorize acidified potassium permanganateTS (0.1 N) (distinction from nitrites).
NitriteNitrites yield brown-red fumes when treated with dilutedmineral acids or acetic acid. A few drops of potassium iodideTS (15%) and a few drops of 2 N sulfuric acid added to asolution of nitrite liberate iodine, which colors starch TS blue.
PeroxideSolutions of peroxides slightly acidified with sulfuric acidyield a deep blue color on the addition of potassiumdichromate TS. On shaking the mixture with an equal volumeof diethyl ether and allowing the liquids to separate, the bluecolor is transferred to the ether layer.
PhosphateNeutral solutions of orthophosphates yield with silver nitrateTS (0.1 N) a yellow precipitate, which is soluble in 1.7 Nnitric acid or in 6 N ammonium hydroxide. With ammoniummolybdate TS, a yellow precipitate, which is soluble in 6 Nammonium hydroxide, is formed.
PotassiumPotassium compounds impart a violet color to a nonluminousflame if not masked by the presence of small quantities ofsodium. In neutral, concentrated or moderately concentratedsolutions of potassium salts, sodium bitartrate TS (10%)slowly produces a white, crystalline precipitate that is solublein 6 N ammonium hydroxide and in solutions of alkali hydrox-ides or carbonates. The precipitation may be accelerated bystirring or rubbing the inside of the test tube with a glass rodor by the addition of a small amount of glacial acetic acid oralcohol.
SodiumDissolve 0.1 g of the sodium compound in 2 mL of water.Add 2 mL of 15% potassium carbonate, and heat to boiling.No precipitate is formed. Add 4 mL of potassium pyroantimo-nate TS, and heat to boiling. Allow to cool in ice water, andif necessary, rub the inside of the test tube with a glass rod.A dense precipitate is formed. Sodium compounds impart anintense yellow color to a nonluminous flame.
SulfateSolutions of sulfates yield with barium chloride TS (10%) awhite precipitate that is insoluble in hydrochloric and nitricacids. Sulfates yield with lead acetate TS (8%) a whiteprecipitate that is soluble in ammonium acetate solution.Hydrochloric acid produces no precipitate when added tosolutions of sulfates (distinction from thiosulfates).
SulfiteWhen treated with 2.7 N hydrochloric acid, sulfites and bisul-fites yield sulfur dioxide, recognizable by its characteristicodor. This gas blackens filter paper moistened with mercurousnitrate TS.
TartrateWhen a few milligrams of a tartrate are added to a mixtureof 15 mL of pyridine and 5 mL of acetic anhydride, an emeraldgreen color is produced.
ThiosulfateWith hydrochloric acid, solutions of thiosulfates yield a whiteprecipitate that soon turns yellow, liberating sulfur dioxide,recognizable by its odor. The addition of ferric chloride TS
FCC V General Tests and Assays / Appendix III / 861
FIGURE 11 General Apparatus for Arsenic Limit Test.(Courtesy of the Fisher Scientific Co., Pittsburgh, PA.)
to solutions of thiosulfates produces a dark violet color thatquickly disappears.
ZincZinc salts, in the presence of sodium acetate, yield a whiteprecipitate with hydrogen sulfide. This precipitate, which isinsoluble in acetic acid, is dissolved by 2.7 N hydrochloricacid. A similar precipitate is produced by ammonium sulfideTS in neutral or alkaline solutions. Solutions of zinc salts yieldwith potassium ferrocyanide TS (10%) a white precipitate thatis insoluble in 2.7 N hydrochloric acid.
B. LIMIT TESTS
ARSENIC LIMIT TEST
Silver Diethyldithiocarbamate Colorimetric Method
Note: All reagents used in this test should be very lowin arsenic content.
Apparatus Use the general apparatus shown in Fig. 11 un-less otherwise specified in an individual monograph. It con-sists of a 125-mL arsine generator flask (a) fitted with ascrubber unit (c) and an absorber tube (e), with a 24/40 stan-dard-taper joint (b) and a ball-and-socket joint (d), securedwith a No. 12 clamp, connecting the units. The tubing betweend and e and between d and c is a capillary having an id of 2mm and an od of 8 mm. Alternatively, an apparatus embodyingthe principle of the general assembly described and illustratedmay be used.
FIGURE 12 Modified Bethge Apparatus for the Distillation ofArsenic Tribromide.
Note: The special assemblies shown in Figs. 12, 13,and 14 are to be used only when specified in certainmonographs.
Standard Arsenic Solution Accurately weigh 132.0 mg ofarsenic trioxide that has been previously dried at 105° for 1h, and dissolve it in 5 mL of a 1:5 sodium hydroxide solution.Neutralize the solution with 2 N sulfuric acid, add 10 mL inexcess, and dilute to 1000.0 mL with recently boiled water.Transfer 10.0 mL of this solution into a 1000-mL volumetricflask, add 10 mL of 2 N sulfuric acid, dilute to volume withrecently boiled water, and mix. Use this final solution, whichcontains 1 �g of arsenic in each milliliter, within 3 days.
Silver Diethyldithiocarbamate Solution Dissolve 1 g ofACS reagent-grade silver diethyldithiocarbamate in 200 mLof recently distilled pyridine. Store this solution in a light-resistant container and use within 1 month.
FIGURE 13 Special Apparatus for the Distillation of ArsenicTrichloride. (Flask A contains 150 mL of hydrochloric acid; flasksD and F contain 20 mL of water. Flask D is placed in an icewater bath, E.)
862 / Appendix III / General Tests and Assays FCC V
FIGURE 14 Special Apparatus for the Determination ofInorganic Arsenic. (A, 250-mL distillation flask; B, receiverchamber, approximately 50-mL capacity; C, reflux condenser;D, splash head.)
Stannous Chloride Solution Dissolve 40 g of stannouschloride dihydrate (SnCl2·2H2O) in 100 mL of hydrochloricacid. Store the solution in glass containers and use within 3months.
Lead Acetate-Impregnated Cotton Soak cotton in a satu-rated solution of lead acetate trihydrate, squeeze out the excesssolution, and dry in a vacuum at room temperature.
Sample Solution Use directly as the Sample Solution in theProcedure the solution obtained by treating the sample asdirected in an individual monograph. Prepare sample solutionsof organic compounds in the generator flask (a), unless other-wise directed, according to the following general procedure:
Caution: Some substances may react unexpectedly withexplosive violence when digested with hydrogen perox-ide. Use appropriate safety precautions at all times.
Note: If halogen-containing compounds are present,use a lower temperature while heating the sample withsulfuric acid; do not boil the mixture; and add theperoxide, with caution, before charring begins to pre-vent loss of trivalent arsenic.
Transfer 1.0 g of sample into the generator flask, add 5 mLof sulfuric acid and a few glass beads, and digest at a tempera-ture not exceeding 120° until charring begins, preferably usinga hot plate in a fume hood. (Additional sulfuric acid may benecessary to completely wet some samples, but the total vol-ume added should not exceed about 10 mL.) After the acid hasinitially decomposed the sample, cautiously add, dropwise,hydrogen peroxide (30%), allowing the reaction to subsideand reheating the sample between drops. Add the first fewdrops very slowly with sufficient mixing to prevent a rapidreaction, and discontinue heating if foaming becomes exces-sive. Swirl the solution in the flask to prevent unreacted
substance from caking on the walls or bottom of the flaskduring digestion.
Note: Maintain oxidizing conditions at all times duringthe digestion by adding small quantities of the peroxidewhenever the mixture turns brown or darkens.
Continue the digestion until the organic matter is destroyed,gradually raising the temperature of the hot plate to 250° to300° until fumes of sulfur trioxide are copiously evolved andthe solution becomes colorless or retains only a light strawcolor. Cool, cautiously add 10 mL of water, heat again tostrong fuming, and cool. Cautiously add 10 mL of water, mix,wash the sides of the flask with a few milliliters of water,and dilute to 35 mL.
Procedure If the Sample Solution was not prepared in thegenerator flask, transfer to the flask a volume of the solution,prepared as directed, equivalent to 1.0 g of the substancebeing tested, and add water to make 35 mL. Add 20 mL of1:5 sulfuric acid, 2 mL of potassium iodide TS, 0.5 mL ofStannous Chloride Solution, and 1 mL of isopropyl alcohol,and mix. Allow the mixture to stand for 30 min at roomtemperature. Pack the scrubber unit (c) with two plugs ofLead Acetate-Impregnated Cotton, leaving a small air spacebetween the two plugs, lubricate joints b and d with stopcockgrease, if necessary, and connect the scrubber unit with theabsorber tube (e). Transfer 3.0 mL of Silver Diethyldithiocar-bamate Solution to the absorber tube, add 3.0 g of granularzinc (20-mesh) to the mixture in the flask, and immediatelyinsert the standard-taper joint (b) into the flask. Allow theevolution of hydrogen and color development to proceed atroom temperature (25° � 3°) for 45 min, swirling the flaskgently at 10-min intervals. Disconnect the absorber tube fromthe generator and scrubber units, and transfer the Silver Dieth-yldithiocarbamate Solution to a 1-cm absorption cell. Deter-mine the absorbance at the wavelength of maximum absorp-tion between 535 nm and 540 nm, with a suitablespectrophotometer or colorimeter, using Silver Diethyldithio-carbamate Solution as the blank. The absorbance due to anyred color from the solution of the sample does not exceedthat produced by 3.0 mL of Standard Arsenic Solution (3 �gAs) when treated in the same manner and under the sameconditions as the sample. The room temperature during thegeneration of arsine from the standard should be held to within�2° of that observed during the determination of the sample.
Interferences Metals or salts of metals such as chromium,cobalt, copper, mercury, molybdenum, nickel, palladium, andsilver may interfere with the evolution of arsine. Antimony,which forms stibine, is the only metal likely to produce apositive interference in the color development with the silverdiethyldithiocarbamate. Stibine forms a red color with silverdiethyldithiocarbamate that has a maximum absorbance at510 nm, but at 535 to 540 nm, the absorbance of the antimonycomplex is so diminished that the results of the determinationwould not be altered significantly.
FCC V General Tests and Assays / Appendix III / 863
CADMIUM LIMIT TEST
Spectrophotometer Use any suitable atomic absorptionspectrophotometer equipped with a Boling-type burner, anair–acetylene flame, and a hollow-cathode cadmium lamp.The instrument should be capable of operating within thesensitivity necessary for the determination.
Standard Solution Transfer 100 mg of cadmium chloridecrystals (CdCl2·2
1⁄2H2O), accurately weighed, into a 1000-mLvolumetric flask, dissolve in and dilute to volume with water,and mix. Pipet 25 mL of this solution into a 100-mL volumetricflask, add 1 mL of hydrochloric acid, dilute to volume withwater, and mix. Each milliliter contains 12.5 �g of cadmium.
Sample Solution Transfer 10 g of sample, accuratelyweighed, into a 50-mL volumetric flask, dissolve in and diluteto volume with water, and mix.
Test Solutions Transfer 5.0 mL of Sample Solution intoeach of five separate 25-mL volumetric flasks. Dilute thecontents of Flask 1 to volume with water, and mix. Add 1.00,2.00, 3.00, and 4.00 mL of Standard Solution, to Flasks 2,3, 4, and 5, respectively, then dilute each flask to volumewith water, and mix. The Test Solutions contain, respectively,0, 0.5, 1.0, 1.5, and 2.0 �g/mL of cadmium.
Procedure Determine the absorbance of each Test Solutionat 228.8 nm, setting the instrument to previously establishedoptimum conditions, using water as a blank. Plot the ab-sorbance of the Test Solutions versus their contents of cad-mium, in micrograms per milliliter. Draw the straight linebest fitting the five points, and extrapolate the line until itintercepts the concentration axis. From the intercept, deter-mine the amount, in micrograms, of cadmium in each milliliterof the Test Solution containing 0 mL of the Standard Prepara-tion. Calculate the quantity, in milligrams per kilogram, ofcadmium in the sample by multiplying this value by 25.
CHLORIDE AND SULFATE LIMITTESTS
Where limits for chloride and sulfate are specified in theindividual monograph, compare the Sample Solution and con-trol in appropriate glass cylinders of the same dimensions andmatched as closely as practicable with respect to their opticalcharacteristics.
If the solution is not perfectly clear after acidification, filterit through filter paper that has been washed free of chlorideand sulfate. Add identical quantities of the precipitant (silvernitrate TS or barium chloride TS) in rapid succession to boththe Sample Solution and the control solution.
Experience has shown that visual turbidimetric comparisonsare best made between solutions containing from 10 to 20 �gof chloride (Cl) ion or from 200 to 400 �g of sulfate (SO4)
ion in 50 mL. Weights of samples are specified on this basis inthe individual monographs in which these limits are included.
Chloride Limit Test
Standard Chloride Solution Dissolve 165 mg of sodiumchloride in water and dilute to 100.0 mL. Transfer 10.0 mLof this solution into a 1000-mL volumetric flask, dilute tovolume with water, and mix. Each milliliter of the final solu-tion contains 10 �g of chloride (Cl) ion.
Procedure Unless otherwise directed, dissolve the specifiedamount of the test substance in 30 to 40 mL of water; neutralizeto litmus external indicator with nitric acid, if necessary; andadd 1 mL in excess. Add 1 mL of silver nitrate TS to theclear solution or filtrate, dilute to 50 mL with water, mix,and allow to stand for 5 min protected from direct sunlight.Compare the turbidity, if any, with that produced similarly ina control solution containing the required volume of StandardChloride Solution and the quantities of the reagents used forthe sample.
Sulfate Limit Test
Standard Sulfate Solution Dissolve 148 mg of anhydroussodium sulfate in water, and dilute to 100.0 mL. Transfer10.0 mL of this solution to a 1000-mL volumetric flask, diluteto volume with water, and mix. Each milliliter of the finalsolution contains 10 �g of sulfate (SO4).
Procedure Unless otherwise directed, dissolve the specifiedamount of the test substance in 30 to 40 mL of water; neutralizeto litmus external indicator with hydrochloric acid, if neces-sary; then add 1 mL of 2.7 N hydrochloric acid. Add 3 mLof barium chloride TS to the clear solution or filtrate, diluteto 50 mL with water, and mix. After 10 min compare theturbidity, if any, with that produced in a solution containingthe required volume of Standard Sulfate Solution and thequantities of the reagents used for the sample.
1,4-DIOXANE LIMIT TEST
Vacuum Distillation Apparatus Assemble a closed-systemvacuum distillation apparatus employing glass vacuum stop-cocks (A, B, and C), as shown in Fig. 15. The concentratortube (D) is made of borosilicate or quartz (not flint) glass,graduated precisely enough to measure the 0.9 mL or moreof distillate and marked so that the analyst can accuratelydilute to 2.0 mL (available as Chromaflex concentrator tube,Kontes Glass Co., Vineland, NJ, Catalog No. K42560-0000).
Standard Preparation Prepare a solution of 1,4-dioxanein water containing 100 �g/mL. Keep the solution refrigerated,and prepare fresh weekly.
Sample Preparation Transfer 20 g of the sample, accu-rately weighed, into a 50-mL round-bottom flask (E) having
864 / Appendix III / General Tests and Assays FCC V
FIGURE 15 Closed-System Vacuum Distillation Apparatus for1,4-Dioxane.
a 24/40 ground-glass neck. Semisolid or waxy samples shouldbe liquefied by heating on a steam bath before making thetransfer. Add 2.0 mL of water to the flask for crystallinesamples, and 1.0 mL for liquid, semisolid, or waxy samples.Place a small Teflon-covered stirring bar in the flask, stopper,and stir to mix. Immerse the flask in an ice bath, and chillfor about 1 min.
Wrap heating tape around the tube connecting the Chro-maflex tube (D) and the round-bottom flask (E), and applyabout 10 V to the tape. Apply a light coating of high-vacuumsilicone grease to the ground-glass joints, and connect theChromaflex tube to the 10/30 joint and the round-bottom flaskto the 24/40 joint. Immerse the vacuum trap in a Dewar flaskfilled with liquid nitrogen, close stopcocks A and B, openstopcock C, and begin evacuating the system with a vacuumpump. Prepare a slush bath from powdered dry ice and metha-nol, and raise the bath to the neck of the round-bottom flask.After freezing the contents of the flask for about 10 min, andwhen the vacuum system is operating at 0.05 mm pressureor lower, open stopcock A for 20 s, and then close it. Removethe slush bath, and allow the flask to warm in air for about1 min. Immerse the flask in a water bath at 20° to 25°, andafter about 5 min warm the water in the bath to 35° to 40°(sufficient to liquefy most samples) while stirring slowly butconstantly with the magnetic bar. Cool the water in the bathby adding ice, and chill for about 2 min. Replace the waterbath with the slush bath, freeze the contents of the flask forabout 10 min, then open stopcock A for 20 s, and close it.Remove the slush bath, and repeat the heating steps as before,this time reaching a final temperature of 45° to 50° or atemperature necessary to melt the sample completely. If thereis any condensation in the tube connecting the round-bottomflask to the Chromaflex tube, slowly increase the voltage tothe heating tape and heat until condensation disappears.
Stir with the magnetic stirrer throughout the followingsteps: Very slowly immerse the Chromaflex tube in the Dewarflask containing liquid nitrogen.
Caution: When there is liquid distillate in the Chro-maflex tube, the tube must be immersed in the nitrogenvery slowly, or the tube will break.
Water will begin to distill into the tube. As ice forms in thetube, raise the Dewar flask to keep the liquid nitrogen levelonly slightly below the level of ice in the tube. When waterbegins to freeze in the neck of the 10/30 joint, or whenliquid nitrogen reaches the 2.0-mL graduation mark on the
Chromaflex tube, remove the Dewar flask and let the ice meltwithout heating. After the ice has melted, check the volumeof water that has distilled, and repeat the sequence of chillingand thawing until at least 0.9 mL of water has been collected.Freeze the tube once again for about 2 min, and release thevacuum first by opening stopcock B, followed by stopcockA. Remove the Chromaflex tube from the apparatus, close itwith a greased stopper, and let the ice melt without heating.Mix the contents of the tube by swirling, note the volume ofdistillate, and dilute to 2.0 mL with water, if necessary. Usethis Sample Preparation as directed under Chromatography(below).
Chromatography (See Chromatography, Appendix IIA.)Use a gas chromatograph equipped with a flame-ionizationdetector. Under typical conditions, the instrument contains a4-mm (id) × 6-ft glass column, or equivalent, packed with80-/100- or 100-/120-mesh Chromosorb 104, or equivalent.The column is maintained isothermally at about 140°, theinjection port at 200°, and the detector at 250°. Nitrogen isthe carrier gas, flowing at a rate of about 35 mL/min. Installan oxygen scrubber between the carrier gas line and the col-umn. The column should be conditioned for about 72 h at250° with 30 to 40 mL/min carrier flow.
Note: Chromosorb 104 is oxygen sensitive. Both newand used columns should be flushed with carrier gasfor 30 to 60 min before heating each time they areinstalled in the gas chromatograph.
Inject a volume of the Standard Preparation, accurately mea-sured, to give about 20% of maximum recorder response.Where possible, keep the injection volume in the range of 2to 4 �L, and use the solvent-flush technique to minimizeerrors associated with injection volumes. In the same manner,inject an identical volume of the Sample Preparation. Theheight of the peak produced by the Sample Preparation doesnot exceed that produced by the Standard Preparation.1
FLUORIDE LIMIT TEST
Method I (Thorium Nitrate Colorimetric Method)
Use this method unless otherwise directed in the individualmonograph.
Caution: When applying this test to organic com-pounds, rigidly control at all times the temperature atwhich the distillation is conducted to the recommendedrange of 135° to 140° to avoid the possibility of ex-plosion.
1If the sample fails the test because of known or suspected interference,another aliquot may be run on a 6-ft × 2-mm (id) column, or equivalent,of 0.2% Carbowax 1500 on Carbopak C, operating at 100° isothermal,with 20 mL/min of helium carrier flow. Under these conditions, the 1,4-dioxane elutes in about 4 min.
FCC V General Tests and Assays / Appendix III / 865
Note: To minimize the distillation blank resulting fromfluoride leached from the glassware, treat the distillationapparatus as follows: Treat the glassware with hot 10%sodium hydroxide solution, followed by flushing withtap water and rinsing with distilled water. At least oncedaily, treat in addition by boiling down 15 to 20 mLof 1:2 sulfuric acid until the still is filled with fumes;cool, pour off the acid, treat again with 10% sodiumhydroxide solution, and rinse thoroughly. For furtherdetails, see AOAC method 944.08.
Unless otherwise directed, place a 5.0-g sample and 30 mLof water in a 125-mL Pyrex distillation flask having a sidearm and trap. The flask is connected with a condenser andcarries a thermometer and a capillary tube, both of whichmust extend into the liquid. Slowly add, with continuousstirring, 10 mL of 70% perchloric acid, and then add 2 or 3drops of a 1:2 solution of silver nitrate and a few glass beads.Connect a small dropping funnel or a steam generator to thecapillary tube. Support the flask on a flame-resistant mat orshielding board, with a hole that exposes about one-third ofthe flask to the low, ‘‘clean’’ flame of a Bunsen burner.
Note: The shielding is essential to prevent the walls ofthe flask from overheating above the level of its liquidcontents.
Distill until the temperature reaches 135°. Add water fromthe funnel or introduce steam through the capillary, main-taining the temperature between 135° and 140° at all times.Continue the distillation until 100 mL of distillate has beencollected. After the 100-mL portion (Distillate A) is collected,collect an additional 50-mL portion of distillate (Distillate B)to ensure that all of the fluorine has been volatilized.
Place 50 mL of Distillate A in a 50-mL Nessler tube. Inanother, similar Nessler tube, place 50 mL of water distilledthrough the apparatus as a control. Add to each tube 0.1 mLof a filtered 1:1000 solution of sodium alizarinsulfonate and1 mL of a freshly prepared 1:4000 solution of hydroxylaminehydrochloride, and mix well. Add, dropwise and with stirring,either 1 N or 0.05 N sodium hydroxide, depending on theexpected volume of volatile acid distilling over, to the tubecontaining the distillate until its color just matches that of thecontrol, which is faintly pink. Then add to each tube 1.0 mLof 0.1 N hydrochloric acid, and mix well. From a buret,graduated in 0.05 mL, add slowly to the tube containing thedistillate enough of a 1:4000 solution of thorium nitrate sothat, after mixing, the color of the liquid just changes to afaint pink. Note the volume of the solution added, then addexactly the same volume to the control, and mix. Now addto the control solution sodium fluoride TS (10 �g F permilliliter) from a buret to make the colors of the two tubesmatch after dilution to the same volume. Mix well, and allowall air bubbles to escape before making the final color compari-son. Check the endpoint by adding 1 or 2 drops of sodiumfluoride TS to the control. A distinct change in color shouldtake place. Note the volume of sodium fluoride TS added.
Dilute Distillate B to 100 mL, and mix well. Place 50 mLof this solution in a 50-mL Nessler tube, and follow theprocedure used for Distillate A. The total volume of sodium
fluoride TS required for the solutions from both Distillate Aand Distillate B should not exceed 2.5 mL.
Method II (Ion-Selective Electrode Method A)
Buffer Solution Dissolve 36 g of cyclohexylenedinitrilote-traacetic acid (CDTA) in sufficient 1 N sodium hydroxide tomake 200 mL. Transfer 20 mL of this solution (equivalentto 4 g of disodium CDTA) into a 1000-mL beaker containing500 mL of water, 57 mL of glacial acetic acid, and 58 g ofsodium chloride, and stir to dissolve. Adjust the pH of thesolution to between 5.0 and 5.5 by the addition of 5 N sodiumhydroxide, then cool to room temperature, dilute to 1000 mLwith water, and mix.
Procedure Unless otherwise directed in the individualmonograph, transfer 8.0 g of sample and 20 mL of water intoa 250-mL distilling flask, cautiously add 20 mL of perchloricacid, and then add 2 or 3 drops of a 1:2 solution of silvernitrate and a few glass beads.
Caution: Handle perchloric acid in an appropriatefume hood.
Following the directions, and observing the Caution andNotes, as given under Method I, distill the solution until 200mL of distillate has been collected.
Transfer a 25.0-mL aliquot of the distillate into a 250-mLplastic beaker, and dilute to 100 mL with the Buffer Solution.Place the fluoride ion and reference electrodes (or a combina-tion fluoride electrode) of a suitable ion-selective electrodeapparatus in the solution. Adjust the calibration control untilthe indicator needle points to the center on the logarithmicconcentration scale, allowing sufficient time for equilibration(about 20 min), and stirring constantly during the equilibrationperiod and throughout the remainder of the procedure. Pipet1.0 mL of a solution containing 100 �g of fluoride (F) ion permilliliter (prepared by dissolving 22.2 mg of sodium fluoride,previously dried at 200° for 4 h, in sufficient water to make100.0 mL) into the beaker, allow the electrode to come toequilibrium, and record the final reading on the logarithmicconcentration scale.
Note: Follow the instrument manufacturer’s instruc-tions regarding precautions and interferences, electrodefilling and check, temperature compensation, and cali-bration.
Calculations Calculate the fluoride content, in milligramsper kilogram, of the sample taken by the formula
[IA/(R – I)] × 100 × (200/25W),
in which I is the initial scale reading before the additionof the sodium fluoride solution; A is the concentration, inmicrograms per milliliter, of fluoride in the sodium fluoridesolution added to the sample solution; R is the final scalereading after addition of the sodium fluoride solution; and Wis the original weight, in grams, of the sample.
Method III (Ion-Selective Electrode Method B)
Sodium Fluoride Solution (5 �g F per milliliter) Transfer2.210 g of sodium fluoride, previously dried at 200° for 4 h
866 / Appendix III / General Tests and Assays FCC V
and accurately weighed, into a 400-mL plastic beaker, add200 mL of water, and stir until dissolved. Quantitatively trans-fer this solution into a 1000-mL volumetric flask with the aidof water, dilute to volume with water, and mix. Store thisstock solution in a plastic bottle. On the day of use, transfer5.0 mL of the stock solution into a 1000-mL volumetric flask,dilute to volume with water, and mix.
Calibration Curve Transfer 1.0, 2.0, 3.0, 5.0, 10.0, and15.0 mL of the Sodium Fluoride Solution into separate 250-mL plastic beakers; add 50 mL of water, 5 mL of 1 N hydro-chloric acid, 10 mL of 1 M sodium citrate, and 10 mL of 0.2M disodium EDTA to each beaker; and mix. Transfer eachsolution into separate 100-mL volumetric flasks, dilute tovolume with water, and mix. Transfer a 50-mL portion of eachsolution into separate 125-mL plastic beakers, and measurethe potential of each solution with a suitable ion-selectiveelectrode apparatus (such as the Orion Model No. 94-09, withsolid-state membrane), using a suitable reference electrode(such as the Orion Model No. 90-01, with single junction).Plot the calibration curve on two-cycle semilogarithmic paper(such as K & E No. 465130), with micrograms of F per 100mL solution on the logarithmic scale.
Procedure Transfer 1.00 g of sample into a 150-mL glassbeaker, add 10 mL of water, and, while stirring continuously,slowly add 20 mL of 1 N hydrochloric acid to dissolve thesample. Boil rapidly for 1 min, then transfer into a 250-mLplastic beaker, and cool rapidly in ice water. Add 15 mL of1 M sodium citrate and 10 mL of 0.2 M disodium EDTA,and mix. Adjust the pH to 5.5 � 0.1 with 1 N hydrochloricacid or 1 N sodium hydroxide, if necessary; transfer into a100-mL volumetric flask; dilute to volume with water; andmix. Transfer a 50-mL portion of this solution into a 125-mL plastic beaker, and measure the potential of the solutionwith the apparatus described under Calibration Curve. Deter-mine the fluoride content, in micrograms, of the sample fromthe Calibration Curve.
Method IV (Ion-Selective Electrode Method C)
Buffer Solution Dissolve 150 g of sodium citrate dihydrateand 10.3 g of disodium EDTA dihydrate in 800 mL of water,adjust the pH to 8.0 with 50% sodium hydroxide solution,and dilute to 1000 mL with water.
Fluoride Standard Solutions1000 mg/kg Fluoride Standard Transfer 2.2108 g of so-
dium fluoride, previously dried at 200° for 4 h, into a 1000-mL volumetric flask and dissolve in and dilute to volumewith water. The resulting solution contains 1000 �g of fluorideper milliliter.
50 mg/kg Fluoride Standard Pipet 50 mL of the 1000mg/kg Fluoride Standard into a 1000-mL volumetric flask.Dilute to volume with water.
10 mg/kg Fluoride Standard Pipet 100 mL of the 50 mg/kg Fluoride Standard into a 500-mL volumetric flask. Diluteto volume with water.
Fluoride Limit Solutions (for a 1-g sample)50 mg/kg Fluoride Limit Solution (1 mg/kg fluoride stan-
dard) Pipet 50 mL of the 10 mg/kg Fluoride Standard intoa 500-mL volumetric flask, and dilute to volume with water.
10 mg/kg Fluoride Limit Solution (0.2 mg/kg fluoride stan-dard) Pipet 10 mL of the 10 mg/kg Fluoride Standard intoa 500-mL volumetric flask, and dilute to volume with water.
Fluoride Limit Solutions (for a 2-g sample)50 mg/kg Fluoride Limit Solution (2 mg/kg fluoride stan-
dard) Pipet 100 mL of the 10 mg/kg Fluoride Standard intoa 500-mL volumetric flask, and dilute to volume with water.
10 mg/kg Fluoride Limit Solution (0.4 mg/kg fluoride stan-dard) Pipet 20 mL of the 10 mg/kg Fluoride Standard intoa 500-mL volumetric flask, and dilute to volume with water.
Note: Store all standard and limit solutions in plasticcontainers.
Sample Preparation Accurately weigh the amount of sam-ple specified in the monograph, transfer it into a 100-mLvolumetric flask, and dissolve it in a minimal amount of water.Add 50.0 mL of the Buffer Solution, dilute to volume withwater, and mix.
Electrode Calibration Pipet 50 mL of the Buffer Solutioninto a plastic beaker. Place the fluoride ion and referenceelectrodes (or a combination fluoride electrode) into the plasticbeaker and stir. At 5-min intervals, add 100 �L and 1000 �Lof the 1000 mg/kg Fluoride Standard and read the potential,in millivolts, after each addition. The difference between thetwo readings is the slope of the fluoride electrode and shouldtypically be in the range of 54 to 60 mV at 25°. If the differencein potential is not within this range, check, and, if necessary,replace the electrode, instrument, or solutions.
Procedure Transfer the entire sample into a plastic beaker.Place the electrode into the beaker, allow the solution toequilibrate for 5 min with stirring, and read the potential, inmillivolts. Remove and rinse the electrode(s) with water. Inanother beaker, using a pipet, add 50 mL of the Buffer Solutionfollowed by 50 mL of the Fluoride Limit Solution that bestreflects the fluoride limit of the sample. Place the electrodein the beaker, equilibrate for 3 min, and read the potential inmillivolts. If the potential of the Fluoride Limit Solution isless than that of the sample, the sample passes the test criterionfor maximum acceptable fluoride level limit.
Method V
Lime Suspension Carefully shake about 56 g of low-fluo-rine calcium oxide (about 2 mg/kg of F) with 250 mL of water,and while stirring, slowly add 250 mL of 60% perchloricacid. Add a few glass beads, and boil until copious fumes ofperchloric acid evolve, then cool, add 200 mL of water, andboil again.
Caution: Handle perchloric acid in an appropriatefume hood.
Repeat the dilution and boiling once more, cool, dilute consid-erably, and if precipitated silicon dioxide forms, filter through
FCC V General Tests and Assays / Appendix III / 867
a fritted-glass filter. While stirring, pour the clear solutioninto 1000 mL of a 1:10 solution of sodium hydroxide, allowthe precipitate to settle, and siphon off the supernatant liquid.Remove the sodium salts from the precipitate by washing fivetimes with water in large centrifuge bottles, shaking the massthoroughly each time. Finally, shake the precipitate into asuspension and dilute with water to 2000 mL. Store in paraffin-lined bottles, and shake well before use.
Note: 100 mL of this suspension should give no appre-ciable fluoride blank when evaporated, distilled, andtitrated as directed under Method I.
Procedure Assemble the distilling apparatus as describedunder Method I, and add 1.67 g of sample, accurately weighed,and 25 mL of 1:2 sulfuric acid to the distilling flask. Distilluntil the temperature reaches 160°, then maintain at 160° to165° by adding water from the funnel, collecting 300 mL ofdistillate. Oxidize the distillate by cautiously adding 2 or3 mL of fluorine-free 30% hydrogen peroxide (to removesulfates), allow to stand for a few minutes, and evaporate ina platinum dish with an excess of Lime Suspension. Ignitebriefly at 600°, then cool and wet the ash with about 10 mLof water. Cover the dish with a watch glass, and cautiouslyintroduce under the watch glass just sufficient 60% perchloricacid to dissolve the ash. Add the contents of the dish throughthe dropping funnel of a freshly prepared distilling apparatus(the distilling flask should contain a few glass beads), usinga total of 20 mL of the 60% perchloric acid to dissolve theash and transfer the solution. Add 10 mL of water and afew drops of a 1:2 solution of silver perchlorate through thedropping funnel, and continue as directed under Method I,beginning with ‘‘Distill until the temperature reaches135°. . . .’’
LEAD LIMIT TEST
Note: Unless otherwise specified in the monograph, usethe Dithizone Method to determine lead levels.
Dithizone Method
Special Reagents Select reagents having as low a lead con-tent as practicable, and store all solutions in containers ofborosilicate glass. Rinse all glassware thoroughly with warm,1:2 nitric acid followed by water.
Ammonia–Cyanide Solution Dissolve 2 g of potassiumcyanide in 15 mL of ammonium hydroxide, and dilute to 100mL with water.
Ammonium Citrate Solution Dissolve 40 g of citric acidin 90 mL of water, add 2 or 3 drops of phenol red TS,then cautiously add ammonium hydroxide until the solutionacquires a red color. Extract it with 20-mL portions of Dithi-
zone Extraction Solution until the dithizone solution retainsits green color or remains unchanged.
Diluted Standard Lead Solution (1 �g Pb in 1 mL)Lead Nitrate Stock Solution Dissolve 159.8 mg of ACS
Reagent-Grade Lead Nitrate [Pb(NO3)2] in 100 mL of watercontaining 1 mL of nitric acid, dilute to 1000.0 mL with water,and mix. Prepare and store this solution in glass containers thatare free from lead salts.
Standard Lead Solution On the day of use, dilute 10.0mL of Lead Nitrate Stock Solution to 100.0 mL with water.Each milliliter of Standard Lead Solution contains the equiva-lent of 10 �g of lead (Pb) ion.
Diluted Standard Lead Solution Immediately before use,transfer 10.0 mL of Standard Lead Solution into a 100-mL volu-metric flask, dilute to volume with 1:100 nitric acid, and mix.
Dithizone Extraction Solution Dissolve 30 mg of dithizonein 1000 mL of chloroform, add 5 mL of alcohol, and mix.Store in a refrigerator. Before use, shake a suitable volumeof the solution with about half its volume of 1:100 nitric acid,discarding the nitric acid. Do not use if more than 1 month old.
Hydroxylamine Hydrochloride Solution Dissolve 20 g ofhydroxylamine hydrochloride in sufficient water to makeabout 65 mL, transfer the solution into a separator, add a fewdrops of thymol blue TS, then add ammonium hydroxide untilthe solution assumes a yellow color. Add 10 mL of a 1:25solution of sodium diethyldithiocarbamate, mix, and allow tostand for 5 min. Extract the solution with successive 10- to15-mL portions of chloroform until a 5-mL test portion ofthe chloroform extract does not assume a yellow color whenshaken with cupric sulfate TS. Add 2.7 N hydrochloric aciduntil the extracted solution is pink, adding 1 or 2 drops moreof thymol blue TS if necessary, then dilute to 100 mL withwater, and mix.
Potassium Cyanide Solution Dissolve 50 g of potassiumcyanide in sufficient water to make 100 mL. Remove the leadfrom the solution by extraction with successive portions ofDithizone Extraction Solution as described under AmmoniumCitrate Solution, then extract any dithizone remaining in thecyanide solution by shaking with chloroform. Finally, dilutethe cyanide solution with sufficient water so that each 100mL contains 10 g of potassium cyanide.
Standard Dithizone Solution Dissolve 10 mg of dithizonein 1000 mL of chloroform, keeping the solution in a glass-stoppered, lead-free bottle suitably wrapped to protect it fromlight and stored in a refrigerator.
Sample Solution Use the solution obtained by treating thesample as directed in an individual monograph as the SampleSolution in the Procedure. Sample solutions of organic com-pounds are prepared, unless otherwise directed, according tothe following general method:
868 / Appendix III / General Tests and Assays FCC V
Caution: Some substances may react unexpectedly withexplosive violence when digested with hydrogen perox-ide. Use appropriate safety precautions at all times.
Transfer 1.0 g of sample into a suitable flask, add 5 mL ofsulfuric acid and a few glass beads, and digest at a temperaturenot exceeding 120° until charring begins, using preferably, ahot plate in a fume hood. (Additional sulfuric acid may benecessary to completely wet some samples, but the total vol-ume added should not exceed about 10 mL.) After the samplehas initially been decomposed by the acid, add with caution,dropwise, hydrogen peroxide (30%), allowing the reaction tosubside and reheating between drops. The first few drops mustbe added very slowly with sufficient mixing to prevent arapid reaction, and heating should be discontinued if foamingbecomes excessive. Swirl the solution in the flask to preventunreacted substance from caking on the walls or bottom ofthe flask during the digestion.
Note: Add small quantities of the peroxide when thesolution begins to darken.
Continue the digestion until the organic matter is destroyed,gradually raising the temperature of the hot plate to 250° to300° until fumes of sulfur trioxide are copiously evolved andthe solution becomes colorless or retains only a light strawcolor. Cool, cautiously add 10 mL of water, again evaporateto strong fuming, and cool. Quantitatively transfer the solutioninto a separator with the aid of small quantities of water.
Procedure Transfer the Sample Solution, prepared as di-rected in the individual monograph, into a separator, andunless otherwise directed, add 6 mL of Ammonium CitrateSolution and 2 mL of Hydroxylamine Hydrochloride Solution.(Use 10 mL of the citrate solution when determining lead iniron salts.) Add 2 drops of phenol red TS to the separator,and make the solution just alkaline (red in color) by theaddition of ammonium hydroxide. Cool the solution, if neces-sary, under a stream of tap water, then add 2 mL of PotassiumCyanide Solution. Immediately extract the solution with 5-mL portions of Dithizone Extraction Solution, draining eachextract into another separator, until the dithizone solutionretains its green color. Shake the combined dithizone solutionsfor 30 s with 20 mL of 1:100 nitric acid; discard the chloroformlayer; add 5.0 mL of Standard Dithizone Solution and 4 mLof Ammonia–Cyanide Solution to the acid solution; and shakefor 30 s. The purple hue in the chloroform solution of thesample caused by any lead dithizonate present does not exceedthat in a control, containing the volume of Diluted StandardLead Solution equivalent to the amount of lead specifiedin the monograph, when treated in the same manner as thesample.
Flame Atomic Absorption Spectrophotometric Method
Select reagents having as low a lead content as practicable,and store all solutions in high-density polyethylene containers.Rinse all plastic and glassware thoroughly with warm, 1:2nitric acid followed by water.
Lead Nitrate Stock Solution (100 �g/mL) Dissolve 159.8mg of reagent-grade lead nitrate [Pb(NO3)2] in 100 mL ofwater containing 1 mL of nitric acid in a 1000-mL volumetricflask, and dilute to volume with water.
Standard Lead Solution (10 �g/mL) On the day of use,transfer 10 mL of Lead Nitrate Stock Solution into a 100-mLvolumetric flask, and dilute to volume with water.
Diluted Standard Lead Solutions On the day of use, pre-pare a set of standard lead solutions that corresponds to thelead limit specified in the monograph:
1 mg/kg Lead Limit (0.5, 1.0, and 1.5 �g/mL standards)On the day of use, transfer 5.0, 10.0, and 15.0 mL of StandardLead Solution into three separate 100-mL volumetric flasks,add 10 mL of 3 N hydrochloric acid to each, and dilute tovolume with water.
5 mg/kg Lead Limit (1.0, 5.0, and 10.0 �g/mL standards)On the day of use, transfer 10.0 and 50.0 mL of StandardLead Solution into two separate 100-mL volumetric flasks,add 10 mL of 3 N hydrochloric acid to each, and dilute tovolume with water. The final standard, 10.0 �g/mL, is takendirectly from the Standard Lead Solution.
10 mg/kg Lead Limit (5.0, 10.0, and 15.0 �g/mL standards)On the day of use, transfer 5.0, 10.0, and 15.0 mL of LeadNitrate Stock Solution into three separate 100-mL volumetricflasks, add 10 mL of 3 N hydrochloric acid to each, and diluteto volume with water.
25% Sulfuric Acid Solution (by volume) Cautiously add100 mL of sulfuric acid to 300 mL of water with constantstirring while cooling in an ice bath.
Sample Preparation Transfer the sample weight as speci-fied in the monograph, weighed to the nearest 0.1 mg, intoan evaporating dish. Add a sufficient amount of 25% SulfuricAcid Solution, and distribute the sulfuric acid uniformlythrough the sample. Within a hood, place the dish on a steambath to evaporate most of the water. Place the dish on a burner,and slowly pre-ash the sample by expelling most of the sulfuricacid. Place the dish in a muffle furnace that has been set at525°, and ash the sample until the residue appears free fromcarbon. Prepare a Sample Blank by ashing 5 mL of 25%sulfuric acid. Cool and cautiously wash down the inside ofeach evaporation dish with water.
Add 5 mL of 1 N hydrochloric acid. Place the dish on asteam bath, and evaporate to dryness. Add 1.0 mL of 3 Nhydrochloric acid and approximately 5 mL of water, and heatbriefly on a steam bath to dissolve any residue. Transfer eachsolution quantitatively to a 10-mL volumetric flask, dilute tovolume, and mix.
Procedure Concomitantly determine the absorbances of theSample Blank, the Diluted Standard Lead Solutions, and theSample Preparation at the lead emission line of 283.3 nm,using a slit-width of 0.7 nm. Use a suitable atomic absorptionspectrophotometer equipped with a lead electrodeless dis-charge lamp (EDL), an air–acetylene flame, and a 4-in. burnerhead. Use water as the blank.
FCC V General Tests and Assays / Appendix III / 869
Calculations Determine the corrected absorbance values bysubtracting the Sample Blank absorbance from each of theDiluted Standard Lead Solutions and from the Sample Prepa-ration absorbances. Prepare a standard curve by plotting thecorrected Diluted Standard Lead Solutions absorbance valuesversus their corresponding concentrations expressed as micro-grams per milliliter. Determine the lead concentration in theSample Preparation by reference to the calibration curve.Calculate the quantity of lead, in milligrams per kilogram, inthe sample taken by the formula
10C/WS,
in which C is the concentration, in micrograms per milliliter,of lead from the standard curve; and WS is the weight, ingrams, of the sample taken.
Atomic Absorption Spectrophotometric GraphiteFurnace Method
The following methods are primarily intended for the analysisof applicable substances containing less than 1 mg/kg of lead.
Method IThis method is intended for the quantitation of lead in sub-stances that are soluble in water, such as sugars and sugarsyrups, at levels as low as 0.03 mg/kg. The method detectionlimit is approximately 5 ng/kg.
Apparatus Use a suitable graphite furnace atomic absorp-tion spectrophotometer set at 283.3 nm and equipped with anautosampler, pyrolytically coated graphite tubes, solid pyro-lytic graphite platforms, and an adequate means of backgroundcorrection. Zeeman effect or Smith-Hieftje background cor-rection is preferred, but deuterium arc background correctionshould be acceptable. (This method was developed on a Per-kin-Elmer Model Z5100, 0.7-nm slit, HGA-600 furnace, AS-60 autosampler with Zeeman background correction.) If theinstrument does not have a well-defined calibration function,a separate calculator or computer is required for linear leastsquares, nonlinear, or quadratic calibrations. Use either a hol-low cathode lamp or an electrode-less discharge lamp as thesource, and use argon as the purge gas and breathing-qualityair (for oxygen ashing to avoid residue build up during thechar step) as the alternate gas. Set up the instrument accordingto the manufacturer’s specifications with consideration of cur-rent good GFAAS practices—addressing such factors as linevoltage, cooling water temperature, graphite part specifica-tions, and furnace temperature. If an optical pyrometer orthermocouple is not available to check the furnace controllertemperature calibration, dim the room lights, and observe thefurnace emission through the sample introduction port whileincreasing the furnace temperature. A characteristic cherryred glow should begin to appear at 800°. If it glows at a lowertemperature, then the furnace is hotter, and temperatures mustbe adjusted downward accordingly.
Use acid-cleaned [in a mixture of 5% sub-boiling, distillednitric acid and 5% sub-boiling, distilled hydrochloric acidmade up in deionized, distilled water (18 megohm), and thor-oughly rinsed with deionized, distilled water (18 megohm)]
autosampler cups (PE B008-7600 Teflon, or equivalent) toavoid contamination. Use micropipets with disposable tipsfree of lead contamination for dilution. Ensure accuracy andprecision of micropipets and tips by dispensing and weighing5 to 10 replicate portions of water onto a microbalance. Useacid-cleaned volumetric glassware to prepare standards anddilute samples to a final volume. For digestion, use acid-cleaned, high-density polyethylene tubes, polypropylenetubes, Teflon tubes, or quartz tubes. Store final diluted samplesin plastic tubes.
Standard Solutions Prepare all lead solutions in 5% sub-boiling distilled nitric acid. Use a single-element 1000- or10,000-�g/mL lead stock to prepare (weekly) an intermediate10-�g/mL standard in 5% nitric acid. Prepare (daily) a LeadStandard Solution (1 �g/mL) by diluting the intermediate10-�g/mL stock solution 1:10. Prepare Working CalibrationStandards of 100.0, 50.0, 25.0, and 10.0 ng/mL from this,using appropriate dilutions. Store standards in acid-cleanedpolyethylene test tubes or bottles. If the GFAAS autosampleris used to automatically dilute standards, ensure calibrationaccuracy by pipetting volumes of 3 �L or greater.
Modifier Stock Solution Weigh 20 g of ultrapure magne-sium nitrate hexahydrate and dilute to 100 mL. Just beforeuse, prepare a Modifier Working Solution by diluting stocksolution 1:10. A volume of 5 �L will provide 0.06 mg ofmagnesium nitrate.
Sample Digestion (Caution: Perform the procedure in afume hood, and wear safety glasses.) Obtain a representativesubsample to be analyzed. For liquid samples such as sugarsyrups, ultrasonicate and/or vortex mix before weighing. Forsolid samples such as crystalline sucrose, make a sugar solu-tion using equal weights of sample (5-g minimum) and deion-ized, distilled (18 megohm) water. Mix samples until com-pletely dissolved. Transfer approximately 1.5 g (record tonearest mg) of sample (or 3.0 g of sugar solution), accuratelyweighed, into a digestion tube. Run a Sample PreparationBlank of 1.5 g of deionized, distilled (18 megohm) waterthrough the entire procedure with each batch of samples. Add0.75 mL of sub-boiling, distilled nitric acid. Heat plastic tubesin a water bath, quartz tubes in a water bath or heating block,warming slowly to between 90° and 95° to avoid spattering.Monitor the temperature by using a ‘‘dummy’’ sample. Heatuntil all brown vapors have dissipated and any rust-coloredtint is gone (20 to 30 min). Cool. Add 0.5 mL of 50% hydrogenperoxide dropwise, heat at 90° to 95° for 5 min, and cool.Add a second 0.5-mL portion of 50% hydrogen peroxide,dropwise, and heat at 90° to 100° for 5 to 10 min until clear.Cool, and dilute to a final volume of 10 mL.
Procedure The furnace program is as follows: (1) dry at200°, using a 20-s ramp and a 30-s hold and a 300-mL/minargon flow; (2) char the sample at 750°, using a 40-s rampand a 40-s hold and a 300-mL/min air flow; (3) cool down,and purge the air from the furnace for 60 s, using a 20° settemperature and a 300-mL/min argon flow; (4) atomize at1800°, using a 0-s ramp and a 10-s hold with the argon flow
870 / Appendix III / General Tests and Assays FCC V
stopped; (5) clean out at 2600°, with a 1-s ramp and a 7-shold; (6) cool down the furnace (if necessary) at 20°, with a1-s ramp and a 5-s hold with a 300-mL/min argon flow.
Use the autosampler to inject 20 �L of blanks, calibrationstandards, and sample solutions and 5 �L of Modifier WorkingSolution. Inject each respective solution in triplicate, and aver-age results. Use peak area measurements for all quantitation.After ensuring that the furnace is clean by running a 5% nitricacid blank, check the instrument sensitivity by running the25-ng/mL calibration standard. If the integrated absorbanceis less than 0.14 abs-sec for a standard, 28-mm × 6-mm, end-heated furnace tube, correct the cause of insufficient sensitiv-ity before proceeding. If the integrated absorbance is greaterthan 0.25 abs-sec, contamination is likely, and the sourceshould be investigated. Calculate the characteristic mass (mo)(mass of Pb pg necessary to produce an integrated absorbanceof 0.0044 abs-sec) as follows:
mo = (0.0044 abs-sec)(25 pg/�L)(20 �L)/(measured 25 pg/�L abs-sec).
Record and track the integrated absorbance and mo for refer-ence and quality assurance.
Standard Curve Inject each calibration standard in tripli-cate. Normal instrument linearity extends to 25 ng/mL. Ifnonlinear calibration capability is not available, limit theworking calibration curve to ≤25 ng/mL. Use the calibrationalgorithms provided in the instrument software. Recheck cali-bration periodically (≤15 samples) by running a 25- or 50-ng/mL calibration standard interspersed with samples. If recheckdiffers from calibration by >10%, recalibrate the instrument.The instrumental detection limit (DL) and quantitation limit(QL), in picograms, may be based on 7 to 10 replicates ofthe Sample Preparation Blank and calculated as follows:
DL = (3)(s.d. blank abs-sec)(10 pg/�L)(20 �L)/(abs-sec 10 ng/mL std),
QL = (10)(s.d. blank abs-sec)(10 pg/�L)(20 �L)/(abs-sec 10 ng/mL std).
During method development, detection limits were typically10 to 14 pg, corresponding to 0.5 to 0.7 ng/mL for 20 �L.This corresponds to a method detection limit of 3.3 to 4.7ng/g of sugar.
Sample Analyses Inject each sample digest in triplicate,and record the integrated absorbance. If instrument responseexceeds that of the calibration curve, dilute with 5% nitricacid to bring the sample response into working range, andnote the dilution factor (DF). Sample solutions having a finalconcentration of >25 ng/mL should be diluted 1:10 to facilitateanalysis in the linear range for systems not equipped withnonlinear calibration. All sample analyses should be blankcorrected using the sample preparation blank. This can typi-cally be done automatically by the software after identifyingand running a representative sample preparation blank. Usethe calibration algorithm provided in the instrument softwareto calculate a blank-corrected, digest lead concentration (innanograms per milliliter).
Calculation of Lead Content Calculate the lead level inthe original sample as follows:
Pb(ng/g) = (blank-corrected Pb ng/mL)(DF)[sample vol(10 mL)][sample wt (approx. 1.5 g)].1
Quality Assurance To ensure analytical accuracy, NISTSRM 1643c acidified water or a similar material should beanalyzed before the unknown samples are. The certified con-tent of SRM 1643c is 35.3 � 0.9 ng/mL. If the concentrationdetermined is not within 10% of the mean reference value(31.8 to 38.8 ng/mL), the reason for inaccuracy should beevaluated, and unknown samples should not be analyzed untilacceptable accuracy is achieved. Also prepare an in-housecontrol solution made from uncontaminated table sugar orreagent-grade sucrose (or other appropriate substance with aPb content <5 ng/g as received) mixed with an equal volumeof water. Spike this solution with Pb to produce a concentra-tion of 100 ng/g. Analyze with each batch of samples. Recov-eries should be 100% � 20%, and the precision for completereplicate digestions should be <5% RSD. Periodically, a sam-ple digest should be checked using the method of standardadditions to ensure that there are no multiplicative or chemicalinterferences. Spiking samples and checking recoveries is al-ways a good practice.
Method IIThis method is primarily intended for the determination oflead at levels of less than 1 mg/kg in substances immisciblewith water, such as edible oils.
Apparatus Use a suitable atomic absorption spectropho-tometer (Perkin-Elmer Model 3100 or equivalent) fitted witha graphite furnace (Perkin-Elmer HGA 600 or equivalent).Use a lead hollow-cathode lamp (Perkin-Elmer or equivalent)with argon as the carrier gas. Follow the manufacturers’ direc-tions for setting the appropriate instrument parameters forlead determination.
Note: For this test, use reagent-grade chemicals withas low a lead content as is practicable, as well as high-purity water and gases. Before use in this analysis, rinseall glassware and plasticware twice with 10% nitricacid and twice with 10% hydrochloric acid, and thenrinse them thoroughly with high-purity water, prefera-bly obtained from a mixed-bed strong-acid, strong-baseion-exchange cartridge capable of producing water withan electrical resistivity of 12 to 15 megohms.
Hydrogen Peroxide–Nitric Acid Solution Dissolve equalvolumes of 10% hydrogen peroxide and 10% nitric acid.
Note: Use caution.
Lead Nitrate Stock Solution Dissolve 159.8 mg of ACSReagent-Grade Lead Nitrate (alternatively, use NIST StandardReference Material, containing 10 mg of lead per kilogram,or equivalent) in 100 mL of Hydrogen Peroxide–Nitric AcidSolution. Dilute to 1000.0 mL with Hydrogen Peroxide–Nitric
1If a sample solution was prepared initially to ensure sample homogene-ity, this is the weight of the original sugar digested (not the weight ofthe solution).
FCC V General Tests and Assays / Appendix III / 871
Acid Solution, and mix. Prepare and store this solution inglass containers that are free from lead salts. Each milliliterof this solution contains the equivalent of 100 �g of lead(Pb) ion.
Standard Lead Solution On the day of use, dilute 10.0 mLof Lead Nitrate Stock Solution to 100.0 mL with HydrogenPeroxide–Nitric Acid Solution, and mix. Each milliliter ofStandard Lead Solution contains the equivalent of 10 �g oflead (Pb) ion.
Butanol–Nitric Acid Solution Slowly add 50 mL of nitricacid to approximately 500 mL of butanol contained in a 1000-mL volumetric flask. Dilute to volume with butanol, and mix.
Standard Solutions Prepare a series of lead standard solu-tions serially diluted from the Standard Lead Solution inButanol–Nitric Acid Solution. Pipet into separate 100-mL vol-umetric flasks 0.2, 0.5, 1, and 2 mL, respectively, of StandardLead Solution, dilute to volume with Butanol–Nitric AcidSolution, and mix. The Standard Solutions contain, respec-tively, 0.02, 0.05, 0.1, and 0.2 �g of lead per milliliter. (Forlead limits greater than 1 mg/kg, prepare a series of standardsolutions in a range encompassing the expected lead concen-tration in the sample.)
Sample Solution (Caution: Perform this procedure in afume hood, and wear safety glasses.) Transfer 1 g of sample,accurately weighed, into a large test tube. Add 1 mL of nitricacid. Place the test tube in a rack in a boiling water bath. Assoon as the rusty tint is gone, add 1 mL of 30% hydrogenperoxide dropwise to avoid a vigorous reaction, and wait forbubbles to form. Stir with an acid-washed plastic spatula ifnecessary. Remove the test tube from the water bath, and letit cool. Transfer the solution into a 10-mL volumetric flask,and dilute to volume with Butanol–Nitric Acid Solution, andmix. Use this solution for analysis.
ProcedureTungsten Solution Transfer 0.1 g of tungstic acid
(H2WO4) and 5 g of sodium hydroxide pellets into a 50-mLplastic bottle. Add 5.0 mL of high-purity water, and mix.Heat the mixture in a hot water bath until complete solutionis achieved. Cool, and store at room temperature.
Procedure Place the graphite tube in the furnace. Injecta 20-�L aliquot of the Tungsten Solution into the graphite tube,using a 300-mL/min argon flow and the following sequence ofconditions: Dry at 110° for 20 s, char at 700° to 900° for 20s, and with the argon flow stopped, atomize at 2700° for 10s; repeat this procedure once more using a second 20-�Laliquot of the Tungsten Solution. Clean the quartz windows.
Standard Curve [Note: The sample injection techniqueis the most crucial step in controlling the precision of theanalysis; the volume of the sample must remain constant.Rinse the �L pipet tip (Eppendorf or equivalent) three timeswith either the Standard Solutions or Sample Solution beforeinjection. Use a fresh pipet tip for each injection, and start theatomization process immediately after injecting the sample.Between injections, flush the graphite tube of any residual
lead by purging at a high temperature as recommended bythe manufacturer.] With the hollow cathode lamp properlyaligned for maximum absorbance and the wavelength set at283.3 nm, atomize 20-�L aliquots of the four Standard Solu-tions, using a 300-mL/min argon flow and the following se-quence of conditions: Dry at 110° for 30 s, with a 20-s rampperiod and a 10-s hold time; then char at 700° for 42 s, witha 20-s ramp period and a 22-s hold time; and then, with theargon flow stopped, atomize at 2300° for 7 s.
Plot a standard curve using the concentration, in micro-grams per milliliter, of each Standard Solution versus itsmaximum absorbance value compensated for background cor-rection as directed for the particular instrument, and draw thebest straight line.
Atomize 20 �L of the Sample Solution under identicalconditions, and measure its corrected maximum absorbance.From the Standard Curve, determine the concentration, C, inmicrograms per milliliter, of the Sample Solution. Calculatethe quantity, in milligrams per kilogram, of lead in the sampleby the formula
10C/W,
in which W is the weight, in grams, of the sample taken.
APDC Extraction Method
Select reagents having as low a lead content as practicable,and store all solutions in high-density polyethylene containers.Rinse all plastic and glassware thoroughly with warm, 1:2nitric acid followed by water.
2% APDC Solution Dissolve 2.0 g of ammonium pyrroli-dinedithiocarbamate (APDC) in 100 mL of water. Filter anyslight residue of insoluble APDC from the solution before use.
Lead Nitrate Stock Solution (100 �g/mL) Dissolve 159.8mg of reagent-grade lead nitrate [Pb(NO3)2] in 100 mL ofwater containing 1 mL of nitric acid in a 1000-mL volumetricflask, and dilute to volume with water.
Standard Lead Solution (2 �g/mL) On the day of use,transfer 2.0 mL of Lead Nitrate Stock Solution into a 100-mL volumetric flask, and dilute to volume with water.
Sample Preparation Transfer a 10.0-g sample to a clean150-mL beaker, and 10 mL of water to a second 150-mLbeaker to serve as the blank. Add to each 30 mL of waterand the minimum amount of hydrochloric acid needed todissolve the sample, plus an additional 1 mL of hydrochloricacid to ensure the dissolution of any lead present. Heat toboiling, and boil for several minutes. Allow to cool, and diluteto about 100 mL with deionized water. Adjust the pH of theresulting solution to between 1.0 and 1.5 with 25% NaOH.Quantitatively transfer the pH-adjusted solution to a clean250-mL separatory funnel, and dilute to about 200 mL withwater. Add 2 mL of 2% APDC Solution, and mix. Extractwith two 20-mL portions of chloroform, collecting the extractsin a clean 50-mL beaker. Evaporate to dryness on a steambath. Add 3 mL of nitric acid to the residue, and heat to near
872 / Appendix III / General Tests and Assays FCC V
dryness. Then add 0.5 mL of nitric acid and 10 mL of deionizedwater to the beaker, and heat until the volume is reduced toabout 3 to 5 mL. Transfer the digested extract to a clean 10-mL volumetric flask, and dilute to volume with water.
Procedure Concomitantly determine the absorbances of theStandard Lead Solution and the Sample Preparation againstthe blank at the lead emission line of 283.3 nm, using a slit-width of 0.7 nm. Use a suitable atomic absorption spectropho-tometer equipped with a lead electrode-less discharge lamp(EDL), or equivalent; an air–acetylene flame; and a 4-in.burner head. Use water as the blank. The absorbance of theSample Preparation is not greater than that of the StandardLead Solution.
MANGANESE LIMIT TEST
Manganese Detection Instrument Use any suitable atomicabsorption spectrophotometer equipped with a fast-responserecorder or other readout device and capable of measuringthe radiation absorbed by manganese atoms at the manganeseresonance line of 279.5 nm.
Standard Preparations Transfer 1000 mg, accuratelyweighed, of manganese metal powder into a 1000-mL volu-metric flask, dissolve by warming in a mixture of 10 mL ofwater and 10 mL of 0.5 N hydrochloric acid, cool, dilute tovolume with water, and mix. Pipet 5.0 mL of this solutioninto a 50-mL volumetric flask, dilute to volume with water,and mix. Finally, pipet 5.0, 10.0, 15.0, and 25.0 mL of thissolution into separate 1000-mL volumetric flasks, dilute eachflask to volume with water, and mix. The final solutionscontain 0.5, 1.0, 1.5, and 2.5 mg/kg of Mn, respectively.
Sample Preparation Transfer 10.000 g of the sample intoa 200-mL Kohlrausch volumetric flask, previously rinsed with0.5 N hydrochloric acid, add 140 mL of 0.5 N hydrochloricacid, and shake vigorously for 15 min, preferably with amechanical shaker. Dilute to volume with 0.5 N hydrochloricacid, and shake. Centrifuge approximately 100 mL of thesample mixture in a heavy-walled centrifuge tube at 2000rpm for 5 min, and use the clear supernatant liquid in thefollowing Procedure.
Procedure Aspirate 0.5 N hydrochloric acid through theair–acetylene burner for 5 min, and obtain a baseline readingat 279.5 nm, following the manufacturer’s instructions foroperating the atomic absorption spectrophotometer being usedfor the analysis. Aspirate a portion of each Standard Prepara-tion in the same manner, note the readings, then aspirate aportion of the Sample Preparation, and note the reading.Prepare a standard curve by plotting the mg/kg of Mn in eachStandard Preparation against the respective readings. Fromthe graph determine the mg/kg of Mn in the Sample Prepara-
tion, and multiply this value by 20 to obtain the mg/kg ofMn in the original sample taken for analysis.
MERCURY LIMIT TEST
Method I
Mercury Detection Instrument Use any suitable atomicabsorption spectrophotometer equipped with a fast-responserecorder and capable of measuring the radiation absorbed bymercury vapors at the mercury resonance line of 253.6 nm.A simple mercury vapor meter or detector equipped with avariable span recorder also is satisfactory.
Note: Wash all glassware associated with the test withnitric acid, and rinse thoroughly with water before use.
Aeration Apparatus The apparatus, shown in Fig. 16, con-sists of a flowmeter (a), capable of measuring flow rates from500 to 1000 mL/min, connected via a three-way stopcock (b),with a Teflon plug, to 125-mL gas washing bottles (c and d),followed by a drying tube (e), and finally a suitable quartzliquid absorption cell (f), terminating with a vent (g) to afume hood.
Note: The absorption cell will vary in optical pathlengthdepending on the type of mercury detection instru-ment used.
Bottle c is fitted with an extra-coarse fritted bubbler (Corning31770 125 EC, or equivalent), and the bottle is marked witha 60-mL calibration line. The drying tube e is lightly packedwith magnesium perchlorate. Bottle c is used for the testsolution, and bottle d, which remains empty throughout theprocedure, is used to collect water droplets.
Alternatively, an apparatus embodying the principle of theassembly described and illustrated may be used. The aeratingmedium may be either compressed air or compressed nitrogen.
Standard Preparation Transfer 1.71 g of mercuric nitrate[Hg(NO3)·H2O] into a 1000-mL volumetric flask, dissolve ina mixture of 100 mL of water and 2 mL of nitric acid, diluteto volume with water, and mix. Discard after 1 month. Transfer10.0 mL of this solution into a second 1000-mL volumetricflask, acidify with 5 mL of a 1:5 sulfuric acid solution, dilute
FIGURE 16 Aeration Apparatus for Mercury Limit Test.
FCC V General Tests and Assays / Appendix III / 873
to volume with water, and mix. Discard after 1 week. On theday of use, transfer 10.0 mL of the second solution into a100-mL volumetric flask, acidify with 5 mL of 1:5 sulfuricacid, dilute to volume with water, and mix. Each milliliter ofthis solution contains 1 �g of mercury. Transfer 2.0 mL ofthis solution (2 �g Hg) into a 50-mL beaker, and add 20 mLof water, 1 mL of a 1:5 sulfuric acid solution, and 1 mL ofa 1:25 solution of potassium permanganate. Cover the beakerwith a watch glass, boil for a few seconds, and cool.
Sample Preparation Prepare as directed in the individualmonograph.
Procedure Assemble the aerating apparatus as shown inFig. 16, with bottles c and d empty and stopcock b in thebypass position. Connect the apparatus to the absorption cell(f) in the instrument, and adjust the air or nitrogen flow rateso that in the following procedure, maximum absorption andreproducibility are obtained without excessive foaming in thetest solution. Obtain a baseline reading at 253.6 nm, followingthe manufacturer’s instructions for operating the instrument.
Treat the Standard Preparation as follows: Destroy theexcess permanganate by adding a 1:10 solution of hydroxyl-amine hydrochloride, dropwise, until the solution is colorless.Immediately wash the solution into bottle c with water, anddilute to the 60-mL mark with water. Add 2 mL of 10%stannous chloride solution (prepared fresh each week by dis-solving 10 g of SnCl2·2H2O in 20 mL of warm hydrochloricacid and diluting with 80 mL of water), and immediatelyreconnect bottle c to the aerating apparatus. Turn stopcock bfrom the bypass to the aerating position, and continue theaeration until the absorption peak has been passed and therecorder pen has returned to the baseline. Disconnect bottle cfrom the aerating apparatus, discard the Standard Preparationmixture, wash bottle c with water, and repeat the foregoingprocedure using the Sample Preparation; any absorbance pro-duced by the Sample Preparation does not exceed that pro-duced by the Standard Preparation.
Method II
Dithizone Extraction Solution Dissolve 30 mg of dithizonein 1000 mL of chloroform, add 5 mL of alcohol, and mix.Store in a refrigerator. Before use, shake a suitable volumeof the solution with about half its volume of 1:100 nitric acid,discarding the nitric acid. Discard the solution after 1 month.
Diluted Dithizone Extraction Solution Just before use, di-lute 5 mL of Dithizone Extraction Solution with 25 mL ofchloroform.
Hydroxylamine Hydrochloride Solution Dissolve 20 g ofhydroxylamine hydrochloride in sufficient water to makeabout 65 mL, transfer the solution into a separator, add a fewdrops of thymol blue TS, and then add ammonium hydroxideuntil a yellow color develops. Add 10 mL of a 1:25 solutionof sodium diethyldithiocarbamate, mix, and allow to standfor 5 min. Extract the solution with successive 10- to 15-mL portions of chloroform until a 5-mL test portion of the
chloroform extract does not develop a yellow color whenshaken with a dilute solution of cupric sulfate. Add 2.7 Nhydrochloric acid until the extracted solution is pink, addingone or two more drops of thymol blue TS, if necessary, thendilute to 100 mL with water, and mix.
Mercury Stock Solution Transfer 135.4 mg of mercuricchloride, accurately weighed, into a 100-mL volumetric flask,dissolve in and dilute to volume with 1 N sulfuric acid, andmix. Dilute 5.0 mL of this solution to 500.0 mL with 1 Nsulfuric acid. Each milliliter contains the equivalent of 10 �gof mercury.
Diluted Standard Mercury Solution On the day of use,transfer 10.0 mL of Mercury Stock Solution into a 100-mLvolumetric flask, dilute to volume with 1 N sulfuric acid,and mix. Each milliliter contains the equivalent of 1 �g ofmercury.
Sodium Citrate Solution Dissolve 250 g of sodium citratedihydrate in 1000 mL of water.
Sample Solution Dissolve 1 g of sample in 30 mL of 1.7N nitric acid by heating on a steam bath. Cool to room tempera-ture in an ice bath, stir, and filter through S and S No. 589,or equivalent, filter paper that has been previously washedwith 1.7 N nitric acid, followed by water. Add 20 mL ofSodium Citrate Solution and 1 mL of Hydroxylamine Hydro-chloride Solution to the filtrate.
Procedure (Note: Because mercuric dithizonate is light sen-sitive, perform this procedure in subdued light.) Prepare acontrol containing 3.0 mL of Diluted Standard Mercury Solu-tion (3 �g Hg), 30 mL of 1.7 N nitric acid, 5 mL of SodiumCitrate Solution, and 1 mL of Hydroxylamine HydrochlorideSolution. Treat the control and the Sample Solution as follows:Using a pH meter, adjust the pH of each solution to 1.8 withammonium hydroxide, and transfer the solutions into differentseparators. Extract each with two 5-mL portions of DithizoneExtraction Solution, and then extract again with 5 mL ofchloroform, discarding the aqueous solutions. Transfer thecombined extracts from each separator into different separa-tors, add 10 mL of 1:2 hydrochloric acid to each, shake well,and discard the chloroform layers. Extract the acid solutionswith about 3 mL of chloroform, shake well, and discard thechloroform layers. Add 0.1 mL of 0.05 M disodium EDTAand 2 mL of 6 N acetic acid to each separator, mix, andthen slowly add 5 mL of ammonium hydroxide. Stopper theseparators, and cool under a stream of cold water, and drythe outside of the separators. To avoid loss, carefully pourthe solutions through the tops of the separators into separatebeakers, and using a pH meter, adjust the pH of both solutionsto 1.8 with 6 N ammonium hydroxide. Return the sample andcontrol solutions to their original separators, add 5.0 mL ofDiluted Dithizone Extraction Solution, and shake vigorously.Any color developed in the Sample Solution does not exceedthat in the control.
874 / Appendix III / General Tests and Assays FCC V
NICKEL LIMIT TEST
Method I
Atomic Absorption System Apparatus Use a suitableatomic absorption spectrometer equipped with a nickel hollowcathode lamp and an air–acetylene flame to measure the ab-sorbance of the Blank Preparation, the Standard Prepara-tions, and the Test Preparation as directed under Procedure(below).
Test Preparation Dissolve 20.0 g of sample in strong aceticacid TS, and dilute to 150.0 mL with the same solvent. Add2.0 mL of a saturated solution of ammonium pyrrolidinedithio-carbamate (about 10 g/L of water), and 10.0 mL of methylisobutyl ketone, and shake for 30 s. Protect from bright light.Allow the two layers to separate, and use the methyl isobutylketone layer.
Blank Preparation Prepare in the same manner as in theTest Preparation, but omit the sample.
Standard Preparations Prepare three Standard Prepara-tions in the same manner as in the Test Preparation, but add0.5 mL, 1.0 mL, and 1.5 mL, respectively, of 10 mg/kg nickelstandard solution TS in addition to 20.0 g of sample.
Procedure Zero the instrument with the Blank Preparation.Concomitantly determine the absorbances of each of the Stan-dard Preparations and of the Test Preparation at least threetimes each, and record the average of the steady readings foreach. Between each measurement, aspirate the Blank Prepara-tion, and ascertain that the reading returns to its initialblank value.
Calculation Calculate the linear equation of the graph usinga least-squares fit, and derive from it the concentration ofnickel in the Test Preparation. Alternatively, plot on a graphthe mean of the readings against the added quantity of nickel.Extrapolate the line joining the points on the graph until itmeets the concentration axis. The distance between this pointand the intersection of the axes represents the concentrationof nickel in the Test Preparation.
Method II
Atomic Absorption System Apparatus Use a suitableatomic absorption spectrometer equipped with a nickel hollowcathode lamp and an air–acetylene flame to measure the ab-sorbance of the Blank Preparation, the Standard Prepara-tions, and the Test Preparation as directed under Procedure(below).
Test Preparation Dissolve 20.0 g of sample in strong aceticacid TS, and dilute to 150.0 mL with the same solvent. Add2.0 mL of a saturated solution of ammonium pyrrolidinedithio-carbamate (about 10 g/L of water) and 10.0 mL of methylisobutyl ketone, and shake for 30 s. Protect from bright light.
Allow the two layers to separate, and use the methyl isobutylketone layer.
Blank Preparation Prepare in the same manner as in theTest Preparation, but omit the sample.
Standard Preparation Prepare three Standard Prepara-tions in the same manner as in the Test Preparation, but add0.5 mL, 1.0 mL, and 1.5 mL, respectively, of 10 mg/kg nickelstandard solution TS in addition to 20.0 g of sample.
Procedure Zero the instrument with the Blank Preparation.Concomitantly determine the absorbances of each of the Stan-dard Preparations and of the Test Preparation at least threetimes each, and record the average of the steady readings foreach. Between each measurement, aspirate the Blank Prepara-tion, and ascertain that the reading returns to its initialblank value.
Calculation Calculate the linear equation of the graph usinga least-squares fit, and derive from it the concentration ofnickel in the Test Preparation. Alternatively, plot on a graphthe mean of the readings against the added quantity of nickel.Extrapolate the line joining the points on the graph until itmeets the concentration axis. The distance between this pointand the intersection of the axes represents the concentrationof nickel in the Test Preparation.
PHOSPHORUS LIMIT TEST
Reagents
Ammonium Molybdate Solution (5%) Dissolve 50 g ofammonium molybdate tetrahydrate, (NH4)6Mo7O24·4H2O, in900 mL of warm water, cool to room temperature, dilute to1000 mL with water, and mix.
Ammonium Vanadate Solution (0.25%) Dissolve 2.5 g ofammonium metavanadate, NH4VO3, in 600 mL of boilingwater, cool to 60° to 70°, and add 20 mL of nitric acid. Coolto room temperature, dilute to 1000 mL with water, and mix.
Zinc Acetate Solution (10%) Dissolve 120 g of zinc ace-tate dihydrate, Zn(C2H3O2)2·2H2O, in 880 mL of water, andfilter through Whatman No. 2V or equivalent filter paperbefore use.
Nitric Acid Solution (29%) Add 300 mL of nitric acid(sp. gr. 1.42) to 600 mL of water, and mix.
Standard Phosphorus Solution (100 �g P in 1 mL) Dis-solve 438.7 mg of monobasic potassium phosphate, KH2PO4,in water in a 1000-mL volumetric flask, dilute to volume withwater, and mix.
Standard Curve Pipet 5.0, 10.0, and 15.0 mL of the Stan-dard Phosphorus Solution into separate 100-mL volumetricflasks. To each of these flasks, and to a fourth, blank flask,add in the order stated 10 mL of Nitric Acid Solution, 10 mL
FCC V General Tests and Assays / Appendix III / 875
of Ammonium Vanadate Solution, and 10 mL of AmmoniumMolybdate Solution, mixing thoroughly after each addition.Dilute to volume with water, mix, and allow to stand for 10min. Determine the absorbance of each standard solution ina 1-cm cell at 460 nm, with a suitable spectrophotometer,using the blank to set the instrument to zero. Prepare a standardcurve by plotting the absorbance of each solution versus itsconcentration, in mg of phosphorus (P) per 100 mL.
Treated Sample Place 20 to 25 g of the starch sample in a250-mL beaker, add 200 mL of a 7:3 methanol:water mixture,disperse the sample, and agitate mechanically for 15 min.Recover the starch by vacuum filtration in a 150-mL medium-porosity fritted-glass or Büchner funnel, and wash the wetcake with 200 mL of the methanol:water mixture. Reslurrythe wet cake in the solvent, and wash it a second time in thesame manner. Dry the filter cake in an air oven at a temperaturebelow 50°, then grind the sample to 20-mesh or finer, andblend thoroughly. Determine the amount of dry substance bydrying a 5-g portion in a vacuum oven, not exceeding 100mm Hg, at 120° for 5 h.
Note: The treatment outlined above is satisfactory forstarch products that are insoluble in cold water. Forpregelatinized starch and other water-soluble starches,prepare a 1% to 2% aqueous paste, place it in a cello-phane tube, and dialyze against running distilled waterfor 30 h to 40 h. Precipitate the starch by pouring thesolution into 4 volumes of acetone per volume of pastewhile stirring. Recover the starch by vacuum filtrationin a medium-porosity fritted-glass or Büchner funnel,and wash the filter cake with absolute ethanol. Dry thefilter cake, and determine the amount of dry substanceas directed for water-insoluble starches.
Sample Preparation Transfer about 10 g of the TreatedSample, calculated on the dry-substance basis and accuratelyweighed, into a Vycor dish, and add 10 mL of Zinc AcetateSolution in a fine stream, distributing the solution uniformlyin the sample. Carefully evaporate to dryness on a hot plate,then increase the heat, and carbonize the sample on the hotplate or over a gas flame. Ignite in a muffle furnace at 550°until the ash is free from carbon (about 1 to 2 h), and cool.Wet the ash with 15 mL of water, and slowly wash down thesides of the dish with 5 mL of Nitric Acid Solution. Heat toboiling, cool, and quantitatively transfer the mixture into a200-mL volumetric flask, rinsing the dish with three 20-mLportions of water and adding the rinsings to the flask. Dilute tovolume with water, and mix. Transfer an accurately measuredaliquot (V, in mL) of this solution, containing not more than1.5 mg of phosphorus, into a 100-mL volumetric flask, andadd 50 mL of water to a second flask to serve as a blank. Toeach flask add in the order stated 10 mL of Nitric Acid Solu-tion, 10 mL of Ammonium Vanadate Solution, and 10 mL ofAmmonium Molybdate Solution, mixing thoroughly after eachaddition. Dilute to volume with water, mix, and allow to standfor 10 min.
Procedure Determine the absorbance of the Sample Prepa-ration in a 1-cm cell at 460 nm, with a suitable spectrophotom-
eter, using the blank to set the instrument at zero. From theStandard Curve, determine the mg of phosphorus in the aliquottaken, recording this value as a. Calculate the amount, in mg/kg, of phosphorus (P) in the original sample by the equation
mg/kg P = (a × 200 × 1000)/(V × W),
in which W is the weight, in g, of the sample taken.
SELENIUM LIMIT TEST
Reagents and Solutions2,3-Diaminonaphthalene Solution On the day of use, dis-
solve 100 mg of 2,3-diaminonaphthalene (C10H10N2) and 500mg of hydroxylamine hydrochloride (NH2OH·HCl) in suffi-cient 0.1 N hydrochloric acid to make 100 mL.
Selenium Stock Solution Transfer 40.0 mg of powderedmetallic selenium into a 1000-mL volumetric flask, and dis-solve in 100 mL of 1:2 nitric acid, warming gently on a steambath to effect solution. Cool, dilute to volume with water,and mix.
Selenium Standard Solution Pipet 5.0 mL of SeleniumStock Solution into a 200-mL volumetric flask, dilute to vol-ume with water, and mix. Each milliliter of this solutioncontains the equivalent of 1 �g of selenium (Se).
Method I
Standard Preparation Pipet 6.0 mL of Selenium StandardSolution into a 150-mL beaker, add 50 mL of 0.25 N nitricacid, and mix.
Sample Preparation Using a 1000-mL combustion flaskand 25 mL of 0.5 N nitric acid as the absorbing liquid, proceedas directed under Oxygen Flask Combustion, Appendix I,using the amount of sample specified in the individual mono-graph (and the magnesium oxide or other reagent, wherespecified).
Note: If the sample contains water of hydration or morethan 1% of moisture, dry it at 140° for 2 h beforecombustion, unless otherwise directed.
Upon completion of combustion, place a few milliliters ofwater in the cup or lip of the combustion flask, loosen thestopper of the flask, and rinse the stopper, sample holder, andsides of the flask with about 10 mL of water. Transfer thesolution, with the aid of about 20 mL of water, into a 150-mL beaker, heat gently to boiling, boil for 10 min, and cool.
Procedure Treat the Sample Preparation, the StandardPreparation, and 50 mL of 0.25 N nitric acid, to serve as theblank, similarly and in parallel as follows: Add a 1:2 solutionof ammonium hydroxide to adjust the pH of the solution to2.0 � 0.2. Dilute with water to 60.0 mL, and transfer to alow-actinic separator with the aid of 10.0 mL of water, addingthe 10.0 mL of rinsings to the separator. Add 200 mg of
876 / Appendix III / General Tests and Assays FCC V
hydroxylamine hydrochloride, swirl to dissolve, immediatelyadd 5.0 mL of 2,3-Diaminonaphthalene Solution, insert thestopper, and swirl to mix. Allow the solution to stand at roomtemperature for 100 min. Add 5.0 mL of cyclohexane, shakevigorously for 2 min, and allow the layers to separate. Discardthe aqueous phases, and centrifuge the cyclohexane extractsto remove any traces of water. Determine the absorbance ofeach extract in a 1-cm cell at the maximum at about 380 nmwith a suitable spectrophotometer, using the extract from theblank to set the instrument. The absorbance of the extractfrom the Sample Preparation is not greater than that fromthe Standard Preparation when a 200-mg sample is tested,or not greater than one-half the absorbance of the extract fromthe Standard Preparation when a 100-mg sample is tested.
Method II
Standard Preparation Pipet 6.0 mL of Selenium StandardSolution into a 150-mL beaker, add 50 mL of 2 N hydrochloricacid, and mix.
Sample Preparation Transfer the amount of sample speci-fied in the individual monograph into a 150-mL beaker, dis-solve in 25 mL of 4 N hydrochloric acid, swirling if necessaryto effect solution, heat gently to boiling, and digest on a steambath for 15 min. Remove from heat, add 25 mL of water, andallow to cool to room temperature.
Procedure Place the beakers containing the Standard Prep-aration and the Sample Preparation in a fume hood, and toa third beaker, add 50 mL of 2 N hydrochloric acid to serveas the blank. Cautiously add 5 mL of ammonium hydroxideto each beaker, mix, and allow the solution to cool. Treateach solution, similarly and in parallel, as directed underProcedure in Method I, beginning with ‘‘Add a 1:2 solutionof ammonium hydroxide. . . . ’’
C. OTHERS
ALGINATES ASSAY
In a suitable closed system, liberate the carbon dioxide fromthe uronic acid groups of about 250 mg of the test sample byheating with hydrochloric acid, and sweep the carbon dioxide,by means of an inert gas, into a titration vessel containingexcess standardized sodium hydroxide. Any suitable systemmay be used as long as it provides precautions against leakageand overheating of the reaction mixture, adequate sweepingtime, avoidance of entrainment of hydrochloric acid, andmeets the requirements of the System Suitability Test. Onesuitable system, with accompanying procedure, is givenbelow.
Apparatus The apparatus is shown in Fig. 17. It consistsessentially of a soda lime column, A, a mercury valve, B,connected through a side arm, C, to a reaction flask, D, bymeans of a rubber connection. Flask D is a 100-mL round-bottom, long-neck boiling flask, resting in a suitable heatingmantle, E.
The reaction flask is provided with a reflux condenser, F,to which is fitted a delivery tube, G, of 40-mL capacity,having a stopcock, H. The reflux condenser terminates in atrap, I, containing 25 g of 20-mesh zinc or tin, which can beconnected with an absorption tower, J.
The absorption tower consists of a 45-cm tube fitted witha medium-porosity fritted glass disk sealed to the inner partabove the side arm and having a delivery tube sealed to itextending down to the end of the tube. A trap, consisting ofa bulb of approximately 100-mL capacity, is blown above thefritted disk and the outer portion of a ground spherical jointis sealed on above the bulb. A 250-mL Erlenmeyer flask, K,is connected to the bottom of the absorption tower. The topof the tower is connected to a soda lime tower, L, which isconnected to a suitable pump to provide vacuum and airsupply, the choice of which is made by a three-way stopcock,M. The volume of air or vacuum is controlled by a capillary-tube regulator or needle valve, N.
All joints are a size 35/25 ground spherical type.
Standard D-Glucurono-6,3-lactone This chemical (C6H8O6)is available as a reference standard with an assay of 100.0 �1.0% (24.99 � 0.25% CO2) from Aldrich Chemical Co.
FIGURE 17 Apparatus for Alginates Assay.
FCC V General Tests and Assays / Appendix III / 877
System Suitability Test Transfer about 250.0 mg of Stan-dard D-Glucurono-6,3-lactone, accurately weighed, into thereaction flask, D, and carry out the Procedure described be-low. The system is considered suitable when the net titrationresults in a calculation of %CO2 in a range of 24.73 to 25.26,which is equivalent to a range of 98.95 to 101.06% D-Glucur-ono-6,3-lactone.
Procedure Transfer about 250 mg of sample, accuratelyweighed, into the reaction flask, D, add 25 mL of 0.1 Nhydrochloric acid, insert several boiling chips, and connectthe flask to the reflux condenser, F, using syrupy phosphoricacid as a lubricant.
Note: Stopcock grease may be used for the other con-nections.
Check the system for air leaks by forcing mercury up intothe inner tube of the mercury valve, B, to a height of about5 cm. Turn off the pressure using the stopcock, M. If themercury level does not fall appreciably after 1 to 2 min, theapparatus may be considered to be free from leaks. Drawcarbon dioxide-free air through the apparatus at a rate of 3000to 6000 mL/h. Raise the heating mantle, E, to the flask, heatthe sample to boiling, and boil gently for 2 min. Turn off andlower the mantle, and allow the sample to cool for 15 min.Charge the delivery tube, G, with 23 mL of hydrochloric acid.Disconnect the absorption tower, J, rapidly transfer 25.0 mLof 0.25 N sodium hydroxide into the tower, add 5 drops ofn-butanol, and reconnect the absorption tower. Draw carbondioxide-free air through the apparatus at the rate of about2000 mL/h, add the hydrochloric acid to the reaction flaskthrough the delivery tube, raise the heating mantle, and heatthe reaction mixture to boiling. After 2 h, discontinue thecurrent of air and heating. Force the sodium hydroxide solutiondown into the flask, K, using gentle air pressure, and thenrinse down the absorption tower with three 15-mL portionsof water, forcing each washing into the flask with air pressure.Remove the flask, and add to it 10 mL of a 10% solution ofbarium chloride (BaCl2·2H2O). Stopper the flask, shake gentlyfor about 2 min, add phenolphthalein TS, and titrate with0.1 N hydrochloric acid. Perform a blank determination (seeGeneral Provisions). Each milliliter of 0.25 N sodium hydrox-ide consumed is equivalent to 5.5 mg of carbon dioxide (CO2).Calculate the results on the dried basis.
�-AMINO NITROGEN (AN)DETERMINATION
Transfer 7 to 25 g of sample, accurately weighed, into a 500-mL volumetric flask with the aid of several 50-mL portionsof warm, ammonia-free water, dilute to volume with water,and mix. Neutralize 20.0 mL of the solution with 0.2 N bariumhydroxide or 0.2 N sodium hydroxide, using phenolphthaleinTS as the indicator, and add 10 mL of freshly prepared phenol-phthalein–formol solution (50 mL of 40% formaldehyde con-
taining 1 mL of 0.05% phenolphthalein in 50% alcohol neu-tralized exactly to pH 7 with 0.2 N barium hydroxide or 0.2N sodium hydroxide). Titrate with 0.2 N barium hydroxideor 0.2 N sodium hydroxide to a distinct red color, add a small,but accurately measured, volume of 0.2 N barium hydroxideor 0.2 N sodium hydroxide in excess, and back titrate toneutrality with 0.2 N hydrochloric acid. Conduct a blanktitration using the same reagents, with 20 mL of water inplace of the test solution. Each milliliter of 0.2 N bariumhydroxide or 0.2 N sodium hydroxide is equivalent to 2.8 mgof �-amino nitrogen.
AMMONIA NITROGEN (NH3-N)DETERMINATION
Caution: Provide adequate ventilation.
Note: Use nitrogen-free reagents, where available, orreagents very low in nitrogen content.
Transfer between 700 mg and 2.2 g of sample into a 500- to800-mL Kjeldahl digestion flask of hard, moderately thick,well-annealed glass. If desired, wrap the sample, if solid orsemisolid, in nitrogen-free filter paper to facilitate the transfer.
Add about 200 mL of water, and mix. Add a few granulesof zinc to prevent bumping, tilt the flask, and cautiously poursodium hydroxide pellets, or a 2:5 sodium hydroxide solution,down the inside of the flask so that it forms a layer under thesolution, using a sufficient amount (usually about 25 g ofsolid sodium hydroxide) to make the mixture strongly alkaline.Immediately connect the flask to a distillation apparatus con-sisting of a Kjeldahl connecting bulb and a condenser thathas a delivery tube extending well beneath the surface of ameasured excess of 0.5 N hydrochloric or sulfuric acid con-tained in a 500-mL flask. Add 5 to 7 drops of methyl redindicator (1 g of methyl red in 200 mL of alcohol) to thereceiver flask. Rotate the Kjeldahl flask to mix its contentsthoroughly, and heat until all of the ammonia has distilled,collecting at least 150 mL of distillate. Wash the tip of thedelivery tube, collecting the washings in the receiving flask,and titrate the excess acid with 0.5 N sodium hydroxide.Perform a blank determination (see General Provisions), sub-stituting 2 g of sucrose for the sample, and make any necessarycorrection. Each milliliter of 0.5 N acid consumed is equivalentto 7.003 mg of ammonia nitrogen.
Note: If it is known that the substance to be determinedhas a low nitrogen content, 0.1 N acid and alkali maybe used, in which case each milliliter of 0.1 N acidconsumed is equivalent to 1.401 mg of nitrogen.
Calculate the percent ammonia nitrogen by the formula
(NH3-N/S) × 100,
in which NH3-N is the weight, in milligrams, of ammonianitrogen, and S is the weight, in milligrams, of sample.
878 / Appendix III / General Tests and Assays FCC V
BENZENE (in Paraffinic Hydrocarbon Solvents)
Apparatus (See Chromatography, Appendix IIA.) Use asuitable gas chromatograph, equipped with a column, orequivalent, that will elute n-decane before benzene under theconditions of the System Suitability Test (below). Columnmaterials and conditions that have been found suitable forthis method are listed in the accompanying tables. See Fig.18 for a typical chromatogram obtained with column No. 5.
ReagentsIsooctane 99 mole percent minimum containing less than
0.05 mole percent aromatic material.Benzene 99.5 mole percent minimum.Internal Standard n-Decane and either n-undecane or n-
dodecane according to the requirement of the System Suitabil-ity Test.
Reference Solution A Prepare a standard solution con-taining 0.5% by weight each of the Internal Standard and ofbenzene in isooctane.
Reference Solution B Prepare a standard solution con-taining about 0.5% by weight each of n-decane, of InternalStandard, and of benzene in isooctane.
Calibration Select the instrument conditions necessary togive the desired sensitivity. Inject a known volume of Refer-ence Solution A, and change the attenuation, if necessary, sothat the benzene peak is measured with a chart deflection ofnot less than 25% or more than 95% of full scale. Whenchoosing the attenuation, consider all unresolved peaks torepresent a single compound. There may be tailing of thenonaromatic peak, but do not use any conditions that lead to
FIGURE 18 Typical Chromatogram for the Determination ofBenzene in Hexanes Using Column No. 5.
FIGURE 19 Illustration of A/B Ratio.
a depth of the valley ahead of the benzene peak (A) less than50% of the weight of the benzene peak (B) as depicted inFig. 19.
If there is tailing of the nonaromatic material, construct abaseline by drawing a line from the bottom of the valleyahead of the benzene peak to the point of tangency after thepeak (see Fig. 20). Measure the areas of the benzene peakand the internal standard peak by any of the following means:triangulation, planimeter, paper cutout, or mechanical or elec-tronic integrator. Do not use integrators on peaks without aconstant baseline, unless the integrator has provision for mak-ing baseline corrections with accuracy at least as good as thatof manual methods.
Calculate a response factor for benzene (Rb) relative to theInternal Standard by the formula
Ai/Wi × Bv/Ab,
in which Ai is the area of the Internal Standard peak inarbitrary units corrected for attenuation; Wi is the weightpercent of Internal Standard in Reference Solution A; Ab isthe area of the benzene peak in arbitrary units corrected forattenuation; and Wb is the weight percent of benzene in Refer-ence Solution A.
Procedure Place approximately 0.1 mL of Internal Stan-dard into a tared 25-mL volumetric flask, weigh on an analyti-cal balance, dissolve in and dilute to volume with the sampleto be analyzed.
FIGURE 20 Illustration of A/B Ratio for a Small ComponentPeak on the Tail of a Large Peak.
FCC V General Tests and Assays / Appendix III / 879
Using the exact instrumental conditions that were used inthe calibration, inject the same volume of sample containingthe Internal Standard. Before measuring the area of the Inter-nal Standard and benzene peaks, change the attenuation toensure at least 25% chart deflection.
Measure the area of the Internal Standard and benzenepeaks in the same manner as was used for the calibration.Calculate the weight percent of benzene in the sample (WB)by the formula
(Ab × Rb × Wi × 100)/(Ai × S),
in which Ab is the area of the benzene peak corrected forattenuation; Rb is the relative response factor for benzene; Wi
Column Materials and Conditions for the Determination of Benzene in Hexanes
Column No. 1 2 3 4 5 6 7
Liquid phase CEF PEF 200 CEF DEGS TCEPE TCEPE DEGSLength, ft 15 6 16 10 15 100 12
m 4.5 2 5 3.1 — 313.7Diameter, in (mm)
Inside 0.07(1.8) — 0.07 0.18(4.5) 0.06(1.5) 0.01(.254)Outside 1⁄8(3.2) 1⁄4(6.4) 1⁄8 — — — 1⁄8
Weight, percent 17 30 20 20 10 — 20Solid support Chromosorb P Chromosorb P Chromosorb P Chromosorb P Chromosorb P Capillary Chromosorb PMesh 60–80 60–80 60–80 80–100 60–80 — 80–100Treatment AW AW AW none AW none AW SilInlet, deg 200 210 250 260 250 275 260Detector, deg 200 155 250 200 175 250 240Column, deg 115 95 90 100 115 95 65Carrier gas N2 He He He N2 N2 HeFlow rate, cm3/min30 60 60 60 1 3 52Detector FI TC FI FI FI FI FIRecorder, mV 5 1 1 1 10 1 1Sample, 1 5 10 1 2 5 0.8 5Split 9 + 1 — — — 100 + 1 100 – 1 —Area Tri EI DI Tri Plan EI EI Tri
Abbreviations Used in TableAW—Acid washed; CEF—N,N-Bis(2-cyanoethyl)formamide; DEGS—Diethylene Glycol Succinate; DI—Disk integrator; EI—Electronicintegrator; FI—Flame ionization; Sil—Silanized; TC—Thermal conductivity; TCEPE—Tetracyanoethylated Pentaerythritrol;Tri—Triangulation.
Retention Times in Minutes for Selected Hydrocarbons Under the Conditions for the Determination of Benzene inHexanes
Column No. 1 2 3 4 5 6 7
Benzene 3.4 2.0 6.5 6.7 5.4 6.1 6.7Toluene 4.4 3.2 9.0 10.3 7.8 7.0 10.3Ethylbenzene 5.4 5.2 11.5 14.8 10.8 8.0 14.8p-m-Xylenes 5.8 — 12.5 — 11.4 8.5 —o-Xylene 7.5 6.8 17.0 16.1 14.5 10.0 —n-Undecane 3.0 2.8 3.5 — — — —n-Dodecane — — — 12.8 8.5 6.5 —
is the weight, in grams, of Internal Standard added; Ai is thearea of the Internal Standard peak corrected for attenuation;and S is the weight, in grams of the sample taken.
System Suitability Test Inject the same volume of Refer-ence Solution B as in the Calibration and record the chromato-gram. n-Decane must be eluted before benzene, and the ratioof A to B (Fig. 19) must be at least 0.5 where A is equal tothe depth of the valley between the n-decane and benzenepeaks and B is equal to the height of the benzene peak.
880 / Appendix III / General Tests and Assays FCC V
COLORS1
Chromium
StandardsStandard Chromium Solution (1000 mg/kg) Transfer
2.829 g of K2Cr2O7, accurately weighed (National Instituteof Standards and Technology No. 136) into a 1-L volumetricflask; dissolve in and dilute to volume with water.
Standard Colorant Solution Transfer 62.5 g of colorantpreviously shown to be free of chromium to a 1-L volumetricflask; dissolve in and dilute to volume with water.
Apparatus Use any suitable atomic absorption spectropho-tometer equipped with a fast response recorder and capableof measuring the radiation absorbed at 357.9 nm.
Instrument Parameters Wavelength setting: 357.9 nm; op-tical passes: 5; lamp current: 8 mA; lamp voltage: 500 v;fuel: hydrogen; oxidant: air; recorder: l mv with a scale expan-sion of 5 or 10. Alternatively, follow the instructions suppliedwith the instrument.
Procedure Set the instrument at the optimum conditionsfor measuring chromium as directed by the manufacturer’sinstructions. Prepare a series of seven standard chromiumsolutions containing Cr at approximately 5, 10, 15, 20, 40,50, and 60 mg/kg by appropriate dilutions of the StandardChromium Solution into 100-mL volumetric flasks; add 80mL of the Standard Colorant Solution, and dilute each flaskto volume with water.
Transfer 5 g of the colorant to be analyzed to a 100-mLvolumetric flask; dissolve in and dilute to volume with water.Prepare a calibration curve using the series of standards, andusing this curve, determine the chromium content of the color-ant samples.
Ether Extracts
Caution: Isopropyl ether forms explosive peroxides.To ensure the absence of peroxides, perform the follow-ing test: Prepare a colorless solution of ferrous thiocya-nate by mixing equal volumes of 0.1 N ferrous sulfateand 0.1 N ammonium thiocyanate. Using titanous chlo-ride, carefully discharge any red coloration due to ferricions. Add 10 mL of ether to 50 mL of the solution, andshake vigorously for 2 to 3 min. A red color indicates thepresence of peroxides. If redistillation is necessary, theusual precautions against peroxide detonation shouldbe observed. Immediately before use, pass the etherthrough a 30-cm column of chromatography-grade alu-minum oxide to remove peroxides and inhibitors.
1To be used or sold for use to color food that is marketed in the UnitedStates, color additives must be from batches that have been certified bythe U.S. Food and Drug Administration (FDA). If color additives are notfrom FDA-certified batches, they are not permitted color additives forfood use in the United States, even if they are compositionally equivalent.The FD&C names can be applied only to FDA-certified batches of thesecolor additives.
FIGURE 21 Upward Displacement-Type Liquid–LiquidExtractor with Sintered-Glass Diffuser.
Apparatus Use an upward displacement-type liquid–liquidextractor, as shown in Fig. 21, with a sintered-glass diffuserand a working capacity of 200 mL. Suspend a piece of brightcopper wire through the condenser, and place a small coil ofcopper wire (about 0.5 g) in the distillation flask.
Alkaline Ether Extract Transfer 5 g of the colorant to abeaker, and dissolve in 150 mL of water. Add 2 mL of 2.5N NaOH solution, transfer the solution into the extractor; anddilute to approximately 200 mL with water. Add 200 mL ofether to the distillation flask, and extract for 2 h with a refluxrate of about 15 mL/min. Set the extracted colorant solutionaside. Transfer the ether extract into a separatory funnel, andwash with two 25-mL portions of 0.1 N NaOH followed bytwo 25-mL portions of water. Reduce the volume of the etherextract to about 5 mL by distillation (in portions) from a taredflask containing a small piece of clean copper coil.
Acid Ether Extract Add 5 mL of 3 N hydrochloric acid tothe extracted colorant solution set aside in the alkaline etherextract procedure above, mix, and extract with ether as di-rected above. Wash the ether extract with two 25-mL portionsof 0.1 N hydrochloric acid and water. Transfer the washedether in portions to the flask containing the evaporated alkalineextract, and carefully remove all the ether by distillation. Drythe residue in an oven at 85° for 20 min. Then allow the flask
FCC V General Tests and Assays / Appendix III / 881
to cool in a desiccator for 30 min, and weigh. Repeat dryingand cooling until a constant weight is obtained. The increasein weight of the tared flask, expressed as a percentage of thesample weight, is the combined ether extract.
Leuco Base
Reagents and SolutionsCupric Chloride Solution Transfer 10.0 g of CuCl2·2H2O
to a 1-L volumetric flask; dissolve in and dilute to volumewith dimethylformamide (DMF).
Sample Solution Prepare as directed in the individualmonograph.
ProcedureSolution 1 Pipet 50 mL of DMF into a 250-mL volumetric
flask, cover, and place in the dark.Solution 2 Pipet 10 mL of the Sample Solution into a
250-mL volumetric flask, add 50 mL of DMF, and place inthe dark.
Solution 3 Pipet 50 mL of Cupric Chloride Solution intoa 250-mL volumetric flask, and gently bubble air through thesolution for 30 min.
Solutions 4a and 4b Pipet 10 mL of the Sample Solutioninto each of two 250-mL volumetric flasks, add 50 mL ofCupric Chloride Solution to each, and bubble air gentlythrough the solutions for 30 min.
Dilute all of the solutions nearly to volume with water;incubate for 5 to 10 min, but no longer, in a water bath cooledwith tap water; and dilute to volume. Record the spectrumfor each solution between 500 nm and 700 nm using anabsorbance range of 0 to 1 and a 1-cm pathlength cell; recordall spectra on the same spectrogram.
Solution in Solution inCurve No. Sample Cell Reference Cell
I 1 1II 1 2III 3 3IVa 3 4aIVb 3 4b
Calculation
% Leuco Base =[(IV − III) − (II − I)] × 2500
a × W × r,
in which the Roman numerals I through IV represent theabsorbance readings for solutions of the corresponding Arabicnumerals (above) at the wavelength maximum; a is the absorp-tivity (for Fast Green, a = 0.156 at 625 nm; for Brillant Blue,a = 0.164 at 630 nm); W is the weight, in grams, of the sampletaken; and r is the ratio of the molecular weights of colorantand leuco base (for Fast Green, r = 0.9712; for Brillant Blue,r = 0.9706).
Mercury
Apparatus The apparatus used for the direct microdetermi-nation of mercury is shown in Fig. 22. It consists of a quartzcombustion tube designed to hold a porcelain combustionboat (60 × 10 × 8 mm) and a small piece of copper oxidewire. The combustion tube is placed in a heavy-duty hingedcombustion tube furnace (Lindburg Type 70T, or equivalent),and it is connected by clamped ball-joints at one end to asource of nitrogen and connected to a series of three traps atthe other. The traps are constructed of a linear array of 18-× 2-mm Pyrex tubes connected by clamped ball-joints andextend from the connection at the combustion tube. Trap Icontains anhydrous calcium sulfate packed between quartz-wool plugs, trap II contains Ascarite packed between cottonplugs, and trap III contains aluminum oxide packed betweencotton plugs. The nitrogen flow forces the mercury throughthe combustion tube, the three traps, and a section of Tygontube to a mercury vapor meter (Beckman model K-23, orequivalent). The mercury released from a sample during com-bustion is quantitated by comparing the recorder responsewith that given by a series of mercury standards.
FIGURE 22 (a) Schematic Diagram of Apparatus forPhotometric Mercury Vapor M Method:
A. Tank of nitrogen G. Dehydrite trapB. Two-stage pressure regulator H. Ascarite trapC. Low-pressure regulator I. Aluminum oxide trapD. Flowmeter J. Mercury vapor meterE. Combustion tube K. AtenuatorF. Combustion-tube furnace L. Recorder
(b) Quartz CombustionTube with Boat andCopper Oxide Packing;(c) Schematic Diagram of Trap Used to Contain Ascarite,Dehydrite, and Aluminum Oxide.
882 / Appendix III / General Tests and Assays FCC V
Reagents and EquipmentAbsorbent CottonAluminum Oxide Anhydrous.Calcium Sulfate Anhydrous, dehydrate, or equivalent.Asbestos Pads, (1 × 0.5 × 1 cm) Preheated at 800° for 1 h.Ascarite 20- to 30-mesh.Copper Oxide Wire Preheated at 850° for 2 h.Nitrogen Purified grade.Quartz WoolSodium Carbonate Anhydrous, fine granular.Standard Solution Transfer approximately 1.35 g of re-
agent-grade mercurous chloride, accurately weighed, into a1-L volumetric flask. Dissolve in and dilute to volume withwater. When diluted 100-fold, the solution contains 0.01 �gHg per microliter (Diluted Standard Solution).
Procedure Preheat the furnace to 650°, and adjust the nitro-gen flow to 1 L/min.
Blank Analysis Place a square piece of preheated asbestospad in the combustion boat, and cover it with sodium carbon-ate. Stop the nitrogen flow, disconnect the ball-joint, quicklyinsert the boat into the combustion tube with large forceps,and reconnect the joint. Note the time, allow the boat to sitin the tube with no nitrogen flow for exactly 1 min, and thenrestart the flow of nitrogen. Mercury elutes almost immedi-ately with the reinstated nitrogen flow; note the recorder re-sponse. Allow about 30 s between runs.
Calibration Determine the recorder response after the ap-plication to the asbestos pad of 1, 2, and 3 �L of the DilutedStandard Solution.
Sample Analysis Transfer 25 mg of colorant, accuratelyweighed, to the combustion boat, and cover the sample com-pletely with sodium carbonate. Follow the procedure used forthe Blank Analysis above, and calculate the mercury contentusing the standard curve.
Trap Problems (1) Some colorants (e.g., Brillant Blue andFast Green) may give a response that is symmetrically dissimi-lar to the Hg peak. If such a response ‘‘carries over’’ to thenext sample, then the aluminum oxide trap may need to bechanged. (2) If the recorder response is of inadequate sensitiv-ity (peak height induced by 0.01 �g less than 0.5 cm), thenthe traps are packed too tightly. Remove or redistribute pack-ing first in the aluminum oxide trap, then try the other traps.(3) The traps will need changing periodically as indicated bya change in the physical appearance of the trap material orby chart responses of different retention times or differentsymmetry from that of mercury standards. (4) If two or morestandards are run in succession, a later sample might give anerroneous mercury response. Run blanks and then repeat thesample analysis to confirm the validity of the response.
Sodium Chloride
Dissolve approximately 2 g of colorant, accurately weighed,in 100 mL of water, and add 10 g of activated carbon that isfree of chloride and sulfate. Boil gently for 2 to 3 min. Coolto room temperature, add 1 mL of 6 N nitric acid, and stir.Dilute to volume with water in a 200-mL volumetric flask,and then filter through dry paper. Repeat the treatment with
2-g portions of carbon until no color is adsorbed onto filterpaper dipped into the filtrate.
Transfer 50 mL of filtrate to a 250-mL flask. Add 2 mLof 6 N nitric acid, 5 mL of nitrobenzene, and 10 mL ofstandardized 0.1 N silver nitrate solution. Shake the flask untilthe silver chloride coagulates. Prepare a saturated solution offerric ammonium sulfate, and add just enough concentratednitric acid to discharge the red color; add 1 mL of this solutionto the 250-mL flask to serve as the indicator. Titrate with 0.1N ammonium thiocyanate solution that has been standardizedagainst the silver nitrate solution until the color persists aftershaking for 1 min. Calculate the weight percent of sodiumchloride, P, by the equation
P = [(V × N)/W] × 22.79,
in which V is the net volume, in milliliters, of silver nitratesolution required; N is the normality of the silver nitratesolution; and W is the weight, in grams, of the sample taken.The factor 22.79 incorporates a total volume of 195 mL be-cause 10 g of activated carbon occupies 5 mL.
Sodium Sulfate
Place 25 mL of the decolorized filtrate obtained from theSodium Chloride test (above) into a 125 mL Erlenmeyer flask,and add 1 drop of a 0.5% phenolphthalein solution in 50%ethanol. Add 0.05 N sodium hydroxide, dropwise, until thesolution is alkaline to pH paper, and then add 0.002 N hydro-chloric acid until the indicator is decolorized. Add 25 mL ofethanol and about 0.2 g of tetrahydroquinone sulfate indicator.Titrate with 0.03 N barium chloride solution to a red endpoint.Make a blank determination.
Calculate the weight percent, P, of sodium sulfate by theequation
P = [(V − B) × N/W] × 55.4,
in which V is the volume, in milliliters, of barium chloridesolution required to titrate the sample; B is the volume, inmilliliters, of barium chloride solution required for the blank;N is the normality of the barium chloride solution; and W isthe weight, in grams, of the sample taken. The factor 55.4incorporates a total volume of 195 mL because 10 g of acti-vated carbon occupies 5 mL.
Total Color
Method I (Spectrophotometric)Pipet 10.0 mL of the dissolved colorant into a 250-mL Erlen-meyer flask containing 90 mL of 0.04 N ammonium acetate,and mix well. Determine the net absorbance of the solutionrelative to water at the wavelength maximum given for eachcolor. Calculate the percentage of colorant present using thefollowing equation, which presumes a 1-cm pathlength cell:
% total color = (A × 100)/(a × W),
in which A is the absorbance; a is the absorptivity; and W isthe weight, in grams, of the sample taken.
Method II (Titration with Titanium Chloride)
Apparatus The apparatus for determining total color bytitration with titanium chloride (TiCl3) is shown in Fig. 23.
FCC V General Tests and Assays / Appendix III / 883
It consists of a storage bottle, A, of 0.1 N titanium chloridetitrant maintained under hydrogen produced by a Kipp genera-tor; an Erlenmeyer flask, B, equipped with a source of CO2
or N2 to maintain an inert atmosphere in which the reactiontakes place; a stirrer; and the buret, C.
Reagents and SolutionsTitanium Chloride Solution (0.1 N) Transfer 73 mL of
commercially prepared 20% TiCl3 solution into a storagebottle, and carefully add 82 mL of concentrated HCl per Lof final solution. Mix well, and bubble CO2 or N2 throughthe solution for 1 h. Before standardizing, maintain the solu-tion under a hydrogen atmosphere for at least 16 h using aKipp generator.
Potassium Dichromate Solution (0.1 N, primary stan-dard) Transfer 4.9032 g of K2Cr2O7 (National Institute ofStandards and Technology No. 136) to a 1-L volumetric flask;dissolve in and dilute to volume with water.
Ammonium Thiocyanate (50%) Transfer 500 g ofNH4SCN, ACS certified, to a 1-L volumetric flask; dissolvein about 600 mL of water, warming if necessary; and diluteto volume.
Ferrous Ammonium Sulfate Fe(NH4)2(SO4)2·6H2O, ACScertified.
Sodium Bitartrate
Standardization of the Titanium Chloride Solution Drainany standing titanium chloride (TiCl3) from the feed lines andburet, and refill with fresh solution. Add 3.0 g of FerrousAmmonium Sulfate to a wide-mouth Erlenmeyer flask fol-lowed by 200 mL of water, 25 mL of 50% sulfuric acid, 25mL of 0.1 N Potassium Dichromate Solution (by pipet), and2 or 3 boiling chips. Boil the solution vigorously on a hotplate for 30 s to remove dissolved air, then quickly transferthe flask to the titration apparatus, securely connect the stopperassembly, and start the carbon dioxide flow and stirrer. Passcarbon dioxide over the solution for 1 min before beginningthe titration.
Add the 0.1 N Titanium Chloride Solution at a fast, steadydrip to within 1 mL of the estimated endpoint (about 20 mL).Reduce the carbon dioxide flow, remove the solid-glass rodfrom the stopper assembly, pipet 10 mL of Ammonium Thiocy-anate (50%) into the flask, insert the glass rod, and increasethe carbon dioxide flow. Continue titrating slowly until theendpoint: A color change from brown-red to light green isobserved. Perform a blank determination using the same re-agents and quantities, and calculate the normality, N, of the 0.1N Titanium Chloride Solution on the basis of three titrations bythe equation
N = (Vr × Nr/Vt − Vb),
in which Vr is the volume, in milliliters, of 0.1 N PotassiumDichromate used; Nr is the normality of the 0.1 N PotassiumDichromate; Vt is the volume, in milliliters, of 0.1 N TitaniumChloride Solution used; and Vb is the volume, in milliliters,of titanium dichloride used in the blank titration.
Procedure Transfer the quantity of colorant prescribed inthe individual monograph into a 500-mL wide-mouth Erlen-
FIGURE 23 Titanous Chloride Titration Apparatus.
meyer flask and add 21 to 22 g of Sodium Bitartrate (sodiumcitrate for Sunset Yellow), 275 mL of water, and two or threeboiling chips. Boil the solution vigorously on a hot plate for30 s to remove dissolved air, then quickly transfer the flaskto the titration apparatus, securely connect the stopper assem-bly, and start the carbon dioxide flow and stirrer. Pass carbondioxide over the solution for 1 min before beginning thetitration.
Titrate the sample until the color lightens, wait 20 s, andthen continue the addition with about 2 s between drops.When the color is almost completely bleached, wait 20 s, andthen continue the addition with 5 s between drops. A completecolor change indicates the endpoint. Perform a blank determi-nation using the same reagents and quantities, and calculatethe total color, T, in percent and on the basis of three titrations,by the equation
T = [(Vt − Vb)/(W × Fs)] × 100 × N,
in which Vt is the volume of titrant used; Vb is the volumeof titrant required to produce the endpoint in a blank; N isthe normality of the titrant; W is the weight, in grams, of thesample taken, and Fs is a factor derived from the stoichiometry
884 / Appendix III / General Tests and Assays FCC V
of the reaction characteristics of each colorant and is givenin the individual monograph.
Method III (Gravimetric)
Transfer approximately 0.5 g of colorant, accurately weighed,to a 400-mL beaker, add 100 mL of water, and heat to boiling.Add 25 mL of 1:50 hydrochloric acid, and bring to a boil.Wash down the sides of the beaker with water, cover, andkeep on a steam bath for several hours or overnight. Cool toroom temperature, and quantitatively transfer the precipitateinto a tared filtering crucible with 1:100 hydrochloric acid.Wash the precipitate with two 15-mL portions of water, anddry the crucible for 3 h at 135°. Cool in a desiccator, andweigh. Calculate the total color, P, in weight percent, by theequation
P = [(Wp × F)/Ws] × 100,
in which Wp is the weight, in grams, of the precipitate; Fis the gravimetric conversion factor given in the individualmonograph; and Ws is the original weight, in grams, of thesample taken.
Uncombined Intermediates and Products of SideReactions
Method I
Sample Solution Transfer approximately 2 g of colorant toa 100-mL volumetric flask; dissolve in and dilute to volumewith water.
Apparatus Pack a 2.5- × 45-cm glass column with approxi-mately 20 g of cellulose (Whatman CF-11 grade, or equiva-lent) that has been slurried in the eluant and from whichthe fines have been removed by decantation. Equilibrate thecolumn thoroughly with the eluant, 35% ammonium sulfate.
Procedure Pipet 5 mL of Sample Solution into a beakercontaining 5 g of cellulose that has been slurried in eluantand from which the fines have been removed by decantation.Stir the mixture thoroughly, add 10 g of ammonium sulfate,and stir until uniformly mixed. Mix the slurry with 15 mL ofeluant, and apply it to the column. Allow the fluid to enterthe column, and wash the beaker with eluant until the sampleis quantitatively transferred. Elute the column with approxi-mately 500 mL of 35% ammonium sulfate, and collect a totalof eight 60-mL fractions. Divide each collected fraction inhalf and add 0.5 mL of NH4OH to one half and 0.5 mL ofHCl to the other.
Calculation After identifying each intermediate and sideproduct by comparing spectra of the fractions with commercialstandards, calculate the concentration, C, of each using theequation
C = A/(a × b),
in which A is the absorbance at the wavelength of maximalabsorption; b is the cell pathlength, in centimeters; and a isthe absorptivity given in the individual monograph.
Method II
Apparatus Use a suitable high-performance liquid chroma-tography system (see Chromatography, Appendix IIA)equipped with a dual wavelength detector system such thatthe effluent can be monitored serially at 254 nm and 325 to385 nm (wide-band pass). Use a 1-m × 2.1-mm (id) column,or equivalent, packed with a strong anion-exchange resin (Du-pont No. 830950405, or equivalent).
Operating Conditions The operating conditions requiredmay vary depending on the system used. The following condi-tions have been shown to give suitable results for Allura Red,Tartrazine, and Sunset Yellow.
Allura RedPrimary Eluant: 0.01 M aqueous Na2B4O7.Secondary Eluant: 0.20 M NaClO4 in aqueous 0.01 M
Na2B4O7.Sample Size: 20 �L of a 0.25% solution.Flow Rate: 0.60 mL/min.Gradient: Linear, in two phases: 0% to 18% in 40 min,
18% to 62% in 8 min more, then hold for 18 min more at 62%.Temperature: 50°.Pressure: 1000 psi.Order of Elution: (1) Cresidinesulfonic acid (CSA); (2)
unknown; (3) Schaeffer’s salt (SS); (4) unknown; (5) 4,4′-diazoaminobis(5-methoxy-2-methylbenzenesulfonic acid)(DMMA); (6) unknown; (7) Allura Red; (8) 6,6′-oxybis(2-naphthalenesulfonic acid) (DONS).
TartrazinePrimary Eluant: 0.01 M aqueous Na2B4O7.Secondary Eluant: 0.10 M NaClO4 in aqueous 0.01 M
Na2B4O7.Sample Size: 50 �L of a 0.15% solution, prepared within
13 min of injection.Flow Rate: 1.00 mL/min.Gradient: Exponential at 4%/min: 0.95%.Temperature: 50°.Pressure: 1000 psi.Order of Elution: (1) Phenylhydrazine-p-sulfonic acid
(PHSA); (2) sulfanilic acid (SA); (3) 1-(4-sulfophenyl)-3-ethylcarboxy-5-hydroxypyrazolone (PY-T); (4) 1-(4-sulfo-phenyl)-3-carboxy-5-hydroxypyrazolone (EEPT); (5) 4,4′-(diazoamino)-dibenzenesulfonic acid (DAADBSA).
Sunset YellowPrimary Eluant: 0.01 M aqueous Na2B4O7.Secondary Eluant: 0.20 M NaClO4 in aqueous 0.01 M
Na2B4O7.Sample Size: 5 �L of a 1% solution.Flow Rate: 0.50 mL/min.Gradient: Linear in four phases: 0% to 11% in 10 min;
hold 25 min; 11% to 38% in 10 min; 38% to 42% in 10 min;42% to 98% in 20 min; hold 20 min.
Temperature: 50°.Pressure: 1000 psi.Order of Elution: (1) Sulfanilic acid (SA); (2) Schaeffer’s
salt (SS); (3) 4,4′-(diazoamino)-dibenzenesulfonic acid(DAADBSA); (4) R-salt dye; (5) Sunset Yellow; (6) 6,6′-oxybis(2-naphthalenesulfonic acid) (DONS).
FCC V General Tests and Assays / Appendix III / 885
Standard SolutionsAllura Red Prepare a solution containing 0.25 g of color-
ant, 0.5 mg of CSA, 0.75 mg of SS, 0.25 mg of DMMA, and1.25 mg of DONS in a 100-mL volumetric flask. Dissolve inand dilute to volume with 0.1 M Na2B4O7.
Tartrazine Prepare a solution containing 0.15 g of color-ant and 0.3 mg each of PHSA, SA, PY-T, EEPT, and DAAD-BSA in a 100-mL volumetric flask. Dissolve in and dilute tovolume with 0.1 M Na2B4O7.
Sunset Yellow Prepare a solution containing 0.25 g ofcolorant, 0.5 mg of SA, 0.75 mg of SS, 0.25 mg of DAADBSA,and 1.25 mg of DONS in a 100-mL volumetric flask. Dissolvein and dilute to volume with 0.1 M Na2B4O7.
Test Solutions Prepare at least four test solutions, eachcontaining the colorant, and one impurity, accurately weighed,dissolved in 0.1 M Na2B4O7, and diluted to volume in a 100-mL volumetric flask. The solutions should encompass therange of concentrations, evenly spaced, given below for eachconstituent:
Allura Red (250 mg) CSA (0.05 to 0.5 mg); SS (0.05 to0.75 mg); DONS (0.5 to 2.5 mg); DMMA (0.025 to 0.25 mg).Inject 20 �L of each solution.
Tartrazine (150 mg) SA (7.5 to 300 �g); PY-T (7.5 to300 �g); EEPT (7.5 to 300 �g); DAADBSA (7.5 to 300 �g).Inject 50 �L of each solution.
Sunset Yellow (250 mg) SA (0.05 to 0.5 mg); SS (0.05to 0.75 mg); DONS (0.5 to 2.5 mg); DAADBSA (0.05 to0.25 mg). Inject 20 �L of each solution.
System SuitabilityResolution Elute the column, or equivalent, with the gra-
dient specified under Operating Conditions until a smoothbaseline is obtained. Inject an aliquot of the Standard Solution.The resolution of the eluted components matches or exceedsthat shown for the corresponding colorant (see Figs. 24, 25,and 26). After determining that the column, or equivalent,will give the required resolution, allow it to rest for 2 weeksbefore use.
Calibration Inject the designated volume of each TestSolution onto a conditioned column, and prepare a standardcurve corresponding to each unreacted intermediate and sidereaction product. Determine the area, A, for each peak fromthe integrator if an automated system is used or by multiplyingthe peak height by the width at one-half the height. The peakheight alone may be used for EEPT, PY-T, and DAADBSA.Calculate the concentration, Ci, of each intermediate or sideproduct using the equation
Ci = mAi + b,
in which Ai is the area of its corresponding chromatographicpeak. Calculate the slope, m, and intercept, b, using the follow-ing linear regression equations:
m = [N�CiAi − �Ci�Ai]/[N�Ai2 − [(�Ai)
2],
b = [A]i − m[C]i,
in which C and A are the calculated averages of the concentra-
FIGURE 24 Allura Red–Top Trace: Eluant Monitored at 254nm; Bottom Trace: Eluant Monitored at 375 to 385 nm.
FIGURE 25 Tartrazine–Top Trace: Eluant Monitored at 254nm; Bottom Trace: Eluant Monitored at 375 to 385 nm.
tions and peak areas, respectively, used to construct the stan-dard curve for one intermediate or side reaction product. Cal-culate the correlation coefficient, r, from the followingequation:
886 / Appendix III / General Tests and Assays FCC V
FIGURE 26 Sunset Yellow–Top Trace: Eluant Monitored at254 nm; Bottom Trace: Eluant Monitored at 375 to 385 nm.
r = [�(Ci − C)(Ai − A)]/[�(Ci − C2) × �(Ai − A)2].
Each time the system is calibrated, add the new data tothose accumulated from previous analyses. The correlationcoefficient must be between 0.95 and 1.00 for any singleexperiment or from accumulated data.
Recalibrate the system after every ten determinations or2 days, whichever occurs first.
Sample Preparation Prepare as directed in the individualmonograph.
Procedure Inject the volume of Sample Preparation as des-ignated in the monograph into the column. Determine theconcentration of intermediates and side reaction products fromthe peak areas using the slope, m, and intercept, b, calculatedunder Calibration by the equation
Cs = mAs + b,
in which Cs is the concentration of the unknown in the SamplePreparation and As its corresponding peak area.
Loss on Drying (Volatile Matter) Transfer 1.5 to 2.5 g ofcolorant, accurately weighed, to a tared crucible. Heat in avacuum oven at 135° for 12 to 15 h. Lower the pressure in theoven to −125 mm Hg, and continue heating for an additional 2h. Cover the crucible, and allow to cool in a desiccator.Reweigh the crucible when cool. The loss of weight is definedas the volatile matter.
Water-Insoluble Matter Transfer about 1 g of colorant,accurately weighed, to a 250-mL beaker, and add 200 mL ofboiling water. Stir to facilitate dissolution of the color.
Tare a filtering crucible equipped with a glass fiber filter(Reeve Angel, No. 5270, or equivalent). Filter the solutionwith the aid of suction when it has cooled to ambient tempera-ture. Rinse the beaker three times, pouring the rinsings throughthe crucible. Wash the filter with water until the filtrate iscolorless.
Dry the crucible and filter in an oven at 135° for at least3 h, cool them in a desiccator and reweigh to the nearest 0.1mg. Calculate the percent water-insoluble matter, I, by theequation
I = (Wc/Ws) × 100,
in which Wc is the difference in crucible weight and Ws isthe sample weight.
GLUTAMIC ACID
Apparatus Use an ion-exchange amino acid analyzer,equipped with sulfonated polystyrene columns, in which theeffluent from the sample is mixed with ninhydrin reagent andthe absorbance of the resultant color is measured continuouslyand automatically at 570 and 440 nm by a recording pho-tometer.
Standard Solution Transfer 1250 � 2 mg of reagent-gradeglutamic acid, accurately weighed, into a 500-mL volumetricflask. Fill the flask half-full with water, add 5 mL of hydro-chloric acid to help dissolve the amino acid, dilute to volumewith water, and mix. Prepare the standard for analysis bydiluting 1 mL of this solution with 4 mL of 0.2 N sodiumcitrate, pH 2.2, buffer. This Standard Solution contains 0.5mg of glutamic acid per milliliter (CS).
Sample Preparation Dilute 5 mg of sample, accuratelyweighed, to exactly 5 mL with 0.2 N sodium citrate, pH 2.2,buffer. Remove any insoluble material by centrifugation orfiltration.
Procedure Using 2-mL aliquots of the Standard Solutionand Sample Preparation, proceed according to the apparatusmanufacturer’s instructions. From the chromatograms thusobtained, match the retention times produced by the StandardPreparation with those produced by the Sample Solution, andidentify the peak produced by glutamic acid. Record the areaof the glutamic acid peak from the sample as AU, and thatfrom the standards as AS.
Calculations Calculate the concentration, CA, in milligramsper milliliter, of glutamic acid in the Sample Preparation bythe formula
AU × CS/AS,
FCC V General Tests and Assays / Appendix III / 887
in which CS is the concentration, in milligrams per milliliter,of glutamic acid in the Standard Solution.
Calculate the percent glutamic acid, on the basis of totalprotein, by the formula
(100 × CA)/(6.25 × NT),
in which NT is the percent total nitrogen determined in themonograph Assay, and 6.25 is the conversion factor for proteinand amino acids.
Calculate the percent glutamic acid in the sample by theformula
100 × CA/SW,
in which SW is the weight, in milligrams, of the sample taken.
HYDROXYPROPOXYL DETERMINATION
Apparatus The apparatus for hydroxypropoxyl group deter-mination is shown in Fig. 27. The boiling flask, D, is fittedwith an aluminum foil-covered Vigreaux column, E, on theside arm and with a bleeder tube through the neck and to thebottom of the flask for the introduction of steam and nitrogen.A steam generator, B, is attached to the bleeder tube throughtube C, and a condenser, F, is attached to the Vigreaux column.The boiling flask and steam generator are immersed in an oilbath, A, equipped with a thermoregulator such that a tempera-ture of 155° and the desired heating rate may be maintained.The distillate is collected in a 150-mL beaker, G, or othersuitable container.
Procedure Unless otherwise directed, transfer about 100 mgof the sample, previously dried at 105° for 2 h and accuratelyweighed, into the boiling flask, and add 10 mL of chromiumtrioxide solution (60 g in 140 mL of water). Immerse thesteam generator and the boiling flask in the oil bath (at roomtemperature) to the level of the top of the chromium trioxide
FIGURE 27 Apparatus for Hydroxypropoxyl Determination.
solution. Start cooling water through the condenser, and passnitrogen gas through the boiling flask at the rate of one bubbleper second. Starting at room temperature, raise the temperatureof the oil bath to 155° over a period of not less than 30 min,and maintain this temperature until the end of the determina-tion. Distill until 50 mL of distillate is collected. Detach thecondenser from the Vigreaux column, and wash it with water,collecting the washings in the distillate container. Titrate thecombined washings and distillate with 0.02 N sodium hydrox-ide to a pH of 7.0, using a pH meter set at the expanded scale.
Note: Phenolphthalein TS may be used for this titrationif it is also used for all standards and blanks.
Record the volume, Va, of the 0.02 N sodium hydroxide used.Add 500 mg of sodium bicarbonate and 10 mL of 2 N sulfuricacid, and then after evolution of carbon dioxide has ceased,add 1 g of potassium iodide. Stopper the flask, shake themixture, and allow it to stand in the dark for 5 min. Titratethe liberated iodine with 0.02 N sodium thiosulfate to the sharpdisappearance of the yellow color, confirming the endpoint bythe addition of a few drops of starch TS. Record the volumeof 0.02 N sodium thiosulfate required as Ya.
Make several reagent blank determinations, using only thechromium trioxide solution in the above procedure. The ratioof the sodium hydroxide titration (Vb) to the sodium thiosulfatetitration (Yb), corrected for variation in normalities, will givethe acidity-to-oxidizing ratio, Vb/Yb = K, for the chromiumtrioxide carried over in the distillation. The factor K shouldbe constant for all determinations.
Make a series of blank determinations using 100 mg ofmethylcellulose (containing no foreign material) in place ofthe sample, recording the average volume of 0.02 N sodiumhydroxide required as Vm and the average volume of 0.02 Nsodium thiosulfate required as Ym.
Calculate the hydroxypropoxyl content of the sample, inmilligrams, by the formula
75.0 × [N1(Va – Vm) – kN2(Ya – Ym)],
in which N1 is the exact normality of the 0.02 N sodiumhydroxide solution, N2 is the exact normality of the 0.02 Nsodium thiosulfate solution, and k = VbN1/YbN2.
The percentage of substitution, by weight, of hydroxypro-poxyl groups, determined as directed above, may be convertedto molecular substitution per glucose unit by reference toFig. 28.
METHOXYL DETERMINATION
Apparatus The apparatus for methoxyl determination, asshown in Fig. 29, consists of a boiling flask, A, fitted with acapillary side arm to provide an inlet for carbon dioxide andconnected to a column, B, which separates aqueous hydriodicacid from the more volatile methyl iodide. After the methyliodide passes through a suspension of aqueous red phosphorus
888 / Appendix III / General Tests and Assays FCC V
FIGURE 28 Chart for Converting Percentage of Substitution, byWeight, of Hydroxypropoxyl Groups to Molecular Substitution perGlucose Unit.
in the scrubber trap, C, it is absorbed in the bromine–aceticacid absorption tube, D. The carbon dioxide is introducedfrom a device arranged to minimize pressure fluctuations andconnected to the apparatus by a small capillary containing asmall plug of cotton.
ReagentsAcetic Potassium Acetate Dissolve 100 g of potassium
acetate in 1000 mL of a mixture consisting of 900 mL ofglacial acetic acid and 100 mL of acetic anhydride.
Bromine–Acetic Acid Solution On the day of use, dissolve5 mL of bromine in 145 mL of the Acetic Potassium Acetatesolution.
Hydriodic Acid Use special-grade hydriodic acid suitablefor alkoxyl determinations, or purify reagent grade as follows:Distill over red phosphorus in an all-glass apparatus, passing
FIGURE 29 DistillationApparatus forMethoxyl Determination.
a slow stream of carbon dioxide through the apparatus untilthe distillation is terminated and the receiving flask has com-pletely cooled.
Caution: Use a safety shield, and conduct the distilla-tion in a fume hood.
Collect the colorless, or almost colorless, constant-boilingacid distilling between 126° and 127°. Store the acid in a cool,dark place in small, brown, glass-stoppered bottles previouslyflushed with carbon dioxide and finally sealed with paraffin.
Procedure Fill trap C half-full with a suspension of about60 mg of red phosphorus in 100 mL of water, introducedthrough the funnel on tube D and the side arm that connectswith the trap at C. Rinse tube D and the side arm with water,collecting the rinsings in trap C, then charge absorption tubeD with 7 mL of Bromine–Acetic Acid Solution. Place thesample, previously accurately weighed in a tared gelatin cap-sule, into the boiling flask A, along with a few glass beadsor boiling stones, then add 6 mL of Hydriodic Acid. Connectthe flask to the condenser, using a few drops of the acid toseal the junction, and begin passing the carbon dioxide throughthe apparatus at the rate of about two bubbles per second.Heat the flask in an oil bath at 150°, continue the reactionfor 40 min, and drain the contents of absorption tube D intoa 500-mL Erlenmeyer flask containing 10 mL of a 1:4 solutionof sodium acetate. Rinse tube D with water, collecting therinsings in the flask, and dilute to about 125 mL with water.Discharge the red-brown color of bromine by adding formicacid, dropwise with swirling, then add 3 drops in excess.Usually a total of 12 to 15 drops of formic acid is required.Allow the flask to stand for 3 min, add 15 mL of 2 N sulfuricacid and 3 g of potassium iodide, and titrate immediately with0.1 N sodium thiosulfate, adding starch TS near the endpoint.Perform a blank determination with the same quantities ofthe same reagents, including the gelatin capsule, and in thesame manner, and make any necessary correction. Each milli-liter of 0.1 N sodium thiosulfate is equivalent to 0.517 mg(517 �g) of methoxyl groups (—OCH3).
NITROGEN DETERMINATION (KjeldahlMethod)
Caution: Provide adequate ventilation in the laboratory,and do not permit accumulation of exposed mercury.
Note: All reagents should be nitrogen free, where avail-able, or otherwise very low in nitrogen content.
Method I
Use this method unless otherwise directed in the individualmonograph. It is not applicable to certain nitrogen-containingcompounds that do not yield their entire nitrogen upon diges-tion with sulfuric acid.
FCC V General Tests and Assays / Appendix III / 889
Nitrites and Nitrates AbsentUnless otherwise directed, transfer from 700 mg to 2.2 g ofsample into a 500- to 800-mL Kjeldahl digestion flask ofhard, moderately thick, well-annealed glass, wrapping thesample, if solid or semisolid, in nitrogen-free filter paper tofacilitate the transfer if desired. Add 700 mg of mercuricoxide or 650 mg of metallic mercury, 15 g of powderedpotassium sulfate or anhydrous sodium sulfate, and 25 mLof 93% to 98% sulfuric acid. (If a sample weight greater than2.2 g is used, increase the sulfuric acid by 10 mL for eachadditional gram of sample.) Place the flask in an inclinedposition, and heat gently until frothing ceases, adding a smallamount of paraffin, if necessary, to reduce frothing.
Caution: The digestion should be conducted in a fumehood, or the digestion apparatus should be equippedwith a fume exhaust system.
Boil briskly until the solution clears, and then continue boilingfor 30 min longer (or for 2 h for samples containing organicmaterial). Cool, add about 200 mL of water, mix, and thencool to below 25°. Add 25 mL of sulfide or thiosulfate solution(40 g of K2S, 40 g of Na2S, or 80 g of Na2S2O3·5H2O in1000 mL of water), and mix to precipitate the mercury. Adda few granules of zinc to prevent bumping, tilt the flask, andcautiously pour sodium hydroxide pellets or a 2:5 solution ofsodium hydroxide down the inside of the flask so that it formsa layer under the acid solution, using a sufficient amount(usually about 25 g of solid NaOH) to make the mixturestrongly alkaline. Immediately connect the flask to a distilla-tion apparatus consisting of a Kjeldahl connecting bulb anda condenser, the delivery tube of which extends well beneaththe surface of a measured excess of 0.5 N hydrochloric orsulfuric acid contained in a 500-mL flask. Add from 5 to 7drops of methyl red indicator (1 g of methyl red in 200 mLof alcohol) to the receiver flask. Rotate the Kjeldahl flask tomix its contents thoroughly, and then heat until all of theammonia has distilled, collecting at least 150 mL of distillate.Wash the tip of the delivery tube, collecting the washings inthe receiving flask, and titrate the excess acid with 0.5 Nsodium hydroxide. Perform a blank determination, substitut-ing 2 g of sucrose for the sample, and make any necessarycorrection (see General Provisions). Each milliliter of 0.5 Nacid consumed is equivalent to 7.003 mg of nitrogen.
Note: If the substance to be determined is known tohave a low nitrogen content, 0.1 N acid and alkali maybe used, in which case each milliliter of 0.1 N acidconsumed is equivalent to 1.401 mg of nitrogen.
Nitrites and Nitrates Present
Note: This procedure is not applicable to liquids or tomaterials having a high chlorine-to-nitrate ratio.
Unless otherwise directed, transfer from 700 mg to 2.2 g ofsample into a Kjeldahl flask, and add 40 mL of 93% to 98%sulfuric acid containing 2 g of salicylic acid. Mix thoroughlyby shaking, and then allow to stand for 30 min or more, withoccasional shaking. Add 5 g of Na2S2O3·5H2O, or 2 g of zincdust (as an impalpable powder, not granules or filings), shake,
and allow to stand for 5 min. Heat over a low flame untilfrothing ceases, then remove the heat, add 700 mg of mercuricoxide (or 650 mg of metallic mercury) and 15 g of powderedpotassium sulfate (or anhydrous sodium sulfate), and boilbriskly until the solution clears. Continue boiling for 30 minlonger (or for 2 h for samples containing organic material),and then continue as directed under Nitrates and NitratesAbsent, beginning with ‘‘Cool, add about 200 mL ofwater. . . .’’
Method II (Semimicro)
Note: Automated instruments may be used in place ofthis manual method, provided the automated equipmenthas been properly calibrated.
Transfer an accurately weighed or measured quantity of sam-ple, equivalent to about 2 or 3 mg of nitrogen, into the digestionflask of a semimicro Kjeldahl apparatus. Add 1 g of a 10:1powdered mixture of potassium sulfate:cupric sulfate, usinga fine jet of water to wash down any material adhering to theneck of the flask, and then pour 7 mL of sulfuric acid downthe inside wall of the flask to rinse it. Cautiously add downthe inside of the flask 1 mL of 30% hydrogen peroxide,swirling the flask during the addition.
Caution: Do not add any peroxide during the digestion.
Heat over a free flame or an electric heater until the solutionhas attained a clear blue color and the walls of the flask arefree from carbonized material. Cautiously add 20 mL of water,cool, then add through a funnel 30 mL of a 2:5 solution ofsodium hydroxide, and rinse the funnel with 10 mL of water.Connect the flask to a steam distillation apparatus, and imme-diately begin the distillation with steam. Collect the distillatein 15 mL of a 1:25 solution of boric acid to which has beenadded 3 drops of methyl red–methylene blue TS and enoughwater to cover the end of the condensing tube. Continuepassing the steam until 80 to 100 mL of distillate has beencollected, then remove the absorption flask, rinse the end ofthe condenser tube with a small quantity of water, and titratewith 0.01 N sulfuric acid. Each milliliter of 0.01 N acid isequivalent to 140 �g of nitrogen.
When more than 2 or 3 mg of nitrogen is present in themeasured quantity of the substance to be determined, 0.02 or0.1 N sulfuric acid may be used in the titration if at least 15mL of titrant is required. If the total dry weight of the materialtaken is greater than 100 mg, increase proportionately thequantities of sulfuric acid and sodium hydroxide added beforedistillation.
SULFUR (by Oxidative Microcoulometry)(Based on ASTM D3120)
Note: All reagents used in this test should be reagentgrade; water should be of high purity, and gases mustbe high-purity grade.
890 / Appendix III / General Tests and Assays FCC V
FIGURE 30 Microcoulometric Titrating System for the Determination of Sulfur in Hexanes.
Apparatus Use the Dohrmann Microcoulometric TitratingSystem (MCTS-30), or equivalent (shown in Fig. 30), unlessotherwise specified in an individual monograph. It consistsof a constant rate injector, A, a pyrolysis furnace, B, a quartzpyrolysis tube, C, a granular-tin scrubber, D, a titration cell,E, and a microcoulometer with a digital readout, F.
Granular-Tin Scrubber Place 5 g of 20- to 30-mesh gran-ular reagent-grade tin between quartz-wool plugs in an elon-gated 18/9-12/5 standard-taper adaptor that connects the py-rolysis tube and the titration cell.
Microcoulometer Must have variable attenuation; gaincontrol; and be capable of measuring the potential of thesensing-reference electrode pair, and comparing this potentialwith a bias potential, amplifying the potential difference, andapplying the amplified difference to the working-auxiliaryelectrode pair to generate a titrant. Also the microcoulometeroutput voltage signal must be proportional to the generatingcurrent.
Pyrolysis Furnace The sample should be pyrolyzed in anelectric furnace having at least two separate and independentlycontrolled temperature zones, the first being an inlet sectionthat can maintain a temperature sufficient to volatilize all theorganic sample. The second zone is a pyrolysis section thatcan maintain a temperature sufficient to pyrolyze the organicmatrix and oxidize all the organically bound sulfur. A thirdoutlet temperature zone is optional.
Pyrolysis Tube Must be fabricated from quartz and con-structed in such a way that a sample, which is vaporizedcompletely in the inlet section, is swept into the pyrolysiszone by an inert gas where it mixes with oxygen and is burned.The inlet end of the tube shall hold a septum for syringe entryof the sample and side arms for the introduction of oxygenand inert gases. The center or pyrolysis section should be ofsufficient volume to ensure complete pyrolysis of the sample.
Sampling Syringe A microlitre syringe of 10-�L capacitycapable of accurately delivering 1 to 10 �L of sample intothe pyrolysis tube. Three-inch × 24-gauge needles are recom-mended to reach the inlet zone of the pyroloysis furnace.
Titration Cell Must contain a sensor-reference pair ofelectrodes to detect changes in triiodide ion concentrationand a generator anode–cathode pair of electrodes to maintain
constant triiodide ion concentration and an inlet for a gaseoussample from the pyrolysis tube. The sensor electrode shall beplatinum foil and the reference electrode platinum wire insaturated triiodide half-cell. The generator anode and cathodehalf-cell shall also be platinum. The titration cell shall beplaced on a suitable magnetic stirrer.
Preparation of Apparatus Carefully insert the quartz py-rolysis tube into the furnace, attach the tin scrubber, andconnect the reactant and carrier-gas lines. Add the Cell Elec-trolyte Solution (see below) to the titration cell, and flush thecell several times. Maintain an electrolyte level of 3.2 to 6.6mm above the platinum electrodes. Place the titration cell ona magnetic stirrer, and connect the cell inlet to the tin scrubberoutlet. Position the platinum-foil electrodes (mounted on themovable cell head) so that the gas-inlet flow is parallel to theelectrodes with the generator anode adjacent to the generatorcathode. Assemble and connect the coulometer in accordancewith the manufacturer’s instructions. Double-wrap the adaptorcontaining the tin scrubber with heating tape and turn theheating tape on. Adjust the flow of the gases, the pyrolysisfurnace temperature, the titration cell, and the coulometer tothe desired operating conditions. Typical operating conditionsare as follows:
Reactant gas flow (oxygen), cm3/min 200Carrier-gas flow (Ar, He), cm3/min 40Furnace temperature, °C
Inlet zone 700 (maximum)Pyrolysis zone 800 to 1000Outlet zone 800 (maximum)
Tin-scrubber temperature, °C 200Titration cell Stirrer speed set to
produce slight vortexCoulometer
Bias voltage, mV 160Gain 50
Constant Rate Injector, �L/s 0.25
The tin scrubber must be conditioned to sulfur, nitrogen,and chlorine before quantitative analysis can be achieved. Asolution containing 10 mg/kg butyl sulfide, 100 mg/kg pyri-dine, and 200 mg/kg chlorobenzene in isooctane has proven
FCC V General Tests and Assays / Appendix III / 891
an effective conditioning agent. With a fresh scrubber installedand heated, two 30-�L samples of this conditioning agentinjected at a flow rate of 0.5 �L/s produce a steadily increasingresponse, with final conditioning indicated by a constant read-ing from the offset during the second injection.
ReagentsArgon or Helium (Argon preferred) High-purity grade,
used as the carrier gas. Two-stage gas regulators must be used.Cell Electrolyte Solution Dissolve 0.5 g of potassium io-
dide and 0.6 g of sodium azide in 500 mL of high-puritywater, add 5 mL of glacial acetic acid and dilute to 1 L. Storein a dark bottle or in a dark place and prepare fresh at leastevery 3 months.
Oxygen High-purity grade, used as the reactant gas.Iodine Resublimed, 20-mesh or less, for saturated refer-
ence electrode.Sulfur Standard (approximately 100 mg/kg) Transfer
0.1569 g of n-butyl sulfide, accurately weighed, into a tared500-mL volumetric flask. Dilute to the mark with isooctane,and reweigh. Calculate the sulfur concentration (S), in percent,by the formula
S = Wb/Ws × 2.192 × 105,
in which Wb is the weight of n-butyl sulfide and Ws is theweight of the solution.
Calibration Prepare a calibration standard (approximately5 mg/kg) by pipetting 5 mL of Sulfur Standard into a 10-mLvolumetric flask and diluting to volume with isooctane. Filland clamp the syringe onto the constant-rate injector, pushthe sliding carriage forward to penetrate the septum with theneedle, and zero the meter in case of long-term drift in theautomatic baseline zero circuitry. Switch S1 automaticallystarts the stepper-motor syringe drive and initiates the analysiscycle. At 2.5 min (before the 3-min meter hold point) set thedigital meter with the scan potentiometer to correspond to thesulfur content of the known standard to the nearest 0.01 mg/kg. At the 3-min point, the number displayed on the meterstops, the plunger drive block is retracted to its original posi-tion, as preset by switch S2, and a baseline re-equilibrationperiod equal to the injection period elapses before a ready
FIGURE 31 Raney Nickel Reduction Apparatus.
light and a beeper indicate that a new sample may be injected.Repeat the Calibration step a total of at least four times.
Procedure Rinse the syringe several times with sample;then fill it, clamp it onto the constant-rate injector, push thesliding carriage forward to penetrate the septum with theneedle, and zero the meter. Turn on switch S1 to startthe stepper-motor syringe drive automatically and initiate theanalysis cycle. After the 3-min hold point, the number dis-played on the meter corresponds to the sulfur content of theinjected sample.
892 / Appendix IV / General Tests and Assays FCC V
APPENDIX IV: CHEWING GUM BASE POLYMERS
BOUND STYRENE
Abbé-Type Refractometer Use an instrument with fourthdecimal place accuracy that can be placed in a nearly hori-zontal position for measurement of the refractive index ofsolids. An Amici-type compensating prism for achromatiza-tion is necessary unless a sodium vapor lamp is used as alight source.
Ethanol–Toluene Azeotrope Mix 70 volumes of ethanolor of formula 2B ethanol with 30 volumes of toluene, refluxfor 4 h over calcium oxide, and then distill, discarding thefirst and last portions and collecting only that portion distillingwithin a range of 1°.
Note: Refluxing and distilling are not necessary if anhy-drous 2B ethanol or absolute grain alcohol is used.
Sample Preparation Sheet out a sample of the polymer toa thickness of 0.5 mm, and cut the sheeted sample into stripsapproximately 13 mm wide and 25 mm long. Fasten one stripto each leg of a ‘‘spider,’’ consisting of a 13-mm square ofsheet aluminum or stainless steel having a Nichrome wire legabout 38 mm long attached to each corner. Place the spiderand strips in a 400-mL flask containing 60 mL of Ethanol–Toluene Azeotrope, positioning the spider so that each samplestrip is in contact on all sides with the solvent. Extract for 1h at a temperature at which the solvent boils gently, thenreplace the solvent with another 60-mL portion of Ethanol–Toluene Azeotrope, and extract for an additional hour. Removethe spider and sample strips from the flask, and dry them at100° to constant weight in a vacuum oven at a pressure ofabout 10 mm Hg.
Caution: The samples must be extracted and dried thor-oughly, but avoid overheating, which would cause plas-ticization.
Remove the extracted and dried strips from the spider, andpress the strips between aluminum foil (0.025 to 0.08 mmthick, having good tear strength) at 100° for 3 to 10 min(preferably not more than 5 min), using any suitable apparatusto produce a uniform thickness not exceeding 0.5 mm. If thepressing is done between flat platens without a cavity, use aforce between about 500 and 1500 lb, increasing the appliedforce proportionally if several strips are pressed at one time.If cavity pressing plates are used, close the platens withoutapplying pressure and preheat for 1 min, then apply a forceof about 11 tons for 3 min, remove the specimens from thepress, and allow them to cool.
Procedure Cut the pressed sample in half with sharp scis-sors, and peel off one piece of the foil. Cut off a strip about6 mm wide and 12 mm long with the scissors so that one ofthe narrower ends is freshly cut.
Check the adjustment of the refractometer by means of aglass test plate pressed firmly against the prism, using a drop
of �-bromo-naphthalene as the contact liquid. The small lightsource should be collimated. The best readings are obtainedwith the glass test piece if the light is diffused through crum-pled tissue paper. After this adjustment, clean the prism wellwith lens paper moistened with alcohol. The refractive indexof the glass test piece and of the test specimen must bemeasured at a known constant temperature, preferably 25°.
Place the test sample on the prism with the cut edge towardthe light source approximately where the edge of the glasstest piece was positioned. Remove the tissue paper from thelight source, press the specimen firmly on the prism, and waitat least 1 min for the sample to attain temperature equilibrium.The upper prism may be closed lightly on the specimen ifadequate light can still be focused on the end of the specimen.Unless the specimen is very thin, however, this operation candamage the prism or its mounting. Adjust the compensatingprism until a sharp dividing line between light and dark fieldswith minimum color is obtained. Test the contact betweenthe specimen and the prism by pressing the test specimenagainst the prism: There should be no change in the positionof the boundary line during this test. Move the hand controlfrom the light into the dark field until the boundary line justreaches the cross hairs, and make at least three readings. Ifthe readings differ by more than 0.0001 refractive index unit,make further readings. Repeat the process of obtaining read-ings with another portion of the sample having a freshly cutedge, and average the mean values of the two sets of readingsthus obtained. If the two mean values do not differ by morethan 0.0002 refractive index unit, report the average as theresults of the calculations. If necessary, correct the refractiveindex measurements to 25° using the equation
n25 = nt + 0.00037(t − 25),
in which n25 is the refractive index at 25°, and nt is therefractive index at the temperature, t, of measurement.
Calculate the percentage of bound styrene in emulsion-polymerized samples by the formula
23.50 + 1164(n25 − 1.53456) − 3497(n25 − 1.53456)2.
Calculate the percentage of bound styrene in solution-poly-merized samples by the formula
(1212.1212)(n25) − 1838.3636.
Alternatively, the percentage of bound styrene may be deter-mined by reference to suitable tables.
MOLECULAR WEIGHT
Polyethylene
Sample Solutions Dissolve 1 g of sample, accuratelyweighed, in 95 mL of tetrahydronaphthalene, filter into a 100-
FCC V General Tests and Assays / Appendix IV / 893
mL volumetric flask, dilute to volume with the solvent, andmix (Solution 1). Transfer 50.0 mL of Solution 1 into a tareddish, evaporate on a steam bath for about 1 h, and thenevaporate to dryness by heating in a vacuum oven at 70° for2 h or to constant weight. Calculate the concentration, C1, ingrams per 100 mL, of Solution 1. Prepare Solutions 2 and 3,respectively, by diluting 5.0-mL and 10.0-mL portions ofSolution 1 to 50.0 mL with the solvent, and then calculatethe concentration of each (C2 and C3, respectively).
Procedure Determine the flow time, in seconds, of the sol-vent (t0) and of the three Sample Solutions (t1, t2, and t3,respectively) in a Cannon-Fenske viscometer immersed in aconstant-temperature bath maintained at 130°. Calculate thespecific viscosity, �sp, of each Sample Solution by the formula
(t/t0) − 1,
and then calculate the reduced viscosity of each by the formula
�sp/C.
Plot the reduced viscosity of each solution against concentra-tion, and extrapolate to zero concentration to obtain the intrin-sic viscosity, [�]. Finally, calculate the molecular weight ofthe polyethylene by the formula
([�]/K)1/a,
in which K is 5.1 × 10−4, and a is 0.725.
Polyisobutylene (Flory Method)
Sample Solutions Dissolve 1 g of sample, accuratelyweighed, in 95 mL of diisobutylene, filter into a 100-mLvolumetric flask, dilute to volume with solvent, and mix (Solu-tion 1). Transfer 50.0 mL of Solution 1 into a tared dish,evaporate on a steam bath for about 1 h, and then evaporateto dryness by heating in a vacuum oven at 70° for 2 h or toconstant weight. Calculate the concentration, C1, in grams per100 mL, of Solution 1. Prepare Solutions 2 and 3, respectively,by diluting 5.0-mL and 10.0-mL portions of Solution 1 to50.0 mL with solvent, and then calculate the concentrationof each (C2 and C3, respectively).
Procedure Determine the flow time, in seconds, of the sol-vent (t0) and of the three Sample Solutions (t1, t2, and t3,respectively) in a Cannon-Fenske viscometer immersed in aconstant-temperature bath maintained at 20°. Calculate thespecific viscosity, �sp, of each Sample Solution by the formula
(t/t0) − 1,
and then calculate the reduced viscosity of each by the formula
�sp/C.
Plot the reduced viscosity of each solution against concentra-tion, and extrapolate to zero concentration to obtain the intrin-sic viscosity, [�]. Finally, calculate the molecular weight ofthe polyisobutylene by the formula
([�]/K)1/a,
in which K is 3.60 × 10−4, and a is 0.64.
Polyvinyl Acetate
Sample Solutions Dissolve 1 g of sample, accuratelyweighed, in 95 mL of acetone, filter into a 100-mL volumetricflask, dilute to volume with the solvent, and mix (Solution1). Transfer 50.0 mL of Solution 1 into a tared dish, evaporateon a steam bath for about 1 h, and then evaporate to drynessby heating in a vacuum oven at 70° for 2 h or to constantweight. Calculate the concentration, C1, in grams per 100 mL,of Solution 1. Prepare Solutions 2 and 3, respectively, bydiluting 5.0-mL and 10.0-mL portions of Solution 1 to 50.0mL with solvent, and then calculate the concentration of each(C2 and C3, respectively).
Procedure Determine the flow time, in seconds, of the sol-vent (t0) and of the three Sample Solutions (t1, t2, and t3,respectively) in a Cannon-Fenske viscometer immersed in aconstant-temperature bath maintained at 25°. Calculate thespecific viscosity, �sp, of each Sample Solution by the formula
(t/t0) − 1,
and then calculate the reduced viscosity of each by the formula
�sp/C.
Plot the reduced viscosity of each solution against concentra-tion, and extrapolate to zero concentration to obtain the intrin-sic viscosity, [�]. Finally, calculate the molecular weight ofthe polyvinyl acetate by the formula
([�]/K)1/a,
in which K is 1.88 × 10−4, and a is 0.69.
QUINONES
Standard Preparations Transfer 25.0 mg of hydroquinoneinto a 100-mL volumetric flask, dissolve in and dilute tovolume with water, and mix. Transfer 1.0-, 2.0-, 3.0-, 4.0-,and 6.0-mL aliquots of this solution into a series of 100-mLvolumetric flasks, dilute each to volume with water, and mix.Transfer 2.0 mL of each of these solutions and 3.0 mL ofwater into a series of 25-mL graduates, add 0.5 mL of 0.1 Nsodium carbonate to each, and continue as directed underSample Preparations (below), beginning with ‘‘. . . shake im-mediately, then add 1.0 mL of 15% sulfuric acid. . . .’’
Sample Preparations Place 30 g of freshly coagulated andwashed sample into a 250-mL two-necked flask, add 100 mLof water, and heat at 66° for 2 h.
Caution: Do not boil.
Cool to room temperature, and transfer 5.0 mL of the extractinto a 25-mL glass-stoppered graduate. Transfer 5.0 mL ofwater into a second graduate to serve as the blank. To eachgraduate add 1.0 mL of 15% sulfuric acid. To the graduatecontaining the sample extract add 0.5 mL of 0.1 N sodium
894 / Appendix IV / General Tests and Assays FCC V
carbonate, shake immediately, and then add 1.0 mL of 15%sulfuric acid.
Note: The elapsed time for this operation should notexceed 15 s.
Add to each graduate 1.0 mL of 2,4-dinitrophenylhydrazinesolution (dissolve 100 mg of 2,4-dinitrophenylhydrazine in50 mL of carbonyl-free methanol, add 4 mL of hydrochloricacid, and dilute to 100 mL with water), stopper, and heat at70° in a water bath for 1 h. Cool to room temperature, thenadd to each graduate 13 mL of water and 5.0 mL of benzene,stopper, and shake vigorously. Allow the phases to separate,and pipet 2.0 mL of the benzene layer from each graduateinto corresponding test tubes containing 10 mL of a 1:100solution of diethanolamine in pyridine. Shake each tube, andallow the color to develop for 10 min.
Procedure Determine the absorbance of the Sample Prepa-ration in a 1-cm cell at 620 nm, with a suitable spectrophotome-ter, against the reagent blank. Determine the absorbance of eachStandard Preparation in the same manner. Prepare a StandardCurve by plotting absorbance of each Standard Preparationagainst micrograms of quinone. From the Standard Curve, readthe quantity, in micrograms, of quinones (as benzoquinone) inthe Sample Preparation, and record the value thus obtained asQ. Calculate the quantity of quinones (as benzoquinone), inparts per million, in the sample by the formula
20Q/W,
in which W is the weight, in grams, of the sample taken.
RESIDUAL STYRENE
Standard Preparation Place 25 mL of carbon disulfide ina 100-mL volumetric flask, cap with a serum stopper, andtare the flask to the nearest 0.1 mg. Using 50-�g syringes,inject 15 �L each of styrene and of alpha-methylstyrene(AMS), reweighing after each addition to obtain the weightof each solution injected. Record the weight, in milligrams,of styrene as w1 and that of AMS as w2. Dilute to volumewith carbon disulfide, and mix. Pipet 2 mL of this solutioninto a second 100-mL volumetric flask, dilute to volume withcarbon disulfide, and mix. Finally, pipet 25 mL of the dilutedsolution into a third 100-mL volumetric flask, dilute to volumewith carbon disulfide, and mix.
AMS–Solvent Solution Place 25 mL of carbon disulfideinto a 100-mL volumetric flask, cap with a serum stopper,and tare the flask to the nearest 0.1 mg. Using a 50-�L syringe,inject 15 �L of AMS, and reweigh to obtain the weight ofAMS injected. Dilute to volume with carbon disulfide, andmix. Pipet 2 mL of this solution into a second 100-mL volu-metric flask, dilute to volume with carbon disulfide, and mix.Finally, pipet 25 mL of the diluted solution into a third 100-
mL volumetric flask, dilute to volume, and mix. Calculatethe weight, in grams, of AMS in each milliliter of the finalsolution, and record the result as w′ (approximately 7.5 × 10−7).
Sample PreparationLatex Samples Add, with agitation, 100 mL of the latex
to a mixture consisting of 15 mL of glacial acetic acid and10 g of sodium chloride in 500 mL of hot water. Coagulationstarts almost immediately. When coagulation is complete,collect the coagulum on a coarse filter or cheesecloth, andwash with 1000 mL of a hot solution prepared with 5.6 g ofsodium hydroxide and 1000 mL of water. Wash with hotwater until the wash water is free of alkali, then cut thecoagulum into small pieces, and dry at 105° for 4 h. Continueas directed under Solid Samples (below), beginning with‘‘Transfer 1.5 g, accurately weighed. . . .’’
Solid Samples Cut a piece approximately 2 in. × 3 in. ×5 in. from the corner of a polymer bale, and pass it througha cold mill, set at least 1⁄4 in. open, four times, reversing thesample on each pass. Cut the sample into two pieces at least1 in. from the edge to expose clean polymer, and then diceapproximately 2 g of the clean polymer or cut into smallstrips. Transfer 1.5 g, accurately weighed, into a 4-oz bottlefitted with a polyethylene cap, add 25.0 mL of the AMS–Solvent Solution, cap tightly, and agitate on a mechanicalshaker until the polymer dissolves.
Note: Some polymers tend to swell and form viscouscements instead of dissolving cleanly. If this occurs,add 5- to 10-mL increments of carbon disulfide to obtaina mobile slurry, and in the next step increase the volumeof methanol by a proportional amount.
Add 25 mL of methanol, cap the bottle, and shake vigorouslyon the shaker for 30 min. After the contents have settled,decant 10 mL of the coagulant serum into a 1-oz bottle, add10 mL of water, and stopper with a serum cap. Shake vigor-ously for 1 min, then turn the bottle upside down, and allowthe layers to separate. Withdraw by syringe 1 to 2 mL of thelower (carbon disulfide) layer, and transfer it into a 10-dramvial filled with 1/4 in. of anhydrous sodium sulfate. Seal witha polyethylene cap, shake to mix, and allow to settle.
Procedure (See Chromatography, Appendix IIA.) Inject a10-�L portion of the Sample Preparation into a suitable gaschromatograph in which the detector is the hydrogen flame-ionization type and the column is 10-ft × 3/16-in. stainlesssteel tubing, or equivalent, packed with 25% Ucon 50 HB2000 on 60- to 80-mesh acid-washed DMCS Chromosorb W,or with equivalent packing materials. Use nitrogen or heliumas the carrier gas, flowing at 40 mL/min. The injection porttemperature is 240°; the column temperature, 170° isothermal;and the detector temperature, 250°. Adjust the sensitivity ofthe instrument to give as large a signal as possible for styreneand AMS as is consistent with an acceptable backgroundlevel. Measure the styrene and AMS peaks by any convenientmethod, recording the area of the styrene peak as A1 and thatof the AMS peak as A2.
In the same manner, inject a 10-�L portion of the StandardPreparation into the chromatograph, obtain the chromato-
FCC V General Tests and Assays / Appendix IV / 895
gram, and record the area of the styrene peak as a1 and thatof the AMS peak as a2. Calculate the styrene factor, F, bythe formula
(w1/w2) × (a2/a1).
Calculate the content of residual styrene in the sample taken,in parts per million, by the formula
(A1/A2) × F × 25 × (w′/W) × 106,
in which W is the weight, in grams, of the sample taken.
SAMPLE SOLUTION FOR ARSENICLIMIT TEST
Transfer 1 g of sample, accurately weighed, into a Kjeldahlflask, rest the open end of the flask in a Kjeldahl fume bulbattached to a water aspirator, add 5 mL of sulfuric acid and4 mL of 30% hydrogen peroxide, and digest over a smallflame. (See Caution statement under Arsenic Limit Test, Ap-pendix IIIB.) Continue adding the peroxide in 2-mL portions,allowing the reaction to subside between additions, until all or-ganic matter is destroyed, fumes of sulfuric acid are copiouslyevolved, and the solution becomes colorless. Maintain oxidiz-ing conditions at all times during the digestion by adding perox-ide whenever the mixture turns brown or darkens. (The amountof peroxide required to completely digest the samples will vary,but as much as 200 mL may be required in some cases, de-pending on the nature of the material.) Cool, cautiously add10 mL of water, again evaporate to strong fuming, and cool.Transfer the solution into an arsine generator flask, wash theKjeldahl flask and bulb with water, adding the washings to thegenerator flask, and dilute to 35 mL with water.
SAMPLE SOLUTION FOR LEAD LIMITTEST
Transfer 3.3 g of sample, accurately weighed, into a porcelaindish or casserole, heat on a hot plate until completely charred,then heat in a muffle furnace at 480° for 8 h or overnight,and cool. Cautiously add 5 mL of nitric acid, evaporate todryness on a hot plate, then heat again in the muffle furnaceat 480° for exactly 15 min, and cool. Extract the ash withtwo 10-mL portions of water, filtering each extract into aseparator. Leach any insoluble material on the filter with 6mL of Ammonium Citrate Solution, 2 mL of HydroxylamineHydrochloride Solution, and 5 mL of water (see Lead LimitTest, Appendix IIIB, for preparation of these solutions), add-ing the filtered washings to the separator. Continue as directedunder Procedure in the Lead Limit Test, Appendix IIIB, begin-ning with ‘‘Add 2 drops of phenol red TS to the separator. . . .’’
TOTAL UNSATURATION
This method measures total unsaturation in a sample by themultivariate analysis of Fourier transform infrared spectra. Itcorrelates the absorbance in the spectral regions correspondingto two major types of unsaturation with their concentrations.This is an extension of univariate least squares analysis thatcorrelates a single band absorbance height or area with concen-tration.
Apparatus Use a Fourier transform infrared spectrometer(FTIR), with its associated computer and peripherals, capableof measuring from 4500 to 500 cm−1 and of acquiring datawith a resolution of at least 2 cm−1. The optics of the instrumentmust be sealed and desiccated, or, like the sample chamber,must be under continuous dry air or nitrogen gas purge. Thespectrometer is equipped with software capable of multicom-ponent analysis using the partial least squares method (PLS-1, or equivalent). This software is commercially available asan accessory to the spectrometer or as an external softwarepackage.
Laboratory Press Use a Carver-type press capable ofpressing polymer films.
Sample Preparation Compression-mold a thin film of thesample to roughly a 500-�m thickness at 10 tons and 90° for30 to 60 s. Do not exceed this time or temperature, as structuralchanges in unsaturation can occur.
Operating Conditions Collect not less than 64 FTIR spec-tral scans of the standards and sample in the absorbance mode.Boxcar apodization and 2 cm−1 resolution are recommendedparameters. Spectral normalization should be done on the4333 cm−1 peak to account for varying sample thicknesses.Use identical operating conditions for the standards and forthe sample.
Calibration Assemble a set of at least ten calibration stan-dards available from the given supplier of food-grade butylrubber (such as Exxon Chemical Co.) that covers the entireunsaturation range expected. Identify characteristic FTIRspectral regions corresponding to the unsaturation componentsby proton magnetic resonance spectroscopy. These spectralregions may include 1700 to 1600 cm−1 CuC stretching, 900to 600 cm−1 vinylic H deformations, and 2200 to 1800 cm−1
overtone regionsCollect not less than 64 spectral scans of the standards.
Construct a calibration matrix containing infrared absorbancevalues for unsaturation types in the standards and their knownconcentrations. Confirm the validity of the calibration matrixmodel as recommended in the software manual. A recom-mended method is cross-validation for all standards by se-quentially excluding one of the standards from the calibrationmatrix, then using the remaining standards to predict theconcentrations. After validation, determine the optimum num-ber of factors, or loading vectors, needed to minimize thedeviation between actual and predicted concentrations. Thisdetermination is automated in most multicomponent analysis
896 / Appendix IV / General Tests and Assays FCC V
packages. For the highest possible precision, a calibration foreach rubber grade for each manufacturer is recommended.
Procedure Obtain the FTIR spectra of the sample underidentical sample preparation and operating conditions as de-
APPENDIX V: ENZYME ASSAYS
A list of the enzymes covered by the general monograph onEnzyme Preparations is shown in the accompanying table.Also incorporated in the table are the trivial names by whicheach is commonly known, as well as the systematic names
Enzyme Preparations Used in Food Processing
TRIVIALNAME CLASSIFICATION SOURCE NAMES (IUB)a NO.a
�-Amylase carbohydrase (1) Aspergillus niger var. 1,4-�-D-glucan 3.2.1.1(2) Aspergillus oryzae var. glucanohydrolase(3) Rhizopus oryzae var.(4) Bacillus subtilis var.(5) barley malt(6) Bacillus licheniformis var.(7) Bacillus stearothermophilus(8) Bacillus subtilis*
d-Bacillus megaterium(9) Bacillus subtilis*
d-Bacillus stearothermophilus(10) Bacillus licheniformis*
d-Bacillus stearothermophilus
�-Amylase carbohydrase (1) barley malt 1,4-�-D-glucan maltohydrolase 3.2.1.2(2) barley
Bromelain protease pineapples: Ananas comosus none 3.4.22.32Ananas bracteatus (L) 3.4.22.33
Catalase oxidoreductase (1) Aspergillus niger var. hydrogen peroxide: hydrogen 1.11.1.6(2) bovine liver peroxide oxidoreductase(3) Micrococcus lysodeikticus
Cellulase carbohydrase (1) Aspergillus niger var. Endo-1,4-(1,3;1,4)-�-D-glucan 4- 3.2.1.4(2) Trichoderma longibrachiatum glucanohydrolase
(formerly reesei)
Chymosin protease (1) Aspergillus niger var. cleaves a single bond in kappaawamori* casein
d-calf prochymosin gene(2) Escherichia coli K-12* cleaves a single bond in kappa 3.4.23.4
d-calf prochymosin gene casein(3) Kluyveromyces marxianus*
Chymotrypsin protease bovine or porcine pancreatic none 3.4.21.1extract
Ficin protease figs: Ficus sp. none 3.4.22.3
�-Galactosidase carbohydrase (1) Mortierella vinacea var. �-D-galactoside galactohydrolase 3.2.1.22raffinoseutilizer
(2) Aspergillus niger
scribed above. Determine the amount of unsaturation in thesample using the same multivariate analysis parameters andoptimal number of factors that were obtained from the calibra-tion matrix. Sum the different unsaturation amounts to obtainthe total unsaturation in the sample.
of the major components or of the enzyme for which thepreparation is standardized, in accordance with the Recom-mendations (1992) of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biologyon the Nomenclature and Classification of Enzymes.
FCC V General Tests and Assays / Appendix V / 897
TRIVIALNAME CLASSIFICATION SOURCE NAMES (IUB)a NO.a
�-Glucanase carbohydrase (1) Aspergillus niger var. 1,3-(1,3;1,4)-�-D-glucan 3.2.1.6(2) Bacillus subtilis var. 3(4)-glucanohydrolase(3) Trichoderma longibrachiatum
Glucoamylase carbohydrase (1) Aspergillus niger var. 1,4-�-D-glucan glucohydrolase 3.2.1.3(Amyloglucosidase) (2) Aspergillus oryzae var.
(3) Rhizopus oryzae var.(4) Rhizopus niveus
Glucose Isomerase isomerase (1) Actinoplanes missouriensis D-xylose ketoisomerase 5.3.1.5(2) Bacillus coagulans(3) Streptomyces olivaceus(4) Streptomyces
olivochromogenus(5) Streptomyces rubiginosus(6) Streptomyces murinus(7) Microbacterium arborescens
Glucose Oxidase oxidoreductase Aspergillus niger var. �-D-glucose: oxygen 1.1.3.41-oxidoreductase
�-D-Glucosidase carbohydrase (1) Aspergillus niger var. �-D-glucoside glucohydrolase 3.2.1.21(2) Trichoderma longibrachiatum
Hemicellulase carbohydrase (1) Aspergillus niger var. (1) �-L-arabinofuranoside 3.2.1.55arabinofuranohydrolase
(2) Trichoderma longibrachiatum (2) 1,4-�-D-mannan 3.2.1.78mannanohydrolase
(3) 1,3-�-D-xylan 3.2.1.32xylanohydrolase
(4) 1,5-�-L-arabinan 3.2.1.99arabinanohydrolase
Invertase carbohydrase Saccharomyces sp. �-D-fructofuranoside 3.2.1.26(Kluyveromyces) fructohydrolase
Lactase carbohydrase (1) Aspergillus niger var. �-D-galactoside 3.2.1.23galactohydrolase
(2) Aspergillus oryzae var.(3) Saccharomyces sp.(4) Candida pseudotropicalis(5) Kluyveromyces marxianus
var. lactis
Lipase lipase (1) edible forestomach tissue of (1) carboxylic-ester hydrolase 3.1.1.1calves, kids, and lambs
(2) animal pancreatic tissues (2) triacylglycerol 3.1.1.3acylhydrolase
(3) Aspergillus oryzae var.(4) Aspergillus niger var.(5) Rhizomucor miehei(6) Candida rugosa
Maltogenic Amylase carbohydrase Bacillus subtilis* 1,4-�-D-glucan 3.2.1.133d-Bacillus stearothermophilus �-maltohydrolase
Pancreatin mixed bovine and porcine pancreatic tissue (1) 1,4-�-D-glucan 3.2.1.1carbohydrase, glucanohydrolaseprotease, and (2) triacylglycerol acylhydrolase 3.1.1.3lipase (3) protease 3.4.21.4
898 / Appendix V / General Tests and Assays FCC V
TRIVIALNAME CLASSIFICATION SOURCE NAMES (IUB)a NO.a
Papain protease papaya: Carica papaya (L) none 3.4.22.23.4.22.6
Pectinaseb carbohydrase (1) Aspergillus niger var. (1) poly(1,4-�-D-galacturonide) 3.2.1.15glycanohydrolase
(2) Rhizopus oryzae var. (2) pectin pectylhydrolase 3.1.1.11(3) poly(1,4-�-D-glacturonide) 4.2.2.2
lyase(4) poly(methoxyl-L- 4.2.2.10
galacturonide) lyase
Pepsin protease porcine or other animal stomach none 3.4.23.1tissue 3.4.23.2
Phospholipase A2 lipase animal pancreatic tissue phosphatidylcholine 3.1.1.42-acylhydrolase
Phytase phosphatase Aspergillus niger var. (1) myo-inositol- 3.1.3.8hexakisphosphate-3-phosphohydrolase
(2) orthophosphoric-mono 3.1.3.2ester phosphohydrolase
Protease (general) protease (1) Aspergillus niger var. none 3.4.23.18(2) Aspergillus oryzae var(3) Bacillus subtilis var. 3.4.24.28(4) Bacillus licheniformis var. 3.4.21.62
Pullulanase carbohydrase Bacillus acidopullulyticus �-dextrin-6-glucanohydrolase 3.2.1.41
Rennet protease (1) fourth stomach of ruminant none 3.4.23.1animals
(2) Endothia parasitica 3.4.23.4(3) Rhizomucor miehei 3.4.23.22(4) Rhizomucor pusillus (Lindt) 3.4.23.23(5) Bacillus cereus
Trypsin protease animal pancreas none 3.4.21.4aEnzyme Nomenclature: recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular
Biology, Academic Press, New York, 1992.bUsually a mixture of pectin depolymerase, pectin methylesterase, pectin lyase, and pectate lyase.*The asterisk indicates a genetically modified organism. The donor organism is listed after ‘‘d-.’’
The following procedures are provided for application as nec-essary in determining compliance with the vendor’s declaredrepresentations for enzyme activity. For all of the proceduresuse filtered, ultra-high purity water with a resistivity of 16 to18 megohms.
ACID PHOSPHATASE ACTIVITY
Application and Principle This procedure is used to deter-mine acid phosphatase activity in preparations derived fromAspergillus niger var. The test is based on the enzymatichydrolysis of p-nitrophenyl phosphate, followed by the mea-surement of the released inorganic phosphate.
Reagents and SolutionsGlycine Buffer (0.2 M, pH 2.5) Dissolve 15.014 g of
glycine (Merck, Catalog No. 4201) in about 800 mL of water.Adjust the pH to 2.5 with 1 M hydrochloric acid (consumptionshould be about 80 mL), and dilute to 1000 mL with water.
Substrate (30 mM) Dissolve 1.114 g of p-nitrophenylphosphate (Boehringer, Catalog No. 738 352) in GlycineBuffer, and adjust the volume to 100 mL with the buffer.Prepare a fresh substrate solution daily.
TCA Solution Dissolve 15 g of trichloroacetic acid inwater, and dilute to 100 mL.
Ascorbic Acid Solution Dissolve 10 g of ascorbic acid inwater, and dilute to 100 mL. Store under refrigeration. Thesolution is stable for 7 days.
Ammonium Molybdate Solution Dissolve 2.5 g of ammo-nium molybdate [(NH4)6MoO24·4H2O] (Merck, Catalog No.1182) in water, and dilute to 100 mL.
FCC V General Tests and Assays / Appendix V / 899
1 M Sulfuric Acid Stir 55.6 mL of concentrated sulfuricacid (H2SO4) (Merck, Catalog No. 731) into about 800 mLof water. Allow to cool, and make up to 1000 mL with water.
Reagent C Mix 3 volumes of 1 M Sulfuric Acid with 1volume of Ammonium Molybdate Solution, then add 1 volumeof Ascorbic Acid Solution, and mix well. Prepare fresh daily.
Standard Phosphate Solution Prepare a 9.0-mM phos-phate stock solution. Dissolve and dilute 612.4 mg of potas-sium dihydrogen phosphate (KH2PO4) (dried in desiccatorwith silica) to 500 mL with water in a volumetric flask. Makethe following dilutions in water from the stock solution, anduse these as standards.
Phosphorus Acid PhosphataseConcentration Activity
Dilution (nmol/mL) (HFU/mL)
1:100 90 24001:200 45 12001:400 22.5 600
Pipet 4.0 mL of each dilution into two test tubes. Also pipet4.0 mL of water into one tube (reagent blank). Add 4.0 mLof Reagent C, and mix. Incubate at 500 for 20 min, and coolto room temperature. Measure the absorbances at 820 nmagainst that of reagent blank. Prepare a standard curve byplotting the absorbances against acid phosphatase activity[HFU (acid phosphatase unit)/mL]. Construct a new standardcurve with each series of assays.
Test Preparation Prepare a solution of the enzyme prepa-ration in the Glycine Buffer so that 1 mL will contain between600 and 2400 HFU/mL.
Procedure Pipet 1.9 mL of Substrate in two test tubes. Add2.0 mL of TCA Solution to one of the tubes (blank), and mix.Put the tubes without TCA Solution in a water bath at 37°and let them equilibrate for 5 min. While using a stopwatch,start the hydrolysis by adding sequentially at proper intervals0.1 mL of Test Preparation to each tube, and mix. Afterexactly 15 min of incubation, stop the reaction by adding 2.0mL of TCA Solution to each tube. Mix, and cool to roomtemperature. Add 0.1 mL of Test Preparation to the reagentblank tube (kept at room temperature), and mix. If precipitateoccurs, separate it by centrifugation for 10 min at 2000 g.
Pipet 0.4 mL of each sample after hydrolysis into separatetest tubes. Add 3.6 mL of water to each tube. Add 4.0 mLof Reagent C, and mix. Incubate at 50° for 20 min, and coolto room temperature. Determine the absorbance against thatof reagent blank at 820 nm.
Calculation One acid phosphatase unit (HFU) is the amountof enzyme that liberates, under the conditions of the assay,inorganic phosphate from p-nitrophenyl phosphate at the rateof 1 nmol/min.
Subtract the blank absorbance from the sample absorbance(the difference should be between 0.100 and 1.000). Deter-mine the acid phosphatase activity (HFU/mL) from the stan-dard curve, and multiply by the dilution factor. For the activityof solid samples, use the following equation:
HFU/g = (HFU/mL × f)/g,
in which f is the dilution factor and g is the weight, in grams,of the sample.
AMINOPEPTIDASE (LEUCINE)ACTIVITY
Application and Principle This procedure is used to deter-mine leucine aminopeptidase activity in enzyme preparationsderived from Lactococcus lactis. The assay is based on therate of absorbance change over 5 min at 30°; the change inabsorbance is due to liberated p-nitroaniline from the hydroly-sis of leucine p-nitroanilide.
ApparatusSpectrophotometer Use a spectrophotometer with temper-
ature control, suitable for measuring absorbancies at 410 nm.Cuvette Use a 10-mm light path, quartz.Thermometer Use a partial immersion thermometer with
a suitable range.Vortex Mixer Use a standard, variable-speed mixer.
Reagents and SolutionspH 7.0 Phosphate Buffer (100 mM) Dissolve 13.6 g of
anhydrous potassium dihydrogen orthophosphate in water,and dilute to 1 L (Solution A). Dissolve 22.8 g of dipotassiumhydrogen orthophosphate trihydrate in water, and dilute to 1L (Solution B). Slowly add approximately 550 mL of SolutionB to approximately 400 mL of Solution A until the pH of thebuffer stabilizes at 7 � 0.02.
Substrate Solution Dissolve 0.0200 g of leucine p-nitroan-ilide hydrochloride (Sigma Chemical Co., Catalog No. L2158)in 100 mL of pH 7.0 Phosphate Buffer.
p-Nitroaniline Stock Solution Transfer 156.9 mg of p-nitroaniline (Aldrich Chemical Co., Catalog No. 18,531-0) toa 1-L volumetric flask, and dilute to volume with water. Thissolution is 1.1136 mM.
Caution: p-Nitroaniline is highly toxic. Avoid breath-ing its dust; avoid contact with skin, eyes, and clothing.Wash the affected area with water; for eyes seek medicalattention.
Standard p-Nitroaniline Solutions Prepare the followingdilutions of p-Nitroaniline Stock Solution: dilute 1 mL of p-Nitroaniline Stock Solution to 100 mL with pH 7.0 PhosphateBuffer (Solution 1, 0.01136 mM); dilute 9 mL of Solution 1with 3 mL of pH 7.0 Phosphate Buffer (Solution 2, 0.00852mM); and dilute 5 mL of Solution 1 with 5 mL of pH 7.0Phosphate Buffer (Solution 3, 0.00568 mM).
Sample Solution Prepare a solution in pH 7.0 PhosphateBuffer that contains between 0.025 and 0.1 unit of aminopepti-dase activity per mL.
900 / Appendix V / General Tests and Assays FCC V
Procedure Determine the absorbance of each of the threestandard p-nitroaniline dilutions (solutions 1, 2, and 3) at 410nm using pH 7.0 Phosphate Buffer as the blank.
Pipet 3 mL of Substrate Solution into a cuvette, insert athermometer in each to ensure that the temperature of thesolution is correct, and equilibrate in the spectrophotometer to30° � 0.2°. Add 150 �L of Sample Solution to the equilibratedSubstrate Solution. Mix, and start recording the absorbance.Continue recording the absorbance for approximately 5 min;it should increase linearly with time. To determine the rateof change of absorbance, ignore the initial 0.5 min of theassay line, and use a period of at least 4 min to estimate therate of change.
Calculation One aminopeptidase activity unit (AP) is de-fined as the quantity of aminopeptidase required to liberate1 �mol/min of leucine from leucine p-nitroanilide under theconditions of the assay at pH 7.0 and 30°.
For each of the diluted Standard Solutions—1, 2, and 3—plot absorbance against p-nitroaniline mM concentration. Theresult is a straight line that passes through the origin. Calculatethe millimolar extinction coefficient (�) of each Standard p-Nitroaniline Solution using the following formula:
� = AN/C,
in which AN is the absorbance of the Standard p-NitroanilineSolution at 410 nm and C is the millimolar concentration ofp-nitroaniline of that solution. Average the three calculatedvalues; this should result in a value of approximately 8.8.Calculate the activity of each sample taken by the equation:
AP/g = (�A × TCV × 1000)/(� × SV × C),
in which �A is the rate of change of absorbance per minute;TCV is the total cuvette volume (3.150 mL); SV is the samplevolume (0.150 mL); and C is the concentration, in milligramsper milliliter, of the sample.
�-AMYLASE ACTIVITY(NONBACTERIAL)
Application and Principle This procedure is used to deter-mine the �-amylase activity of enzyme preparations derivedfrom Aspergillus niger var.; Aspergillus oryzae var.; Rhizopusoryzae var.; and barley malt. The assay is based on the timerequired to obtain a standard degree of hydrolysis of a starchsolution at 30° � 0.1°. The degree of hydrolysis is determinedby comparing the iodine color of the hydrolysate with thatof a standard.
ApparatusReference Color Standard Use a special Alpha-Amylase
Color Disk (Orbeco Analytical Systems, 185 Marine Street,Farmingdale, NY 11735, Catalog No. 620-S5). Alternatively,prepare a color standard by dissolving 25.0 g of cobaltous
chloride (CoCl2·6H2O) and 3.84 g of potassium dichromatein 100 mL of 0.01 N hydrochloric acid. This standard is stableindefinitely when stored in a stoppered bottle or compara-tor tube.
Comparator Use either the standard Hellige comparator(Orbeco, Catalog No. 607) or the pocket comparator withprism attachment (Orbeco, Catalog No. 605AHT). The com-parator should be illuminated with a 100-W frosted lampplaced 6 in. from the rear opal glass of the comparator andmounted so that direct rays from the lamp do not shine intothe operator’s eyes.
Comparator Tubes Use the precision-bored square tubeswith a 13-mm viewing depth that are supplied with the Helligecomparator. Suitable tubes are also available from other appa-ratus suppliers (e.g., Thomas Scientific).
Reagents and SolutionsBuffer Solution (pH 4.8) Dissolve 164 g of anhydrous
sodium acetate in about 500 mL of water, add 120 mL ofglacial acetic acid, and adjust the pH to 4.8 with glacial aceticacid. Dilute to 1000 mL with water, and mix.
�-Amylase Solution Dissolve into 5 mL of water a quan-tity of �-amylase, free from �-amylase activity (Sigma Chemi-cal Co., Catalog No. A7005), equivalent to 250 mg of �-amylase with 2000° diastatic power.
Special Starch Use starch designated as ‘‘Starch (Lintner)Soluble’’ (Baker Analyzed Reagent, Catalog No. 4010). Be-fore using new batches, test them in parallel with previouslots known to be satisfactory. Variations of more than �3°diastatic power in the averages of a series of parallel testsindicate an unsuitable batch.
Buffered Substrate Solution Disperse 10.0 g (dry-weightbasis) of Special Starch in 100 mL of cold water, and slowlypour the mixture into 300 mL of boiling water. Boil and stirfor 1 to 2 min, then cool, and add 25 mL of Buffer Solution,followed by all of the �-Amylase Solution. Quantitativelytransfer the mixture into a 500-mL volumetric flask with theaid of water saturated with toluene, dilute to volume with thesame solvent, and mix. Store the solution at 30° � 2° for notless than 18 h nor more than 72 h before use. (This solutionis also known as ‘‘buffered limit dextrin substrate.’’)
Stock Iodine Solution Dissolve 5.5 g of iodine and 11.0g of potassium iodide in about 200 mL of water, dilute to250 mL with water, and mix. Store in a dark bottle, and makea fresh solution every 30 days.
Dilute Iodine Solution Dissolve 20 g of potassium iodidein 300 mL of water, and add 2.0 mL of Stock Iodine Solution.Quantitatively transfer the mixture into a 500-mL volumetricflask, dilute to volume with water, and mix. Prepare daily.
Sample Preparation Prepare a solution of the sample sothat 5 mL of the final dilution will give an endpoint between10 and 30 min under the conditions of the assay.
For barley malt, finely grind 25 g of the sample in a Miag-Seck mill (Buhler-Miag, Inc., P.O. Box 9497, Minneapolis,MN 55440). Quantitatively transfer the powder into a 1000-mL Erlenmeyer flask, add 500 mL of a 0.5% solution ofsodium chloride, and allow the infusion to stand for 2.5 h at30° � 0.2°, agitating the contents by gently rotating the flaskat 20-min intervals.
FCC V General Tests and Assays / Appendix V / 901
Caution: Do not mix the infusion by inverting the flask.The quantity of the grist left adhering to the inner wallsof the flask as a result of agitation must be as small aspossible.
Filter the infusion through a 32-cm fluted filter of WhatmanNo. 1, or equivalent, paper on a 20-cm funnel, returning thefirst 50 mL of filtrate to the filter. Collect the filtrate until 3h have elapsed from the time the sodium chloride solutionand the sample were first mixed. Pipet 20.0 mL of the filteredinfusion into a 100-mL volumetric flask, dilute to volumewith the 0.5% sodium chloride solution, and mix.
Procedure Pipet 5.0 mL of Dilute Iodine Solution into aseries of 13- × 100-mm test tubes, and place them in a waterbath maintained at 30° � 0.1°, allowing 20 tubes for eachassay.
Pipet 20.0 mL of the Buffered Substrate Solution, previouslyheated in the water bath for 20 min, into a 50-mL Erlenmeyerflask, and add 5.0 mL of 0.5% sodium chloride solution, alsopreviously heated in the water bath for 20 min. Place the flaskin the water bath.
At zero time, rapidly pipet 5.0 mL of the Sample Prepara-tion into the equilibrated substrate, mix immediately by swirl-ing, stopper the flask, and place it back in the water bath.After 10 min, transfer 1.0 mL of the reaction mixture fromthe 50-mL flask into one of the test tubes containing the DiluteIodine Solution, shake the tube, then pour its contents into aComparator Tube, and immediately compare with the Refer-ence Color Standard in the Comparator, using a tube of waterbehind the color disk.
Note: Be certain that the pipet tip does not touch theiodine solution; carryback of iodine to the hydrolyzingmixture will interfere with enzyme action and will affectthe results of the determination.
In the same manner, repeat the transfer and comparison proce-dure at accurately timed intervals until the �-amylase coloris reached, at which time record the elapsed time. In caseswhere two comparisons 30 s apart show that one is darkerand the other lighter than the Reference Color Standard, recordthe endpoint to the nearest quarter min. Shake out the 13-mm Comparator Tube between successive readings. Minimizeslight differences in color discrimination between operatorsby using a prism attachment and by maintaining a 6- to 10-in. distance between the Comparator and the operator’s eye.
Calculation One �-amylase dextrinizing unit (DU) is de-fined as the quantity of �-amylase that will dextrinize solublestarch in the presence of an excess of �-amylase at the rateof 1 g/h at 30°.
Calculate the �-amylase dextrinizing units in the sampleas follows:
DU (solution) = 24/(W × T),
and
DU (dry basis) = DU (solution) × 100/(100 − M),
in which W is the weight, in grams, of the enzyme sampleadded to the incubation mixture in the 5-mL aliquot of the
Sample Preparation used; T is the elapsed dextrinizing time,in minutes; 24 is the product of the weight of the starchsubstrate (0.4 g) and 60 min; and M is the percent moisturein the sample, determined by suitable means.
�-AMYLASE ACTIVITY (BACTERIAL)
Application and Principle This procedure is used to deter-mine the �-amylase activity, expressed as bacterial amylaseunits (BAU), of enzyme preparations derived from Bacillussubtilis var., Bacillus licheniformis var., and Bacillus stearoth-ermophilus. It is not applicable to products that contain �-amylase. The assay is based on the time required to obtain astandard degree of hydrolysis of a starch solution at 30° �0.1°. The degree of hydrolysis is determined by comparingthe iodine color of the hydrolysate with that of a standard.
Apparatus Use the Reference Color Standard, the Compa-rator, and the Comparator Tubes as described under �-Amy-lase Activity (Nonbacterial), described in this Appendix, butuse either daylight or daylight-type fluorescent lamps as thelight source for the Comparator. (Incandescent lamps giveslightly lower results.)
Reagents and SolutionspH 6.6 Buffer Dissolve 9.1 g of potassium dihydrogen
phosphate (KH2PO4) in sufficient water to make 1000 mL(Solution A). Dissolve 9.5 g of dibasic sodium phosphate(Na2HPO4) in sufficient water to make 1000 mL (SolutionB). Add 400 mL of Solution A to 600 mL of Solution B, mix,and adjust the pH to 6.6, if necessary, by the addition ofSolution A or Solution B as required.
Dilute Iodine Solution Prepare as directed under �-Amy-lase Activity (Nonbacterial).
Special Starch Use the material described under �-Amy-lase Activity (Nonbacterial).
Starch Substrate Solution Disperse 10.0 g (dry-weightbasis) of Special Starch in 100 mL of cold water, and slowlypour the mixture into 300 mL of boiling water. Boil and stirfor 1 to 2 min, and then cool while continuously stirring.Quantitatively transfer the mixture into a 500-mL volumetricflask with the aid of water, add 10 mL of pH 6.6 Buffer,dilute to volume with water, and mix.
Sample Preparation Prepare a solution of the sample sothat 10 mL of the final dilution will give an endpoint between15 and 35 min under the conditions of the assay.
Procedure Pipet 5.0 mL of Dilute Iodine Solution into aseries of 13- × 100-mm test tubes, and place them in a waterbath maintained at 30° � 0.1°, allowing 20 tubes for eachassay.
Pipet 20.0 mL of the Starch Substrate Solution into a 50-mL Erlenmeyer flask, stopper, and allow to equilibrate for20 min in the water bath at 30°.
902 / Appendix V / General Tests and Assays FCC V
At zero time, rapidly pipet 10.0 mL of the Sample Prepara-tion into the equilibrated mixture, and continue as directedin the Procedure under �-Amylase Activity (Nonbacterial),beginning with ‘‘. . . mix immediately by swirling, stopperthe flask. . . .’’
Calculation One bacterial amylase unit (BAU) is definedas that quantity of enzyme that will dextrinize starch at therate of 1 mg/min under the specified test conditions.
Calculate the �-amylase activity of the sample, expressedas BAU, by the formula
BAU/g = 40F/T,
in which 40 is a factor (400/10) derived from the 400 mg ofstarch (20 mL of a 2% solution) and the 10-mL aliquot ofSample Preparation used; F is the dilution factor (total dilutionvolume/sample weight, in grams); and T is the dextrinizingtime, in minutes.
CATALASE ACTIVITY
Application and Principle This procedure is used to deter-mine the catalase activity, expressed as Baker Units, of prepa-rations derived from Aspergillus niger var., bovine liver, orMicrococcus lysodeikticus. The assay is an exhaustion methodbased on the breakdown of hydrogen peroxide by catalaseand the simultaneous breakdown of the catalase by the perox-ide under controlled conditions.
Reagents and SolutionsAmmonium Molybdate Solution (1%) Dissolve 1.0 g of
ammonium molybdate [(NH4)6MoO24·4H2O] (Merck, CatalogNo. 1182) in water, and dilute to 100 mL.
0.250 N Sodium Thiosulfate Dissolve 62.5 g of sodiumthiosulfate (Na2S2O3·5H2O) in 750 mL of recently boiled andcooled water, add 3.0 mL of 0.2 N sodium hydroxide as astabilizer, dilute to 1000 mL with water, and mix. Standardizeas directed for 0.1 N Sodium Thiosulfate (see Solutions andIndicators), and, if necessary, adjust to exactly 0.250 N.
Peroxide Substrate Solution Dissolve 25.0 g of anhydrousdibasic sodium phosphate (Na2HPO4), or 70.8 g of Na2H-PO4·12H2O, in about 1500 mL of water, and adjust to pH 7.0� 0.1 with 85% phosphoric acid. Cautiously add 100 mL of30% hydrogen peroxide, dilute to 2000 mL in a graduate, andmix. Store in a clean amber bottle, loosely stoppered. Thesolution is stable for more than 1 week if kept at 5° in a fullcontainer. (With freshly prepared substrate, the blank willrequire about 16 mL of 0.250 N Sodium Thiosulfate. If theblank requires less than 14 mL, the substrate solution is unsuit-able and should be prepared fresh again. The sample titrationmust be between 50% and 80% of that required for the blank.)
Procedure Pipet an aliquot of not more than 1.0 mL of thesample, previously diluted to contain approximately 3.5 Baker
Units of catalase, into a 200-mL beaker. Rapidly add 100 mLof Peroxide Substrate Solution, previously adjusted to 25°,and stir immediately for 5 to 10 s. Cover the beaker, andincubate at 25° � 1° until the reaction is completed. Stirvigorously for 5 s, and then pipet 4.0 mL from the beakerinto a 50-mL Erlenmeyer flask. Add 5 mL of 2 N sulfuricacid to the flask, mix, then add 5.0 mL of 40% potassiumiodide solution, freshly prepared, and 1 drop of AmmoniumMolybdate Solution (1%), and mix. While continuing to mix,titrate rapidly to a colorless endpoint with 0.250 N SodiumThiosulfate, recording the volume, in milliliters, required asS. Perform a blank determination with 4.0 mL of PeroxideSubstrate Solution, and record the volume required, in millili-ters, as B.
Note: When preparations derived from beef liver aretested, the reaction is complete within 30 min. Prepara-tions derived from Aspergillus and other sources mayrequire up to 1 h. In assaying an enzyme of unknownorigin, run a titration after 30 min and then at 10-minintervals thereafter. The reaction is complete when twoconsecutive titrations are the same.
Calculation One Baker Unit is defined as the amount ofcatalase that will decompose 264 mg of hydrogen peroxideunder the conditions of the assay.
Calculate the activity of the sample by the equation
Baker Units/g or mL = 0.4(B − S) × (1/C),
in which C is the milliliters of aliquot of original enzymepreparation added to each 100 mL of Peroxide SubstrateSolution, or when 1 mL of diluted enzyme is used, C is thedilution factor; B is the volume, in milliliters, as definedabove; and S is the milliliters of 0.250 N Sodium Thiosulfate,as defined above.
CELLULASE ACTIVITY
Application and Principle This assay is based on the enzy-matic hydrolysis of the interior �-1,4-glucosidic bonds of adefined carboxymethyl cellulose substrate at pH 4.5 and at40°. The corresponding reduction in substrate viscosity isdetermined with a calibrated viscometer.
ApparatusCalibrated Viscometer Use a size 100 Calibrated Cannon-
Fenske Type Viscometer, or its equivalent (Scientific Prod-ucts, Catalog No. P2885-100).
Constant-Temperature Glass Water Bath (40° � 0.1°)Use a constant-temperature glass water bath, or its equivalent(Scientific Products, Catalog No. W3520-10).
Stopwatches Use two stopwatches, Stopwatch No. 1, cali-brated in 1⁄10 min for determining the reaction time (Tr), andStopwatch No. 2, calibrated in 1⁄5 s for determining the effluxtime (Tt).
FCC V General Tests and Assays / Appendix V / 903
Waring Blender Use a two-speed Waring blender, or itsequivalent (Scientific Products, Catalog No. 58350-1).
Reagents and SolutionsAcetic Acid Solution (2 N) While agitating a 1-L beaker
containing 800 mL of water, carefully add 116 mL of glacialacetic acid. Cool to room temperature. Quantitatively transferthe solution to a 1-L volumetric flask, and dilute to volumewith water.
Sodium Acetate Solution (2 N) Dissolve 272.16 g of so-dium acetate trihydrate in approximately 800 mL of watercontained in a 1-L beaker. Quantitatively transfer to a 1-Lvolumetric flask, and dilute to volume with water.
Acetic Acid Solution (0.4 N) Transfer 200 mL of AceticAcid Solution (2 N) into a 1-L volumetric flask, and dilute tovolume with water.
Sodium Acetate Solution (0.4 N) Transfer 200 mL of So-dium Acetate Solution (2 N) into a 1-L volumetric flask, anddilute to volume with water.
Acetate Buffer (pH 4.5) Using a standardized pH meter,add Sodium Acetate Solution (0.4 N) with continuous agitationto 400 mL of Acetic Acid Solution (0.4 N) in a suitable flaskuntil the pH is 4.5 � 0.05.
Sodium Carboxymethylcellulose Use sodium carboxy-methylcellulose (Hercules, Inc., CMC Type 7HF).
Sodium Carboxymethylcellulose Substrate (0.2% w/v)Transfer 200 mL of water into the bowl of the Waring blender.With the blender on low speed, slowly disperse 1.0 g (mois-ture-free basis) of the Sodium Carboxymethylcellulose intothe bowl, being careful not to splash out any of the liquid.Using a rubber policeman, wash down the sides of the glassbowl with water. Place the top on the bowl and blend athigh speed for 1 min. Quantitatively transfer to a 500-mLvolumetric flask, and dilute to volume with water. Filter thesubstrate through gauze before use.
Sample Preparation Prepare an enzyme solution so that1 mL of the final dilution will produce a relative fluiditychange between 0.18 and 0.22 in 5 min under the conditionsof the assay. Weigh the enzyme, and quantitatively transferit to a glass mortar. Triturate with water and quantitativelytransfer the mixture to an appropriate volumetric flask. Diluteto volume with water, and filter the enzyme solution throughWhatman No. 1 filter paper before use.
Procedure Place the Calibrated Viscometer in the 40° �0.1° water bath in an exactly vertical position. Use only ascrupulously clean viscometer. (To clean the viscometer, drawa large volume of detergent solution followed by water throughthe viscometer by using an aspirator with a rubber tube con-nected to the narrow arm of the viscometer.)
Pipet 20 mL of filtered Sodium CarboxymethylcelluloseSubstrate and 4 mL of Acetate Buffer into a 50-mL Erlenmeyerflask. Allow at least two flasks for each enzyme sample andone flask for a substrate blank. Stopper the flasks, and equili-brate them in the water bath for 15 min.
At zero time, pipet 1 mL of the enzyme solution into theequilibrated substrate. Start stopwatch no. 1, and mix thesolution thoroughly. Immediately pipet 10 mL of the reactionmixture into the wide arm of the viscometer.
After approximately 2 min, apply suction with a rubbertube connected to the narrow arm of the viscometer, drawingthe reaction mixture above the upper mark into the drivingfluid head. Measure the efflux time by allowing the reactionmixture to freely flow down past the upper mark. As themeniscus of the reaction mixture falls past the upper mark,start stopwatch no. 2. At the same time, record the reactiontime, in minutes, from stopwatch no. 1 (Tr). As the meniscusof the reaction mixture falls past the lower mark, record thetime, in seconds, from stopwatch no. 2 (Tt).
Repeat the final step until a total of four determinations isobtained over a reaction time (Tr) of not more than 15 min.
Prepare a substrate blank by pipetting 1 mL of water into24 mL of buffered substrate. Pipet 10 mL of the reactionmixture into the wide arm of the viscometer. Determine thetime (Ts) in seconds required for the meniscus to fall betweenthe two marks. Use an average of five determinations for (Ts).
Prepare a water blank by pipetting 10 mL of equilibratedwater into the wide arm of the viscometer. Determine thetime (Tw) in seconds required for the meniscus to fall betweenthe two marks. Use an average of five determinations for (Tw).
Calculations One Cellulase Unit (CU) is defined as theamount of activity that will produce a relative fluidity changeof 1 in 5 min in a defined carboxymethyl cellulose substrateunder the conditions of the assay.
Calculate the relative fluidities (Fr) and the (Tn) values foreach of the four efflux times (Tt) and reaction times (Tr) asfollows:
Fr = (Ts − Tw)/(Tt − Tw),
Tn = 1⁄2(Tt/60 s/min) + Tr = (Tt/120) + Tr,
in which Fr is the relative fluidity for each reaction time; Ts
is the average efflux time, in seconds, for the substrate blank;Tw is the average efflux time, in seconds, for the water blank;Tt is the efflux time, in seconds, of reaction mixture; Tr is theelapsed time, in minutes, from zero time, that is, the timefrom addition of the enzyme solution to the buffered substrateuntil the beginning of the measurement of efflux time (Tt);and Tn is the reaction time, in minutes (Tr), plus one-half ofthe efflux time (Tt), converted to minutes.
Plot the four relative fluidities (Fr) as the ordinate againstthe four reaction times (Tn) as the abscissa. A straight lineshould be obtained. The slope of this line corresponds to therelative fluidity change per minute and is proportional to theenzyme concentration. The slope of the best line through aseries of experimental points is a better criterion of enzymeactivity than is a single relative fluidity value. From the graph,determine the Fr values at 10 and 5 min. They should havea difference in fluidity of not more than 0.22 or less than0.18. Calculate the activity of the enzyme unknown as follows:
CU/g = [1000(Fr10 − Fr5)]/W,
in which Fr5 is the relative fluidity at 5 min of reaction time;Fr10 is the relative fluidity at 10 min of reaction time; 1000is the milligrams per gram; and W is the weight, in milligrams,of enzyme added to the reaction mixture in a 1-mL aliquotof enzyme solution.
904 / Appendix V / General Tests and Assays FCC V
CHYMOTRYPSIN ACTIVITY
Application and Principle This procedure is used to deter-mine chymotrypsin activity in chymotrypsin preparations de-rived from purified extracts of porcine or bovine pancreas.
Reagents and Solutions0.15 M Phosphate Buffer (pH 7.0) Dissolve 4.54 g of
monobasic potassium phosphate in water, and dilute to 500mL. Dissolve 4.73 g of anhydrous dibasic sodium phosphatein water, and dilute to 500 mL. Mix 38.0 mL of the monobasicpotassium phosphate solution with 61.1 mL of the dibasicsodium phosphate solution. Adjust the pH of the mixture to7.0 by the dropwise addition of the dibasic sodium phosphatesolution, if necessary.
Substrate Solution Dissolve 23.7 mg of N-acetyl-L-tyro-sine ethyl ester in about 50 mL of the 0.15 M PhosphateBuffer with warming. When the solution has cooled, diluteto 100.0 mL with the 0.15 M Phosphate Buffer.
Sample Preparation Dissolve a sufficient amount of sam-ple, accurately weighed, in 0.001 N hydrochloric acid to pro-duce a solution containing between 12 and 16 USP Chymo-trypsin Units per milliliter. This solution should cause achange in absorbance between 0.008 and 0.012 in a 30-sinterval.
Procedure Conduct the assay in a suitable spectrophotome-ter equipped to maintain a temperature of 24° � 0.1° in thecell compartment. Determine the temperature before and aftermeasuring the absorbance to ensure that the temperature doesnot change more than 0.5° during the assay. Pipet 0.2 mL ofthe 0.001 N hydrochloric acid and 3.0 mL of the SubstrateSolution into a 1-cm cell. Place the cell in the spectrophotome-ter, and adjust the instrument so that the absorbance will read0.200 at 237 nm. Pipet 0.2 mL of the Sample Preparationinto a second cell, add 3.0 mL of the Substrate Solution, andplace the cell in the spectrophotometer. Begin timing thereaction from the addition of the Substrate Solution. Read theabsorbance at 30-s intervals for at least 5 min. Repeat theprocedure at least once. If the rate of change fails to remainconstant for at least 3 min, repeat the test, and if necessary, usea lower sample concentration. The duplicate determinations atthe same sample concentration should match the first determi-nation in rate of absorbance change.
Calculations One USP Chymotrypsin Unit is defined as theactivity causing a change in absorbance at the rate 0.0075/min under the conditions of the assay. Determine the averageabsorbance change per min using only those values withinthe 3-min portion of the curve where the rate of change isconstant. Plot a curve of absorbance against time.
Calculate the number of Chymotrypsin Units per milligramby the formula
(A2 – A1)/(0.0075TW),
in which A2 is the straight-line initial absorbance reading; A1
is the straight-line final absorbance reading; T is the elapsed
time, in minutes; and W is the weight, in milligrams, ofthe sample in the volume of solution used to determine theabsorbance.
DIASTASE ACTIVITY (DIASTATICPOWER)
Application and Principle This procedure is used to deter-mine the amylase activity of barley malt and other enzymepreparations. The assay is based on a 30-min hydrolysis of astarch substrate at pH 4.6 and 20°. The reducing sugar groupsproduced on hydrolysis are measured in a titrimetric procedureusing alkaline ferricyanide.
ApparatusMill Use a laboratory mill of the type Miag-Seck, for fine
grinding of malt (Buhler Miag, Inc.).
Reagents and SolutionsAcetate Buffer Solution Dissolve 68 g of sodium acetate
(NaC2H3O2·3H2O) in 500 mL of 1 N acetic acid in a 1000-mL volumetric flask, dilute to volume with water, and mix.
Special Starch Use the material described under �-Amy-lase Activity (Nonbacterial).
Starch Substrate Solution Disperse 20.0 g (dry-weightbasis) of Special Starch in 50 mL of water, mix to a finepaste, and pour slowly into 750 mL of boiling water. Boiland stir for 2 min, cool, add 20 mL of Acetate Buffer Solution,and mix. Quantitatively transfer into a 1000-mL volumetricflask, dilute to volume with water, and mix.
Acetic Acid–Potassium Chloride–Zinc Sulfate Solution (A-P-Z) Dissolve 70 g of potassium chloride and 20 g of zincsulfate (ZnSO4·7H2O) in 700 mL of water in a 1000-mLvolumetric flask, add 200 mL of glacial acetic acid, dilute tovolume with water, and mix.
Alkaline Ferricyanide Solution (0.05 N) Dissolve 16.5 gof potassium ferricyanide [K3Fe(CN)6] and 22 g of anhydroussodium carbonate in 800 mL of water in a 1000-mL volumetricflask, dilute to volume with water, and mix.
Potassium Iodide Solution Dissolve 50 g of potassiumiodide in 50 mL of water in a 100-mL volumetric flask, diluteto volume with water, and mix. Add 2 drops of 50% sodiumhydroxide solution, and mix. The solution should be colorless.
Sample PreparationMalt Samples Grind 30 g of the sample to a fine powder
in a Maig-Seck mill. Accurately weigh 25 g of the powder,and transfer it into a 1000-mL Erlenmeyer flask. Add 500mL of a 0.5% sodium chloride solution, and allow the infusionto stand for 2.5 h at 20° � 0.2°, agitating the contents bygently rotating the flask at 20-min intervals.
Note: Do not mix the infusion by inverting the flask.The quantity of grist left adhering to the inner walls of
FCC V General Tests and Assays / Appendix V / 905
the flask as a result of agitation must be as small aspossible. Gently swirl the contents of the flask withoutsplashing them against the walls to mix sufficiently.
Filter the infusion through a 32-cm fluted filter of WhatmanNo. 1, or equivalent, paper on a 20-cm funnel, returning thefirst 50 mL of filtrate to the filter. Place a watch glass overthe funnel, and use a suitable cover around the stem and overthe receiver to reduce evaporation losses during filtration.Collect the filtrate until 30 min of filtration time have elapsed.Pipet 20.0 mL of the filtrate into a 100-mL volumetric flask,dilute to volume with 0.5% sodium chloride solution, and mix.
Other Enzyme Preparations Prepare a solution so that 10mL of the final dilution will give a diastatic power (DP) valuebetween 2° and 150°.
Procedure Pipet 10.0 mL of the Sample Preparation intoa 250-mL volumetric flask, and at zero time, add 200 mL ofStarch Substrate Solution, previously equilibrated for 30 minin a water bath maintained at 20° � 0.2°. Start the stopwatchat zero time.
Place the mixture in the water bath at 20°, and allow it tocool for exactly 30 min, then add 20.0 mL of 0.5 N sodiumhydroxide, dilute to volume with water, and mix.
Prepare a blank by adding 20.0 mL of 0.5 N sodium hydrox-ide to a 250-mL volumetric flask, followed by 10.0 mL ofthe Sample Preparation. Swirl to mix, add 200 mL of StarchSubstrate Solution, dilute to volume with water, and mix.
Pipet 5.0 mL of the sample digestion mixture into a 125-mL Erlenmeyer flask, add 10.0 mL of Alkaline FerricyanideSolution, and swirl to mix. Heat the flask for exactly 20 minin a boiling water bath, and then cool to room temperature.Add 25 mL of A-P-Z Solution, followed by 1 mL of PotassiumIodide Solution, and swirl to mix. Titrate with 0.05 N sodiumthiosulfate to the complete disappearance of the blue color,recording the volume, in milliliters, of 0.05 N sodium thiosul-fate required as S.
Treat the blank solution in the same manner as describedfor the sample, recording the volume, in milliliters, of 0.05N sodium thiosulfate required as B.
Calculation One unit of diastase activity, expressed as de-grees diastatic power (DP°), is defined as that amount ofenzyme contained in 0.1 mL of a 5% solution of the sampleenzyme preparation that will produce sufficient reducing sug-ars to reduce 5 mL of Fehling’s solution when the sample isincubated with 100 mL of the substrate for 1 h at 20°.
Note: The definition of the unit does not correspond tothe method of the determination.
Calculate the diastase activity, expressed as DP°, of thesample by the formulas
DP°, as-is basis = (B − S) × 23,
and
DP°, dry basis = DP°, as-is basis × 100/(100 − M),
in which 23 is a factor, determined by collaborative study,required to convert to the units of the definition, and M is
the percent moisture of the sample, determined by suitablemeans.
�-GALACTOSIDASE ACTIVITY
Application and Principle Use this procedure to determine�-galactosidase activity in enzyme preparations derived fromAspergillus niger var. The assay is based on a 15-min hydroly-sis of p-nitrophenyl-�-D-galactopyranoside followed by spec-trophotometric measurement of the liberated p-nitrophenol.
Reagents and SolutionsAcetate Buffer Dissolve 11.55 mL of glacial acetic acid
in water, and dilute to 1 L (Solution A). Dissolve 16.4 g ofsodium acetate in water, and dilute to 1 L (Solution B). Mix7.5 mL of Solution A and 42.5 mL of Solution B, and diluteto 200 mL with water. Adjust the pH of this solution to 5.5with either Solution A or Solution B as necessary.
Substrate Solution Dissolve 0.210 g of p-nitrophenyl-�-D-galactopyranoside (Sigma Chemical Co., Catalog No. 877,or equivalent) in and dilute to 100 mL with Acetate Buffer.
Borax Buffer Dissolve 47.63 g of sodium borate decahy-drate in warm water. Cool to room temperature. Add 20 mLof 4 N sodium hydroxide solution, adjust the pH of the solutionto 9.7 with 4 N sodium hydroxide, and dilute to 2 L with water.
p-Nitrophenol Stock Solution Dissolve 0.0334 g of p-ni-trophenol (Aldrich Chemical Co., Catalog No. 24,132-6, orequivalent) in and dilute to 1 L with water. This solutioncontains 0.24 �mol of p-nitrophenol per milliliter of water.
Preparation of Standards and SamplesStandards Prepare the following dilutions of p-Nitrophe-
nol Stock Solution with water: 100:50 (v/v) (0.16 �mol/mL);50:100 (v/v) (0.08 �mol/mL); and 25:125 (v/v) (0.04 �mol/mL). Transfer 2.0 mL of the Substrate Solution to each offive separate test tubes. Add 1 mL of the p-Nitrophenol StockSolution to the first tube, 1.0 mL of each dilution to the nextthree tubes, and 1.0 mL of water to the fifth tube. Add 5.0mL of Borax Buffer to each tube, and mix.
Samples Prepare a solution of �-galactosidase sample inAcetate Buffer that contains between 0.008 and 0.024 galactos-idase units of activity per milliliter.
Procedure Equilibrate the Substrate Solution in a waterbath at 37° � 0.2° for at least 15 min. For active samples,transfer 1.0 mL of each sample to separate test tubes andequilibrate in the 37° � 0.2° water bath. At zero time, add2.0 mL of Substrate Solution, mix, and return to the waterbath. After exactly 15.0 min, add 5.0 mL of Borax Buffer toeach tube, mix, and remove from the water bath.
For sample blanks, transfer, in sequence, 1.0 mL of eachsample to separate test tubes, add 5.0 mL of Borax Buffer,and mix. Add 2.0 mL of Substrate Solution to each tube,and mix.
906 / Appendix V / General Tests and Assays FCC V
Measure the absorbance of each standard sample and blankat 405 nm versus that of water. Determine the absorbancesof all solutions within 30 min of completing the tests.
Calculations One galactosidase activity unit (GalU) is de-fined as the quantity of the enzyme that will liberate p-ni-trophenol at the rate of 1 �mol/min under the conditions ofthe assay.
Calculate the factor � for the p-nitrophenol standards usingthe following equation:
� = AN/C,
in which AN is the absorbance of the p-nitrophenol standardsat 405 nm, and C is the concentration, in millimoles permilliliter, of p-nitrophenol.
Because the averaged millimolar extinction coefficient ofp-nitrophenol at 405 nm is 18.3, � should be approximately2.29 [or (18.3)/8].
GalU/g = [(AS − AB) × F]/(� × T × M),
in which AS is the sample absorbance; AB is the blank ab-sorbance; F is the appropriate dilution factor; T is the reactiontime, in minutes; M is the weight, in grams, of the sample; and� is a factor calculated above for the p-nitrophenol standards(proportional to the millimolar extinction coefficient for p-nitrophenol).
�-GLUCANASE ACTIVITY
Application and Principle This procedure is used to deter-mine ß-glucanase activity of enzyme preparations derivedfrom Aspergillus niger var. and Bacillus subtilis var. Theassay is based on a 15-min hydrolysis of lichenin substrateat 40° and at pH 6.5. The increase in reducing power due toliberated reducing groups is measured by the neocuproinemethod.
Reagents and SolutionsPhosphate Buffer Dissolve 13.6 g of monobasic potas-
sium phosphate in about 1900 mL of water, add 70% sodiumhydroxide solution until the pH is 6.5 � 0.05, then transferthe solution into a 2000-mL volumetric flask, dilute to volumewith water, and mix.
Neocuproine Solution A Dissolve 40.0 g of anhydroussodium carbonate, 16.0 g of glycine, and 450 mg of cupricsulfate pentahydrate in about 600 mL of water. Transfer thesolution into a 1000-mL volumetric flask, dilute to volumewith water, and mix.
Neocuproine Solution B Dissolve 600 mg of neocuproinehydrochloride in about 400 mL of water, transfer the solutioninto a 500-mL volumetric flask, dilute to volume with water,and mix. Discard when a yellow color develops.
Lichenin Substrate Grind 150 mg of lichenin (SigmaChemical Co., Catalog No. L-6133, or equivalent) to a fine
powder in a mortar, and dissolve it in about 50 mL of waterat about 85°. After solution is complete (20 to 30 min), add90 mg of sodium borohydride and continue heating belowthe boiling point for 1 h. Add 15 g of Amberlite MB-3, oran equivalent ion-exchange resin, and stir continuously for30 min. Filter with the aid of a vacuum through WhatmanNo. 1 filter paper, or equivalent, in a Büchner funnel, andwash the paper with about 20 mL of water. Add 680 mg ofmonobasic potassium phosphate to the filtrate, and refilterthrough a 0.22-�m Millipore filter pad, or equivalent. Washthe pad with 10 mL of water, and adjust the pH of the filtrateto 6.5 � 0.05 with 1 N sodium hydroxide or 1 N hydrochloricacid. Transfer the filtrate into a 100-mL volumetric flask,dilute to volume with water, and mix. Store at 2° to 4° fornot more than 3 days.
Glucose Standard Solution Dissolve 36.0 mg of anhy-drous dextrose in Phosphate Buffer in a 1000-mL volumetricflask, dilute to volume with water, and mix.
Test Preparation Prepare a solution from the enzymepreparation sample so that 1 mL of the final dilution willcontain between 0.01 and 0.02 �-glucanase units. Weigh thesample, transfer it into a volumetric flask of appropriate size,dilute to volume with Phosphate Buffer, and mix.
Procedure Pipet 2 mL of Lichenin Substrate into each offour separate test tubes graduated at 25 mL, and heat the tubesin a water bath at 40° for 10 to 15 min to equilibrate.
After equilibration, add 1 mL of Phosphate Buffer to tube1 (substrate blank), 1 mL of Glucose Standard Solution totube 2 (glucose standard), 4 mL of Neocuproine Solution Aand 1 mL of the Test Preparation to tube 3 (enzyme blank),and 1 mL of the Test Preparation to tube 4 (sample). Preparea fifth tube for the buffer blank, and add 3 mL of PhosphateBuffer.
Incubate the five tubes at 40° for exactly 15 min, and thenadd 4 mL of Neocuproine Solution A to tubes 1, 2, 4, and 5.Add 4 mL of Neocuproine Solution B to all five tubes, andcap each with a suitably sized glass marble.
Caution: Do not use rubber stoppers.
Heat the tubes in a vigorously boiling water bath for exactly12 min to develop color, then cool to room temperature incold water, and adjust the volume of each to 25 mL withwater. Cap the tubes with Parafilm, or other suitable closure,and mix by inverting several times.
Determine the absorbance of each solution at 450 nm in1-cm cells, with a suitable spectrophotometer, against thebuffer blank in tube 5.
Calculation One �-glucanase unit (BGU) is defined as thatquantity of enzyme that will liberate reducing sugar (as glu-cose equivalence) at a rate of 1 �mol/min under the conditionsof the assay.
Calculate the activity of the enzyme preparation taken foranalysis as follows:
BGU = [(A4 − A3) × 36 × 106]/[(A2 − A1) ×180 × 15 × �g sample],
in which A4 is the absorbance of the sample (tube 4), A3 is
FCC V General Tests and Assays / Appendix V / 907
the absorbance of the enzyme blank (tube 3), A2 is the ab-sorbance of the glucose standard (tube 2), A1 is the absorbanceof the substrate blank (tube 1), 36 is the micrograms of glucosein the Glucose Standard Solution, 106 is the factor convertingmicrograms to grams, 180 is the weight of 1 �mol of glucose,and 15 is the reaction time, in minutes.
GLUCOAMYLASE ACTIVITY(AMYLOGLUCOSIDASE ACTIVITY)
Application and Principle This procedure is used to deter-mine the glucoamylase activity of preparations derived fromAspergillus niger var., but it may be modified to determinepreparations derived from Aspergillus oryzae var. and Rhizo-pus oryzae var. (as indicated by the variations in the textbelow). The sample hydrolyzes p-nitrophenyl-�-D-glucopyra-noside (PNPG) to p-nitrophenol (PNP) and glucose at pH 4.3and 50°.
Use the quantity of PNP liberated per unit of time to calcu-late the enzyme activity. Measure the PNP liberated againsta quantity of a standard preparation of PNP by measuring theabsorbance of the solutions at 400 nm after adjusting the pHof the reaction mixture to pH 8.0.
Note: Use a pH of 5.0 when testing preparations derivedfrom Aspergillus oryzae var. or Rhizopus oryzae var.
ApparatusWater Bath Use an open, circulating water bath with con-
trol accuracy of at least �0.1°.Spectrophotometer Use a spectrophotometer suitable for
measuring absorbances at 400 nm.Cuvettes Use 10-mm light-path fused quartz.Thermometer Use a partial immersion thermometer with
a suitable range, graduated in 1/10°.Timer Use a solid-state timer, model 69240 (GCS Corpo-
ration, Precision Scientific Group), or equivalent, accurate to�0.01 min in 240 min.
Vortex Mixer Use a standard variable-speed mixer.
Reagents and Solutionsp-Nitrophenol Stock Solution (PNP) (0.001 M) Dissolve
139.11 mg of p-nitrophenol previously dried (60°, maximum4 h) into water, and dilute to 1000 mL.
Caution: Avoid contact with skin. If contact occurs,wash the affected area with water. Work in a well-ventilated area.
Acetate Buffer Solution (0.1 M) Dissolve 4.4 g of sodiumacetate trihydrate (NaC2H3O2·3H2O) in approximately 800mL of water, add 4.5 mL of acetic acid (C2H4O2). Adjust topH 4.5 � .05 by adding either sodium acetate or glacial aceticacid as required. Dilute to 1 L.
Note: Use a pH of 5.0 when testing preparations derivedfrom Aspergillus oryzae var. or Rhizopus oryzae var.
The pH optimum is 5.0 for Aspergillus oryzae var.—or Rhizo-pus oryzae var.—derived preparations.
Sodium Carbonate Solution (0.3 M) Dissolve 15.9 g ofsodium carbonate (Na2CO3) in water, and dilute to 500 mL.
p-Nitrophenyl-�-D-glucopyranoside Solution (PNPG)Dissolve 100.0 mg of PNPG (Sigma Chemical Co., CatalogNo. N1377) in acetate buffer, and dilute to 100 mL.
Preparation of Standards and SamplesStandards Dilute three portions of PNP Stock Solution to
produce standards for the standard curve. Add 3 mL of thePNP Stock Solution to 125 mL of Sodium Carbonate Solution,and dilute to 500 mL with water to produce the first standard,containing 0.006 �mol/mL. Add 2 mL of PNP Stock Solutionto 25 mL of Sodium Carbonate Solution, and dilute to 100mL with water to produce the second standard, containing0.02 �mol/mL. Add 5 mL of PNP Stock Solutions to 25 mLof Sodium Carbonate Solution, and dilute to 100 mL withwater to produce the third standard, containing 0.05 �mol/mL.
Sample Solution Dilute 1.00 � 0.01 g of sample in suffi-cient Acetate Buffer Solution to produce a solution that con-tains between 0.1 and 0.3 glucoamylase units of activity permilliliter.
Procedure Measure absorbances of each of the three PNPStandard Solutions to calculate the molar extinction coeffi-cient. Equilibrate the PNPG Solution in a 50° water bath forat least 15 min. For active samples, transfer 2.0 mL of theSample Solution to a test tube. Loosely stopper, and place thetube in the water bath to equilibrate for 5 min. At zero time,add 2.0 mL of PNPG Solution, and mix at moderate speedon a vortex mixer. Return the mixture to the water bath.Exactly 10.0 min later, add 3.0 mL of the Sodium CarbonateSolution, mix on the vortex, and remove from the water bath.
For sample blanks, transfer 2.0 mL of the Sample Solutionand 3.0 mL of the Sodium Carbonate Solution into a test tube,and mix. Add 2.0 mL of PNPG Solution, and mix. Measurethe absorbance of each sample and the blank versus water ina 10-mm cell.
Note: Determine the absorbance of the sample andblank solutions not more than 20 min after adding So-dium Carbonate Solution.
Calculations One unit of glucoamylase activity is definedas the amount of glucoamylase that will liberate 0.1 �mol/min of p-nitrophenol from the PNPG Solution under the condi-tions of the assay.
Calculate the millimolar extinction of the PNP standardsusing the following equation:
� = An/C,
in which An is the absorbance of the p-nitrophenol standard, at400 nm, and C is concentration, in �mol/mL, of p-nitrophenol.
The averaged millimolar extinction coefficient, M, shouldbe approximately 18.2.
Glucoamylase �/g = [(AS − AB) × 7 × F]/� ×10 × 0.10 × W × 2,
908 / Appendix V / General Tests and Assays FCC V
in which AS is the sample absorbance; AB is the blank ab-sorbance; F is the appropriate dilution factor; W is the weightof sample, in grams; 7 is the final volume of the test solutions;10 is the reaction time, in minutes; 0.10 is the amount of PNPliberated, in �mol/min/unit of enzyme; 2 is the sample aliquot,in milliliters, and M is the millimolar extinction coefficient.
GLUCOSE ISOMERASE ACTIVITY
Note: Glucose isomerase activity of the commercialenzyme is usually determined on the enzyme that hasbeen immobilized by binding with a polymer matrixor other suitable material. The following method isdesigned for use with such preparations.
Application and Principle Use this procedure to determineglucose isomerase preparations derived from Actinoplanesmissouriensis, Bacillus coagulans, Microbacterium arbo-rescens, Streptomyces murinus, Streptomyces olivaceus,Streptomyces olivochromogenes, and Streptomyces rubigino-sus. It is based on measurement of the rate of conversion ofglucose to fructose in a packed-bed reactor. The procedureas outlined approximates an initial velocity assay method.Specific conditions are glucose concentration, 45% w/w; pH(inlet), measured at room temperature in the 7.0 to 8.5 range, asspecified; temperature, 60.0°; and magnesium concentration, 4× 10-3 M.
The optimum conditions for enzymes from different micro-bial sources and methods of preparation may vary; therefore,if the manufacturer recommends different pH conditions, buff-ering systems, or methods of sample preparation, use suchvariations in the instructions given in the text.
ApparatusColumn Assembly and Apparatus (Note: Make all connec-
tions with inert tubing, glass, or plastic as appropriate.) Thecolumn assembly is shown in Fig. 32. Use a 2.5- × 40-cmglass column provided with a coarse, sintered-glass bottomand a water jacket connected to a constant-temperature waterbath, maintained at 60.0°, by means of a circulating pump.Connect the top of the column to a variable-speed peristalticpump having a maximum flow rate of 800 mL/h. The diameterof the tubing with which the peristaltic pump is fitted shouldpermit variation of the pumping volume from 60 to 150 mL/h. Connect the outlet of the column with a collecting vessel.
Reagents and SolutionsGlucose Substrate Dissolve 539 g of anhydrous glucose
and 1.0 g of magnesium sulfate (MgSO4·7H2O) in 700 mL ofwater or the manufacturer’s recommended buffer, previouslyheated to 50° to 60°. Cool the solution to room temperature,and adjust the pH as specified by the enzyme manufacturer.Transfer the solution to a 1000-mL volumetric flask, diluteto volume with water or the specified buffer, and mix. Transferto a vacuum flask, and de-aerate for 30 min.
FIGURE 32 Column Assembly for Assay of ImmobilizedGlucose Isomerase.
Magnesium Sulfate Solution Dissolve 1.0 g of magnesiumsulfate (MgSO4·7H2O) in 700 mL of water. Adjust the pH to7.5 to 8.0 as specified by the manufacturer, using 1 N sodiumhydroxide, dilute to 1000 mL with water, and mix.
Sample Preparation Transfer to a 500-mL vacuum flaskan amount of the sample, accurately weighed in grams ormeasured in milliliters, as appropriate, sufficient to obtain2000 to 8000 glucose isomerase units (GIcU). Add 200 mLof Glucose Substrate, stir gently for 15 s, and repeat thestirring every 5 min for 40 min. De-aerate by vacuum for30 min.
Column Preparation Quantitatively transfer the SamplePreparation to the column with the aid of Magnesium SulfateSolution as necessary. Allow the enzyme granules to settle,and then place a porous disk so that it is even with, and incontact with, the top of the enzyme bed. Displace all of theair from the disk. Place a cotton plug about 1 or 2 cm abovethe disk. (This plug acts as a filter. It ensures proper heatingof the solution and traps dissolved gases that may be present inthe Glucose Substrate.) Connect the tubing from the peristalticpump with the top of the column, and seal the connectionby suitable means to protect the column contents from theatmosphere. Place the inlet tube of the peristaltic pump intothe Glucose Substrate solution, and begin a downward flowof the Glucose Substrate into the column at a rate of at least80 mL/h. Maintain the flow rate for 1 h at room temperature.
Assay Adjust the flow of the Glucose Substrate to such arate that a fractional conversion of 0.2 to 0.3 will be produced,based on the estimated activity of the sample. Calculate thefractional conversion from optical rotation values obtainedon the starting Glucose Substrate and the sample effluent, asspecified under Calculations, below. After establishing thecorrect flow rate, run the column overnight (16 h minimum),then check the pH of the Glucose Substrate, and readjust ifnecessary to the specified pH. Measure the flow rate, andcollect a sample of the column effluent. Cover the effluentsample, allow it to stand for 30 min at room temperature, andthen determine the fractional conversion of glucose to fructose(see Calculations, below). If the conversion is less than 0.2
FCC V General Tests and Assays / Appendix V / 909
or more than 0.3, adjust the flow rate to bring the conversioninto this range. If a flow rate adjustment is required, collectan additional effluent sample after allowing the column to re-equilibrate for at least 2 h, and then determine the fractionalconversion. Measure the flow rate, and collect an effluentsample. Cover the sample, let it stand at room temperaturefor 30 min, and determine the fractional conversion.
CalculationsSpecific Rotation Measure the optical rotation of the efflu-
ent sample and of the starting Glucose Substrate at 25.0°,and calculate their specific rotations [see Optical (Specific)Rotation, Appendix IIB] by the equation
[�] = 100a/lpd,
in which a is the corrected observed rotation, in degrees; l isthe length of the polarimeter tube, in decimeters; p is theconcentration of the test solution, expressed as grams of soluteper 100 g of solution; and d is the specific gravity of thesolution at 25°.
Fractional Conversion Calculate the fractional conver-sion, X, by the equation
X = (�E – �S)/(�F – �S),
in which �E is the specific rotation of the column effluent,�S is the specific rotation of the Glucose Substrate, and �F
is the specific rotation of fructose (which, in this case, hasbeen calculated to be −94.54).
Activity The enzyme activity is expressed in glucose iso-merase units (GIcU, the subscript c signifying column pro-cess). One GIcU is defined as the amount of enzyme thatconverts glucose to fructose at an initial rate of 1 �mol/min,under the conditions specified.
Calculate the glucose isomerase activity by the equation:
GIcU/g or mL = (FS/W) × XE × ln[XE/(XE − X)],
in which F is the flow rate, in milliliters per minute; S is theconcentration of the Glucose Substrate, in micromoles permilliliter; X is the fractional conversion, as determined above;XE is the fractional conversion at equilibrium, or 0.51; andW is the weight or volume of the sample taken, in grams ormilliliters, respectively.
GLUCOSE OXIDASE ACTIVITY
Application and Principle This procedure is used to deter-mine glucose oxidase activity in preparations derived fromAspergillus niger var. The assay is based on the titrimetricmeasurement of gluconic acid produced in the presence ofexcess substrate and excess air.
Reagents and SolutionsChloride–Acetate Buffer Solution Dissolve 2.92 g of so-
dium chloride and 4.10 g of sodium acetate in about 900 mL
of water. Adjust the pH to 5.1 with either dilute acetic acidor dilute sodium hydroxide solution and dilute to 1000.0 mL.
Sodium Hydroxide Solution (0.1 N)Hydrochloric Acid Solution (0.05 N) Standardized.Phenolphthalein Solution (2% w/v) Solution in methanol.Octadecanol Solution Saturated solution in methanol.Substrate Solution Dissolve 30.00 g of anhydrous glucose
in 1000 mL of the Chloride–Acetate Buffer Solution.Sample Preparation Dissolve an accurately weighed
amount of enzyme preparation in the Chloride–Acetate BufferSolution, and dilute in the buffer solution to obtain an enzymeactivity of 5 to 7 activity units per milliliter.
Procedure Transfer 25.0 mL of the Substrate Solution toa 32- × 200-mm test tube. To a second 32- × 200-mm testtube transfer 25.0 mL of the Chloride–Acetate Buffer Solution(blank). Equilibrate both tubes in a 35° � 0.1° water bath for20 min. Add 3.0 mL of the Sample Preparation to each testtube, mix, and insert a glass sparger into each tube with apreadjusted air flow of 700 to 750 mL/min. If excessivefoaming occurs, add 3 drops of the Octadecanol Solution toeach tube. After exactly 15 min, remove the sparge and rinseany adhering reaction mixture back into the tube with water.Immediately add 10 mL of the Sodium Hydroxide Solutionand 3 drops of the Phenolphthalein Solution to each tube.Insert a small magnetic stirrer bar, stir, and titrate to thephenolphthalein endpoint with the standardized 0.05 N Hydro-chloric Acid Solution.
Calculation One Glucose Oxidase Titrimetric unit of activ-ity (GOTu) is the quantity of enzyme that will oxidize 3 mgof glucose to gluconic acid under the conditions of the assay.Determine the enzyme activity using the following equation:
GOTu/g = [(B − T) × N × 180 × F]/[3 × W],
in which B is the titration volume, in milliliters, of the blank;T is the titration volume, in milliliters, of the sample; N isthe normality of the titrant; 180 is the molecular weight ofglucose; F is the sample dilution factor; 3 is from the unitdefinition; and W is the weight, in grams, of the enzymepreparation contained in each milliliter of the sample solution.
HEMICELLULASE ACTIVITY
Application and Principle This procedure is used to deter-mine hemicellulase activity of preparations derived from As-pergillus niger var. The test is based on the enzymatic hydroly-sis of the interior glucosidic bonds of a defined locust (carob)bean gum substrate at 40° and pH 4.5. Determine the corres-ponding reduction in substrate viscosity with a calibratedviscometer.
ApparatusViscometer Use a size 100 calibrated Cannon-Fenske
Type Viscometer, or equivalent (Scientific Products, CatalogNo. 2885-100).
910 / Appendix V / General Tests and Assays FCC V
Glass Water Bath Use a constant-temperature glass waterbath, maintained at 40° � 0.1° (Scientific Products, CatalogNo. W3520-10).
Stopwatches Use two stopwatches—Stopwatch No. 1, cal-ibrated in 1⁄10 min for determining the reaction time (Tr), andStopwatch No. 2, calibrated in 1⁄5 s for determining the effluxtime (Tt).
Reagents and SolutionsAcetate Buffer (pH 4.5) Add 0.2 N sodium acetate, with
continuous agitation, to 400 mL of 0.2 N acetic acid until thepH is 4.5 � 0.05, as determined by a pH meter.
Locust Bean Gum Use Powdered Type D-200 locust beangum, or its equivalent (Meer Corp.). Because the substratemay vary from lot to lot, test each lot in parallel with aprevious lot known to be satisfactory. Variations of more than�5% viscosity in the average of a series of parallel testsindicate an unsuitable lot.
Substrate Solution Place 12.5 mL of 0.2 N hydrochloricacid and 250 mL of warm water (72° to 75°) in the bowl ofa power blender (Waring two-speed, or equivalent, ScientificProducts, Catalog No. 58350-1), and set the blender on lowspeed. Slowly disperse 2.0 g of Locust Bean Gum, on amoisture-free basis, into the bowl, taking care not to splashout any of the liquid in the bowl. Wash down the sides ofthe bowl with warm water, using a rubber policeman, coverthe bowl, and blend at high speed for 5 min. Quantitativelytransfer the mixture to a 1000-mL beaker, and cool to roomtemperature. Using a pH meter, adjust the mixture to pH6.0 with 0.2 N sodium hydroxide. Quantitatively transfer themixture to a 1000-mL volumetric flask, dilute to volume withwater, and mix. Filter the substrate through gauze before use.
Sample Preparation Prepare a solution of the sample inwater so that 1 mL of the final dilution will produce a changein relative fluidity between 0.18 and 0.22 in 5 min under theconditions specified in the Procedure.
Weigh the enzyme preparation, quantitatively transfer it toa glass mortar, and triturate with water. Quantitatively transferthe mixture to an appropriately sized volumetric flask, diluteto volume with water, and mix. Filter through Whatman No.1 filter paper, or equivalent, before use.
Procedure Scrupulously clean the viscometer by drawinga large volume of detergent solution, followed by water,through the instrument, and place the viscometer, previouslycalibrated, in the glass water bath in an exactly vertical posi-tion. Pipet 20.0 mL of Substrate Solution and 4.0 mL ofAcetate Buffer into a 50-mL Erlenmeyer flask, allowing atleast two flasks for each enzyme sample and one flask for asubstrate blank. Stopper the flasks, and equilibrate them inthe water bath for 15 min. At zero time, pipet 1.0 mL ofthe Sample Preparation into the equilibrated substrate, starttiming with stopwatch no. 1, and mix thoroughly. Immediatelypipet 10.0 mL of this mixture into the wide arm of the viscome-ter. After about 2 min, draw the reaction mixture above the up-per mark into the driving fluid head by applying suction witha rubber tube connected to the narrow arm of the instrument.Measure the efflux time by allowing the reaction mixture toflow freely down past the upper mark. As the meniscus falls
past the upper mark, start stopwatch no. 2, and at the same time,record the reaction time (TR), in minutes, from stopwatch no.1. As the meniscus of the reaction mixture falls past the lowermark, record the time (TT), in seconds, from stopwatch no. 2.Immediately re-draw thereaction mixtureabove theupper markand into the driving fluid head. As the meniscus falls freely pastthe upper mark, restart stopwatch no. 2, and at the same timerecord the reaction time (TR), in minutes, from stopwatch no.1. As the meniscus falls past the lower mark, record the time(TT), in seconds, from stopwatch no. 2.
Repeat the latter operation, beginning with ‘‘Immediatelyre-draw the reaction mixture. . . ,’’ until a total of four determi-nations is obtained over a reaction time (TR) of not more than15 min.
Prepare a substrate blank by pipetting 1.0 mL of water intoa mixture of 20.0 mL of Substrate Solution and 4.0 mL ofAcetate Buffer, and then immediately pipet 10.0 mL of thismixture into the wide arm of the viscometer. Determine thetime (TS), in seconds, required for the meniscus to fall betweenthe two marks. Use an average of five determinations as TS.
Prepare a water blank by pipetting 10.0 mL of water, pre-viously equilibrated to 40° � 0.1°, into the wide arm of theviscometer. Determine the time (TW), in seconds, required forthe meniscus to fall between the two marks. Use an averageof five determinations as TW.
Calculation One hemicellulase unit (HCU) is defined asthat activity that will produce a relative fluidity change of 1over a period of 5 min in a locust bean gum substrate underthe conditions specified. Calculate the relative fluidities (FR)and TN values (see definition below) for each of the fourefflux times (TT) and reaction times (TR) as follows:
FR = (TS − TW)/(TT − TW),
and
TN = 1⁄2(TT/60 s/min) + TR = (TT/120) + TR,
in which FR is the relative fluidity for each reaction time; TS
is the average efflux time, in seconds, for the substrate blank;TW is the average efflux time, in seconds, for the water blank;TT is the efflux time, in seconds, of the sample reaction mixture;TR is the elapsed time, in minutes, from zero time, that is, thetime from addition of the enzyme solution to the buffered sub-strate until the beginning of the measurement of the efflux time(TT); and TN is the reaction time (TR), in minutes, plus one-halfof the efflux time (TT) converted to minutes.
Plot the four relative fluidities (FR) as the ordinate againstthe four reaction times (TN) as the abscissa. This should resultin a straight line. The slope of the line corresponds to therelative fluidity change per minute and is proportional to theenzyme concentration. The slope of the best line through aseries of experimental points is a better criterion of enzymeactivity than is a single relative fluidity value. From the curve,determine the FR values at 10 and 5 min. They should havea difference in fluidity of not more than 0.22 and not lessthan 0.18. Calculate the activity of the enzyme sample asfollows:
HCU/g = 1000(FR10 − FR5/W),
FCC V General Tests and Assays / Appendix V / 911
in which FR10 is the relative fluidity at 10 min reaction time;FR5 is the relative fluidity at 5 min reaction time; 1000 ismilligrams per gram; and W is the weight, in milligrams, ofthe enzyme sample contained in the 1.0-mL aliquot of SamplePreparation added to the equilibrated substrate in the Pro-cedure.
INVERTASE ACTIVITY
Application and Principle This procedure is used to deter-mine the invertase activity of enzyme preparations from yeastSaccharomyces sp (Kluyveromyces). The assay is based on a30-min hydrolysis of sucrose at 30° � 0.1° and at pH 4.62.The degree of hydrolysis is determined by measuring theoptical rotation of the solution with a polarimeter.
Reagents and SolutionsAcetate Buffer Dissolve 4.0 g of sodium hydroxide
(NaOH) in about 900 mL of water, and carefully neutralizewith 12.0 g of acetic acid 98% to 100% (CH3COOH). Coolto room temperature. Transfer the solution into a 1000-mLvolumetric flask, dilute to volume with water, and mix. ThepH should be 4.62 � 0.05.
Sucrose Substrate Solution Dissolve 82.152 g of sucrosein about 900 mL of water. Transfer the solution into a 1000-mL volumetric flask, dilute to volume with water, and mix.Use a freshly prepared solution only.
Sodium Carbonate Solution Dissolve 53.0 g of sodiumcarbonate (Na2CO3) in about 400 mL of water, then transferthe solution into a 500-mL volumetric flask, dilute to volumewith water, and mix.
Test Preparation Using a 100-mL volumetric flask, pre-pare a solution from the starting enzyme preparation byweighing a minimum of 1 g of sample accurately to within1 mg. Dilute with water so that the final solution will containbetween 1.3 and 5.3 Invertase Units per 20 mL. Pipet 20.0mL of this solution into a 100-mL Erlenmeyer flask.
Blank Preparation Pipet 20.0 mL water in a 100-mL Er-lenmeyer flask.
Procedure To the flasks containing 20.0 mL of each TestPreparation and to the Blank Preparation, add 5.00 mL ofAcetate Buffer. At zero time, and at regular time intervals sothat each test sample is analyzed in the same elapsed time,place the flasks containing the Test Preparations and theBlank Preparation in a circulating water bath maintained at30.0° � 0.1°. Equilibrate the samples for 10 min in the waterbath. In the same order and with the same time intervals,rapidly pipet 25.00 mL of equilibrated Sucrose Substrate Solu-tion into the test flasks. Incubate for 30.0 min, and stir continu-ously. Terminate the reaction by adding 10.00 mL of SodiumCarbonate Solution, and swirl to mix. Place the flasks con-taining the Test Preparations and the Blank Preparation ina water bath maintained at 20.0° � 0.1° for 30 min. Use a
polarimeter with an accuracy of at least 0.001 degrees of arc.With the same precision, determine the optical rotation ofeach solution at 589 nm (sodium lamp), using a 10-cm path-length cell with the thermostat set at 20.0° � 0.1°. Use waterto blank the polarimeter initially.
Calculation One Invertase Unit (INVU) is defined as thequantity of enzyme that will hydrolyze 1.142 �mol of sucroseper minute under the conditions of the assay.
Calculate the invertase units per 20 mL as follows:
(Rbt − Rtest) × 2 × 1,000,000/[66.77 − (Glu + Fru)](342.3)(1.142) = INVU/20 mL,
where Glu is (0.525)(52.5) and Fru is (0.525)(−91.315), orsimplified:
(Rbt + Rtest) × 58.71 = INVU/20 mL,
in which Rbt is the rotation of Blank Preparation; Rtest is therotation of the Test Preparation; 66.77 is the specific rotationof sucrose; 52.50 is the specific rotation of glucose; −91.315is the specific rotation of fructose; 0.525 is 0.5 corrected for5% weight increase by hydrolysis; 342.3 is the molecularmass (grams per mole) of sucrose; and 1.142 is the unitdefinition factor. Specific rotations are valid at the averageconcentrations in this test.
Invertase Activity in Weighed Samples:
INVU/20 mL × d/w = INVU/g,
Invertase Activity in Pipetted Samples:
INVU/20 mL × d = INVU/mL,
in which d is the total dilution factor and w is the weight ofthe sample.
LACTASE (NEUTRAL)(�-GALACTOSIDASE) ACTIVITY
Application and Principle This procedure is used to deter-mine the neutral lactase activity of enzyme preparations de-rived from Kluyveromyces marxianus var. lactis and Sacchar-omyces sp. The assay is based on a 10-min hydrolysis ofan o-nitrophenyl-�-D-galactopyranoside (ONPG) substrate at30.0° � 0.1° and at pH 6.50.
Reagents and SolutionsMagnesium Solution Dilute 24.65 g of magnesium sulfate
heptahydrate (MgSO4·7H2O) in about 950 mL of water. Trans-fer the solution into a 1000-mL volumetric flask, dilute tovolume with water, and mix.
EDTA Solution Dissolve 1.86 g of disodium EDTA dihy-drate (C10H14N2Na2O8·2H2O) in about 950 mL of water.Transfer the solution into a 1000-mL volumetric flask, diluteto volume with water, and mix.
912 / Appendix V / General Tests and Assays FCC V
P-E-M Buffer Dissolve 8.8 g of potassium dihydrogenphosphate (KH2PO4) and 8.0 g of dipotassium hydrogen phos-phate trihydrate (K2HPO4·3H2O) in about 900 mL of water.Add 10.0 mL of Magnesium Solution and 10.0 mL of EDTASolution. Transfer the solution into a 1000-mL volumetricflask, dilute to volume with water, and mix. The pH shouldbe 6.50 � 0.05.
Lactase Reference Preparation (Highly concentrated lac-tase preparation) This preparation can be obtained from Gist-Brocades, Delft, The Netherlands.
ONPG (o-nitrophenyl-�-D-galactopyranoside) is vali-dated according to the following procedure:
Validation of New ONPG Transfer 150, 250, and 375mg of the new ONPG into separate 100-mL volumetricflasks, dilute to volume with P-E-M Buffer, and mix. Pre-pare solutions of the Lactase Reference Preparation byweighing an amount of Lactase Reference Preparation cor-responding to 5000 � 250 Neutral Lactase Units (NLU)accurately to within 1 mg in duplicate in 50-mL volumetricflasks, dissolve in P-E-M Buffer, dilute to volume with thesame, and mix. Prepare dilutions of this initial solutionwith P-E-M Buffer so that 1 mL of the final dilution willcontain 0.0375, 0.0750, and 0.1125 NLU of activity. Induplicate, determine the enzyme activity of the three en-zyme concentrations using each of the new ONPG Substratesolutions corresponding to 150, 250, and 375 mg and theold ONPG Substrate at 250 mg by following the steps inthe Procedure, below.
Calculation Calculate the enzyme activity followingthe steps indicated under Calculation for NLU Activity,below. Determine the average of the duplicates for eachenzyme concentration at each level of ONPG Substrate (themaximum allowable difference between these duplicates is6.5%). Determine the overall average for the three enzymeconcentrations (0.0375, 0.0750, and 0.1125) for each ONPGSubstrate level (150, 250, and 375 mg of ONPG).
To determine the overall average of three enzyme con-centrations at 150 mg of ONPG:
X = (A + B + C)/3,
in which A is the average result of 0.0375 at 150 mg ofONPG, B is the average result of 0.0750 at 150 mg ofONPG, and C is the average result of 0.1125 at 150 mgof ONPG.
To determine the overall average of three enzyme con-centrations at 250 mg of ONPG:
Y = (D + E + F)/3,
in which D is the average result of 0.0375 at 250 mg ofONPG, E is the average result of 0.0750 at 250 mg ofONPG, and F is the average result of 0.1125 at 250 mg ofONPG.
To determine the overall average of three enzyme con-centrations at 375 mg of ONPG:
Z = (G + H + I)/3,
in which G is the average result of 0.0375 at 375 mg ofONPG, H is the average result of 0.0750 at 375 mg of
ONPG, and I is the average result of 0.1125 at 375 mg ofONPG.
The ONPG analyzed is suitable for use when the follow-ing specifications are met for each ONPG concentration:
1. The average result of each enzyme concentration foreach ONPG level does not deviate more than 3% from theoverall average of the three enzyme concentrations for thatlevel of ONPG. For example, A or B or C should not deviatemore than 3% from X; D or E or F should not deviate morethan 3% from Y; G or H or I should not deviate more than3% from Z.
2. The overall average of the three enzyme concentra-tions found for 150 mg of ONPG (X) should not vary morethan 81% to 99% of the overall average of the three enzymesconcentrations found for 250 mg of ONPG (Y). The overallaverage of the three enzyme concentrations found for 375mg of ONPG (Z) should not vary more than 96% to 114%of the overall average of the three enzyme concentrationsof 250 mg of ONPG (Y).
3. The absorbance of each blank is less than 0.050.4. For each new lot of ONPG, the overall average of
the three enzyme concentrations found for 250 mg of ONPG(Y) per 100 mL should be within 5% of the overall averageof the three enzyme concentrations found for 250 mg ofONPG of the lot in use at that moment.
ONPG Substrate Dissolve 250.0 mg ONPG (use lot cur-rently in use) in about 80 mL of P-E-M Buffer. Transfer thesolution to a 100-mL volumetric flask, dilute to volume withP-E-M Buffer, and mix. Prepare, at most, 2 h before incu-bation.
Sodium Carbonate Solution Dissolve 50.0 g of sodiumcarbonate anhydrous (Na2CO3) and 37.2 g of disodium EDTAdihydrate (C10H14N2Na2O8·2H2O) in about 900 mL of water.Transfer the solution into a 1000-mL volumetric flask, diluteto volume with water, and mix.
Standard o-Nitrophenol Solution Transfer 139.0 mg ofo-nitrophenol into a 1000-mL volumetric flask, dissolve in10 mL of 96% ethanol, dilute to volume with water, and mix.Pipet 2-, 4-, 6-, 8-, 10-, 12-, and 14-mL portions of thissolution into a series of 100-mL volumetric flasks, add 25mL of Sodium Carbonate Solution to each, dilute each tovolume with P-E-M Buffer, and mix. The dilutions contain,respectively, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 and 0.14 �mol/mL of o-nitrophenol.
Determine the absorbance of each dilution at 420 nm in a 1-cm path-length cell, with a suitable spectrophotometer, usingwater as the blank. For each dilution, plot absorbance against�mol of o-nitrophenol (this must result in a straight linethrough the origin). Divide the absorbance of each dilutionby �mol of o-nitrophenol to obtain the extinction coefficient(M) at that dilution (the slope of the line is the extinctioncoefficient). Average the seven values thus calculated (thisshould result in a value of 4.60 � 0.05).
Test Preparation Using a volumetric flask, prepare a testsolution from the starting enzyme preparation by accuratelyweighing out a minimum of 1 g of sample to the nearestmilligram. Dissolve in P-E-M Buffer so that 1 mL of the final
FCC V General Tests and Assays / Appendix V / 913
dilution will contain between 0.027 and 0.095 NLU. Transfer1 mL of this final dilution to a 15- × 150-mm test tube asthe Test Preparation. Perform in duplicate.
Procedure Equilibrate the test tubes containing each TestPreparation in a water bath maintained at 30.0° � 0.1° forat least 5 but not more than 15 min. At zero time, in the orderof the series and at regular time intervals, rapidly pipet 5.00mL of ONPG Substrate, equilibrated at 30.0° � 0.1°, intothe test tubes, and mix by shaking. After a 10.0-min incubation(reaction) time, in the same order and with the same regularintervals, pipet 2.00 mL of Sodium Carbonate Solution intoeach, mix by shaking, and hold at room temperature. Deter-mine the absorbance of each solution within 30 min at 420nm in a 1-cm cell with a suitable spectrophotometer, usingas the blank a solution prepared in the same manner as for thesample except adding ONPG Substrate and Sodium CarbonateSolution in reverse order.
Calculation for NLU Activity One Neutral Lactase Unit(NLU) is defined as that quantity of enzyme that will liberate1.30 �mol/min of o-nitrophenol under the conditions of theassay. Calculate the activity of the enzyme preparation takenfor the analysis as follows:
NLU/g = [(A × 8 × f)/(M × 10 × W)]/1.30,
in which A is the average of the absorbance readings for thesample, corrected for the sample blank; 8 is the volume, inmilliliters, of the incubation mixture after termination; f isthe total dilution factor of the sample; M is the extinctioncoefficient, determined as directed under Standard o-Ni-trophenol Solution; 10 is the incubation time, in minutes; Wis the sample weight, in grams; and 1.30 is the factor usedin the unit definition.
LACTASE (ACID) (ß-GALACTOSIDASE)ACTIVITY
Application and Principle This procedure is used to deter-mine lactase activity of enzyme preparations derived fromAspergillus oryzae var. The assay is based on a 15-min hydro-lysis of an o-nitrophenyl-�-D-galactopyranoside substrate at37° and pH 4.5.
Reagents and Solutions2.0 N Acetic Acid Dilute 57.5 mL of glacial acetic acid
to 500 mL with water. Mix well, and store in a refrigerator.4.0 N Sodium Hydroxide Dissolve 40.0 g of sodium hy-
droxide in sufficient water to make 250 mL.Acetate Buffer Combine 50 mL of 2.0 N Acetic Acid and
11.3 mL of 4.0 N Sodium Hydroxide in a 1000-mL volumetricflask, and dilute to volume with water. Verify that the pH is4.50 � 0.05, using a pH meter, and adjust, if necessary, with2.0 N Acetic Acid or 4.0 N Sodium Hydroxide.
2.0 mM o-Nitrophenol Stock Transfer 139.0 mg of o-nitrophenol to a 500-mL volumetric flask, dissolve in 10 mLof USP alcohol (95% ethanol) by swirling, and dilute tovolume with 1% sodium carbonate.
o-Nitrophenol Standards0.10 mM Standard Solution Pipet 5.0 mL of the 2.0 mM
o-Nitrophenol Stock solution into a 100-mL volumetric flask,and dilute to volume with 1% sodium carbonate solution.
0.14 mM Standard Solution Pipet 7.0 mL of the 2.0 mMo-Nitrophenol Stock solution into a 100-mL volumetric flask,and dilute to volume with 1% sodium carbonate solution.
0.18 mM Standard Solution Pipet 9.0 mL of the 2.0 mMo-Nitrophenol Stock solution into a 100-mL volumetric flask,and dilute to volume with 1% sodium carbonate solution.
Substrate Transfer 370.0 mg of o-nitrophenyl-�-D-galac-topyranoside to a 100-mL volumetric flask, and add 50 mLof Acetate Buffer. Swirl to dissolve, and dilute to volume withAcetate Buffer.
Note: Perform the assay procedure within 2 h of Sub-strate preparation.
Test Preparation Prepare a solution from the test samplepreparation such that 1 mL of the final dilution will containbetween 0.15 and 0.65 lactase unit. Weigh, and quantitativelytransfer the enzyme to a volumetric flask of appropriate size.Dissolve the enzyme in water, swirling gently, and dilute withwater if necessary.
Note: Perform the assay procedure within 2 h of dissolu-tion of the Test Preparation.
System Suitability Determine the absorbance of the threeo-Nitrophenol Standards at 420 nm in a 1-cm cell, using asuitable spectrophotometer. Use water to zero the instrument.Calculate the millimolar extinction, M, for each of the o-Nitrophenol Standards (0.10, 0.14, and 0.18 mM) by theequation
� = An/C,
in which An is the absorbance of each o-Nitrophenol Standardat 420 nm and C is the corresponding concentration of o-nitrophenol in the standard. M for each standard should beapproximately 4.60/mM. Perform a linear regression analysisof the absorbance readings of the three o-Nitrophenol Stan-dards versus the o-nitrophenol concentration in each (0.10,0.14, and 0.18 mM). The r2 should not be less than 0.99.Determine the mean M of the three o-Nitrophenol Standardsfor use in the calculations below.
Procedure For each sample or blank, pipet 2.0 mL of theSubstrate solution into a 25- × 150-mm test tube, and equili-brate in a water bath maintained at 37.0° � 0.1° for approxi-mately 10 min. At zero time, rapidly pipet 0.5 mL of theTest Preparation (or 0.5 mL of water as a blank) into theequilibrated substrate, mix by brief (1 s) vortex, and immedi-ately return the tubes to the water bath. After exactly 15 minof incubation, rapidly add 2.5 mL of 10% sodium carbonatesolution, and vortex the tube to stop the enzyme reaction.Dilute the samples and blanks to 25.0 mL by adding 20.0 mLof water, and thoroughly mix. Determine the absorbance of
914 / Appendix V / General Tests and Assays FCC V
the diluted samples and blanks at 420 nm in a 1-cm cell,using a suitable spectrophotometer. Use water to zero theinstrument.
Calculation One lactase unit (ALU) is defined as that quan-tity of enzyme that will liberate o-nitrophenol at a rate of 1�mol/min under the conditions of the assay.
Calculate the activity (lactase activity per gram) of theenzyme preparation taken for analysis as follows:
ALU/g = [(AS − B)(25)]/[(�)(15)(W)],
in which AS is the average of absorbance readings for theTest Preparation; B is the average of absorbance readings forthe blank; 25 is the final volume, in milliliters, of the dilutedincubation mixture; � is the mean absorptivity of the o-Ni-trophenol Standards per micromole, 15 is the incubation time,in minutes, and W is the weight, in grams, of original enzymepreparation contained in the 0.5-mL aliquot of Test Prepara-tion used in the incubation.
LIPASE ACTIVITY
Application and Principle This procedure is used to deter-mine the lipase activity in preparations derived from microbialsources and animal pancreatic tissues. The assay is basedon the potentiometric measurement of the rate at which thepreparations will catalyze the hydrolysis of tributyrin.
ApparatusAutomatic Recording Titrimeter Use an instrument op-
erating in the pH stat mode and equipped with a jacketedtitration cell (Radiometer Titralab, or equivalent).
Constant Temperature Bath Operated at 30° � 0.1°.Blender
Reagents and Solutions0.05 N Sodium Hydroxide Dissolve 2.0 g of sodium hy-
droxide in water, and dilute to 100 mL. Standardize withNIST grade potassium hydrogen phthalate.
Emulsification Reagent Dissolve 17.9 g of sodium chlo-ride and 0.41 g of monobasic potassium phosphate in about400 mL of water. Add 540 mL of glycerol and, with vigorousstirring, add 6.0 g of gum arabic (Sigma, Catalog No. G 9752).Stir until dissolved. Dilute to 1000 mL.
Glycine Buffer (0.1 M) Dissolve 7.50 g of glycine (Sigma,Catalog No. G 126) and 3.8 g of sodium hydroxide in about900 mL water. Adjust the pH to 10.8, and dilute to 1000 mL.
Note: Instead of the Glycine Buffer, some enzyme prep-arations may require the use of 0.01 M pH 8.0 TrisBuffer prepared as directed for Tris Buffer under Proteo-lytic Activity, Bacterial (PC), except to titrate with 1N hydrochloric acid to pH 8.0.
Substrate Emulsion Transfer 15.9 mL of tributyrin(Sigma, Catalog No. T 8626) to a blender, add 50 mL Emulsifi-
cation Reagent and 235 mL water. Blend for 15 min at maxi-mum speed. Equilibrate in the 30° constant temperature bathfor at least 15 min before use. Use within 4 h.
Sample Preparation Dissolve an accurately weighedamount of the enzyme preparation in Glycine Buffer (or pH8.0 Tris Buffer if specified) so that each milliliter containsbetween 2000 and 5000 lipase units per milliliter. Accuratelydilute a portion of this solution with water to obtain a finalsolution containing between 0.5 and 1.5 lipase units per milli-liter.
Procedure Fill the titrator buret with the 0.05 N SodiumHydroxide solution, and following the manufacturer’s instruc-tions, set the temperature to 30° and the pH set point to 7.0.Transfer 15.0 mL of the Substrate Emulsion to the titrationcell, and add a small stirrer bar. Add 1.0 mL of the dilutedSample Preparation, and actuate the titrator. Record the rateof 0.05 N Sodium Hydroxide addition. Stop the titration aftera constant (linear) rate of addition has been observed for 5min. Determine the addition rate, in milliliters per minute,from the linear portion of the recording and record this valueas R.
Calculation One lipase unit (LU) is defined as the quantityof enzyme that will liberate 1 �mol of butyric acid per minunder the conditions of the test.
Calculate the activity of the enzyme preparation by theformula
LU/g = R × N × 1000/W,
in which R is the addition rate, in milliliters per minute; N isthe normality of the Sodium Hydroxide solution; 1000 convertsmM to �M; and W is the weight, in grams, of the enzymepreparation contained in 1 mL of the diluted Sample Prepa-ration.
LIPASE (MICROBIAL) ACTIVITY FORMEDIUM- AND LONG-CHAIN FATTYACIDS
Application and PrincipleThis procedure is used to determine the lipase activity inpreparations derived from microbial sources. The assay isbased on the measurement of the amount of free fatty acidsformed from an olive oil emulsion in the presence of sodiumtaurocholate over a fixed time interval. This assay is particu-larly used for measuring lipase activity in foods.
Reagents and SolutionsGum Arabic Solution Dissolve 110 g of gum arabic (aca-
cia) (Sigma, Catalog No. G-9752, or equivalent) and 12.5 gof analytical-grade calcium chloride (CaCl2·2H2O) in 800 mLof water in a 1000-mL volumetric flask, and dilute to volumewith water. Shake or stir for 30 min at room temperature to
FCC V General Tests and Assays / Appendix V / 915
dissolve completely. Centrifuge at 4000 × g for 20 min orfilter through a Büchner funnel using Celite as a filter aid.Store the supernatant or filtrate at 4°. Divide into single-use,24-mL aliquots. The solution is stable for 6 months at −20°.
Substrate Emulsion Place 130 mL of olive oil (Sigma,Catalog No. O-1500, or equivalent) and 400 mL of GumArabic Solution in a mixer bowl, and cool the mixture to 5°to 10° on ice. Emulsify the mixture with a Waring Blender,or equivalent, operated at high speed for 30 min, keeping thetemperature below 30° by repeatedly mixing at high speedfor 5 min and turning the blender off for 1 min. Check thequality of the emulsion microscopically: 90% of the dropletsshould have a diameter equal to or less than 3 �m, and theremaining 10% should not exceed 10 �m. The emulsion isstable for 3 days at 4°.
Reference Standard Solution Dissolve an aliquot of FungiLipase-International FIP Standard (International Commissionon Pharmaceutical Enzymes F.I.P., Center for Standards ofthe Federation Internationale Pharmaceutique, Harelbekes-traat 72, B-9000 Gent, Belgium) in a 1% sodium chloridesolution and dilute it to obtain a solution of 2.4 to 3.6 FIPmicrobial lipase units per milliliter. Prepare this solution fresh.
0.02 N Sodium Hydroxide Solution Prepare daily by dilut-ing 10 mL of analytical-grade 1 N sodium hydroxide to 500mL with recently boiled water.
0.5% Sodium Taurocholate Solution Dissolve 0.5 g ofsodium taurocholate (DIFCO, Catalog No. 0278-15-8) in 100mL of water. Prepare this solution fresh.
Sample Preparation Dissolve an accurately weighedamount of the enzyme preparation in a 1% sodium chloridesolution, and dilute to obtain a solution of 2.4 to 3.6 FIPmicrobial lipase units per milliliter. Prepare this solution fresh.
Procedure (Note: Assay the Fungi Lipase-International FIPStandard as an internal standard each time.)
Automatic Titration Use an automatic titration devicewith a 25 mL � 0.02 mL buret, a pH meter giving a resolutionto 0.01, and a reaction vessel with a capacity of 100 mL. Add24 mL of Substrate Emulsion, 9 mL of water, and 2 mL of 0.5%Sodium Taurocholate Solution to the reaction vessel. Place thereaction vessel in a water bath preheated to 37° � 0.5° over ahot plate provided with magnetic stirring, and add a magnet tothe reaction vessel. Pre-incubate the reaction vessel at 37° �0.5° for 10 to 15 min while stirring at about 300 rpm. Immerse apH-electrode and the tip of the buret into the solution. If desired,gently blow nitrogen gas onto the solution. Adjust the pH of thesolution to 7.0 with 0.02 N Sodium Hydroxide Solution. Set theautomatic buret to zero. Add 5.0 mL of the enzyme solutionwhile simultaneously starting a timer. Maintain the pH at 7.0by automatic titration. After 10.0 min, abruptly (within 30 s)bring the pH to 9.0 by manually adding additional 0.02 N So-dium Hydroxide Solution. Record the volume of 0.02 N SodiumHydroxide Solution consumed as N1. Run the test with a blankby setting up the titration in the same manner, except after ad-justing the pH to 7.0 with 0.02 N Sodium Hydroxide Solution,set the automatic buret to zero, and maintain the pH at 7.0 byautomatic titration. After 10.0 min, abruptly (within 30 s) bringthe pH to 9.0 as before, and then add 5.0 mL of enzyme solution.Because the enzyme lowers the pH, return the pH to 9.0 by
adding 0.02 N Sodium Hydroxide Solution. Record the volumeof 0.02 N Sodium Hydroxide Solution consumed as N2.
Manual Titration Follow the same procedure as with Au-tomatic Titration, but keep the pH at 7.0 with 0.02 N SodiumHydroxide Solution from a 25-mL buret, demarked in 0.02-mL units.
Calculation One unit of enzyme activity (FIP Unit) is de-fined as that quantity of a standard lipase preparation (FungiLipase-International FIP Standard) that liberates the equiva-lent of 1 �mol of fatty acid per minute from the SubstrateEmulsion under the described assay conditions. The specificactivity is expressed in international FIP units per milligramof the Sample Preparation.
The use of an enzyme reference standard of known activity,controlled by the Center for Standards of the Commission,eliminates difficulties from interlaboratory differences in qual-ity of reagents such as the Gum Arabic Solution, olive oil, orSubstrate Emulsion or in the set-up of the experiment. Theactivity (FIP U/mg) using an enzyme reference standard iscalculated by the formula
(A × C)/B,
in which A is the specific activity, in units/mg, of the testsample (measured); B is the specific activity, in units/mg, ofFungi Lipase-International FIP Standard (measured); and Cis the number of FIP units/mg of Fungi Lipase-InternationalFIP Standard as indicated on the container.
One milliliter of the 0.02 N Sodium Hydroxide Solution cor-responds with the neutralization of 20 �mol of fatty acids. Fivemilliliters of enzyme solution liberates (N1 − N2) mL × 20 �molof fatty acids over a 10-min time interval. If the enzyme solutioncontains W mg of enzyme preparation per milliliter, the specificactivity, in units/mg, is calculated as follows:
[(N1 − N2) × 20]/(10 × 5 × W),
in which (N1 − N2) is the volume, in milliliters, of the 0.02N Sodium Hydroxide Solution used for the titration.
LYSOZYME ACTIVITY1
Application and Principle The purpose of this procedureis to determine the lysozyme activity in purified lysozymepreparations derived from animal or microbial sources. Theassay is based on the rate of decrease in absorbance at 450nm, attributed to the lysis of Micrococcus lysodeikticus bylysozyme. The decrease in absorbance is measured using aUV/V spectrophotometer equipped to control the sample tem-perature at 25°.
Note: Ensure that all glassware and supplies are heatsterilized. The work area should be aseptically clean.
1Shugar, D. 1952. The measurement of lysozyme activity and theultra-violet inactivation of lysozyme. Biochimica et Biophysica Acta.8:302–309.
916 / Appendix V / General Tests and Assays FCC V
Any residual lysozyme contamination will adverselyaffect the results of the assay.
Reagents and SolutionsSodium Phosphate Buffer Solution Dissolve 10.4 g of
monobasic sodium phosphate (NaH2PO4·H2O) in 500 mL ofsterile, deionized water in a 1000-mL volumetric flask, anddilute to volume. Similarly, dissolve 9.465 g of anhydrousdibasic sodium phosphate (Na2HPO4) in sterile, deionizedwater, and dilute to 1000 mL. Mix 815 mL of the monobasicsodium phosphate solution with 185 mL of the dibasic sodiumphosphate solution. Adjust the pH of the buffer to 6.2; whenchecking the pH, use an aliquot of the buffer to preventcontamination of the solution. Adjust the pH by adding moremonobasic or dibasic sodium phosphate solution as needed.The buffer solution may be stored under refrigeration for upto 1 month.
Substrate Solution Add 30 to 40 mg of Micrococcus lyso-deikticus (Sigma M-3770, or equivalent) to 100 mL of SodiumPhosphate Buffer Solution in a 250-mL Erlenmeyer flask, tiltgently to mix, and do not shake. Allow the substrate to incubateat 37° for 30 min before using it. The substrate solution isstable for 2 h at room temperature. Zero a spectrophotometeragainst air, then measure the absorbance of the substrate solu-tion, which should give a reading of 1.7 � 0.1 at 450 nm.
Note: If the absorbance is significantly lower than 1.7,do not adjust the concentration. Run the analysis, andcheck the rate of the reaction. The rate of the decreasein absorbance should range between 0.03 and 0.06 unitsper min.
Standard Preparation Use a commercial reference stan-dard lysozyme of a specified strength from an animal ormicrobial source in accordance with the origin of the prepara-tion being measured. Measure 50 mg of the reference standardlysozyme into a 50-mL volumetric flask, and dissolve, withstirring, in approximately 25-mL of Sodium Phosphate BufferSolution. Dilute to volume with Sodium Phosphate BufferSolution, and mix thoroughly. If desired, freeze aliquots of thisStandard Preparation for subsequent assays. Quantitativelytransfer 3 mL of the Standard Preparation to a 100-mL volu-metric flask, and dilute to volume with Sodium PhosphateBuffer Solution.
Sample Preparation Measure 50 mg of sample into a 50-mL volumetric flask. Dissolve the sample, with stirring, inapproximately 25 mL of Sodium Phosphate Buffer Solution.Dilute to volume with Sodium Phosphate Buffer Solution, andmix the solution thoroughly. Quantitatively transfer 3 mL ofthe solution to a 100-mL volumetric flask, and dilute to volumewith Sodium Phosphate Buffer Solution.
Procedure Conduct the test in a spectrophotometerequipped to maintain a temperature of 25° in the cell compart-ment. Perform the test in triplicate for the Standard Prepara-tion and for the Sample Preparation.
Place a 1-cm cell into the spectrophotometer, and adjustthe absorbance to zero. Pipet 2.9 mL of Substrate Solutioninto the cell; the initial absorbance of the solution should be1.7 � 0.1 at 450 nm (see Note above). Pipet 0.1 mL of the
Standard Preparation into the substrate, and mix well. Recordthe decrease in absorbance over 3 min, recording the ab-sorbance value approximately every 15 s. The rate of thedecrease in absorbance should be linear, and range between0.03 and 0.06 per min. Repeat the procedure with the SamplePreparation.
Calculation One lysozyme unit is defined as the amount oflysozyme that causes a decrease in absorbance of 0.001 permin at 450 nm, 25°, and pH 6.2, using a suspension of Micro-coccus lysodeikticus as the substrate.
The assay stabilizes over the first min; disregard the firstmin of readings in the calculation. Determine the averageabsorbance change per min using only the linear portion ofthe curve where the rate of change is constant, usually thefinal 2 min.
Calculate the number of lysozyme units per mg by theequation
lysozyme units = (A1 − A2)/(T × W × 0.001),
in which A1 is the initial absorbance reading in the straight-line portion of the curve; A2 is the final absorbance readingin the straight-line portion of the curve; T is the elapsed time,in min, between the initial and final absorbance readings; Wis the weight, in mg, of the lysozyme in the volume of SamplePreparation used in the Assay; and 0.001 is the decrease inabsorbance caused by one unit of lysozyme per min.
MALTOGENIC AMYLASE ACTIVITY
Application and Principle This procedure is used to deter-mine maltogenic amylase activity in preparations derived fromBacillus subtilis containing a Bacillus stearothermophilus am-ylase gene. The test is based on a 30-min hydrolysis of malto-triose under controlled conditions and measurement of theglucose formed by high-performance liquid chromatography(HPLC).
Reagents and SolutionsCitrate Buffer, 0.1 M Dissolve 5.255 g of citric acid
(C6H8O7·H2O) in about 150 mL of water. Adjust the pH to5.0 with 1 N sodium hydroxide, and dilute to 250 mL.
Substrate Solution Dissolve 1.00 g of maltotriose (SigmaChemical Co., Catalog No. M 8378) in Citrate Buffer in a50-mL volumetric flask, and dilute to volume with CitrateBuffer.
Sodium Chloride Solution, 1 M Dissolve 29.22 g of so-dium chloride in water, and dilute to 500 mL.
Amberlite MB-1 Ion Exchange Resin Air dry at roomtemperature for about 1 week. Protect from contamination.
Glucose Standards Dissolve 1.80 g of anhydrous glucosein water, and dilute to 1000 mL. Transfer 20.0, 50.0, 75.0,and 100.0 mL to separate 100-mL volumetric flasks, anddilute to volume with water. These solutions contain 0.36,
FCC V General Tests and Assays / Appendix V / 917
0.9, 1.35, and 1.80 mg of glucose per mL. Using filtered,degassed water as the mobile phase, equilibrate an HPX 87Ccolumn, or equivalent, in a high-performance liquid chromato-graph equipped with a differential refractometer. Chromato-graph 5-�L portions of the glucose standards, and recordthe chromatograms. Prepare a standard curve of the glucoseconcentration versus the peak height.
Sample Preparation Prepare a solution of each sample tocontain approximately 7.5 Maltogenic Amylase Units(MANU) per mL. Further dilute an aliquot of each sampleso that the final dilution contains 1% by volume of the SodiumChloride Solution, 1 M and contains between 0.150 and 0.600MANU per mL.
Procedure Transfer 2.00 mL of each sample to separatetest tubes, and equilibrate in the 37° water bath for at least10 min. At the same time, equilibrate the Substrate Solutionin the same water bath. At zero time, transfer 2.0 mL of theequilibrated Substrate Solution to the first sample tube, mixthoroughly, and return the tube to the 37° bath. Repeat theprocess for each sample. After exactly 30.0 min, transfer thetest tube to a boiling water bath for 15 min, then remove andcool to room temperature. Add approximately 100 mg ofAmberlite MB-1 Ion Exchange Resin to each tube, place thetubes on the shaker, and mix for at least 15 min. Filter thetreated solution through a 0.45-�m filter. Use a separate filterfor each sample. Inject a 5-�L portion of each filtered sampleinto a previously equilibrated high-performance liquid chro-matograph equipped with an HPX 87C column (Biorad, orequivalent) and a differential refractometer. Filtered, degassedwater is the mobile phase. Record the elution curve.
Calculation One Maltogenic Amylase Unit (MANU) is de-fined as the amount of enzyme that will cleave maltotrioseat a rate of 1 �mol/min under the conditions of the test.From the elution curve of each sample, determine the glucoseconcentration (G) in the sample from the previously preparedstandard curve. Calculate the MANU/g by the equation
MANU/g = G × 4 × F/180.1 × 30 × W,
in which G is the glucose concentration in the test solution;4 is the total test solution volume; 30 is the reaction time, inmin; F is the dilution factor; and W is the sample weight, in g.
MILK-CLOTTING ACTIVITY
Application and Principle This procedure is to be appliedto enzyme preparations derived from either animal or micro-bial sources.
ApparatusBottle-Rotating Apparatus Use a suitable assembly, de-
signed to rotate at a rate of 16 to 18 rpm.Sample Bottles Use 125-mL, squat, round, wide-mouth
bottles (such as Scientific Products, Catalog No. B-7545-125).
Substrate Solution Dissolve 60 g of low-heat, nonfat drymilk (such as Galloway West, Peake Grade A) in 500 mL ofa solution, adjusted to pH 6.3 if necessary, containing in eachmL 2.05 mg of sodium acetate (NaC2H3O2) and 1.11 mg ofcalcium chloride (CaCl2).
Standard Preparation Use a standard-strength rennet, bo-vine rennet, microbial rennet (Endothia parasitica), or micro-bial rennet (Mucor species), as appropriate for the preparationto be assayed. Such standards, which are available from com-mercial coagulant manufacturers, should be of known activity.Dilute the standard-strength material 1 to 200 with water, andmix. Equilibrate to 300 before use, and prepare no more than2 h before use.
Sample Preparation Prepare aqueous solutions or dilu-tions of the sample to produce a final concentration such thatthe clotting time, as determined in the Procedure below, willbe within 1 min of that of the Standard Preparation. Prepareno more than 1 h before use.
Procedure Transfer 50.0 mL of the Substrate Solution intoeach of four 125-mL Sample Bottles. Place the bottles on theBottle-Rotating Apparatus, and suspend the apparatus in awater bath, maintained at 30° � 0.5°, so that the bottles areat an angle of approximately 20° to 30° to the horizontal.Immerse the bottles so that the water level in the bath is aboutequal to the substrate level in the bottles. Begin rotating theapparatus at 16 to 18 rpm, then add 1.0 mL of the SamplePreparation to each of two bottles, and record the exact timeof addition. Add 1.0 mL of the Standard Preparation to eachof the other two bottles, recording the exact time.
Observe the rotating bottles, and record the exact time ofthe first evidence of clotting (i.e., when fine granules or flecksadhere to the sides of the bottle). Variations in the responseof different lots of the substrate may cause variations in clot-ting time; therefore, measure the test samples and standardssimultaneously on the same substrate. Average the clottingtime, in s, of the duplicate samples, recording the time forthe Standard Preparation as TS and that for the Sample Prepa-ration as TU.
Calculation Calculate the activity of the enzyme prepara-tion by the equation
Milk-clotting units/mL = 100 × (TS/TU) × (DS/DU),
in which 100 is the activity assigned to the Standard Prepara-tion, DS is the dilution factor for the Standard Preparation,and DU is the dilution factor for the Sample Preparation.
Note: The dilution factors should be expressed as frac-tions; for example, a dilution of 1 to 200 would beexpressed as 1⁄200.
PANCREATIN ACTIVITY
Application and Principle These procedures are used todetermine the primary enzyme activities in pancreatin prepara-tions.
918 / Appendix V / General Tests and Assays FCC V
Reference StandardsUSP Sodium Taurocholate Reference Standard (Caution:
Avoid inhaling airborne particles.) Keep container tightlyclosed. Dry at 105° for 4 h before using.
USP Pancreatin Reference Standard Keep containertightly closed, and store in a refrigerator. Do not open whilecold, and do not dry before using.
Amylase Activity
pH 6.8 Phosphate Buffer On the day of use, dissolve 13.6g of monobasic potassium phosphate in water to make 500mL of solution. Dissolve 14.2 g of anhydrous dibasic sodiumphosphate in water to make 500 mL of solution. Mix 51 mLof the monobasic potassium phosphate solution with 49 mLof the dibasic sodium phosphate solution. If necessary, adjustby the dropwise addition of the appropriate solution to a pHof 6.8
Substrate Solution On the day of use, stir a portion ofpurified soluble starch equivalent to 2.0 g of dried substancewith 10 mL of water, and add this mixture to 160 mL ofwater, add it to the hot solution, and heat to boiling, withcontinuous mixing. Cool to room temperature, and add waterto make 200 mL.
Standard Preparation Weigh accurately about 20 mg ofUSP Pancreatin Reference Standard into a suitable mortar.Add about 30 mL of pH 6.8 Phosphate Buffer, and trituratefor 5 to 10 min. Transfer the mixture with the aid of pH 6.8Phosphate Buffer to a 50-mL volumetric flask, dilute withpH 6.8 Phosphate Buffer to volume, and mix. Calculate theactivity, in USP Units of amylase activity per mL, of theresulting solution from the declared potency on the label ofthe Reference Standard.
Assay Preparation For Pancreatin having about the sameamylase activity as the USP Pancreatin Reference Standard,weigh accurately about 40 mg of Pancreatin into a suitablemortar.
Note: For Pancreatin having a different amylase activ-ity, weigh accurately the amount necessary to obtainan Assay Preparation having amylase activity per mLcorresponding approximately to that of the StandardPreparation.
Add about 3 mL of pH 6.8 Phosphate Buffer, and trituratefor 5 to 10 min. Transfer the mixture with the aid of pH 6.8Phosphate Buffer to a 100-mL volumetric flask, dilute withpH 6.8 Phosphate Buffer to volume, and mix.
Procedure Prepare four stoppered, 250-mL conical flasks,and mark them S, U, BS, and BU. Pipet into each flask 25mL of Substrate Solution, 10 mL of pH 6.8 Phosphate Buffer,and 1 mL of sodium chloride solution (11.7 in 1000), insertthe stoppers, and mix. Place the flasks in a water bath main-tained at 25° � 0.1°, and allow them to equilibrate. To flasksBU and BS add 2 mL of 1 N hydrochloric acid, mix, andreturn the flasks to the water bath. To flasks U and BU add
1.0-mL portions of the Assay Preparation, and to flasks Sand BS add 1.0 mL of the Standard Preparation. Mix each,and return the flasks to the water bath. After 10 min, accuratelytimed from the addition of the enzyme, add 2-mL portionsof 1 N hydrochloric acid to flasks S and U, and mix. To eachflask, with continuous stirring, add 10.0 mL of 1 N iodineVS, and immediately add 45 mL of 0.1 N sodium hydroxide.Place the flasks in the dark at a temperature between 15° and25° for 15 min. To each flask add 4 mL of 2 N sulfuricacid, and titrate with 0.1 N sodium thiosulfate VS to thedisappearance of the blue color. Calculate the amylase activity,in USP Units per mg, taken by the formula
100(CS/WU)(VBU − VU)/(VBS − VS),
in which CS is the amylase activity of the Standard Prepara-tion, in USP Units per mL; WU is the amount, in mg, ofPancreatin taken; and VU, VS, VBU and VBS are the volumes,in mL, of 0.1 N sodium thiosulfate consumed in the titrationof the solutions in flasks, U, S, BU, and BS, respectively.
Lipase Activity
Gum Arabic Solution Centrifuge a 1:10 solution of gumarabic until clear. Use only the clear solution.
Olive Oil Substrate Combine 165 mL of the Gum ArabicSolution, 20 mL of olive oil, and 15 g of crushed ice in thecup of an electric blender. Cool the mixture in an ice bath to5°, and homogenize at high speed for 15 min, intermittentlycooling in an ice bath to prevent the temperature from ex-ceeding 30°. Test for suitability of mixing as follows: Placea drop of the homogenate on a microscope slide and gentlypress a cover slide in place to spread the liquid. Examine theentire field under high power (43 × magnification objectivelens and 5 × magnification ocular), using an eyepiece equippedwith a calibrated micrometer. The substrate is satisfactory if90% of the particles do not exceed 2 �m in diameter andnone exceeds 10 �m in diameter.
Buffer Solution Dissolve 60 mg of tris(hydroxymethyl)-aminomethane and 234 mg of sodium chloride in water tomake 100 mL.
Sodium Taurocholate Solution Prepare a solution to con-tain 80.0 mg of USP Sodium Taurocholate Reference Standardin each mL.
Standard Test Dilution Suspend about 200 mg of USPPancreatin Reference Standard, accurately weighed, in about3 mL of cold water in a mortar, triturate for 10 min, and addcold water to a volume necessary to produce a concentrationof 8 to 16 USP Units of lipase activity per mL, based on thedeclared potency on the label of the Reference Standard.Maintain the suspension at 4°, and mix before using. For eachdetermination, withdraw 5 to 10 mL of the cold suspension,and allow the temperature to rise to 200 before pipeting theexact volume.
Assay Test Dilution Suspend about 200 mg of the Pancre-atin sample, accurately weighed, in about 3 mL of cold water
FCC V General Tests and Assays / Appendix V / 919
in a mortar, triturate for 10 min, and add cold water to avolume necessary to produce a concentration of 8 to 16 USPUnits of lipase activity per mL, based on the estimated potencyof the test material. Maintain the suspension at 4°, and mixbefore using. For each determination, withdraw 5 to 10 mLof the cold suspension, and allow the temperature to rise to20° before pipeting the exact volume.
Procedure Mix 10.0 mL of Olive Oil Substrate, 8.0 mL ofBuffer Solution, 2.0 mL of Sodium Taurocholate Solution,and 9.0 mL of water in a jacketed glass vessel of about 50-mL capacity, the outer chamber of which is connected to athermostatically controlled water bath. Cover the mixture, andstir continuously with a mechanical stirring device.
With the mixture maintained at a temperature of 37° �0.1°, add 0.1 N sodium hydroxide, from a microburet insertedthrough an opening in the cover, to adjust potentiometricallythe pH to 9.20, using a calomel-glass electrode system. Add1.0 mL of Assay Test Dilution, and then continue adding the0.1 N sodium hydroxide for 5 min to maintain the pH at 9.0.Determine the volume of 0.1 N sodium hydroxide added aftereach min.
In the same manner, titrate 1.0 mL of Standard Test Di-lution.
Calculation From the Standard Test Dilution, plot the vol-ume of 0.1 N sodium hydroxide titrated against time. Usingonly the points that fall on the straight-line segment of thecurve, calculate the mean acidity released per min by theAssay Test Dilution. Taking into consideration dilution factors,calculate the lipase activity of the Standard Test Dilution,using the lipase activity of the USP Pancreatin ReferenceStandard stated on the label.
Protease Activity
Casein Substrate Place 1.25 g of finely powdered caseinin a 100-mL conical flask containing 5 mL of water, shaketo form a suspension, add 10 mL of 0.1 N sodium hydroxide,shake for 1 min, add 50 mL of water, and shake for about 1h to dissolve the casein. Adjust the pH to about 8.0 � 0.1,using 1 N sodium hydroxide or 1 N hydrochloric acid. Transferthe solution to a 100-mL volumetric flask, dilute with waterto volume, and mix. Use this substrate on the day it is prepared.
Buffer Solution Dissolve 6.8 g of monobasic potassiumphosphate and 1.8 g of sodium hydroxide in 950 mL of waterin a 1000-mL volumetric flask, adjust to a pH of 7.5 � 0.2,using 0.2 N sodium hydroxide, dilute with water to volume,and mix. Store this solution in a refrigerator.
Trichloroacetic Acid Solution Dissolve 50 g of trichloro-acetic acid in 1000 mL of water. Store this solution at roomtemperature.
Filter Paper Determine the suitability of the filter paperby filtering a 5-mL portion of Trichloroacetic Acid Solutionthrough the paper and measuring the absorbance of the filtrateat 280 nm, using an unfiltered portion of the same Trichloro-
acetic Acid Solution as the blank. The absorbance is not morethan 0.04. If the absorbance is more than 0.04, the filter papermay be washed repeatedly with Trichloroacetic Acid Solutionuntil the absorbance of the filtrate, determined as above, isnot more than 0.04.
Standard Test Dilution Add about 100 mg of USP Pancre-atin Reference Standard, accurately weighed, to 100.0 mL ofBuffer Solution, and mix by shaking intermittently at roomtemperature for about 25 min. Dilute quantitatively with BufferSolution to produce a concentration of about 2.5 USP Unitsof protease activity per mL, based on the potency declaredon the label of the Reference Standard.
Assay Test Dilution Add about 100 mg of USP PancreatinReference Standard, accurately weighed, to 100.0 mL ofBuffer Solution, and mix by shaking intermittently at roomtemperature for 25 min. Dilute quantitatively with Buffer Solu-tion to obtain a dilution that corresponds in activity to theStandard Test Dilution.
Procedure Label test tubes in duplicate S1, S2, and S3 forthe standard series, and U for the sample. Pipet into tubes S1
2.0 mL, into S2 and U 1.5 mL, and into S3 1.0 mL of BufferSolution. Pipet into tubes S1 1.0 mL, into S2 1.5 mL, and intoS3 2.0 mL of the Standard Test Dilution. Pipet into tubes U1.5 mL of the Assay Test Dilution. Pipet into one tube eachof S1, S2, S3, and U 5.0 mL of Trichloroacetic Acid Solution,and mix. Designate these tubes as S1B, S2B, S3B, and UB,respectively. Prepare a blank by mixing 3 mL of Buffer Solu-tion and 5 mL of Trichloroacetic Acid Solution in a separatetest tube marked B. Place all the tubes in a 40° water bath,insert a glass stirring rod into each tube, and allow temperatureequilibration. At zero time, add to each tube, at timed intervals,2.0 mL of the Casein Substrate, preheated to the bath tempera-ture, and mix. Accurately timed, 60 min after the addition ofthe Casein Substrate, stop the reaction in tubes S1, S2, S3, andU by adding 5.0 mL of Trichloroacetic Acid Solution at thecorresponding time intervals, stir, and remove all the tubesfrom the bath. Allow to stand for 10 min at room temperatureto complete protein precipitation, and filter. The filtrates mustbe free from haze. Determine the absorbances of each filtrate,in a 1-cm cell, at 280 nm, with a suitable spectrophotometer,using the intake from the blank (tube B) to set the instrument.
Calculation Correct the absorbance values for the filtratesfrom tubes S1, S2, and S3 by subtracting the absorbance valuesfor the filtrates from tubes S1B, S2B, and S3B, respectively, andplot the corrected absorbance values against the correspondingvolumes of the Standard Test Dilution used. From the curve,using the corrected absorbance value (U − UB for the USPPancreatin Reference Standard taken), and taking into consid-eration the dilution factors, calculate the protease activity, inUSP Units, of the USP Pancreatin Reference Standard takenby comparison with that of the standard, using the proteaseactivity stated on the label of USP Pancreatin ReferenceStandard.
920 / Appendix V / General Tests and Assays FCC V
PEPSIN ACTIVITY
Application This procedure is to be applied to preparationsderived from porcine or other animal stomachs.
ApparatusMeasuring Vessels Use 100-mL conically shaped measur-
ing vessels complying with the following descriptions: (1)diameters not exceeding 1 cm at the bottom; (2) comply inother respects with the water and sediment tube ASTM Stan-dard Method D96-68; (3) graduated from 0 to 0.5 mL in 0.05-mL graduations, from 2 to 3 mL in 0.1-mL graduations, from3 to 5 mL in 0.2-mL graduations, from 5 to 10 mL in 1-mLgraduations, from 10 to 25 mL in 5-mL graduations, and withgraduation marks at 50, 75, and 100 mL.
Note: Measuring vessels other than the type describedherein may be used if they are of such design andgraduation to permit measurement of the residue withequivalent accuracy.
Reagents and SolutionsHydrochloric Acid Solution Mix 35 mL of 1.0 N hydro-
chloric acid with 385 mL of water.Substrate Boil one or more hen eggs for 15 min to provide
coagulated albumen (Miles, Inc.), and cool rapidly by immer-sion in cold water. Remove the shell and pellicle and all of theyolk, and at once rub the albumen through a clean, dry No. 40sieve, rejecting the first portion that passes through the sieve.
Substrate Preparation Place 10 g of the Substrate in eachof as many 100-mL wide-mouth bottles as needed for the test,and immediately add 35 mL of Hydrochloric Acid Solution (allat one time or in portions). By suitable means, thoroughlydisintegrate the particles of albumen. Equilibrate to 52° beforeuse in the Procedure, below.
Standard Preparation Dissolve 100 mg of USP PepsinReference Standard in 150 mL of Hydrochloric Acid Solution.Use this solution within 1 h.
Sample Preparation Dissolve 100 mg of the pepsin sam-ple, or an amount of the enzyme preparation that will providea solution similar to or slightly stronger than the StandardPreparation, in 150 mL of Hydrochloric Acid Solution. Usethis solution within 1 h.
Procedure Pipet 5.0 mL of the Standard Preparation intoeach of two bottles containing the Substrate Preparation. Totwo or more additional substrate bottles add graduated aliquotsof the Sample Preparation so that one bottle will containapproximately the same amount, and the others will containsuccessively lesser amounts, of pepsin as is contained in the5.0 mL of the Standard Preparation, using, for example, 5.0,4.9, and 4.8 mL. When less than 5.0 mL of the SamplePreparation is used, add sufficient Hydrochloric Acid Solutionto make 5.0 mL of combined Sample Preparation plus acidadded. At once stopper the bottles securely, invert them threetimes, and heat in a water bath, maintained at 52° � 0.5°,for 2.5 h, agitating the contents equally every 10 min by
inverting the bottles once. Remove the bottles from the bath,and pour the contents of each into separate measuring vessels.
Transfer the undigested albumen that adheres to the sidesof the bottles into the respective measuring vessel with theaid of small portions of water until 50 mL has been used foreach. Mix the contents of each vessel, allow them to standfor 30 min, and then read for each the volume of undigestedalbumen. Average the sediment volumes in the two standardvessels, and note which of the sample vessels contains undi-gested albumen closest to the average for the standards. Fi-nally, record as v the volume, in mL, of Sample Preparationthat produced the undigested albumen closest to the averageproduced by the Standard Preparations.
Calculation One pepsin unit is defined as that quantity ofenzyme that digests 3000 times its weight of coagulated eggalbumen under the conditions of the assay.
Calculate the activity of the enzyme preparation by theequation
Pepsin units/mg = 3000 × (S/u) × (5.0/v),
in which S is the weight, in mg, of USP Pepsin ReferenceStandard used to make the Standard Preparation; u is theweight, in mg, of enzyme preparation taken for analysis; andv is as defined in the Procedure.
PHOSPHOLIPASE A2 ACTIVITY
Application and Principle This procedure is used to deter-mine the phospholipase A2 activity from extracts of porcinepancreatic tissue. The analysis is performed by potentiometrictitration.
ApparatusAutomatic Titrator Use a suitable automatic recording
titrator equipped with a stirred, thermostated, controlled-atmo-sphere titration cell (e.g., Radiometer Autotitrator).
Homogenizer Use a suitable homogenizer (e.g., Biomixer;Fisher Scientific, Catalog No. 11-504-2-4, or equivalent).
Constant-Temperature Water Bath Set at 40° � 0.1°.
Reagents and SolutionsCalcium Chloride Solution (0.3 M) Transfer 4.41 g of
calcium chloride dihydrate to a 100-mL volumetric flask,dissolve in, and dilute to volume with water.
Sodium Deoxycholate Solution (0.016 M) Dissolve 0.67g of sodium deoxycholate (Sigma Chemical Co., Catalog No.D6750) in 100 mL of water.
Sodium Hydroxide Solution (0.1 N) Use a standardizedsolution.
Substrate Solution Add the yolk of one fresh egg to 100mL of deionized water and homogenize until a stable emulsionis obtained. Add 5 mL of the Calcium Chloride Solution,and mix.
FCC V General Tests and Assays / Appendix V / 921
Sample Preparation Dissolve an accurately weighedamount of enzyme preparation in 0.001 N hydrochloric acid,and dilute to obtain an enzyme activity of 10 to 80 units ofactivity per mL.
Procedure Pre-equilibrate the Substrate Solution, the So-dium Deoxycholate Solution, and about 50 mL of water to40° in the water bath. Transfer 10 mL of the Substrate Solutionto the thermostated titration vessel. Add 5 mL of the SodiumDeoxycholate Solution and 10 mL of deionized water. Blanketthe cell with nitrogen and equilibrate for approximately 5min. Using the Automatic Titrator filled with 0.1 N SodiumHydroxide Solution, adjust the pH of the solution to 8.0 �0.05. Monitor the consumption (if any) of sodium hydroxidefor 5 min as a blank. Refill the Automatic Titrator. Add 0.1mL of Sample Solution containing between 1 and 8 units ofactivity and start the Automatic Titrator. Record the sodiumhydroxide consumption for at least 5 min.
Calculation One phospholipase unit is defined as the quan-tity of enzyme that produces 1 microequivalent of free fattyacid per min under the conditions of the test. Determine therate, R, of titrant consumption during 0 to 3 min of the reaction.
Note: The recorder trace must be linear during the first3 min of the reaction.
Determine the rate of titrant consumption (if any) duringequilibration (blank) (RB):
Units/g = (R × N) − (RB × N)/W,
in which R and RB are the rates of titrant consumption of thesample and blank, respectively, in �L/min; N is the normalityof the titrant; and W is the weight, in g, contained in 0.1 mLof the Sample Preparation taken for the test.
PHYTASE ACTIVITY
Application and Principle This procedure is used to deter-mine the activity of enzymes releasing phosphate from phy-tate. The assay is based on enzymatic hydrolysis of sodiumphytate under controlled conditions by measurement of theamount of ortho phosphate released.
Reagents and Solutions
Note: All glassware must be acid washed, rinsed, andscrupulously cleaned to ensure the absence of phos-phate.
Acetate Buffer (pH 5.5) Dissolve 1.76 g of 100% aceticacid (C2H4O2), 30.02 g of sodium acetate trihydrate (C2H3O2-Na·3H2O), and 0.147 g of calcium chloride dihydrate in about900 mL of water. Transfer the solution into a 1000-mL volu-metric flask, dilute to volume with water, and mix. The pHshould be 5.50 � 0.05.
Substrate Solution Dissolve 8.40 g of sodium phytate dec-ahydrate (C6H6O24P6Na12·10H2O) (Sigma Chemical Co.) in900 mL of Acetate Buffer. Adjust the pH to 5.50 � 0.05 at37.0° � 0.1° by adding 4 M acetic acid. Cool to ambienttemperature. Quantitatively transfer the mixture to a 1000-mL volumetric flask, dilute to volume with Acetate Buffer,and mix. Prepare fresh daily.
Nitric Acid Solution (27%) While stirring, slowly add 70mL of 65% nitric acid to 130 mL of water.
Ammonium Heptamolybdate Solution Dissolve 100 g ofammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24
·4H2O] in 900 mL of water in a 1000-mL volumetric flask.Add 10 mL of 25% ammonia solution, dilute to volume withwater, and mix. This solution is stable for 4 weeks whenstored at ambient temperature and shielded from light.
Ammonium Vanadate Solution Dissolve 2.35 g of ammo-nium monovanadate (NH4VO3) in 400 mL of warm (60°)water. While stirring, slowly add 20 mL of Nitric Acid Solution(27%). Cool to ambient temperature. Quantitatively transferthe mixture to a 1000-mL volumetric flask, dilute to volumewith water, and mix. This solution is stable for 4 weeks whenstored at ambient temperature and shielded from light.
Color/Stop Solution While stirring, add 250 mL of Ammo-nium Vanadate Solution to 250 mL of Ammonium Heptamo-lybdate Solution. Slowly add 165 mL of 65% nitric acid. Coolto ambient temperature. Quantitatively transfer the mixtureto a 1000-mL volumetric flask, dilute to volume with water,and mix. Prepare fresh daily.
Potassium Dihydrogen Phosphate Solution Dry a suffi-cient amount of potassium dihydrogen phosphate (KH2PO4)in a vacuum oven at 100° to 104° for 2 h. Cool to ambienttemperature in a desiccator over dried silica gel.
In duplicate (solutions A and B), weigh approximately0.245 g of dried potassium dihydrogen phosphate accuratelyto within 1 mg and dilute with Acetate Buffer to 1 L to obtainsolutions containing 1.80 mmol/L of potassium dihydrogenphosphate.
Phytase Reference Preparation (Highly concentrated phy-tase preparation) This preparation can be obtained fromGist-Brocades, Delft, The Netherlands, with an assigned activ-ity (by collaborative assay), or the activity of the referencepreparation can be determined according to Procedure 2.
Phytase Reference Solutions, Procedure 1 Weigh anamount of Phytase Reference Preparation corresponding with20,000 phytase units accurately to within 1 mg in duplicatein 200-mL volumetric flasks. Dissolve in and dilute to volumewith Acetate Buffer, and mix. Dilute with Acetate Buffer toobtain dilutions containing approximately 0.01, 0.02, 0.04,0.06, and 0.08 phytase units per 2.0 mL of the final dilution.
Sample Preparation, Procedure 1 Suspend or dissolveand dilute accurately weighed amounts of sample in AcetateBuffer so that 2.0 mL of the final dilution will contain between0.02 and 0.08 phytase units.
Sample Preparation, Procedure 2 In duplicate, accuratelyweigh amounts of Phytase Reference Preparation and dissolveand dilute in Acetate Buffer to obtain dilutions containing0.06 � 0.006 phytase units per 2.0 mL of the final dilution.
922 / Appendix V / General Tests and Assays FCC V
ProceduresProcedure 1 (Determination of the phytase activity)
Transfer 2.00 mL of the Sample Preparation, Procedure 1,and the Phytase Reference Solutions, Procedure 1, into sepa-rate 20- × 150-mm glass test tubes. Using a stopwatch andstarting at time equals zero, in the order of the series andwithin regular time intervals, place the tubes into a 37.0° �0.1° water bath and allow their contents to equilibrate for 5min. At time equals 5 min, in the same order of the seriesand with the same time intervals, add 4.0 mL of SubstrateSolution (previously equilibrated to 37.00 � 0.10) to eachtest tube. Mix, and replace in the 37.0° � 0.1° water bath.At time equals 65 min, in the same order and within the sametime intervals, terminate the incubation by adding 4.0 mL ofColor/Stop Solution. Mix, and cool to ambient temperature.
Prepare blanks by transferring 2.00 mL of the Sample Prep-aration, Procedure 1, and the Phytase Reference Solutions,Procedure 1, into separate 20- × 150-mm glass test tubes.Using a stopwatch and starting at time equals zero, in theorder of the series and within regular time intervals, placethe tubes into a 37.0° � 0.1° water bath and allow them toequilibrate for 5 min. At time equal 5 min, in the same orderof the series and within the same time intervals, add 4.0 mLof Color/Stop Solution. Mix, and cool to ambient temperature.Next add 4.00 mL of Substrate Solution to the blank tubes,and mix.
Centrifuge all test tubes for 5 min at 3000 × g. Determinethe absorbance of each solution at 415 nm in a 1-cm path-length cell with a suitable spectrophotometer, using water tozero the instrument.
Procedure 2 (Determination of the phytase activity of thePhytase Reference Preparation) Transfer 2.00 mL of Sam-ple Preparation, Procedure 2, and 2.00 mL (three times fromPotassium Dihydrogen Phosphate Solution A and two timesfrom B) of Potassium Dihydrogen Phosphate Solutions intoseparate 20- × 150-mm glass test tubes. Using a stopwatchand starting at time equals zero, in the order of the series andwithin regular time intervals, place the tubes into a 37.0° �0.1° water bath and allow their contents to equilibrate for 5min. At time equals 5 min, in the same order of the seriesand within the same time intervals, add 4.0 mL of SubstrateSolution (previously equilibrated to 37.0° � 0.1°) to the testtubes. Mix, and replace in the 37.0° � 0.1° water bath. Attime equals 35 min, in the same order and within the sametime intervals, terminate the incubation by adding 4.0 mL ofColor/Stop Solution. Mix, and cool to ambient temperature.
Prepare blanks by transferring 2.00 mL of Sample Prepara-tion, Procedure 2, into separate 20- × 150-mm glass test tubes.Prepare Reagent Blanks by transferring 2.00 mL of water intoa series of five separate 20- × 150-mm glass test tubes. Add4.0 mL of Color/Stop Solution to all blank tubes and mix.Next add 4.0 mL of Substrate Solution, and mix. Determinethe absorbance of each solution at 415 nm in a 1-cm path-length cell with a suitable spectrophotometer, using water tozero the instrument.
CalculationsCalculation, Procedure 1 One Phytase (fytase) unit
(FTU) is the amount of enzyme that liberates inorganic phos-
phate at 1 �mol/min from sodium phytate 0.0051 mol/L at37.00 at pH 5.50 under the conditions of the test. Calculate thecorrected absorbance (sample minus blank) for each SamplePreparation and Phytase Reference Solution. Plot the accu-rately calculated phytase activity (FTU per 2 mL) of eachPhytase Reference Solution against the corresponding ab-sorbance. From the curve, determine the phytase activity ineach Sample Preparation (FTU per 2 mL):
Activity (FTU/g) = (FTU per 2 mL × dilution)/sample weight (g).
Calculation, Procedure 2 Calculate the corrected ab-sorbances AR for each Sample Preparation (absorbance Phy-tase Reference Solution minus corresponding absorbanceblank) and for each Potassium Dihydrogen Phosphate Solu-tion, Ap (absorbance Potassium Dihydrogen Phosphate Solu-tion minus average absorbance reagent blank). Calculate C,the phosphate concentration of each Potassium DihydrogenPhosphate Solution:
(W × 1000 × 2)/MW = C (mmol/2 mL).
Calculate the absorbances D for each Potassium DihydrogenPhosphate Solution after correction for the amount of potas-sium dihydrogen phosphate weighed:
Ap/C = D(absorbance units/mmol of phosphate per 2 mL).
Calculate the average of results D, giving E (maximum allow-able difference, 5%).
Calculate the activity for each Phytase Reference Prepa-ration:
(AR × f)/(30 × R × E) = FTU/g,
in which AR equals the corrected absorbance of the PhytaseStandard Solution; f equals the total dilution factor of thereference preparation; 30 equals the incubation time, in min;R equals sample weight, in g; E equals average of D factors;W equals the weight of potassium dihydrogen phosphate, in g;and MW equals the molecular weight of potassium dihydrogenphosphate, 136.09 (g/mol).
PLANT PROTEOLYTIC ACTIVITY
Application and Principle This procedure is used to deter-mine the proteolytic activity of papain, ficin, and bromelain.The assay is based on a 60-min proteolytic hydrolysis of acasein substrate at pH 6.0 and 40°. Unhydrolyzed substrateis precipitated with trichloroacetic acid and removed by filtra-tion; solubilized casein is then measured spectrophotometri-cally.
Reagents and SolutionsSodium Phosphate Solution (0.05 M) Transfer 7.1 g of
anhydrous dibasic sodium phosphate into a 1000-mL volumet-ric flask, dissolve in about 500 mL of water, dilute to volumewith water, and mix. Add 1 drop of toluene as a preservative.
FCC V General Tests and Assays / Appendix V / 923
Citric Acid Solution (0.05 M) Transfer 10.5 g of citricacid monohydrate into a 1000-mL volumetric flask, dissolvein about 500 mL of water, dilute to volume with water, andmix. Add 1 drop of toluene as a preservative.
Phosphate–Cysteine–EDTA Buffer Solution Dissolve 7.1g of anhydrous dibasic sodium phosphate in about 800 mLof water, and then dissolve in this solution 14.0 g of disodiumEDTA dihydrate and 6.1 g of cysteine hydrochloride monohy-drate.
Adjust to pH 6.0 � 0.1 with 1 N hydrochloric acid or 1 Nsodium hydroxide, then transfer into a 1000-mL volumetricflask, dilute to volume with water, and mix.
Trichloroacetic Acid Solution Dissolve 30 g of trichloro-acetic acid in 100 mL of water.
Casein Substrate Solution Disperse 1 g (moisture-freebasis) of Hammarsten-grade casein (United States Biochemi-cal Corp., Catalog No. 12840, or equivalent) in 50 mL ofSodium Phosphate Solution, and heat for 30 min in a boilingwater bath, with occasional agitation. Cool to room tempera-ture, and with rapid and continuous agitation, adjust to pH6.0 � 0.1 by the addition of Citric Acid Solution.
Note: Rapid and continuous agitation during the addi-tion prevents casein precipitation.
Quantitatively transfer the mixture into a 100-mL volumetricflask, dilute to volume with water, and mix.
Stock Standard Solution Transfer 100.0 mg of USP Pa-pain Reference Standard into a 100-mL volumetric flask, dis-solve, and dilute to volume with Phosphate–Cysteine–EDTABuffer Solution, and mix.
Diluted Standard Solutions Pipet 2, 3, 4, 5, 6, and 7 mLof Stock Standard Solution into a series of 100-mL volumetricflasks, dilute each to volume with Phosphate–Cysteine–EDTABuffer Solution, and mix by inversion.
Test Solution Prepare a solution from the enzyme prepara-tion so that 2 mL of the final dilution will give a �A in theProcedure between 0.2 and 0.5. Weigh the sample accurately,transfer it quantitatively to a glass mortar, and triturate withPhosphate–Cysteine–EDTA Buffer Solution. Transfer the mix-ture quantitatively into a volumetric flask of appropriate size,dilute to volume with Phosphate–Cysteine–EDTA Buffer So-lution, and mix.
Procedure Pipet 5 mL of Casein Substrate Solution intoeach of a series of 25- × 150-mm test tubes, allowing threetubes for the enzyme unknown, six for a papain standardcurve, and nine for enzyme blanks. Equilibrate the tubes for15 min in a water bath maintained at 40° � 0.1°. Startingthe stopwatch at zero time, rapidly pipet 2 mL of each of theDiluted Standard Solutions, and 2-mL portions of the TestSolution, into the equilibrated substrate. Mix each by swirling,stopper, and place the tubes back in the water bath. After60.0 min, add 3 mL of Trichloroacetic Acid Solution to eachtube. Immediately mix each tube by swirling.
Prepare enzyme blanks containing 5.0 mL of Casein Sub-strate Solution, 3.0 mL of Trichloroacetic Acid Solution, and2.0 mL of one of the appropriate Diluted Standard Solutionsor the Test Solution.
Return all tubes to the water bath, and heat for 30.0 min,allowing the precipitated protein to coagulate completely. Fil-ter each mixture through Whatman No. 42, or equivalent,filter paper, discarding the first 3 mL of filtrate. The subse-quent filtrate must be perfectly clear. Determine the ab-sorbance of each filtrate in a 1-cm cell at 280 nm, with asuitable spectrophotometer, against its respective blank.
Calculation One papain unit (PU) is defined in this assayas that quantity of enzyme that liberates the equivalent of 1�g of tyrosine per h under the conditions of the assay.
Prepare a standard curve by plotting the absorbances offiltrates from the Diluted Standard Solutions against the cor-responding enzyme concentrations, in mg/mL. By interpola-tion from the standard curve, obtain the equivalent concentra-tion of the filtrate from the Test Solution.
Calculate the activity of the enzyme preparation taken foranalysis as follows:
PU/mg = (A × C × 10)/W,
in which A is the activity of USP Papain Reference Standard,in PU per mg; C is the concentration, in mg/mL, of ReferenceStandard from the standard curve, equivalent to the enzymeunknown; 10 is the total volume, in mL, of the final incubationmixture; and W is the weight, in mg, of original enzymepreparation in the 2-mL aliquot of Test Solution added to theincubation mixture.
PROTEOLYTIC ACTIVITY, BACTERIAL(PC)
Application and Principle This procedure is used to deter-mine protease activity, expressed as PC units, of preparationsderived from Bacillus subtilis var. and Bacillus licheniformisvar. The assay is based on a 30-min proteolytic hydrolysis ofcasein at 37° and pH 7.0. Unhydrolyzed casein is removedby filtration, and the solubilized casein is determined spectro-photometrically.
Reagents and SolutionsCasein Use Hammarsten-grade casein (United States Bio-
chemical Corp., Catalog No. 12840, or equivalent).Tris Buffer (pH 7.0) Dissolve 12.1 g of enzyme-grade (or
equivalent) tris(hydroxymethyl)aminomethane in 800 mL ofwater, and titrate with 1 N hydrochloric acid to pH 7.0. Trans-fer into a 1000-mL volumetric flask, dilute to volume withwater, and mix.
TCA Solution Dissolve 18 g of trichloroacetic acid and19 g of sodium acetate trihydrate in 800 mL of water in a1000-mL volumetric flask, add 20 mL of glacial acetic acid,dilute to volume with water, and mix.
Substrate Solution Dissolve 6.05 g of enzyme-grade tris-(hydroxymethyl)aminomethane in 500 mL of water, add 8mL of 1 N hydrochloric acid, and mix. Dissolve 7 g of Casein
924 / Appendix V / General Tests and Assays FCC V
in this solution, and heat for 30 min in a boiling water bath,stirring occasionally.
Cool to room temperature, and adjust to pH 7.0 with 0.2N hydrochloric acid, adding the acid slowly, with vigorousstirring, to prevent precipitation of the casein. Transfer themixture into a 1000-mL volumetric flask, dilute to volumewith water, and mix.
Sample Preparation Using Tris Buffer, prepare a solutionof the sample enzyme preparation so that 2 mL of the finaldilution will contain between 10 and 44 bacterial proteaseunits.
Procedure Pipet 10.0 mL of the Substrate Solution intoeach of a series of 25- × 150-mm test tubes, allowing onetube for each enzyme test, one tube for each enzyme blank,and one tube for a substrate blank. Equilibrate the tubes for15 min in a water bath maintained at 37° � 0.1°.
Starting the stopwatch at zero time, rapidly pipet 2.0 mLof the Sample Preparation into the equilibrated substrate.Mix, and replace the tubes in the water bath. Add 2 mL ofTris Buffer (instead of the Sample Preparation) to the substrateblank. After exactly 30 min, add 10 mL of TCA Solution toeach enzyme incubation and to the substrate blank to stop thereaction. Heat the tubes in the water bath for an additional30 min to allow the protein to coagulate completely.
At the end of the second heating period, shake each tubevigorously, and filter through 11-cm Whatman No. 42, orequivalent, filter paper, discarding the first 3 mL of filtrate.
Note: The filtrate must be perfectly clear.
Determine the absorbance of each sample filtrate in a 1-cmcell, at 275 nm, with a suitable spectrophotometer, using thefiltrate from the substrate blank to set the instrument at zero.Correct each reading by subtracting the appropriate enzymeblank reading, and record the value so obtained as AU.
Standard Curve Transfer 100.0 mg of L-tyrosine, chro-matographic-grade or equivalent (Aldrich Chemical Co.), pre-viously dried to constant weight, to a 1000-mL volumetricflask. Dissolve in 60 mL of 0.1 N hydrochloric acid. Whencompletely dissolved, dilute the solution to volume with water,and mix thoroughly. This solution contains 100 �g of tyrosinein 1.0 mL. Prepare three more dilutions from this stock solu-tion to contain 75.0, 50.0, and 25.0 �g of tyrosine per mL.Determine the absorbance of the four solutions at 275 nm ina 1-cm cell on a suitable spectrophotometer versus 0.006 Nhydrochloric acid. Prepare a plot of absorbance versus tyrosineconcentration.
Calculation One bacterial protease unit (PC) is defined asthat quantity of enzyme that produces the equivalent of 1.5�g/mL of L-tyrosine per min under the conditions of the assay.
From the Standard Curve, and by interpolation, determinethe absorbance of a solution having a tyrosine concentrationof 60 �g/mL. A figure close to 0.0115 should be obtained.Divide the interpolated value by 40 to obtain the absorbanceequivalent to that of a solution having a tyrosine concentrationof 1.5 �g/mL, and record the value thus derived as AS.
Calculate the activity of the sample enzyme preparation bythe equation
PC/g = (AU/AS) × (22/30W),
in which 22 is the final volume, in mL, of the reaction mixture;30 is the time, in min, of the reaction; and W is the weight,in g, of the original sample taken.
PROTEOLYTIC ACTIVITY, FUNGAL(HUT)
Application and Principle This procedure is used to deter-mine the proteolytic activity, expressed as hemoglobin unitson the tyrosine basis (HUT), of preparations derived fromAspergillus oryzae var. and Aspergillus niger var., and it maybe used to determine the activity of other proteases at pH 4.7.The test is based on the 30-min enzymatic hydrolysis of ahemoglobin substrate at pH 4.7 and 40°. Unhydrolyzed sub-strate is precipitated with trichloroacetic acid and removedby filtration. The quantity of solubilized hemoglobin in thefiltrate is determined spectrophotometrically.
Reagents and SolutionsHemoglobin Use Hemoglobin Substrate Powder (Sigma
Chemical Co., Catalog No. H2625) or a similar high-gradematerial that is completely soluble in water.
Acetate Buffer Solution Dissolve 136 g of sodium acetate(NaC2H3O2·3H2O) in sufficient water to make 500 mL. Mix25.0 mL of this solution with 50.0 mL of 1 M acetic acid,dilute to 1000 mL with water, and mix. The pH of this solutionshould be 4.7 � 0.02.
Substrate Solution Transfer 4.0 g of the Hemoglobin intoa 250-mL beaker, add 100 mL of water, and stir for 10 minto dissolve. Immerse the electrodes of a pH meter in thesolution, and while stirring continuously, adjust the pH to 1.7by adding 0.3 N hydrochloric acid. After 10 min, adjust thepH to 4.7 by adding 0.5 M sodium acetate. Transfer thesolution into a 200-mL volumetric flask, dilute to volumewith water, and mix. This solution is stable for about 5 dayswhen refrigerated.
Trichloroacetic Acid Solution Dissolve 140 g of trichloro-acetic acid in about 750 mL of water. Transfer the solutionto a 1000-mL volumetric flask, dilute to volume with water,and mix thoroughly.
Sample Preparation Dissolve an amount of the samplein the Acetate Buffer Solution to produce a solution containing,in each mL, between 9 and 22 HUT. (Such a concentrationwill produce an absorbance reading, in the procedure below,within the preferred range of 0.2 to 0.5.)
Procedure Pipet 10.0 mL of the Substrate Solution intoeach of a series of 25- × 150-mm test tubes: one for eachenzyme test and one for the substrate blank. Heat the tubesin a water bath at 40° for about 5 min. To each tube, exceptthe substrate blank, add 2.0 mL of the Sample Preparation,and begin timing the reaction at the moment the solution is
FCC V General Tests and Assays / Appendix V / 925
added; add 2.0 mL of the Acetate Buffer Solution to thesubstrate blank tube. Close the tubes with No. 4 rubber stop-pers, and tap each tube gently for 30 s against the palm ofthe hand to mix. Heat each tube in a water bath at 40° forexactly 30 min, and then rapidly pipet 10.0 mL of the Trichlo-roacetic Acid Solution into each tube. Shake each tube vigor-ously against the stopper for about 40 s, and then allow tocool to room temperature for 1 h, shaking each tube againstthe stopper at 10- to 12-min intervals during this period.Prepare enzyme blanks as follows: Heat, in separate tubes,10.0 mL of the Substrate Solution and about 5 mL of theSample Preparation in the water bath for 30 min, then add10.0 mL of the Trichloroacetic Acid Solution to the SubstrateSolution, shake well for 40 s, and to this mixture add 2.0 mLof the preheated Sample Preparation. Shake again, and coolat room temperature for 1 h, shaking at 10- to 12-min intervals.
At the end of 1 h, shake each tube vigorously, and filterthrough 11-cm Whatman No. 42, or equivalent, filter paper,refiltering the first half of the filtrate through the same paper.Determine the absorbance of each filtrate in a 1-cm cell, at275 nm, with a suitable spectrophotometer, using the filtratefrom the substrate blank to set the instrument to zero. Correcteach reading by subtracting the appropriate enzyme blankreading, and record the value so obtained as AU.
Note: If a corrected absorbance reading between 0.2and 0.5 is not obtained, repeat the test using more orless of the enzyme preparation as necessary.
Standard Curve Transfer 100.0 mg of L-tyrosine, chro-matographic-grade, or equivalent (Aldrich Chemical Co.),previously dried to constant weight, to a 1000-mL volumetricflask. Dissolve in 60 mL of 0.1 N hydrochloric acid. Whenthe L-tyrosene is completely dissolved, dilute the solution tovolume with water, and mix thoroughly. This solution contains100 �g of tyrosine in 1.0 mL. Prepare three more dilutionsfrom this stock solution to contain 75.0, 50.0, and 25.0 �gof tyrosine per mL. Determine the absorbance of the foursolutions at 275 nm in a 1-cm cell on a suitable spectrophotom-eter versus 0.006 N hydrochloric acid. Prepare a plot of ab-sorbance versus tyrosine concentration. Determine the slopeof the curve in terms of absorbance per �g of tyrosine. Multi-ply this value by 1.10, and record it as AS. A value of approxi-mately 0.0084 should be obtained.
Calculation One HUT unit of proteolytic (protease) activityis defined as that amount of enzyme that produces, in 1 minunder the specified conditions, a hydrolysate whose ab-sorbance at 275 nm is the same as that of a solution containing1.10 �g/mL of tyrosine in 0.006 N hydrochloric acid.
Calculate the HUT per g of the original enzyme preparationby the equation
HUT/g = (AU/AS) × (22/30W),
in which 22 is the final volume of the test solution; 30 is thereaction time, in min; and W is the weight, in g, of the originalsample taken.
Note: The value for AS, under carefully controlled andstandardized conditions, is 0.0084; this value may be
used for routine work in lieu of the value obtained fromthe standard curve, but the exact value calculated fromthe standard curve should be used for more accurateresults and in cases of doubt.
PROTEOLYTIC ACTIVITY, FUNGAL(SAP)
Application and Principle This procedure is used to deter-mine proteolytic activity, expressed in spectrophotometricacid protease units (SAP), of preparations derived from Asper-gillus niger var. and Aspergillus oryzae var. The test is basedon a 30-min enzymatic hydrolysis of a Hammarsten CaseinSubstrate at pH 3.0 and at 37°. Unhydrolyzed substrate isprecipitated with trichloroacetic acid and removed by filtra-tion. The quantity of solubilized casein in the filtrate is deter-mined spectrophotometrically.
Reagents and SolutionsCasein Use Hammarsten-grade casein (United States Bio-
chemical Corp., Catalog No. 12840, or equivalent).Glycine–Hydrochloric Acid Buffer (0.05 M) Dissolve
3.75 g of glycine in about 800 mL of water. Add 1 N hydro-chloric acid until the solution is pH 3.0, determined with apH meter. Quantitatively transfer the solution to a 1000-mLvolumetric flask, dilute to volume with water, and mix.
TCA Solution Dissolve 18.0 g of trichloroacetic acid and11.45 g of anhydrous sodium acetate in about 800 mL ofwater, and add 21.0 mL of glacial acetic acid. Quantitativelytransfer the solution to a 1000-mL volumetric flask, dilute tovolume with water, and mix.
Substrate Solution Pipet 8 mL of 1 N hydrochloric acidinto about 500 mL of water, and with continuous agitation,disperse 7.0 g (moisture-free basis) of Casein into this solu-tion. Heat for 30 min in a boiling water bath, stirring occasion-ally, and cool to room temperature. Dissolve 3.75 g of glycinein the solution, and using a pH meter, adjust to pH 3.0 with0.1 N hydrochloric acid. Quantitatively transfer the solutionto a 1000-mL volumetric flask, dilute to volume with water,and mix.
Sample Preparation Using Glycine–Hydrochloric AcidBuffer, prepare a solution of the sample enzyme preparationso that 2 mL of the final dilution will give a corrected ab-sorbance of enzyme incubation filtrate at 275 nm (�A, asdefined in the Procedure) between 0.200 and 0.500. Weighthe enzyme preparation, quantitatively transfer it to a glassmortar, and triturate with Glycine–Hydrochloric Acid Buffer.Quantitatively transfer the mixture to an appropriately sizedvolumetric flask, dilute to volume with Glycine–HydrochloricAcid Buffer, and mix.
Procedure Pipet 10.0 mL of Substrate Solution into eachof a series of 25- × 150-mm test tubes, allowing at least twotubes for each sample, one for each enzyme blank, and one
926 / Appendix V / General Tests and Assays FCC V
for a substrate blank. Stopper the tubes, and equilibrate themfor 15 min in a water bath maintained at 37° � 0.1°.
At zero time, start the stopwatch, and rapidly pipet 2.0 mLof the Sample Preparation into the equilibrated substrate. Mixby swirling, and replace the tubes in the water bath.
Note: Keep the tubes stoppered during incubation.
Add 2 mL of Glycine–Hydrochloric Acid Buffer (instead ofthe Sample Preparation) to the substrate blank. After exactly30 min, add 10 mL of TCA Solution to each enzyme incubationand to the substrate blank to stop the reaction. In the followingorder, prepare an enzyme blank containing 10 mL of SubstrateSolution, 10 mL of TCA Solution, and 2 mL of the SamplePreparation. Heat all tubes in the water bath for 30 min,allowing the precipitated protein to coagulate completely.
At the end of the second heating period, cool the tubes inan ice bath for 5 min, and filter through Whatman No. 42filter paper, or equivalent. The filtrates must be perfectly clear.Determine the absorbance of each filtrate in a 1-cm cell at275 nm with a suitable spectrophotometer, against the sub-strate blank. Correct each absorbance by subtracting the ab-sorbance of the respective enzyme blank.
Standard Curve Transfer 181.2 mg of L-tyrosine, chro-matographic-grade or equivalent (Sigma Chemical Co.), pre-viously dried to constant weight, to a 1000-mL volumetricflask. Dissolve in 60 mL of 0.1 N hydrochloric acid. Whenthe L-tyrosine is completely dissolved, dilute the solution tovolume with water, and mix thoroughly. This solution contains1.00 �mol of tyrosine per 1.0 mL. Prepare dilutions from thisstock solution to contain 0.10, 0.20, 0.30, 0.40, and 0.50 �mol/mL. Determine against a water blank the absorbance of eachdilution in a 1-cm cell at 275 nm. Prepare a plot of absorbanceversus �mol of tyrosine per mL. A straight line must beobtained. Determine the slope and intercept for use in theCalculation below. A value close to 1.38 should be obtained.The slope and intercept may be calculated by the least squaresmethod as follows:
Slope = [n� (MA) − � (M) �(A)]/[n� (M2) − (�M)2],
Intercept = [�(A) �(M2) − � (M) �(MA)]/[n� (M2) −(�M)2],
in which n is the number of points on the standard curve, Mis the �mol of tyrosine per mL for each point on the standardcurve, and A is the absorbance of the sample.
Calculation One spectrophotometric acid protease unit isthat activity that will liberate 1 �mol of tyrosine per minunder the conditions specified. The activity is expressed asfollows:
SAP/g = (�A − I) × 22/(S × 30 × W),
in which �A is the corrected absorbance of the enzyme incuba-tion filtrate; I is the intercept of the Standard Curve; 22 isthe final volume of the incubation mixture, in mL; S is theslope of the Standard Curve; 30 is the incubation time, inmin; and W is the weight, in g, of the enzyme sample containedin the 2.0-mL aliquot of Sample Preparation added to theincubation mixture in the Procedure.
PULLULANASE ACTIVITY
Application and Principle This procedure is used to deter-mine pullulanase activity derived from Bacillus acidopullulyt-icus. The method is based on measuring the increase in reduc-ing sugars formed by a 30-min hydrolysis of pullulan at 40°and pH 5.0. The increase in reducing sugars is measuredspectrophotometrically at 520 nm using a modified Nelson–Somogyi procedure.
Reagents and SolutionsCitrate Buffer (pH 5.0) Dissolve 10.5 g of citric acid
monohydrate in 950 mL of water, adjust the pH to 5.0 �0.05 using 5 N sodium hydroxide, and dilute to 1000 mL.
Nelson’s Color Reagent Dissolve 25.0 g of ammoniummolybdate tetrahydrate in 300 mL of water. Carefully add20.0 mL of concentrated sulfuric acid while stirring. Dissolve3.0 g of sodium arsenate heptahydrate in 25 mL of water.Slowly add this solution to the ammonium molybdate solutionwith stirring. Dilute this solution to 500 mL with water.
Somogyi’s Copper Reagent Dissolve 14.0 g of anhydrousdibasic sodium phosphate and 20.0 g of potassium sodiumtartrate tetrahydrate into 250 mL of water. Add 60.0 g of 1M sodium hydroxide solution. Dissolve 4.0 g of cupric sulfatepentahydrate into 25 mL of water. Add this solution to thetartrate solution. Add 90.0 g of anhydrous sodium sulfatewhile stirring. Dilute the final solution to 500 mL.
Glucose Standards Dissolve 800 mg of previously driedanhydrous D-glucose in 100 mL of the Citrate Buffer. Prepareglucose standards containing 16, 40, 80, and 120 �g/mL ofglucose.
Pullulan Substrate Dissolve 150 mg of pullulan (SigmaChemical Co., Catalog No. P-4516, or equivalent) in 49.80g of the Citrate Buffer. Prepare daily.
Sample Preparation Dissolve an accurately weighedamount of the enzyme preparation in Citrate Buffer and dilutein Citrate Buffer to obtain an enzyme activity of 0.01 to 0.03activity units per mL.
Procedure Transfer 1.0-mL aliquots of Pullulan Substrateto separate 15- × 150-mm test tubes. Insert a one-hole stopperin each tube, and equilibrate for 15 min in a 40° � 0.1° waterbath. At time equals zero and at 30-s intervals, add 1 mL of therespective samples, and mix. Exactly 30.0 min after addition ofthe samples, add 2.0 mL of Somogyi’s Copper Reagent toeach tube to terminate the reaction. Mix thoroughly, and allowthe tubes to come to room temperature. To obtain sampleblanks, add the Somogyi’s Copper Reagent to the samplebefore adding the substrate.
Prepare a standard curve by adding 2.0 mL of each glucosestandard and 2.0 mL of Somogyi’s Copper Reagent to a testtube, and mix. The blanks and Glucose Standards should notbe incubated at 40°.
Loosely stopper samples, blanks, and Glucose Standardscontaining Somogyi’s Copper Reagent, and place them in avigorously boiling water bath for exactly 25.0 min. Cool inan ice-water bath for approximately 1 min.
FCC V General Tests and Assays / Appendix V / 927
Add 2.00 mL of Nelson’s Color Reagent to each tube, andmix thoroughly to dissolve any red precipitate that might bepresent. Let the solutions stand for 5 min. Add 2.0 mL ofwater to each tube, and mix.
Measure the absorbance of all solutions at 520 nm, usingwater as the reference. Mix the contents of each tube beforetransferring them to the cuvette.
Calculations One pullulanase unit (PUN) is the amount ofactivity that under the conditions of the test, will liberatereducing sugars equivalent to 1 �mol of glucose per min.Determine the linear regression line for absorbance versustwo times the glucose concentration (�g/mL) in the standards.Use the slope, I, in the following equation to determine theactivity in the enzyme preparation:
PUN/g = (AS – AB)/I × W × 180 × 30,
in which AS is the absorbance of the sample; AB is the ab-sorbance of the blank; W is the weight, in g, of the enzymepreparation contained in the 1.0 mL of Sample Preparationtaken for analysis; 180 is the molecular weight of glucose;and 30 is the incubation time, in min.
TRANSGLUTAMINASE ACTIVITY(Glutaminyl-peptide -Glutaminyltransferase)
Application and Principle This procedure is used to deter-mine transglutaminase activity in preparations derived fromStreptoverticillium mobaraense var. The assay is based onthe enzymatic formation of a glutamic acid -hydroxamatein a glutaminyl residue in the substrate peptide with anothersubstrate, hydroxylamine. The amount of the glutamic acid-hydroxamate formed as a red complex with ferric ion inacidic conditions at 37° is measured spectrophotometrically.
Reagents and SolutionsSubstrate Solution Transfer 12.110 g of Tris[tri(hydroxy-
methyl)aminomethane], 3.475 g of hydroxylamine hydrochlo-ride, 1.624 g of glutathione, and 5.060 g of carbobenzyloxy-glutaminylglycine into a 500-mL beaker. Add 350 mL ofwater, and using a magnetic stirrer, mix well. Adjust the pHto 6.0 with appropriate concentrations (usually 1 N and 6 N)of hydrochloric acid. Quantitatively transfer this mixture intoa 500-mL volumetric flask, and dilute to volume with water.
Stopping Solution Prepare a 3 N hydrochloric acid solu-tion by diluting concentrated hydrochloric acid (ca. 36%)four-fold with water. Make a 12% trichloroacetic acid (TCA)solution by transferring 12.0 g of TCA into a 100-mL volumet-ric flask, adding water to dissolve the TCA, and diluting tovolume with water. Prepare a 5% solution of ferric chloride(FeCl3) in 0.1 N hydrochloric acid by transferring 5.0 g offerric chloride hexahydrate (FeCl3·6H2O) into a 100-mL volu-metric flask, adding 0.1 N hydrochloric acid to dissolve theferric chloride, and diluting to volume with 0.1 N hydrochloric
acid. On the day of use, combine all three solutions (3 Nhydrochloric acid, 12% TCA, and 5% ferric chloride) in equalvolumes in a beaker, and using a magnetic stirrer, mix well.
0.2 M Tris-HCl Buffer (pH 6.0) Transfer 18.11 g of Tris-[tri(hydroxymethyl)aminomethane] into a 500-mL beaker.Add 350 mL of water, and using a magnetic stirrer, mix well.Adjust the pH to 6.0 with appropriate concentrations (usually1 N and 6 N) of hydrochloric acid. Quantitatively transfer thismixture into a 500-mL volumetric flask, and dilute to volumewith water.
Sample Solution Place 100 mg of sample, accuratelyweighed, into a 100-mL beaker, and add about 45 mL of 0.2M Tris-HCl Buffer. Using a magnetic stirrer, mix well at roomtemperature for 30 min. Quantitatively transfer the mixtureinto a 50-mL volumetric flask, and dilute to volume with 0.2M Tris-HCl Buffer.
ProcedureCalibration Curve Transfer 64.8 mg of L-glutamic acid
-monohydroxamate standard, accurately weighed, into a suit-able flask, and add 10 mL of 0.2 M Tris-HCl Buffer. Dilutethis solution sequentially in five steps each by a geometricfactor of 2 with 0.2 M Tris-HCl Buffer. Transfer 200 �L ofeach dilution by pipet into individual test tubes, and incubateat 37° for 1 min. Add 2 mL of Substrate Solution, previouslyincubated at 37° for 10 min, to each tube, and mix vigorouslywith a vortex mixer. Further incubate the mixtures for exactly10 min, add 2 mL of Stopping Solution to each tube, and starta stopwatch. Mix vigorously with the vortex mixture, andseparate any insoluble material by centrifugation at 1500 ×g for 10 min at about 25°. Measure the absorbance of thesupernatant in each tube at 525 nm exactly 30 min after theaddition of the Stopping Solution. Plot the absorbance againstthe amount of L-glutamic acid -monohydroxamate, and ob-tain a standard calibration curve used to calculate the amountof glutamic acid -monohydroxamate in carbobenzyloxy-glutaminylglycine from the absorbance obtained in the analy-sis of the samples.
Analysis of Samples Transfer 200 �L of Sample Solutionby pipet into a test tube, and incubate at 37° for 1 min. Add2 mL of Substrate Solution, previously incubated at 37° for10 min, and mix vigorously using a vortex mixer. Furtherincubate the mixture for exactly 10 min, add 2 mL of StoppingSolution, and start a stopwatch. Mix vigorously using a vortexmixer, and separate any insoluble material by centrifugationat 1500 × g for 10 min at about 25°. Measure the absorbanceof the supernatant at 525 nm exactly 30 min after adding theStopping Solution.
For the blank, place 200 �L of Sample Solution into a testtube, and incubate at 37° for 1 min. Add 2 mL of StoppingSolution, and mix vigorously using a vortex mixer. Furtherincubate for exactly 10 min, and add 2 mL of SubstrateSolution, previously incubated at 37° for 10 min, and start astopwatch. Mix vigorously using a vortex mixer. Separate anyinsoluble material by centrifugation at 1500 × g for 10 min.Measure the absorbance of the supernatant at 525 nm exactly30 min after adding the Substrate Solution.
928 / Appendix V / General Tests and Assays FCC V
Calculation One unit of enzyme activity is defined as theamount of enzyme that catalyzes the transglutamination of 1�mol of substrate into product in 1 min under the conditionsof the assay. The specific activity of Transglutaminase isdefined as
Transglutaminase activity (U/g) = C × TS × 1/S × 1⁄10,
in which C is the concentration, in micromoles per milliliter,of hydroxamate in the Sample Solution (obtained from thestandard calibration curve); TS is the total volume, in millili-ters, of Sample Solution; S is the mass, in grams, of the sampletaken; and 10 is the reaction time in minutes.
Transglutaminase Transfer of acyl groups between the -carboxyamide group of peptide-bound glutamine residues andvarious amines, including the �-amino group of peptide-boundlysine, to form intra- and inter-molecular �-(-glutamyl)lysinecrosslinks.
Trivial Classifi- SystematicName cation Source Name (IUB) IUB No.
Transglutam- acyltransferase Streptoverticil- R-glutaminyl- 2.3.2.13inase or aminotrans- lium mobara- peptide: amine
ferase ense var. -glutamyltrans-ferase
TRYPSIN ACTIVITY
Application and Principle This procedure is used to deter-mine the trypsin activity of trypsin preparations derived frompurified extracts of porcine or bovine pancreas.
Reagents and SolutionsFifteenth Molar Phosphate Buffer (pH 7.6) Dissolve 4.54
g of monobasic potassium phosphate in sufficient water tomake 500 mL of solution. Dissolve 4.73 g of anhydrousdibasic sodium phosphate in sufficient water to make 500 mLof solution.
Mix 13 mL of the monobasic potassium phosphate solutionwith 87 mL of the anhydrous dibasic sodium phosphate so-lution.
Substrate Solution Dissolve 85.7 mg of N-benzoyl-l-argi-nine ethyl ester hydrochloride, suitable for use in assayingtrypsin, in sufficient water to make 100 mL.
Note: Determine the suitability of the substrate andcheck the adjustment of the spectrophotometer by per-forming the assay using USP Trypsin ReferenceStandard.
Dilute 10.0 mL of this solution to 100.0 mL with FifteenthMolar Phosphate Buffer. Determine the absorbance of thissolution at 253 nm in a 1-cm cell, with a suitable spectropho-tometer, using water as the blank and maintaining the celltemperature at 25° � 0.1°. Adjust the absorbance of the solu-tion, if necessary, by the addition of Fifteenth Molar Phos-phate Buffer so that it measures not less than 0.575 and notmore than 0.585. Use this solution within a period of 2 h.
Sample Preparations Dissolve a sufficient amount ofsample, accurately weighed, in 0.001 N hydrochloric acid toproduce a solution containing about 3000 USP trypsin unitsin each mL. Prepare three dilutions using 0.001 N hydrochloricacid so that the final solutions will contain 12, 18, and 24USP trypsin units in each 0.2 mL. Use these concentrationsin the Procedure below.
Procedure Conduct the test in a spectrophotometerequipped to maintain a temperature of 25° � 0.1° in the cellcompartment.
Determine the temperature in the reaction cell before andafter the measurement of absorbance to ensure that the temper-ature does not change by more than 0.5°.
Pipet 0.2 mL of 0.001 N hydrochloric acid and 3.0 mL ofSubstrate Solution into a 1-cm cell. Place this cell in thespectrophotometer, and adjust the instrument so that the ab-sorbance will read 0.050 at 253 nm. Pipet 0.2 mL of theSample Preparation containing 12 USP units into another 1-cm cell. Add 3.0 mL of Substrate Solution, and place the cellin the spectrophotometer. At the same time the SubstrateSolution is added, start a stopwatch, and read the absorbanceat 30-s intervals for 5 min. Repeat the procedure with theSample Preparations containing 18 and 24 USP units. Plotcurves of absorbance versus time for each concentration, anduse only those values that form a straight line to determinethe activity of the trypsin. Discard the values on the plateau,and take the average of the results from the three concentrationlevels as the actual activity of the trypsin.
Calculations One USP trypsin unit is the activity causinga change in the absorbance of 0.003/min under the conditionsspecified in this assay.
Calculate the number of USP trypsin units per mg at eachlevel by the equation
USP trypsin units = (A1 − A2)/(T × W × 0.003),
in which A1 is the absorbance straight-line final reading; A2
is the absorbance straight-line initial reading; T is the elapsedtime, in min, between the initial and final readings; and W isthe weight, in mg, of trypsin in the volume of solution usedin determining the absorbance.
FCC V General Tests and Assays / Appendix VI / 929
APPENDIX VI: ESSENTIAL OILS AND FLAVORS
ACETALS
Hydroxylamine Hydrochloride Solution Prepare as di-rected under Aldehydes, this Appendix.
Procedure Weigh accurately the quantity of the samplespecified in the monograph, and transfer it into a 125-mLErlenmeyer flask. Add 30 mL of Hydroxylamine Hydrochlo-ride Solution, and reflux on a steam bath for exactly 60 min.Allow the condenser to drain into the flask for 5 min afterremoving the flask from the steam bath. Detach, and rapidlycool the flask to room temperature. Add bromophenol blueTS as the indicator, and titrate with 0.5 N alcoholic potassiumhydroxide to pH 3.4, or to the same light color as producedin the original hydroxylamine hydrochloride solution on add-ing the indicator. Calculate the mL of 0.5 N alcoholic potas-sium hydroxide consumed per g of sample (A).
Using a separate portion of the sample, proceed as directedunder Aldehydes, this Appendix. Calculate the mL of 0.5 Nalcoholic potassium hydroxide consumed per g of sample (B).
Calculate the percentage of acetals by the formula
(A – B) × f,
in which f is the equivalence factor given in the monograph.
ACID VALUE
Dissolve about 10 g of the sample, accurately weighed, in 50mL of alcohol, previously neutralized to phenolphthalein with0.1 N sodium hydroxide. (Add 50 g of ice when testing cinna-myl formate, citronellyl formate, geranyl formate, isoamylformate, or linalyl formate.) Add 1 mL of phenolphthaleinTS, and titrate with 0.1 N sodium hydroxide until the solutionremains faintly pink after shaking for 10 s, unless otherwisedirected in the individual monograph. Calculate the acid value(AV) by the formula
AV = (5.61 × S)/W,
in which S is the number of mL of 0.1 N sodium hydroxideconsumed in the titration of the sample, and W is the weight,in g, of the sample.
ALDEHYDES
Hydroxylamine Hydrochloride Solution Dissolve 50 g ofhydroxylamine hydrochloride (preferably reagent grade orfreshly recrystallizedbefore using) in 90 mLof water, and dilute
to 1000 mL with aldehyde-free alcohol. Adjust the solution toa pH of 3.4 with 0.5 N alcoholic potassium hydroxide.
Procedure Weigh accurately the quantity of sample speci-fied in the monograph, and transfer it into a 125-mL Erlenmeyerflask. Add 30 mL of Hydroxylamine Hydrochloride Solution,mix thoroughly, and allow to stand at room temperature for 10min, unless otherwise specified in the monograph. Titrate with0.5 N alcoholic potassium hydroxide to a greenish yellow end-point that matches the color of 30 mL of Hydroxylamine Hydro-chloride Solution in a 125-mL flask when the same volume ofbromophenol blue TS has been added to each flask, or prefera-bly titrate to a pH of 3.4 using a suitable pH meter. Calculatethe percentage of aldehyde (A) by the equation
A = (S – b)(100e)/W,
in which S is the number of mL of 0.5 N alcoholic potassiumhydroxide consumed in the titration of the sample, b is the num-ber of mL of 0.5 N alcoholic potassium hydroxide consumedin the titration of the blank, e is the equivalence factor given inthe monograph, and W is the weight, in mg, of the sample.
ALDEHYDES AND KETONES
Hydroxylamine Method
Hydroxylamine Solution Dissolve 20 g of hydroxylaminehydrochloride (reagent grade or, preferably, freshly crystal-lized) in 40 mL of water, and dilute to 400 mL with alcohol.Add, with stirring, 300 mL of 0.5 N alcoholic potassiumhydroxide, and filter. Use this solution within 2 days.
Procedure Weigh accurately the quantity of the samplespecified in the individual monograph, and transfer it into a250-mL glass-stoppered flask. Add 75.0 mL of Hydroxyl-amine Solution to this flask and to a similar flask for a residualblank titration (see General Provisions). If the component tobe determined is an aldehyde, stopper the flasks and allowthem to stand at room temperature for 1 h unless otherwisestated in the monograph. If the component to be determinedis a ketone, attach the flask to a suitable condenser, and refluxthe mixture for 1 h unless otherwise stated in the monograph,and then cool to room temperature. Titrate both flasks to thesame greenish yellow endpoint using bromophenol blue TSas the indicator or, preferably, to a pH of 3.4 using a pHmeter. (If the indicator is used, the endpoint color must bethe same as that produced when the blank is titrated to a pHof 3.4.) Calculate the percentage of aldehyde or ketone bythe equation
AK = (b – S)(100e)/W,
930 / Appendix VI / General Tests and Assays FCC V
in which AK is the percentage of aldehyde or ketone, b is thenumber of mL of 0.5 N hydrochloric acid consumed in theresidual blank titration, S is the number of mL of 0.5 Nhydrochloric acid consumed in the titration of the sample, eis the equivalence factor given in the monograph, and W isthe weight, in mg, of the sample.
Hydroxylamine tert-Butyl Alcohol Method
Hydroxylamine Solution Dissolve 45 g of reagent-gradehydroxylamine hydrochloride in 130 mL of water, add 850mL of tert-butyl alcohol, mix, and using a pH meter, neutralizeto a pH of 3.0 to 3.5 with sodium hydroxide.
Caution: Do not heat the solution.
Procedure Weigh accurately the quantity of the samplespecified in the individual monograph, and transfer it into a250-mL glass-stoppered flask. Add 50 mL of the Hydroxyl-amine Solution, or the volume specified in the monograph,mix thoroughly, and allow to stand at room temperature forthe time specified in the monograph. Titrate with 0.5 N sodiumhydroxide to the same pH as the Hydroxylamine Solutionused. Calculate the percentage of aldehyde or ketone by theequation
AK = (S)(100e)/W,
in which AK is the percentage of aldehyde or ketone, S is thenumber of mL of 0.5 N sodium hydroxide consumed in thetitration of the sample, e is the equivalence factor given inthe monograph, and W is the weight, in mg, of the sample.
Neutral Sulfite Method
Pipet a 10-mL sample into a 100-mL cassia flask fitted witha stopper, and add 50 mL of a freshly prepared 30 in 100solution of sodium sulfite. Add 2 drops of phenolphthaleinTS, and neutralize with 50% (by volume) acetic acid solution.Heat the mixture in a boiling water bath, and shake the flaskrepeatedly, neutralizing the mixture from time to time by theaddition of a few drops of the 50% acetic acid solution,stoppering the flask to prevent loss of volatile material. Afterno coloration appears upon the addition of a few more dropsof phenolphthalein TS and heating for 15 min, cool to roomtemperature. When the liquids have separated completely, addsufficient sodium sulfite solution to raise the lower level ofthe oily layer within the graduated portion of the neck of theflask. Calculate the percentage, by volume, of the aldehydeor ketone by the equation
AK = 100 – (V × 10),
in which AK is the percentage, by volume, of the aldehydeor ketone in the sample, and V is the number of mL ofseparated oil in the graduated neck of the flask.
CHLORINATED COMPOUNDS
Wind a 1.5- × 5-cm strip of 20-mesh copper gauze aroundthe end of a copper wire. Heat the gauze in a nonluminousflame of a Bunsen burner until it glows without coloring the
flame green. Permit the gauze to cool, and re-ignite it severaltimes until a good coat of oxide has formed. With a medicinedropper, apply 2 drops of the sample to the cooled gauze,ignite, and permit it to burn freely in the air. Again cool thegauze, add 2 more drops, and burn as before. Continue thisprocess until a total of 6 drops have been added and ignited.Then hold the gauze in the outer edge of a Bunsen flameadjusted to a height of 4 cm. Not even a transient green coloris imparted to the flame. If at any of the additions the sampleappears to be instantly vaporized, the test must be repeatedfrom the beginning.
ESTERS
Ester Determination Weigh accurately the quantity of thesample specified in the monograph, and transfer it into a 125-mL Erlenmeyer flask containing a few boiling stones. Addto this flask and, simultaneously, to a similar flask for aresidual blank titration (see General Provisions) 25.0 mL of0.5 N alcoholic potassium hydroxide. Connect each flask toa reflux condenser, and reflux the mixtures on a steam bathfor exactly 1 h, unless otherwise directed in the monograph.Allow the mixtures to cool, add 10 drops of phenolphthaleinTS to each flask, and titrate the excess alkali in each flaskwith 0.5 N hydrochloric acid. Calculate the percentage ofesters (E) in the sample by the equation
E = (b – S)(100e)/W,
in which b is the number of mL of 0.5 N hydrochloric acidconsumed in the residual blank titration, S is the number ofmL of 0.5 N hydrochloric acid consumed in the titration ofthe sample, e is the equivalence factor given in the monograph,and W is the weight, in mg, of the sample.
Ester Determination (High-Boiling Solvent)0.5 N Potassium Hydroxide Solution Dissolve about 35
g of potassium hydroxide in 75 mL of water, add 1000 mLof a suitable grade of monoethyl ether of diethylene glycol,and mix.
Procedure Weigh accurately the quantity of the samplespecified in the monograph, and transfer it into a 200-mLErlenmeyer flask having a standard-taper joint. To this flaskand to a similar flask for a residual blank titration (see GeneralProvisions) add two glass beads and 25.0 mL of 0.5 N Potas-sium Hydroxide Solution, allowing exactly 1 min for drainagefrom the buret or pipet. Attach an air condenser to each flask,reflux gently for 1 h, and cool. Rinse down the condenserswith about 50 mL of water, then add phenolphthalein TS toeach flask, and titrate the excess alkali with 0.5 N sulfuricacid to the disappearance of the pink color. Calculate thepercentage of esters (E) in the sample by the equation
E = (b – S)(100e)/W,
in which b is the number of mL of 0.5 N sulfuric acid consumedin the blank determination, S is the number of mL of 0.5 N
FCC V General Tests and Assays / Appendix VI / 931
sulfuric acid required in the titration of the sample, e is theequivalence factor given in the monograph, and W is theweight, in mg, of the sample.
Saponification Value Proceed as directed for Ester Deter-mination or Ester Determination (High-Boiling Solvent), asspecified in the monograph. Calculate the saponification value(SV) by the equation
SV = (b – S)(28.05)/W,
in which b and S are as defined under Ester Determination,and W is the weight, in g, of the sample.
Ester Value If the sample contains no free acids, the saponi-fication value and the ester value are identical. If a determina-tion of the Acid Value (AV) is specified in the monograph,calculate the ester value (EV) by the equation
EV = SV – AV,
in which SV is the saponification value.
LINALOOL DETERMINATION
Transfer a 10-mL sample, previously dried with sodium sul-fate, into a 125-mL glass-stoppered Erlenmeyer flask pre-viously cooled in an ice bath. Add to the cooled oil 20 mLof dimethyl aniline (monomethyl-free), and mix thoroughly.To the mixture add 8 mL of acetyl chloride and 5 mL ofacetic anhydride, cool for several min, permit to stand at roomtemperature for another 30 min, then immerse the flask in awater bath maintained at 40° � 1° for 16 h. Wash the ace-tylated oil with three 75-mL portions of ice water, followedby successive washes with 25-mL portions of 5% sulfuricacid, until the separated acid layer no longer becomes cloudyor emits an odor of dimethyl aniline when made alkaline.After removal of the dimethyl aniline, wash the acetylatedoil first with 10 mL of sodium carbonate TS and then withsuccessive portions of water until the washings are neutral tolitmus. Finally, dry the acetylated oil with anhydrous sodiumsulfate, and proceed as directed for Ester Determination underEsters, this Appendix. Calculate the percentage of linalool(C10H18O) by the equation
L = [7.707(b – S)]/[W – 0.021(b – S)],
in which L is the percentage of linalool, b is the number ofmL of 0.5 N hydrochloric acid consumed in the residual blanktitration, S is the number of mL of 0.5 N hydrochloric acidconsumed in the titration of the sample, and W is the weight,in g, of the sample.
Note: When this method is applied to essential oilscontaining appreciable amounts of esters, perform anEster Determination, this appendix, on a sample of theoriginal oil and calculate the percentage of total linaloolby the equation
L = [7.707(b – S)(1 – 0.0021E)]/[W – 0.21(b – S)],
in which L is the percentage of linalool, E is the percent-age of esters, calculated as linalyl acetate (C12H20O2)in the sample of the original oil, and b, S, and W areas defined in the preceding paragraph.
Note: This entire procedure is applicable only to linalooland linalool-containing oils. It is not intended for thedetermination of other tertiary alcohols.
PERCENTAGE OF CINEOLE
Temper-ature 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
24 45.6 45.7 45.9 46.0 46.1 46.3 46.4 46.5 46.6 46.825 46.9 47.0 47.2 47.3 47.4 47.6 47.7 47.8 47.9 48.126 48.2 48.3 48.5 48.6 48.7 48.9 49.0 49.1 49.2 49.427 49.5 49.6 49.8 49.9 50.0 50.2 50.3 50.4 50.5 50.728 50.8 50.9 51.1 51.2 51.3 51.5 51.6 51.7 51.8 52.029 52.1 52.2 52.4 52.5 52.6 52.8 52.9 53.0 53.1 53.330 53.4 53.5 53.7 53.8 53.9 54.1 54.2 54.3 54.4 54.631 54.7 54.8 55.0 55.1 55.2 55.4 55.5 55.6 55.7 55.932 56.0 56.1 56.3 56.4 56.5 56.7 56.8 56.9 57.0 57.233 57.3 57.4 57.6 57.7 57.8 58.0 58.1 58.2 58.3 58.534 58.6 58.7 58.9 59.0 59.1 59.3 59.4 59.5 59.6 59.835 59.9 60.0 60.2 60.3 60.4 60.6 60.7 60.8 60.9 61.136 61.2 61.3 61.5 61.6 61.7 61.9 62.0 62.1 62.2 62.437 62.5 62.6 62.8 62.9 63.0 63.2 63.3 63.4 63.5 63.738 63.8 63.9 64.1 64.2 64.4 64.5 64.6 64.8 64.9 65.139 65.2 65.4 65.5 65.7 65.8 66.0 66.2 66.3 66.5 66.640 66.8 67.0 67.2 67.3 67.5 67.7 67.9 68.1 68.2 68.441 68.6 68.8 69.0 69.2 69.4 69.6 69.7 69.9 70.1 70.342 70.5 70.7 70.9 71.0 71.2 71.4 71.6 71.8 71.9 72.143 72.3 72.5 72.7 72.9 73.1 73.3 73.4 73.6 73.8 74.044 74.2 74.4 74.6 74.8 75.0 75.2 75.3 75.5 75.7 75.945 76.1 76.3 76.5 76.7 76.9 77.1 77.2 77.4 77.6 77.846 78.0 78.2 78.4 78.6 78.8 79.0 79.2 79.4 79.6 79.847 80.0 80.2 80.4 80.6 80.8 81.1 81.3 81.5 81.7 81.948 82.1 82.3 82.5 82.7 82.9 83.2 83.4 83.6 83.8 84.049 84.2 84.4 84.6 84.8 85.0 85.3 85.5 85.7 85.9 86.150 86.3 86.6 86.8 87.1 87.3 87.6 87.8 88.1 88.3 88.651 88.8 89.1 89.3 89.6 89.8 90.1 90.3 90.6 90.8 91.152 91.3 91.6 91.8 92.1 92.3 92.6 92.8 93.1 93.3 93.653 93.8 94.1 94.3 94.6 94.8 95.1 95.3 95.6 95.8 96.154 96.3 96.6 96.9 97.2 97.5 97.8 98.1 98.4 98.7 99.055 99.3 99.7 100.0
PHENOLS
Pipet 10 mL of the oil, which has been subjected to anytreatment specified in the monograph, into a 100-mL cassiaflask, add 75 mL of 1 N potassium hydroxide, and shakevigorously for 5 min to ensure complete extraction of thephenol by the alkali solution. Allow the mixture to stand forabout 30 min, then add sufficient 1 N potassium hydroxideto raise the oily layer into the graduated portion of the flask,stopper the flask, and allow it to stand overnight. Read thevolume of insoluble oil to 0.05 mL. Calculate the percentage,by volume, of phenols by the equation
P = (10 – V) × 10,
932 / Appendix VI / General Tests and Assays FCC V
in which P is the percentage, by volume, of phenols, and Vis the observed volume, in mL, of insoluble oil.
PHENOLS, FREE
Transfer about 5 g, accurately weighed, of the sample into a150-mL flask having a standard-taper neck. Pipet exactly 10mL of a 1:10 solution of acetic anhydride in anhydrous pyri-dine into the flask, and pipet exactly 10 mL of this solution,preferably measured with the same pipet, into a second 150-mL flask for the residual blank titration (see General Provi-sions). Connect the flasks to condensers, reflux for 1 h, andcool to a temperature below 100°. Add 25 mL of water toeach flask through the condensers, and reflux again for 10min. Cool the flasks, add phenolphthalein TS, and titrate with0.5 N potassium hydroxide. Calculate the percentage of freephenols by the equation
Percentage of Free Phenols = (b – S) × 100f/W,
in which b is the number of mL of 0.5 N potassium hydroxideconsumed in the residual blank titration, s is the number ofmL of 0.5 N potassium hydroxide consumed in the titrationof the sample, f is the equivalence factor given in the mono-graph, and W is the weight, in mg, of the sample.
RESIDUE ON EVAPORATION
Weigh accurately the quantity of sample specified in themonograph, and transfer it into a suitable evaporating dishthat has previously been heated on a steam bath, cooled toroom temperature in a desiccator, and accurately weighed.Weigh the sample in the dish. Heat the evaporating dishcontaining the sample on the steam bath for the period oftime specified in the monograph. Cool the dish and its contentsto room temperature in a desiccator, and weigh accurately.Calculate the residue as percentage of the sample used.
SOLUBILITY IN ALCOHOL
Transfer a 1.0-mL sample into a calibrated 10-mL glass-stoppered cylinder graduated in 0.1-mL subdivisions, and addslowly, in small portions, alcohol of the concentration speci-fied in the monograph. Maintain the temperature at 25°, andshake the cylinder thoroughly after each addition of alcohol.When a clear solution is first obtained, record the number ofmL of alcohol required. Continue the addition of the alcoholuntil a total of 10 mL has been added. If opalescence or
cloudiness occurs during these subsequent additions of alco-hol, record the number of mL of alcohol at which the phenome-non occurs.
TOTAL ALCOHOLS
Unless otherwise stated in the monograph, transfer 10 g of asolid sample, or 10 mL of a liquid sample, accurately weighed,into a 100-mL flask having a standard-taper neck. Add 10mL of acetic anhydride and 1 g of anhydrous sodium acetate,mix these materials, attach a reflux condenser to the flask,and reflux the mixture for 1 h. Cool, and through the con-denser, add 50 mL of water at a temperature between 50°and 60°. Shake intermittently for 15 min, cool to room temper-ature, transfer the mixture completely to a separator, allowthe layers to separate, and then remove and reject the lower,aqueous layer. Wash the oil layer successively with 50 mLof a saturated sodium chloride solution, 50 mL of a 10%sodium carbonate solution, and 50 mL of saturated sodiumchloride solution. If the oil is still acid to moistened litmuspaper, wash it with additional portions of sodium chloridesolution until it is free from acid. Drain off the oil, dry it withanhydrous sodium sulfate, and then filter it.
Transfer the quantity of acetylated oil specified in the mono-graph, and accurately weighed, into a tared 125-mL Erlen-meyer flask, and add 10 mL of neutral alcohol, 10 drops ofphenolphthalein TS, and 0.1 N alcoholic potassium hydroxide,dropwise, until a pink endpoint is obtained. If more than0.20 mL is needed, reject the sample, and wash and test theremaining acetylated oil until its acid content is below thislevel. Prepare a blank for residual titration (see General Provi-sions), using the same volume of alcohol and indicator, andadd 1 drop of 0.1 N alkali to produce a pink endpoint. Transfer25.0 mL of 0.5 N alcoholic potassium hydroxide into eachof the flasks, reflux them simultaneously for 1 h, cool, andtitrate the contents of each flask with 0.5 N hydrochloricacid to the disappearance of the pink color. Calculate thepercentage of Total Alcohols (A) by the equation
A = [b − S)(100e)]/[W − 21(b − S)],
in which b is he number of milliliters of 0.5 N hydrochloricacid consumed in the residual blank titration; S is the numberof milliliters of 0.5 N hydrochloric acid consumed in thetitration of the sample; e is the equivalence factor given inthe monograph; and W is the weight, in milligrams, of thesample of the acetylated oil.
ULTRAVIOLET ABSORBANCE OFCITRUS OILS
Transfer the quantity of the sample specified in the monographinto a 100-mL volumetric flask, add alcohol to volume, and
FCC V General Tests and Assays / Appendix VI / 933
FIGURE 33 Typical Spectrogram of Lemon Oil.
mix. Determine the ultraviolet absorption spectrum of thesolution in the range of 260 to 400 nm in a 1-cm cell with asuitable recording or manual spectrophotometer, using alcoholas the blank. If a manual instrument is used, read absorbancesat 5-nm intervals from 260 nm to a point about 12 nm fromthe expected maximum absorbance, then at 3-nm intervalsfor three readings, and at 1-nm intervals to a point about 5nm beyond the maximum, and then at 10-nm intervals to 400nm. From these data, plot the absorbances as ordinates againstwavelength on the abscissa, and draw the spectrogram. Drawa baseline tangent to the areas of minimum absorbance, asshown in Fig. 33 (which is typical of lemon oil), joining pointA in the region of 280 to 300 nm and a second point, B, inthe region of 355 to 380 nm. Locate the point of maximumabsorbance, C, and from it drop a vertical line, perpendicularto the abscissa, that intersects line AB at D. Read from theordinate the absorbances corresponding to points C and D,subtract the latter from the former, and correct the differencefor the actual weight of oil taken, calculating on the basis ofthe sample weight specified in the monograph.
VOLATILE OIL CONTENT
This procedure is used, when specified in the individual mono-graph, for determining the volatile oil content of gums, resins,and essential oils.
Apparatus The apparatus is shown in Fig. 34. It consistsof a 1000-mL boiling flask, A, attached through a trap, D, to
FIGURE 34 Apparatus for Determination of Volatile OilContent.
a Liebig condenser, C, which is connected to a 25-mL collectortube, B, graduated in 0.10-mL units.
Procedure Place 750 mL of water in the boiling flask, boilfor 10 min, and cool to 50°. Transfer the specified volumeof the sample, prepared as directed in the monograph, intothe flask, then immediately attach the remainder of the appara-tus to the flask, and boil until the volume of distilled oilcollected in the graduated collector tube remains constant.Avoid splashing the contents of the flask in order to preventcontamination of the distillate with nonvolatile material, anddo not continue distillation for an extended time after thevolume of distillate becomes constant. If the distilled oil isheavier than water, set the stopcock in the closed position toprevent return of the heavy distillate to the flask.
When distillation is complete, allow the contents of thecollection tube to settle until the oil and water layers areseparated completely. Allow the distillate to cool to roomtemperature, read its volume, and calculate therefrom thepercentage of volatile oil.
Note: When the volatile oil thus collected is to beused in additional tests, as may be specified in themonograph, the oil should be drained off, dried, andfiltered before use.
934 / Appendix VII / General Tests and Assays FCC V
APPENDIX VII: FATS AND RELATED SUBSTANCES
ACETYL VALUE(Based on AOCS Method Cd 4-40)
The acetyl value is defined as the number of mg of potassiumhydroxide required to neutralize the acetic acid obtained bysaponifying 1 g of the acetylated sample.
Acetylation Boil 50 mL of the oil or melted fat with 50mL of freshly distilled acetic anhydride for 2 h under a refluxcondenser. Pour the mixture into a beaker containing 500 mLof water, and boil for 15 min, bubbling a stream of nitrogenor carbon dioxide through the mixture to prevent bumping.Cool slightly, remove the water, add another 500 mL of water,and boil again. Repeat for a third time with another 500-mLportion of water, and remove the wash water, which shouldbe neutral to litmus. Transfer the acetylated fat to a separator,and wash with two 200-mL portions of warm water, separatingas much as possible of the wash water each time. Transferthe washed sample to a beaker, add 5 g of anhydrous sodiumsulfate, and let stand for 1 h, agitating occasionally to assistdrying. Filter the oil through a dry filter paper, preferably inan oven at 100° to 110°, and keep the filtered oil in the ovenuntil it is completely dry. The acetylated product should bea clear, brilliant oil.
Saponification Weigh accurately from 2 to 2.5 g each ofthe acetylated oil and of the original, untreated sample intoseparate 250-mL Erlenmeyer flasks. Add to each flask 25.0mL of 0.5 N alcoholic potassium hydroxide, and continue asdirected in the Procedure under Saponification Value, in thisAppendix, beginning with ‘‘Connect an air condenser. . . .’’Record the saponification value of the untreated sample as S,and that of the acetylized oil as S′, then calculate the acetylvalue of the sample by the formula
(S′ – S)/(1.000 – 0.00075S).
ACID VALUE(Based on AOCS Methods Te 1a-64 and Cd 3d-63)
The acid value is defined as the number of mg of potassiumhydroxide required to neutralize the fatty acids in 1 g of thetest substance.
Method I (Commercial Fatty Acids)
Unless otherwise directed, weigh accurately about 5 g of thesample into a 500-mL Erlenmeyer flask, and dissolve it in75 to 100 mL of hot alcohol, previously boiled and neutralizedto phenolphthalein TS with sodium hydroxide. Agitation andfurther heating may be necessary to effect complete solution
of the sample. Add 0.5 mL of phenolphthalein TS, and titrateimmediately, while shaking, with 0.5 N sodium hydroxide tothe first pink color that persists for at least 30 s. Calculatethe acid value by the formula
56.1V × N/W,
in which V is the volume, in mL, and N is the normality,respectively, of the sodium hydroxide solution; and W is theweight, in g, of the sample taken.
Method II (Animal Fats and Vegetable and Marine Oils)
Prepare a solvent mixture consisting of equal parts, by volume,of isopropyl alcohol and toluene. Add 2 mL of a 1% solutionof phenolphthalein in isopropyl alcohol to 125 mL of themixture, and neutralize with alkali to a faint but permanentpink color. Weigh accurately the appropriate amount of well-mixed liquid sample indicated in the table below, dissolve itin the neutralized solvent mixture, warming if necessary, andshake vigorously while titrating with 0.1 N potassium hydrox-ide to the first permanent pink color of the same intensity asthat of the neutralized solvent before mixing with the sample.Calculate the acid value by the formula
56.1V × N/W,
in which V is the volume, in mL, and N is the normality,respectively, of the potassium hydroxide solution; and W isthe weight, in g, of the sample taken.
Acid Value Sample Weight (g)
0–1 201–4 104–15 2.515–75 0.575 and over 0.1
CHLOROPHYLL(Based on AOCS Method Cc 13d-55)
Use a reliable spectrophotometer with a sample holder equili-brated at 44° � 3° to obtain absorbance values at 630, 670,and 710 nm. Calculate the concentration of chlorophyll (C)using the following equation:
C = [A670 – (A630/2) – (A710/2)]/(K × b),
in which C is the concentration of chlorophyll, in mg/kg; Ais the absorbance at the wavelength indicated by the subscript;K is the constant for the specific spectrophotometer beingused and is equal to 0.1016 for the Beckman Model DU; andb is the optical pathlength through the sample, in cm.
FCC V General Tests and Assays / Appendix VII / 935
COLD TEST(Based on AOCS Method Cc 11-53)
Filter a sample (200 to 300 mL), and transfer to a clean, drybottle. Fill the bottle completely, and insert a cork stopper.Seal with paraffin, and equilibrate at 25° in a water bath sothat it is completely covered. Next, immerse the bottle in anice and water bath so it is completely covered. Monitor thebath during the test and replenish the ice frequently to keepthe bath at 0°.
After 5.5 h remove the bottle from the bath. The samplemust be clear; fat crystals or cloudiness must be totally absent.
COLOR (AOCS-Wesson)(Based on AOCS Method Cc 13b-45)
Apparatus Use a Lovibond tintometer or the equivalent anda set of color comparison glasses that conform to the AOCS-Wesson Tintometer Color Scale (available from the NationalInstitute of Standards and Technology). A minimum set ofglasses consists of
Red 0.1 0.2 0.3 0.4 0.5 0.6 0.8 0.91.0 2.0 2.5 3.0 3.5 4.0 5.0 6.07.0 7.6 8.0 9.0 10.0 11.0 12.0 16.0
20.0Yellow 1.0 2.0 3.0 5.0 10.0 15.0 20.0 35.0
50.0 70.0
For making color comparisons, use color tubes of clear, color-less glass with a smooth, flat, polished bottom (length 154mm; id 19 mm; od 22 mm), and marked to indicate liquidcolumns of 25.4 and 133.35 mm.
Procedure Add 0.1 g of diatomaceous earth to a 60-g sam-ple, agitate for 2.5 min at room temperature (or 10° to 15°above the melting point if the sample is not liquid), and filter.Adjust the temperature to 25° to 35° (or not more than 100above the melting point), and fill the color tube to the desiredmark. Place the tube in the tintometer (in a dark booth orcabinet), and match the sample color as closely as possiblewith a standard glass.
FATTY ACID COMPOSITION(Based on AOCS Methods Ce 1-62, Ce 1b-89, Ce1e-91)
Apparatus Use a suitable gas chromatograph (see AppendixIIA) equipped with a flame ionization detector (FID) and
containing either a 3.05-m × 2- or 4-mm id glass columnpacked with preconditioned 10%, by weight, DEGS-PS on100- to 120-mesh diatomaceous earth (Chromosorb WHP, orequivalent) or a 30-m × 0.20- to 0.35-mm id capillary fusedsilica column, or equivalent, containing a suitable station-ary phase.
Operating Conditions The operating conditions may varywith the instrument used, but a suitable chromatogram maybe obtained using a temperature program 180° to 215°; inlettemperature (injector), 300°; detector, 300°; and a suitablecarrier gas flow.
Standard Solutions Run through the chromatograph a com-mercially available standard containing a mixture of fatty-acid methyl esters. Fatty acids and methyl esters with a widerange of carbon numbers and double-bond configurations canbe purchased. The calculated concentration should compareto that claimed within �2 , where is the standard deviationcalculated from at least 10 replicate determinations, preferablymade over a period of several days.
Determine that the system is functioning properly: injectinto the chromatograph a suitable number of samples of thestandard to ensure that the resolution factor, R, defining theefficiency of the separation between methyl stearate andmethyl oleate is 0.9 or greater. Calculate R by the equation
R = 2(t2 – t1)/(w2 + w1),
in which t2 and t1 are the retention times of peak 2 and peak1, respectively, and w2 and w1 are the corresponding widthsof the bases of the peaks obtained by extrapolating relativelystraight sides of the peaks to the baseline. Baseline separationof the various components in both the standard and the samplepreparations is desirable.
Sample Preparation (for fats and oils) (Based on AOCSMethod Ce 2-66) Introduce 100 to 1000 mg of the fat intoa 50- or 125-mL reaction flask. Add 4 to 10 mL of 0.5 Nmethanolic sodium hydroxide, and add a boiling chip. Attacha condenser, and heat the mixture on a steam bath until thefat globules go into solution. This step should take 5 to 10min. Add 5 to 12 mL of 12.5% boron fluoride–methanolreagent (this reagent contains 125 g of boron fluoride perL of methanol and is available commercially) through thecondenser, and boil for 2 min. Add 2 to 5 mL of heptanethrough the condenser, and boil for 1 min longer. Removefrom heat, remove condenser, and add about 15 mL of satu-rated sodium chloride solution. Stopper the flask, and shakevigorously for 15 s. Transfer about 1 mL of the heptanesolution into a test tube and add a small amount of anhydroussodium sulfate. The dry heptane solution may then be injecteddirectly into a gas chromatograph.
The methyl esters should be analyzed as soon as possible.They may be kept in an atmosphere of nitrogen in a screw-cap vial at 2° for 24 h. For longer storage, they should besealed in a glass ampule, subjected first to a vacuum and thenbackfilled with nitrogen and stored at −20° (freezer).
936 / Appendix VII / General Tests and Assays FCC V
Procedure Inject an appropriate volume (0.1 �L to 1.0 �L)of sample into the chromatograph. If an automated system isused, follow the manufacturer’s instructions; if calculationsare to be done manually, proceed as follows:
Calculate the area percent of each component (CN) by theequation
CN = [AN/TS] × 100,
in which AN is the area of the peak corresponding to componentCN, and TS is the total area for all detected components[TS = �AN].
FREE FATTY ACIDS(Based on AOCS Method Ca 5a-40)
Unless otherwise directed, accurately weigh the appropriateamount of the sample, indicated in the table below, into a250-mL Erlenmeyer flask or other suitable container. Add 2mL of phenolphthalein TS to the specified amount of hotalcohol, neutralize with alkali to the first faint, but permanent,pink color, and then add the hot, neutralized alcohol to thesample container. Titrate with the appropriate normality ofsodium hydroxide, shaking vigorously, to the first permanentpink color of the same intensity as that of the neutralizedalcohol. The color must persist for at least 30 s. Calculate thepercentage of free fatty acids (FFA) in the sample by theformula
VNe/W,
in which V is the volume and N is the normality of the sodiumhydroxide used; W is the weight of the sample, in g; and eis the equivalence factor given in the monograph.
FFA Range Grams of Milliliters of Strength(%) Sample Alcohol of NaOH
0.00–0.2 56.4 � 0.2 50 0.1 N0.2–1.0 28.2 � 0.2 50 0.1 N1.0–30.0 7.05 � 0.05 75 0.25 N30.0–50.0 7.05 � 0.05 100 0.25–1.0 N50.0–100 3.525 � 0.001 100 1.0 N
FREE GLYCERIN OR PROPYLENEGLYCOL(Based on AOCS Method Ca 14-56)
Reagents and Solutions Use the Periodic Acid Solution,Potassium Iodide Solution, and Chloroform as described under1-Monoglycerides, in this Appendix.
Procedure To the combined aqueous extracts obtained asdirected under 1-Monoglycerides, add 50.0 mL of PeriodicAcid Solution. Run two blanks by adding 50.0 mL of this
reagent solution to two 500-mL glass-stoppered Erlenmeyerflasks, each containing 75 mL of water. Continue as directedin the Procedure under 1-Monoglycerides, beginning with‘‘. . . and allow to stand for at least 30 min but no longer than90 min.’’
Calculation Calculate the percentage of free glycerin in theoriginal sample by the formula
(b – S) × N × 2.30/W,
or calculate the percentage of free propylene glycol by theformula
(b – S) × N × 3.81/W,
in which b is the number of mL of sodium thiosulfate con-sumed in the blank determination; S is the number of mLrequired in the titration of the aqueous extracts from thesample; N is the exact normality of the sodium thiosulfate;W is the weight, in g, of the original sample taken; 2.30 isthe molecular weight of glycerin divided by 40; and 3.81 isthe molecular weight of propylene glycol divided by 20.
Note: If the aqueous extract contains more than 20 mgof glycerin or more than 30 mg of propylene glycol,dilute the extract in a volumetric flask and transfer asuitable aliquot into a 500-mL glass-stoppered Erlen-meyer flask before proceeding with the test. The weightof the sample should be corrected in the calculation.
HEXANE-INSOLUBLE MATTER
If the sample is plastic or semisolid, soften a portion bywarming it at a temperature not exceeding 60°, and then mixit thoroughly. Transfer 100 g of well-mixed sample into a1500-mL wide-mouth Erlenmeyer flask, add 1000 mL of sol-vent hexane, and shake until the sample is dissolved. Filter theresulting solution through a 600-mL Corning ‘‘C’’ porosity, orequivalent, filtering funnel that previously has been dried at105° for 1 h, cooled in a desiccator, and weighed. Wash theflask with two successive 250-mL portions of solvent hexane,and pass the washings through the filter. Dry the funnel at105° for 1 h, cool to room temperature in a desiccator, andweigh. From the gain in weight of the funnel, calculate thepercentage of the hexane-insoluble matter in the sample.
HYDROXYL VALUE(Based on AOCS Methods Cd 4-40 and Cd 13-60)
The hydroxyl value is defined as the number of mg of potas-sium hydroxide equivalent to the hydroxyl content of 1 g ofthe unacetylated sample.
FCC V General Tests and Assays / Appendix VII / 937
Method I
Proceed as directed under Acetyl Value, in this Appendix, butcalculate the hydroxyl value by the formula
(S′ – S)/(1.000 – 0.00075S′).
Method II
Unless otherwise directed, accurately weigh the appropriateamount of the sample indicated in the table below, transferit into a 250-mL glass-stoppered Erlenmeyer flask, and add5.0 mL of pyridine–acetic anhydride reagent (mix 3 volumesof freshly distilled pyridine with 1 volume of freshly distilledacetic anhydride).
Hydroxyl Value Sample Weight (g)
0–20 1020–50 550–100 3
100–150 2150–200 1.50200–250 1.25250–300 1300–350 0.75
Pipet 5 mL of the pyridine–acetic anhydride reagent into asecond 250-mL flask for the reagent blank. Heat the flasksfor 1 h on a steam bath under reflux condensers, then add 10mL of water through each condenser, heat for 10 min longer,and allow the flasks to cool to room temperature. Add 15 mLof n-butyl alcohol, previously neutralized to phenolphthaleinTS with 0.5 N alcoholic potassium hydroxide, through thecondenser, then remove the condensers, and wash the sidesof the flasks with 10 mL of n-butyl alcohol. To each flaskadd 1 mL of phenolphthalein TS, and titrate to a faint pinkendpoint with 0.5 N alcoholic potassium hydroxide, recordingthe mL required for the sample as S and that for the blankas B. To correct for free acid, mix about 10 g of the sample,accurately weighed, with 10 mL of freshly distilled pyridine,previously neutralized to phenolphthalein, add 1 mL of phe-nolphthalein TS, and titrate to a faint endpoint with 0.5 Nalcoholic potassium hydroxide, recording the mL required asA. Calculate the hydroxyl value by the formula
[B + (WA/C) – S] × 56.1N/W,
in which W and C are the weights, in g, of the samples takenfor acetylation and for the free acid determination, respec-tively; and N is the exact normality of the alcoholic potassiumhydroxide.
IODINE VALUE(Based on AOCS Method Cd 1d-92)
The iodine value is a measure of unsaturation and is expressedas the number of g of iodine absorbed, under the prescribedconditions, by 100 g of the test substance.
Modified Wijs Method (Acetic Acid/Cyclohexane Method)
Wijs Solution Dissolve 13 g of resublimed iodine in 1000mL of glacial acetic acid. Pipet 10.0 mL of this solution intoa 250-mL flask, add 20 mL of potassium iodide TS and 100mL of water, and titrate with 0.1 N sodium thiosulfate, addingstarch TS near the endpoint. Record the volume required asA. Set aside about 100 mL of the iodine–acetic acid solutionfor future use. Pass chlorine gas, washed and dried with sulfu-ric acid, through the remainder of the solution until a 10.0-mL portion requires not quite twice the volume of 0.1 Nsodium thiosulfate consumed in the titration of the originaliodine solution. A characteristic color change occurs whenthe desired amount of chlorine has been added. Alternatively,Wijs Solution may be prepared by dissolving 16.5 g of iodinemonochloride, ICl, in 1000 mL of glacial acetic acid. Storethe solution in amber bottles sealed with paraffin until readyfor use, and use within 30 days.
Total Halogen Content Pipet 10.0 mL of Wijs Solutioninto a 500-mL Erlenmeyer flask containing 150 mL of recentlyboiled and cooled water and 15 mL of potassium iodide TS.Titrate immediately with 0.1 N sodium thiosulfate, recordingthe volume required as B.
Halogen Ratio Calculate the I/Cl ratio by the formula
A/(B – A).
The halogen ratio must be between 1.0 and 1.2. If the ratiois not within this range, the halogen content can be adjustedby adding the original solution or by passing more chlorinethrough the solution.
Note: Wijs Solution is commercially available.
Procedure The appropriate weight of the sample, in g, iscalculated by dividing the number 25 by the expected iodinevalue. Melt the sample, if necessary, and filter it through adry filter paper. Transfer the accurately weighed quantity ofsample into a clean, dry, 500-mL glass-stoppered bottle orflask containing 20 mL of glacial acetic acid/cyclohexane,1:1, v/v, and pipet 25.0 mL of Wijs Solution into the flask.The excess of iodine should be between 50% and 60% of thequantity added, that is, between 100% and 150% of the quan-tity absorbed. Swirl, and let stand in the dark for 1.0 h wherethe iodine value is <150 and for 2.0 h where the iodine valueis ≥150. Add 20 mL of potassium iodide TS and 100 mL ofrecently boiled and cooled water, and titrate the excess iodinewith 0.1 N sodium thiosulfate, adding the titrant graduallyand shaking constantly until the yellow color of the solutionalmost disappears. Add starch TS, and continue the titrationuntil the blue color disappears entirely. Toward the end ofthe titration, stopper the container and shake it violently sothat any iodine remaining in solution in the glacial acetic acid/cyclohexane, 1:1, solution may be taken up by the potassiumiodide solution. Concomitantly, conduct two determinationson blanks in the same manner and at the same temperature.Calculate the iodine value by the formula
(B – S) × 12.69N/W,
in which B – S represents the difference between the volumesof sodium thiosulfate required for the blank and for the sample,
938 / Appendix VII / General Tests and Assays FCC V
respectively; N is the normality of the sodium thiosulfate; andW is the weight, in g, of the sample taken.
MELTING RANGE
Fats of animal and vegetable origin do not exhibit a sharpmelting point. For the purpose of this test, melting range isdefined as the range of temperature in which the samplebecomes a perfectly clear liquid after first passing througha stage of gradual softening, during which it may becomeopalescent.
Apparatus Use any suitable commercial or other apparatus.Use melting-point capillary tubes—id, 1 mm; od, 2 mm;length, 50 to 80 mm; and open at both ends.
ProcedureCapillary Method (Based on AOCS Method Cc 1-25)
Melt the sample and filter it through filter paper; the samplemust be absolutely dry. Dip three capillary tubes in the liquidsample so that the oil stands approximately 10 mm high inthe tubes, and fuse the end of the tube containing the samplewithout burning it. Place the tubes containing the liquid samplein a beaker, and equilibrate them at least 16 h at 4° to 10° in arefrigerator. Determine the melting range, using a temperatureincrease of 0.5° per min when within 10° of the anticipatedmelting point. The melting ranges of the three samples shouldbe no more than 0.5° apart.
1-MONOGLYCERIDES(Based on AOCS Method Cd 11-57)
Reagents and SolutionsPeriodic Acid Solution Dissolve 5.4 g of periodic acid,
H5IO6, in 100 mL of water, add 1900 mL of glacial aceticacid, and mix. Store in a light-resistant, glass-stoppered bottleor in a clear, glass-stoppered bottle protected from light.
Chloroform Use chloroform meeting the following test:To each of three 500-mL flasks add 50.0 mL of Periodic AcidSolution, then add 50 mL of chloroform and 10 mL of waterto two of the flasks and 50 mL of water to the third. To eachflask add 20 mL of potassium iodide TS, mix gently, andcontinue as directed in the Procedure, beginning with‘‘. . . allow to stand at least 1 min. . . . ’’ The difference be-tween the volume of 0.1 N sodium thiosulfate required in thetitrations with and without the chloroform is not greater than0.5 mL.
Procedure Melt the sample, if not liquid, at a temperaturenot higher than 10° above its melting point, and mix thor-oughly. Transfer an accurately weighed portion of the sample,
equivalent to about 150 mg of 1-monoglycerides, into a 100-mL beaker (or weigh a sample equivalent to 20 mg of glycerinor 30 mg of propylene glycol if only Free Glycerin or Propyl-ene Glycol is to be determined), and dissolve in 25 mL ofchloroform. Transfer the solution, with the aid of an additional25 mL of chloroform, into a separator, wash the beaker with25 mL of water, and add the washing to the separator. Stopperthe separator tightly, shake vigorously for 30 to 60 s, andallow the layers to separate. (Add 1 to 2 mL of glacial aceticacid to break emulsions formed due to the presence of soap.)Collect the aqueous layer in a 500-mL glass-stoppered Erlen-meyer flask, and extract the chloroform solution again usingtwo 25-mL portions of water. Retain the combined aqueousextracts for the determination of Free Glycerin or PropyleneGlycol (in this Appendix). Transfer the chloroform to a 500-mL glass-stoppered Erlenmeyer flask, and add 50.0 mL ofPeriodic Acid Solution to this flask and to each of two blankflasks containing 50 mL of chloroform and 10 mL of water.Swirl the flasks during the addition of the reagent, and allowto stand for at least 30 min, but no longer than 90 min. Toeach flask, add 20 mL of potassium iodide TS, and allow tostand at least 1 min, but no longer than 5 min, before titrating.Add 100 mL of water, and titrate with 0.1 N sodium thiosul-fate, using a magnetic stirrer to keep the solution thoroughlymixed, to the disappearance of the brown iodine color, thenadd 2 mL of starch TS and continue the titration to the disap-pearance of the blue color. Calculate the percentage of 1-monoglycerides1 in the sample by the formula
(B – S) × N × 17.927/W,
in which B is the number of mL of sodium thiosulfate con-sumed in the blank determination; S is the number of mLrequired in the titration of the sample; N is the exact normalityof the sodium thiosulfate; W is the weight, in g, of the sampletaken; and 17.927 is the molecular weight of glyceryl monoste-arate divided by 20.
TOTAL MONOGLYCERIDES
Preparation of Silica Gel Place about 10 g of 100- to 200-mesh silica gel of a grade suitable for chromatographic workin a tared weighing bottle, cap immediately, and weigh accu-rately. Remove the cap, dry at 200° for 2 h, cap immediately,and cool for 30 min. Raise the cap momentarily to equalizethe pressure, then weigh again, reheat for 5 min at 200°,cool, and reweigh. Repeat this 5-min drying cycle until twoconsecutive weights agree within 10 mg. Calculate the per-centage of water in the original silica gel (A) by the formula
(loss in wt/sample wt) × 100,
1The monoglyceride may be calculated to some monoester other thanglyceryl monostearate by dividing the molecular weight of the monoglyc-eride by 20 and substituting the value so obtained for 17.927 in theformula, using 17.80, for example, in calculating to the monooleate.
FCC V General Tests and Assays / Appendix VII / 939
then calculate the amount of water required to adjust the watercontent to 5% by the formula
W × (5 – A)/95,
in which W is the weight, in g, of the undried sample to be used.Accurately weigh the appropriate amount of the undried
silica gel to be used in the determination, transfer to a suitableblender or mixer, and add the calculated amount of water togive a final water content of 5% � 0.1%. Blend for 1 hto ensure complete water distribution, and store in a sealedcontainer. Determine the water content of the adjusted silicagel as directed above, and readjust if necessary.
Note: Each new lot of silica gel should be checked forsuitability by the analysis of a monoglyceride of knowncomposition.
Sample Preparation (Caution: To avoid rearrangement ofpartial glycerides, use extreme caution in applying heat tosamples, and do not heat above 50°.)
Samples Melting Below 50° Melt the sample, if necessary,by warming for short periods below 50°, not exceeding a totalof 30 min.
Samples Melting Above 50° Grind about 10 g in a mortarand pestle, chilling solid samples, if necessary, in carbondioxide.
Weigh accurately about 1 g of the prepared sample into a100-mL beaker, add 15 mL of chloroform, and warm, ifnecessary, to effect solution. Use only minimal heat, and donot heat above 40°.
Preparation of Chromatographic Column Connect a 19-× 290-mm chromatographic tube, equipped with an outer 19/22 standard-taper joint at the top and a coarse, fritted-glassdisk and inner 19/22 standard-taper joint at the bottom, withan adapter consisting of an outer 19/22 joint connected to aTeflon stopcock. Do not grease the joints. Weigh 30 g of theprepared silica gel into a 150-mL beaker, add 50 to 60 mLof petroleum ether, and stir slowly with a glass rod until allair bubbles are expelled. Transfer the slurry to the columnthrough a powder funnel, and open the stopcock, allowingthe liquid level to drop to about 2 cm above the silica gel.Transfer any silica gel slurry remaining in the beaker into thecolumn with a minimum amount of petroleum ether, thenrinse the funnel and sides of the column. Drain the solventthrough the stopcock until the level drops to 2 cm above thesilica gel, and remove the powder funnel.
Procedure Carefully add the Sample Preparation to theprepared column. Open the stopcock, and adjust the flow rateto about 2 mL/min, discarding the eluate. Rinse the samplebeaker with 5 mL of chloroform, and add the rinsing to thecolumn when the level drops to 2 cm above the silica gel.Never allow the column to become dry on top, and maintaina flow rate of 2 mL/min throughout the elution. Avoid inter-ruptions during elution as they may cause pressure buildupand result in leakage through the stopcock or cracks in thesilica gel packing.
Attach a 250-mL reservoir separator, provided with a Teflonstopcock and a 19/22 standard-taper drip tip inner joint, to
the column. Add 200 mL of benzene, elute, and discard theeluate, which contains the triglycerides fraction. When thelevel of benzene drops to 2 cm above the silica gel, add 200mL of a 1:10 mixture of ether in benzene, elute, and discardthe eluate, which contains the diglycerides and the free fattyacid fraction. When all of the ether–benzene solvent has beenadded from the separator and the level in the column dropsto 2 cm above the silica gel, add from 250 to 300 mL ofether, and collect the monoglyceride fraction in a tared flask.Rinse the tip of the column into the flask with a few mL ofether, and evaporate to dryness on a steam bath under a streamof nitrogen or dry air. Cool for at least 15 min, weigh, thenreheat on the steam bath for 5 min in the same manner.Cool, reweigh, and repeat the 5-min evaporation, cooling, andreweighing procedures until two consecutive weights agreewithin 2 mg. The weight of the residue represents the totalmonoglycerides in the sample taken.
OXYETHYLENE DETERMINATION
Apparatus The apparatus for oxyethylene group determina-tion is shown in Fig. 35. It consists of a boiling flask, A, fittedwith a capillary side tube to provide an inlet for carbon dioxideand connected by a condenser with trap B, which contains anaqueous suspension of red phosphorus. The first absorptiontube, C, contains a silver nitrate solution to absorb ethyl iodide.Absorption tube D is fitted with a 1.75-mm spiral rod (23turns, 8.5-mm rise per turn), which is required to provide alonger contact of the evolved ethylene with the bromine solu-tion. A standard-taper adapter and stopcock are connected totube D to permit the transfer of the bromine solution into atitration flask without loss. A final trap, E, containing a potas-sium iodide solution, collects any bromine swept out by theflow of carbon dioxide.
Dimensions of the apparatus not readily determined fromFig. 35 are as follows: carbon dioxide inlet capillary, 1-mmid; flask A, 28-mm diameter, 12/18 standard-taper joint; con-denser, 9-mm id; inlet to trap B, 2-mm id; inlet to trap C, 7/15 standard-taper joint, 2-mm id; trap C, 14-mm id; trap D,inner tube, 8-mm od, 2-mm opening at bottom of spiral; outertube, approximately 12.5-mm id; side arm 7 cm from top ofinserted spiral, 3.5-mm id, 2-mm opening at bottom.
ReagentsHydriodic Acid Use special-grade hydriodic acid suitable
for alkoxyl determinations, or purify reagent-grade as follows:Distill over red phosphorus in an all-glass apparatus, passinga slow stream of carbon dioxide through the apparatus untilthe distillation is terminated and the receiving flask has com-pletely cooled.
Caution: Use a safety shield, and conduct the distilla-tion in a hood.
Silver Nitrate Solution Dissolve 15 g of silver nitrate in50 mL of water, mix with 400 mL of alcohol, and add a fewdrops of nitric acid.
940 / Appendix VII / General Tests and Assays FCC V
FIGURE 35 Apparatus for Oxyethylene Determination.
Bromine–Bromide Solution Add 1 mL of bromine to 300mL of glacial acetic acid saturated with dry potassium iodide(about 5 g). Fifteen mL of this solution requires about 40 mLof 0.05 N sodium thiosulfate. Store in a brown bottle in adark place, and standardize at least once a day during use.
Procedure Fill trap B with enough of a suspension of 60mg of red phosphorus in 100 mL of water to cover the inlettube. Pipet 10 mL of the Silver Nitrate Solution into tube Cand 15 mL of the Bromine–Bromide Solution into tube D,and place 10 mL of a 1:10 solution of potassium iodide intrap E. Transfer an accurately weighed quantity of the samplespecified in the monograph into the reaction flask, A, and add10 mL of Hydriodic Acid along with a few glass beads orboiling stones. Connect the flask to the condenser, and beginpassing carbon dioxide through the apparatus at the rate ofabout one bubble per s. Heat the flask in an oil bath at 140°to 145°, and continue the reaction at this temperature for atleast 40 min. Heating should be continued until the cloudyreflux in the condenser becomes clear and until the supernatantliquid in the silver nitrate tube, C, is almost completely clari-fied. Five min before the reaction is terminated, heat the SilverNitrate Solution in tube C in a hot water bath at 50° to60° to expel any dissolved olefin. At the completion of thedecomposition, cautiously disconnect tubes D and C in theorder named, then disconnect the carbon dioxide source andremove the oil bath. Connect tube D to a 500-mL iodine flaskcontaining 150 mL of water and 10 mL of a 1:10 solution ofpotassium iodide, run the Bromine–Bromide Solution into
the flask, and rinse the tube and spiral with water. Add thepotassium iodide solution from trap E to the flask, rinsingthe side arm and tube with a few mL of water, stopper theflask, and allow to stand for 5 min. Add 5 mL of 2 N sulfuricacid, and titrate immediately with 0.05 N sodium thiosulfate,using 2 mL of starch TS for the endpoint. Transfer the silvernitrate solution from tube C into a flask, rinsing the tube withwater, dilute to 150 mL with water, and heat to boiling. Cool,and titrate with 0.05 N ammonium thiocyanate, using 3 mLof ferric ammonium sulfate TS as the indicator. Perform ablank determination. Calculate the percentage of oxyethylenegroups (—CH2CH2O—), as ethylene, by the formula
(B – S) × N × 2.203/W,
in which B – S represents the difference between the volumesof sodium thiosulfate required for the blank and the samplesolution, respectively; N is the normality of the sodium thiosul-fate; W is the weight, in g, of the sample taken; and 2.203 isan equivalence factor for oxyethylene. Calculate the percent-age of oxyethylene groups, as ethyl iodide, by the formula
(B′ – S′) × N′ × 4.405/W,
in which B′ – S′ represents the difference between the volumesof ammonium thiocyanate required for the blank and thesample solution, respectively; N′ is the normality of the ammo-nium thiocyanate; and 4.405 is an equivalence factor for oxy-ethylene. The sum of the values so obtained represents thepercentage of oxyethylene groups in the sample taken.
PEROXIDE VALUE
Transfer about 10 g of sample, accurately weighed, into asuitable container, add 30 mL of a 3:2 mixture of glacialacetic acid:chloroform, and mix. Add 1 mL of a saturatedsolution of potassium iodide, and mix for 1 min. Add 100mL of water, begin titrating with 0.05 N sodium thiosulfate,adding starch TS as the endpoint is approached, and continuethe titration until the blue starch color has just disappeared.Perform a blank determination (see General Provisions), andmake any necessary correction. Calculate the peroxide value,as milliequivalents of peroxide per kilogram of sample, bythe formula
(S × N × 1000)/W,
in which S is the net volume, in milliliters, of sodium thiosul-fate solution required for the sample; N is the exact normalityof the sodium thiosulfate solution; and W is the weight, ingrams, of the sample taken.
REICHERT-MEISSL VALUE(Based on AOCS Method Cd 5-40)
The Reichert-Meissl value is a measure of soluble volatilefatty acids (chiefly butyric and caproic). It is expressed in
FCC V General Tests and Assays / Appendix VII / 941
FIGURE 36 Reichert-Meissl Distillation Apparatus. [Note: Asuitable heating mantle may be substituted for the burner.]
terms of the number of mL of 0.1 N sodium hydroxide requiredto neutralize the fatty acids obtained from a 5-g sample underthe specified conditions of the method.
Apparatus Use a glass distillation apparatus of the samedimensions and construction as that shown in Fig. 36.
ReagentsSodium Hydroxide Solution Prepare a solution containing
50.0% by weight of NaOH, and protect from contact withcarbon dioxide. Allow the solution to settle, and use only theclear liquid.
Glycerin–Sodium Hydroxide Mixture Add 20 mL of theSodium Hydroxide Solution to 180 mL of glycerin.
Procedure Unless otherwise directed, accurately weighabout 5 g of the sample, previously melted if necessary, intothe 300-mL distillation flask. Add 20.0 mL of the Glycerin–Sodium Hydroxide Mixture, and heat until the sample is com-pletely saponified, as indicated by the mixture becoming per-fectly clear. Shake the flask gently if any foaming occurs.Add 135 mL of recently boiled and cooled water, dropwiseat first to prevent foaming, then add 6 mL of 1:5 sulfuric acidand a few pieces of pumice stone or silicon carbide. Rest theflask on a piece of heat-proof board having a center hole 5cm in diameter, and begin the distillation, regulating the flameso as to collect 110 mL of distillate in 30 � 2 min (measuretime from the passage of the first drop of distillate from thecondenser to the receiving flask), letting the distillate dripinto the flask at a temperature not higher than 20°.
When 110 mL has distilled, disconnect the receiving flask,and remove the flame. Mix the contents of the flask withgentle shaking, and immerse almost completely for 15 minin water cooled to 15°. Filter the distillate through dry, 9-cm,moderately retentive paper (S & S No. 589 White Ribbon,or equivalent), add phenolphthalein TS, and titrate 100 mLof the filtrate with 0.1 N sodium hydroxide to the first pinkcolor that remains unchanged for 2 to 3 min. Perform a blankdetermination using the same quantities of the same reagents,and calculate the Reichert-Meissl value by the formula
1.1 × (S – B),
in which S is the volume of 0.1 N sodium hydroxide requiredfor the sample, and B is the volume required for the blank.
SAPONIFICATION VALUE(Based on AOCS Methods Tl 1a-64 and Cd 3-25)
The saponification value is defined as the number of mg ofpotassium hydroxide required to neutralize the free acids andsaponify the esters in 1 g of the test substance.
Procedure Melt the sample, if necessary, and filter itthrough a dry filter paper to remove any traces of moisture.Unless otherwise directed, weigh accurately into a 250-mLflask a sample of such size that the titration of the samplesolution after saponification will require between 45% and55% of the volume of 0.5 N hydrochloric acid required forthe blank, and add to the flask 50.0 mL of 0.5 N alcoholicpotassium hydroxide. Connect an air condenser, at least 65cm in length, to the flask, and reflux gently until the sampleis completely saponified (usually 30 min to 1 h). Cool slightly,wash the condenser with a few mL of water, add 1 mL ofphenolphthalein TS, and titrate the excess potassium hydrox-ide with 0.5 N hydrochloric acid. Heat the contents of theflask to boiling, again titrate to the disappearance of any pinkcolor that may have developed, and record the total volumeof acid required. Perform a blank determination using the sameamount of 0.5 N alcoholic potassium hydroxide. Calculate thesaponification value by the formula
56.1(B – S) × N/W,
in which B – S represents the difference between the volumesof 0.5 N hydrochloric acid required for the blank and thesample, respectively; N is the normality of the hydrochloricacid; and W is the weight, in g, of the sample taken.
Note: A ‘‘masked phenolphthalein indicator’’ may beused with off-color materials. Prepare the indicator bydissolving 1.6 g of phenolphthalein and 2.7 g of methyl-ene blue in 500 mL of alcohol, and adjust the pH withalcoholic alkali solution so that the greenish blue coloris faintly tinged with purple. The color change, whengoing from acid to alkali, is from green to purple.
942 / Appendix VII / General Tests and Assays FCC V
SOAP
Prepare a solvent mixture consisting of equal parts, by volume,of benzene and methanol, add bromophenol blue TS, andneutralize with 0.5 N hydrochloric acid, or use neutralizedacetone as the solvent. Accurately weigh the amount of samplespecified in the individual monograph, dissolve it in 100 mLof the neutralized solvent mixture, and titrate with 0.5 Nhydrochloric acid to a definite yellow endpoint. Calculate thepercentage of soap in the sample by the formula
VNe/W,
in which V and N are the volume and normality, respectively,of the hydrochloric acid; W is the weight of the sample, ing; and e is the equivalence factor given in the monograph.
SPECIFIC GRAVITY
The specific gravity of a fat or oil is determined at 25°, exceptwhen the substance is a solid at that temperature, in whichcase the specific gravity is determined at the temperaturespecified in the monograph, and is referred to water at 25°.
Clean a suitable pycnometer by filling it with a saturatedsolution of chromic acid (CrO3) in sulfuric acid and allowingit to stand for at least 4 h. Empty the pycnometer, rinse itthoroughly, then fill it with recently boiled water, previouslycooled to about 20°, and place in a constant-temperature bathat 25°. After 30 min, adjust the level of water to the properpoint on the pycnometer, and stopper. Remove the pycnometerfrom the bath, wipe dry with a clean cloth free from lint, andweigh. Empty the pycnometer, rinse several times with alcoholand then with ether, allow to dry completely, remove anyether vapor, and weigh. Determine the weight of the containedwater at 25° by subtracting the weight of the pycnometer fromits weight when full.
Filter the oil or melted sample through filter paper to removeany impurities and the last traces of moisture, and cool to afew degrees below the temperature at which the determinationis to be made. Fill the clean, dry pycnometer with the sample,and place it in the constant-temperature bath at the specifiedtemperature. After 30 min, adjust the level of the oil to themark on the pycnometer, insert the stopper, wipe dry, andweigh. Subtract the weight of the empty pycnometer from itsweight when filled with the sample, and divide the differenceby the weight of the water contained at 25°. The quotient isthe specific gravity at the temperature of observation, referredto water at 25°.
STABILITY (Active Oxygen Method)(Based on AOCS Method Cd 12-57)
Fat stability is the time, in h, required for a sample of fat oroil to attain a peroxide value of 100. This period of time is
determined by interpolation between two measurements andis assumed to be an index of resistance to rancidity.
Caution: All equipment must be scrupulously clean (foran acceptable cleaning procedure, see AOCS OfficialMethod Cd 12-57). Do not use chromic acid or otheracidic cleaning agents. All receptacles in the heatermust be calibrated for temperature under the exact con-ditions of the test. During the test, the temperaturemust be monitored in a sample tube containing therecommended quantity of oil.
Apparatus Use a suitable heating block and aeration appa-ratus, such as shown in the Official and Tentative Methodsof the AOCS or in JAOCS 33 (1956), pp. 628–630.
Sampling Remove samples from large containers or pro-cessing equipment with sampling devices only of stainlesssteel, aluminum, nickel, or glass. Solid fat samples should betaken at least 5 cm from the walls of large containers and 2.5cm from the walls of small containers. If liquid oil is to bepoured from a container, clean the spout or lip with an acetone-moistened cloth. Under no circumstances should samples betaken from containers equipped with plastic or enameled topsor paper or wax liners.
Procedure Unless already completely liquid, the sampleshould be melted at a temperature not more than 10° aboveits melting point. Pour 20 mL into each of two or more sampletubes ensuring that the sample does not contact the tube wherethe stopper will later fit. Insert the aeration tube assembly sothat the end of the air delivery tube is 5 cm below the surface ofthe sample. Place the sample tube in a container of vigorouslyboiling water for 5 min (during this time adjust the air flowrate from the manifold). Remove the tube, wipe dry, andtransfer immediately to the constant-temperature heater, main-tained at 97.8° � 0.2°, and connect the aeration tube to themanifold. Determine to the nearest h the time required forthe sample to attain a Peroxide Value (in this appendix) of100 milliequivalents (meq) as follows: With 1-g samples de-termine when the peroxide value is approximately 75 meqand 125 meq, then perform the test on four 5-g samplesdetermining the peroxide value in duplicate at the times corres-ponding to 75 and 125 meq. Make a second determinationon two 5-g samples exactly 1 h after the first pair. Plot thesevalues against aeration time; the AOM stability value in h isgiven where the line crosses 100 meq.
UNSAPONIFIABLE MATTER(Based on AOCS Method Ca 6a-40)
This procedure determines those substances frequently founddissolved in fatty materials that cannot be saponified by alkalihydroxides but that are soluble in the ordinary fat solvents.
FCC V General Tests and Assays / Appendix VII / 943
Procedure Accurately weigh 5.0 g of the sample into a 250-mL flask, add a solution of 2 g of potassium hydroxide in 40mL of alcohol, and boil gently under a reflux condenser for1 h or until saponification is complete. Transfer the contentsof the flask to a glass-stoppered extraction cylinder (approxi-mately 30 cm in length, 3.5 cm in diameter, and graduatedat 40, 80, and 130 mL). Wash the flask with sufficient alcoholto make a volume of 40 mL in the cylinder, and completethe transfer with warm and then cold water until the totalvolume is 80 mL. Finally, wash the flask with a few mL ofpetroleum ether, add the washings to the cylinder, cool thecontents of the cylinder to room temperature, and add 50 mLof petroleum ether.
Insert the stopper, shake the cylinder vigorously for at least1 min, and allow both layers to become clear. Siphon theupper layer as completely as possible without removing anyof the lower layer, collecting the ether fraction in a 500-mLseparator. Repeat the extraction and siphoning at least sixtimes with 50-mL portions of petroleum ether, shaking vigor-ously each time. Wash the combined extracts, with vigorousshaking, with 25-mL portions of 10% alcohol until the washwater is neutral to phenolphthalein, and discard the washings.Transfer the ether extract to a tared beaker, and rinse theseparator with 10 mL of ether, adding the rinsings to thebeaker. Evaporate the ether on a steam bath just to dryness,and dry the residue to constant weight, preferably at 75° to80° under a vacuum of not more than 200 mm Hg, or at 100°for 30 min. Cool in a desiccator, and weigh to obtain theuncorrected weight of unsaponifiable matter.
Determine the quantity of fatty acids in the residue asfollows: Dissolve the residue in 50 mL of warm alcohol(containing phenolphthalein TS and previously neutralizedwith sodium hydroxide to a faint pink color), and titrate with0.02 N sodium hydroxide to the same color. Each mL of 0.02N sodium hydroxide is equivalent to 5.659 mg of fatty acids,calculated as oleic acid.
Subtract the calculated weight of fatty acids from the weightof the residue to obtain the corrected weight of unsaponifiablematter in the sample.
VOLATILE ACIDITY
Modified Hortvet-Sellier MethodApparatus Assemble a modified Hortvet-Sellier distillationapparatus as shown in Fig. 37, using a sufficiently large (ap-proximately 38- × 203-mm) inner Sellier tube and large dis-tillation trap.
FIGURE 37 Modified Hortvet-Sellier Distillation Apparatus.
Procedure Transfer the amount of sample, accuratelyweighed, specified in the monograph into the inner tube ofthe assembly, and insert the tube in the outer flask containingabout 300 mL of recently boiled hot water. To the sampleadd 10 mL of approximately 4 N perchloric acid [35 mL (60g) of 70% perchloric acid in 100 mL of water], and connect theinner tube to a water-cooled condenser through the distillationtrap. Distill by heating the outer flask so that 100 mL ofdistillate is collected within 20 to 25 min. Collect the distillatein 100-mL portions, add phenolphthalein TS to each portion,and titrate with 0.5 N sodium hydroxide. Continue the distilla-tion until a 100-mL portion of the distillate requires no morethan 0.5 mL of 0.5 N sodium hydroxide for neutralization.
Caution: Do not distill to dryness.
Calculate the weight, in mg, of volatile acids in the sampletaken by the formula
V × e,
in which V is the total volume, in mL, of 0.5 N sodiumhydroxide consumed in the series of titrations and e is theequivalence factor given in the monograph.
944 / Appendix VIII / General Tests and Assays FCC V
APPENDIX VIII: OLEORESINS
COLOR VALUE
Sample Preparation Transfer 70 to 100 mg of the sample,previously mixed well by shaking and accurately weighed,into a 100-mL volumetric flask, dissolve in acetone, dilute tovolume with acetone, and mix. Allow the solution to standfor 2 min, then pipet 10 mL into a second 100-mL volumetricflask, dilute to volume with acetone, and mix.
Procedure Determine the absorbance of the Sample Prepa-ration with a suitable spectrophotometer in a 1-cm cell at 460nm, using acetone as the blank. Record the value obtained asAS. In the same manner, determine the absorbance of a Na-tional Institute of Standards and Technology Standard GlassFilter 930, and record the value obtained as AF.
Note: The recommended range for absorbance valuesis between 0.30 and 0.70. Solutions having absorbancesgreater than 0.70 should be diluted with acetone toone-half the original concentration, and those havingabsorbances less than 0.30 should be discarded andthe Sample Preparation prepared with a larger sample.Appropriate adjustments should be made in the sampleweight (W) used in the Calculation below.
Calculation Determine the instrument correction factor, F,by the formula
AN/AF,
in which AN is the absorbance of the filter as stated by theNational Institute of Standards and Technology. Calculate thecolor value of the sample by the formula
(AS × 164 × F)/W,
in which W is the weight, in g, of sample taken.
CURCUMIN CONTENT
Sample Preparation Transfer about 500 mg of sample,accurately weighed, into a 100-mL volumetric flask, and rec-ord the weight, in milligrams, as W. Dissolve the sample inabout 75 mL of acetone, dilute to volume with acetone, andmix. Pipet a 5-mL portion of this solution into a second 100-mL volumetric flask, dilute to volume with acetone, and mix.Finally, pipet a 1-mL portion of the last solution into a 50-mL volumetric flask, dilute to volume with acetone, and mix.
Note: Protect all solutions from light by using activeglassware or by covering the glassware with aluminum
foil. Make the absorbance readings as soon as possibleafter the solutions are prepared.
Procedure Determine the absorbance of the Sample Prepa-ration in a 1-cm cell at the wavelength of maximum absorptionbetween 420 and 425 nm with a suitable spectrophotometer,using acetone as the blank. Calculate the percent curcuminin the sample by the formula
(A × 100)(165 × b × c),
in which A is the absorbance of the Sample Preparation; 100is the conversion to percent; 165 is the absorptivity factor, inliters per gram-centimeter, for curcumin; b is the path lengthof the cell; and c is the concentration, in grams per liter, ofthe solution presented to the spectrophotometer.
Calculate c by the formula
W × 5 × 10 6,
in which W is the starting weight, in milligrams, of the sample,and 5 × 10 6 is the conversion factor for the dilution schedule.
PIPERINE CONTENT
Stock Standard Solution Purify piperine by repeated crys-tallization from isopropanol until a product having a meltingrange of 129° to 130° is obtained. Transfer 100.0 mg of thecrystals, accurately weighed, into a 100-mL volumetric flask,dissolve in ethylene dichloride, dilute to volume with ethylenedichloride, and mix. Pipet 10.0 mL of this solution into asecond 100-mL volumetric flask, dilute to volume with ethyl-ene dichloride, and mix.
Standard Dilutions Pipet 1.0, 3.0, 5.0, and 10.0 mL of theStock Standard Solution (corresponding to 0.1, 0.3, 0.5, and1.0 mg of piperine, respectively) into separate 100-mL volu-metric flasks, dilute each flask to volume with ethylene dichlo-ride, and mix. Determine the absorbance of each dilution atonce, as directed in the Procedure.
Sample Preparation Heat a portion of the sample to 100°on a steam bath or in an oven (but not on a hot plate), mixwith a glass stirring rod, and transfer 100 mg, accuratelyweighed, into a 100-mL volumetric flask. Dissolve in ethylenedichloride, dilute to volume with ethylene dichloride, andmix. Pipet 1.0 mL of this solution into a second 100-mLvolumetric flask, dilute to volume with ethylene dichloride,and mix. Determine the absorbance of the solution at once,as directed in the Procedure.
Procedure Determine the absorbance of the Sample Prepa-ration and of each of the Standard Dilutions in 1-cm cells at
FCC V General Tests and Assays / Appendix VIII / 945
the wavelength of maximum absorption at about 342 nm witha suitable spectrophotometer, using ethylene dichloride as theblank. Prepare a standard curve of concentration, in mg per100 mL, versus absorbance for the four Standard Dilutions,including the absorbance at zero concentration obtained withthe blank. From the standard curve, determine the concentra-tion of piperine in the Sample Preparation, and record thevalue as C, in mg per 100 mL. Calculate the percentage ofpiperine in the sample by the formula
100 × (100C/W),
in which W is the weight, in mg, of sample taken.
RESIDUAL SOLVENT
This procedure is for the determination of acetone, ethylenedichloride, hexane, isopropanol, methanol, methylene chlo-ride, and trichloroethylene residues.
Distilling Head Use a Clevenger trap designed for use withoils heavier than water. A suitable design is shown in Fig. 38a.
Toluene The toluene used for this analysis should not con-tain any of the solvents determined by this method. The puritymay be determined by gas chromatographic analysis, usingone of the following columns or their equivalent: (1) 17% byweight of Ucon 75-H-90,000 on 35/80-mesh Chromosorb W;(2) 20% Ucon LB-135 on 35/80-mesh Chromosorb W; (3)15% Ucon LB-1715 on 60/80-mesh Chromosorb W; or (4)
FIGURE 38 Clevenger Traps (all measurements are in mm) (a)Oils Heavier Than Water; (b) Oils Lighter Than Water.
Porapak Q 50/60 mesh. Follow the conditions described underProcedure, and inject the same amount of toluene as will beinjected in the analysis of the solvents. If impurities interferingwith the test are present, they will appear as peaks occurringbefore the toluene peak and should be removed by fractionaldistillation.
Benzene The benzene used for this analysis should be freefrom interfering impurities. The purity may be determined asdescribed under Toluene.
Detergent and Antifoam Any such products that are freefrom volatile compounds may be used. If volatile compoundsare present, they may be removed by prolonged boiling ofthe aqueous solutions of the products.
Reference Solution A Prepare a solution in Toluene con-taining 2500 ppm of benzene. If the toluene available containsbenzene as the only impurity, the benzene level can be deter-mined by gas chromatography and sufficient benzene addedto bring the level to 2500 ppm.
Reference Solution B Prepare a solution containing 0.63%v/w of acetone in water.
Sample Preparation A (all solvents except methanol) Place50.0 g of the sample, 1.00 mL of Reference Solution A, 10g of anhydrous sodium sulfate, 50 mL of water, and a smallamount each of Detergent and Antifoam in a 250-mL round-bottom flask with a 24/40 ground-glass neck. Attach the Dis-tilling Head, a 400-mm water-cooled condenser, and a re-ceiver, and collect approximately 15 mL of distillate. Add 15g of anhydrous potassium carbonate to the distillate, coolwhile shaking, and allow the phases to separate. All of thesolvents except methanol will be present in the toluene layer,which is used in the Procedure. Draw off the aqueous layerfor use in Sample Preparation B.
Sample Preparation B (methanol only) Place the aqueouslayer obtained from Sample Preparation A in a 50-mL round-bottom distilling flask with a 24/40 ground-glass neck, adda few boiling chips and 1.00 mL of Reference Solution B,and collect approximately 1 mL of distillate, which will con-tain any methanol from the sample, together with acetone asthe internal standard. The distillate is used in the Procedure.
Procedure Use a gas chromatograph equipped with a hot-wire detector and a suitable sample-injection system or on-column injection. Under typical conditions, the instrumentcontains a 1/4-in. (od) × 6- to 8-ft column, or equivalent,maintained isothermally at 70° to 80°. The flow rate of drycarrier gas is 50 to 80 mL/min, and the sample size is 15 to20 �L (for the hot-wire detector). The column selected foruse in the chromatograph depends on the components to beanalyzed and, to a certain extent, on the preference of theanalyst. The columns 1, 2, 3, and 4, as described under Tolu-ene, may be used as follows: (1) This column separates acetoneand methanol from their aqueous solution. It may be used for
946 / Appendix VIII / General Tests and Assays FCC V
the separation and analysis of hexane, acetone, and trichloro-ethylene in the toluene layer from Sample Preparation A. Theelution order is acetone, methanol, and water, or hexane,acetone, isopropanol plus methylene chloride, benzene, tri-chloroethylene, and ethylene dichloride plus toluene. (2) Thiscolumn separates methylene chloride and isopropanol, andethylene dichloride. The elution order is hexane plus acetone,methylene chloride, isopropanol, benzene, ethylene dichlo-ride, trichloroethylene, and toluene. (3) This is the best gen-eral-purpose column, except for the determination of metha-nol. The elution order is hexane, acetone, benzene, ethylenedichloride, and toluene. (4) This column is used for the deter-mination of methanol, which elutes just after the largewater peak.
Calibration Determine the response of the detector forknown ratios of solvents by injecting known mixtures ofsolvents and benzene in toluene. The levels of the solventsand benzene in toluene should be of the same magnitude asthey will be present in the sample under analysis.
Calculate the areas of the solvents with respect to benzene,and then calculate the calibration factor, F, as follows:
F (solvent) = (wt % solvent/wt % benzene) ×(area of benzene/area of solvent).
The recovery of the various solvents from the oleoresin sam-ple, with respect to the recovery of benzene, is as follows:hexane, 52%; acetone, 85%; isopropanol, 100%; methylenechloride, 87.5%; trichloroethylene, 113%; ethylene dichloride,102%; and methanol, 87%.
Calculation Calculate the ppm of residual solvent (exceptmethanol) by the equation
Res. solv. = {[43.4 × F (solvent) × 100]/[% recovery of solvent]× (area of solvent/area of benzene),
in which 43.4 is the ppm of benzene internal standard, relatedto the 50-g oleoresin sample taken for analysis. Calculate theppm of residual methanol by the equation
Methanol = {[100 × F (methanol)]/0.87} ×(area of methanol/area of benzene),
in which 100 is the ppm of acetone internal standard, relatedto the 50-g oleoresin sample taken for analysis.
SCOVILLE HEAT UNITS
Sample Preparation Transfer 200 mg of the sample intoa 50-mL volumetric flask, dilute to volume with alcohol, andmix thoroughly by shaking. Allow the insolubles to settlebefore use.
Sucrose Solution Prepare a suitable volume of a 10% w/vsolution of sucrose in water.
Standard Solution Add 0.15 mL of the Sample Preparationto 140 mL of the Sucrose Solution, and mix. This solutioncontains the equivalent of 240,000 Scoville Heat Units.
Test Solutions If the oleoresin sample is claimed to containmore than 240,000 Scoville Heat Units, prepare one or moredilutions according to the following table:
Scoville Standard SucroseHeat Units Solution (mL) Solution (mL)
360,000 20 10480,000 20 20600,000 20 30720,000 20 40840,000 20 50960,000 20 60
1,080,000 20 701,200,000 20 801,320,000 20 901,440,000 20 1001,560,000 20 1101,680,000 20 1201,800,000 20 1301,920,000 20 1402,040,000 20 150
If the oleoresin sample is claimed to contain less than 240,000Scoville Heat Units, prepare one or more dilutions accordingto the following table:
Scoville Sample SucroseHeat Units Preparation (mL) Solution (mL)
100,000 0.15 60117,500 0.15 70170,000 0.15 100205,000 0.15 120
Procedure Select five panel members who are thoroughlyexperienced with this method. Instruct the panelists to swallow5 mL of the solution corresponding to the claimed content ofScoville Heat Units. The sample passes the test if three of thefive panel members perceive a pungent or stinging sensation inthe throat.
VOLATILE OIL CONTENT
Weigh accurately an amount of sample sufficient to yield 2to 5 mL of volatile oil, and transfer with the aid of water intoa 1000- or 2000-mL round-bottom shortneck flask with a 24/40 ground-glass neck. Add a magnetic stirring bar and about
FCC V General Tests and Assays / Appendix IX / 947
500 mL of water, and connect a Clevenger trap of the propertype (see Figs. 38a and 38b) and a 400-mm water-cooledcondenser. Heat the flask with stirring, and distill at a rate of1 to 1.5 drops per s until two consecutive readings taken at1-h intervals show no change of oil volume in the trap. Coolto room temperature, allow to stand until the oil layer is clear,and read the volume of oil collected, estimating to the nearest
APPENDIX IX: ROSINS AND RELATED SUBSTANCES
ACID NUMBER
The acid number is the number of mg of potassium hydroxiderequired to neutralize the free acids in 1 g of the test substance.
Procedure Unless otherwise directed in the individualmonograph, transfer about 4 g of the sample, previouslycrushed into small lumps and accurately weighed, into a 250-mL Erlenmeyer flask, and add 100 mL of a 1:3 mixture oftoluene–isopropyl alcohol, previously neutralized to phenol-phthalein TS with sodium hydroxide. Dissolve the sample byshaking or heating gently, if necessary, then add about 0.5mL of phenolphthalein TS, and titrate with 0.5 N or 0.1 Nalcoholic potassium hydroxide to the first pink color thatpersists for 30 s. Calculate the acid number by the formula
56.1V × N/W,
in which V is the exact volume, in mL, and N is the exactnormality, respectively, of the potassium hydroxide solution,and W is the weight, in g, of the sample.
SOFTENING POINT
Drop MethodThe drop softening point is that temperature at which a givenweight of rosin or rosin derivative begins to drop from thebulb of a special thermometer mounted in a test tube that isimmersed in a constant-temperature bath.
Apparatus The apparatus illustrated in Fig. 39 consists ofthe components described in the following paragraphs.
Thermometer Use a special total-immersion softeningpoint thermometer,1 covering the range from 0° to 250° andgraduated in 10 divisions. The bulb should be 15.9 � 0.8 mmin length and 6.35 � 0.4 mm in diameter.
Heating Bath Use an 800- to 2000-mL beaker containinga suitable heating medium. For rosins having a softening pointbelow 80°, use water; for those having softening points above
1 Available from the Walter K. Kessler Co., Inc.
0.02 mL. Calculate the percentage (v/w) of volatile oil in thesample by the formula
100(V/W),
in which V is the volume, in mL, of oil collected, and W isthe weight, in g, of sample taken.
FIGURE 39 Apparatus for Drop Softening Point Determination.
80°, use glycerin or silicone oil, depending upon the tempera-ture range required. Maintain the temperature of the heatingmedium within �1° of the temperature specified in the indi-vidual monograph. Stir the bath medium constantly duringthe test with a suitable mechanical stirrer to ensure uniformheating of the medium.
Test Tube Use a standard 22-mm od × 200- to 250-mmtest tube with a rim, fitted with a cork stopper as shown inFig. 39.
948 / Appendix IX / General Tests and Assays FCC V
Sample Preparation Place about 20 g of the sample in a50-mL beaker, and heat it in an oven, on a sand bath or hotplate, or in an oil bath until the sample becomes soft enoughto mold on the thermometer bulb. Tare the softening pointthermometer, and cautiously warm the bulb over a hot plateuntil it registers 15° to 20° above the expected softening pointof the sample. Immediately dip the thermometer bulb into themelted sample, withdraw, and rotate it to deposit a uniformfilm of the molten sample over the surface of the bulb, takingcare not to extend the film higher than the top of the bulb.Quickly place the thermometer on a balance, and weigh. Theweight of the sample on the thermometer bulb should bebetween 0.5 and 0.55 g. If the weight is low, again dip thebulb in the molten sample; if the weight is high, pull off someof the sample with the fingers. When the correct sample weighthas been obtained, mold the sample uniformly around thebulb by rolling it on the palm of the hand or between thefingers. The sample must be of uniform thickness over thebulb, and it must not extend up onto the thermometer stem(see Fig. 39). (If the film of the sample is not uniform whencooled, remove it completely from the bulb and apply a newone. Do not reheat the film and try to remold it.) Allow thefilm and thermometer to cool to approximately 35° or lower,allowing about 15 min for cooling.
Note: If samples having high softening points crack or‘‘check’’ on the thermometer bulb upon cooling to roomtemperature, prepare another sample film and cool onlyto about 50° below the expected softening point.
Procedure Fill the glass beaker to a depth of not less than101.6 mm or more than 108 mm with a suitable heatingmedium; support the beaker over a Bunsen burner, hot plate,or other suitable source of heat; and insert the bath stirrer anda bath temperature thermometer. Place the stirrer to one sideso that the impeller clears the side of the beaker and is about12.7 mm above the bottom of the beaker. Start the stirrer,heat the bath to the temperature specified in the individualmonograph, and maintain this temperature within �1°throughout the test.
Insert the prepared sample thermometer in the test tube,supporting it with a notched cork stopper so that the lowerend of the bulb is 25.4 mm from the bottom of the test tube.Place the test tube in the bath so that the bottom of thethermometer bulb is 50.8 mm from the bottom of the beaker;the top of the bulb should be about 25.4 to 38.1 mm belowthe liquid level of the bath. Stir the bath to keep its temperatureuniform throughout. Observe the sample thermometer, andrecord as the softening point the reading at which the elongateddrop of sample on the end of the bulb first becomes constricted(see Fig. 39). Report the softening point to the nearest 1.0°.
Caution: If the rosin crystallizes, thus making it diffi-cult to obtain the correct softening point, prepare a newsample by heating the rosin rapidly, yet cautiously, overa flame to a temperature of 160° to 170° to destroy allcrystal nuclei. Dip the thermometer bulb into the moltenresin, remove it momentarily, and rotate the thermome-ter to provide a uniform resin film on the bulb as itpartially cools in the air. Dip the bulb in the melted
sample repeatedly until the proper amount of resin isdeposited on the bulb. Do not report results if a crystal-free sample cannot be obtained.
Ring-and-Ball MethodThe ring-and-ball softening point is the temperature at whicha disk of the sample held within a horizontal ring is forceddownward a distance of 25.4 mm under the weight of a steelball as the sample is heated at a prescribed rate in a water,glycerin, or silicone oil (Dow Corning 200 fluid 50 cs or anequivalent is suitable) bath.
Apparatus Ring-and-ball softening point may be deter-mined manually using the apparatus described below. Auto-mated apparatus may be used provided equivalent results areobtained. The calibration of any automated apparatus shouldbe monitored on a regular basis because accurate temperaturecontrol is required. The apparatus illustrated in Figs. 40 and41 consists of the components described in the followingparagraphs.
Ring Use a brass-shouldered ring conforming to the di-mensions shown in Fig. 40a. If desired, the ring may beattached by brazing or other convenient manner to a brasswire of about 13 B & S gauge (1.52 to 2.03 mm in diameter)as shown in Fig. 41a.
Ball Use a steel ball, 9.53 mm in diameter, weighingbetween 3.45 and 3.55 g.
Ball-Centering Guide If desired, center the ball by usinga guide constructed of brass and having the general shapeand dimensions illustrated in Fig. 40c.
Container Use a heat-resistant glass vessel, such as an800-mL low-form Griffin beaker, not less than 85 mm indiameter and not less than 127 mm in depth from the bottomof the flare.
Support for Ring and Thermometer Use any convenientdevice for supporting the ring and thermometer, provided thatit meets the following requirements: (1) the ring is supportedin a substantially horizontal position; (2) when the apparatusshown in Fig. 40d is used, the bottom of the ring is 25.4 mmabove the horizontal plate below it, the bottom surface of thehorizontal plate is 12.7 to 19 mm above the bottom of thecontainer, and the depth of the liquid in the container is notless than 101.6 mm; (3) if the apparatus shown in Fig. 41eis used, the bottom of the ring is 25.4 mm above the bottomof the container, with the bottom end of the rod resting onthe bottom of the container, and the depth of the liquid in thecontainer is not less than 101.6 mm, as shown in Figs. 41a,b, and c; and (4) in both assemblies, the thermometer issuspended so that the bottom of the bulb is level with thebottom of the ring and within 12.7 mm of, but not touching,the ring.
Thermometers Depending on the expected softening pointof the sample, use either an ASTM 15C or 15F low-softening-point thermometer (–2° to 80°) or an ASTM 16C or 16F high-softening-point thermometer (30° to 200°), as described underThermometers, Appendix I.
Stirrer Use a suitable mechanical stirrer rotating between500 and 700 rpm. To ensure uniform heat distribution in theheating medium, the direction of the shaft rotation should
FCC V General Tests and Assays / Appendix IX / 949
FIGURE 40 Shouldered Ring, Ring Holder, Ball-CenteringGuide, and Assembly of Apparatus Showing Two Rings.
move the liquid upward. (See Fig. 41d for recommendeddimensions.)
FIGURE 41 Assembly of Apparatus Showing Stirrer andSingle-Shouldered Ring.
Sample Preparation Select a representative sample of thematerial under test consisting of freshly broken lumps freeof oxidized surfaces. Immediately before use, scrape off thesurface layer of samples received as lumps, avoiding inclusionof finely divided material or dust. The amount of sampletaken should be at least twice that necessary to fill the desirednumber of rings, but in no case less than 40 g. Immediatelymelt the sample in a clean container, using an oven, hotplate, or sand or oil bath to prevent local overheating. Avoidincorporating air bubbles in the melting sample, which mustnot be heated above the temperature necessary to pour thematerial readily without inclusion of air bubbles. The timefrom the beginning of heating to the pouring of the sampleshall not exceed 15 min. Immediately before filling the rings,preheat them to approximately the same temperature at whichthe sample is to be poured. While being filled, the rings shouldrest on an aluminum or steel plate. Pour a sufficient amountof the sample into the rings to leave an excess on cooling.Cool for at least 30 min, and then cut the excess material offcleanly with a slightly heated knife or spatula. Use a cleancontainer and a fresh sample if the test is repeated.
ProcedureMaterials Having Softening Points above 80° Fill the
glass vessel with glycerin to a depth of not less than 101.6mm and not more than 107.95 mm. The starting temperatureof the bath shall be 32°. For resins (including rosin), cool thebath liquid to not less than 27° below the anticipated softeningpoint, but in no case lower than 35°. Position the axis of thestirrer shaft near the back wall of the container, with the
950 / Appendix IX / General Tests and Assays FCC V
blades clearing the wall and with the bottom of the blades 19mm above the top of the ring. Unless the ball-centering guideis used, make a slight indentation in the center of the sampleby pressing the ball or a rounded rod, slightly heated for hardmaterials, into the sample at this point. Suspend the ringcontaining the sample in the bath so that the lower surfaceof the filled ring is 25.4 mm above the upper surface of thelower horizontal plate (see Fig. 40d), which is at least 12.7mm and not more than 19 mm above the bottom of the glassvessel, or 25.4 mm above the bottom of the container (seeFig. 41e). Place the ball in the bath but not on the test specimen.Suspend an ASTM high-softening-point thermometer (16Cor 16F) in the bath so that the bottom of its bulb is level withthe bottom of the ring and within 12.7 mm of, but not touching,the ring. Maintain the initial temperature of the bath for 15min. Begin stirring, and continue stirring at 500 to 700 rpmuntil the determination is complete. Apply heat in such amanner that the temperature of the bath liquid is raised 5°per min, avoiding the effects of drafts by using shields ifnecessary.
Note: The rate of rise of the temperature should beuniform and should not be averaged over the test period.Reject all tests in which the rate of rise exceeds �0.5°for any min period after the first three.
Record as the softening point the temperature of the thermom-eter at the instant the sample touches the lower horizontalplate (see Fig. 40d) or the bottom of the container (see Fig.41e). Make no correction for the emergent stem of the ther-mometer.
Materials Having Softening Points of 80° or Below Fol-low the above procedure, except use an ASTM low-softening-
point thermometer (15C or 15F) and use freshly boiled watercooled to 5° as the heating medium. For resins (includingrosins), use water cooled to not less than 27° below the antici-pated softening point, but in no case lower than 5°. Reportthe softening point to the nearest 1.0°.
VISCOSITY
Unless otherwise directed in the individual monograph, trans-fer the prepared sample into an 8-oz wide-mouth glass jar,10.8 cm high and 7 cm in inside diameter, equipped with ascrew lid. Condition the sample in a water bath at 25° �0.2°for 30 min (�5 min), taking care to prevent water from cominginto contact with the sample. Insert a No. 4 spindle in aBrookfield Model RVF viscometer,2 or equivalent, and movethe jar into place under the spindle, adjusting the elevationof the jar so that the upper surface of the sample is in thecenter of the shaft indentation and the spindle is in the centerof the jar.
Note: Keep the viscometer level at all times during thetest procedure.
Set the viscometer to rotate at 20 rpm, and allow the spindleto rotate until a constant dial reading is obtained. The viscosity,in centipoise, is the dial reading on the 0 to 100 scale multipliedby the appropriate factor (for a Brookfield RVF, spindle No.4, 20 rpm, the factor is 100).
2Available from Brookfield Engineering Laboratories, Inc., Stough-ton, MA.
FCC V General Tests and Assays / Appendix X / 951
APPENDIX X: CARBOHYDRATES (STARCHES, SUGARS, AND RELATED SUBSTANCES)
ACETYL GROUPS
Transfer about 5 g of the sample, accurately weighed, into a250-mL Erlenmeyer flask, suspend in 50 mL of water, add afew drops of phenolphthalein TS, and titrate with 0.1 N sodiumhydroxide to a permanent pink endpoint. Add 25.0 mL of0.45 N sodium hydroxide, stopper the flask, and shake vigor-ously for 30 min, preferably with a mechanical shaker. Re-move the stopper, wash the stopper and sides of the flaskwith a few mL of water, and titrate the excess alkali with 0.2N hydrochloric acid to the disappearance of the pink color,recording the volume, in mL, of 0.2 N hydrochloric acidrequired as S. Perform a blank titration of 25.0 mL of 0.45N sodium hydroxide, and record the volume, in mL, of 0.2N hydrochloric acid required as B. Calculate the percentageof acetyl groups by the formula
% Acetyl Groups = (B – S) × N × 0.043 × 100/W,
in which N is the exact normality of the hydrochloric acidsolution, and W is the weight, in g, of the sample.
CRUDE FAT
Apparatus The apparatus consists of a Butt-type extractor,1
as shown in Fig. 42, having a standard-taper 34/45 female
FIGURE 42 Butt-Type Extractor for Crude Fat Determination.
joint at the upper end, to which is attached a Friedrichs- orHopkins-type condenser, and a 24/40 male joint at the lowerend, to which is attached a 125-mL Erlenmeyer flask.
Procedure Transfer about 10 g of the sample, previouslyground to 20-mesh or finer and accurately weighed, to a 15-cm filter paper, roll the paper tightly around the sample, andplace it in a suitable extraction shell. Plug the top of the shellwith cotton previously extracted with hexane, and place theshell in the extractor. Attach the extractor to a dry 125-mLErlenmeyer flask containing about 50 mL of hexane and toa water-cooled condenser, apply heat to the flask to produce150 to 200 drops of condensed solvent per min, and extractfor 16 h. Disconnect the flask, and filter the extract to removeany insoluble residue. Rinse the flask and filter with a fewmL of hexane, combine the washings and filtrate in a taredflask, and evaporate on a steam bath until no odor of solventremains. Dry in a vacuum for 1 h at 100°, cool in a desiccator,and weigh.
INVERT SUGAR2
AssayApparatus Mount a ring support on a ringstand 1 to 2 in.above a gas burner, and mount a second ring 6 to 7 in. abovethe first. Place a 6-in. open-wire gauze on the lower ring tosupport a 400-mL Erlenmeyer flask, and place a 4-in. watchglass with a center hole on the upper ring to deflect heat.Attach a 50-mL buret to the ringstand so that the tip justpasses through the watch glass centered above the flask. Alter-natively, a buret with an offset tip may be used in place ofa buret with a straight tip extending through the hole in thewatch glass. Place an indirectly lighted white surface behindthe assembly for observing the endpoint. Alternatively, use ahot titrator/illuminator available from ICUMSA (c/o CentralScientific Laboratories, 445 New Cross Road, London SE 146 TA, England).
Mixed Fehling’s SolutionCopper Sulfate Solution Dissolve 34.639 g of CuSO4-
·5H2O in water; dilute to 500 mL, and filter.Alkaline Tartrate Solution Dissolve 173 g of potassium
sodium tartrate (KNaC4H4O6·4H2O) and 50 g of NaOH inwater, and dilute to 500 mL; allow to stand 2 days, and filterbefore use.
1Available from H.S. Martin & Co., Evanston, IL.2Based on ICUMSA Method GS 4/3-3 (1994).
952 / Appendix X / General Tests and Assays FCC V
Just before use, prepare the Mixed Fehling’s Solution bymixing equal volumes of Copper Sulfate Solution and AlkalineTartrate Solution.
Stock Standard Solution Transfer approximately 9.5 g ofNF-grade sucrose, accurately weighed, to a 1000-mL volumet-ric flask; dissolve in 100 mL of water, add 5 mL of hydrochlo-ric acid, and store 3 days at 20° to 25°. Dilute to volume withwater. This solution is stable for several months.
Sample Solution Transfer 10 g or a suitable weight of sam-ple, accurately weighed, to a 1-L volumetric flask; dissolvein and dilute to volume with water so that the final SampleSolution contains between 250 and 400 mg of Invert Sugarper 100 mL.
Invert Sugar Solution (0.25 g per 100 mL) Immediatelybefore use in standardizing the Mixed Fehling’s Solution,pipet 25 mL of Stock Standard Solution into a 100-mL volu-metric flask, dilute to volume with water, and mix.
Standardized Fehling’s Solution To 20 mL of Mixed Feh-ling’s Solution in a 400-mL flask containing a few boilingchips add 15 mL of water and 39 mL of Invert Sugar Solution.Mix by swirling, heat, and titrate with the Invert Sugar Solu-tion as directed under Procedure. Adjust the Mixed Fehling’sSolution for the correct amount of copper (equivalent to 100mg of invert sugar), and restandardize if the total volume ofInvert Sugar Solution is more or less than 40 mL.
ProcedureInvert Sugar Conduct a preliminary test to ascertain the
volume of water to be added to the 20 mL of StandardizedFehling’s Solution to obtain a final total volume of 75 mLwhen the endpoint of the titration is reached. The invert sugarcontent of the Sample Solution should be between 250 and400 mg per 100 mL so that a titer between 25 and 40 mL isneeded to achieve the endpoint. Calculate the amount of waterto be added to the Mixed Fehling’s Solution as the difference
75 – [20 (mL of Mixed Fehling’s Solution) + (number ofmL of preliminary titer)].
Pipet 20 mL of Mixed Fehling’s Solution in a 400-mL flaskcontaining a few glass beads or boiling chips, add the requiredamount of water and mix. Rinse a 50-mL buret, and fill withSample Solution. Rapidly add the Sample Solution within 0.5mL of the endpoint, mix by swirling at room temperature.Immediately place the flask on the wire gauze, adjust theburner flame so that the boiling point of the solution is reachedin 2 min. Boil gently but steadily for 2 min. As boilingcontinues, add 3 to 4 drops of 1% aqueous methylene blueindicator. Complete the titration within 1 min by adding theSample Solution dropwise or in small increments until theblue color disappears. Allow a 5-s reaction time betweendrops at the end of the titration. Calculate the percent of invertsugar, PI, in the sample by using the following equation:
PI = f × 10000/CSV,
in which f is the correction factor for the apparent reducingpower of sucrose as seen from the table immediately following
this Assay (f = 1 if no sucrose is present), CS is the concentra-tion (dry basis), in mg/mL, of the Sample Solution, and V isthe volume, in mL, of the Sample Solution used in the titration.
Sucrose Pipet 100 mL of Sample Solution into a 200-mLvolumetric flask, and add slowly 10 mL of 2.7 N hydrochloricacid, diluted 1:1, while gently swirling the solution; place ina constant-temperature bath maintained at 60°; agitate contin-uously for 3 min; and allow to sit in the bath for an additional7 min. Remove the flask from the bath, and cool to 20° asrapidly as possible; dilute to volume with water, and mixwell. Continue as directed in the Procedure (above) underInvert Sugar. Calculate the percent invert sugar present afterhydrolysis (PH) using the equation
PH = 20000/CSVH,
in which CS is the concentration, in mg/mL, of sample in theSample Solution, as defined above, and VH is the volume, inmL, of the hydrolyzed Sample Solution used in the titration.If VH falls outside the limits of 25 to 40 mL, repeat thehydrolysis with a different volume of Sample Solution. Calcu-late the percent sucrose by using the following equation:
PS = 0.95(PH – PI),
in which PH and PI are the percentages of invert sugar deter-mined after and before hydrolysis, respectively.
ICUMSA Table: Sucrose Correction to Be Applied inLane & Eynon Constant Volume Method
Sucrose in Sucrose inBoiling Correction Boiling CorrectionMixture, g Factor (f) Mixture, g Factor (f)
0.5 0.988 5.5 0.9001.0 0.975 6.0 0.8941.5 0.962 6.5 0.8892.0 0.950 7.0 0.8842.5 0.942 7.5 0.8793.0 0.934 8.0 0.8743.5 0.925 8.5 0.8704.0 0.917 9.0 0.8654.5 0.912 9.5 0.8615.0 0.906 10.0 0.856
LACTOSE
AssayApparatus Use a suitable high-performance liquid chro-matographic system (see Chromatography, Appendix IIA)equipped with a differential refractometer detector, a precol-
FCC V General Tests and Assays / Appendix X / 953
umn, an online 0.45-�m filter, and a 250-mm × 4.6-mm (id)stainless steel column, or equivalent.
Solid Phase Microparticle silica gel with siloxane bondedcyano-amino moieties (Whatman P-10 carbohydrate, or equiv-alent) equilibrated and operated at room temperature.
Mobile Phase Acetonitrile–water (80:20) at a flow rateof 2 mL/min.
ReagentsAcetonitrile An appropriate grade for liquid chromatog-
raphy.Fructose Internal Standard Solution Prepare a solution
of fructose to be used as an internal standard by transferring50 g of commercial grade �-D(–)fructose powder to a 500-mL volumetric flask, and dissolve in and dilute to volumewith water.
Standard Solution Transfer about 2 g of NF-grade anhy-drous lactose, accurately weighed, to a 100-mL volumetricflask, add 10 mL of Fructose Internal Standard Solution, anddilute to volume with water. Prepare fresh daily.
Water An appropriate grade for liquid chromatography.
System Suitability (See Chromatography, Appendix IIA.)Repeatability Allow the chromatographic system to equil-
ibrate at a flow rate of 2 mL/min, then inject 25-�L aliquotsof the Standard Solution. The chromatogram should showbaseline resolution and a retention time for water of 1 to 2 min;fructose, 2 to 3 min; and lactose, 5 to 6 min. The coefficient ofvariation for the relative peak heights (lactose peak height/fructose peak height) for ten injections should be ≤0.6% whencolumn equilibration is complete.
Linearity of Detector Response On a monthly basis (orwhen changes in the system are made), monitor the linearityof detector response by injecting standard lactose solutionscontaining 1.4%, 1.8%, 2.0%, 2.2%, and 2.6% lactose. Linearregression of the curve generated by plotting peak heightversus concentration should give a correlation coefficient ofat least 0.999.
Sample Preparation Prepare the sample as directed in theindividual monograph. Analysis must be performed within24 h.
Procedure Inject triplicate 25-�L aliquots of sample andstandard solutions. If more than one sample is to be analyzed,inject the standard solution after every third sample. Calculateresults using average standard response factors bracketingevery three samples (see Chromatography, Appendix IIA).
Calculation Calculate the % Lactose (dry basis) by theformula
(RL/RF) × (WL/WS) × (100 – ML/100 – MS) × P,
in which RL and RF are the response factors for lactose andfructose; WS and WL are the weights, in g, of the sample andlactose standard in their respective solutions; MS and ML arethe percentages of moisture in the sample and lactose standard;and P is the purity, in percent, of the lactose standard. Deter-mine the moisture content by drying at 120° for 16 h.
PROPYLENE CHLOROHYDRIN(2-Chloro-1-propanol)
Special ApparatusGas Chromatograph (See Chromatography, Appendix
IIA.) Use a suitable gas chromatograph. A dual-column, orequivalent, instrument equipped with a flame-ionization de-tector and an integrator is preferred.
Concentrator Use a Kuderna-Danish concentrator havinga 500-mL flask, available from Kontes Glass Co., Vineland,NJ (Catalog No. K-57000), or equivalent.
Pressure Bottles Use 200-mL pressure bottles, with aNeoprene washer, glass stopper, and attached wire clamp,available from Fisher Scientific Co. (Vitro 400, Catalog No.3-100), or equivalent.
Gas Chromatography Column Use a stainless steel col-umn, or equivalent, 3 m × 3.2 mm (od), packed with 10%Carbowax 20 M on 80/100-mesh Gas Chrom 2, or equivalent.After packing and before use, condition the column overnightat 200°, using a helium flow of 25 mL/min.
ReagentsDiethyl Ether Use anhydrous, analytical reagent-grade di-
ethyl ether, available from Fisher Scientific Co. or J. T. BakerCo., or other suitable sources.
Note: Some lots of diethyl ether contain foreign residuesthat interfere with the analysis and/or the interpretationof the chromatograms. If the ether quality is unknownor suspect, concentrate 50 mL to a volume of about 1mL in the concentrator, and then chromatograph a 2.0-�L portion using the conditions outlined under the Pro-cedure. If the chromatogram is excessively noisy andcontains signal peaks that overlap or interfere in themeasurement of the peaks produced by the propylenechlorohydrin isomers, the ether should be redistilled.
Florisil PR Use 60/100-mesh material, available fromFloridin Co., 3 Penn Center, Pittsburgh, PA 15235, or anequivalent product available from Supelco, Bellefonte, PA16823.
Propylene Chlorohydrins Use 1-Chloro-2-propanol Prac-tical Grade, containing 25% 2-Chloro-1-propanol, availablefrom Aldrich Chemical Company, Milwaukee, WI 53233.
Standard Preparation Draw 25 �L of Propylene Chloro-hydrins into a 50-�L syringe, weigh accurately, and dischargethe contents into a 500-mL volumetric flask partially filledwith water. Reweigh the syringe, and record the weight ofthe chlorohydrins taken. Dilute to volume with water, andmix. This solution contains about 27.5 mg of mixed chlorohy-drins, or about 55 �g/mL. Prepare this solution fresh daily.
Sample Preparation Transfer a blended representative50.0-g sample into a pressure bottle, and add 125 mL of 2 Nsulfuric acid. Clamp the top in place, and swirl the contentsuntil the sample is completely dispersed. Place the bottle ina boiling water bath, heat for 10 min, then swirl the bottle to
954 / Appendix X / General Tests and Assays FCC V
mix the contents, and heat in the bath for an additional 15min. Cool in air to room temperature, then neutralize thehydrolyzed sample to pH 7 with 25% sodium hydroxide solu-tion, and filter through Whatman No. 1 paper, or equivalent,in a Büchner funnel, using suction. Wash the bottle and filterpaper with 25 mL of water, and combine the washings withthe filtrate. Add 30 g of anhydrous sodium sulfate, and stirwith a magnetic stirring bar for 5 to 10 min, or until thesodium sulfate is completely dissolved. Transfer the solutioninto a 500-mL separator equipped with a Teflon plug, rinsethe flask with 25 mL of water, and combine the washingswith the sample solution. Extract with five 50-mL portionsof Diethyl Ether, allowing at least 5 min in each extractionfor adequate phase separation. Transfer the combined etherextracts in a concentrator, place the graduated receiver of theconcentrator in a water bath maintained at 50° to 55°, andconcentrate the extract to a volume of 4 mL.
Note: Ether extracts of samples may contain foreignresidues that interfere with the analysis and/or interpre-tation of the chromatograms. These residues are be-lieved to be degradation products arising during thehydrolysis treatment. Analytical problems created bytheir presence can be avoided through application of acleanup treatment performed as follows: Concentratethe ether extract to about 8 mL, instead of 4 mL specifiedabove. Add 10 g of Florisil PR, previously heated to130° for 16 h just before use, to a chromatographictube of suitable size, then tap gently, and add 1 g ofanhydrous sodium sulfate to the top of the column.Wet the column with 25 mL of Diethyl Ether, andquantitatively transfer the concentrated extract to thecolumn with the aid of small portions of the ether. Elutewith three 25-mL portions of the ether, collect all ofthe eluate, transfer it to a concentrator, and concentrateto a volume of 4 mL.
Cool the extract to room temperature, transfer it quantita-tively to a 5.0-mL volumetric flask with the aid of smallportions of Diethyl Ether, dilute to volume with the ether,and mix.
Control Preparations Transfer 50.0-g portions of unmodi-fied (underivatized) waxy corn starch into five separate pres-sure bottles, and add 125 mL of 2 N sulfuric acid to eachbottle. Add 0.0, 0.5, 1.0, 2.0, and 5.0 mL of the StandardPreparation to the bottles, respectively, giving propylenechlorohydrin concentrations, on the starch basis, of 0, 0.5, 1,2, and 5 mg/kg, respectively. Calculate the exact concentrationin each bottle from the weight of Propylene Chlorohydrinsused in making the Standard Preparation. Clamp the tops inplace, swirl until the contents of each bottle are completelydissolved, and proceed with the hydrolysis, neutralization,filtration, extraction, extract concentration, and final dilutionas directed under Sample Preparation.
Procedure Perform the analysis by gas chromatographywith the gas chromatograph and gas chromatography columnpreviously described. The operating conditions may be varied,depending on the column and instrument used. A suitable
chromatogram was obtained using a column oven temperatureof 110°, isothermal; injection port temperature of 210°; detec-tor temperature of 240°; and hydrogen (30 mL/min), air (350mL/min), or helium (25 mL/min), as the carrier gas.
Inject 2.0-�L aliquots of each of the concentrated extracts,prepared as directed under Control Preparations, allowingsufficient time between injections for signal peaks correspond-ing to the two chlorohydrin isomers to be recorded (and inte-grated) and for the column to be purged. Record and sum thesignal areas (integrator outputs) from the two chlorohydrinisomers for each of the controls.
Using identical operating conditions, inject a 2.0-�L aliquotof the concentrated extract prepared as directed under SamplePreparation, and record and sum the signal areas (integratoroutputs) from the sample.
Calculation Prepare a standard curve for the summed signalareas for each of the controls against the calculated propylenechlorohydrin concentrations, in mg/kg, derived from the actualweight of chlorohydrin isomers used. Using the summed sig-nal areas corresponding to the 1-chloro-2-propanol and 2-chloro-1-propanol from the sample, determine the concentra-tion of mixed propylene chlorohydrins, in mg/kg, in the sampleby reference to the calibration plot.
Note: After gaining experience with the procedure anddemonstrating that the calibration plot derived from thecontrol samples is linear and reproducible, the numberof controls can be reduced to one containing about 5mg/kg of mixed propylene chlorohydrin isomers. Thepropylene chlorohydrin level in the sample can then becalculated as follows:
Propylene chlorohydrins, mg/kg = (C × a)/A,
in which C is the concentration, in mg/kg, of propylene chloro-hydrins (sum of isomers) in the control; a is the sum of thesignal areas produced by the propylene chlorohydrin isomersin the sample; and A is the sum of the signal areas producedby the propylene chlorohydrin isomers in the control.
REDUCING SUGARS ASSAY
Apparatus Mount a ring support on a ringstand 1 to 2 in.above a gas burner, and mount a second ring 6 to 7 in. abovethe first. Place a 6-in. open-wire gauze on the lower ring tosupport a 250-mL Erlenmeyer flask, and place a 4-in. watchglass with a center hole on the upper ring to deflect heat.Attach a 25-mL buret to the ringstand so that the tip justpasses through the watch glass centered above the flask. Placean indirectly lighted white surface behind the assembly forobserving the endpoint.
Standardized Fehling’s Solution Measure a quantity ofFehling’s Solution A, add an equal quantity of Fehling’s Solu-tion B, and mix (see Cupric Tartrate TS, Alkaline in the
FCC V General Tests and Assays / Appendix X / 955
section on Solutions and Indicators). Immediately before use,standardize as follows: Transfer 3.000 g of primary standarddextrose (NIST Standard Reference Material, or equivalent),previously dried in vacuum at 100° for 2 h, into a 500-mLvolumetric flask, dissolve in and dilute to volume with water,and mix. Pipet 25 mL of the mixed Fehling’s solution into a200-mL Erlenmeyer flask containing a few glass beads, andtitrate with the standard dextrose solution as directed underProcedure. Adjust the concentration of Fehling’s Solution Aby dilution or the addition of copper sulfate, so that the titrationrequires 20.0 mL of the standard dextrose solution.
Procedure Transfer about 3 g of the sample, accuratelyweighed, into a 500-mL volumetric flask, dissolve in anddilute to volume with water, and mix. Pipet 25.0 mL ofStandardized Fehling’s Solution into a 200-mL Erlenmeyerflask containing a few glass beads, and add the sample solutionfrom a buret to within 0.5 mL of the anticipated endpoint(determined by preliminary titration). Immediately place theflask on the wire gauze of the Apparatus, and adjust the burnerso that the boiling point will be reached in about 2 min. Bringto a boil, and boil gently for 2 min. As boiling continues, add2 drops of a 1% aqueous solution of methylene blue, andcomplete the titration within 1 min by adding the samplesolution dropwise or in small increments until the blue colordisappears. Record the volume, in mL, of sample solutionrequired as V. Calculate the percentage of reducing sugars,as D-glucose on the dried basis, by the equation
% Reducing Sugars = (500 × 0.12 × 100)/(V × W),
in which W is the weight, in g, of the sample of dry substance.
SULFUR DIOXIDE DETERMINATION(Based on AOAC Method 962.16)
Reagents3% Hydrogen Peroxide Solution Dilute 30% hydrogen
peroxide to 3% with water. Just before use, add 3 drops ofmethyl red TS, and titrate to a yellow endpoint using 0.01 Nsodium hydroxide. If the endpoint is exceeded, discard thesolution and prepare another 3% hydrogen peroxide solution.
Standardized Titrant Prepare a solution of 0.01 N sodiumhydroxide.
Nitrogen A source of high-purity nitrogen is required witha flow regulator that will maintain a flow of 200 � 10 mL/min. To guard against the presence of oxygen in the nitrogen,an oxygen scrubbing apparatus or solution such as an alkalinepyrogallol trap may be used. Prepare the pyrogallol trap asfollows: Add 4.5 g of pyrogallol to the trap, purge the trapwith nitrogen for 2 to 3 min, and add potassium hydroxidesolution (65 g of potassium hydroxide added to 85 mL ofwater) to the trap while maintaining an atmosphere of nitrogenin the trap.
Caution: Exothermic reaction.
FIGURE 43 The Optimized Monier-Williams Apparatus;Component Identification Is Given in Text (component F isdepicted in FIGURE 44).
Sample Preparation (for solids) Transfer 50 g of the sam-ple, or a quantity of the sample with a known quantity ofsulfur dioxide (500 to 1500 �g of SO2), to a food processoror blender, if necessary. Add 50 mL of 5% ethanol in water,and briefly grind the mixture, reserving another 50 mL of 5%ethanol in water to rinse the blender jar. Grinding or blendingshould be continued only until the food is chopped into piecessmall enough to pass through the 24/40 joint of a flask (seeFig. 43).
Sample Preparation (for liquids) Mix 50 g of the sample,or a quantity with a known amount of sulfur dioxide (500 to1500 �g of SO2), with 100 mL of 5% ethanol in water.
Apparatus The apparatus shown diagrammatically (Fig.43) is designed to accomplish the selective transfer of sulfurdioxide from the sample in boiling aqueous hydrochloric acidto the 3% Hydrogen Peroxide Solution. This apparatus iseasier to assemble than the official apparatus, and the back-
956 / Appendix X / General Tests and Assays FCC V
FIGURE 44 Diagram of Bubbler (F in FIGURE 43) (lengthsare given in mm).
pressure inside the apparatus is limited to the unavoidablepressure due to the height of the 3% Hydrogen PeroxideSolution above the tip of the bubbler, F. Keeping the back-pressure as low as possible reduces the likelihood that sulfurdioxide will be lost through leaks.
Note: Tygon and silicon tubing should be preboiledbefore use in this procedure.
The apparatus should be assembled as shown in Fig. 43with a thin film of stopcock grease on the sealing surfacesof all the joints except the joint between the separatory funneland the flask. Each joint should be clamped together to ensurea complete seal throughout the analysis. The separatory funnel,B, should have a capacity of 100 mL or greater. An inletadapter, A, with a hose connector (Kontes K-183000, or equiv-alent) is required to provide a means of applying a head ofpressure above the solution. (A pressure-equalizing droppingfunnel is not recommended because condensate, perhaps withsulfur dioxide, is deposited in the funnel and the side arm.)The round-bottom flask, C, is a 1000-mL flask with three 24/40 tapered joints. The gas inlet tube, D (Kontes K-179000,or equivalent), should be of sufficient length to permit intro-duction of the nitrogen within 2.5 cm of the bottom of theflask. The Allihn condenser, E (Kontes K-431000-2430, orequivalent), has a jacket length of 300 mm. The bubbler, F,is fabricated from glass according to the dimensions given inFig. 44, and it has the same dimensions as a 50-mL graduatedcylinder (see Fig. 44). The 3% Hydrogen Peroxide Solutioncan be contained in a receiving vessel, G, with an id of about2.5 cm and a depth of 18 cm.
Buret Use a 10-mL buret with overflow tube and hoseconnections for an Ascarite tube or equivalent air-scrubbingapparatus. This will permit the maintenance of a carbon diox-ide-free atmosphere over the Standardized Titrant.
Chilled Water Circulator The condenser must be chilledwith a coolant, such as 20% methanol–water, at a flow rateso that the condenser outlet temperature is maintained at 5°.A circulating pump equivalent to the Neslab Coolflow 33 issuitable.
Determination Assemble the apparatus as shown in Fig.43. The flask must be positioned in a heating mantle that iscontrolled by a power-regulating device such as Variac, orequivalent. Add 400 mL of distilled water to the flask. Closethe stopcock of the separatory funnel, and add 90 mL of 4 Nhydrochloric acid to the separatory funnel. Begin the flow ofnitrogen at a rate of 200 � 10 mL/min. The condenser coolantflow must be initiated at this time. Add 30 mL of 3% HydrogenPeroxide Solution, which has been titrated to a yellow end-point with the Standardized Titrant, to the receiving vessel,G. After 15 min, the apparatus and the water will be thoroughlydeoxygenated, and the apparatus will be ready for sampleintroduction.
Sample Introduction and Distillation Remove the separa-tory funnel, and quantitatively transfer the sample in aqueousethanol to the flask. Wipe the tapered joint clean with alaboratory tissue, apply stopcock grease to the outer joint ofthe separatory funnel, and return the separatory funnel to thetapered joint flask. The nitrogen flow through the 3% Hydro-gen Peroxide Solution should resume as soon as the funnelis reinserted into the appropriate joint in the flask. Examineeach joint to ensure that it is sealed.
Apply a head pressure above the hydrochloric acid solutionin the separatory funnel with a rubber bulb equipped with avalve. Open the stopcock in the separatory funnel, and permitthe hydrochloric acid solution to flow into the flask. Continueto maintain sufficient pressure above the acid solution to forcethe solution into the flask. The stopcock may temporarily beclosed, if necessary, to pump up the pressure above the acid.To guard against the escape of sulfur dioxide into the separa-tory funnel, close the stopcock before the last few mL drainout of the separatory funnel.
Apply the power to the heating mantle. Use a power settingthat will cause 80 to 90 drops of condensate to return to theflask from the condenser per min. After 1.75 h of boiling,cool the contents of the 1000-mL flask at the condensationrate stated above, and remove the contents of the receivingvessel, G.
Titration Add 3 drops of Methyl Red Indicator, and titratethe above-mentioned contents with the Standardized Titrantto a yellow endpoint that persists for at least 20 s. Calculatethe sulfur dioxide content, expressed as �g of sulfur dioxideper g of sample (�g/g or mg/kg) as follows:
mg/kg = (32.03 × VB × N × 1000)/Wt,
in which 32.03 is the milliequivalent weight, in mg, of sulfurdioxide; VB is the volume, in mL, of sodium hydroxide titrantof normality, N, required to reach the endpoint; the factor1000 converts mg to �g; and Wt is the weight, in g, of sampleintroduced into the 1000-mL flask.
FCC V General Tests and Assays / Appendix X / 957
TOTAL SOLIDS
Note: The refractive index, RI, of solutions of variouscarbohydrates at specific temperatures is directly corre-lated with the solutions’ concentrations (in g/100 g orpercent dried solids). The following tables, as requiredin some monographs in this edition, are provided forthe user’s convenience.
Apparatus Use a suitable refractometer (see Refractive In-dex, Appendix IIB) equipped with a jacket for water circula-tion or some other mechanism for maintaining the sample at20.0° � 0.1° or some other fixed temperature. Before proceed-ing with measurements, ensure that the prism has reached theequilibrium temperature.
Standardization To achieve the theoretical accuracy of�0.0001, calibrate the instrument daily by determining therefractive index of distilled water, which is 1.3330 at 20°,and 1.3325 at 25°.
Procedure Determine the refractive index after ensuringthat the sample and prism have reached the equilibrium tem-perature.
For Corn Syrups, High-Fructose Corn Syrups, Liquid Fruc-tose, and Maltodextrin, convert the refractive index to approxi-mate percent solids using the accompanying tables.
Note: These tables cover the approximate total solidslevels of these products in commerce. If the ash ordextrose equivalent of the sample differs from the prod-uct in the table, use the accompanying ash and dextroseequivalent correction table.
Glucose Syrup (Corn Syrup)28 DEa Glucose Syrup—0.3% Ash
Ric RI °Baumé at 140°F%DSb 20°C 45°C (60°C) + 1
76.0 1.4888 1.4837 40.9877.0 1.4915 1.4864 41.4978.0 1.4943 1.4892 42.0079.0 1.4971 1.4919 42.5180.0 1.4999 1.4947 43.01
aDextrose EquivalentbDry SubstancecRefractive Index
36 DE Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
78.4 1.4938 1.4887 42.0179.4 1.4965 1.4914 42.5280.4 1.4993 1.4941 43.0281.4 1.5021 1.4969 43.5282.4 1.5049 1.4997 44.02
34 DE High-Maltose Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
78.6 1.4933 1.4882 41.9979.6 1.4960 1.4909 42.4980.6 1.4988 1.4936 42.9981.6 1.5015 1.4964 43.4982.6 1.5043 1.4992 43.99
43 DE High-Maltose Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
78.9 1.4934 1.4883 42.0079.9 1.4961 1.4910 42.5180.9 1.4988 1.4937 43.0181.9 1.5016 1.4964 43.5182.9 1.5044 1.4992 44.01
43 DE Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
78.7 1.4933 1.4882 42.0179.7 1.4960 1.4909 42.5180.7 1.4988 1.4936 43.0281.7 1.5015 1.4964 43.5282.7 1.5043 1.4992 44.01
43 DE (Ion-Exchanged) Glucose Syrup—0.03% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
78.8 1.4935 1.4884 41.9979.8 1.4962 1.4911 42.5080.8 1.4990 1.4938 43.0081.8 1.5018 1.4966 43.5082.8 1.5045 1.4994 43.99
53 DE Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
80.5 1.4962 1.4911 42.6481.5 1.4989 1.4938 43.1482.5 1.5016 1.4965 43.6483.5 1.5044 1.4992 44.1384.5 1.5072 1.5020 44.63
958 / Appendix X / General Tests and Assays FCC V
63 DE Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
81.0 1.4955 1.4904 42.5382.0 1.4982 1.4931 43.0283.0 1.5009 1.4958 43.5284.0 1.5037 1.4985 44.0185.0 1.5064 1.5012 44.50
63 DE (Ion-Exchanged) Glucose Syrup—0.03% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
81.3 1.4963 1.4912 42.6082.3 1.4990 1.4939 43.1083.3 1.5017 1.4965 43.5984.3 1.5044 1.4993 44.0985.3 1.5072 1.5020 44.58
66 DE Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
81.0 1.4949 1.4898 42.3682.0 1.4975 1.4924 42.8683.0 1.5002 1.4951 43.3684.0 1.5029 1.4978 43.8585.0 1.5056 1.5005 44.35
95 DE Glucose Syrup—0.3% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
69.0 1.4598 1.4550 35.4670.0 1.4621 1.4573 35.9671.0 1.4644 1.4596 36.4672.0 1.4668 1.4619 36.9673.0 1.4692 1.4643 37.45
95 DE (Ion-Exchanged) Glucose Syrup—0.03% Ash
RI RI °Baumé at 140°F%DS 20°C 45°C (60°C) + 1
69.0 1.4597 1.4549 35.3970.0 1.4620 1.4572 35.8971.0 1.4644 1.4595 36.3972.0 1.4667 1.4619 36.8973.0 1.4691 1.4642 37.38
High-Fructose Corn Syrup Solids
42% High-Fructose Corn Syrup—0.03% Ash
RIb RI%DSa 20°C 45°C
69.0 1.4597 1.454370.0 1.4620 1.456571.0 1.4643 1.458972.0 1.4667 1.461273.0 1.4691 1.4635aDry SubstancebRefractive Index
55% High-Fructose Corn Syrup—0.05% Ash
RIb RI%DSa 20°C 45°C
75.0 1.4738 1.468076.0 1.4762 1.470477.0 1.4786 1.472878.0 1.4811 1.475279.0 1.4835 1.4776
Liquid Fructose
RI RI%DS 20°C 45°C
75.0 1.4732 1.466776.0 1.4756 1.469177.0 1.4780 1.471578.0 1.4805 1.473979.0 1.4829 1.4763
FCC V General Tests and Assays / Appendix X / 959
Maltodextrin
12 DEa Maltodextrin—0.3% Ash
CommercialRIc RI °Baumé 140°F
%DSb 20°C 45°C (60°C) + 1
45.0 1.4149 1.4105 24.5746.0 1.4171 1.4126 25.1347.0 1.4193 1.4148 25.6848.0 1.4215 1.4170 26.2449.0 1.4237 1.4192 26.7950.0 1.4260 1.4214 27.3451.0 1.4282 1.4237 27.8952.0 1.4305 1.4259 28.4453.0 1.4328 1.4282 28.9954.0 1.4351 1.4305 29.5355.0 1.4375 1.4328 30.0856.0 1.4398 1.4351 30.6257.0 1.4422 1.4375 31.1658.0 1.4446 1.4399 31.7159.0 1.4470 1.4422 32.2460.0 1.4494 1.4446 32.7861.0 1.4519 1.4471 33.3262.0 1.4544 1.4495 33.8563.0 1.4569 1.4520 34.3964.0 1.4594 1.4545 34.9265.0 1.4619 1.4570 35.4566.0 1.4644 1.4595 35.9867.0 1.4670 1.4621 36.5168.0 1.4696 1.4646 37.0469.0 1.4722 1.4672 37.5670.0 1.4748 1.4698 38.0871.0 1.4775 1.4724 38.6172.0 1.4801 1.4751 39.1373.0 1.4828 1.4778 39.6574.0 1.4855 1.4805 40.1675.0 1.4883 1.4832 40.6876.0 1.4910 1.4859 41.1977.0 1.4938 1.4887 41.7178.0 1.4966 1.4915 42.2279.0 1.4994 1.4943 42.7380.0 1.5023 1.4971 43.2481.0 1.5051 1.4999 43.7482.0 1.5080 1.5028 44.2583.0 1.5110 1.5057 44.7584.0 1.5139 1.5086 45.2685.0 1.5168 1.5116 45.7686.0 1.5198 1.5145 46.2687.0 1.5228 1.5175 46.7688.0 1.5259 1.5206 47.2589.0 1.5289 1.5236 47.7590.0 1.5320 1.5267 48.2491.0 1.5351 1.5298 48.7392.0 1.5382 1.5329 49.2393.0 1.5414 1.5360 49.7294.0 1.5446 1.5392 50.2195.0 1.5478 1.5424 50.69aDextrose EquivalentbDry SubstancecRefractive Index
Ash and DEa Corrections for Corn Syrup and Maltodextrin:b
Changes in Refractive Index for an increase of. . .
%DSc 1% Ash 1 DE
2 0.000000 –0.0000014 0.000000 –0.0000036 0.000001 –0.0000058 0.000002 –0.00000710 0.000003 –0.00001012 0.000004 –0.00001214 0.000006 –0.00001516 0.000008 –0.00001718 0.000010 –0.00002020 0.000013 –0.00002322 0.000016 –0.00002624 0.000019 –0.00002926 0.000022 –0.00003328 0.000026 –0.00003630 0.000030 –0.00004032 0.000034 –0.00004434 0.000039 –0.00004836 0.000044 –0.00005238 0.000049 –0.00005740 0.000055 –0.00006142 0.000061 –0.00006644 0.000068 –0.00007146 0.000074 –0.00007648 0.000082 –0.00008150 0.000089 –0.00008752 0.000097 –0.00009354 0.000105 –0.00009956 0.000114 –0.00010558 0.000123 –0.00011260 0.000133 –0.00011862 0.000143 –0.00012564 0.000153 –0.00013266 0.000164 –0.00014068 0.000175 –0.00014770 0.000187 –0.00015572 0.000199 –0.00016374 0.000212 –0.00017276 0.000225 –0.00018178 0.000239 –0.00019080 0.000253 –0.00019982 0.000268 –0.00020884 0.000283 –0.000218aDextrose EquivalentbWartman, A. M., et al. J. Chemical and Engineering Data 21:467,1976.cDry Substance
960 / Appendix X / General Tests and Assays FCC V
Invert Sugar
For invert sugar, convert the refractive index to approximatepercent solids (uncorrected for invert sugar) using the accom-panying sucrose table. Correct for invert sugar by using thefollowing formula:
D = (S + C) + (P1 × 0.022),
Sucrose
International Refractive Index Scale of ICUMSAa (1974) for Pure Sucrose Solutions at 20°C and 589 nmb
Sucrose g/100 g 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
56 1.4329 4332 4334 4336 4338 4340 4343 4345 4347 434957 1.4352 4354 4356 4358 4360 4363 4365 4367 4369 437258 1.4374 4376 4378 4380 4383 4385 4387 4389 4392 439459 1.4396 4398 4401 4403 4405 4407 4410 4412 4414 441760 1.4419 4421 4423 4426 4428 4430 4432 4435 4437 4439
61 1.4442 4444 4446 4448 4451 4453 4455 4458 4460 446262 1.4464 4467 4469 4471 4474 4476 4478 4481 4483 448563 1.4488 4490 4492 4495 4497 4499 4502 4504 4506 450964 1.4511 4513 4516 4518 4520 4523 4525 4527 4530 453265 1.4534 4537 4539 4541 4544 4546 4548 4551 4553 4556
66 1.4558 4560 4563 4565 4567 4570 4572 4575 4577 457967 1.4582 4584 4586 4589 4591 4594 4596 4598 4601 460368 1.4606 4608 4610 4613 4615 4618 4620 4623 4625 462769 1.4630 4632 4635 4637 4639 4642 4644 4647 4649 465270 1.4654 4657 4659 4661 4664 4666 4669 4671 4674 4676
71 1.4679 4681 4683 4686 4688 4691 4693 4696 4698 470172 1.4703 4706 4708 4711 4713 4716 4718 4721 4723 472673 1.4728 4730 4733 4735 4738 4740 4743 4745 4748 475074 1.4753 4756 4758 4761 4763 4766 4768 4771 4773 477675 1.4778 4781 4783 4786 4788 4791 4793 4796 4798 4801
76 1.4804 4806 4809 4811 4814 4816 4819 4821 4824 482677 1.4829 4832 4834 4837 4839 4842 4844 4847 4850 485278 1.4855 4857 4860 4862 4865 4868 4870 4873 4875 487879 1.4881 4883 4886 4888 4891 4894 4896 4899 4901 490480 1.4907 4909 4912 4914 4917 4920 4922 4925 4928 4930
81 1.4933 4935 4938 4941 4943 4946 4949 4951 4954 495782 1.4959 4962 4964 4967 4970 4972 4975 4978 4980 498383 1.4986 4988 4991 4994 4996 4999 5002 5004 5007 501084 1.5012 5015 5018 5020 5023 5026 5029 5031 5034 503785 1.5039
aAdapted from ‘‘Refractometry and Tables—Official’’ (ICUMSA SPS-3 1994), International Commission for Uniform Methods of Sugar Analysis(ICUMSA), c/o British Sugar Technical Centre, Colney, Norwich NR4 7UB, England.bNo rounding has been carried out; therefore, values given may be too low by a maximum of 1 × 10–4.
in which S is the approximate percent solids determined fromthe refractive index table for sucrose, C is the temperaturecorrection derived from the accompanying temperature cor-rection table if the refractometer was operated at other than20°, and P1 is the percent invert sugar determined as directedunder Assay for Invert Sugar in this Appendix.
FCC V General Tests and Assays / Appendix X / 961
Temperature Corrections for Refractometric Sucrose Solutions with Measurements at 20° and 589 nm
Temper- Measured Sucrose (% solids)ature(°C) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Subtract from the measured value
15 0.29 0.30 0.32 0.33 0.34 0.35 0.36 0.37 0.37 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.37 0.3716 0.24 0.25 0.26 0.27 0.28 0.28 0.29 0.30 0.30 0.30 0.31 0.31 0.31 0.31 0.31 0.30 0.30 0.3017 0.18 0.19 0.20 0.20 0.21 0.21 0.22 0.22 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.2218 0.12 0.13 0.13 0.14 0.14 0.14 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.1519 0.06 0.06 0.07 0.07 0.07 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07
Add to the measured value
21 0.06 0.07 0.07 0.07 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.0722 0.13 0.14 0.14 0.14 0.15 0.15 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.15 0.15 0.1523 0.20 0.21 0.21 0.22 0.22 0.23 0.23 0.23 0.23 0.24 0.24 0.24 0.24 0.23 0.23 0.23 0.23 0.2224 0.27 0.28 0.29 0.29 0.30 0.30 0.31 0.31 0.31 0.32 0.32 0.32 0.32 0.31 0.31 0.31 0.30 0.3025 0.34 0.35 0.36 0.37 0.38 0.38 0.39 0.39 0.40 0.40 0.40 0.40 0.40 0.39 0.39 0.38 0.38 0.37
26 0.42 0.43 0.44 0.45 0.46 0.46 0.47 0.47 0.48 0.48 0.48 0.48 0.48 0.47 0.47 0.46 0.46 0.4527 0.50 0.51 0.52 0.53 0.54 0.55 0.55 0.56 0.56 0.56 0.56 0.56 0.56 0.55 0.55 0.54 0.53 0.5228 0.58 0.59 0.60 0.61 0.62 0.63 0.64 0.64 0.64 0.65 0.65 0.64 0.64 0.63 0.63 0.62 0.61 0.6029 0.66 0.67 0.68 0.70 0.71 0.71 0.72 0.73 0.73 0.73 0.73 0.73 0.72 0.72 0.71 0.70 0.69 0.6730 0.74 0.76 0.77 0.78 0.79 0.80 0.81 0.81 0.82 0.82 0.81 0.81 0.80 0.80 0.79 0.78 0.76 0.75
31 0.83 0.84 0.85 0.87 0.88 0.89 0.89 0.90 0.90 0.90 0.90 0.89 0.89 0.88 0.87 0.86 0.84 0.8232 0.92 0.93 0.94 0.96 0.97 0.98 0.98 0.99 0.99 0.99 0.99 0.98 0.97 0.96 0.95 0.93 0.92 0.9033 1.01 1.02 1.03 1.05 1.06 1.07 1.07 1.08 1.08 1.08 1.07 1.07 1.06 1.04 1.03 1.01 1.00 0.9834 1.10 1.11 1.13 1.14 1.15 1.16 1.16 1.17 1.17 1.16 1.16 1.15 1.14 1.13 1.11 1.09 1.07 1.0535 1.19 1.21 1.22 1.23 1.24 1.25 1.25 1.26 1.26 1.25 1.25 1.24 1.23 1.21 1.19 1.17 1.15 1.13
36 1.29 1.30 1.31 1.33 1.34 1.34 1.35 1.35 1.35 1.34 1.34 1.33 1.31 1.29 1.28 1.25 1.23 1.2037 1.39 1.40 1.41 1.42 1.43 1.44 1.44 1.44 1.44 1.43 1.43 1.41 1.40 1.38 1.36 1.33 1.31 1.2838 1.49 1.50 1.51 1.52 1.53 1.53 1.54 1.54 1.53 1.53 1.52 1.50 1.48 1.46 1.44 1.42 1.39 1.3639 1.59 1.60 1.61 1.62 1.63 1.63 1.63 1.63 1.63 1.62 1.61 1.59 1.57 1.55 1.52 1.50 1.47 1.4340 1.69 1.70 1.71 1.72 1.73 1.73 1.73 1.73 1.72 1.71 1.70 1.68 1.66 1.63 1.61 1.58 1.54 1.51
SOURCE: Adapted from ‘‘Refractometry and Tables—Official’’ (ICUMSA SPS-3 1994), International Commission for Uniform Methods of SugarAnalysis (ICUMSA), c/o British Sugar Technical Centre, Colney, Norwich NR4 7UB, England.
962 / Solutions and Indicators / General Tests and Assays FCC V
SOLUTIONS AND INDICATORS
The directions given for the preparation of solutions andindicators are for guidance; the use of commercially availableones is acceptable.
COLORIMETRIC SOLUTIONS (CS)
Colorimetric solutions are used in the preparation of colori-metric standards for certain chemicals and for the carboniza-tion tests with sulfuric acid that are specified in several mono-graphs. Directions for the preparation of the primarycolorimetric solutions and Matching Fluids are given underthe test for Readily Carbonizable Substances, Appendix IIB.Store the solutions in suitably resistant, tight containers.
Comparison of colors as directed in the Food ChemicalsCodex tests is preferably made in matched color-comparisontubes or in a suitable colorimeter under conditions that ensurethat the colorimetric reference solution and that of the speci-men under test are treated alike in all respects.
STANDARD BUFFER SOLUTIONS
Reagent Solutions Before mixing, dry the crystalline re-agents, except the boric acid, at 110° to 120°, and use water
Composition of Standard Buffer Solutions
Hydrochloric Acid Phthalate Neutralized Phosphate AlkalineAcid Buffer Buffer Phthalate Buffer Buffer Borate Buffer
To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M To 50.0 mL of 0.2 MKCl add the mL of HCl KHC6H4(COO)2 add the KHC6H4(COO)2 add the KH2PO4 add the mL of H3BO3-KCl add the mLspecified mL of HCl specified mL of NaOH specified NaOH specified of NaOH specified
pH 0.2 M HCl (mL) pH 0.2 M HCl (mL) pH 0.2 M NaOH (mL) pH 0.2 M NaOH (mL) pH 0.2 M NaOH (mL)
1.2 85.0 2.2 49.5 4.2 3.0 5.8 3.6 8.0 3.91.3 67.2 2.4 42.2 4.4 6.6 6.0 5.6 8.2 6.01.4 53.2 2.6 35.4 4.6 11.1 6.2 8.1 8.4 8.61.5 41.4 2.8 28.9 4.8 16.5 6.4 11.6 8.6 11.81.6 32.4 3.0 22.3 5.0 22.6 6.6 16.4 8.8 15.81.7 26.0 3.2 15.7 5.2 28.8 6.8 22.4 9.0 20.81.8 20.4 3.4 10.4 5.4 34.1 7.0 29.1 9.2 26.41.9 16.2 3.6 6.3 5.6 38.8 7.2 34.7 9.4 32.12.0 13.0 3.8 2.9 5.8 42.3 7.4 39.1 9.6 36.92.1 10.2 4.0 0.1 — — 7.6 42.4 9.8 40.62.2 7.8 — — — — 7.8 44.5 10.0 43.7
8.0 46.1 — —
Dilute all final solutions to 200.0 mL (see Procedure). The standard pH values given in this table are considered to be reproducible towithin �0.02 of the pH unit specified at 25°.
that has been previously boiled and cooled in preparing thesolutions. Store the prepared reagent solutions in chemicallyresistant glass or polyethylene bottles, and use within 3months. Discard if molding is evident.
Potassium Chloride, 0.2 M Dissolve 14.91 g of potassiumchloride (KCl) in sufficient water to make 1000.0 mL.
Potassium Biphthalate, 0.2 M Dissolve 40.84 g of potas-sium biphthalate [KHC6H4(COO)2] in sufficient water to make1000.0 mL.
Potassium Phosphate, Monobasic, 0.2 M Dissolve 27.22g of monobasic potassium phosphate (KH2PO4) in sufficientwater to make 1000.0 mL.
Boric Acid–Potassium Chloride, 0.2 M Dissolve 12.37 gof boric acid (H3BO3) and 14.91 g of potassium chloride(KCl) in sufficient water to make 1000.0 mL.
Hydrochloric Acid, 0.2 M, and Sodium Hydroxide, 0.2M Prepare and standardize as directed under VolumetricSolutions in this section.
Procedure To prepare 200 mL of a standard buffer solutionhaving a pH within the range 1.2 to 10.0, place 50.0 mL ofthe appropriate 0.2 M salt solution, prepared as above, in a200-mL volumetric flask, add the volume of 0.2 M hydrochlo-ric acid or of sodium hydroxide specified for the desiredpH in the accompanying table, dilute to volume with water,and mix.
FCC V General Tests and Assays / Solutions and Indicators / 963
STANDARD SOLUTIONS FOR THEPREPARATION OF CONTROLS ANDSTANDARDS
The following solutions are used in tests for impurities thatrequire the comparison of the color or turbidity produced ina solution of the test substance with that produced by a knownamount of the impurity in a control. Directions for the prepara-tion of other standard solutions are given in the monographsor under the general tests in which they are required (see alsoIndex).
Ammonium Standard Solution (10 �g NH4 in 1 mL) Dis-solve 296.0 mg of ammonium chloride (NH4Cl) in sufficientwater to make 100.0 mL, and mix. Transfer 10.0 mL of thissolution into a 1000-mL volumetric flask, dilute to volumewith water, and mix.
Barium Standard Solution (100 �g Ba in 1 mL) Dissolve177.9 mg of barium chloride (BaCl2·2H2O) in water in a 1000-mL volumetric flask, dilute to volume with water, and mix.
Iron Standard Solution (10 �g Fe in 1 mL) Dissolve 702.2mg of ferrous ammonium sulfate [Fe(NH4)2(SO4)2·6H2O] in10 mL of 2 N sulfuric acid in a 100-mL volumetric flask,dilute to volume with water, and mix. Transfer 10.0 mL ofthis solution into a 1000-mL volumetric flask, add 10 mL of2 N sulfuric acid, dilute to volume with water, and mix.
Magnesium Standard Solution (50 �g Mg in 1 mL) Dis-solve 50.0 mg of magnesium metal (Mg) in 1 mL of hydrochlo-ric acid in a 1000-mL volumetric flask, dilute to volume withwater, and mix.
Phosphate Standard Solution (10 �g PO4 in 1 mL) Dis-solve 143.3 mg of monobasic potassium phosphate (KH2PO4)in water in a 100-mL volumetric flask, dilute to volume withwater, and mix. Transfer 10.0 mL of this solution into a 1000-mL volumetric flask, dilute to volume with water, and mix.
TEST SOLUTIONS (TS) AND OTHERREAGENTS
Certain of the following test solutions are intended for useas acid–base indicators in volumetric analyses. Such solutionsshould be adjusted so that when 0.15 mL of the indicatorsolution is added to 25 mL of carbon dioxide-free water, 0.25mL of 0.02 N acid or alkali, respectively, will produce thecharacteristic color change.
In general, the directive to prepare a solution ‘‘fresh’’ indi-cates that the solution is of limited stability and must beprepared on the day of use.
Acetic Acid (approximately 17.5 N) Use ACS reagent-gradeAcetic Acid, Glacial (99.7% of CH3COOH).
Acetic Acid TS, Diluted (1 N) A solution containing about6% (w/v) of CH3COOH. Prepare by diluting 60.0 mL ofglacial acetic acid, or 166.6 mL of 36% acetic acid (6 N),with sufficient water to make 1000 mL.
Acetic Acid TS, Strong (5 N) A solution containing 30%(v/v) of CH3COOH. Prepare by diluting 300.0 mL of glacialacetic acid with sufficient water to make 1000 mL.
Alcohol (Ethanol; Ethyl Alcohol; C2H5OH) Use ACS re-agent-grade Ethyl Alcohol (not less than 95.0%, by volume,of C2H5OH).
Note: For use in assays and tests involving ultravioletspectrophotometry, use ACS reagent-grade Ethyl Alco-hol Suitable for Use in Ultraviolet Spectrophotometry.
Alcohol, Absolute (Anhydrous Alcohol; Dehydrated Alco-hol) Use ACS reagent-grade Ethyl Alcohol, Absolute (notless than 99.5%, by volume, of C2H5OH).
Alcohol, Diluted A solution containing 41.0% to 42.0%,by weight, corresponding to 48.4% to 49.5%, by volume, at15.56°, of C2H5OH.
Alcohol, 70% (at 15.56°) A 38.6:15 mixture (v/v) of 95%alcohol and water, having a specific gravity of 0.884 at 25°.To prepare 100 mL, dilute 73.7 mL of alcohol to 100 mLwith water at 25°.
Alcohol, 80% (at 15.56°) A 45.5:9.5 mixture (v/v) of 95%alcohol and water, having a specific gravity of 0.857 at 25°.To prepare 100 mL, dilute 84.3 mL of alcohol to 100 mLwith water at 25°.
Alcohol, 90% (at 15.56°) A 51:3 mixture (v/v) of 95%alcohol and water, having a specific gravity of 0.827 at 25°.To prepare 100 mL, dilute 94.8 mL of alcohol to 100 mLwith water at 25°.
Alcohol, Aldehyde-Free Dissolve 2.5 g of lead acetate in5 mL of water, add the solution to 1000 mL of alcohol con-tained in a glass-stoppered bottle, and mix. Dissolve 5 g ofpotassium hydroxide in 25 mL of warm alcohol, cool, andadd slowly, without stirring, to the alcoholic solution of leadacetate. Allow to stand for 1 h, then shake the mixture vigor-ously, allow to stand overnight, decant the clear liquid, andrecover the alcohol by distillation. Ethyl Alcohol FCC, AlcoholUSP, or USSD #3A or #30 may be used. If the titration of a250-mL sample of the alcohol by Hydroxylamine Hydrochlo-ride TS does not exceed 0.25 mL of 0.5 N alcoholic potassiumhydroxide, the above treatment may be omitted.
Alcoholic Potassium Hydroxide TS See Potassium Hy-droxide TS, Alcoholic.
964 / Solutions and Indicators / General Tests and Assays FCC V
Alkaline Cupric Tartrate TS (Fehling’s Solution) See Cu-pric Tartrate TS, Alkaline.
Alkaline Mercuric Potassium Iodide TS (Nessler’s Re-agent) See Mercuric Potassium Iodide TS, Alkaline.
Ammonia–Ammonium Chloride Buffer TS (approximatelypH 10) Dissolve 67.5 g of ammonium chloride (NH4Cl) inwater, add 570 mL of ammonium hydroxide (28%), and dilutewith water to 1000 mL.
Ammonia TS (6 N in NH3) A solution containing between9.5% and 10.5% of NH3. Prepare by diluting 400 mL ofammonium hydroxide (28%) with sufficient water to make1000 mL.
Ammonia TS, Stronger (15.2 N in NH3) (Ammonium Hy-droxide; Stronger Ammonia Water) Use ACS reagent-gradeAmmonium Hydroxide, which is a practically saturated solu-tion of ammonia in water, containing between 28% and 30%of NH3.
Ammoniacal Silver Nitrate TS Add 6 N ammonium hy-droxide, dropwise, to a 1:20 solution of silver nitrate untilthe precipitate that first forms is almost, but not entirely,dissolved. Filter the solution, and place in a dark bottle.
Caution: Ammoniacal Silver Nitrate TS forms explo-sive compounds on standing. Do not store this solution,but prepare a fresh quantity for each series of determina-tions. Neutralize the excess reagent and rinse all glass-ware with hydrochloric acid immediately after complet-ing a test.
Ammonium Acetate TS Dissolve 10 g of ammonium ace-tate (NH4C2H3O2) in sufficient water to make 100 mL.
Ammonium Carbonate TS Dissolve 20 g of ammoniumcarbonate and 20 mL of Ammonia TS in sufficient water tomake 100 mL.
Ammonium Chloride TS Dissolve 10.5 g of ammoniumchloride (NH4Cl) in sufficient water to make 100 mL.
Ammonium Molybdate TS Dissolve 6.5 g of finely pow-dered molybdic acid (85%) in a mixture of 14 mL of waterand 14.5 mL of ammonium hydroxide. Cool the solution, andadd it slowly, with stirring, to a well-cooled mixture of 32mL of nitric acid and 40 mL of water. Allow to stand for 48h, and filter through a fine-porosity, sintered-glass cruciblelined at the bottom with a layer of glass wool. This solutiondeteriorates upon standing and is unsuitable for use if, uponthe addition of 2 mL of Sodium Phosphate TS to 5 mL of thesolution, an abundant yellow precipitate does not form at onceor after slight warming. Store it in the dark. If a precipitateforms during storage, use only the clear, supernatant solution.
Ammonium Oxalate TS Dissolve 3.5 g of ammonium oxa-late [(NH4)2C2O4·H2O] in sufficient water to make 100 mL.
Ammonium Sulfanilate TS To 2.5 g of sulfanilic acid add15 mL of water and 3 mL of 6 N ammonium hydroxide, andmix. Add, with stirring, more 6 N ammonium hydroxide, ifnecessary, until the acid dissolves, adjust the pH of the solutionto about 4.5 with 2.7 N hydrochloric acid, using BromocresolGreen TS as an outside indicator, and dilute to 25 mL.
Ammonium Sulfide TS Saturate 6 N ammonium hydroxidewith hydrogen sulfide (H2S), and add two-thirds of its volumeof 6 N ammonium hydroxide. Residue upon ignition: not morethan 0.05%. The solution is not rendered turbid either byMagnesium Sulfate TS or by Calcium Chloride TS (carbon-ate). This solution is unsuitable for use if an abundant precipi-tate of sulfur is present. Store it in small, well-filled, darkamber-colored bottles in a cold, dark place.
Ammonium Thiocyanate TS (1 N) Dissolve 8 g of ammo-nium thiocyanate (NH4SCN) in sufficient water to make100 mL.
Anthrone TS Carefully dissolve about 0.1 g of anthrone in100 g of sulfuric acid. Use a freshly prepared solution.
Antimony Trichloride TS Dissolve 20 g of antimony tri-chloride (SbCl3) in chloroform to make 100 mL. Filter ifnecessary.
Barium Chloride TS Dissolve 12 g of barium chloride(BaCl2·2H2O) in sufficient water to make 100 mL.
Barium Diphenylamine Sulfonate TS Dissolve 300 mg ofp-diphenylamine sulfonic acid barium salt in 100 mL of water.
Barium Hydroxide TS Use a saturated solution of bariumhydroxide in recently boiled water. Use a freshly preparedsolution.
Benedict’s Qualitative Reagent See Cupric Citrate TS, Al-kaline.
Benzidine TS Dissolve 50 mg of benzidine in 10 mL ofglacial acetic acid, dilute to 100 mL with water, and mix.
Bismuth Nitrate TS Reflux 5 g of bismuth nitrate [Bi-(NO3)3·5H2O] with 7.5 mL of nitric acid and 10 mL of wateruntil dissolved, cool, filter, and dilute to 250 mL with water.
Bromine TS (Bromine Water) Prepare a saturated solutionof bromine by agitating 2 to 3 mL of bromine (Br2) with 100mL of cold water in a glass-stoppered bottle, the stopper ofwhich should be lubricated with petrolatum. Store it in a coldplace protected from light.
Bromocresol Blue TS Use Bromocresol Green TS.
Bromocresol Green TS Dissolve 50 mg of bromocresolgreen in 100 mL of alcohol, and filter if necessary.
FCC V General Tests and Assays / Solutions and Indicators / 965
Bromocresol Purple TS Dissolve 250 mg of bromocresolpurple in 20 mL of 0.05 N sodium hydroxide, and dilute withwater to 250 mL.
Bromophenol Blue TS Dissolve 100 mg of bromophenolblue in 100 mL of 1:2 alcohol, and filter if necessary.
Bromothymol Blue TS Dissolve 100 mg of bromothymolblue in 100 mL of 1:2 alcohol, and filter if necessary.
Calcium Chloride TS Dissolve 7.5 g of calcium chloride(CaCl2·2H2O) in sufficient water to make 100 mL.
Calcium Hydroxide TS A solution containing approxi-mately 140 mg of Ca(OH)2 in each 100 mL. To prepare, add3 g of calcium hydroxide [Ca(OH)2] to 1000 mL of water,and agitate the mixture vigorously and repeatedly for 1 h.Allow the excess calcium hydroxide to settle, and decant ordraw off the clear, supernatant liquid.
Calcium Sulfate TS A saturated solution of calcium sulfatein water.
Carr-Price Reagent See Antimony Trichloride TS.
Ceric Ammonium Nitrate TS Dissolve 6.25 g of cericammonium nitrate [(NH4)2Ce(NO3)6] in 100 mL of 0.25 Nnitric acid. Prepare the solution fresh every third day.
Chlorine TS (Chlorine Water) A saturated solution of chlo-rine in water. Place the solution in small, completely filled,light-resistant containers. Chlorine TS, even when kept fromlight and air, is apt to deteriorate. Store it in a cold, darkplace. For full strength, prepare this solution fresh.
Chromotropic Acid TS Dissolve 50 mg of chromotropicacid or its sodium salt in 100 mL of 75% sulfuric acid (madeby cautiously adding 75 mL of 95% to 98% sulfuric acid to33.3 mL of water).
Cobaltous Chloride TS Dissolve 2 g of cobaltous chloride(CoCl2·6H2O) in 1 mL of hydrochloric acid and sufficientwater to make 100 mL.
Cobalt–Uranyl Acetate TS Dissolve, with warming, 40 gof uranyl acetate [UO2(C2H3O2)2·2H2O] in a mixture of 30 gof glacial acetic acid and sufficient water to make 500 mL.Similarly, prepare a solution containing 200 g of cobaltousacetate [Co(C2H3O2)2·4H2O] in a mixture of 30 g of glacialacetic acid and sufficient water to make 500 mL. Mix thetwo solutions while still warm, and cool to 20°. Maintain thetemperature at 20° for about 2 h to separate the excess saltsfrom solution, and then filter through a dry filter.
Congo Red TS Dissolve 500 mg of congo red in a mixtureof 10 mL of alcohol and 90 mL of water.
Copper Sulfate TS Dissolve 12.5 g of cupric sulfate insufficient water to make 100 mL.
Cresol Red TS Triturate 100 mg of cresol red in a mortarwith 26.2 mL of 0.01 N sodium hydroxide until solution iscomplete, then dilute the solution with water to 250 mL.
Cresol Red–Thymol Blue TS Add 15 mL of Thymol BlueTS to 5 mL of Cresol Red TS, and mix.
Crystal Violet TS Dissolve 100 mg of crystal violet in 10mL of glacial acetic acid.
Cupric Citrate TS, Alkaline (Benedict’s Qualitative Re-agent) With the aid of heat, dissolve 173 g of sodium citrate(C6H5Na3O7·2H2O) and 117 g of sodium carbonate (Na2-CO3·H2O) in about 700 mL of water, and filter through paper,if necessary. In a separate container, dissolve 17.3 g of cupricsulfate (CuSO4·5H2O) in about 100 mL of water, and slowlyadd this solution, with constant stirring, to the first solution.Cool the mixture, dilute to 1000 mL, and mix.
Cupric Nitrate TS Dissolve 2.4 g of cupric nitrate [Cu-(NO3)2·3H2O] in sufficient water to make 100 mL.
Cupric Sulfate TS Dissolve 12.5 g of cupric sulfate (Cu-SO4·5H2O) in sufficient water to make 100 mL, and mix.
Cupric Tartrate TS, Alkaline (Fehling’s Solution) TheCopper Solution (A): Dissolve 34.66 g of carefully selected,small crystals of cupric sulfate, CuSO4·5H2O, showing notrace of efflorescence or of adhering moisture, in sufficientwater to make 500 mL. Store this solution in small, tightcontainers. The Alkaline Tartrate Solution (B): Dissolve 173 gof crystallized potassium sodium tartrate (KNaC4H4O6·4H2O)and 50 g of sodium hydroxide (NaOH) in sufficient water tomake 500 mL. Store this solution in small, alkali-resistantcontainers. For use, mix exactly equal volumes of solutionsA and B at the time required.
Cyanogen Bromide TS Dissolve 5 g of cyanogen bromidein water to make 50 mL.
Caution: Prepare this solution in a hood, as cyanogenbromide volatilizes at room temperature, and the vaporis highly irritating and poisonous.
Denigès’ Reagent See Mercuric Sulfate TS.
Dichlorophenol–Indophenol TS Warm 100 mg of 2,6-di-chlorophenol–indophenol sodium with 100 mL of water. Filterand use within 3 days.
2,7-Dihydroxynaphthalene TS Dissolve 100 mg of 2,7-dihydroxynaphthalene in 1000 mL of sulfuric acid, and allowthe solution to stand until the initial color disappears. If thesolution is very dark, discard it and prepare a new solutionfrom a different supply of sulfuric acid. This solution is stablefor approximately 1 month if stored in a dark bottle.
Diphenylamine TS Dissolve 1 g of diphenylamine in 100mL of sulfuric acid. The solution should be colorless.
966 / Solutions and Indicators / General Tests and Assays FCC V
Diphenylcarbazone TS Dissolve about 1 g of diphenylcar-bazone (C13H12N4O) in sufficient alcohol to make 100 mL.Store this solution in a brown bottle.
�,�-Dipyridyl TS Dissolve 100 mg of �,�-dipyridyl(C10H8N2) in 50 mL of absolute alcohol.
Dithizone TS Dissolve 25.6 mg of dithizone in 100 mL ofalcohol.
Eosin Y TS (adsorption indicator) Dissolve 50 mg of eosinY in 10 mL of water.
Eriochrome Black TS Dissolve 200 mg of eriochromeblack T and 2 g of hydroxylamine hydrochloride(NH2OH·HCl) in sufficient methanol to make 50 mL, andfilter. Store the solution in a light-resistant container and usewithin 2 weeks.
p-Ethoxychrysoidin TS Dissolve 50 mg of p-ethoxychry-soidin monohydrochloride in a mixture of 25 mL of waterand 25 mL of alcohol, add 3 drops of hydrochloric acid, stirvigorously, and filter if necessary to obtain a clear solution.
Fehling’s Solution See Cupric Tartrate TS, Alkaline.
Ferric Ammonium Sulfate TS Dissolve 8 g of ferric am-monium sulfate [FeNH4(SO4)2·12H2O] in sufficient water tomake 100 mL.
Ferric Chloride TS Dissolve 9 g of ferric chloride(FeCl3·6H2O) in sufficient water to make 100 mL.
Ferric Chloride TS, Alcoholic Dissolve 100 mg of ferricchloride (FeCl3·6H2O) in 50 mL of absolute alcohol. Preparethis solution fresh.
Ferric Sulfate TS, Acid Add 7.5 mL of sulfuric acid to100 mL of water, and dissolve 80 g of ferrous sulfate in themixture with the aid of heat. Mix 7.5 mL of nitric acid and20 mL of water, warm, and add to this the ferrous sulfatesolution. Concentrate the mixture until, upon the sudden disen-gagement of ruddy vapors, the black color of the liquidchanges to red. Test for the absence of ferrous iron, and, ifnecessary, add a few drops of nitric acid and heat again. Whenthe solution is cold, add sufficient water to make 110 mL.
Ferrous Sulfate TS Dissolve 8 g of clear crystals of ferroussulfate (FeSO4·7H2O) in about 100 mL of recently boiled andthoroughly cooled water. Prepare this solution fresh.
Formaldehyde TS A solution containing approximately37.0% (w/v) of HCHO. It may contain methanol to preventpolymerization.
Fuchsin–Sulfurous Acid TS Dissolve 200 mg of basicfuchsin in 120 mL of hot water, and allow the solution tocool. Add a solution of 2 g of anhydrous sodium sulfite in
20 mL of water, and then add 2 mL of hydrochloric acid.Dilute the solution with water to 200 mL, and allow to standfor at least 1 h. Prepare this solution fresh.
Hydrochloric Acid (approximately 12 N) Use ACS reagent-grade Hydrochloric Acid (36.5% to 38.0% of HCl).
Hydrochloric Acid TS, Diluted (2.7 N) A solution con-taining 10% (w/v) of HCl. Prepare by diluting 226 mL ofhydrochloric acid (36%) with sufficient water to make1000 mL.
Hydrogen Peroxide TS A solution containing between 2.5and 3.5 g of H2O2 in each 100 mL. It may contain suitablepreservatives, totaling not more than 0.05%.
Hydrogen Sulfide TS A saturated solution of hydrogensulfide made by passing H2S into cold water. Store it in small,dark, amber-colored bottles, filled nearly to the top. It isunsuitable unless it possesses a strong odor of H2S, and unlessit produces at once a copious precipitate of sulfur when addedto an equal volume of Ferric Chloride TS. Store in a cold,dark place.
Hydroxylamine Hydrochloride TS Dissolve 3.5 g of hy-droxylamine hydrochloride (NH2OH·HCl) in 95 mL of 60%alcohol, and add 0.5 mL of a 1:1000 solution of bromophenolblue and 0.5 N alcoholic potassium hydroxide until a greentint develops in the solution. Then add sufficient 60% alcoholto make 100 mL.
8-Hydroxyquinoline TS Dissolve 5 g of 8-hydroxyquino-line (oxine) in sufficient alcohol to make 100 mL.
Indigo Carmine TS (Sodium Indigotindisulfonate TS) Dis-solve a quantity of sodium indigotindisulfonate, equivalent to180 mg of C16H8N2O2(SO3Na)2, in sufficient water to make100 mL. Use within 60 days.
Iodine TS Dissolve 14 g of iodine (I2) in a solution of 36g of potassium iodide (KI) in 100 mL of water, add 3 dropsof hydrochloric acid, dilute with water to 1000 mL, and mix.
Isopropanol [Isopropyl Alcohol; 2-Propanol; (CH3)2CHOH]Use ACS reagent-grade Isopropyl Alcohol.
Note: For use in assays and tests involving ultravioletspectrophotometry, use ACS reagent-grade IsopropylAlcohol Suitable for Use in Ultraviolet Spectropho-tometry.
Isopropanol, Anhydrous (Dehydrated Isopropanol) Useisopropanol that has been previously dried by shaking withanhydrous calcium chloride, followed by filtering.
Lead Acetate TS Dissolve 9.5 g of clear, transparent crys-tals of lead acetate [Pb(C2H3O2)2·3H2O] in sufficient recentlyboiled water to make 100 mL. Store in well-stoppered bottles.
FCC V General Tests and Assays / Solutions and Indicators / 967
Lead Subacetate TS Triturate 14 g of lead monoxide (PbO)to a smooth paste with 10 mL of water, and transfer themixture to a bottle, using an additional 10 mL of water forrinsing. Dissolve 22 g of lead acetate [Pb(C2H3O2)2·3H2O] in70 mL of water, and add the solution to the lead oxide mixture.Shake it vigorously for 5 min, then set it aside, shaking itfrequently during 7 days. Finally, filter, and add enough re-cently boiled water through the filter to make 100 mL.
Lead Subacetate TS, Diluted Dilute 3.25 mL of Lead Sub-acetate TS with sufficient water, recently boiled and cooled,to make 100 mL. Store in small, well-fitted, tight containers.
Litmus TS Digest 25 g of powdered litmus with three suc-cessive 100-mL portions of boiling alcohol, continuing eachextraction for about 1 h. Filter, wash with alcohol, and discardthe alcohol filtrate. Macerate the residue with about 25 mLof cold water for 4 h, filter, and discard the filtrate. Finally,digest the residue with 125 mL of boiling water for 1 h, cool,and filter.
Magnesia Mixture TS Dissolve 5.5 g of magnesium chlo-ride (MgCl2·6H2O) and 7 g of ammonium chloride (NH4Cl)in 65 mL of water, add 35 mL of 6 N ammonium hydroxide,set the mixture aside for a few days in a well-stopperedbottle, and filter. If the solution is not perfectly clear, filterit before using.
Magnesium Sulfate TS Dissolve 12 g of crystals of magne-sium sulfate (MgSO4·7H2O), selected for freedom from efflo-rescence, in water to make 100 mL.
Malachite Green TS Dissolve 1 g of malachite green oxa-late in 100 mL of glacial acetic acid.
Mayer’s Reagent See Mercuric–Potassium Iodide TS.
Mercuric Acetate TS Dissolve 6 g of mercuric acetate[Hg(C2H3O2)2] in sufficient glacial acetic acid to make 100mL. Store in tight containers protected from direct sunlight.
Mercuric Chloride TS Dissolve 6.5 g of mercuric chloride(HgCl2) in water to make 100 mL.
Mercuric–Potassium Iodide TS (Mayer’s Reagent) Dis-solve 1.358 g of mercuric chloride (HgCl2) in 60 mL of water.Dissolve 5 g of potassium iodide (KI) in 10 mL of water.Mix the two solutions, and add water to make 100 mL.
Mercuric–Potassium Iodide TS, Alkaline (Nessler’s Reagent)Dissolve 10 g of potassium iodide (KI) in 10 mL of water, andadd slowly, with stirring, a saturated solution of mercuric chlorideuntil a slight red precipitate remains undissolved. To this mixtureadd an ice-cold solution of 30 g of potassium hydroxide (KOH)in 60 mL of water, then add 1 mL more of the saturated solutionof mercuric chloride. Dilute with water to 200 mL. Allow theprecipitate to settle, and draw off the clear liquid. A 2-mL portionof this reagent, when added to 100 mL of a 1:300,000 solution
of ammonium chloride in ammonia-free water, instantly producesa yellow-brown color.
Mercuric Sulfate TS (Denigès’ Reagent) Mix 5 g of yellowmercuric oxide (HgO) with 40 mL of water, and while stirring,slowly add 20 mL of sulfuric acid, then add another 40 mLof water, and stir until completely dissolved.
Mercurous Nitrate TS Dissolve 15 g of mercurous nitratein a mixture of 90 mL of water and 10 mL of 2 N nitric acid.Store in dark, amber-colored bottles in which a small globuleof mercury has been placed.
Methanol (Methyl Alcohol) Use ACS reagent-gradeMethanol.
Methanol, Anhydrous (Dehydrated Methanol) UseMethanol.
p-Methylaminophenol Sulfate TS Dissolve 2 g of p-meth-ylaminophenol sulfate [(HOC6H4NHCH3)2·H2SO4] in 100 mLof water. To 10 mL of this solution add 90 mL of water and20 g of sodium bisulfite. Confirm the suitability of this solutionby the following test: Add 1 mL of the solution to each offour tubes containing 25 mL of 0.5 N sulfuric acid and 1 mLof Ammonium Molybdate TS. Add 5 �g of phosphate (PO4)to one tube, 10 �g to a second, and 20 �g to a third, using0.5, 1.0, and 2.0 mL, respectively, of Phosphate StandardSolution, and allow to stand for 2 h. The solutions in the threetubes should show readily perceptible differences in blue colorcorresponding to the relative amounts of phosphate added,and the one to which 5 �g of phosphate was added shouldbe perceptibly bluer than the blank.
Methylene Blue TS Dissolve 125 mg of methylene blue in100 mL of alcohol, and dilute with alcohol to 250 mL.
Methyl Orange TS Dissolve 100 mg of methyl orange in100 mL of water, and filter if necessary.
Methyl Red TS Dissolve 100 mg of methyl red in 100 mLof alcohol, and filter if necessary.
Methyl Red–Methylene Blue TS Add 10 mL of MethylRed TS to 10 mL of Methylene Blue TS, and mix.
Methylrosaniline Chloride TS See Crystal Violet TS.
Methyl Violet TS See Crystal Violet TS.
Millon’s Reagent To 2 mL of mercury in an Erlenmeyerflask add 20 mL of nitric acid. Shake the flask in a hood tobreak the mercury into small globules. After about 10 minadd 35 mL of water, and if a precipitate or crystals appear,add sufficient 1:5 nitric acid (prepared from nitric acid fromwhich the oxides have been removed by blowing air throughit until it is colorless) to dissolve the separated solid. Add a1:10 solution of sodium hydroxide, dropwise, with thorough
968 / Solutions and Indicators / General Tests and Assays FCC V
mixing, until the curdy precipitate that forms after the additionof each drop no longer redissolves but is dispersed to forma suspension. Add 5 mL more of the dilute nitric acid, andmix well. Prepare this solution fresh.
�-Naphtholbenzein TS Dissolve 0.2 g of �-naphtholbenz-ein in glacial acetic acid to make 100 mL. Sensitivity: Add100 mL of freshly boiled and cooled water to 0.2 mL of a1:1000 solution of �-naphtholbenzein in ethanol, and add 0.1mL of 0.1 N sodium hydroxide: a green color develops. Addsubsequently 0.2 mL of 0.1 N hydrochloric acid: the color ofthe solution changes to yellow-red.
Naphthol Green TS Dissolve 500 mg of naphthol green Bin water to make 1000 mL.
Nessler’s Reagent See Alkaline Mercuric–Potassium Io-dide TS.
Neutral Red TS Dissolve 100 mg of neutral red in 100 mLof 50% alcohol.
Nickel Standard Solution TS (10 mg/kg) Prepare a 0.40%(w/v) solution of analytical reagent-grade nickel chloride(NiCl2·6H2O) with water. Pipet 1.0 mL of the solution intoa 100-mL volumetric flask, and dilute to volume with water.
Ninhydrin TS See Triketohydrindene Hydrate TS.
Nitric Acid (approximately 15.7 N) Use ACS reagent-gradeNitric Acid (69.0% to 71.0% of HNO3).
Nitric Acid TS, Diluted (1.7 N) A solution containing about10% (w/v) of HNO3. Prepare by diluting 105 mL of nitricacid (70%) with water to make 1000 mL.
Orthophenanthroline TS Dissolve 150 mg of orthophen-anthroline (C12H8N2·H2O) in 10 mL of a solution of ferroussulfate, prepared by dissolving 700 mg of clear crystals offerrous sulfate (FeSO4·7H2O) in 100 mL of water. The ferroussulfate solution must be prepared immediately before dissolv-ing the orthophenanthroline. Store the solution in well-closedcontainers.
Oxalic Acid TS Dissolve 6.3 g of oxalic acid (H2C2O4-·2H2O) in water to make 100 mL.
Phenol Red TS (Phenolsulfonphthalein TS) Dissolve 100mg of phenolsulfonphthalein in 100 mL of alcohol, and filterif necessary.
Phenolphthalein TS Dissolve 1 g of phenolphthalein in100 mL of alcohol.
Phenolsulfonphthalein TS See Phenol Red TS.
p-Phenylphenol TS On the day of use, dissolve 750 mg ofp-phenylphenol in 50 mL of Sodium Hydroxide TS.
Phosphoric Acid Use ACS reagent-grade Phosphoric Acid(not less than 85.0% of H3PO4).
Phosphotungstic Acid TS Dissolve 1 g of phosphotungsticacid (approximately 24WO3·2H3PO4·48H2O) in water to make100 mL.
Picric Acid TS See Trinitrophenol TS.
Potassium Acetate TS Dissolve 10 g of potassium acetate(KC2H3O2) in water to make 100 mL.
Potassium Chromate TS Dissolve 10 g of potassium chro-mate (K2CrO4) in water to make 100 mL.
Potassium Dichromate TS Dissolve 7.5 g of potassiumdichromate (K2Cr2O7) in water to make 100 mL.
Potassium Ferricyanide TS (10%) Dissolve 1 g of potas-sium ferricyanide [K3Fe(CN)6] in 10 mL of water. Preparethis solution fresh.
Potassium Ferrocyanide TS Dissolve 1 g of potassiumferrocyanide [K4Fe(CN)6·3H2O] in 10 mL of water. Preparethis solution fresh.
Potassium Hydroxide TS (1 N) Dissolve 6.5 g of potassiumhydroxide (KOH) in water to make 100 mL.
Potassium Hydroxide TS, Alcoholic Use 0.5 N AlcoholicPotassium Hydroxide (see Volumetric Solutions in thissection).
Potassium Iodide TS Dissolve 16.5 g of potassium iodide(KI) in water to make 100 mL. Store in light-resistant con-tainers.
Potassium Permanganate TS Use 0.1 N Potassium Per-manganate (see Volumetric Solutions in this section).
Potassium Pyroantimonate TS Dissolve 2 g of potassiumpyroantimonate in 95 mL of hot water. Cool quickly, and adda solution containing 2.5 g of potassium hydroxide in 50 mLof water and 1 mL of an 8.5:100 solution of sodium hydroxide.Allow to stand for 24 h, filter, and dilute with water to 150 mL.
Potassium Sulfate TS Dissolve 1 g of potassium sulfate(K2SO4) in sufficient water to make 100 mL.
Quimociac TS Dissolve 70 g of sodium molybdate (Na2-MoO4·2H2O) in 150 mL of water (Solution A). Dissolve 60g of citric acid in a mixture of 85 mL of nitric acid and 150mL of water, and cool (Solution B). Gradually add SolutionA to Solution B, with stirring, to produce Solution C. Dissolve5.0 mL of natural or synthetic quinoline in a mixture of 35mL of nitric acid and 100 mL of water (Solution D). Graduallyadd Solution D to Solution C, mix well, and allow to standovernight. Filter the mixture, add 280 mL of acetone to the
FCC V General Tests and Assays / Solutions and Indicators / 969
filtrate, dilute to 1000 mL with water, and mix. Store in apolyethylene bottle.
Caution: This reagent contains acetone. Do not use itnear an open flame. Operations involving heating orboiling should be conducted in a well-ventilated hood.
Quinaldine Red TS Dissolve 100 mg of quinaldine red in100 mL of glacial acetic acid.
Schiff’s Reagent, Modified Dissolve 200 mg of rosanilinehydrochloride (C20H20ClN3) in 120 mL of hot water. Cool,add 2 g of sodium bisulfite (NaHSO3) followed by 2 mL ofhydrochloric acid, and dilute to 200 mL with water. Store ina brown bottle at 15° or lower.
Silver Nitrate TS Use 0.1 N Silver Nitrate (see VolumetricSolutions in this section).
Sodium Bisulfite TS Dissolve 10 g of sodium bisulfite(NaHSO3) in water to make 30 mL. Prepare this solution fresh.
Sodium Bitartrate TS Dissolve 1 g of sodium bitartrate(NaHC4H4O6·H2O) in water to make 10 mL. Prepare thissolution fresh.
Sodium Borate TS Dissolve 2 g of sodium borate (Na2-B4O7·10H2O) in water to make 100 mL.
Sodium Carbonate TS Dissolve 10.6 g of anhydrous so-dium carbonate (Na2CO3) in water to make 100 mL.
Sodium Cobaltinitrite TS Dissolve 10 g of sodium cobalti-nitrite [Na3Co(NO2)6] in water to make 50 mL, and filter ifnecessary.
Sodium Fluoride TS Dry about 500 mg of sodium fluoride(NaF) at 200° for 4 h. Weigh accurately 222 mg of the driedsodium fluoride, and dissolve it in sufficient water to makeexactly 100 mL. Transfer 10.0 mL of this solution into a1000-mL volumetric flask, dilute to volume with water, andmix. Each mL of this final solution corresponds to 10 �g offluorine (F).
Sodium Hydroxide TS (1 N) Dissolve 4.3 g of sodiumhydroxide (NaOH) in water to make 100 mL.
Sodium Indigotindisulfonate TS See Indigo Carmine TS.
Sodium Nitroferricyanide TS Dissolve 1 g of sodium ni-troferricyanide [Na2Fe(NO)(CN)5·2H2O] in water to make 20mL. Prepare this solution fresh.
Sodium Phosphate TS Dissolve 12 g of clear crystals ofdibasic sodium phosphate (Na2HPO4·7H2O) in water to make100 mL.
Sodium Sulfide TS Dissolve 1 g of sodium sulfide(Na2S·9H2O) in water to make 10 mL. Prepare this solutionfresh.
Sodium Tetraphenylborate TS Dissolve 1.2 g of sodiumtetraphenylborate in water to make 200 mL. If necessary, stirfor 5 min with 1 g of freshly prepared hydrous aluminumoxide, and filter to clarify.
Sodium Thiosulfate TS Use 0.1 N Sodium Thiosulfate (seeVolumetric Solutions in this section).
Stannous Chloride TS Dissolve 40 g of reagent-grade stan-nous chloride dihydrate (SnCl2·2H2O) in 100 mL of hydro-chloric acid.
Starch TS Mix 1 g of a suitable starch with 10 mg of redmercuric oxide and sufficient cold water to make a thin paste.Add 20 mL of boiling water, boil for 1 min with continuousstirring, and cool. Use only the clear solution. Test the sensitiv-ity of the Starch TS by adding 5 mL of Starch TS to 100 mL ofwater. Add 0.05 mL of freshly prepared 0.1 N potassium iodidesolution and 1 drop of 50 mg/kg chlorine solution, made bydiluting 1 mL of a commercial 5% sodium hypochlorite(NaOCl) solution in 1000 mL of water. The deep blue colorproduced is discharged by 0.05 mL of 0.1 N sodium thiosulfate.
Starch Iodide Paste TS Heat 100 mL of water in a 250-mL beaker to boiling, add a solution of 750 mg of potassiumiodide (KI) in 5 mL of water, then add 2 g of zinc chloride(ZnCl2) dissolved in 10 mL of water, and while the solutionis boiling, add with stirring a smooth suspension of 5 g ofpotato starch in 30 mL of cold water. Continue to boil for 2min, then cool. Store in well-closed containers in a cool place.This mixture must show a definite blue streak when a glassrod dipped in a mixture of 1 mL of 0.1 M sodium nitrite, 500mL of water, and 10 mL of hydrochloric acid is streaked ona smear of the paste.
Sulfanilic Acid TS Dissolve 800 mg of sulfanilic acid (p-NH2C6H4SO3H·H2O) in 100 mL of acetic acid. Store in tightcontainers.
Sulfuric Acid (approximately 36 N) Use ACS reagent-gradeSulfuric Acid (95.0% to 98.0% of H2SO4).
Sulfuric Acid TS (95%) Add a quantity of sulfuric acid ofknown concentration to sufficient water to adjust the finalconcentration to between 94.5% and 95.5% of H2SO4. Sincethe acid concentration may change upon standing or uponintermittent use, the concentration should be checked fre-quently and solutions assaying more than 95.5% or less than94.5% discarded or adjusted by adding either diluted or fumingsulfuric acid, as required.
Sulfuric Acid TS, Diluted (2 N) A solution containing 10%(w/v) of H2SO4. Prepare by cautiously adding 57 mL of sulfu-ric acid (95% to 98%) or Sulfuric Acid TS to about 100 mLof water, then cool to room temperature, and dilute with waterto 1000 mL.
Tannic Acid TS Dissolve 1 g of tannic acid (tannin) in 1mL of alcohol, and add water to make 10 mL. Prepare thissolution fresh.
970 / Solutions and Indicators / General Tests and Assays FCC V
Thymol Blue TS Dissolve 100 mg of thymol blue in 100mL of alcohol, and filter if necessary.
Thymolphthalein TS Dissolve 100 mg of thymolphthaleinin 100 mL of alcohol, and filter if necessary.
Triketohydrindene Hydrate TS (Ninhydrin TS) Dissolve200 mg of triketohydrindene hydrate (C9H4O3·H2O) in waterto make 100 mL. Prepare this solution fresh.
Trinitrophenol TS (Picric Acid TS) Dissolve the equivalentof 1 g of anhydrous trinitrophenol in 100 mL of hot water.Cool the solution, and filter if necessary.
Xylenol Orange TS Dissolve 100 mg of xylenol orange in100 mL of alcohol.
VOLUMETRIC SOLUTIONS
Normal Solutions A normal solution contains 1 g equiva-lent weight of the solute per L of solution. The normalitiesof solutions used in volumetric determinations are designatedas 1 N, 0.1 N, 0.05 N, etc., in this Codex.
Molar Solutions A molar solution contains 1 g molecularweight of the solute per L of solution. The molarities of suchsolutions are designated as 1 M, 0.1 M, 0.05 M, etc., in thisCodex.
Preparation and Methods of Standardization The detailsfor the preparation and standardization of solutions used inseveral normalities are usually given only for the one mostfrequently required. Solutions of other normalities are pre-pared and standardized in the same general manner as de-scribed. Solutions of lower normalities may be prepared accu-rately by making an exact dilution of a stronger solution,but solutions prepared in this way should be restandardizedbefore use.
Dilute solutions that are not stable, such as 0.01 N potassiumpermanganate and sodium thiosulfate, are preferably preparedby diluting exactly the higher normality with thoroughlyboiled and cooled water on the same day they are to be used.
All volumetric solutions should be prepared, standardized,and used at the standard temperature of 25°, if practicable.When a titration must be carried out at a markedly differenttemperature, the volumetric solution should be standardizedat that same temperature, or a suitable temperature correctionshould be made. Since the strength of a standard solution maychange upon standing, the normality or molarity factor shouldbe redetermined frequently.
Although the directions provide only one method of stan-dardization, other methods of equal or greater accuracy maybe used. For substances available as certified primary stan-dards, or of comparable quality, the final standard solution
may be prepared by weighing accurately a suitable quantityof the substance and dissolving it to produce a specific volumesolution of known concentration. Hydrochloric and sulfuricacids may be standardized against a certified primary standard.
In volumetric assays described in this Codex, the numberof mg of the test substance equivalent to 1 mL of the primaryvolumetric solution is given. In general, these equivalentsmay be derived by simple calculation (see also Solutions, inthe General Provisions).
Ammonium Thiocyanate, 0.1 N (7.612 g NH4SCN per 1000mL) Dissolve about 8 g of ammonium thiocyanate(NH4SCN) in 1000 mL of water, and standardize by titratingthe solution against 0.1 N Silver Nitrate as follows: Transferabout 30 mL of 0.1 N Silver Nitrate, accurately measured,into a glass-stoppered flask. Dilute with 50 mL of water, thenadd 2 mL of Ferric Ammonium Sulfate TS and 2 mL of nitricacid, and titrate with the ammonium thiocyanate solution to thefirst appearance of a red-brown color. Calculate the normality,and, if desired, adjust the solution to exactly 0.1 N. If desired,0.1 N Ammonium Thiocyanate may be replaced by 0.1 Npotassium thiocyanate where the former is directed in varioustests and assays.
Barium Hydroxide, 0.2 N [17.14 g Ba(OH)2 per 1000 mL]Dissolve about 36 g of barium hydroxide [Ba(OH)2·8H2O] in1 L of recently boiled and cooled water, and quickly filterthe solution. Keep this solution in bottles with well-fittedrubber stoppers with a soda–lime tube attached to each bottleto protect the solution from carbon dioxide in the air. Standard-ize as follows: Transfer quantitatively about 60 mL of 0.1 Nhydrochloric acid, accurately measured, to a flask; add 2 dropsof Phenolphthalein TS; and slowly titrate with the bariumhydroxide solution, with constant stirring, until a permanentpink color is produced. Calculate the normality of the bariumhydroxide solution and, if desired, adjust to exactly 0.2 Nwith freshly boiled and cooled water.
Note: Solutions of alkali hydroxides absorb carbon di-oxide when exposed to air. Connect the buret used fortitrations with barium hydroxide solution directly to thestorage bottle, and provide the bottle with a soda–limetube so that air entering must pass through this tube,which will absorb carbon dioxide. Frequently restan-dardize standard solutions of barium hydroxide.
Bromine, 0.1 N (7.990 g Br per 1000 mL) Dissolve 3 g ofpotassium bromate (KBrO3) and 15 g of potassium bromide(KBr) in sufficient water to make 1000 mL, and standardizethe solution as follows: Transfer about 25 mL of the solution,accurately measured, into a 500-mL iodine flask, and dilutewith 120 mL of water. Add 5 mL of hydrochloric acid, stopperthe flask, and shake it gently. Then add 5 mL of PotassiumIodide TS, restopper, shake the mixture, allow it to stand for5 min, and titrate the liberated iodine with 0.1 N SodiumThiosulfate, adding Starch TS near the end of the titration.Calculate the normality. Store this solution in dark, amber-colored, glass-stoppered bottles.
FCC V General Tests and Assays / Solutions and Indicators / 971
Ceric Sulfate, 0.1 N [33.22 g Ce(SO4)2 per 1000 mL]Transfer 59 g of ceric ammonium nitrate [Ce(NO3)4·2NH4-NO3·2H2O] to a beaker, add 31 mL of sulfuric acid, mix, andcautiously add water, in 20-mL portions, until solution iscomplete. Cover the beaker, let stand overnight, filter througha sintered-glass crucible of fine porosity, add water to make1000 mL, and mix. Standardize the solution as follows: Weighaccurately 200 mg of primary standard arsenic trioxide(As2O3) previously dried at 100° for 1 h, and transfer to a500-mL Erlenmeyer flask. Wash down the inner walls of theflask with 25 mL of a 2:25 solution of sodium hydroxide,swirl to dissolve the sample, and when solution is complete,add 100 mL of water, and mix. Add 10 mL of 1:3 sulfuric acidand 2 drops each of Orthophenanthroline TS and a solution ofosmium tetroxide in 0.1 N sulfuric acid (1:400), and slowlytitrate with the ceric sulfate solution until the pink color ischanged to a very pale blue. Calculate the normality. Each4.946 mg of As2O3 is equivalent to 1 mL of 0.1 N CericSulfate.
Ceric Sulfate, 0.01 N [3.322 g Ce(SO4)2 per 1000 mL]Dissolve 4.2 g of ceric sulfate [Ce(SO4)2·4H2O] or 5.5 gof the acid sulfate [Ce(HSO4)4] in about 500 mL of watercontaining 28 mL of sulfuric acid, and dilute to 1000 mL.Allow the solution to stand overnight, and filter. Standardizethis solution daily as follows: Weigh accurately about 275mg of hydroquinone (C6H6O2), dissolve it in sufficient 0.5 NAlcoholic Sulfuric Acid to make 500.0 mL, and mix. To 25.0mL of this solution add 75 mL of 0.5 N sulfuric acid, 20 mLof water, and 2 drops of Diphenylamine TS. Titrate with theceric sulfate solution at a rate of about 25 drops per 10 s untilan endpoint is reached that persists for 10 s. Perform a blankdetermination using 100 mL of 0.5 N Alcoholic Sulfuric Acid,20 mL of water, and 2 drops of Diphenylamine TS, and makeany necessary correction. Calculate the normality of the cericsulfate solution by the formula
0.05W/55.057V,
in which W is the weight, in mg, of the hydroquinone sampletaken, and V is the volume, in mL, of the ceric sulfate solutionconsumed in the titration.
Disodium EDTA, 0.05 M (16.81 g C10H14N2Na2O8 per 1000mL) Dissolve 18.6 g of disodium ethylenediaminetetraace-tate (C10H14N2Na2O8·2H2O) in sufficient water to make 1000mL, and standardize the solution as follows: Weigh accuratelyabout 200 mg of chelometric standard calcium carbonate(CaCO3), transfer to a 400-mL beaker, add 10 mL of water,and swirl to form a slurry. Cover the beaker with a watchglass, and introduce 2 mL of 2.7 N hydrochloric acid from apipet inserted between the lip of the beaker and the edge ofthe watch glass. Swirl the contents of the beaker to dissolvethe calcium carbonate. Wash down the sides of the beaker,the outer surface of the pipet, and the watch glass, and diluteto about 100 mL with water. While stirring, preferably witha magnetic stirrer, add about 30 mL of the disodium EDTAsolution from a 50-mL buret, then add 15 mL of 1 N SodiumHydroxide and 300 mg of Hydroxy Naphthol Blue Indicator,
and continue the titration to a blue endpoint. Calculate themolarity by the formula
W/100.09V,
in which W is the weight, in mg, of CaCO3 in the sample ofcalcium carbonate taken, and V is the volume, in mL, ofdisodium EDTA solution consumed. Each 5.004 mg of CaCO3
is equivalent to 1 mL of 0.05 M Disodium EDTA.For the determination of aluminum in its salts, use 0.05
M Disodium EDTA standardized as follows: Transfer 2 g,accurately weighed, of aluminum wire to a 1000-mL volumet-ric flask, and add 50 mL of a 1:1 hydrochloric acid–watermixture. Swirl the flask to ensure complete wetting of thewire, and allow the reaction to proceed. When dissolution iscomplete, dilute with water to volume, and mix. Transfer 10.0mL of this solution to a 250-mL beaker, add 25.0 mL of thedisodium EDTA solution, boil gently for 5 min, and cool.Add in the order given, and with continuous stirring, 20 mLof pH 4.5 buffer solution (77.1 g of ammonium acetate and57 mL of glacial acetic acid in 1000 mL of solution), 50 mLof alcohol, and 2 mL of Dithizone TS. Titrate with 0.05 MZinc Sulfate to a bright rose pink color, and perform a blankdetermination, substituting 10 mL of water for the 10.0 mLof aluminum solution. Each mL of disodium EDTA solutionis equivalent to 1.349 mg of aluminum (Al).
Ferrous Ammonium Sulfate, 0.1 N [39.21 g Fe(NH4)2-(SO4)2·6H2O per 1000 mL] Dissolve 40 g of ferrous ammo-nium sulfate hexahydrate in a previously cooled mixture of40 mL of sulfuric acid and 200 mL of water, dilute to 1000mL with water, and mix. On the day of use, standardize thesolution as follows: Transfer from 25 to 30 mL of the solution,accurately measured, into a flask, add 2 drops of Orthophenan-throline TS, and titrate with 0.1 N Ceric Sulfate until the redcolor is changed to pale blue. From the volume of 0.1 N CericSulfate consumed, calculate the normality.
Hydrochloric Acid, 1 N (36.46 g HCl per 1000 mL) Dilute85 mL of hydrochloric acid with water to make 1000 mL,and standardize the solution as follows: Accurately weighabout 1.5 g of primary standard anhydrous sodium carbonate(Na2CO3) that has been heated at a temperature of about 270°for 1 h. Dissolve it in 100 mL of water, and add 2 drops ofMethyl Red TS. Add the acid slowly from a buret, with constantstirring, until the solution becomes faintly pink. Heat thesolution to boiling, and continue the titration until the faintpink color is no longer affected by continued boiling. Calculatethe normality. Each 52.99 mg of Na2CO3 is equivalent to 1mL of 1 N Hydrochloric Acid.
Hydroxylamine Hydrochloride, 0.5 N (35 g NH2OH·HCl per1000 mL) Dissolve 35 g of hydroxylamine hydrochloride in150 mL of water, and dilute to 1000 mL with anhydrousmethanol. To 500 mL of this solution add 15 mL of a 0.04%solution of bromophenol blue in alcohol, and titrate with 0.5N Triethanolamine until the solution appears green-blue bytransmitted light. Prepare this solution fresh before each se-ries of analyses.
972 / Solutions and Indicators / General Tests and Assays FCC V
Iodine, 0.1 N (12.69 g I per 1000 mL) Dissolve about 14g of iodine (I) in a solution of 36 g of potassium iodide (KI)in 100 mL of water, add 3 drops of hydrochloric acid, dilutewith water to 1000 mL, and standardize as follows: Weighaccurately about 150 mg of primary standard arsenic trioxide(As2O3) previously dried at 105° for 1 h, and dissolve it in20 mL of 1 N Sodium Hydroxide by warming if necessary.Dilute with 40 mL of water, add 2 drops of Methyl OrangeTS, and follow with 2.7 N hydrochloric acid until the yellowcolor is changed to pink. Then add 2 g of sodium bicarbonate(NaHCO3), dilute with 50 mL of water, add 3 mL of StarchTS, and slowly add the iodine solution from a buret until apermanent blue color is produced. Calculate the normality.Each 4.946 mg of As2O3 is equivalent to 1 mL of 0.1 NIodine. Store this solution in glass-stoppered bottles.
Lithium Methoxide, 0.1 N (3.797 g CH3OLi per 1000 mL)Dissolve 600 mg of freshly cut lithium metal in a mixture of150 mL of anhydrous methanol and 850 mL of benzene. Filterthe resulting solution if it is cloudy, and standardize it asfollows: Dissolve about 80 mg of benzoic acid (NationalInstitute of Standards and Technology primary standard), ac-curately weighed, in 35 mL of dimethylformamide, add 5drops of Thymol Blue TS, and titrate with the lithium methox-ide solution to a dark blue endpoint.
Caution: Protect the solution from absorption of carbondioxide and moisture by covering the titration vesselwith aluminum foil while dissolving the benzoic acidsample and during the titration.
Each mL of 0.1 N Lithium Methoxide is equivalent to 12.21mg of benzoic acid.
Mercuric Nitrate, 0.1 M [32.46 g Hg(NO3)2 per 1000 mL]Dissolve about 35 g of mercuric nitrate [Hg(NO3)2·H2O] ina mixture of 5 mL of nitric acid and 500 mL of water, anddilute with water to 1000 mL. Standardize the solution asfollows: Transfer an accurately measured volume of about 20mL of the solution into an Erlenmeyer flask, and add 2 mLof nitric acid and 2 mL of Ferric Ammonium Sulfate TS. Coolto below 20°, and titrate with 0.1 N Ammonium Thiocyanateto the first appearance of a permanent brown color. Calculatethe molarity.
Oxalic Acid, 0.1 N (4.502 g H2C2O4 per 1000 mL) Dissolve6.45 g of oxalic acid (H2C2O4·2H2O) in sufficient water tomake 1000 mL. Standardize by titration against freshly stan-dardized 0.1 N Potassium Permanganate as directed underPotassium Permanganate, 0.1 N. Store this solution in glass-stoppered bottles, protected from light.
Perchloric Acid, 0.1 N (10.046 g HClO4 per 1000 mL) Mix8.5 mL of perchloric acid (70%) with 500 mL of glacial aceticacid and 30 mL of acetic anhydride.
Caution: Handle perchloric acid in an appropriatefume hood.
Cool, and add glacial acetic acid to make 1000 mL. Allowthe prepared solution to stand for 1 day for the excess acetic
anhydride to be combined, and determine the water contentby the Karl Fischer Titrimetric Method, Appendix IIB. If thewater content exceeds 0.05%, add more acetic anhydride, butif the solution contains no titratable water, add sufficient waterto make the content between 0.02% and 0.05%. Allow tostand for 1 day, and again determine the water content bytitration. Standardize the solution as follows: Weigh accu-rately about 700 mg of primary standard potassium biphthalate[KHC6H4(COO)2], previously dried at 105° for 2 h, and dis-solve it in 50 mL of glacial acetic acid in a 250-mL flask.Add 2 drops of Crystal Violet TS, and titrate with the perchloricacid solution until the violet color changes to emerald green.Deduct the volume of the perchloric acid consumed by 50mL of the glacial acetic acid, and calculate the normality.Each 20.42 mg of KHC6H4(COO)2 is equivalent to 1 mL of0.1 N Perchloric Acid.
Perchloric Acid, 0.1 N, in Dioxane Mix 8.5 mL of perchlo-ric acid (70%) with sufficient dioxane, which has been espe-cially purified by adsorption, to make 1000 mL.
Caution: Handle perchloric acid in an appropriatefume hood.
Standardize the solution as follows: Weigh accurately about700 mg of primary standard potassium biphthalate[KHC6H4(COO)2], previously dried at 105° for 2 h, and dis-solve in 50 mL of glacial acetic acid in a 250-mL flask. Add2 drops of Crystal Violet TS, and titrate with the perchloricacid solution until the violet color changes to blue-green.Deduct the volume of the perchloric acid consumed by 50mL of the glacial acetic acid, and calculate the normality.Each 20.42 mg of KHC6H4(COO)2 is equivalent to 1 mL of0.1 N Perchloric Acid.
Potassium Acid Phthalate, 0.1 N [20.42 g KHC6H4(COO)2
per 1000 mL] Dissolve 20.42 g of primary standard potas-sium biphthalate [KHC6H4(COO)2], previously dried at 105°for 2 h, in glacial acetic acid in a 1000-mL volumetric flask,warming on a steam bath if necessary to effect solution andprotecting the solution from contamination by moisture. Coolto room temperature, dilute to volume with glacial acetic acid,and mix.
Potassium Dichromate, 0.1 N (4.903 g K2Cr2O7 per 1000mL) Dissolve about 5 g of potassium dichromate (K2Cr2O7)in 1000 mL of water, transfer quantitatively 25 mL of thissolution to a 500-mL glass-stoppered flask, add 2 g of potas-sium iodide (free from iodate) (KI), dilute with 200 mL ofwater, add 5 mL of hydrochloric acid, and mix. Allow tostand for 10 min in a dark place, and titrate the liberatediodine with 0.1 N Sodium Thiosulfate, adding Starch TS asthe endpoint is approached. Correct for a blank run on the samequantities of the same reagents, and calculate the normality.
Potassium Hydroxide, 1 N (56.11 g KOH per 1000 mL)Prepare and standardize 1 N potassium hydroxide by the pro-cedure set forth for 1 N Sodium Hydroxide, using 74 g of thepotassium hydroxide (KOH) to prepare the solution. Each
FCC V General Tests and Assays / Solutions and Indicators / 973
204.2 mg of KHC6H4(COO)2 is equivalent to 1 mL of 1 NPotassium Hydroxide.
Potassium Hydroxide, 0.5 N, Alcoholic (Caution: The so-lution may become very hot. Allow it to cool before addingthe aldehyde-free alcohol.) Dissolve about 35 g of potassiumhydroxide (KOH) in 20 mL of water, and add sufficient alde-hyde-free alcohol to make 1000 mL. Allow the solution tostand in a tightly stoppered bottle for 24 h. Then quickly decantthe clear supernatant liquid into a suitable, tight container, andstandardize as follows: Transfer quantitatively 25 mL of 0.5N hydrochloric acid into a flask, dilute with 50 mL of water,add 2 drops of Phenolphthalein TS, and titrate with the alco-holic potassium hydroxide solution until a permanent, palepink color is produced. Calculate the normality. Store thissolution in tightly stoppered bottles protected from light.
Potassium Iodate, 0.05 M (10.70 g KIO3 per 1000 mL) Dis-solve 10.700 g of potassium iodate of primary standard quality(KIO3), previously dried at 110° to constant weight, in suffi-cient water to make 1000.0 mL.
Potassium Permanganate, 0.1 N (3.161 g KMnO4 per 1000mL) Dissolve about 3.3 g of potassium permanganate(KMnO4) in 1000 mL of water in a flask, and boil the solutionfor about 15 min. Stopper the flask, allow it to stand for atleast 2 days, and filter through a fine-porosity, sintered-glasscrucible. If necessary, the bottom of the crucible may be linedwith a pledget of glass wool. Standardize the solution asfollows: Weigh accurately about 200 mg of sodium oxalateof primary standard quality (Na2C2O4), previously dried at100° to constant weight, and dissolve it in 250 mL of water.Add 7 mL of sulfuric acid, heat to about 70°, and then slowlyadd the permanganate solution from a buret, with constantstirring, until a pale pink color that persists for 15 s is produced.The temperature at the conclusion of the titration should benot less than 60°. Calculate the normality. Each 6.700 mg ofNa2C2O4 is equivalent to 1 mL of 0.1 N Potassium Permanga-nate. Potassium permanganate is reduced on contact withorganic substances such as rubber; therefore, the solution mustbe handled in apparatus made entirely of glass or other suitablyinert material. Store it in glass-stoppered, amber-colored bot-tles, and restandardize frequently.
Silver Nitrate, 0.1 N (16.99 g AgNO3 per 1000 mL) Dis-solve about 17.5 g of silver nitrate (AgNO3) in 1000 mL ofwater, and standardize the solution as follows: Weigh accu-rately 100 mg of primary standard sodium chloride, previouslydried at 120° for 16 h, into a 150-mL beaker, and dissolve itin 5 mL of water. Add 5 mL of acetic acid, 50 mL of methanol,and 2 or 3 drops of Eosin Y TS, and titrate with the silvernitrate solution to the endpoint. Calculate the normality.
Sodium Acetate, 0.1 N (8.203 g CH3COONa per 1000 mL)Dissolve 8.20 g of anhydrous sodium acetate in glacial aceticacid to make 1000 mL, and standardize the solution as follows:To 25.0 mL of the prepared sodium acetate solution, add 50mL of glacial acetic acid and 1 mL of �-Naphtholbenzein TS.
Titrate with 0.1 N Perchloric Acid until a yellow-brown colorchanges through yellow to green.
Caution: Handle perchloric acid in an appropriatefume hood.
Perform a blank determination, and make any necessary cor-rection. Calculate the normality factor.
Sodium Arsenite, 0.05 N (3.248 g NaAsO2 per 1000 mL)Transfer 2.4725 g of arsenic trioxide, which has been pulver-ized and dried at 100° to constant weight, to a 1000-mLvolumetric flask, dissolve it in 20 mL of 1 N Sodium Hydrox-ide, and add 1 N Sulfuric Acid or 1 N Hydrochloric Acid untilthe solution is neutral or only slightly acid to litmus. Add 15g of sodium bicarbonate, dilute to volume with water, and mix.
Sodium Hydroxide, 1 N (40.00 g NaOH per 1000 mL) Dis-solve about 40 g of sodium hydroxide (NaOH) in about 1000mL of carbon dioxide-free water. Shake the mixture thor-oughly, and allow it to stand overnight in a stoppered bottle.Standardize the clear liquid as follows: Transfer about 5 g ofprimary standard potassium biphthalate [KHC6H4(COO)2],previously dried at 105° for 2 h and accurately weighed, toa flask, and dissolve it in 75 mL of carbon dioxide-free water.If the potassium biphthalate is in the form of large crystals,crush it before drying. To the flask add 2 drops of Phenol-phthalein TS, and titrate with the sodium hydroxide solutionto a permanent pink color. Calculate the normality. Each 204.2mg of potassium biphthalate is equivalent to 1 mL of 1 NSodium Hydroxide.
Note: Solutions of alkali hydroxides absorb carbon di-oxide when exposed to air. Therefore, store them inbottles with well-fitted, suitable stoppers provided witha tube filled with a mixture of sodium hydroxide andlime so that air entering the container must pass throughthis tube, which will absorb the carbon dioxide. Fre-quently restandardize standard solutions of sodium hy-droxide.
Sodium Hydroxide, 0.5 N, Alcoholic (22.5 g NaOH per 1000mL) (Caution: The following solution may become veryhot. Allow it to cool before adding the aldehyde-free alcohol.)Dissolve about 22.5 g of sodium hydroxide (NaOH) in 20mL of water, and add sufficient aldehyde-free alcohol to make1000 mL. Allow the solution to stand in a tightly stopperedbottle for 24 h. Then quickly decant the clear supernatantliquid into a suitable, tight container, and standardize asfollows:
Quantitatively transfer 25 mL of 0 5 N hydrochloric acidinto a flask, dilute with 50 mL of water, add 2 drops ofPhenolphthalein TS, and titrate with the alcoholic sodiumhydroxide solution until a permanent, pale pink color appears.Calculate the normality. Store this solution in tightly stopperedbottles protected from light.
Sodium Methoxide, 0.1 N, in Pyridine (5.40 g CH3ONa per1000 mL) Weigh 14 g of freshly cut sodium metal, and cutinto small cubes. Place about 0.5 mL of anhydrous methanol
974 / Solutions and Indicators / General Tests and Assays FCC V
in a round-bottom 120-mL flask equipped with a ground-glass joint, add 1 cube of the sodium metal, and when thereaction subsides, add the remaining sodium metal to theflask. Connect a water-cooled condenser to the flask, andslowly add 100 mL of anhydrous methanol, in small portions,through the top of the condenser. Regulate the addition ofthe methanol so that the vapors are condensed and do notescape through the top of the condenser. After addition ofthe methanol is complete, connect a drying tube to the top ofthe condenser, and allow the solution to cool. Transfer 17.5mL of this solution (approximately 6 N) into a 1000-mLvolumetric flask containing 70 mL of anhydrous methanol,and dilute to volume with freshly distilled pyridine. Storepreferably in the reservoir of an automatic buret suitablyprotected from carbon dioxide and moisture. Standardize thesolution as follows: Weigh accurately about 400 mg of primarystandard benzoic acid, transfer it into a 250-mL wide-mouthErlenmeyer flask, and dissolve it in 50 mL of freshly distilledpyridine. Add a few drops of Thymolphthalein TS, and titrateimmediately with the sodium methoxide solution to a blueendpoint. During the titration, direct a gentle stream of nitro-gen into the flask through a short piece of 6-mm glass tubingfastened near the tip of the buret. Perform a blank determina-tion (see the General Provisions), correct for the volumeof sodium methoxide solution consumed by the blank, andcalculate the normality. Each 12.21 mg of benzoic acid isequivalent to 1 mL of 0.1 N Sodium Methoxide in Pyridine.
Sodium Methoxide, 0.02 N, in Toluene (1.08 g CH3ONaper 1000 mL) Weigh 2.5 g of freshly cut sodium metal,and cut into small cubes. Place about 200 mL of anhydrousmethanol in a 1000-mL volumetric flask, chill in an ice bath,and add the cubes one at a time to the methanol. When thelast cube is dissolved, dilute to the mark with toluene, andmix. Standardize the solution as follows: Weigh accuratelyabout 20 mg of primary standard benzoic acid, transfer itinto a 50-mL conical flask, and dissolve it in 25 mL ofdimethylformamide. Add 2 drops of a solution of 100 mgof thymol blue in 10 mL of dimethylformamide, and titrateimmediately with the sodium methoxide solution to a blueendpoint. Titrate a blank solution of dimethylformamide inthe same manner, correct the volume of sodium methoxidesolution consumed by the blank, and calculate the normality.Each 2.442 mg of benzoic acid is equivalent to 1 mL of 0.02N Sodium Methoxide in Toluene.
Sodium Thiosulfate, 0.1 N (15.81 g Na2S2O3 per 1000 mL)Dissolve about 26 g of sodium thiosulfate (Na2S2O3·5H2O)and 200 mg of sodium carbonate (Na2CO3) in 1000 mL ofrecently boiled and cooled water. Standardize the solution asfollows: Weigh accurately about 210 mg of primary standardpotassium dichromate, previously pulverized and dried at 120°for 4 h, and dissolve in 100 mL of water in a 500-mL glass-stoppered flask. Swirl to dissolve the sample, remove thestopper, and quickly add 2 g of sodium bicarbonate, 3 g ofpotassium iodide, and 5 mL of hydrochloric acid. Stopper theflask, swirl to mix, and let stand in the dark for 10 min. Rinsethe stopper and inner walls of the flask with water, and titratethe liberated iodine with the sodium thiosulfate solution until
the solution is only faint yellow. Add Starch TS, and continuethe titration to the discharge of the blue color. Calculate thenormality.
Sulfuric Acid, 1 N (49.04 g H2SO4 per 1000 mL) Addslowly, with stirring, 30 mL of sulfuric acid to about 1020mL of water, allow to cool to 25°, and standardize by titrationagainst primary standard sodium carbonate (Na2CO3) as di-rected under 1 N Hydrochloric Acid. Each 52.99 mg ofNa2CO3 is equivalent to 1 mL of 1 N Sulfuric Acid.
Sulfuric Acid, Alcoholic, 5 N (245.2 g H2SO4 per 1000 mL)Add cautiously, with stirring, 139 mL of sulfuric acid to asufficient quantity of absolute alcohol to make 1000.0 mL.
Sulfuric Acid, Alcoholic, 0.5 N Add cautiously, with stir-ring, 13.9 mL of sulfuric acid to a sufficient quantity ofabsolute alcohol to make 1000.0 mL. Alternatively, preparethis solution by diluting 100.0 mL of 5 N Sulfuric Acid withabsolute alcohol to make 1000.0 mL.
Thorium Nitrate, 0.1 M [48.01 g Th(NO3)4 per 1000 mL]Weigh accurately 55.21 g of thorium nitrate [Th(NO3)4-·4H2O], dissolve it in water, dilute to 1000.0 mL, and mix.Standardize the solution as follows: Transfer 50.0 mL into a500-mL volumetric flask, dilute to volume with water, andmix. Transfer 50.0 mL of the diluted solution into a 400-mLbeaker, add 150 mL of water and 5 mL of hydrochloric acid,and heat to boiling. While stirring, add 25 mL of a saturatedsolution of oxalic acid, then digest the mixture for 1 h justbelow the boiling point, and allow to stand overnight. Decantthrough Whatman No. 42, or equivalent, filter paper, andtransfer the precipitate to the filter using about 100 mL of awash solution consisting of 70 mL of the saturated oxalicacid solution, 430 mL of water, and 5 mL of hydrochloricacid. Transfer the precipitate and filter paper to a tared tall-form porcelain crucible, dry, char the paper, and ignite at950° for 1.5 h or to constant weight. Cool in a desiccator,weigh, and calculate the molarity of the solution by theformula
200W/264.04,
in which W is the weight, in g, of thorium oxide obtained.
Triethanolamine, 0.5 N [74 g N(CH2CH2OH)3 per 1000 mL]Transfer 65 mL (74 g) of 98% triethanolamine into a 1000-mL volumetric flask, dilute to volume with water, stopper theflask, and mix thoroughly.
Zinc Sulfate, 0.05 M (8.072 g ZnSO4 per 1000 mL) Dis-solve about 15 g of zinc sulfate (ZnSO4·7H2O) in sufficientwater to make 1000 mL, and standardize the solution asfollows: Dilute about 35 mL, accurately measured, with 75mL of water, add 5 mL of Ammonia–Ammonium ChlorideBuffer TS and 0.1 mL of Eriochrome Black TS, and titratewith 0.05 M Disodium EDTA until the solution is deep blue.Calculate the molarity.
FCC V General Tests and Assays / Solutions and Indicators / 975
INDICATORS
The necessary solutions of indicators may be prepared asdirected under Test Solutions (TS) and Other Reagents. Thesodium salts of many indicators are commercially availableand may be used interchangeably in water solutions with thealcohol solutions specified for the free indicators.
Useful pH indicators, listed in ascending order of the lowerlimit of their range, are methyl yellow (pH 2.9 to 4.0), bromo-phenol blue (pH 3.0 to 4.6), bromocresol green (pH 4.0 to5.4), methyl red (pH 4.2 to 6.2), bromocresol purple (pH 5.2to 6.8), bromothymol blue (pH 6.0 to 7.6), phenol red (pH6.8 to 8.2), thymol blue (pH 8.0 to 9.2), and thymolphthalein(pH 9.3 to 10.5).
Alphazurine 2G Use a suitable grade.
Azo Violet [4-(p-Nitrophenylazo) Resorcinol] A red pow-der, melting at about 193° with decomposition.
Bromocresol Blue Use Bromocresol Green.
Bromocresol Green (Bromocresol Blue; Tetrabromo-m-cre-solsulfonphthalein) A white or pale buff-colored powder;slightly soluble in water; soluble in alcohol and in solutionsof alkali hydroxides. Transition interval: from pH 3.8 (yellow)to 5.4 (blue).
Bromocresol Purple (Dibromo-o-cresolsulfonphthalein) Awhite to pink, crystalline powder; insoluble in water; solublein alcohol and in solutions of alkali hydroxides. Transitioninterval: from pH 5.2 (yellow) to 6.8 (purple).
Bromophenol Blue (Tetrabromophenolsulfonphthalein)Pink crystals, soluble in alcohol. Insoluble in water; solublein solutions of alkali hydroxides. Transition interval: from pH3.0 (yellow) to 4.6 (blue).
Bromothymol Blue (Dibromothymolsulfonphthalein) Arose red powder. Insoluble in water; soluble in alcohol andin solutions of alkali hydroxides. Transition interval: from pH6.0 (yellow) to 7.6 (blue).
Cresol Red (o-Cresolsulfonphthalein) A red-brown pow-der. Slightly soluble in water; soluble in alcohol and in dilutesolutions of alkali hydroxides. Transition interval: from pH7.2 (yellow) to 8.8 (blue).
Crystal Violet (Hexamethyl-p-rosaniline Chloride) Darkgreen crystals. Slightly soluble in water; sparingly soluble inalcohol and in glacial acetic acid. Its solutions are deep violet.
Sensitiveness Dissolve 100 mg in 100 mL of glacial aceticacid, and mix. Pipet 1 mL of the solution into a 100-mLvolumetric flask, and dilute with glacial acetic acid to volume.The solution is violet-blue and does not show a red tint. Pipet20 mL of the diluted solution into a beaker, and titrate with0.1 N Perchloric Acid, adding the perchloric acid slowly from
a microburet. Not more than 0.1 mL of 0.1 N Perchloric Acidis required to produce an emerald green color.
Caution: Handle perchloric acid in an appropriatefume hood.
Dithizone (Diphenylthiocarbazone) A blue-black powder.Insoluble in water; soluble in alcohol and in chloroform,yielding intensely green solutions even in high dilutions.
Eriochrome Black T [Sodium 1-(1-Hydroxy-2-naphthylazo)-5-nitro-2-naphthol-4-sulfonate] A brown-black powderhaving a faint metallic sheen. Soluble in alcohol, in methanol,and in hot water.
Sensitiveness To 10 mL of a 1:200,000 solution in a mix-ture of equal parts (v/v) of methanol and water add a 1:100solution of sodium hydroxide until the pH is 10. The solution ispure blue and free from cloudiness. Add 0.2 mL of MagnesiumStandard Solution (10 �g Mg ion). The color of the solutionchanges to red-violet, and with the continued addition ofmagnesium ion, it becomes wine red.
p-Ethoxychrysoidin Monohydrochloride [4-(p-Ethoxyphe-nylazo)-m-phenylenediamine Monohydrochloride; 4′-Ethoxy-2,4-diaminoazobenzene Monohydrochloride] A red powder,insoluble in water. Transition interval: from pH 3.5 (red) to5.5 (yellow).
Hydroxy Naphthol Blue The disodium salt of 1-(2-naph-tholazo-3,6-disulfonic acid)-2-naphthol-4-sulfonic acid de-posited on crystals of sodium chloride. Small blue crystals,freely soluble in water. In the pH range between 12 and 13,its solution is red-pink in the presence of calcium ion anddeep blue in the presence of excess disodium EDTA.
Suitability for Calcium Determinations Dissolve 300 mgin 100 mL of water, add 10 mL of 1 N Sodium Hydroxideand 1.0 mL of a 1:200 calcium chloride solution, and dilutewith water to 165 mL. The solution is red-pink. Add 1.0 mLof 0.05 M Disodium EDTA. The solution becomes deep blue.
Litmus A blue powder, cubes, or pieces. Partly soluble inwater and in alcohol. Transition interval: from approximatelypH 4.5 (red) to 8 (blue). Litmus is unsuitable for determiningthe pH of solutions of carbonates or bicarbonates.
Methylene Blue [3,7-Bis(dimethylamino)phenazathioniumChloride] Dark green crystals or a crystalline powder havinga bronzelike luster. Soluble in water and in chloroform; spar-ingly soluble in alcohol.
Methyl Orange (Helianthin; Tropaeolin D; 4′-Dimethylami-noazobenzene-4-sodium Sulfonate) An orange-yellow pow-der or crystalline scales. Slightly soluble in cold water; readilysoluble in hot water; insoluble in alcohol. Transition interval:from pH 3.2 (pink) to 4.4 (yellow).
Methyl Red (o-Carboxybenzeneazodimethylaniline Hydro-chloride) A dark red powder or violet crystals. Sparingly
976 / Solutions and Indicators / General Tests and Assays FCC V
soluble in water; soluble in alcohol. Transition interval: frompH 4.2 (red) to 6.2 (yellow).
Methyl Red Sodium The sodium salt of o-carboxybenzen-eazo-dimethylaniline. An orange-brown powder. Freely solu-ble in cold water and in alcohol. Transition interval: from pH4.2 (red) to 6.2 (yellow).
Methyl Yellow (p-Dimethylaminoazobenzene) Yellowcrystals, melting between 114° and 117°. Insoluble in water;soluble in alcohol, in benzene, in chloroform, in ether, indilute mineral acids, and in oils. Transition interval: from pH2.9 (red) to 4.0 (yellow).
Murexide Indicator Preparation Add 400 mg of murexideto 40 g of powdered potassium sulfate (K2SO4), and grind ina glass mortar to a homogeneous mixture. Alternatively, usetablets containing 0.4 mg of murexide admixed with potassiumsulfate or potassium chloride, available commercially.
Naphthol Green B The ferric salt of 6-sodium sulfo-1-isonitroso-1,2-naphthoquinone. A dark green powder, insolu-ble in water.
Neutral Red (3-Amino-7-dimethylamino-2-methylphenazineChloride) A coarse, red to olive green powder. Sparinglysoluble in water and in alcohol. Transition interval: from pH6.8 (red) to 8.0 (orange).
Phenol Red (Phenolsulfonphthalein) A bright to dark red,crystalline powder. Very slightly soluble in water; sparinglysoluble in alcohol; soluble in solutions of alkali hydroxides.Transition interval: from pH 6.8 (yellow) to 8.2 (red).
Phenolphthalein White or yellow-white crystals. Practi-cally insoluble in water; soluble in alcohol and in solutionsof alkali hydroxides. Transition interval: from pH 8.0 (color-less) to 10.0 (red).
Quinaldine Red (5-Dimethylamino-2-strylethylquinoliniumIodide) A dark, blue-black powder, melting at about 260°with decomposition. Sparingly soluble in water; freely solublein alcohol. Transition interval: from pH 1.4 (colorless) to3.2 (red).
Thymol Blue (Thymolsulfonphthalein) A dark, brown-green, crystalline powder. Slightly soluble in water; solublein alcohol and in dilute alkali solutions. Acid transition inter-val: from pH 1.2 (red) to 2.8 (yellow). Alkaline transitioninterval: from pH 8.0 (yellow) to 9.2 (blue).
Thymolphthalein A white to slightly yellow, crystallinepowder. Insoluble in water; soluble in alcohol and in solutionsof alkali hydroxides. Transition interval: from pH 9.3 (color-less) to 10.5 (blue).
Xylenol Orange [3,3′-Bis-di(carboxymethyl)aminomethyl-o-cresolsulfonphthalein] An orange powder. Soluble in water
and in alcohol. In acid solution it is lemon yellow, and itsmetal complexes are intensely red. It gives a distinct endpointin the direct EDTA titration of metals such as bismuth, tho-rium, scandium, lead, zinc, lanthanum, cadmium, andmercury.
INDICATOR PAPERS AND TESTPAPERS
Indicator papers and test papers are strips of paper of suitabledimension and grade (usually Swedish O filter paper or othermakes of like surface, quality, and ash) impregnated with asufficiently stable indicator solution or reagent.
Treat strong, white filter paper with hydrochloric acid, andwash with water until the last washing shows no acid reactionto Methyl Red TS. Then treat with 6 N ammonium hydroxide,wash again with water until the last washing is not alkalinetoward Phenolphthalein TS, and dry thoroughly. Saturate thedry paper with the appropriate indicator solution prepared asdirected below, and dry carefully by suspending from glassrods or other inert material in still air free from acid, alkali,and other fumes. Cut the paper into strips of convenient size,and store in well-closed containers protected from light andmoisture.
Indicator papers and test papers that are available commer-cially may be used, if desired.
Acetaldehyde Test Paper Use a solution prepared by mix-ing equal volumes of a 20% solution of morpholine and a5% solution of sodium nitroferricyanide. Saturate the preparedfilter paper in the mixture, and use the moistened paper withoutdrying.
Cupric Sulfate Test Paper Use Cupric Sulfate TS.
Lead Acetate Test Paper Usually about 6 × 80 mm in size.Use Lead Acetate TS, and dry the paper at 100°, avoidingcontact with metal.
Litmus Paper, Blue Usually about 6 × 50 mm in size. Itmeets the requirements of the following tests.
Phosphate Place 10 strips in 10 mL of water to whichhave been added 1 mL of nitric acid and 0.5 mL of 6 Nammonium hydroxide. Allow to stand for 10 min, then decantthe solution, warm, and add 5 mL of Ammonium MolybdateTS. Shake at about 40° for 5 min. No precipitate of phospho-molybdate is formed.
Residue on Ignition Ignite carefully 10 strips of the paperto constant weight. The weight of the residue corresponds tonot more than 400 �g per strip of about 3 cm2.
Rosins, Acids, etc. Immerse a strip of the blue paper ina solution of 100 mg of silver nitrate (AgNO3) in 50 mL ofwater. The color of the paper does not change in 30 s.
FCC V General Tests and Assays / Solutions and Indicators / 977
Sensitiveness Drop a 10- to 12-mm strip in 100 mL of0.0005 N hydrochloric acid contained in a beaker, and stircontinuously. The color of the paper is changed within 45 s.
Litmus Paper, Red Usually about 6 × 50 mm in size. Redlitmus meets the requirements for Phosphate, Residue on Igni-tion, and Rosins, Acids, etc., under Litmus Paper, Blue.
Sensitiveness Drop a 10- × 12-mm strip into 100 mL of0.0005 N sodium hydroxide contained in a beaker, and stircontinuously. The color of the paper changes within 30 s.
Phenolphthalein Paper Use a 1:1000 solution of phenol-phthalein in 1:2 alcohol.
Starch Iodate Paper Use a mixture of equal volumes ofStarch TS and potassium iodate solution (1:20).
Starch Iodide Paper Use a solution of 500 mg of potassiumiodide (KI) in 100 mL of freshly prepared Starch TS.
DETECTOR TUBES
Ammonia Detector Tube A fuse-sealed glass tube (Draegeror equivalent) that is designed to allow gas to be passedthrough it and that contains suitable absorbing filters andsupport media for the indicator bromophenol blue. TheDraeger Reference Number is CH 20501; the measuring rangeis 5 to 70 ppm.
Note: Suitable detector tubes are available from Na-tional Draeger, Inc., P.O. Box 120, Pittsburgh, PA15205-0120. Tubes other than those specified in themonograph may be used in accordance with the sectionentitled Codex Specifications in the General Provisions.
Carbon Dioxide Detector Tube A fuse-sealed glass tube(Draeger or equivalent) that is designed to allow gas to bepassed through it and that contains suitable absorbing filtersand support media for the indicators hydrazine and crystalviolet. The Draeger Reference Number is CH 30801; themeasuring range is 0.01% to 0.30%.
Carbon Monoxide Detector Tube A fuse-sealed glass tube(Draeger or equivalent) that is designed to allow gas to bepassed through it and that contains suitable absorbing filtersand support media for the indicators iodine pentoxide, sele-nium dioxide, and fuming sulfuric acid. The Draeger Refer-ence Number is CH 25601; the measuring range is 5 to150 ppm.
Chlorine Detector Tube A fuse-sealed glass tube (Draegeror equivalent) that is designed to allow gas to be passedthrough it and that contains suitable absorbing filters andsupport media for the indicator o-toluidine. The Draeger Ref-erence Number is CH 24301; the measuring range is 0.2 to3 ppm.
Hydrogen Sulfide Detector Tube A fuse-sealed glass tube(Draeger or equivalent) that is designed to allow gas to bepassed through it and that contains suitable absorbing filtersand support media for the indicator, which is a suitable leadsalt. The Draeger Reference Number is 6719001; the measur-ing range is 1 to 20 ppm.
Nitric Oxide–Nitrogen Dioxide Detector Tube A fuse-sealed glass tube (Draeger or equivalent) that is designed toallow gas to be passed through it and that contains suitableabsorbing filters and support media for an oxidizing layerand the indicator diphenylbenzidine. The Draeger ReferenceNumber is CH 29401; the measuring range is 0.5 to 10 ppm.
Sulfur Dioxide Detector Tube A fuse-sealed glass tube(Draeger or equivalent) that is designed to allow gas to bepassed through it and that contains suitable absorbing filtersand support media for an iodine–starch indicator. The DraegerReference Number is CH 31701; the measuring range is 1 to25 ppm.
Water Vapor Detector Tube A fuse-sealed glass tube(Draeger or equivalent) that is designed to allow gas to bepassed through it and that contains suitable absorbing filtersand support media for the indicator, which consists of a sele-nium sol in suspension in sulfuric acid. The Draeger ReferenceNumber is CH 67 28531; the measuring range is 5 to 200mg/m3.
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
1 Acesulfame Potassium 55589-62-3 C4H4KNO4S 201.24 <= 1
Nonnutritivesweetener;
flavorenhancer
2 Acetic Acid, Glacial 64-19-7 C2H4O2 60.05 <= 0.5 Acidifier;flavoring agent
3 Acetone 67-64-1 C3H6O 58.08 <= 1 Extractionsolvent
4 Acetone Peroxides 1336-17-0 <= 4
Bleachingagent;
maturingagent; doughconditioner
5 Acetylated Monoglycerides <= 2
Emulsifier;coating agent;
texture-modifying
agent; solvent;lubricant
6 N-Acetyl-L-Methionine 65-82-7 C7H13NO3S 191.25 <= 5 Nutrient
7 Acid Hydrolysates of Proteins <= 3Flavoring
agent; flavorenhancer
8 Acidified Sodium ChloriteSolutions <= 1
Antimicrobialagent in
processingwater used to
spray, dip,rinse, or storefood before
processing, tobe followed by
rinsing inpotable water
or byblanching,cooking, orcanning;
sanitizer forhard surfaces;
broad-spectrum
bactericide,virucide,
fungicide, andsporicide
9 Aconitic Acid 499-12-7 C6H6O6 174.11 <= 0.5Flavoring
substance andadjuvant
page 2. Food Chemicals Codex (5th Edition) © 2003
10 Adipic Acid 124-04-9 C6H10O4 146.14 <= 2Buffer;
neutralizingagent
11 Agar 9002-18-0 <= 5Stabilizer;emulsifier;thickener
12 DL-Alanine 302-72-7 C3H7NO2 89.09 <= 5 Nutrient
13 L-Alanine 56-41-7 C3H7NO2 89.09 <= 5 Nutrient
14 Alginic Acid 9005-32-7 (C6H8O6)n 176.13 (calculated) <= 5Stabilizer;thickener;emulsifier
15 Alginic Acid 9005-32-7 (C6H8O6)n 200.00 (avg) <= 5Stabilizer;thickener;emulsifier
16 Allura Red (1) 25956-17-6 C18H14N2O8S2Na2
496.43 <= 10 Color
17 Almond Oil, Bitter, FFPA 8013-76-1 Flavoringagent
18 Aluminum Ammonium Sulfate 7784-25-0 AlNH4(SO4)2 ·12H2O 453.32 <= 3
Buffer;neutralizing
agent
19 Aluminum Potassium Sulfate 7784-24-9 AlK(SO4)2 ·12H2O 474.38 <= 3
Buffer;neutralizing
agent; firmingagent
20 Aluminum Sodium Sulfate(anhydrous) 10102-71-3 AlNa(SO4)2 242.09 <= 3
Buffer;neutralizing
agent; firmingagent
21 Aluminum Sodium Sulfate(dodecahydrate) 7784-28-3 AlNa(SO4)2 ·
12H2O 458.29 <= 3
Buffer;neutralizing
agent; firmingagent
22 Aluminum Sulfate (anhydrous) 10043-01-3 Al2(SO4)3 342.14 <= 3 Firming agent
23 Aluminum Sulfate(octadecahydrate) 7784-31-8 Al2(SO4)3 ·
18H2O 666.41 <= 3 Firming agent
24 Ambrette Seed Oil 8015-62-1 Flavoringagent
25 Ammonia Solution 7664-41-7 NH3 17.03 <= 0.5
pH controlagent; surface
finishingagent; boiler
water additive
26 Ammoniated Glycyrrhizin 1407-03-0Flavoring
agent; flavorenhancer
27 Ammonium Alginate 9005-34-9 (C6H7O6NH4)n 193.16 (calculated) <= 5Stabilizer;thickener;emulsifier
page 3. Food Chemicals Codex (5th Edition) © 2003
28 Ammonium Alginate 9005-34-9 (C6H7O6NH4)n 217.00 (avg) <= 5Stabilizer;thickener;emulsifier
29 Ammonium Bicarbonate 1066-33-7 NH4HCO3 79.06 <= 3Alkali;
leaveningagent
30 Ammonium Carbonate 10361-29-2 <= 3
Buffer;leavening
agent;neutralizing
agent
31 Ammonium Chloride 12125-02-9 NH4Cl 53.49 <= 4Yeast food;
doughconditioner
32 Ammonium Phosphate, Dibasic 7783-28-0 (NH4)2HPO4 132.06 <= 4
Buffer; doughconditioner;leavening
agent; yeastfood
33 Ammonium Phosphate,Monobasic 7722-76-1 NH4H2PO4 115.03 <= 4
Buffer; doughconditioner;leavening
agent; yeastfood
34 Ammonium Saccharin C7H8N2O3S 200.21 <= 2 Nonnutritivesweetener
35 Ammonium Sulfate 7783-20-2 (NH4)2SO4 132.14 <= 3Dough
conditioner;yeast nutrient
36 Amyris Oil, West Indian Type Flavoringagent
37 Angelica Root Oil 8015-64-3 Flavoringagent
38 Angelica Seed Oil Flavoringagent
39 Anise Oil 8007-70-3 Flavoringagent
40 Annatto Extracts 1393-63-1 <= 10 Color
41 β-Apo-8'-Carotenal 1107-26-2 C30H40O 416.65 <= 10 Color
42 Arabinogalactan 9036-66-2 <= 0.1Dietary fiber;humectant;stabilizer
43 L-Arginine 74-79-3 C6H14N4O2 174.20 <= 5 Nutrient
44 L-Arginine Monohydrochloride 1119-34-2 C6H14N4O2 ·HCl 210.66 <= 5 Nutrient
45 Ascorbic Acid 50-81-7 C6H8O6 176.13 <= 2Antioxidant;meat-curingaid; nutrient
46 Ascorbyl Palmitate 137-66-6 C22H38O7 414.54 <= 2 Antioxidant
page 4. Food Chemicals Codex (5th Edition) © 2003
47 L-Asparagine (anhydrous) 70-47-3 C4H8N2O3 132.12 <= 5 Nutrient
48 L-Asparagine (monohydrate) 5794-13-8 C4H8N2O3 ·H2O 150.13 <= 5 Nutrient
49 Aspartame 22839-47-0 C14H18N2O5 294.31 <= 1
Sweetener;sugar
substitute;flavor
enhancer
50 Aspartame-Acesulfame Salt 106372-55-8 C18H23O9N3S 457.45 <= 1 Sweetener
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
451 Polydextrose Solution <= 0.5Bulking agent;
humectant;texturizer
452 Polyethylene 9002-88-4 <= 3
Masticatorysubstance inchewing gum
base
453 Polyethylene Glycols 25322-68-3 <= 1
Dispersing,coating,binding,
plasticizingagent;
lubricant;flavoringadjuvant
454 Polyglycerol Esters of FattyAcids <= 2 Emulsifier
455 Polyglycerol Polyricinoleic Acid <= 1 Emulsifier
456 Polyisobutylene 9003-27-4 <= 3
Masticatorysubstance inchewing gum
base
457 Polypropylene Glycol 25322-69-4 <= 1 Defoamingagent
458 Polysorbate 20 9005-64-5 <= 2 Emulsifier;stabilizer
459 Polysorbate 60 9005-67-8 <= 2 Emulsifier;stabilizer
460 Polysorbate 65 9005-71-4 <= 2 Emulsifier;stabilizer
461 Polysorbate 80 9005-65-6 <= 2 Emulsifier;stabilizer
462 Polyvinyl Acetate 9003-20-7 <= 3
Masticatorysubstance inchewing gum
base
463 Polyvinylpolypyrrolidone <= 2Clarifying
agent;stabilizer
page 2. Food Chemicals Codex (5th Edition) © 2003
464 Polyvinylpyrrolidone 9003-39-8 (C6H9NO)x ~40,000 <= 2
Clarifyingagent;
separation/filtration aid;stabilizer;
bodying agent;tableting aid;dispersant;coating onfresh fruit
465 Polyvinylpyrrolidone 9003-39-8 (C6H9NO)x ~360,000 <= 2
Clarifyingagent;
separation/filtration aid;stabilizer;
bodying agent;tableting aid;dispersant;coating onfresh fruit
466 Pork Collagen <= 1 Binder; purgereduction
467 Potassium Acid Tartrate 868-14-4 C4H5KO6 188.18 <= 2 Acidifier;buffer
468 Potassium Alginate 9005-36-1 (C6H7O6K)n 214.22 (calculated) <= 5Stabilizer;thickener;
gelling agent
469 Potassium Alginate 9005-36-1 (C6H7O6K)n 238.00 (avg) <= 5Stabilizer;thickener;
gelling agent
470 Potassium Benzoate 582-25-2 C7H5KO2 160.22 <= 2Preservative;antimicrobial
agent
471 Potassium Bicarbonate 298-14-6 KHCO3 100.12 <= 2pH control;leavening
agent
472 Potassium Bromate 7758-01-2 KBrO3 167.00 <= 4
Maturingagent;
oxidizingagent
473 Potassium Carbonate(anhydrous) 584-08-7 K2CO3 138.21 <= 2 pH control
474 Potassium Carbonate (hydrated) 584-08-7 K2CO3 ·1½H2O 165.23 <= 2 pH control
475 Potassium Carbonate Solution
<= 2 , calculated on thebasis of potassiumcarbonate (K2CO3)
determined in the Assay(below)
pH control
476 Potassium Chloride 7447-40-7 KCl 74.55
Nutrient;gelling agent;salt substitute;
yeast food
page 3. Food Chemicals Codex (5th Edition) © 2003
477 Potassium Citrate 6100-05-6 C6H5K3O7 ·H2O 324.41 <= 2
Buffer;sequestrant;
stabilizer
478 Potassium Gibberellate 125-67-7 C19H21KO6 384.47 <= 5 Enzymeactivator
479 Potassium Gluconate(anhydrous) 299-27-4 C6H11KO7 234.25 <= 2 Nutrient;
sequestrant
480 Potassium Gluconate(monohydrate) 35398-15-3 C6H11KO7 ·
H2O 252.26 <= 2 Nutrient;sequestrant
481 Potassium Glycerophosphate 1319-70-6 C3H7K2O6P ·3H2O 302.30 <= 4 Nutrient
482 Potassium Hydroxide 1310-58-3 KOH 56.11 <= 2 pH control
483 Potassium Hydroxide Solution
<= 2 , calculated on thebasis of PotassiumHydroxide (KOH)
determined in the Assay
pH control
484 Potassium Iodate 7758-05-6 KIO3 214.00 <= 4
Maturingagent;
oxidizingagent; doughconditioner
485 Potassium Iodide 7681-11-0 KI 166.00 <= 4 Nutrient
486 Potassium Lactate Solution 996-31-6 C3H5KO3 128.17 <= 2
Emulsifier;flavor
enhancer;flavoring agent
or adjuvant;humectant; pHcontrol agent
487 Potassium Metabisulfite 16731-55-8 K2S2O5 222.31 <= 2
Preservative;antioxidant;bleaching
agent
488 Potassium Nitrate 7757-79-1 KNO3 101.10 <= 4Antimicrobial
agent;preservative
489 Potassium Nitrite 7758-09-0 KNO2 85.10 <= 4
Color fixativein meat and
meatproducts;
antimicrobialagent
490 Potassium Phosphate, Dibasic 7758-11-4 K2HPO4 174.18 <= 2Buffer;
sequestrant;yeast food
491 Potassium Phosphate,Monobasic 7778-77-0 KH2PO4 136.09 <= 2
Buffer;sequestrant;yeast food
492 Potassium Phosphate, Tribasic 7778-53-2 K3PO4 <= 2 Emulsifier
page 4. Food Chemicals Codex (5th Edition) © 2003
493 Potassium Polymetaphosphate 7790-53-6 (KPO3)n <= 2
Emulsifier;moisture-retaining
agent
494 Potassium Pyrophosphate 7320-34-5 K4P2O7 330.34 <= 2 Emulsifier;texturizer
495 Potassium Sorbate 590-00-1 C6H7KO2 150.22 <= 2Antimicrobial
agent;preservative
496 Potassium Sulfate 7778-80-5 K2SO4 174.26 <= 2 pH control
497 Potassium Sulfite 10117-38-1 K2SO3 158.26 <= 2 Preservative;antioxidant
498 Potassium Tripolyphosphate 13845-36-8 448.41 <= 2 Texturizer
499 L-Proline 147-85-3 C5H9NO2 115.13 <= 5 Nutrient
500 Propane 74-98-6 C3H8 44.10 Propellant;aerating agent
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
501 Propionic Acid 79-09-4 C3H6O2 74.08 <= 2 Preservative;mold inhibitor
502 Propylene Glycol 57-55-6 C3H8O2 76.10 <= 1Solvent;
wetting agent;humectant
503 Propylene Glycol Alginate 9005-37-2 (C9H14O7)n(esterified) 234.21 (calculated) <= 5
Stabilizer;thickener;emulsifier
504 Propylene Glycol Mono- andDiesters <= 2 Emulsifier;
stabilizer
505 Propyl Gallate 121-79-9 C10H12O5 212.20 <= 1 Antioxidant
506 Propylparaben 94-13-3 C10H12O3 180.20 <= 2Preservative;antimicrobial
agent
507 Pyridoxine Hydrochloride 58-56-0 C8H11NO3 · HCl 205.64 <= 2 Nutrient
508 Quinine Hydrochloride 130-89-2 C20H24N2O2 ·HCl · 2H2O 396.91 Flavoring
agent
509 Quinine Sulfate (anhydrous) 804-63-7 (C20H24N2O2)2· H2SO4 · 2H2O 782.96 Flavoring
agent
510 Rapeseed Oil, FullyHydrogenated 84681-71-0 <= 0.1
Cooking orsalad oil;
component ofmargarine orshortening;
coating agent;emulsifying
agent;stabilizer;thickener;texturizer
511 Rapeseed Oil,Superglycerinated <= 0.1
Cooking orsalad oil;
component ofmargarine orshortening;
coating agent;emulsifying
agent;texturizer.
512 Riboflavin 83-88-5 C17H20N4O6 376.37 Nutrient
513 Riboflavin 5'-Phosphate Sodium 130-40-5 C17H20N4NaO9P · 2H2O 514.36 <= 2 Nutrient
page 2. Food Chemicals Codex (5th Edition) © 2003
514 Rice Bran Wax 8016-60-2 <= 3
Masticatorysubstance inchewing gumbase; coating
agent
515 Rosemary Oil 8000-25-7 Flavoringagent
516 Rose Oil 8007-01-0 Flavoringagent
517 Rue Oil 8014-29-7 Flavoringagent
518 Saccharin 81-07-2 C7H5NO3S 183.18 <= 2 Nonnutritivesweetener
519 Safflower Oil (Unhydrogenated) 8001-23-8 <= 0.1 Coating agent;texturizer
520 Sage Oil, Dalmatian Type 8022-56-8 Flavoringagent
521 Sage Oil, Spanish Type 8016-65-7 Flavoringagent
522 Salatrim 177403-56-4 <= 0.1
Reduced-energy fat
replacementfor
conventionalfats and oils
523 Sandalwood Oil, East IndianType 84787-70-2 Flavoring
agent
524 Savory Oil (Summer Variety) 8016-68-0 Flavoringagent
525 DL-Serine 302-84-1 C3H7NO3 105.09 <= 5 Nutrient
526 L-Serine 56-45-1 C3H7NO3 105.09 <= 5 Nutrient
527 Sheanut Oil, Refined <= 0.1
Component ofa mixture of
oils used as acocoa buttersubstitute; as
a coatingagent; and in
margarine andshortening
528 Shellac, Bleached 9000-59-3 <= 2
Coating agent;surface-finishing
agent; glaze
529 Shellac, Bleached, Wax-Free 9000-59-3 <= 2
Coating agent;surface-finishing
agent; glaze
page 3. Food Chemicals Codex (5th Edition) © 2003
530 Silicon Dioxide 7631-86-9 SiO2 60.08 <= 5
Anticakingagent;
defoamingagent; carrier;conditioning
agent;chillproofingagent in maltbeverages;
filter aid
531 Sodium Acetate (anhydrous) 127-09-3 C2H3NaO2 82.03 <= 2 Buffer
532 Sodium Acetate (trihydrate) 6131-90-4 C2H3NaO2 ·3H2O 136.08 <= 2 Buffer
533 Sodium Acid Pyrophosphate 7758-16-9 Na2H2P2O7 221.94 <= 2
Buffer;emulsifier;leavening
agent;sequestrant
534 Sodium Alginate 9005-38-3 (C6H7O6Na)n 198.11 (calculated) <= 5
Stabilizer;thickener;emulsifier;
gelling agent
535 Sodium Alginate 9005-38-3 (C6H7O6Na)n 222.00 (avg) <= 5
Stabilizer;thickener;emulsifier;
gelling agent
536 Sodium Aluminosilicate 1344-00-9 <= 5 Anticakingagent
537 Sodium Aluminum Phosphate,Acidic (anhydrous) 7785-88-8 Na3Al2H15(PO4)
8897.82 <= 2 Leavening
agent
538 Sodium Aluminum Phosphate,Acidic (dihydrate) 15136-87-5 Na3Al3H14(PO4)
8 · 2H2O 959.83 <= 2 Leaveningagent
539 Sodium Aluminum Phosphate,Acidic (tetrahydrate) 10305-76-7 Na3Al3H14(PO4)
8 · 4H2O 993.84 <= 2 Leaveningagent
540 Sodium Aluminum Phosphate,Basic 7785-88-8 <= 2 Emulsifier
541 Sodium Aluminum Phosphate,Basic 10279-59-1 <= 2 Emulsifier
542 Sodium Ascorbate 134-03-2 C6H7NaO6 198.11 <= 2Antioxidant;meat curingaid; nutrient
543 Sodium Benzoate 532-32-1 C7H5NaO2 144.11 <= 2Preservative;antimicrobial
agent
544 Sodium Bicarbonate 144-55-8 NaHCO3 84.01 <= 2
pH controlagent;
leaveningagent
545 Sodium Bisulfate 7681-38-1 NaHSO4 120.06 <= 2 Acidifier
page 4. Food Chemicals Codex (5th Edition) © 2003
546 Sodium Bisulfite 7631-90-5 NaHSO3 104.06 <= 2 Preservative
547 Sodium Carbonate (anhydrous) 497-19-8 Na2CO3 105.99 <= 4 pH control
548 Sodium Carbonate(monohydrate) 5968-11-6 Na2CO3 · H2O 124.00 <= 4 pH control
549 Sodium Carbonate(decahydrate) 6132-02-1 Na2CO3 · 10H2O 286.14 <= 4 pH control
550 Sodium Chloride 7647-14-5 NaCl 58.44
Nutrient;preservative;
flavoring agentand intensifier;curing agent;
doughconditioner
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
551 Sodium Citrate (anhydrous) 68-04-2 C6H5Na3O7 258.07 <= 2
Buffer;sequestrant;
emulsionstabilizer;
nutrient forcultured
buttermilk
552 Sodium Citrate (dihydrate) 6132-04-3 C6H5Na3O7 ·2H2O 294.10 <= 2
Buffer;sequestrant;
emulsionstabilizer;
nutrient forcultured
buttermilk
553 Sodium Dehydroacetate 4418-26-2 C8H7NaO4 ·H2O 208.15 <= 2 Preservative
554 Sodium Diacetate (anhydrous) 126-96-5 C4H7NaO4 ·xH2O 142.09 <= 2
Sequestrant;preservative;antimicrobialagent; mold
inhibitor
555 Sodium Erythorbate 6381-77-7 C6H7NaO6 ·H2O 216.12 <= 5 Preservative;
antioxidant
556 Sodium Ferric Pyrophosphate(anhydrous) 1332-96-3 Na8Fe4(P2O7)5 ·
xH2O 1277.02 <= 4 Nutrient
557 Sodium Ferrocyanide 13601-19-9 Na4Fe(CN)6 ·10H2O 484.07
Anticakingagent forsodiumchloride
558 Sodium Gluconate 527-07-1 C6H11NaO7 218.14 <= 2 Nutrient;sequestrant
559 Sodium Hydroxide 1310-73-2 NaOH 40.00 <= 2 pH controlagent
560 Sodium Hydroxide Solutions<= 2 , calculated on the
basis of NaOH determinedin the Assay
pH controlagent
561 Sodium Hypophosphite 7681-53-0 NaH2PO2 · H2O 105.99 <= 4 Preservative;antioxidant
562 Sodium Lactate Solution(anhydrous) 72-17-3 C3H5NaO3 112.06 <= 2
Emulsifier;flavor
enhancer;flavoring agent
or adjuvant;humectant; pHcontrol agent
page 2. Food Chemicals Codex (5th Edition) © 2003
563 Sodium Lauryl Sulfate 151-21-3 <= 2 Surface-activeagent
564 Sodium Lignosulfonate 8061-51-6 <= 1
Binder;dispersant;boiler water
additive
565 Sodium MagnesiumAluminosilicate 12040-43-6 <= 5 Anticaking
agent
566 Sodium Metabisulfite 7681-57-4 Na2S2O5 190.11 <= 2 Preservative;antioxidant
567 Sodium Metaphosphate,Insoluble 50813-16-6 <= 4
Emulsifier;sequestrant;
texturizer
568 Sodium Metasilicate (anhydrous) 6834-92-0 Na2O · SiO2 ·xH2O 122.06 <= 5
Saponifyingagent; boiler
water additive
569 Sodium Methylate 124-41-4 CH3ONa 54.02 <= 5
Catalyst forthe
transesterification of fats
570 Sodium Nitrate 7631-99-4 NaNO3 85.00 <= 4Antimicrobial
agent;preservative
571 Sodium Nitrite 7632-00-0 NaNO2 69.00 <= 4
Color fixativein meat and
meatproducts;
antimicrobialagent;
preservative
572 Sodium Phosphate, Dibasic(anhydrous) 7558-79-4 Na2HPO4 141.96 <= 4
Emulsifier;texturizer;
buffer; nutrient
573 Sodium Phosphate, Dibasic(dihydrate) 10028-24-7 Na2HPO4 ·
2H2O 177.99 <= 4Emulsifier;texturizer;
buffer; nutrient
574 Sodium Phosphate, Monobasic(anhydrous) 7558-80-7 NaH2PO4 119.98 <= 4
Buffer;emulsifier;
nutrient
575 Sodium Phosphate, Monobasic(monohydrate) 10049-21-5 NaH2PO4 · H2O 137.99 <= 4
Buffer;emulsifier;
nutrient
576 Sodium Phosphate, Tribasic(anhydrous) 7601-54-9 Na3PO4 163.94 <= 4
Antimicrobial;buffer;
emulsifier;nutrient
577 Sodium Phosphate, Tribasic(dodecahydrate) 10101-89-0 Na3PO4 · 12H2O 380.12 <= 4
Antimicrobial;buffer;
emulsifier;nutrient
578 Sodium Polyphosphates, Glassy 68915-31-1 <= 4Emulsifier;
sequestrant;texturizer
page 3. Food Chemicals Codex (5th Edition) © 2003
579 Sodium Polyphosphates, Glassy 10361-03-2 <= 4Emulsifier;
sequestrant;texturizer
580 Sodium Potassium Tartrate 304-59-6 C4H4KNaO6 ·4H2O 282.22 <= 2 Buffer;
sequestrant
581 Sodium PotassiumTripolyphosphate 24315-83-1 Na3K2P3O10 400.08 <= 2 Texturizer;
sequestrant
582 Sodium Propionate 137-40-6 C3H5NaO2 96.06 <= 2 Preservative;mold inhibitor
583 Sodium Pyrophosphate(anhydrous) 7722-88-5 Na4P2O7 265.90 <= 4
Emulsifier;buffer;
nutrient;sequestrant;
texturizer
584 Sodium Pyrophosphate(decahydrate) 13472-36-1 Na4P2O7 ·
10H2O 446.06 <= 4
Emulsifier;buffer;
nutrient;sequestrant;
texturizer
585 Sodium Saccharin 128-44-9 C7H4NNaO3S ·2H2O 241.19 <= 2 Nonnutritive
sweetener
586 Sodium Sesquicarbonate 533-96-0 Na2CO3 ·NaHCO3 · 2H2O 226.03 <= 2
pH controlagent;
neutralizer indairy products;
buffer
587 Sodium Stearoyl Lactylate 25383-99-7 <= 2
Emulsifier;dough
conditioner;stabilizer;whipping
agent
588 Sodium Stearyl Fumarate 4070-80-8 C22H39NaO4 390.54 <= 2 Doughconditioner
589 Sodium Sulfate (anhydrous) 7757-82-6 Na2SO4 142.04 <= 2Agent incaramel
production
590 Sodium Sulfate (decahydrate) 7727-73-3 Na2SO4 · 10H2O 322.19 <= 2Agent incaramel
production
591 Sodium Sulfite 7757-83-7 Na2SO3 126.04 <= 2
Preservative;antioxidant;bleaching
agent
592 Sodium Tartrate 868-18-8 C4H4Na2O6 ·2H2O 230.08 <= 2 Sequestrant
593 Sodium Thiosulfate 10102-17-7 <= 2 Sequestrant;antioxidant
594 Sodium Trimetaphosphate 7785-84-4 (NaPO3)3 305.89 <= 4Starch-
modifyingagent
page 4. Food Chemicals Codex (5th Edition) © 2003
595 Sodium Tripolyphosphate(anhydrous) 7758-29-4 Na5P3O10 367.86 <= 2 Emulsifier;
sequestrant
596 Sodium Tripolyphosphate(hexahydrate) 15091-98-2 Na5P3O10 ·
6H2O 475.96 <= 2 Emulsifier;sequestrant
597 Solin Oil <= 0.1 Coating agent;texturizer
598 Sorbic Acid 110-44-1 C6H8O2 112.13 <= 2 Preservative;mold inhibitor
599 Sorbitan Monostearate 1338-41-6 <= 2
Emulsifier;stabilizer;defoaming
agent
600 Sorbitol 50-70-4 C6H14O6 182.17 <= 2
Humectant;texturizing
agent; nutritivesweetener
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
601 Sorbitol Solution <= 2 , calculated on theanhydrous basis
Humectant;texturizing
agent; nutritivesweetener
602 Soybean Oil (Unhydrogenated) 8001-22-7 <= 0.1 Coating agent;texturizer
603 Soy Protein Concentrate 9010-10-0 <= 1
Proteinsupplement;water and fat
binder;stabilizer and
thickener;texturizing
agent
604 Spearmint Oil 8008-79-5 Flavoringagent
605 Spice Oleoresins
Flavoringagent; color(oleoresinspaprika and
turmeric only)
606 Spike Lavender Oil 84837-04-7 Flavoringagent
607 Stannous Chloride (anhydrous) 7772-99-8 SnCl2 189.60 <= 4Reducing
agent;antioxidant
608 Stannous Chloride (dihydrate) 10025-0969-091 SnCl2 · 2H2O 225.63 <= 4Reducing
agent;antioxidant
609 Starter Distillate Flavoringagent
610 Stearic Acid 57-11-4 C18H36O2 284.48 <= 2
Component inthe
manufactureof other food-
gradeadditives;lubricant;
defoamingagent
611 Stearyl Monoglyceridyl Citrate <= 2 Emulsionstabilizer
612 Succinic Acid 110-15-6 C4H6O4 118.09 <= 2Buffer;
neutralizingagent
page 2. Food Chemicals Codex (5th Edition) © 2003
613 Succinylated Monoglycerides <= 2Emulsifier;
doughconditioner
614 Sucralose 56038-13-2 C12H19Cl3O8 397.64 <= 1
Nonnutritivesweetener;
flavorenhancer
615 Sucrose 57-50-1 C12H22O11 342.30 <= 0.1
Nutritivesweetener;formulation
and texturizingaid
616 Sucrose Acetate Isobutyrate 27216-37-1 C40H62O19 846.9 (832-856) <= 1 Stabilizer
617 Sucrose Acetate Isobutyrate 123-13-6 C40H62O19 846.9 (832-856) <= 1 Stabilizer
618 Sucrose Fatty Acid Esters <= 2Emulsifier;stabilizer;texturizer
619 Sugar Beet Fiber <= 1
Anticakingagent; bindingagent; bulking
agent;dispersing
agent; sourceof dietary
fiber;stabilizing
agent;texturizing
agent;thickening
agent
620 Sulfur Dioxide 7446-09-5 SO2 64.06 <= 2 by weight
Antioxidant;bleaching
agent;preservative
621 Sulfuric Acid 7664-93-9 H2SO4 98.07 <= 5 Acidifier
622 Sunflower Oil (Unhydrogenated) 8008-31-9 <= 0.1 Coating agent;texturizer
623 Sunset Yellow (1) 2783-94-0 C16H10N2O7S2Na2
452.38 <= 10 Color
624 Talc 14807-96-6 <= 5
Anticakingagent; coating
agent;lubricating andrelease agent;
surface-finishingagent;
texturizingagent
625 Tallow <= 0.1 Coating agent;texturizer
page 3. Food Chemicals Codex (5th Edition) © 2003
626 Tangerine Oil, Coldpressed 8008-31-9 Flavoringagent
627 Tannic Acid 1401-55-4 <= 2
Clarifyingagent;
flavoringagent; flavorenhancer;flavoringadjuvant
628 Tarragon Oil 8016-88-4 Flavoringagent
629 Tartaric Acid 87-69-4 C4H6O6 150.09 <= 2Acidifier;
sequestrant;flavoring agent
630 Tartrazine (1) 1934-21-0 C16H9N4O9S2Na3
534.37 <= 10 Color
631 TBHQ 1948-33-0 C10H14O2 166.22 <= 2 Antioxidant
632 Terpene Resin, Natural 9003-74-1 <= 3
Masticatorysubstance inchewing gum
base
633 Terpene Resin, Synthetic <= 3
Masticatorysubstance inchewing gum
base
634 Thiamine Hydrochloride 67-03-8 C12H17ClN4OS ·HCl 337.27 <= 2 Nutrient
635 Thiamine Mononitrate 532-43-4 C12H17N5O4S 327.36 <= 2 Nutrient
636 L-Threonine 72-19-5 C4H9NO3 119.12 <= 5 Nutrient
637 Thyme Oil 8007-46-3 Flavoringagent
638 Titanium Dioxide 13463-67-7 TiO2 79.90 <= 10 Color
639 All-rac-a-Tocopherol 10191-41-0 C29H50O2 430.71 <= 2 Nutrient;antioxidant
640 RRR-α-Tocopherol Concentrate 59-02-9 C29H50O2 430.71 <= 2 Nutrient;antioxidant
641 RRR-Tocopherols Concentrate,Mixed <= 2
High-AlphaType: Nutrient;
antioxidant.Low-Alpha
Type:Antioxidant
642 RRR-α-Tocopheryl Acetate 58-95-7 C31H52O3 472.75 <= 2 Nutrient
643 All-rac-a-Tocopheryl Acetate 7695-91-2 C31H52O3 472.75 <= 2 Nutrient
644 RRR-α-Tocopheryl AcetateConcentrate <= 2 Nutrient
page 4. Food Chemicals Codex (5th Edition) © 2003
645 RRR-α-Tocopheryl AcidSuccinate 4345-03-3 C33H54O5 530.79 <= 2 Nutrient
646 Tragacanth 9000-65-1 <= 2Stabilizer;thickener;emulsifier
647 Trehalose (anhydrous) 99-20-7 C12H22O11 ·2H2O 378.33 <= 0.1
Humectant;nutritive
sweetener;stabilizer;thickener;texturizer
648 Trehalose (dihydrate) 6138-23-4 C12H22O11 ·2H2O 378.33 <= 0.1
Humectant;nutritive
sweetener;stabilizer;thickener;texturizer
649 Triacetin 102-76-1 C9H14O6 218.21 <= 1 Humectant;solvent
650 Trichloroethylene 79-01-6 C2HCl3 131.39 <= 1 Extractionsolvent
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
651 Triethyl Citrate 77-93-0 C12H20O7 276.29 <= 2 Solvent
652 DL-Tryptophan 54-12-6 C11H12N2O2 204.22 <= 5 Nutrient
653 L-Tryptophan 73-22-3 C11H12N2O2 204.22 <= 5 Nutrient
654 L-Tyrosine 60-18-4 C9H11NO3 181.19 <= 5 Nutrient
655 Urea 57-13-6 CH4N2O 60.06 <= 5 Fermentationaid
656 L-Valine 72-18-4 C5H11NO2 117.15 <= 5 Nutrient
657 Vegetable Oil Phytosterol Esters <= 0.1 Source ofphytosterols
658 Vitamin A 68-26-8 <= 2 Nutrient
659 Vitamin B12 68-19-9 C63H88CoN14O14P 1355.38 Nutrient
660 Vitamin D2 50-14-6 C28H44O 396.66 Nutrient
661 Vitamin D3 67-97-0 C27H44O 384.65 Nutrient
662 Vitamin K 84-80-0 C31H46O2 450.71 <= 2 Nutrient
663 Wheat Gluten 8002-80-0 <= 1
Doughstrengthener;
nutrient;stabilizer and
thickener;surface-finishing
agent; andtexturizing
agent
664 Wheat Protein Isolate <= 0.5
Texturizer;nutrient;
emulsifier;water-binding
aid; gellingagent;
foaming agent
665 Whey <= 0.5 Texturizer;nutrient
666 Whey Protein Concentrate <= 0.5
Texturizer;nutrient;
emulsifier;water-binding
aid; gellingagent
page 2. Food Chemicals Codex (5th Edition) © 2003
667 Whey Protein Isolate <= 0.5
Source of high-quality
protein; gellingagent; water-binding aid;foaming or
whipping aid;emulsifier;
edible coatingused as amoisturebarrier
668 Whey, Reduced Lactose <= 0.5Texturizer;nutrient;
emulsifier
669 Whey, Reduced Minerals <= 0.5 Texturizer;nutrient
670 Wintergreen Oil 68917-75-9 Flavoringagent
671 Xanthan Gum 11138-66-2 <= 2
Stabilizer;thickener;emulsifier;
suspendingagent; bodying
agent; foamenhancer
672 Xylitol 87-99-0 C5H12O5 152.15 <= 1 Nutritivesweetener
673 Yeast, Autolyzed <= 2
Flavoringagent; flavorenhancer;
proteinsource; binder
674 Yeast, Dried <= 1 Carrier; flavorenhancer
675 Yeast Extract <= 2Flavoring
agent; flavorenhancer
676 Zein 9010-66-6 <= 2
Surface-finishingagent;
texturizingagent
677 Zinc Gluconate 4468-02-4 C12H22O14Zn 455.68 <= 2 Nutrient
678 Zinc Oxide 1314-13-2 ZnO 81.38 <= 10 Nutrient
679 Zinc Sulfate (monohydrate) 7446-19-7 ZnSO4 · H2O 179.45 <= 4 Nutrient
680 Zinc Sulfate (heptahydrate) 7446-20-0 ZnSO4 · 7H2O 287.54 <= 4 Nutrient
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
51 DL-Aspartic Acid 617-45-8 C4H7NO4 133.10 <= 5 Nutrient
52 L-Aspartic Acid 56-84-8 C4H7NO4 133.10 <= 5 Nutrient
53 Azodicarbonamide 123-77-3 C2H4N4O2 116.08 <= 5 Maturingagent for flour
54 Balsam Peru Oil 8007-00-9 Flavoringagent
55 Basil Oil, Comoros Type Flavoringagent
56 Basil Oil, European Type 8015-73-4 Flavoringagent
57 Bay Oil Flavoringagent
58 Beeswax, White <= 5
Surface-finishing(glazing)
agent; releaseagent; rawmaterial for
flavoring agent
59 Beeswax, Yellow 8012-89-3 <= 5
Candy glazeand polish;
raw materialfor flavoring
agent
60 Bentonite 1302-78-9 <= 0.004% Clarifying,filter agent
61 Benzoic Acid 65-85-0 C7H6O2 122.12 <= 2.0Preservative;antimicrobial
agent
62 Benzoyl Peroxide 94-36-0 C14H10O4 242.23 <= 4 Bleachingagent
63 Bergamot Oil, Coldpressed 8007-75-8 Flavoringagent
64 BHA 25013-16-5 C11H16O2 180.25 Antioxidant
65 BHT 128-37-0 C15H24O 220.35 Antioxidant
66 Biotin 58-85-5 C10H16N2O3S 244.31 <= 2 Nutrient
67 Birch Tar Oil, Rectified Flavoringagent
68 Black Pepper Oil 8006-82-4 Flavoringagent
page 2. Food Chemicals Codex (5th Edition) © 2003
69 Bohenin <= 0.5
Tempering aidand antibloomagent in themanufactureof chocolate
and chocolatecoatings
70 Bois de Rose Oil Flavoringagent
71 Brilliant Blue (1) 3844-45-9 C37H34N2O9S3Na2
792.86 <= 10 Color
72 Brominated Vegetable Oil
Flavoringagent;
beveragestabilizer
73 Butadiene-Styrene Rubber <= 3
Masticatorysubstance inchewing gum
base
74 Butane 106-97-8 C4H10 58.12 Propellant;aerating agent
75 Butylated Hydroxymethylphenol C15H24O2 236.35 Antioxidant
76 1,3-Butylene Glycol 107-88-0 C4H10O2 90.12 <= 2Solvent forflavoringagents
77 Caffeine (anhydrous) 58-08-2 C8H10N4O2 194.19 <= 1 Flavoringagent
78 Caffeine (monohydrate) 58-08-2 C8H10N4O2 ·H2O 212.21 <= 1 Flavoring
agent
79 Calcium Acetate 62-54-4 Ca(C2H3O2)2 158.17 <= 2Buffer;
stabilizer;firming agent
80 Calcium Acid Pyrophosphate 35405-51-7 CaH2P2O7 216.04 <= 2 Leaveningagent; nutrient
81 Calcium Alginate 9005-35-0 [(C6H7O6)2Ca]n 195.16 (calculated) <= 5Stabilizer;thickener;emulsifier
82 Calcium Alginate 9005-35-0 [(C6H7O6)2Ca]n 219.00 (avg) <= 5Stabilizer;thickener;emulsifier
83 Calcium Ascorbate 5743-27-1 C12H14CaO12 ·2H2O 426.34 <= 2 Antioxidant
84 Calcium Bromate 10102-75-7 Ca(BrO3)2 · H2O 313.90 <= 4
Maturingagent;
oxidizingagent
page 3. Food Chemicals Codex (5th Edition) © 2003
85 Calcium Carbonate 471-34-1 CaCO3 100.09 <= 3
pH controlagent;
nutrient;dough
conditioner;firming agent;yeast nutrient
86 Calcium Chloride (anhydrous) 10043-52-4 CaCl2 110.98 <= 5 Firming agent
87 Calcium Chloride (dihydrate) 10035-04-8 CaCl2 · 2H2O 147.01 <= 5 Firming agent
88 Calcium Chloride Solution<= 4 , calculated on the
amount of CaCl2 asdetermined in the Assay
Sequestrant;firming agent
89 Calcium Citrate 5785-44-4 Ca3(C6H5O7)2 ·4H2O 570.50 <= 2
Sequestrant;buffer; firming
agent
90 Calcium Disodium EDTA 23411-34-9 C10H12CaN2Na2O8 · 2H2O 410.30 <= 4 Preservative;
sequestrant
91 Calcium Gluconate (anhydrous) 299-28-5 C12H22CaO14 430.38 <= 2Firming agent;
stabilizer;texturizer
92 Calcium Gluconate(monohydrate)
C12H22CaO14 ·H2O 448.39 <= 2
Firming agent;stabilizer;texturizer
93 Calcium Glycerophosphate 27214-00-2 C3H7CaO6P 210.14 <= 4 Nutrient
94 Calcium Hydroxide 1305-62-0 Ca(OH)2 74.10 <= 2
Buffer;neutralizing
agent; firmingagent
95 Calcium Iodate 7789-80-2 Ca(IO3)2 · H2O 407.90 <= 4Maturing
agent; doughconditioner
96 Calcium Lactate (anhydrous) 814-80-2 C6H10CaO6 ·xH2O 218.22 <= 2
Buffer; doughconditioner;
yeast nutrient
97 Calcium Lactobionate(anhydrous) 5001-51-4 C24H42CaO24 754.66 <= 2
Firming agentin dry puddingmixes; nutrient
98 Calcium Lignosulfonate 8061-52-7 <= 1 Binder;dispersant
99 Calcium Oxide 1305-78-8 CaO 56.08 <= 2
pH controlagent;
nutrient;dough
conditioner;yeast food
100 Calcium Pantothenate 137-08-6 C18H32CaN2O10
476.54 <= 2 Nutrient
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
101 Calcium Pantothenate, CalciumChloride Double Salt 6363-38-8 C18H32CaN2O1
0 · CaCl2587.52 <= 2 Nutrient
102 Calcium Pantothenate, Racemic 6381-63-1 C18H32CaN2O10
476.54 <= 2 Nutrient
103 Calcium Peroxide 1305-79-9 CaO2 72.08 <= 4
Doughconditioner;
oxidizingagent
104 Calcium Phosphate, Dibasic(anhydrous) 7757-93-9 CaHPO4 136.06 <= 2
Leaveningagent; doughconditioner;
nutrient; yeastfood
105 Calcium Phosphate, Dibasic(dihydrate) 7789-77-7 CaHPO4 · 2H2O 172.09 <= 2
Leaveningagent; doughconditioner;
nutrient; yeastfood
106 Calcium Phosphate, Monobasic(anhydrous) 7758-23-8 Ca(H2PO4)2 234.05 <= 2
Buffer; doughconditioner;
firming agent;leavening
agent;nutrient; yeast
food;sequestrant
107 Calcium Phosphate, Monobasic(monohydrate) 10031-30-8 Ca(H2PO4)2 ·
H2O 252.07 <= 2
Buffer; doughconditioner;
firming agent;leavening
agent;nutrient; yeast
food;sequestrant
108 Calcium Phosphate, Tribasic 7758-87-4 Ca3(PO4)2 310.18 <= 2
Anticakingagent; buffer;
nutrient;clouding agent
109 Calcium Phosphate, Tribasic 1306-06-5 Ca5OH(PO4)3 502.31 <= 2
Anticakingagent; buffer;
nutrient;clouding agent
110 Calcium Phosphate, Tribasic 62974-97-4 Ca10(OH)2(PO4)6
1004.61 <= 2
Anticakingagent; buffer;
nutrient;clouding agent
111 Calcium Propionate 4075-81-4 C6H10CaO4 186.22 <= 2 Preservative;mold inhibitor
page 2. Food Chemicals Codex (5th Edition) © 2003
112 Calcium Pyrophosphate 7790-76-3 Ca2P2O7 254.10 <= 2Buffer;
neutralizingagent; nutrient
113 Calcium Saccharin (anhydrous) 6485-34-3 C14H8CaN2O6S2 · 3½H2O 467.48 <= 2 Nonnutritive
sweetener
114 Calcium Silicate 1344-95-2 <= 5 Anticakingagent; filter aid
115 Calcium Sorbate 7492-55-9 C12H14CaO4 262.32 <= 2Antimicrobial
agent;preservative
116 Calcium Stearate 1592-23-0 <= 2Anticaking
agent; binder;emulsifier
117 Calcium Stearoyl Lactylate 5793-94-2 <= 2
Doughconditioner;stabilizer;whipping
agent
118 Calcium Sulfate (anhydrous) 7778-18-9 CaSO4 136.14 <= 2
Nutrient; yeastfood; doughconditioner;
firming agent;sequestrant
119 Calcium Sulfate (dihydrate) 10101-41-4 CaSO4 · 2H2O 172.18 <= 2
Nutrient; yeastfood; doughconditioner;
firming agent;sequestrant
120 Cananga Oil 68606-83-7 Flavoringagent
121 Candelilla Wax 8006-44-8 <= 3
Masticatorysubstance inchewing gumbase; surface-finishing agent
122 Canola Oil 120962-03-0 <= 0.1
Cooking orsalad oil;
component ofmargarine orshortening;
coating agent;texturizer
123 Canthaxanthin 514-78-3 C40H52O2 564.85 <= 10 Color
124 Caramel 8028-89-5 <= 2 Color
125 Caraway Oil 8000-42-8 Flavoringagent
page 3. Food Chemicals Codex (5th Edition) © 2003
126 Carbon, Activated <= 10
Decolorizingagent; taste-
and odor-removing
agent;purification
agent in foodprocessing
127 Carbon Dioxide 124-38-9 CO2 44.01
Propellant andaeratingagent;
carbonatingagent; direct-
contactfreezing agent
128 Cardamom Oil 8000-66-6 Flavoringagent
129 Carmine 1390-65-4 C22H20O13 492.39 <= 2 Color
130 Carnauba Wax 8015-86-9 <= 5
Anticakingagent; surface
-finishing(glazing)
agent; releaseagent; carrier
for flavors
131 L-Carnitine 541-15-1 C7H15NO3 161.20 <= 1 Nutrient
132 β-Carotene 7235-40-7 C40H56 536.88 <= 5 Nutrient; color
133 Carrot Seed Oil 8015-88-1 Flavoringagent
134 Cascarilla Oil 8007-06-5 Flavoringagent
135 Casein and Caseinate Salts 9000-71-9 <= 1
Binder;extender;clarifying
agent;emulsifier;stabilizer
136 Cassia Oil 8007-80-5 Flavoringagent
137 Castor Oil 8001-79-4 <= 0.1
Antistickingagent; release
agent;component of
protectivecoatings
138 Cedar Leaf Oil 8007-20-3 Flavoringagent
139 Celery Seed Oil Flavoringagent
page 4. Food Chemicals Codex (5th Edition) © 2003
140 Cellulose Gel 9004-34-6 <= 2
Anticakingagent; binding
agent;dispersing
agent
141 Cellulose Gum 9004-32-4 <= 3 Thickener;stabilizer
142 Cellulose, Powdered 9004-34-6 <= 3
Anticakingagent; bindingagent; bulking
agent;dispersingagent; filter
aid; texturizingagent;
thickeningagent
143 Chamomile Oil, English Type 8015-92-7 Flavoringagent
144 Chamomile Oil, German Type Flavoringagent
145 Chlorine 7782-50-5 Cl2 70.91 <= 10
Antimicrobialagent;
bleachingagent;
oxidizingagent
146 Cholic Acid 81-25-4 C24H40O5 408.58 <= 4 Emulsifier
147 Choline Bitartrate 87-67-2 C9H19NO7 253.25 <= 2 Nutrient
148 Choline Chloride 67-48-1 C5H14ClNO 139.65 <= 2 Nutrient
149 Cinnamon Bark Oil, Ceylon Type 8015-91-6 Flavoringagent
150 Cinnamon Leaf Oil 8015-91-6 Flavoringagent
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
151 Citric Acid (anhydrous) 77-92-9 C6H8O7 192.13 <= 0.5
Sequestrant;dispersing
agent;acidifier;
flavoring agent
152 Citric Acid (monohydrate) 5949-29-1 C6H8O7 · H2O 210.14 <= 0.5
Sequestrant;dispersing
agent;acidifier;
flavoring agent
153 Clary Oil 8016-63-5 Flavoringagent
154 Clove Leaf Oil 8015-97-2 Flavoringagent
155 Clove Oil 8000-34-8 Flavoringagent
156 Clove Stem Oil 8015-98-3 Flavoringagent
157 Cocoa Butter Substitute <= 0.1 Coating agent;texturizer
158 Coconut Oil (Unhydrogenated) 8001-31-8 <= 0.1
Coating agent;emulsifying
agent;texturizer
159 Cognac Oil, Green 8016-21-5 Flavoringagent
160 Copaiba Oil 8013-97-6 Flavoringagent
161 Copper Gluconate 527-09-3 C12H22CuO14 453.84 <= 5 Nutrient
162 Copper Sulfate (anydrous) 7758-98-7 CuSO4 159.6 <= 4 Nutrient
163 Copper Sulfate (pentahydrate) 7758-99-8 CuSO4 · 5H2O 249.68 <= 4 Nutrient
164 Coriander Oil 8008-52-4 Flavoringagent
165 Corn Oil (Unhydrogenated) 8001-30-7 <= 0.1
Coating agent;emulsifying
agent;texturizer
166 Costus Root Oil 8023-88-9 Flavoringagent
page 2. Food Chemicals Codex (5th Edition) © 2003
167 Cottonseed Oil(Unhydrogenated) 8001-29-4 <= 0.1
Cooking orsalad oil;
component ofmargarine orshortening;tenderizer;
carrier;stabilizer;thickener;
coating agent;texturizer
168 Cubeb Oil 8007-87-2 Flavoringagent
169 Cumin Oil 8014-13-9 Flavoringagent
170 Curdlan 54724-00-4 (C6H10O5)n <= 0.5
Firming agent;gelling agent;
stabilizer;thickener
171 beta-Cyclodextrin 7585-39-9 (C6H10O5)7 1135.0 <= 1Encapsulating
agent;stabilizer
172 gamma-Cyclodextrin 17465-86-0 (C6H10O5)8 1297.14 <= 1Stabilizer;emulsifier;
carrier
173 L-Cysteine Monohydrochloride(anhydrous) 52-89-1 C3H7NO2S · HCl 157.62 <= 5 Nutrient
174 L-Cysteine Monohydrochloride(monohydrate) 7048-04-6 C3H7NO2S · HCl
· H2O 175.63 <= 5 Nutrient
175 L-Cystine 56-89-3 C6H12N2O4S2 240.30 <= 5 Nutrient
176 Dammar Gum 9000-16-2 <= 5 Stabilizer;glazing agent
177 Decanoic Acid 334-48-5 C10H20O2 172.27
Component inthe
manufactureof other food-
gradeadditives;defoaming
agent;flavoring agent
178 Dehydroacetic Acid 520-45-6 C8H8O4 168.15 <= 0.5Antimicrobial
agent;preservative
179 Desoxycholic Acid 83-44-3 C24H40O4 392.58 <= 4 Emulsifier
180 Dexpanthenol 81-13-0 C9H19NO4 205.25 <= 5 Nutrient
181 Dextrin 9004-53-9 <= 1
Thickener;colloidal
stabilizer;binder;surface-
finishing agent
page 3. Food Chemicals Codex (5th Edition) © 2003
182 Dextrose 50-99-7 180.16 <= 0.1
Nutritivesweetener;humectant;texturizing
agent
183 Diacetyl Tartaric Acid Esters ofMono- and Diglycerides 91052-83-4 <= 2 Emulsifier
184 Diacetyl Tartaric Acid Esters ofMono- and Diglycerides 100085-39-0 <= 2 Emulsifier
185 Diatomaceous Earth (naturalpowder and calcined powder) 61790-53-2 <= 10
Filter aid infood
processing
186 Diatomaceous Earth (flux-calcined powder) 68855-54-9 <= 10
Filter aid infood
processing
187 Dilauryl Thiodipropionate 123-28-4 C30H58O4S 514.85 <= 10 Antioxidant
188 Dill Seed Oil, European Type Flavoringagent
189 Dill Seed Oil, Indian Type Flavoringagent
190 Dillweed Oil, American Type 8006-75-5 Flavoringagent
191 Dimethyl Dicarbonate 4525-33-1 C4H6O5 134.09 <= 1 Preservative;antimicrobial
192 Dimethylpolysiloxane 9006-65-9 <= 5 Defoamingagent
193 Dioctyl Sodium Sulfosuccinate 577-11-7 C20H37NaO7S 444.56 <= 2 Emulsifier;wetting agent
194 Disodium EDTA 6381-92-6 C10H14N2Na2O8 · 2H2O 372.24 <= 10
Preservative;sequestrant;
stabilizer
195 Disodium Guanylate (anhydrous) 5550-12-9 C10H12N5Na2O8P · xH2O 407.19 <= 5 Flavor
enhancer
196 Disodium Inosinate (anhydrous) 4691-65-0 C10H11N4Na2O8P · xH2O 392.17 <= 5 Flavor
enhancer
197 Enzyme-Modified Fats <= 1 Flavoringagent
198 Enzyme Preparations <= 5
Enzyme (seediscussion
underClassification,
below)
199 Erythorbic Acid 89-65-6 C6H8O6 176.13 <= 2 Preservative;antioxidant
page 4. Food Chemicals Codex (5th Edition) © 2003
200 Erythritol 149-32-6 C4H10O4 122.12 <= 1
Flavorenhancer;humectant;
nutritivesweetener;texturizing
agent;stabilizer
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
201 Erythrosine (1) 16423-68-0 C20H6O5I4Na2 879.86 <= 10 Color
202 Ethoxylated Mono- andDiglycerides <= 1
Doughconditioner;emulsifier
203 Ethoxyquin (monomer) 91-53-2 C14H19NO 217.31 <= 2 Antioxidant
204 Ethyl Alcohol 64-17-5 C2H6O 46.07 <= 0.5Extraction
solvent; carriersolvent
205 Ethyl Cellulose 9004-57-3 <= 3Protectivecoating;
binder; filler
206 Ethylene Dichloride 107-06-2 C2H4Cl2 98.96 <= 1 Extractionsolvent
207 Ethyl Maltol 4940-11-8 C7H8O3 140.14Flavoring
agent; flavorenhancer
208 Eucalyptus Oil 8000-48-4 Flavoringagent
209 Fast Green (1) 2353-45-9 C37H34N2O10S3Na2
808.86 <= 10 Color
210 FD&C Blue No. 1 (1) 3844-45-9 C37H34N2O9S3Na2
792.86 (as Pb) <= 10 Color
211 FD&C Blue No. 2 (1) 860-22-0 C16H8N2O8S2Na2
466.36 (as Pb) <= 10 Color
212 FD&C Green No. 3 (1) 2353-45-9 C37H34N2O10S3Na2
808.86 (as Pb) <= 10 Color
213 FD&C Red No. 3 (1) 16423-68-0 C20H6O5I4Na2 879.86 (as Pb) <= 10 Color
214 FD&C Red No. 40 (1) 25956-17-6 C18H14N2O8S2Na2
496.43 <= 10 Color
215 FD&C Yellow No. 5 (1) 1934-21-0 C16H9N4O9S2Na3
534.37 <= 10 Color
216 FD&C Yellow No. 6 (1) 2783-94-0 C16H10N2O7S2Na2
452.37 <= 10 Color
217 Fennel Oil 8006-84-6 Flavoringagent
218 Ferric Ammonium Citrate, Brown 1185-57-5 <= 2 Nutrient
page 2. Food Chemicals Codex (5th Edition) © 2003
219 Ferric Ammonium Citrate, Green 1185-57-5 <= 2
Nutrient;anticakingagent forsodiumchloride
220 Ferric Citrate (anhydrous) 2338-05-8 FeC6H5O7 ·xH2O 244.95 <= 2 Nutrient
221 Ferric Phosphate (anhydrous) 10045-86-0 FePO4 · xH2O 150.82 <= 4 Nutrient
222 Ferric Pyrophosphate(anhydrous) 10058-44-3 Fe4(P2O7)3 ·
xH2O 745.22 <= 4 Nutrient
223 Ferrous Citrate 23383-11-1 FeC6H6O7 245.95 <= 2 Nutrient
224 Ferrous Fumarate 141-01-5 C4H2FeO4 169.90 <= 2 Nutrient
225 Ferrous Gluconate 299-29-6 C12H22FeO14 ·2H2O 482.18 <= 2 Nutrient; color
adjunct
226 Ferrous Glycinate 20150-34-9 Fe(OH2)2(OOCCH2NH2)2
239.99 <= 1 Source ofdietary iron
227 Ferrous Lactate (anhydrous) 5905-52-2 C6H10FeO6 ·xH2O 233.99 <= 1 Nutrient
228 Ferrous Sulfate 7782-63-0 FeSO4 · 7H2O 278.02 <= 2 Nutrient
229 Ferrous Sulfate, Dried(anhydrous) 7720-78-7 FeSO4 · xH2O 151.91 <= 2 Nutrient
230 Fir Needle Oil, Canadian Type Flavoringagent
231 Fir Needle Oil, Siberian Type Flavoringagent
232 Folic Acid 59-30-3 C19H19N7O6 441.40 <= 2 Nutrient
233 Food Starch, Modified <= 1
Thickener;colloidal
stabilizer;binder
234 Food Starch, Unmodified <= 1
Thickener;colloidal
stabilizer;binder
235 Formic Acid 64-18-6 CH2O2 46.03Flavoringadjunct;
preservative
236 Fructose 57-48-7 C6H12O6 180.16 <= 0.1 Nutritivesweetener
237 Fumaric Acid 110-17-8 C4H4O4 116.07 <= 2 Acidifier;flavoring agent
238 Furcelleran 9000-21-9 <= 5Stabilizer;thickener;
gelling agent
page 3. Food Chemicals Codex (5th Edition) © 2003
239 Garlic Oil 8000-78-0 Flavoringagent
240 Gelatin 9000-70-8 <= 1.5
Firming agent;stabilizer and
thickener;surface-activeagent; surface
-finishingagent
241 Gellan Gum 71010-52-1 <= 2 Stabilizer;thickener
242 Geranium Oil, Algerian Type 8000-46-2 Flavoringagent
243 Gibberellic Acid 77-06-5 C19H22O6 346.38 <= 5 Enzymeactivator
244 Ginger Oil 8007-08-7 Flavoringagent
245 Glucono Delta-Lactone 90-80-2 C6H10O6 178.14 <= 4
Acidifier;leavening
agent;sequestrant
246 Glucose Syrup <= 0.1 Nutritivesweetener
247 Glucose Syrup, Dried <= 0.1 Nutritivesweetener
248 L-Glutamic Acid 56-86-0 C5H9NO4 147.13 <= 5 Salt substitute;nutrient
249 L-Glutamic Acid Hydrochloride 138-15-8 C5H9NO4 · HCl 183.59 <= 5Salt substitute;
flavoringagent; nutrient
250 L-Glutamine 56-85-9 C5H10N2O3 146.15 <= 5 Nutrient
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
251 Glutaraldehyde 111-30-8 C5H8O2 100.12 <= 2
Fixing agent inthe
immobilizationof enzyme
preparations;cross-linking
agent formicroencapsulating flavoringsubstances;antimicrobial
for sugarmilling
252 Glycerin 56-81-5 C3H8O3 92.09 <= 1
Humectant;solvent;
bodying agent;plasticizer
253 Glycerol Ester of Gum Rosin <= 1
Masticatorysubstance inchewing gum
base
254 Glycerol Ester of PartiallyDimerized Rosin <= 1
Masticatorysubstance inchewing gum
base
255 Glycerol Ester of PartiallyHydrogenated Gum Rosin <= 1
Masticatorysubstance inchewing gum
base
256 Glycerol Ester of PartiallyHydrogenated Wood Rosin <= 1
Masticatorysubstance inchewing gum
base
257 Glycerol Ester of PolymerizedRosin <= 1
Masticatorysubstance inchewing gum
base
258 Glycerol Ester of Tall Oil Rosin <= 1
Masticatorysubstance inchewing gum
base
259 Glycerol Ester of Wood Rosin 8050-30-4 <= 1
Masticatorysubstance inchewing gum
base;beveragestabilizer
260 Glyceryl Behenate 30233-64-8 C69H134O6 1059.83 <= 1 Emulsifier;texturizer
page 2. Food Chemicals Codex (5th Edition) © 2003
261 Glyceryl-Lacto Esters of FattyAcids <= 0.5 Emulsifier;
stabilizer
262 Glyceryl Monooleate 25496-72-4 C21H40O4 356.54 <= 1 Emulsifier;flavoring agent
263 Glyceryl Monostearate 31566-31-1 <= 1 Emulsifier
264 Glyceryl Palmitostearate <= 1 Emulsifier
265 Glyceryl Tristearate 555-43-1 C57H110O6 891.49 <= 1
Crystallizationaccelerator;
lubricant;surface-
finishing agent
266 Glycine 56-40-6 C2H5NO2 75.07 <= 5 Nutrient
267 Grapefruit Oil, Coldpressed 8016-20-4 Flavoringagent
268 Grape Skin Extract 11029-12-2 <= 5 Color
269 Guar Gum 9000-30-0 <= 2Stabilizer;thickener;emulsifier
270 Gum Arabic 9000-01-5 <= 5 Stabilizer;emulsifier
271 Gum Ghatti 9000-28-6 <= 5 Emulsifier
272 Gum Guaiac 9000-29-7 <= 2 Antioxidant
273 Helium 7440-59-7 He 4.00 Processing aid
274 Heptylparaben 1085-12-7 C14H20O3 236.31 <= 2Preservative;antimicrobial
agent
275 Hexanes 110-54-3 C6H14 86.18 <= 1 Extractionsolvent
276 4-Hexylresorcinol 136-77-6 C12H18O2 194.27 <= 2
Colorstabilizer;enzymaticbrowninginhibitor
277 High-Fructose Corn Syrup <= 0.1 Nutritivesweetener
278 L-Histidine 71-00-1 C6H9N3O2 155.16 <= 5 Nutrient
279 L-Histidine Monohydrochloride 5934-29-2 C6H9N3O2 · HCl· H2O 209.63 <= 5 Nutrient
280 Hops Oil 8007-04-3 Flavoringagent
281 Hydrochloric Acid 7647-01-0 HCl 36.46 <= 1 Acidifier
page 3. Food Chemicals Codex (5th Edition) © 2003
282 Hydrogenated StarchHydrolysate (Sorbitol) 68425-17-2 C6H14O6 182.17 <= 1
Humectant;texturizing
agent;stabilizer;thickener;
crystalmodification
agent
283 Hydrogenated StarchHydrolysate (Maltitol) 68425-17-2 C12H24O11 344.31 <= 1
Humectant;texturizing
agent;stabilizer;thickener;
crystalmodification
agent
284Hydrogenated StarchHydrolysate (Dextrose
Monomer)C12H24O11 plus 162.14 <= 1
Humectant;texturizing
agent;stabilizer;thickener;
crystalmodification
agent
285 Hydrogenated StarchHydrolysate C6H10O5 162.14 <= 1
Humectant;texturizing
agent;stabilizer;thickener;
crystalmodification
agent
286 Hydrogen Peroxide 7722-84-1 H2O2 34.01 <= 4
Bleaching,oxidizing
agent; starchmodifier; anti-
microbialagent
287 Hydroxylated Lecithin 8029-76-3 <= 1 Emulsifier;clouding agent
288 Hydroxypropyl Cellulose 9004-64-2 <= 3
Emulsifier; filmcoating;
protectivecolloid;
stabilizer;suspending
agent;thickener
289 Hydroxypropyl Methylcellulose 9004-65-3 <= 3
Thickeningagent;
stabilizer;emulsifier
290 Indigotine (1) 860-22-0 C16H8N2O8S2Na2
466.36 <= 10 Color
291 Inositol 87-89-8 C6H12O6 180.16 <= 4 Nutrient
page 4. Food Chemicals Codex (5th Edition) © 2003
292 Invert Sugar 8013-17-0 <= 0.5 Nutritivesweetener
293 Iron, Carbonyl 37220-42-1 Fe 55.85 <= 4 Nutrient
294 Iron, Electrolytic 7439-89-6 Fe 55.85 <= 4 Nutrient
295 Iron, Reduced 7439-89-6 Fe 55.85 <= 10 Nutrient
296 Isobutane 75-28-5 C4H10 58.12 Propellant;aerating agent
297 Isobutylene-Isoprene Copolymer 9010-85-9 <= 3
Masticatorysubstance inchewing gum
base
298 DL-Isoleucine 443-79-8 C6H13NO2 131.17 <= 5 Nutrient
299 L-Isoleucine 73-32-5 C6H13NO2 131.17 <= 5 Nutrient
300 Isopropyl Alcohol 67-63-0 C3H8O 60.10 <= 1 Extractionsolvent
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
301 Juniper Berries Oil 8012-91-7 Flavoringagent
302 Kaolin 1332-58-7 <= 10 Anticakingagent
303 Karaya Gum 9000-36-6 <= 2Stabilizer;thickener;emulsifier
304 Kelp <= 2Nutrient
(source ofiodine)
305 Konjac Flour 37220-17-0 <= 2
Gelling agent;thickener; film
former;stabilizer
306 Labdanum Oil 8016-26-0 Flavoringagent
307 Lactic Acid (L(+)-Lactic Acid) 79-33-4 C3H6O3 90.08 <= 0.5 Acidifier
308 Lactic Acid (DL-Lactic Acid) 598-82-3 C3H6O3 90.08 <= 0.5 Acidifier
309 Lactose (anhydrous) 63-42-3 C12H22O11 342.30 <= 0.5
Nutritivesweetener;processing
aid; humectant(anhydrous
form);texturizer
310 Lactose (monohydrate) 5989-81-1 C12H22O11 ·H2O 360.32 <= 0.5
Nutritivesweetener;processing
aid; humectant(anhydrous
form);texturizer
311 Lactylated Fatty Acid Esters ofGlycerol and Propylene Glycol <= 2
Emulsifier;stabilizer;whipping
agent;plasticizer
312 Lactylic Esters of Fatty AcidsEmulsifier;
surface-activeagent
313 Lanolin, Anhydrous 8006-54-0 <= 3
Masticatorysubstance inchewing gum
base
314 Lard (Unhydrogenated) <= 0.1 Coating agent;texturizer
page 2. Food Chemicals Codex (5th Edition) © 2003
315 Laurel Leaf Oil 8006-78-8 Flavoringagent
316 Lauric Acid 143-07-7 C12H24O2 200.32 <= 0.1
Component inthe
manufactureof other food-
gradeadditives;defoaming
agent
317 Lavandin Oil, Abrial Type 8022-15-9 Flavoringagent
318 Lavender Oil 8000-28-0 Flavoringagent
319 Lecithin 8002-43-5 <= 1 Antioxidant;emulsifier
320 Lemongrass Oil 8007-02-1 Flavoringagent
321 Lemon Oil, Coldpressed 8008-56-8 Flavoringagent
322 Lemon Oil, Desert Type,Coldpressed
Flavoringagent
323 Lemon Oil, Distilled Flavoringagent
324 DL-Leucine 328-39-2 C6H13NO2 131.17 <= 5 Nutrient
325 L-Leucine 61-90-5 C6H13NO2 131.17 <= 5 Nutrient
326 Lime Oil, Coldpressed 8008-26-2 Flavoringagent
327 Lime Oil, Distilled Flavoringagent
328 Limestone, Ground <= 3
Texturizingand releaseagent andmodifier for
chewing gumbase and
chewing gum
329 Linaloe Wood Oil 8006-86-8 Flavoringagent
330 Linoleic Acid 60-33-3 C18H32O2 280.45 <= 2Flavoringadjuvant;nutrient
331 Locust (Carob) Bean Gum 9000-40-2 <= 5 Stabilizer;thickener
332 Lovage Oil 8016-31-7 Flavoringagent
333 L-Lysine Monohydrochloride 657-27-2 C6H14N2O2 ·HCl 182.65 <= 5 Nutrient
page 3. Food Chemicals Codex (5th Edition) © 2003
334 Mace Oil 8007-12-3 Flavoringagent
335 Magnesium Carbonate(anhydrous) 546-93-0 MgCO3 84.31 <= 2
pH control;drying agent;
color-retentionagent;
anticakingagent; carrier
336 Magnesium Carbonate (basic) 39409-82-0 4MgCO3 ·Mg(OH)2 · 5H2O 485.65 <= 2
pH control;drying agent;
color-retentionagent;
anticakingagent; carrier
337 Magnesium Carbonate(monohydrate) 23389-33-5 MgCO3 · H2O 102.33 <= 2
pH control;drying agent;
color-retentionagent;
anticakingagent; carrier
338 Magnesium Chloride 7791-18-6 MgCl2 · 6H2O 203.30 <= 4
Color-retention
agent; firmingagent
339 Magnesium Gluconate(anhydrous) 3632-91-5 C12H22MgO14 414.60 <= 2 Nutrient
340 Magnesium Gluconate(dihydrate) 59625-89-7 C12H22MgO14 ·
2H2O 450.63 <= 2 Nutrient
341 Magnesium Hydroxide 1309-42-8 Mg(OH)2 58.32 <= 2
pH control;drying agent;
color-retentionagent
342 Magnesium Oxide 1309-48-4 MgO 40.30 <= 4
pH control;neutralizer;anticaking
agent; free-flowing agent;firming agent
343 Magnesium Phosphate, Dibasic,Mixed Hydrates MgHPO4 · xH2O <= 2
Nutrient;leaveningagent; pH
control agent
344 Magnesium Phosphate, Dibasic,Trihydrate 7782-75-4 MgHPO4 · 3H2O 174.33 <= 2
Nutrient;leaveningagent; pH
control agent
345 Magnesium Phosphate, Tribasic(anhydrous) 7757-87-1 Mg3(PO4)2 ·
xH2O 262.86 <= 2 Nutrient
346 Magnesium Silicate 1343-88-0 <= 5 Anticakingagent; filter aid
347 Magnesium Stearate 557-04-0 <= 5Anticaking
agent; binder;emulsifier
page 4. Food Chemicals Codex (5th Edition) © 2003
348 Magnesium Sulfate(monohydrate) 14168-73-1 MgSO4 · xH2O 138.38 <= 4 Nutrient
349 Magnesium Sulfate(heptahydrate) 10034-99-8 MgSO4 · xH2O 246.47 <= 4 Nutrient
350 Magnesium Sulfate (dried) 15244-36-7 MgSO4 · xH2O <= 4 Nutrient
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
351 Malic Acid 617-48-1 C4H6O5 134.09 <= 2 Acidifier;flavoring agent
352 Malt Syrup 8002-48-0 <= 0.5
Color;enzyme;flavoringagent;
humectant;nutritive
sweetener;stabilizer;
thickener; andtexturizer
353 Maltitol 585-88-6 C12H24O11 344.31 <= 1Sweetener;humectant;stabilizer
354 Maltitol Syrup <= 1 , calculated on theanhydrous basis
Humectant;texturizing
agent;stabilizer;sweetener
355 Maltodextrin 9050-36-6 <= 0.5
Anticaking andfree-flowing
agent; bulkingagent;
stabilizer andthickener;surface-
finishing agent
356 Maltol 118-71-8 C6H6O3 126.11 Flavoringagent
357 Mandarin Oil, Coldpressed 8008-31-9 Flavoringagent
358 Manganese Chloride(anhydrous) 7773-01-5 MnCl2 125.84 <= 4 Nutrient
359 Manganese Chloride(tetrahydrate) 13446-34-9 MnCl2 · 4H2O 197.91 <= 4 Nutrient
360 Manganese Citrate(decahydrate) 10024-66-5 Mn3(C6H5O7)2 ·
10H2O 723.17 <= 2 Nutrient
361 Manganese Citrate (anhydrous) 10024-66-5 Mn3(C6H5O7)2 543.02 <= 2 Nutrient
362 Manganese Gluconate(dihydrate) 6485-39-8 C12H22MnO14 ·
2H2O 481.27 <= 2 Nutrient
363 Manganese Gluconate(anhydrous) 6485-39-8 C12H22MnO14 445.24 <= 2 Nutrient
364 Manganese Glycerophosphate(anhydrous) 1320-46-3 C3H7MnO6P ·
xH2O 225.00 <= 4 Nutrient
page 2. Food Chemicals Codex (5th Edition) © 2003
365 Manganese Hypophosphite(anhydrous) 10043-84-2 Mn(H2PO2)2 ·
xH2O 184.92 <= 4 Nutrient
366 Manganese Sulfate 7785-87-7 MnSO4 · H2O 169.02 <= 4 Nutrient
367 Mannitol 69-65-8 C6H14O6 182.17 <= 1
Nutritivesweetener;texturizing
agent
368 Marjoram Oil, Spanish Type 8015-01-8 Flavoringagent
369 Marjoram Oil, Sweet Flavoringagent
370 Masticatory Substances, Natural <= 3
Masticatorysubstance inchewing gum
base
371 Menhaden Oil, Hydrogenated 8016-14-6 <= 0.1
Coating agent;crystal
stabilizer.Used as ablend with
other fats andoils
372 Menhaden Oil, Refined 8002-50-4 <= 0.1
A source oflong-chain
(greater thanC18) ω-3
polyunsaturated fatty acids. It
is used as ablend with
other fats andoils
373 Mentha Arvensis Oil, PartiallyDementholized 68919-18-0 Flavoring
agent
374 DL-Methionine 59-51-8 C5H11NO2S 149.21 <= 5 Nutrient
375 L-Methionine 63-68-3 C5H11NO2S 149.21 <= 5 Nutrient
376 Methyl Alcohol 67-56-1 CH3OH 32.04 <= 1 Extractionsolvent
377 Methylcellulose 9004-67-5 <= 3
Thickener;stabilizer;emulsifier;
bodying agent;bulking agent;
binder; filmformer
378 Methylene Chloride 75-09-2 CH2Cl2 84.93 <= 1 Extractionsolvent
379 Methyl Ester of Rosin, PartiallyHydrogenated <= 1
Masticatorysubstance inchewing gum
base
page 3. Food Chemicals Codex (5th Edition) © 2003
380 Methyl Ethyl Cellulose 9004-69-7 <= 3Emulsifier;stabilizer;
foaming agent
381 Methylparaben 99-76-3 C8H8O3 152.15 <= 2Preservative;antimicrobial
agent
382 Mineral Oil, White 8042-47-5 <= 1
Defoamingagent;
lubricant;release agent;
protectivecoating;
glazing agent;sealing agent
383 Monoammonium L-Glutamate 7558-63-6 C5H12N2O4 ·H2O 182.18 <= 5
Flavorenhancer; salt
substitute
384 Monoammonium Glycyrrhizinate(anhydrous)
C42H65NO16 ·5H2O 839.98 Flavoring
agent
385 Mono- and Diglycerides <= 2 Emulsifier;stabilizer
386 Monoglyceride Citrate 36291-32-4 <= 2 Solubilizer forantioxidants
387 Monopotassium L-Glutamate(anhydrous) 19473-49-5 C5H8KNO4 ·
H2O 203.24 <= 5Flavor
enhancer; saltsubstitute
388 Monosodium L-Glutamate 6106-04-3 C5H8NNaO4 ·H2O 187.13 <= 5 Flavor
enhancer
389 Morpholine 110-91-8 C4H9NO 87.12 <= 1
Boiler wateradditive;
component ofcoatings forfruits and
vegetables
390 Mustard Oil Flavoringagent
391 Myristic Acid 544-63-8 C14H28O2 228.37 <= 2
Component inthe
manufactureof other food-
gradeadditives;defoaming
agent
392 Myrrh Oil 9000-45-7 Flavoringagent
393 Natamycin 7681-93-8 C33H47NO13 665.73 <= 2 Antimycotic
394 Niacin 59-67-6 C6H5NO2 123.11 Nutrient
395 Niacinamide 98-92-0 C6H6N2O 122.13 <= 2 Nutrient
396 Niacinamide Ascorbate 1987-71-9 <= 2 Nutrient
page 4. Food Chemicals Codex (5th Edition) © 2003
397 Nickel 7440-02-0 Ni 58.69Catalyst for
hydrogenationreactions
398 Nisin Preparation 1414-45-5 C143H230O37N42S7
~3348 <= 2 Antimicrobialagent
399 Nitrogen 7727-37-9 N2 28.01
Gas: air andoxygen
displacer;propellant and
aeratingagent;
packaginggas; Liquid:
direct-contactfreezing agent
400 Nitrogen Enriched Air N2 28.01Air andoxygen
displacer
page 1. Food Chemicals Codex (5th Edition) © 2003
Title: Food Chemicals Codex (5th Edition)
Table: Interactive Table - Monographs
No. material or substance name CAS Registry No. mol. formula mol. weight (g/mol) lead content (mg/kg) function
401 Nitrous Oxide 10024-97-2 N2O 44.01
Propellant;aeratingagent;
packaging gas
402 Nutmeg Oil 8008-45-5 Flavoringagent
403 Octanoic Acid 124-07-2 C8H16O2 144.21 <= 0.1
Component inthe
manufactureof other food-
gradeadditives;defoaming
agent;flavoring agent
404 Oleic Acid 112-80-1 C18H34O2 282.47 <= 0.1
Component inthe
manufactureof other food-
gradeadditives;defoaming
agent;lubricant;
binder
405 Olestra <= 0.1Calorie-freesubstitute forfats and oils
406 Olibanum Oil 8016-36-2 Flavoringagent
407 Onion Oil 8002-72-0 Flavoringagent
408 Orange Oil, Bitter, Coldpressed Flavoringagent
409 Orange Oil, Coldpressed 8028-48-6 Flavoringagent
410 Orange Oil, Distilled Flavoringagent
411 Origanum Oil, Spanish Type 8007-11-2 Flavoringagent
412 Orris Root Oil Flavoringagent
413 Ox Bile Extract C24H39NaO5 430.56 Surfactant
page 2. Food Chemicals Codex (5th Edition) © 2003
414 Oxystearin 8028-45-3 <= 2
Crystallizationinhibitor insalad and
cooking oils;sequestrant;defoaming
agent
415 Ozone 10028-15-6 O3 48.00
Antimicrobialin the
treatment,processing,
and storage ordisplay of fish,
meat, andpoultry and in
preparing,packing, orholding rawagricultural
commodities.Disinfectant
for water to beused for directconsumption
or to make ice
416 Palmarosa Oil 8014-19-5 Flavoringagent
417 Palmitic Acid 57-10-3 C16H32O2 256.43 <= 0.1
Component inthe
manufactureof other food-
gradeadditives;defoaming
agent
418 Palm Kernel Oil(Unhydrogenated) 8023-79-8 <= 0.1 Coating agent;
texturizer
419 Palm Oil (Unhydrogenated) 8002-75-3 <= 0.1
Coating agent;emulsifying
agent;texturizer
420 DL-Panthenol 16485-10-2 C9H19NO4 205.25 <= 2 Nutrient
421 Paraffin, Synthetic 8002-74-2 <= 3
Masticatorysubstance inchewing gum
base
422 Parsley Herb Oil 8000-68-8 Flavoringagent
423 Parsley Seed Oil 8000-68-8 Flavoringagent
page 3. Food Chemicals Codex (5th Edition) © 2003
424 Partially Hydrolyzed Proteins <= 2
Binder; doughconditioner;
emulsifier andemulsifier salt;
flavoringagent; flavorenhancer;nutrient;
fermentationaid; surface-active agent;
texturizer
425 Peanut Oil (Unhydrogenated) 8002-03-7 <= 0.1 Coating agent;texturizer
426 Pectins 9000-69-5 <= 5Gelling agent;
thickener;stabilizer
427 Pennyroyal Oil 8013-99-8 Flavoringagent
428 Pentaerythritol Ester of PartiallyHydrogenated Wood Rosin <= 1
Masticatorysubstance inchewing gum
base
429 Pentaerythritol Ester of WoodRosin <= 1
Masticatorysubstance inchewing gum
base
430 Peppermint Oil 8006-90-4 Flavoringagent
431 Perlite <= 10Filter aid in
foodprocessing
432 Petitgrain Oil, Paraguay Type 8014-17-3 Flavoringagent
433 Petrolatum 8009-03-8 <= 1
Defoamingagent;
lubricant;protectivecoating;
release agent
434 Petrolatum 92045-77-7 <= 1
Defoamingagent;
lubricant;protectivecoating;
release agent
435 Petrolatum 100684-33-1 <= 1
Defoamingagent;
lubricant;protectivecoating;
release agent
page 4. Food Chemicals Codex (5th Edition) © 2003
436 Petroleum Wax 8002-74-2 <= 1
Masticatorysubstance inchewing gum
base;protectivecoating;
defoamingagent;
microcapsulesfor spices and
flavoringagents
437 Petroleum Wax (HydrotreatedParaffin Wax) 64742-51-4 <= 1
Masticatorysubstance inchewing gum
base;protectivecoating;
defoamingagent;
microcapsulesfor spices and
flavoringagents
438 Petroleum Wax (HydrotreatedMicrocrystalline Wax) 64742-60-5 <= 1
Masticatorysubstance inchewing gum
base;protectivecoating;
defoamingagent;
microcapsulesfor spices and
flavoringagents
439 Petroleum Wax (MicrocrystallineWax) 63231-60-7 <= 1
Masticatorysubstance inchewing gum
base;protectivecoating;
defoamingagent;
microcapsulesfor spices and
flavoringagents
440 Petroleum Wax, Synthetic <= 1
Masticatorysubstance inchewing gum
base;protectivecoating;
defoamingagent
441 DL-Phenylalanine 150-30-1 C9H11NO2 165.19 <= 5 Nutrient
442 L-Phenylalanine 63-91-2 C9H11NO2 165.19 <= 5 Nutrient
page 5. Food Chemicals Codex (5th Edition) © 2003
443 Phosphoric Acid 7664-38-2 H3PO4 98.00 <= 3 Acidifier;sequestrant
444 Pimenta Leaf Oil 8016-45-3 Flavoringagent
445 Pimenta Oil Flavoringagent
446 Pine Needle Oil, Dwarf 8000-26-8 Flavoringagent
447 Pine Needle Oil, Scotch Type 8023-99-2 Flavoringagent
448 Poloxamer 331 3800 (avg) <= 2
Solubilizingand stabilizingagent in flavorconcentrates
449 Poloxamer 407 12,500 (avg) <= 2
Solubilizingand stabilizingagent in flavorconcentrates
450 Polydextrose 68424-04-4 <= 0.5Bulking agent;
humectant;texturizer
IndexTitles of monographs in Section 2 and those of flavor chemicals in Section 3 are shown inblue type and are linked. An asterisk indicates a new monograph for the Fifth Edition. For titles of new monographs introduced in the three supplements to the Fourth Edition, please consult the listings under General Information.
A
Abbreviations, 6–7, 517Absolute Alcohol, 5Acacia, 210–211Accuracy of Analytical Methods, xxv‘‘Accurately,’’ Defined, 4, 833Acesulfame K, 9–10Acesulfame Potassium, 9–10Acetal, 518–519Acetaldehyde, 518–519
Infrared Spectrum, 642Acetaldehyde Diethyl Acetal, 518–519
Infrared Spectrum, 642Acetaldehyde Test Paper, 976Acetals Determination, 929Acetanisole, 518–519
Infrared Spectrum, 642Acetate Identification Test, 859Aceteugenol, 562–563Acetic Acid, Glacial, 10Acetic Acid, Glacial (reagent), 5, 963Acetic Acid TS, Diluted, 963Acetic Acid TS, Strong, 963Acetic Aldehyde, 518–519Acetic and Fatty Acid Esters of Glycerol, 12Acetoacetic Ester, 550–551Acetoglycerides, 12Acetoin, 518–519
Infrared Spectrum, 643�-Acetolactatedecarboxylase (Bacillus subtilis
containing a Bacillus brevis gene), 148,150
2-Acetonaphthone, 596–597Acetone, 10–11Acetone Peroxides, 11–12Acetophenone, 518–519
Infrared Spectrum, 643N-Acetyl-L-2-amino-4-(methylthio)butyric
Acid, 12–134-Acetylanisole, 518–519Acetylated Distarch Adipate, 182Acetylated Distarch Phosphate, 183Acetylated Mono- and Diglycerides, 12Acetylated Monoglycerides, 12Acetylbenzene, 518–5193-Acetyl-2,5-dimethyl Furan, 518–519
Infrared Spectrum, 643Acetyl Eugenol, 562–563Acetyl Groups, 951
979
N-Acetyl-L-Methionine, 12–13Infrared Spectrum, 644
Acetyl Methyl Carbinol, 518–5193-Acetyl-6-methyl-1,2-pyran-2,4(3H)-dione,
132Acetyl Propionyl, 608–6092-Acetylpyrazine, 520–521
Infrared Spectrum, 6443-Acetylpyridine, 520–521
Infrared Spectrum, 6442-Acetylpyrrole, 520–5212-Acetyl Thiazole, 520–521
Infrared Spectrum, 645Acetyl Valeryl, 568–569Acetyl Value, 934Achilleic Acid, 16Acid Base Titrations, 631Acid Calcium Phosphate, 76–77Acid Ferric Sulfate TS, 966Acid Hydrolysates of Proteins, 13–15Acid-Hydrolyzed Milk Protein, 13–15Acid-Hydrolyzed Proteins, 13–15Acidified Sodium Chlorite Solutions, 15–16Acid-Insoluble Ash, 854Acidity Determination by Iodometric Method,
632Acid-Modified Starch, 181–182Acid Number (Rosins and Related
Substances), 947Acid Phosphatase Activity, 898–899Acid Phthalate Buffer, 962Acid Reagents, 5Acid Sodium Pyrophosphate, 400–401Acid Value
Essential Oils and Flavors, 929Fats and Related Substances, 934Flavor Chemicals, 634
Aconitic Acid, 16Activated Carbon, 94–96Added Substances Policy, 2Adipic Acid, 17Agar, 17–18DL-Alanine, 18
Infrared Spectrum, 645L-Alanine, 18–19
Infrared Spectrum, 645Alcohol, 157–158Alcohol, Absolute, 963Alcohol, Aldehyde-Free, 963
Alcohol, Anhydrous, 963Alcohol C-6, 572–573Alcohol C-8, 608–609Alcohol C-9, 606–607Alcohol C-10, 542–543Alcohol C-11, 626–627Alcohol C-12, 582–583Alcohol Content of Ethyl Oxyhydrate, 631–
632Alcohol, Dehydrated, 963Alcohol, Diluted, 963Alcohol, Enanthic, 568–569Alcohol, Ethyl, 157–158Alcohol, Isobutyl, 578-579Alcohol, Methyl, 286–287Alcohol, Solubility in, 932Alcohol, 70%, 963Alcohol, 80%, 963Alcohol, 90%, 963Alcoholic Ferric Chloride TS, 966Alcoholic Potassium Hydroxide TS, 963Alcoholic Potassium Hydroxide, 0.5 N, 973Alcoholic Sulfuric Acid, 0.5 N, 974Alcoholic Sulfuric Acid, 5 N, 974Alcohols, Total, in Essential Oils and Flavors,
932Aldehyde C-6, 568–569Aldehyde C-7, 568–569Aldehyde C-8, 606–607Aldehyde C-9, 604–605Aldehyde C-10, 542–543Aldehyde C-11 Undecyclic, 626–627Aldehyde C-11 Undecylenic, 626–627Aldehyde C-12, 582–583Aldehyde C-12 MNA, 600–601Aldehyde C-14 Pure, So-Called, 626–627Aldehyde C-16, 558–559Aldehyde C-18, So-Called, 604–605Aldehyde-Free Alcohol, 963Aldehydes and Ketones
Essential Oils and Flavors Assays, 929–930Flavor Chemical Assays, 630–631
Algin, 354–355, 401Alginates Assay, 876–877Algin Derivative, 376–377Alginic Acid, 19Alkaline Borate Buffer, 962Alkaline Cupric Citrate TS, 965Alkaline Cupric Tartrate TS, 964
980 / Alkaline Mercuric–Potassium Iodide TS / Index FCC V
Alkaline Mercuric–Potassium Iodide TS, 964Allergens, 2Allspice Oil, 332–333
* Allura Red, 20–21, 165Allura Red (AC), 20–21p-Allylanisole, 550–551Allyl Caproate, 520–521Allyl Cyclohexanepropionate, 520–521
Infrared Spectrum, 646Allyl-3-cyclohexanepropionate, 520–5214-Allyl-1,2-dimethoxy Benzene, 596–5974-Allylguaiacol, 562–563Allyl Heptanoate, 520–521
Infrared Spectrum, 646Allyl Heptoate, 520–521Allyl Hexanoate, 520–521
Infrared Spectrum, 646Allyl Ionone, 520–521Allyl �-Ionone, 520–521
Infrared Spectrum, 647Allyl Isopentanoate, 522–523Allyl Isothiocyanate, 522–523
Infrared Spectrum, 647Qualitative Testing for Phenols, 634
Allyl Isovalerate, 522–523Infrared Spectrum, 647
4-Allyl-2-methoxyphenol, 562–5634-Allyl-2-methoxyphenol Acetate, 562–563Allyl Phenoxy Acetate, 522–523
Infrared Spectrum, 648Allyl Propionate, 522–523
Infrared Spectrum, 648Almond Oil, Bitter, FFPA, 21
Infrared Spectrum, 648Alphazurine 2G, 975Aluminum Ammonium Sulfate, 21–22Aluminum Identification Test, 859Aluminum Potassium Sulfate, 22Aluminum Silicate, 45–46Aluminum Sodium Sulfate, 22–23Aluminum Sulfate, 22–23Ambrette Seed Liquid, 24Ambrette Seed Oil, 24
Infrared Spectrum, 649Aminoacetic Acid, 208–209N-[4-[[(2-Amino-1,4-dihydro-4-oxo-6-
pteridinyl)methyl]amino]benzoyl]-L-glutamic Acid, 180–181
L-2-Aminoglutaramic Acid, 197L-2-Amino-5-guanidinovaleric Acid, 5L-2-Amino-5-guanidinovaleric Acid
Monohydrochloride, 35L-2-Amino-3-hydroxybutyric Acid, 4744-Amino-3-hydroxybutyric Acid
Trimethylbetaine, 100–101DL-2-Amino-3-hydroxypropanoic Acid, 396L-2-Amino-3-hydroxypropanoic Acid, 396–
397N-[p-[[(2-Amino-4-hydroxy-6-pteridinyl)-
methyl]amino]benzoyl]glutamic Acid,180–181
L-�-Amino-4(or 5)-imidazolepropionic Acid,217
L-�-Amino-4(or 5)-imidazolepropionic AcidMonohydrochloride, 217–218
DL-�-Amino-3-indolepropionic Acid, 490L-�-Amino-3-indolepropionic Acid, 489–490L-2-Amino-3-mercaptopropanoic Acid
Monohydrochloride, 130L-2-Amino-3-methylbutyric Acid, 492DL-2-Amino-4-(methylthio)butyric Acid, 285–
286L-2-Amino-4-(methylthio)butyric Acid, 286DL-2-Amino-3-methylvaleric Acid, 234L-2-Amino-3-methylvaleric Acid, 234–235DL-2-Amino-4-methylvaleric Acid, 252L-2-Amino-4-methylvaleric Acid, 252–253�-Amino Nitrogen Determination, 877
L-2-Aminopentanedioic Acid, 1962-Aminopentanedioic Acid Hydrochloride,
196–197Aminopeptidase (Leucine) Activity, 899–900Aminopeptidase, Leucine (Aspergillus niger
var., Aspergillus oryzae var., and othermicrobial species), 148, 150
DL-�-Amino-�-phenylpropionic Acid, 330L-�-Amino-�-phenylpropionic Acid, 330–331DL-2-Aminopropanoic Acid, 18L-2-Aminopropanoic Acid, 18–19L-�-Aminosuccinamic Acid, 37DL-Aminosuccinic Acid, 40–41L-Aminosuccinic Acid, 41Ammonia–Ammonium Chloride Buffer TS,
964Ammoniacal Silver Nitrate TS, 964Ammonia Detector Tube, 977Ammonia Nitrogen Determination, 877Ammonia Solution, 24–25Ammoniated Glycyrrhizin, 25Ammonia TS, 964Ammonia TS, Stronger, 964Ammonia Water, Stronger, 24–25Ammonium Acetate TS, 964Ammonium Alginate, 26Ammonium Alum, 21–22Ammonium Bicarbonate, 26–27Ammonium Carbonate, 27Ammonium Carbonate TS, 964Ammonium Chloride, 27–28Ammonium Chloride TS, 964Ammonium Dihydrogen Phosphate, 28Ammonium Glutamate, 292–293Ammonium Glycyrrhizinate, 293Ammonium Glycyrrhizinate, Pentahydrate,
293Ammonium Hydroxide, 24–25Ammonium Hydroxide (reagent), 5Ammonium Identification Test, 859Ammonium Molybdate TS, 964Ammonium Oxalate TS, 964Ammonium Phosphate, Dibasic, 28Ammonium Phosphate, Monobasic, 28Ammonium Saccharin, 28–30Ammonium Standard Solution, 963Ammonium Sulfanilate TS, 964Ammonium Sulfate, 30Ammonium Sulfide TS, 964Ammonium Thiocyanate TS, 964Ammonium Thiocyanate, 0.1 N, 970Amyl Acetate, 576–5771-Amyl Alcohol, 522–523Amylase, 147�-Amylase, 150, 896�-Amylase Activity
Bacterial, 901–902Nonbacterial, 900–901
�-Amylase, 150, 896Amylase, Maltogenic, 151Amyl Butyrate, 522–523Amyl Butyrate, 576–577Amyl Caprylate, 524–525Amylcinnamaldehyde, 522–523�-Amylcinnamaldehyde, 522–523
Infrared Spectrum, 649Amyl Cinnamate, 522–523
Infrared Spectrum, 649Amyl Formate, 522–523Amyl Formate, 576–577Amyl Heptanoate, 524–525Amyl Hexanoate, 576–577Amyl Isovalerate, 578–579Amyl Octanoate, 524–525
Infrared Spectrum, 650Amyloglucosidase, 151Amyloglucosidase Activity, 907–908
Amyl Propionate, 524–525Infrared Spectrum, 650
Amyl Salicylate, 578–579Amyl Valerate, 578–579Amyl Vinyl Carbinol, 606–607Amyris Oil, West Indian Type, 30–31
Infrared Spectrum, 650Analytical Samples, 4Anethole, 524–525
Infrared Spectrum, 651Qualitative Testing for Phenols, 634
trans-Anethole, 524–525Aneurine Hydrochloride, 472–473Angelica Root Oil, 31
Infrared Spectrum, 651Angelica Seed Oil, 31–32
Infrared Spectrum, 651Angelica Seed Oleoresin, 446, 447Angular Rotation, 844Anhydrous Alcohol, 5Anhydrous Isopropanol, 966Anhydrous Lanolin, 245Anhydrous Methanol, 967Animal Lipase, 147p-Anisaldehyde, 588–589Anise Oil, 32
Infrared Spectrum, 652Anise Oleoresin, 446, 447Anisic Alcohol, 524–525Anisic Aldehyde, 588–589Anisole, 524–525
Infrared Spectrum, 652Qualitative Testing for Phenols, 634
Anisyl Acetate, 524–525Infrared Spectrum, 652
Anisyl Acetone, 588–589Anisyl Alcohol, 524–525
Infrared Spectrum, 653Anisyl Formate, 524–525
Infrared Spectrum, 653Annatto Extracts, 32–33Anthrone TS, 964Antimony Trichloride TS, 964Antioxidants in Ethyl Acrylate, Limit Test for,
632–633APDC Extraction Method, 871–872APM, 37–38APM-Ace, 39–40APO, 33–34Apocarotenal, 33–34�-Apo-8′-Carotenal, 33–34Apparatus for Tests and Assays, 4, 831–833
* Arabinogalactan, 34D-Araboascorbic Acid, 152L-Arginine, 34–35
Infrared Spectrum, 653L-Arginine Monohydrochloride, 35
Infrared Spectrum, 654Arsenic Limit Test, 861–862
Chewing Gum Base Polymers, 895Arsenic Specifications Policy, xv, 2Ascorbic Acid, 36L-Ascorbic Acid, 36Ascorbyl Palmitate, 36–37Ash (Acid-Insoluble), 854Ash (Total), 854–855L-Asparagine, 37
Infrared Spectrum, 654Aspartame, 37–38Aspartame-Acesulfame Salt, 39–40
Infrared Spectrum, 654DL-Aspartic Acid, 40–41
Infrared Spectrum, 655L-Aspartic Acid, 41
Infrared Spectrum, 655N-L-�-Aspartyl-L-phenylalanine 1-Methyl
Ester, 37–38
FCC V Index / Calcium Chloride TS / 981
Assays and TestsApparatus, 831–833Carbohydrates, 951–956Chemical Tests and Determinations, 859–
891Chewing Gum Base Polymers, 892–896Data Elements Required for Validation of,
xxviii–xxxixEnzyme Assays, 896–928Essential Oils and Flavors, 929–933Fats and Related Substances, 934–944Flavor Chemicals, 630–636General Requirements and Provisions, 4–7Oleoresins, 944–950Physical Tests and Determinations, 834–858Solutions and Indicators, 962–967
Assay Tolerances, Maximum, 6Atomic Absorption Spectrophotometric
Graphite Furnace Method, 869–871Atomic Weights and Chemical Formulas, 4Autolyzed Yeast, 507–508Autolyzed Yeast Extract, 510–511Azodicarbonamide, 41–42Azodicarboxylic Acid Diamide, 41–42Azo Violet, 975
B
Bacterial �-Amylase Activity, 901–902Bacterial Proteolytic Activity, 923–924Baking Soda, 405Balsam Fir Oil, 179Balsam Peru Oil, 42
Infrared Spectrum, 655Barium Chloride TS, 964Barium Diphenylamine Sulfonate TS, 964Barium Hydroxide TS, 964Barium Hydroxide, 0.2 N, 970Barium Standard Solution, 963Basil Oil, Comoros Type, 43
Infrared Spectrum, 656Basil Oil, European Type, 43–44
Infrared Spectrum, 656Basil Oil Exotic, 43Basil Oil, Italian Type, 43–44Basil Oil, Réunion Type, 43Basil Oleoresin, 446, 447Bay Leaf Oil, 246Bay Oil, 44
Infrared Spectrum, 656BCD, 126–128Beeswax, White, 44–45Beeswax, Yellow, 45Beet Fiber, 458–460Beet Sugar, 455–4561,3-Behenic-2-oleic Glyceride, 51Benedict’s Qualitative Reagent, 964Bentonite, 45–46Benzaldehyde, 526–527
Hydrogen Acid in, Limit Test for, 633Infrared Spectrum, 657
Benzaldehyde Glyceryl Acetal, 526–527Infrared Spectrum, 657
Benzene (in Paraffinic Hydrocarbon Solvents),878–879
Benzidine TS, 9641,2-Benzisothiazole-3(2H)-one-1,1-Dioxide,
388–3891,2-Benzisothiazole-3(2H)-one-1,1-Dioxide
Sodium Salt, 432–4331,2-Benzisothiazolin-3-one 1,1-Dioxide
Ammonium Salt, 28–301,2-Benzisothiazolin-3-one 1,1-Dioxide
Calcium Salt, 79–80Benzoate Identification Test, 8591,2-Benzodihydropyrone, 526–527
Infrared Spectrum, 657
Benzoic Acid, 46–47Benzophenone, 526–527
Infrared Spectrum, 658o-Benzosulfimide, 388–389Benzoylbenzene, 526–527Benzoyl Peroxide, 47Benzyl Acetate, 526–527
Infrared Spectrum, 658Benzyl Alcohol, 526–527
Infrared Spectrum, 658Benzyl Benzoate, 526–527
Infrared Spectrum, 659Benzyl Butyrate, 526–527
Infrared Spectrum, 659Benzyl n-Butyrate, 526–527Benzyl Cinnamate, 528–529
Infrared Spectrum, 659Benzyl Formate, 528–529
Infrared Spectrum, 660Benzyl Isobutyrate, 528–529
Infrared Spectrum, 660Benzyl Isovalerate, 528–529
Infrared Spectrum, 660Benzyl 3-Methyl Butyrate, 528–529Benzyl 2-Methyl Propionate, 528–529Benzyl Phenylacetate, 528–529
Infrared Spectrum, 661Benzyl Propanoate, 528–529Benzyl Propionate, 528–529
Infrared Spectrum, 661Benzyl Salicylate, 528–529
Infrared Spectrum, 661Bergamot Oil, Coldpressed, 48
Infrared Spectrum, 662Beta-1,3-glucan, 125–126BHA, 48–49BHT, 49Bicarbonate Identification Test, 859Biotechnology, xxxi–xxxiiBiotin, 49–50D-Biotin, 49–50Birch Tar Oil, Rectified, 50
Infrared Spectrum, 662Bismuth Nitrate TS, 964Bisulfite Identification Test, 859Bitter Almond Oil Free from Prussic Acid, 21Black Pepper Oil, 50
Infrared Spectrum, 662Black Pepper Oleoresin, 446, 447Blank Tests, 4Bleached Starch, 182Blue Litmus Paper, 976–977
* Bohenin, 51Bois de Rose Oil, 51–52
Infrared Spectrum, 663Boric Acid–Potassium Chloride, 0.2 M, 962Borneol, 528–529
Infrared Spectrum, 663Bornyl Acetate, 530–531
Infrared Spectrum, 663l-Bornyl Acetate, 530–531Bound Styrene in Chewing Gum Base, 892Bovine Liver Catalase, 147Bovine Rennet, 147, 151Brewer’s Yeast, 508–510
* Brilliant Blue, 52–53Brilliant Blue (FCF), 52–53, 162Bromelain, 147, 150, 896Bromide Identification Test, 859Brominated Vegetable Oil, 53–54Bromine TS, 964Bromine, 0.1 N, 970Bromocresol Blue, 975Bromocresol Blue TS, 964Bromocresol Green, 975Bromocresol Green TS, 964Bromocresol Purple, 975Bromocresol Purple TS, 965
Bromophenol Blue, 975Bromophenol Blue TS, 965Bromothymol Blue, 975Bromothymol Blue TS, 965Buffer Solutions, Standard, 962Butadiene-Styrene Rubber, 54–57
Infrared Spectra, 664, 665Butane, 57–58n-Butane, 57–581,4-Butanedicarboxylic Acid, 17Butanedioic Acid, 4522,3-Butanedione, 544–5451,2,3,4-Butanetetrol, 1531-Butanol, 530–5312-Butanone, 530–531
Infrared Spectrum, 665Butan-3-one-2-yl Butanoate, 530–531
Infrared Spectrum, 665(E)-Butenedioic Acid, 186–187Butter Starter Distillate, 449–450Butyl Acetate, 530–531
Infrared Spectrum, 666n-Butyl Acetate, 530–531Butyl Alcohol, 530–531
Infrared Spectrum, 666Butyl Aldehyde, 532–533Butylated Hydroxyanisole, 48–49Butylated Hydroxymethylphenol, 58Butylated Hydroxytoluene, 49Butyl Butyrate, 530–531
Infrared Spectrum, 666n-Butyl n-Butyrate, 530–531Butyl Butyryllactate, 530–531
Infrared Spectrum, 6672-sec-Butyl Cyclohexanone, 532–533
Infrared Spectrum, 6671,3-Butylene Glycol, 58–59Butyl Ester, 530–531tert-Butylhydroquinone, 469–471Butyl Isobutyrate, 532–533
Infrared Spectrum, 667Butyl Isovalerate, 532–533
Infrared Spectrum, 668Butyl 2-Methyl Butyrate, 532–533
Infrared Spectrum, 668Butyl Octadecanoate, 532–533Butyl Phenylacetate, 532–533
Infrared Spectrum, 668Butyl Rubber, 233–234Butyl Stearate, 532–533Butyraldehyde, 532–533
Infrared Spectrum, 669Butyrate, 530–531Butyric Acid, 532–533
Infrared Spectrum, 669Butyrin, 624–625�-Butyrolactone, 534–535
Infrared Spectrum, 669Butyryllactic Acid, 530–531
C
Cadmium Limit Test, 863Caffeine, 59
Infrared Spectrum, 670Calcium Acetate, 59–60Calcium Acid Pyrophosphate, 60–61Calcium Alginate, 61Calcium Ascorbate, 61–62Calcium Biphosphate, 76–77Calcium Bromate, 62Calcium Carbonate, 62–63Calcium Chloride, 63–64Calcium Chloride Double Salt of DL- or D-
Calcium Pantothenate, 73–74Calcium Chloride Solution, 64Calcium Chloride TS, 965
982 / Calcium Citrate / Index FCC V
Calcium Citrate, 64–65Calcium Disodium Edetate, 65–66Calcium Disodium EDTA, 65–66Calcium Disodium Ethylene-
diaminetetraacetate, 65–66Calcium Disodium (Ethylenedinitrilo)-
tetraacetate, 65–66Calcium 4-(�,D-Galactosido)-D-gluconate, 69–
70Calcium Gluconate, 66–67Calcium Glycerophosphate, 67Calcium Hydroxide, 67–68Calcium Hydroxide TS, 965Calcium Hydroxyapatite, 77Calcium Identification Test, 859Calcium Iodate, 68–69Calcium Lactate, 69Calcium Lactobionate, 69–70Calcium Lignosulfonate, 70–72Calcium Oxide, 72Calcium Pantothenate, 72–73D-Calcium Pantothenate, 72–73Calcium Pantothenate, Calcium Chloride
Double Salt, 73–74Calcium Pantothenate, Racemic, 74–75Calcium Peroxide, 75Calcium Phosphate, Dibasic, 75–76Calcium Phosphate, Monobasic, 76–77Calcium Phosphate, Tribasic, 77Calcium Propionate, 78Calcium Pyrophosphate, 78–79Calcium Saccharin, 79–80Calcium Silicate, 80–82Calcium Sorbate, 82Calcium Stearate, 82–83Calcium Stearoyl Lactate, 83–84Calcium Stearoyl Lactylate, 83–84Calcium Stearoyl-2-Lactylate, 83–84Calcium Sulfate, 84–85Calcium Sulfate TS, 965Calf Rennet, 147, 151Camphene, 534–535d-Camphor, 534–535
Infrared Spectrum, 670Cananga Oil, 85
Infrared Spectrum, 670Candelilla Wax, 85–86
Infrared Spectrum, 671Cane Sugar, 455–456Canola Oil, 86–88Cantha, 88Canthaxanthin, 88Capacity factor (k), 834Capraldehyde, 542–543Capric Acid, 131–132Caproic Acid, 568–569Caproic Aldehyde, 568–569Capryl Alcohol, 608–609Caprylic Acid, 307Caprylic Aldehyde, 606–607Capsicum Oleoresin, 446, 447Caramel, 88–93Caramel Color, 88–93Caraway Oil, 93–94
Infrared Spectrum, 671Caraway Oleoresin, 446, 447Carbamide, 491Carbohydrase and Protease, Mixed (Bacillus
licheniformis var.), 149Carbohydrase and Protease, Mixed (Bacillus
subtilis var. including Bacillusamyloliquefaciens), 149
Carbohydrase (Aspergillus niger var.,including Aspergillus aculeatus), 148
Carbohydrase (Aspergillus oryzae var.), 148Carbohydrase (Bacillus acidopullulyticus var.),
148
Carbohydrase (Bacillus stearothermophilus),148
Carbohydrase (Bacillus subtilis containing aBacillus megaterium �-amylase gene),149
Carbohydrase (Bacillus subtilis containing aBacillus stearothermophilus �-amylasegene), 149
Carbohydrase (Candida pseudotropicalis), 148Carbohydrase (Kluyveromyces marxianus var.
lactis), 148Carbohydrase (Martierella vinaceae var.
raffinoseutilizer), 148Carbohydrase (Rhizopus niveus), 148Carbohydrase (Rhizopus oryzae var.), 148Carbohydrase (Saccharomyces species), 148Carbohydrase [(Trichoderma longibrachiatum
var.) (formerly reesei)], 149Carbohydrates and Related Substances, Tests
and Assays forAcetyl Groups, 951Crude Fat, 951Invert Sugar, 951–952Lactose, 952–953Propylene Chlorohydrin, 953–954Reducing Sugars Assay, 954–955Sulfur Dioxide Determination, 955–956Total Solids, 957–961
Carbon, Activated, 94–96Carbonate Identification Test, 859Carbon Dioxide, 96–98Carbon Dioxide Detector Tube, 977Carbon Dioxide-Free Water, 5Carbon Monoxide Detector Tube, 977Carbonyl Iron, 229–230(R)-3-Carboxy-2-hydroxy-N,N,N-trimethyl-1-
propanaminium Hydroxide, Inner Salt,100–101
[2-Carboxy-�-(N-(b-methoxycarbonyl-2-phenyl)ethylcarbamoyl)]ethanaminium 6-methyl-4-oxo-1,2,3-oxathiazin-3-ide-2,2-dioxide, 39–40
Cardamom Oil, 98Infrared Spectrum, 671
Cardamom Oleoresin, 446, 447Carmine, 98–99Carminic Acid, 98–99Carnauba Wax, 99–100L-Carnitine, 100–101Carob Bean Gum, 256Carotene, 101–102�-Carotene, 101–102�-Carotene-4,4′-dione, 88Carrot Seed Oil, 102
Infrared Spectrum, 672Carr-Price Reagent, 965Carvacrol, 534–535
Infrared Spectrum, 672l-Carveol, 534–535
NF, 6724-Carvomenthenol, 620–621d-Carvone, 534–535
Infrared Spectrum, 673dextro-Carvone, 534–535l-Carvone, 534–535
Infrared Spectrum, 673levo-Carvone, 534–535l-Carvyl Acetate, 536–537
Infrared Spectrum, 673�-Caryophyllene, 536–537
Infrared Spectrum, 674Cascarilla Oil, 102–103
Infrared Spectrum, 674Casein and Caseinate Salts, 103–104Cassia Oil, 104–105
Infrared Spectrum, 674Castor Oil, 105Catalase, 150, 896
Catalase Activity, 902Catalase (Aspergillus niger var.), 149Catalase (bovine liver), 147Catalase (Micrococcus lysodeikticus), 149Caustic Potash, 362Caustic Soda, 416Caustic Soda Solution, 416–417gamma-CD, 129–130Cedar Leaf Oil, 105–106
Infrared Spectrum, 675Cedar Leaf Oil, White, 105–106Celery Oleoresin, 447Celery Seed Oil, 106
Infrared Spectrum, 675Cellulase, 150, 896Cellulase Activity, 902–903Cellulose Gel, 106–107Cellulose Gum, 107–108
Viscosity of, 850–851Cellulose, Hydroxypropyl, 225Cellulose, Methyl Ester, 290Cellulose, Microcrystalline, 106–107Cellulose, Modified, 107–108, 225, 290Cellulose, Modified (EC), 158Cellulose, Modified (HPMC), 225–227Cellulose, Modified (MC), 287–288Cellulose, Powdered, 109–110Centrifuges, 4Ceric Ammonium Nitrate TS, 965Ceric Sulfate, 0.01 N, 971Ceric Sulfate, 0.1 N, 971Chamomile Oil, English Type, 110
Infrared Spectrum, 675Chamomile Oil, German Type, 111
Infrared Spectrum, 676Chamomile Oil, Hungarian Type, 111Chemical Formulas and Atomic Weights, 4Chemical Tests and Determinations, 859–891Chewing Gum Base Polymers
Arsenic Limit Test, 895Bound Styrene, 892Lead Limit Test, 895Molecular Weight, 892–893Quinones, 893–894Residual Styrene, 894–895Total Unsaturation, 895–896
Chicle, 280Chicle, Venezuelan, 280Chilte, 280China Clay, 236Chiquibul, 280Chloride and Sulfate Limit Tests, 863Chloride Identification Test, 859Chlorinated Compounds Determination, 930Chlorine, 111–112Chlorine Detector Tube, 977Chlorine TS, 965Chlorophyll, 934Cholalic Acid, 112Cholecalciferol, 498–499Cholic Acid, 112Choline Bitartrate, 112–113Choline Chloride, 113Chromatography, 834
Column, 834–835Gas, 630, 635–636, 836–838High-Performance Liquid, 838–841Technology Development, xv–xviThin-Layer, 835–836
Chromium, Color Additive Assays, 880Chromotropic Acid TS, 965Chymosin, 150, 896Chymosin (Aspergillus niger var. awamori,
Escherichia coli K-12, andKluyveromyces marxianus, eachmicroorganism containing a calfprochymosin gene), 149
Chymotrypsin, 147, 896
FCC V Index / Dicabonic Acid / 983
Chymotrypsin Activity, 904CI Food Yellow 4, 468–469Cinene, 584–5491,8-Cineol, 562–563Cineole, Percentage of, 931Cinnamal, 536–537Cinnamaldehyde, 536–537
Infrared Spectrum, 676Cinnamic Acid, 536–537
Infrared Spectrum, 676Cinnamic Alcohol, 536–537Cinnamic Aldehyde, 536–537Cinnamon Bark Oil, Ceylon Type, 114
Infrared Spectrum, 677Cinnamon Leaf Oil, 114–115
Infrared Spectrum, 677Cinnamon Oil, 104–105Cinnamyl Acetate, 536–537
Infrared Spectrum, 678Cinnamyl Alcohol, 536–537Cinnamyl Butyrate, 536–537Cinnamyl Cinnamate, 536–537Cinnamyl Formate, 538–539
Infrared Spectrum, 679Cinnamyl Isobutyrate, 538–539
Infrared Spectrum, 679Cinnamyl Isovalerate, 538–539
Infrared Spectrum, 679Cinnamyl Propionate, 538–539
Infrared Spectrum, 680Citral, 538–539
Infrared Spectrum, 680Citrate Identification Test, 859Citric Acid, 115–116Citridic Acid, 16Citronellal, 538–539
Infrared Spectrum, 680Citronellol, 538–539
Infrared Spectrum, 681Citronellyl Acetate, 538–539
Infrared Spectrum, 681Citronellyl Butyrate, 540–541
Infrared Spectrum, 681Citronellyl Formate, 540–541
Infrared Spectrum, 682Citronellyl Isobutyrate, 540–541
Infrared Spectrum, 682Citronellyl Propanoate, 540–541Citronellyl Propionate, 540–541
Infrared Spectrum, 682Citrus Oils, Ultraviolet Absorbance of, 932–
933Clary Oil, 116
Infrared Spectrum, 683Clary Sage Oil, 116Clove Bud Oil, 117Clove Leaf Oil, 116–117
Infrared Spectrum, 683Clove Oil, 117
Infrared Spectrum, 683Clove Stem Oil, 117–118
Infrared Spectrum, 684CMC, 107–108Coagulated or Concentrated Latices of
Vegetable Origin, 280Cobalt Identification Test, 859Cobaltous Chloride CS, 845Cobaltous Chloride TS, 965Cobalt–Uranyl Acetate TS, 965Cocoa Butter Substitute, 118–119Coconut Oil (Unhydrogenated), 119–120Cognac Oil, Green, 120
Infrared Spectrum, 684Cold Test, 935Color Additive Assays
Chromium, 880Ether Extracts, 880–881Leuco Base, 881
Mercury, 881–882Total Color, 882–884Uncombined Intermediates and Products of
Side Reactions, 884–886Color (AOCS-Wesson), 935Colorimetric Solutions, 962Color Value, Oleoresins, 944Column Chromatography, 834–835Committee on Food Chemicals Codex, xiii,
xix–xx, xxi–xxii, xxivCongo Red TS, 965‘‘Constant Weight,’’ Defined, 6Containers, 8
Light-Resistant, 8Tared, 8Tight, 8Well-Closed, 8
‘‘Cool Place,’’ Defined, 8Copaiba Oil, 120
Infrared Spectrum, 684Copper Gluconate, 121Copper Identification Test, 859Copper Sulfate, 121–122Copper Sulfate TS, 965Coriander Oil, 122
Infrared Spectrum, 685Coriander Oleoresin, 447, 448Cornmint Oil, Partially Dementholized, 285Corn Oil (Unhydrogenated), 122–123Corn Sugar, 135–136Corn Syrup, 194–195Corn Syrup, High-Fructose, 215–217Corn Syrup Solids, 957–958Costus Root Oil, 123
Infrared Spectrum, 685Cottonseed Oil (Unhydrogenated), 123–124Coulometric Titration, 853Cream of Tartar, 354Cresol Red, 975Cresol Red–Thymol Blue TS, 965Cresol Red TS, 965Cresol, Test for Free, 634p-Cresyl Acetate, 540–541
Infrared Spectrum, 685Test for, 634
p-Cresyl Isobutyrate, 624–625p-Cresyl Methyl Ether, 590–591Crospovidone, 350Crown Gum, 280Crude Fat Determination, 951Crystal Violet, 975Crystal Violet TS, 965Cubeb Oil, 124–125
Infrared Spectrum, 686Cubeb Oleoresin, 447, 448Cuminal, 540–541Cuminaldehyde, 540–541Cuminic Aldehyde, 540–541
Infrared Spectrum, 686p-Cuminic Aldehyde, 540–541Cumin Oil, 125
Infrared Spectrum, 686Cumin Oleoresin, 447, 448Cupric Citrate TS, Alkaline, 965Cupric Nitrate TS, 965Cupric Sulfate, 121–122Cupric Sulfate CS, 845Cupric Sulfate Test Paper, 976Cupric Sulfate TS, 965Cupric Tartrate TS, Alkaline, 965Curcumin Content, 944
* Curdlan, 125–126Cyanocobalamin, 496–497Cyanogen Bromide TS, 965Cyclamen Aldehyde, 540–541
Infrared Spectrum, 687beta-Cyclodextrin, 126–128�-Cyclodextrin, 126–128
�-Cyclodextrin, 129–130gamma-Cyclodextrin, 129–130
Infrared Spectrum, 6871,2,3,5/4,6-Cyclohexanehexol, 228–229Cyclohexyl Acetate, 540–541Cyclomaltooctaose, 129–130Cyclooctaamylose, 129–130Cyclopentadecanolide, 608–609p-Cymene, 542–543
Infrared Spectrum, 687L-Cysteine Monohydrochloride, 130
Infrared Spectrum, 688L-Cystine, 130–131
Infrared Spectrum, 688
D
Damar Gum, 131Damar Resin, 131Dammar, 131Dammar Gum, 131Dammar Resin, 131Danish Agar, 187–189DATEM, 136–137D.E., 137–138‘‘Deaerated Water,’’ Defined, 5(E),(E)-2,4-Decadienal, 542–543
Infrared Spectrum, 688trans,trans-2,4-Decadienal, 542–543�-Decalactone, 542–543
Infrared Spectrum, 669�-Decalactone, 542–543
Infrared Spectrum, 689Decanal, 542–543
Infrared Spectrum, 689Decanoic Acid, 131–132
Infrared Spectrum, 6901-Decanol, 542–543(E)-2-Decenal, 542–543
Infrared Spectrum, 690trans-2-Decenal, 542–543cis-4-Decenal, 542–543(Z)-4-Decenal, 542–543
Infrared Spectrum, 690Decyl Alcohol, 542–543
Infrared Spectrum, 691Dehydrated Alcohol, 5Dehydroacetic Acid, 132Denigès’ Reagent, 965Deoxycholic Acid, 132–133Desiccators and Desiccants, 4Desoxycholic Acid, 132–133Detector Tubes, 977Devitalized Wheat Gluten, 500Dexpanthenol, 133–134Dextrin, 134–135Dextro Calcium Pantothenate, 72–73Dextrose, 135–136Diacetyl, 544–545
Infrared Spectrum, 691Diacetyl Tartaric Acid Esters of Mono- and
Diglycerides, 136–1372,6-Diaminohexanoic Acid Hydrochloride,
257Diammonium Hydrogen Phosphate, 28Diammonium Phosphate, 28Diaquo bis (glycinato) iron (II), 176–177Diastase Activity (Diastatic Power), 904–905Diatomaceous Earth, 137–138Diatomaceous Silica, 137–138Diatomite, 137–138Dibasic Ammonium Phosphate, 28Dibenzyl Ether, 544–545
Infrared Spectrum, 6912,6-Di-tert-butyl-p-cresol, 49Dicalcium Phosphate, 75–76Dicabonic Acid, 140–141
984 / 1,6-Dichloro-1,6-dideoxy-�-D-fructofuranosyl-4-chloro-4-deoxy-�-D-galactopyranoside / Index FCC V
1,6-Dichloro-1,6-dideoxy-�-D-fructofuranosyl-4-chloro-4-deoxy-�-D-galactopyranoside,453–455
1,2-Dichloroethane, 158–159Dichloromethane, 288–289Dichlorophenol–Indophenol TS, 965Dietary Fiber from Beets, 458–460Dietary Supplements, 81,2-Di[(1′-ethoxy)ethoxy]propane, 544–545Diethylene Imidoxide, 296Diethylene Oximide, 296Diethyl Malonate, 544–545
Infrared Spectrum, 692Diethyl Sebacate, 544–545
Infrared Spectrum, 692Diethyl Succinate, 544–545
Infrared Spectrum, 692Dihydroanethole, 616–617Dihydrocarveol, 544–545d-Dihydrocarvone, 546–547
Infrared Spectrum, 693Dihydrocoumarin, 526–52713�,12�-Dihydroxycholanic Acid, 132–1332,7-Dihydroxynaphthalene TS, 9651,2-Dihydroxypropane, 3764,4′-Diketo-�-carotene, 88Dilauryl Thiodipropionate, 138–139Dill Herb Oil, American Type, 140Dill Oil, 140Dill Oil, Indian Type, 139Dill Seed Oil, European Type, 139
Infrared Spectrum, 693Dill Seed Oil, Indian, 139Dill Seed Oil, Indian Type, 139
Infrared Spectrum, 693Dillseed Oleoresin, 447, 448Dillweed Oil, American Type, 140
Infrared Spectrum, 694Diluted Acetic Acid TS, 963Diluted Alcohol, 963Diluted Hydrochloric Acid TS, 966Diluted Lead Subacetate TS, 967Diluted Nitric Acid TS, 968Diluted Sulfuric Acid TS, 969Dimagnesium Phosphate, 262–2631,2-Dimethoxy-4-allylbenzene, 594–5953,4-Dimethoxybenzaldehyde, 628–629
* 2,6-Dimethoxy Phenol, 546–547Infrared Spectrum, 694
2,5-Dimethyl-3-acetylfuran, 518–519Dimethyl Anthranilate, 546–547Dimethyl Benzyl Carbinol, 546–547
Infrared Spectrum, 694Dimethyl Benzyl Carbinyl Acetate, 546–547
Infrared Spectrum, 695Dimethyl Benzyl Carbinyl Butyrate, 546–
547Infrared Spectrum, 695
* 3,4-Dimethyl 1,2-Cyclopentandione, 546–547
Infrared Spectrum, 695Dimethyl Dicarbonate, 140–141
Infrared Spectrum, 696Dimethyldiketone, 544–545Dimethyl Ester, 140–141Dimethylglyoxal, 544–5452,6-Dimethyl-5-heptenal, 548–549
Infrared Spectrum, 696Dimethylketol, 518–519Dimethyl Ketone, 10–116,6-Dimethyl-2-methylene-bicyclo-
[3.1.1]heptane, 616–6173,7-Dimethyl-1,6-octadien-3-ol, 584–585cis-3,7-Dimethyl-2,6-octadien-1-ol, 602–603(E)-3,7-Dimethyl-2,6-octadien-1-ol, 564–565trans-3,7-Dimethyl-2,6-octadien-1-ol, 564–5653,7-Dimethyl-1,6-octadien-3-yl Acetate, 584–
585
3,7-Dimethyl-2,6-octadien-1-yl Acetate, 564–565
3,7-Dimethyl-1,6-octadien-3-yl Benzoate,584–585
3,7-Dimethyl-2,6-octadien-1-yl-Benzoate,564–565
3,7-Dimethyl-2,6-octadien-1-yl Butyrate, 564–565
3,7-Dimethyl-1,6-octadien-3-yl Formate, 586–587
3,7-Dimethyl-2,6-octadien-1-yl Formate, 566–567
3,7-Dimethyl-2,6-octadien-3-yl Isobutyrate,586–587
3,7-Dimethyl-2,6-octadien-1-yl Phenylacetate,566–567
3,7-Dimethyl-2,6-octadien-1-yl Propionate,566–567
3,7-Dimethyl-2,6-octadien-3-yl Propionate,586–587
Dimethyl Octanol, 548–5493,7-Dimethyl-1-octanol, 548–549
Infrared Spectrum, 6963,7-Dimethyl-3-octanol, 622–6233,7-Dimethyl-6-octen-1-al, 538–5393,7-Dimethyl-6-octen-1-ol, 538–5393,7-Dimethyl-6-octen-1-yl Acetate, 538–5393,7-Dimethyl-6-octen-1-yl Butyrate, 540–5413,7-Dimethyl-6-octen-1-yl Formate, 540–5413,7-Dimethyl-6-octen-1-yl Isobutyrate, 540–
5413,7-Dimethyl-6-octen-1-yl Propionate, 540–
541�,�-Dimethylphenethyl Acetate, 546–547�,�-Dimethylphenethyl Alcohol, 546–547�,�-Dimethylphenethyl Butyrate, 546–547Dimethylpolysiloxane, 141
Viscosity of, 848–8492,3-Dimethylpyrazine, 548–549
Infrared Spectrum, 6972,5-Dimethylpyrazine, 548–549
Infrared Spectrum, 6972,6-Dimethylpyrazine, 548–549
Infrared Spectrum, 697Dimethyl Pyrocarbonate, 140–1412,5-Dimethylpyrrole, 548–549
Infrared Spectrum, 698Dimethyl Silicone, 141Dimethyl Succinate, 548–549Dimethyl Sulfide, 548–549
Infrared Spectrum, 698Dioctyl Sodium Sulfosuccinate, 141–1431,4-Dioxane Limit Test, 863–864Diphenylamine TS, 965Diphenylcarbazone TS, 966Diphenyl Ether, 550–551
Infrared Spectrum, 698Diphenyl Ketone, 526–527Diphenyl Oxide, 550–551Dipotassium Monophosphate, 367–368Dipotassium Phosphate, 367–368�,�′-Dipyridyl TS, 966Disodium Dihydrogen Diphosphate, 400–401Disodium Dihydrogen Pyrophosphate, 400–
401Disodium Edetate, 143–144Disodium EDTA, 143–144Disodium EDTA, 0.05 M, 971Disodium Ethylenediaminetetraacetate,
143–144Disodium (Ethylenedinitrilo)tetraacetate,
143–144Disodium Guanosine-5′-monophosphate, 144–
145Disodium Guanylate, 144–145Disodium Inosinate, 145–146Disodium Inosine-5′-monophosphate, 145–146Disodium Monohydrogen Phosphate, 127
Disodium Phosphate, 427Disodium Pyrophosphate, 400–401Disodium Tartrate, 438Disodium D-Tartrate, 438Distarch Phosphate, 182–183Distillation Range Testing, 841–8423,3′-Dithiobis(2-aminopropanoic acid), 130–
131Dithizone, 975Dithizone Method, 867–868Dithizone TS, 966DMDC, 140–141Docusate Sodium, 141–143�-Dodecalactone, 550–551
Infrared Spectrum, 699�-Dodecalactone, 550–551Dodecanal, 582–583Dodecanoic Acid, 246–2471-Dodecanol, 582–583(E)-2-Dodecen-1-al, 550–551
Infrared Spectrum, 699trans-2-Dodecen-1-al, 550–551DOSS, 141–143Dried Glucose Syrup, 195Dried Yeast, 508–510Drop Method, 947–948
E
Edible Gelatin, 189–191Electrolytic Iron, 230–231Enanthic Alcohol, 568–569Enocianina, 209–210Enzyme Assays, 896–898
Acid Phosphatase Activity, 898–899Aminopeptidase Activity, 899–900�-Amylase Activity, 900–902Catalase Activity, 902Cellulase Activity, 902–903Chymotrypsin Activity, 904Diastase Activity, 904–905�-Galactosidase Activity, 905–906�-Glucanase Activity, 906–907Glucoamylase Activity, 907–908Glucose Isomerase Activity, 908–909Glucose Oxidase Activity, 909Hemicellulase Activity, 909–911Invertase Activity, 911Lactase Activity (Acid), 913–914Lactase Activity (Neutral), 911–913Lipase Activity, 914–915Lysozyme Activity, 915–916Maltogenic Amylase Activity, 916–917Milk-Clotting Activity, 917Pancreatin Activity, 917–919Pepsin Activity, 920Phospholipase A2 Activity, 920–921Phytase Activity, 921–922Plant Proteolytic Activity, 922–923Proteolytic Activity, Bacterial, 923–924Proteolytic Activity, Fungal, 924–926Pullulanase Activity, 926–927Transglutaminase Activity, 927–928Trypsin Activity, 928
Enzyme-Hydrolyzed (Source) Protein, 319–321
Enzyme-Modified Fats, 146Enzyme-Modified (Source) Protein, 319–321Enzyme Preparations, 146–147, 151–152
Animal-Derived Preparations, 147Plant-Derived Preparations, 147–148Microbially Derived Preparations, 148–150
Eosin Y TS, 9661:8-Epoxy-p-menthane, 562–563Epsom Salt, 266Equisetic Acid, 16Ergocalciferol, 497–498
FCC V Index / Ficin / 985
Eriochrome Black T, 975Eriochrome Black TS, 966Erythorbic Acid, 152Erythritol, 153meso-Erythritol, 153
* Erythrosine, 154–155, 164Essential Oils and Flavors, Tests and Assays
Acetals, 929Acid Value, 929Aldehydes, 929Aldehydes and Ketones, 929–930Chlorinated Compounds, 930Esters, 930–931Linalool Determination, 931Phenols, 931–932Residue on Evaporation, 932Solubility in Alcohol, 932Total Alcohol, 932Ultraviolet Absorption of Citrus Oils, 932–
933Volatile Oils Content, 933
Ester Assays, 930–931Ester Gum, 201–202Estragole, 550–551
Infrared Spectrum, 699Estragon Oil, 467Ethanal, 518–519Ethanol, 157–158Ethanol (reagent), 5Ether Extracts, Color Additive Assays, 880–
881Ethone, 550–551
Infrared Spectrum, 700p-Ethoxychrysoidin Monohydrochloride, 975p-Ethoxychrysoidin TS, 9666-Ethoxy-1,2-dihydro-2,2,4-trimethylquinoline,
156–1573-Ethoxy-4-hydroxybenzaldehyde, 560–5611-Ethoxy-2-hydroxy-4-propenylbenzene, 616–
617Ethoxylated Mono- and Diglycerides, 155–
156Ethoxyquin, 156–157Ethyl Acetate, 550–551
Carbonized Substances in, Limit Test for,633
Infrared Spectrum, 700Methyl Compounds in, Limit Test for, 633
Ethyl Acetoacetate, 550–551Infrared Spectrum, 700
Ethyl Acrylate, 550–551Infrared Spectrum, 701Antioxidants in, Limit Test for, 632–633
Ethyl Alcohol, 157–158Ethyl Alcohol (reagent), 5Ethyl o-Aminobenzoate, 552–553Ethyl p-Anisate, 552–553
Infrared Spectrum, 701Ethyl Anthranilate, 552–553
Infrared Spectrum, 701Ethyl Benzoate, 552–553
Infrared Spectrum, 702Ethyl Benzoyl Acetate, 552–553Ethyl-(E)-2-butenoate, 552–553
Infrared Spectrum, 702Ethyl-trans-2-butenoate, 552–553Ethyl Butyl Ketone, 568–5692-Ethylbutyraldehyde, 552–553
Infrared Spectrum, 702Ethyl Butyrate, 552–553
Infrared Spectrum, 7032-Ethylbutyric Acid, 552–553
Infrared Spectrum, 703Ethyl Caprate, 554–555Ethyl Caproate, 556–557Ethyl Capronate, 556–557Ethyl Caprylate, 558–559Ethyl Cellulose, 158
Ethyl Cinnamate, 552–553Infrared Spectrum, 703
Ethyl Citrate, 489Ethyl Crotonate, 552–553Ethyl Decanoate, 554–555
Infrared Spectrum, 7042-Ethyl-3,5(6)-dimethylpyrazine, 554–555
Infrared Spectrum, 704Ethyl Dodecanoate, 556–557Ethylene Brassylate, 554–555
Infrared Spectrum, 704trans-1,2-Ethylenedicarboxylic Acid, 186–187Ethylene Dichloride, 158–159Ethylene Trichloride, 488–4892-Ethyl Fenchol, 554–555
Infrared Spectrum, 705Ethyl Formate, 554–555
in Acidity Determination by IodometricMethod, 632
Infrared Spectrum, 7054-Ethyl Guaiacol, 554–555
Infrared Spectrum, 705Ethyl Heptanoate, 554–555
Infrared Spectrum, 706Ethyl Heptoate, 554–555Ethyl Hexanoate, 556–557
Infrared Spectrum, 7062-Ethyl Hexanol, 556–5572-Ethyl-1-hexanol, 556–557
* 5-Ethyl 3-Hydroxy 4-Methyl 2(5H)-Furanone, 556–557
Infrared Spectrum, 706Ethyl 2-Hydroxypropionate, 556–5572-Ethyl-3-hydroxy-4-pyrone, 159Ethyl Isobutyrate, 556–557
Infrared Spectrum, 707Ethyl Isovalerate, 556–557
Infrared Spectrum, 707Ethyl Lactate, 556–557
Infrared Spectrum, 707Ethyl Laurate, 556–557
Infrared Spectrum, 708Ethyl Levulinate, 556–557
Infrared Spectrum, 708Ethyl Malonate, 544–545Ethyl Maltol, 159Ethyl p-Methoxybenzoate, 552–553Ethyl 2-Methylbutyrate, 556–557Ethyl 3-Methylbutyrate, 556–557Ethyl 2-Methylpentanoate, 558–559
Infrared Spectrum, 708Ethyl Methylphenylglycidate, 558–559
Infrared Spectrum, 7092-Ethyl-3-methylpyrazine, 558–559
Infrared Spectrum, 709Ethyl 3-Methylthiopropionate, 558–559
Infrared Spectrum, 709Ethyl Myristate, 558–559
Infrared Spectrum, 710Ethyl Nonanoate, 558–559
Infrared Spectrum, 710Ethyl 9-Octadecenoate, 558–559Ethyl Octanoate, 558–559
Infrared Spectrum, 710Ethyl Octoate, 558–559Ethyl Oleate, 558–559Ethyl 3-Oxybutanoate, 550–551Ethyl Oxyhydrate, Alcohol Content Testing,
631–632Ethyl Oxyhydrate (so-called), 558–559Ethyl Pelargonate, 558–559Ethyl n-Pentanoate, 560–561Ethyl Phenylacetate, 560–561
Infrared Spectrum, 711Ethyl Phenylglycidate, 560–561
Infrared Spectrum, 711Ethyl 3-Phenylpropenate, 552–553
Ethyl Propionate, 560–561Infrared Spectrum, 711
* 3-Ethyl Pyridine, 560–561Infrared Spectrum, 712
Ethyl Salicylate, 560–561Infrared Spectrum, 712
Ethyl Sebacate, 560–561Ethyl Succinate, 544–545Ethyl 10-Undecanoate, 560–561Ethyl Valerate, 560–561Ethyl Vanillin, 560–561
Infrared Spectrum, 712Eucalyptol, 562–563
Infrared Spectrum, 713Eucalyptus Oil, 159–160
Infrared Spectrum, 713Eugenic Acid, 562–563Eugenol, 562–563
Hydrocarbons in, Limit Test for, 633Infrared Spectrum, 713
Eugenol Acetate, 562–563Eugenyl Acetate, 562–563
Infrared Spectrum, 714Eugenyl Methyl Ether, 594–595Exaltolide, 608–609‘‘Excessive Heat,’’ Defined, 8Expanded Perlite, 326–327
F
Farnesol, 562–563Infrared Spectrum, 714
* Fast Green, 160–161Fast Green FCF, 160–161, 163–164Fats and Related Substances, Tests and
Assays, 934–943Fats, Enzyme-Modified, 146Fatty Acid Composition, 935–936Fatty Acids, Free, 936FD&C Blue No. 1, 162FD&C Blue No. 2, 162–163FD&C Green No. 3, 163–164FD&C Red No. 3, 164FD&C Red No. 40, 165FD&C Yellow No. 5, 165–166FD&C Yellow No. 6, 166–167Fehling’s Solution, 966d-Fenchone, 562–563
Infrared Spectrum, 714Fenchyl Alcohol, 562–563
Infrared Spectrum, 715Fennel Oil, 167
Infrared Spectrum, 715Fennel Oleoresin, 447, 448Ferric Ammonium Citrate, Brown, 167–168Ferric Ammonium Citrate, Green, 169Ferric Ammonium Sulfate TS, 966Ferric Chloride CS, 845Ferric Chloride TS, 966Ferric Chloride TS, Alcoholic, 966Ferric Citrate, 169Ferric Orthophosphate, 169–172Ferric Phosphate, 169–172Ferric Pyrophosphate, 172Ferric Sulfate TS, Acid, 966Ferrous Ammonium Sulfate, 0.1 N, 971Ferrous Bisglycinate, 176–177Ferrous Citrate, 172–173Ferrous Fumarate, 173–174Ferrous Gluconate, 174–175
* Ferrous Glycinate, 176–177Ferrous Lactate, 177–178Ferrous Sulfate, 178Ferrous Sulfate, Dried, 178–179Ferrous Sulfate TS, 966FHMO, 280–284Ficin, 50, 147, 896
986 / ‘‘Filtration,’’ Defined / Index FCC V
‘‘Filtration,’’ Defined, 4Fir Needle Oil, Canadian Type, 179
Infrared Spectrum, 715Fir Needle Oil, Siberian Type, 179–180
Infrared Spectrum, 716Fischer-Tropsch Paraffin, 318Flame Atomic Absorption Spectrophotometric
Method, 868–869Flavin Mononucleotide, Sodium Salt, 384–386Flavor Chemicals
Acetaldehyde, 518–519Acetaldehyde Diethyl Acetal, 518–519Acetanisole, 518–519Acetoin, 518–519Acetophenone, 518–5193-Acetyl-2,5-dimethyl Furan, 518–5192-Acetylpyrazine, 520–5213-Acetylpyridine, 520–5212-Acetylpyrrole, 520–5212-Acetyl Thiazole, 520–521Allyl Cyclohexanepropionate, 520–521Allyl Heptanoate, 520–521Allyl Hexanoate, 520–521Allyl �-Ionone, 520–521Allyl Isothiocyanate, 522–523Allyl Isovalerate, 522–523Allyl Phenoxy Acetate, 522–523Allyl Propionate, 522–5231-Amyl Alcohol, 522–523Amyl Butyrate, 522–523�-Amylcinnamaldehyde, 522–523Amyl Cinnamate, 522–523Amyl Formate, 522–523Amyl Heptanoate, 524–525Amyl Octanoate, 524–525Amyl Propionate, 524–525Anethole, 524–525Anisole, 524–525Anisyl Acetate, 524–525Anisyl Alcohol, 524–525Anisyl Formate, 524–525Benzaldehyde, 526–527Benzaldehyde Glyceryl Acetal, 526–5271,2-Benzodihydropyrone, 526–527Benzophenone, 526–527Benzyl Acetate, 526–527Benzyl Alcohol, 526–527Benzyl Benzoate, 526–527Benzyl Butyrate, 526–527Benzyl Cinnamate, 528–529Benzyl Formate, 528–529Benzyl Isobutyrate, 528–529Benzyl Isovalerate, 528–529Benzyl Phenylacetate, 528–529Benzyl Propionate, 528–529Benzyl Salicylate, 528–529Borneol, 528–529Bornyl Acetate, 530–5312-Butanone, 530–531Butan-3-one-2-yl Butanoate, 530–531Butyl Acetate, 530–531Butyl Alcohol, 530–531Butyl Butyrate, 530–531Butyl Butyryllactate, 530–5312-sec-Butyl Cyclohexanone, 532–533Butyl Isobutyrate, 532–533Butyl Isovalerate, 532–533Butyl 2-Methyl Butyrate, 532–533Butyl Phenylacetate, 532–533Butyl Stearate, 532–533Butyraldehyde, 532–533Butyric Acid, 532–533�-Butyrolactone, 534–535Camphene, 534–535d-Camphor, 534–535Carvacrol, 534–535l-Carveol, 534–535d-Carvone, 534–535
l-Carvone, 534–535l-Carvyl Acetate, 536–537�-Caryophyllene, 536–537Cinnamaldehyde, 536–537Cinnamic Acid, 536–537Cinnamyl Acetate, 536–537Cinnamyl Alcohol, 536–537Cinnamyl Butyrate, 536–537Cinnamyl Cinnamate, 536–537Cinnamyl Formate, 538–539Cinnamyl Isobutyrate, 538–539Cinnamyl Isovalerate, 538–539Cinnamyl Propionate, 538–539Citral, 538–539Citronellal, 538–539Citronellol, 538–539Citronellyl Acetate, 538–539Citronellyl Butyrate, 540–541Citronellyl Formate, 540–541Citronellyl Isobutyrate, 540–541Citronellyl Propionate, 540–541p-Cresyl Acetate, 540–541Cuminic Aldehyde, 540–541Cyclamen Aldehyde, 540–541Cyclohexyl Acetate, 540–541p-Cymene, 542–543(E),(E)-2,4-Decadienal, 542–543�-Decalactone, 542–543�-Decalactone, 542–543Decanal, 542–543(E)-2-Decenal, 542–543(Z)-4-Decenal, 542–543Decyl Alcohol, 542–543Diacetyl, 544–545Dibenzyl Ether, 544–5451,2-Di[(1′-ethoxy)ethoxy]propane, 544–545Diethyl Malonate, 544–545Diethyl Sebacate, 544–545Diethyl Succinate, 544–545Dihydrocarveol, 544–545d-Dihydrocarvone, 546–547
* 2,6-Dimethoxy Phenol, 546–547Dimethyl Anthranilate, 546–547Dimethyl Benzyl Carbinol, 546–547Dimethyl Benzyl Carbinyl Acetate, 546–
547Dimethyl Benzyl Carbinyl Butyrate, 546–
547* 3,4-Dimethyl 1,2-Cyclopentandione,
546–5472,6-Dimethyl-5-heptenal, 548–5493,7-Dimethyl-1-octanol, 548–5492,3-Dimethylpyrazine, 548–5492,5-Dimethylpyrazine, 548–5492,6-Dimethylpyrazine, 548–5492,5-Dimethylpyrrole, 548–549Dimethyl Succinate, 548–549Dimethyl Sulfide, 548–549Diphenyl Ether, 550–551�-Dodecalactone, 550–551�-Dodecalactone, 550–551(E)-2-Dodecen-1-al, 550–551Estragole, 550–551Ethone, 550–551Ethyl Acetate, 550–551Ethyl Acetoacetate, 550–551Ethyl Acrylate, 550–551Ethyl p-Anisate, 552–553Ethyl Anthranilate, 552–553Ethyl Benzoate, 552–553Ethyl Benzoyl Acetate, 552–553Ethyl-(E)-2-butenoate, 552–5532-Ethylbutyraldehyde, 552–553Ethyl Butyrate, 552–5532-Ethylbutyric Acid, 552–553Ethyl Cinnamate, 552–553Ethyl Decanoate, 554–5552-Ethyl-3,5(6)-dimethylpyrazine, 554–555
Ethylene Brassylate, 554–5552-Ethyl Fenchol, 554–555Ethyl Formate, 554–5554-Ethyl Guaiacol, 554–555Ethyl Heptanoate, 554–555Ethyl Hexanoate, 556–5572-Ethyl Hexanol, 556–557
* 5-Ethyl 3-Hydroxy 4-Methyl 2(5H)-Furanone, 556–557
Ethyl Isobutyrate, 556–557Ethyl Isovalerate, 556–557Ethyl Lactate, 556–557Ethyl Laurate, 556–557Ethyl Levulinate, 556–557Ethyl 2-Methylbutyrate, 556–557Ethyl 2-Methylpentanoate, 558–559Ethyl Methylphenylglycidate, 558–5592-Ethyl-3-methylpyrazine, 558–559Ethyl 3-Methylthiopropionate, 558–559Ethyl Myristate, 558–559Ethyl Nonanoate, 558–559Ethyl Octanoate, 558–559Ethyl Oleate, 558–559Ethyl Oxyhydrate (so-called), 558–559Ethyl Phenylacetate, 560–561Ethyl Phenylglycidate, 560–561Ethyl Propionate, 560–561
* 3-Ethyl Pyridine, 560–561Ethyl Salicylate, 560–561Ethyl 10-Undecenoate, 560–561Ethyl Valerate, 560–561Ethyl Vanillin, 560–561Eucalyptol, 562–563Eugenol, 562–563Eugenyl Acetate, 562–563Farnesol, 562–563d-Fenchone, 562–563Fenchyl Alcohol, 562–563Furfural, 562–563
* Furfural Mercaptan, 564–565Furfuryl Alcohol, 564–5652-Furyl Methyl Ketone, 564–565Fusel Oil, Refined, 564–565Geraniol, 564–565Geranyl Acetate, 564–565Geranyl Benzoate, 564–565Geranyl Butyrate, 564–565Geranyl Formate, 566–567
* Geranyl Isovalerate, 566–567Geranyl Phenylacetate, 566–567Geranyl Propionate, 566–567Glyceryl Tripropanoate, 566–567(E),(E)-2,4-Heptadienal, 566–567�-Heptalactone, 566–567Heptanal, 568–569
* 2,3-Heptandione, 568–5692-Heptanone, 568–5693-Heptanone, 568–569(Z)-4-Hepten-1-al, 568–569Heptyl Alcohol, 568–569�-Hexalactone, 568–569Hexanal, 568–569Hexanoic Acid, 568–569(E)-2-Hexen-1-al, 570–571(E)-2-Hexen-1-ol, 570–571(Z)-3-Hexenol, 570–571(E)-2-Hexenyl Acetate, 570–571(Z)-3-Hexenyl Acetate, 570–571
* (Z)-3-Hexenyl Butyrate, 570–571* (Z)-3-Hexenyl Formate, 570–571
(Z)-3-Hexenyl Isovalerate, 570–571(Z)-3-Hexenyl 2-Methylbutyrate, 572–573n-Hexyl Acetate, 572–573Hexyl Alcohol, 572–573Hexyl-2-butenoate, 572–573
* Hexyl Butyrate, 572–573* Hexyl Hexanoate, 572–573
�-Hexylcinnamaldehyde, 572–573
FCC V Index / Flaxseed Oil (Unhydrogenated), Low Linoleic Acid / 987
Hexyl Isovalerate, 572–573Hexyl 2-Methylbutyrate, 572–573Hydroxycitronellal, 574–575Hydroxycitronellal Dimethyl Acetal, 574–
5754-Hydroxy-2,5-dimethyl-3(2H)-furanone,
574–5756-Hydroxy-3,7-dimethyloctanoic Acid
Lactone, 574–5754-(p-Hydroxyphenyl)-2-butanone, 574–575Indole, 574–575�-Ionone, 574–575�-Ionone, 576–577Isoamyl Acetate, 576–577Isoamyl Alcohol, 576–577Isoamyl Benzoate, 576–577Isoamyl Butyrate, 576–577Isoamyl Formate, 576–577Isoamyl Hexanoate, 576–577
* Isoamyl Isobutyrate, 578–579Isoamyl Isovalerate, 578–579Isoamyl Phenyl Acetate, 578–579Isoamyl Salicylate, 578–579Isoborneol, 578–579Isobornyl Acetate, 578–579Isobutyl Acetate, 578–579Isobutyl Alcohol, 578–579Isobutyl-2-butenoate, 580–581Isobutyl Butyrate, 580–581Isobutyl Cinnamate, 580–581
* Isobutyl Formate, 580–581* Isobutyl Hexanoate, 580–581
Isobutyl Phenylacetate, 580–581Isobutyl Salicylate, 580–581Isobutyraldehyde, 580–581Isobutyric Acid, 580–581Isoeugenol, 582–583Isoeugenyl Acetate, 582–583Isopropyl Acetate, 582–583Isopulegol, 582–583Isovaleric Acid, 582–583Lauryl Alcohol, 582–583Lauryl Aldehyde, 582–583Levulinic Acid, 582–583d-Limonene, 584–585l-Limonene, 584–585Linalool, 584–585
* Linalool Oxide, 584–585Linalyl Acetate, 584–585Linalyl Benzoate, 584–585Linalyl Formate, 586–587Linalyl Isobutyrate, 586–587Linalyl Propionate, 586–587
* Maltol Isobutyrate, 586–587Menthol, 586–587l-Menthone, 586-587dl-Menthyl Acetate, 588–589l-Menthyl Acetate, 588–5892-Mercaptopropionic Acid, 588–589p-Methoxybenzaldehyde, 588–589
* 2-Methoxy 3-(or 5- or 6-) IsopropylPyrazine, 588–589
2-Methoxy-3(5)-methylpyrazine, 588–5894-p-Methoxyphenyl-2-butanone, 588–5892-Methoxypyrazine, 590–591Methyl Acetate, 590–5914-Methyl Acetophenone, 590–591p-Methyl Anisole, 590–591Methyl Anthranilate, 590–591Methyl Benzoate, 590–591�-Methylbenzyl Acetate, 590–591�-Methylbenzyl Alcohol, 590–5912-Methyl Butanal, 590–5913-Methyl Butanal, 590–5912-Methylbutyl Acetate, 592–5932-Methylbutyl Isovalerate, 592–593Methyl Butyrate, 592–5932-Methylbutyric Acid, 592–593
�-Methylcinnamaldehyde, 592–593Methyl Cinnamate, 592–5936-Methylcoumarin, 592–593Methyl Cyclopentenolone, 592–593
* 5H-5-Methyl-6,7-dihydrocyclopenta[6]pyrazine, 594–595
Methyl Eugenol, 594–595* 5-Methyl Furfural, 594–595* Methyl Furoate, 594–595
6-Methyl-5-hepten-2-one, 594–595* Methyl Hexanoate, 594–595
Methyl Hexyl Ketone, 595–595Methyl Ionones, 594–595Methyl Isobutyrate, 596–597Methyl Isoeugenol, 596–5975-Methyl-2-isopropyl-2-hexenal, 596–597
* Methyl Isovalerate, 596–597Methyl 2-Methylbutyrate, 596–597Methyl-3-methylthiopropionate, 596–597Methyl beta-Naphthyl Ketone, 596–597Methyl 2-Octynoate, 596–5972-Methylpentanoic Acid, 596–5974-Methylpentanoic Acid, 598–5994-Methyl-2-pentanone, 598–5992-Methyl-2-pentenoic Acid, 598–599Methyl Phenylacetate, 598–599Methyl Phenylcarbinyl Acetate, 598–599
* 5-Methyl 2-Phenyl 2-Hexenal, 598–599Methyl Propyl 3-Methyl Butyrate, 598–5992-Methylpyrazine, 598–599Methyl Salicylate, 600–6014-Methyl-5-thiazole Ethanol, 600–601
* Methyl Thiobutyrate, 600–6013-Methylthiopropionaldehyde, 600–6012-Methylundecanal, 600–601
* Methyl Valerate, 600–601Myrcene, 600–601Myristaldehyde, 600–601Myristyl Alcohol, 602–603
* �-Naphthyl Ethyl Ether, 602–603Nerol, 602–603Nerolidol, 602–603Neryl Acetate, 602–603(E),(E)-2,4-Nonadienal, 602–603(E),(Z)-2,6-Nonadienal, 602–603(E),(Z)-2,6-Nonadienol, 604–605�-Nonalactone, 604–605�-Nonalactone, 604–605Nonanal, 604–605Nonanoic Acid, 604–6052-Nonanone, 604–605(E)-2-Nonenal, 604–605(E)-2-Nonen-1-ol, 604–605(Z)-6-Nonen-1-ol, 604–605Nonyl Acetate, 606–607Nonyl Alcohol, 606–607�-Octalactone, 606–607�-Octalactone, 606–607Octanal, 606–6073-Octanol, 606–607(E)-2-Octen-1-al, 606–6071-Octen-3-ol, 606–607(Z)-3-Octen-1-ol, 606–6071-Octen-3-yl Acetate, 608–6091-Octen-3-yl Butyrate, 608–609Octyl Acetate, 608–6093-Octyl Acetate, 608–609Octyl Alcohol, 608–609Octyl Formate, 608–609Octyl Isobutyrate, 608–609�-Pentadecalactone, 608–6092,3-Pentanedione, 608–6092-Pentanone, 610–611�-Phellandrene, 610–611Phenethyl Acetate, 610–611Phenethyl Alcohol, 610–611Phenethyl Isobutyrate, 610–611
Phenethyl Isovalerate, 610–6112-Phenethyl 2-Methylbutyrate, 610–611Phenethyl Phenylacetate, 610–611Phenethyl Salicylate, 610–611Phenoxyethyl Isobutyrate, 612–613Phenylacetaldehyde, 612–613Phenylacetaldehyde Dimethyl Acetal, 612–
613Phenylacetic Acid, 612–613Phenylethyl Anthranilate, 612–613Phenylethyl Butyrate, 612–613
* Phenyl Ethyl Cinnamate, 612–613* Phenyl Ethyl Propionate, 614–615
3-Phenyl-1-propanol, 614–6152-Phenylpropionaldehyde, 614–6153-Phenylpropionaldehyde, 614–6152-Phenylpropionaldehyde Dimethyl Acetal,
614–6153-Phenylpropyl Acetate, 614–615�-Pinene, 614–615�-Pinene, 616–617Piperidine, 616–617Piperonal, 616–617Propenylguaethol, 616–617Propionaldehyde, 616–617Propyl Acetate, 616–617Propyl Alcohol, 616–617p-Propyl Anisole, 616–617
* Propyl Formate, 618–619* Propyl Mercaptan, 618–619
Propyl Propionate, 618–619Pyrrole, 618–619Rhodinol, 618–619Rhodinyl Acetate, 618–619Rhodinyl Formate, 618–619
* Salicylaldehyde, 618–619Santalol, 620–621Santalyl Acetate, 620–621Specifications for, 517�-Terpinene, 620–621�-Terpinene, 620–621Terpinen-4-ol, 620–621�-Terpineol, 620–621Terpinyl Acetate, 622–623Terpinyl Propionate, 622–623
* �-Tetradecalactone, 622–623Tetrahydrofurfuryl Alcohol, 622–623Tetrahydrolinalool, 622–6232,3,5,6-Tetramethylpyrazine, 622–623Thymol, 622–623Tolualdehyde, Mixed isomers, 624–625p-Tolualdehyde, 624–625p-Tolyl Isobutyrate, 624–625Tributyrin, 624–625
* 2-Tridecanone, 624–6252-Tridecenal, 624–625Trimethylamine, 624–6253,5,5-Trimethyl Hexanal, 624–6252,4,5-Trimethyl �-3-Oxazoline, 626–6272,3,5-Trimethylpyrazine, 626–627�-Undecalactone, 626–627�-Undecalactone, 626–627Undecanal, 626–6272-Undecanone, 626–6271,3,5-Undecatriene, 626–62710-Undecenal, 626-627(E)-2-Undecenol, 626–627Undecyl Alcohol, 626–627Valeraldehyde, 628–629Valeric Acid, 628–629�-Valerolactone, 628–629Vanillin, 628–629Veratraldehyde, 628–629Zingerone, 628–629
Flaxseed Oil (Unhydrogenated), Low LinoleicAcid, 441
988 / Fluoride Limits / Index FCC V
Fluoride LimitsPolicy, xivTests, 864–867
Folic Acid, 180–181Food-Grade Gelatin, 189–191Food Starch, Modified, 181–183Food Starch-Modified, 181–183Food Starch, Unmodified, 183–184Formaldehyde TS, 966Former and Current Titles of Food Chemicals
Codex Monographs, xxxivFormic Acid, 184–185Free Fatty Acids, 936Free Glycerin or Propylene Glycol, 936Free Phenols, 932Freskomenthe, 532–533�-D-Fructofuranosyl-�-D-glucopyranoside,
455–456�-Fructofuranosidase, 151Fructose, 185–186d-Fructose, 185–186Fruit Sugar, 185–186Fuchsin–Sulfurous Acid TS, 966Fully Hydrogenated Rapeseed Oil, 382Fumaric Acid, 186–187Functional Use in Foods, 8Function, Statement of, 8Fungal Proteolytic Activity
HUT, 924–925SAP, 925–926
2-Furaldehyde, 562–563Furcelleran, 187–189Furfural, 562–563
Infrared Spectrum, 716Furfuryl Alcohol, 564–565
Infrared Spectrum, 716* Furfuryl Mercaptan, 564–565
Infrared Spectrum, 7172-Furyl Methyl Ketone, 564–565
Infrared Spectrum, 717Fusel Oil, Refined, 564–565
G
4-O-�-Galactopyranosyl-D-glucose, 241�-Galactosidase, 150, 896�-Galactosidase Activity, 905–906, 911–914�-Galactosidase, 151Galam, 397Gallic Acid, Propyl Ester, 378Gallotannic Acid, 466–467Garlic Oil, 189
Infrared Spectrum, 718Gas Chromatography, 836–838
Flavor Chemicals Assays, 630, 635–636Gaultheria Oil, 504Gelatin, 189–191Gelatinized Starch, 181–182Gellan Gum, 191–192General Provisions and Requirements, 1–8Genetic Science, xxxi–xxxiiGeraniol, 564–565
Infrared Spectrum, 718Geranium Oil, Algerian Type, 192
Infrared Spectrum, 718Geranium Oil, East Indian Type, 315Geranium Oil, Turkish Type, 315Geranyl Acetate, 564–565
Infrared Spectrum, 719Geranyl Benzoate, 564–565
Infrared Spectrum, 719Geranyl Butyrate, 564–565
Infrared Spectrum, 719Geranyl Formate, 566–567
Infrared Spectrum, 720* Geranyl Isovalerate, 566–567
Infrared Spectrum, 720
Geranyl Phenylacetate, 566–567Infrared Spectrum 720
Geranyl Propionate, 566–567Infrared Spectrum, 721
Gibberellic Acid, 193Ginger Oil, 193–194
Infrared Spectrum, 721Ginger Oleoresin, 447, 448Glacial Acetic Acid, 10Glacial Acetic Acid (reagent), 5, 963�-Glucanase, 150, 897�-Glucanase Activity, 906–907D-Glucitol, 443–444Glucoamylase, 151, 897Glucoamylase Activity, 907–908D-Gluconic Acid, Monopotassium Salt, 361Glucono Delta-Lactone, 194�-D-Glucopyranosyl-1,4-D-glucitol, 270–271Glucose, 135–136D-Glucose, 135–136Glucose Isomerase (Actinoplanes
missouriensis, Bacillus coagulans,Streptomyces olivaceus, Streptomycesolivochromogenes, Microbacteruimarborescens, Streptomyces rubiginosusvar., or Streptomyces murinus), 149, 151,897
Glucose Isomerase Activity, 908–909Glucose Oxidase, 151, 897Glucose Oxidase Activity, 909Glucose Oxidase (Aspergillus niger var.), 149Glucose Syrup, 194–195Glucose Syrup, Dried, 195Glucose Syrup Solids, 195, 957–958�-D-Glucosidase, 151, 897Gluside, 388–389Glutamic Acid, 196Glutamic Acid Determination, 886–887L-Glutamic Acid, 196
Infrared Spectrum, 721L-Glutamic Acid Hydrochloride, 196–197
Infrared Spectrum, 722L-Glutamine, 197
Infrared Spectrum, 722Glutaral, 197–198Glutaraldehyde, 197–198Gluten, Wheat, 200Glycerin, 198–199Glycerol, 198–199Glycerol, Acetic and Fatty Acid Esters of, 12Glycerol Ester of Gum Rosin, 199
Infrared Spectrum, 722Glycerol Ester of Partially Dimerized
Rosin, 200Infrared Spectrum, 723
Glycerol Ester of Partially HydrogenatedGum Rosin, 200
Infrared Spectrum, 723Glycerol Ester of Partially Hydrogenated
Wood Rosin, 200–201Infrared Spectrum, 723
Glycerol Ester of Polymerized Rosin, 201Infrared Spectrum, 724
Glycerol Ester of Tall Oil Rosin, 201Infrared Spectrum, 724
Glycerol Ester of Wood Rosin, 201–202Infrared Spectrum, 724
Glycerol Esters of Condensed Castor Oil FattyAcids, 343–344
Glyceryl Behenate, 202–203Glyceryl-Lacto Esters of Fatty Acids, 203–
204Glyceryl Monooleate, 204–205Glyceryl Monostearate, 205–207Glyceryl Palminostearate, 207–208Glyceryl Triacetate, 487–488Glyceryl Tribehenate, 202–203Glyceryl Tributyrate, 624–625
Glyceryl Tridocosanoate, 202–203Glyceryl Tripropanoate, 566–567Glyceryl Tristearate, 208Glycine, 208–209
Infrared Spectrum, 725Glycocoll, 208–209Glycyrrhizin, ammoniated, 25Good Manufacturing Practices Guidelines for
Food Chemicals, xx, xxix–xxxii, 3Graham’s Salt, 429–430Granular Metal Powders, Sieve Analysis of,
858Granulated Sugar, 455–456Grapefruit Oil, Coldpressed, 209
Infrared Spectrum, 725Grapefruit Oil, Expressed, 209Grape Skin Extract, 209–210Guaiac Resin, 212Guar Gum, 210Gum Arabic, 210–211Gum Ghatti, 211–212Gum Guaiac, 212Gum Tragacanth, 486Gutta hang kang, 280Gutta Katiau, 280
H
Hazard Analysis and Critical Control Points,xxix, 3
Heat, Excessive, 8Heavy Metals Limit Policy, xiv, 3Heliotropine, 616–617Helium, 212–213Hemicellulase, 151, 897Hemicellulase Activity, 909–911(E),(E)-2,4-Heptadienal, 566–567
Infrared Spectrum, 725trans, trans-2,4-Heptadienal, 566–567�-Heptalactone, 566–567
Infrared Spectrum, 726Heptaldehyde, 568–569Heptanal, 568–569
Infrared Spectrum, 726* 2,3-Heptandione, 568–569
Infrared Spectrum, 7262-Heptanone, 568–569
Infrared Spectrum, 7273-Heptanone, 568–569
Infrared Spectrum, 727cis-4-Hepten-1-al, 568–569(Z)-4-Hepten-1-al, 568–569
Infrared Spectrum, 727Heptyl Alcohol, 568–569
Infrared Spectrum, 728n-Heptyl-p-hydroxybenzoate, 213Heptylparaben, 213Hexadecanoic Acid, 315–3162,4-Hexadienoic Acid, 441–4422,4-Hexadienoic Acid, Calcium Salt, 822,4-Hexadienoic Acid, Potassium Salt, 371cis-Hexahydro-2-oxo-1H-thieno[3,4]imidazole-
4-valeric Acid, 49–50Hexahydropyridine, 616–617�-Hexalactone, 568–569
Infrared Spectrum, 728Hexaldehyde, 568–569Hexanal, 568–569Hexanedioic Acid, 171,2,3,4,5,6-Hexanehexol, 278, 443–444Hexane-Insoluble Matter, 936Hexanes, 213–214
Determination of Benzenes in, 878–879Hexanoic Acid, 568–569
Infrared Spectrum, 7281-Hexanol, 572–573
FCC V Index / Iron Standard Solution / 989
(E)-2-Hexen-1-al, 570–571Infrared Spectrum, 729
trans-2-Hexen-1-al, 570–571(E)-2-Hexen-1-ol, 570–571trans-2-Hexen-1-ol, 570–571cis-3-Hexen-1-ol, 570–571(Z)-3-Hexenol, 570–571
Infrared Spectrum, 729(E)-2-Hexenyl Acetate, 570–571
Infrared Spectrum, 729trans-2-Hexen-1-yl Acetate, 570–571cis-3-Hexen-1-yl Acetate, 570–571(Z)-3-Hexenyl Acetate, 570–571
Infrared Spectrum, 730* (Z)-3-Hexenyl Butyrate, 570–571
Infrared Spectrum, 730* (Z)-3-Hexenyl Formate, 570–571
Infrared Spectrum, 730cis-3-Hexen-1-yl Isovalerate, 570–571(Z)-3-Hexenyl Isovalerate, 570–571cis-3-Hexenyl 2-Methylbutyrate, 572–573(Z)-3-Hexenyl 2-Methylbutyrate, 572–573Hexyl Acetate, 572–573
Infrared Spectrum, 731Hexyl Alcohol, 572–573
Infrared Spectrum, 7314-Hexyl-1,3-benzenediol, 214–215Hexyl-2-Butenoate, 572–573
Infrared Spectrum, 731* Hexyl Butyrate, 572–573
Infrared Spectrum, 731�-Hexylcinnamaldehyde, 572–573
Infrared Spectrum, 732* Hexyl Hexanoate, 572–573
Infrared Spectrum, 732Hexyl Isovalerate, 572–573Hexyl 2-Methylbutyrate, 572–573Hexylresorcinol, 214–2154-Hexylresorcinol, 214–215
Infrared Spectrum, 733High-Fructose Corn Syrup, 215–217High-Fructose Corn Syrup Solids, 958High-Performance Liquid Chromatography,
838–841L-Histidine, 217
Infrared Spectrum, 733L-Histidine Monohydrochloride, 217–218
Infrared Spectrum, 733HMO, 280–284Hop Oleoresin, 447, 448Hops Oil, 218
Infrared Spectrum, 734Hydratropic Aldehyde, 614–615Hydratropic Aldehyde Dimethyl Acetal, 614–
615Hydrocarbons in Eugenol, Limit Test for, 633Hydrocarbon Solvents, Determination of
Benzenes in, 878–879Hydrochloric Acid, 218–221Hydrochloric Acid Buffer, 962Hydrochloric Acid (reagent), 5, 966Hydrochloric Acid Table, 854–855Hydrochloric Acid TS, Diluted, 966Hydrochloric Acid 0.2 M, 962Hydrochloric Acid, 1 N, 971Hydrocinnamaldehyde, 614–615Hydrocinnamyl Alcohol, 614–615Hydrocyanic Acid in Benzaldehyde, Limit
Test for, 633Hydrofluoric Acid (reagent), 5Hydrogenated Glucose Syrup, 271–272Hydrogenated Maltose, 270–271Hydrogenated Menhaden Oil, 280–284
* Hydrogenated Starch Hydrolysate, 221–222Hydrogen Peroxide, 222–224Hydrogen Peroxide TS, 966Hydrogen Sulfide Detector Tube, 977Hydrogen Sulfide TS, 966
�-Hydro-omega-hydroxy-poly(oxyethylene)-poly(oxypropylene)(51-57 moles)poly-(oxyethylene) Block Copolymer, 334–335
�-Hydro-omega-hydroxy-poly(oxyethylene)-poly(oxypropylene)(63-71 moles)poly-(oxyethylene) Block Copolymer, 335–336
Hydrolyzable Gallotannin, 466–467Hydrolyzed Plant Protein (HPP), 13–15Hydrolyzed (Source) Protein Extract, 13–15Hydrolyzed Vegetable Protein (HVP), 13–15Hydroquinone Monomethyl Ether Testing,
632–633Hydroquinone Testing, 632Hydroxyanisole, Butylated, 48–492-Hydroxybutanedioic Acid, 266–2673-Hydroxy-2-butanone, 518–519Hydroxycitronellal, 574–575
Infrared Spectrum, 734Hydroxycitronellal Dimethyl Acetal, 574–
575Infrared Spectrum, 734
4-Hydroxydecanoic Acid Lactone, 542–5434-Hydroxy-2,5-dimethyl-3(2H)-furanone,
574–575Infrared Spectrum, 735
7-Hydroxy-3,7-dimethyl Octanal, 574–5757-Hydroxy-3,7-dimethyl Octanal: Acetal, 574–
5756-Hydroxy-3,7-dimethyloctanoic Acid
Lactone, 574–5754-Hydroxydodecanoic Acid Lactone, 550–551(2-Hydroxyethyl)trimethyl-ammonium
Chloride, 113(2-Hydroxyethyl)trimethyl-ammonium-l-(+)-
tartrate salt, 112–1134-Hydroxyhexanoic Acid Lactone, 568–569Hydroxylamine Hydrochloride TS, 966Hydroxylamine Hydrochloride, 0.5 N, 971Hydroxylamine Method, 630–631, 929–930Hydroxylamine/Tert-Butyl Alcohol Method,
630, 930Hydroxylated Lecithin, 224–225Hydroxyl Value, 936–9374-Hydroxy-3-methoxybenzaldehyde, 628–6294-Hydroxy-3-methylethylbenzene, 554–555Hydroxymethylphenol, Butylated, 585-Hydroxy-6-methyl-3,4-pyridinedimethanol
Hydrochloride, 379–3803-Hydroxy-2-methyl-4-pyrone, 273Hydroxy Naphthol Blue, 9755-Hydroxynonanoic Acid Lactone, 604–6055-Hydroxyoctanoic Acid Lactone, 606–607L-�-(p-Hydroxyphenyl)alanine, 490–4914-(p-Hydroxyphenyl)-2-butanone, 574–575
Infrared Spectrum, 7352-Hydroxypropanoic Acid, Calcium Salt, 692-Hydroxypropanoic Acid, Monopotassium
Salt, 364–3662-Hydroxypropanoic Acid, Monosodium Salt,
418–4192-Hydroxypropionic Acid, 239–240�-Hydroxypropionic Acid, 239–240Hydroxypropoxyl Determination, 887–888Hydroxypropyl Alginate, 376–377Hydroxypropyl Cellulose, 225Hydroxypropyl Distarch Phosphate, 182Hydroxypropyl Methylcellulose, 225–227Hydroxypropyl Starch, 1838-Hydroxyquinoline TS, 966Hydroxysuccinic Acid, 266–267Hydroxytoluene, Butylated, 495-Hydroxyundecanoic Acid Lactone, 626–627Hypophosphite Identification Test, 859
I
Identification Tests, 4
Acetate, 859Aluminum, 859Ammonium, 859Benzoate, 859Bicarbonate, 859Bisulfite, 859Bromide, 859Calcium, 859Carbonate, 859Chloride, 859Citrate, 859Cobalt, 859Copper, 859Hypophosphite, 859Iodide, 859–860Iron, 860Lactate, 860Magnesium, 860Manganese, 860Nitrate, 860Nitrite, 860Peroxide, 860Phosphate, 860Potassium, 860Sodium, 860Sulfate, 860Sulfite, 860Tartrate, 860Thiosulfate, 860–861Zinc, 861
‘‘Ignite to Constant Weight,’’ Defined, 6IMP, 424Indian Gum, 211–212Indicators
Papers and Test Papers, 975–976Quantity Used, 5
Indigo Carmine, 162–163, 227–228Indigo Carmine TS, 966
* Indigotine, 162–163, 227–228Indigotine Disulfonate, 227–228Indole, 574–575
Infrared Spectrum, 735Infrared Spectra, 637Inositol, 228–229i-Inositol, 228–229meso-Inositol, 228–229myo-Inositol, 228–229Insoluble Sodium Polyphosphate, 424International Harmonization of Specifications,
xvInternational System of Units, 6–7Invertase, 151, 897Invertase Activity, 911Invert Sugar, 229Invert Sugar Assays, 455–456, 951–952, 960Invert Sugar Syrup, 229Iodide Identification Test, 859–860Iodine TS, 966Iodine Value, 937–938Iodine, 0.1 N, 972�-Ionone, 574–575
Infrared Spectrum, 736�-Ionone, 576–577
Infrared Spectrum, 736Ion-Selective Electrode Test, 865–866Iron Ammonium Citrate, 167–168, 169Iron, Carbonyl, 229–230Iron, Electrolytic, 230–231Iron, Reduced, 231–232Iron (II) Fumarate, 173–174Iron (II) Gluconate, 174–175Iron (II) 2-Hydroxypropionate, 177–178Iron Identification Test, 860Iron (II) Lactate, 177–178Iron Phosphate, 169–172Iron Pyrophosphate, 172Iron Standard Solution, 963
990 / Isoamyl Acetate / Index FCC V
Isoamyl Acetate, 576–577Infrared Spectrum, 736
Isoamyl Alcohol, 576–577Isoamyl Benzoate, 576–577
Infrared Spectrum, 737Isoamyl Butyrate, 576–577
Infrared Spectrum, 737Isoamyl Caproate, 576–577Isoamyl Caprylate, 524–525Isoamyl Cinnamate, 522–523Isoamyl Formate, 576–577
Infrared Spectrum, 737Isoamyl Hexanoate, 576–577
Infrared Spectrum, 738* Isoamyl Isobutyrate, 578–579
Infrared Spectrum, 738Isoamyl Isovalerate, 578–579Isoamyl Octanoate, 524–525Isoamyl Phenyl Acetate, 578–579
Infrared Spectrum, 738Isoamyl 3-Phenyl Propenate, 522–523Isoamyl Propionate, 524–525Isoamyl Salicylate, 578–579
Infrared Spectrum, 739Isoborneol, 578–579
Infrared Spectrum, 739Isobornyl Acetate, 578–579
Infrared Spectrum, 739Isobutane, 232–233Isobutyl Acetate, 578–579
Infrared Spectrum, 740Isobutyl Alcohol, 578–579
Infrared Spectrum, 740Isobutyl-2-butenoate, 580–581
Infrared Spectrum, 740Isobutyl Butyrate, 580–581
Infrared Spectrum, 741Isobutyl Cinnamate, 580–581
Infrared Spectrum, 741Isobutylene-Isoprene Copolymer, 233–234
Infrared Spectrum, 741* Isobutyl Formate, 580–581
Infrared Spectrum, 742* Isobutyl Hexanoate, 580–581
Infrared Spectrum, 742Isobutyl Isovalerate, 598–599Isobutyl Phenylacetate, 580–581
Infrared Spectrum, 742Isobutyl Salicylate, 580–581
Infrared Spectrum, 743Isobutyraldehyde, 580–581
Infrared Spectrum, 743Isobutyric Acid, 580–581
Infrared Spectrum, 743Isodihydrolavandulal, 596–597Isoestragole, 524–525Isoeugenol, 582–583
Infrared Spectrum, 744Isoeugenyl Acetate, 582–583
Infrared Spectrum, 744Isoeugenyl Methyl Ether, 596–597DL-Isoleucine, 234
Infrared Spectrum, 744L-Isoleucine, 234–235
Infrared Spectrum, 745Isopropanol, 235, 966Isopropanol, Anhydrous, 235Isopropyl Acetate, 582–583
Infrared Spectrum, 640Isopropylacetic Acid, 582–583Isopropyl Alcohol, 235p-Isopropylbenzaldehyde, 540–541Isopropylformic Acid, 580–581Isopulegol, 582–583
Infrared Spectrum, 745Isovaleraldehyde, 590–591Isovaleric Acid, 582–583
Infrared Spectrum, 746
J
Jelutong, 280Juniper Berries Oil, 235–236
Infrared Spectrum, 746
K
Kaolin, 236Karaya Gum, 237Karite, 397Karl Fischer Titrimetric Method for Water, 5,
851–852KASAL, 403–404Kelp, 237–238Ketones, Assays for, 631, 929–930Kjeldahl Method for Nitrogen Determination,
888–889Konjac, 238–239Konjac Flour, 238–239Konjac Gum, 238–239Konnyaku, 238–239
L
Labdanum Oil, 239Infrared Spectrum, 746
Labeling Policy, 2–3Lactase, 151, 897Lactase Activity
Acid �-Galactosidase, 911–913Neutral �-Galactosidase, 913–914
Lactated Mono-Diglycerides, 203–204Lactate Identification Test, 860Lactic Acid, 239–240Lactic Acid, 530–531Lactic and Fatty Acid Esters of Glycerol,
203–204Lactose, 241Lactose Determination, 952–953Lactylated Fatty Acid Esters of Glycerol
and Propylene Glycol, 241–243Lactylic Esters of Fatty Acids, 243–244Lanolin, Anhydrous, 245Larch Fiber, 34Larch Gum, 34Lard (Unhydrogenated), 245–246Laurel Leaf Oil, 246
Infrared Spectrum, 747Laurel Leaf Oleoresin, 447, 448Lauric Acid, 246–247Lauryl Alcohol, 582–583
Infrared Spectrum, 747Lauryl Aldehyde, 582–583
Infrared Spectrum, 747Lavandin Oil, Abrial Type, 247
Infrared Spectrum, 748Lavender Oil, 247–248
Infrared Spectrum, 748Lavender Oil, Spike, 448–449Lead Acetate Test Paper, 976Lead Acetate TS, 966Lead Limits
Chewing Gum Base, 895Policy, xiv, xv, 3Test, 633, 867–872
Lead Subacetate TS, 967Lead Subacetate TS, Diluted, 967LEAR Oil, 86–88Leche caspi (sorva), 280Leche de vaca, 280Lecithin, 248–249Lecithin, Hydroxylated, 224–225Legal Status of FCC, xviLemongrass Oil, 249–250
Infrared Spectrum, 748Lemon Oil Arizona, 251
Lemon Oil, Coldpressed, 250–251Infrared Spectra, 749
Lemon Oil, Desert Type, Coldpressed, 251Infrared Spectrum, 749
Lemon Oil, Distilled, 251–252Infrared Spectrum, 749
Lemon Oil, Expressed, 250–251DL-Leucine, 252
Infrared Spectrum, 750L-Leucine, 252–253
Infrared Spectrum, 750Leucine Aminopeptidase, 148, 150, 899–900Leuco Base, Color Additive Assay, 881Levocarnitine, 100–101Levulinic Acid, 582–583
Infrared Spectrum, 750Levulose, 185–186Light-Resistant Container, 8Lime, 72Lime Oil, Coldpressed, 253
Infrared Spectrum, 751Lime Oil, Distilled, 253–254
Infrared Spectrum, 751Lime Oil, Expressed, 253Lime, Slaked, 67–68Limestone, Ground, 254–255Limit of Detection, xxviLimit of Quantitation, xxviiLimit Tests
Antioxidants in Ethyl Acrylate, 632–633Arsenic, 861–862Cadmium, 863Carbonizable Substances in Ethyl Acetate,
633Chloride and Sulfate, 8631,4-Dioxane, 863–864Fluoride, 864–867Hydrocarbons in Eugenol, 633Hydrocyanic Acid in Benzaldehyde, 633Lead, 633, 867–872Manganese, 872Mercury, 872–873Methyl Compounds in Ethyl Acetate, 633Nickel, 874Oxidizable Substances in dl-Menthol, 633–
634Peroxide Value, 633Phosphorus, 874–875Reducing Substances, 634Selenium, 875–876
d-Limonene, 584–585Infrared Spectrum, 752
l-Limonene, 584–585Linaloe Wood Oil, 255
Infrared Spectrum, 752Linalool, 584–585
Infrared Spectrum, 752Linalool Determination, 931
* Linalool Oxide, 584–585Infrared Spectrum, 753
Linalyl Acetate, 584–585Infrared Spectrum, 753
Linalyl Benzoate, 585–585Infrared Spectrum, 753
Linalyl Formate, 586–587Infrared Spectrum, 754
Linalyl Isobutyrate, 586–587Infrared Spectrum, 754
Linalyl Propionate, 586–587Infrared Spectrum, 754
Linearity of Analytical Methods, xxviiLinoleic Acid, 255–256Linseed Oil, Low Linolenic Acid, 441Lipase, 151Lipase Activity, 914
for Medium- and Long-Chain Fatty Acids(Microbial), 914–915
Lipase, Animal, 147
FCC V Index / Methyl Hexanoate / 991
Lipase (Aspergillus niger var.), 149Lipase (Aspergillus oryzae var.), 149Lipase [(Candida rugosa) (formerly Candida
cylindracea)], 150Lipase (Rhizomucor (Mucor) miehei), 150Liquid Paraffin, 291–292Liquid Petrolatum, 291–292Lithium Methoxide, 0.1 N, 972Litmus, 975Litmus Paper, Blue, 976–977Litmus Paper, Red, 977Litmus TS, 967Locust Bean Gum, 256Locust (Carob) Bean Gum, 256Loss on Drying, 4, 6, 855Lovage Oil, 256–257
Infrared Spectrum, 755Low Erucic Acid Rapeseed Oil, 86–88Low Linolenic Acid Flaxseed Oil
(Unhydrogenated), 441Low Linolenic Acid Linseed Oil, 441Lye, 416Lye Solutions, 416–417L-Lysine Monohydrochloride, 257
Infrared Spectrum, 755Lysozyme, 147, 151Lysozyme Activity, 915–916
M
Mace Oil, 257–258Infrared Spectrum, 755
Maddrell’s Salt, 424Magadi Soda, 434Magnesia Mixture TS, 967Magnesium Carbonate, 258–259Magnesium Chloride, 259Magnesium Gluconate, 259–260Magnesium Hydroxide, 260Magnesium Identification Test, 860Magnesium Oxide, 261Magnesium Phosphate, Dibasic, Mixed
Hydrates, 261–262Magnesium Phosphate, Dibasic, Trihydrate,
262–263Magnesium Phosphate, Tribasic, 263Magnesium Silicate, 263–265Magnesium Standard Solution, 963Magnesium Stearate, 265Magnesium Sulfate, 266Magnesium Sulfate TS, 967Malachite Green TS, 967Malic Acid, 266–267DL-Malic Acid, 266–267Malonic Ester, 544–545Malt, 147Malt Extract, 267–270Maltitol, 270–271D-Maltitol, 270–271Maltitol Syrup, 271–272Maltodextrin, 272–273Maltodextrin Solids, 959Maltogenic Amylase, 151, 897Maltogenic Amylase Activity, 916–917Maltol, 273
* Maltol Isobutyrate, 586–587Infrared Spectrum, 756
Malt Syrup, 267–270Mandarin Oil, Coldpressed, 273–274
Infrared Spectrum, 756Mandarin Oil, Expressed, 273–274Manganese Chloride, 274–275Manganese Citrate, 275Manganese Gluconate, 275–276Manganese Glycerophosphate, 276Manganese Hypophosphite, 277Manganese Identification Test, 860
Manganese Limit Test, 872Manganese Sulfate, 277Mannite, 278Mannitol, 278D-Mannitol, 278Maple Furanone, 556–557Marjoram Oil, Spanish Type, 279
Infrared Spectrum, 756Marjoram Oil, Sweet, 279
Infrared Spectrum, 757Marjoram Oleoresin, Sweet, 447, 448Massaranduba balata, 280Massaranduba chocolate, 280Masticatory Substances, Natural, 280Mayer’s Reagent, 967Maximum Assay Tolerances, 6Melting Range
Fats and Related Substances, 938Tests, 842–843
Menhaden Oil, Hydrogenated, 280–284Menhaden Oil, Refined, 284–285Mentha Arvensis Oil, Partially
Dementholized, 285Infrared Spectrum, 757
p-Mentha-1,5-diene, 610–611d-p-Mentha-1,8-diene, 584–585l-p-Mentha-1,8-diene, 584–585p-Mentha-6,8-dien-2-ol, 534–535p-Mentha-6,8-dien-2-yl Acetate, 536–5373-p-Menthanol, 586–587l-p-Menthan-3-one, 586–587dl-p-Menthan-3-yl Acetate, 588–589l-p-Menthan-3-yl Acetate, 588–589p-Menth-1-en-8-ol, 620–621p-Menth-4-en-3-ol, 582–583Menthen-1-yl-8 Acetate, 622–623Menthen-1-yl-8 Propionate, 622–623Menthol, 586–587
Infrared Spectrum, 757dl-Menthol, Limit Test for Oxidizable
Solutions in, 633–634Menthone, 586–587dl-Menthyl Acetate, 588–589l-Menthyl Acetate, 588–5892-Mercaptopropionic Acid, 588–589
Infrared Spectrum, 758Mercuric Acetate TS, 967Mercuric Chloride TS, 967Mercuric Nitrate, 0.1 M, 972Mercuric-Potassium Iodide TS, 967Mercuric-Potassium Iodide TS, Alkaline, 967Mercuric Sulfate TS, 967Mercurous Nitrate TS, 967Mercury
Color Additive Assays, 881–882Limit Test, 872–873
Metal Powders, Sieve Analysis of, 858Methanol, 286–287Methanol, Anhydrous, 967Methional, 600–601DL-Methionine, 285–286
Infrared Spectrum, 758L-Methionine, 286
Infrared Spectrum, 758p-Methoxyacetophenone, 518–519p-Methoxybenzaldehyde, 588–589
Infrared Spectrum, 759p-Methoxybenzyl Acetate, 524–525p-Methoxybenzyl Alcohol, 524–525p-Methoxybenzyl Formate, 524–525
* 2-Methoxy 3-(or 5- or 6-) IsopropylPyrazine, 588–589
Infrared Spectrum, 759Methoxyl Determination, 887–8882-Methoxy-3(5)-methylpyrazine, 588–589
Infrared Spectrum, 7594-p-Methoxyphenyl-2-butanone, 588–589
Infrared Spectrum, 760
1-(p-Methoxyphenyl)-1-penten-3-one, 550–5512-Methoxy-4-propenylphenol, 582–5832-Methoxy-4-propenyl Phenyl Acetate, 582–
5832-Methoxypyrazine, 590–591
Infrared Spectrum, 760Methyl Acetate, 590–591
Infrared Spectrum, 7604′-Methyl Acetophenone, 590–591
Infrared Spectrum, 761Methylacetopyronone, 132Methyl Alcohol, 286–287p-Methylaminophenol Sulfate TS, 967Methyl Amyl Ketone, 568–569p-Methyl Anisole, 590–591
Infrared Spectrum, 761Methyl Anthranilate, 590–591
Infrared Spectrum, 761Methylbenzaldehyde, 624p-Methylbenzaldehyde, 624Methyl Benzoate, 590–591
Infrared Spectrum, 762�-Methylbenzyl Acetate, 590–591
Infrared Spectrum, 762�-Methylbenzyl Alcohol, 590–591
Infrared Spectrum, 7622-Methyl Butanal, 590–591
Infrared Spectrum, 7633-Methyl Butanal, 592–593�-Methyl Butyl Acetate, 576–5772-Methylbutyl Acetate, 592–5932-Methylbutyl Isovalerate, 592–5932-Methylbutyl-3-methylbutanoate, 592–593Methyl Butyrate, 592–5932-Methylbutyric Acid, 592–593
Infrared Spectrum, 763Methylcellulose, 287–288
Viscosity of, 849–850Methyl Chavicol, 550–551�-Methylcinnamaldehyde, 592–593
Infrared Spectrum, 763Methyl Cinnamate, 592–593
Infrared Spectrum, 764Methyl Compounds in Ethyl Acetate, Limit
Test for, 6336-Methylcoumarin, 592–593
Infrared Spectrum, 764Methyl p-Cresol, 590–5913-Methylcyclopentane-1,2-dione, 592–593Methyl Cyclopentenolone, 592–593
Infrared Spectrum, 764* 5H-5-Methyl-6,7-dihydrocyclopenta[6]-
pyrazine, 594–595Methylene Blue, 975Methylene Blue TS, 967Methylene Chloride, 288–289Methylene Dichloride, 288-2893,4-(Methylenedioxy)benzaldehyde, 616–617Methyl Ester of Rosin, Partially
Hydrogenated, 289–290Infrared Spectrum, 765
Methyl Ethyl Cellulose, 290Methyl Ethyl Ketone, 530–531Methyl Eugenol, 594–595
Infrared Spectrum, 765* 5-Methyl Furfural, 594–595
Infrared Spectrum, 765* Methyl Furoate, 594–595
Infrared Spectrum, 766Methyl Glycol, 376Methyl Heptenone, 594–5956-Methyl-5-hepten-2-one, 594–595
Infrared Spectrum, 766Methyl Heptine Carbonate, 596–597Methyl Heptyl Ketone, 604–605
* Methyl Hexanoate, 594–595Infrared Spectrum, 766
992 / Methyl Hexyl Ketone / Index FCC V
Methyl Hexyl Ketone, 594–595Infrared Spectrum, 767
Methyl p-Hydroxybenzoate, 290–291Methyl Ionones, 594–595Methyl Isobutyl Ketone, 598–599Methyl Isobutyrate, 596–597
Infrared Spectrum, 767Methyl Isoeugenol, 596–597
Infrared Spectrum, 767d-1-Methyl-4-isopropenyl-6-cyclohexen-2-one,
534–535l-1-Methyl-4-isopropenyl-6-cyclohexen-2-one,
534–5355-Methyl-2-isopropyl-2-hexenal, 596–597
Infrared Spectrum, 7682-Methyl-3-(p-isopropylphenyl)-
propionaldehyde, 540–541* Methyl Isovalerate, 596–597
Infrared Spectrum, 768Methyl N-Methyl Anthranilate, 546–547Methyl 2-Methyl-butanoate, 596–597Methyl 2-Methylbutyrate, 596–5977-Methyl-3-methylene-1,6-octadiene, 600–
601d-2-Methyl-5-(1-methylethenyl)-
cyclohexanone, 546–5471-Methyl-4-(1-methylethyl)-1,3-
cyclohexadiene, 620–6211-Methyl-4-(1-methylethyl)-1,4-
cyclohexadiene, 620–621Methyl-3-methylthiopropionate, 596–597
Infrared Spectrum, 768Methyl �-Naphthyl Ketone, 596–597
Infrared Spectrum, 769Methyl n-Nonyl Acetaldehyde, 600–601Methyl Nonyl Ketone, 626–627Methyl 2-Octynoate, 596–597
Infrared Spectrum, 769Methyl Orange, 975Methyl Orange TS, 9676-Methyl-1,2,3-oxathiazine-4(3H)-one-2,2
Dioxide Potassium Salt, 9Methylparaben, 290–2912-Methylpentanoic Acid, 596–597
Infrared Spectrum, 7694-Methylpentanoic Acid, 598–599
Infrared Spectrum, 7704-Methyl-2-pentanone, 598–5992-Methyl-2-pentenoic Acid, 598–599
Infrared Spectrum, 770�-Methyl Phenylacetaldehyde, 614–615Methyl Phenylacetate, 598–599
Infrared Spectrum, 770p-Methylphenyl Acetate, 540–541Methyl Phenylcarbinol, 590–591Methyl Phenylcarbinyl Acetate, 598–599
Infrared Spectrum, 771Methylphenyl Ether, 524–525
* 5-Methyl 2-Phenyl 2-Hexenal, 598–599Infrared Spectrum, 771
Methyl Phenyl Ketone, 518–5192-Methyl Propanoic Acid, 580–5812-Methyl Propanyl Butyrate, 580–581Methyl Propyl Ketone, 610–611Methyl Propyl 3-Methyl Butyrate, 598–5992-Methylpyrazine, 598–599
Infrared Spectrum, 771Methyl Pyrazinyl Ketone, 520–521Methyl Pyridyl Ketone, 520–521Methyl 2-Pyrrolyl Ketone, 520–521Methyl Red, 975–976Methyl Red–Methylene Blue TS, 967Methyl Red Sodium, 976Methyl Red TS, 967Methylrosaniline Chloride TS, 967Methyl Salicylate, 600–601
Infrared Spectrum, 772Methyl Sulfide, 548–549
2-Methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl), 499–500
4-Methyl-5-thiazole Ethanol, 600–601Infrared Spectrum, 772
* Methyl Thiobutyrate, 600–601Infrared Spectrum, 772
3-Methylthiopropionaldehyde, 600–601Infrared Spectrum, 773
Methyl p-Tolyl Ketone, 590–5912-Methylundecanal, 600–601
Infrared Spectrum, 773* Methyl Valerate, 600–601
Infrared Spectrum, 773Methyl Vanillin, 628–629Methyl Violet TS,Methyl Yellow, 976Mg/Kg and Percent Policy, 4Microbially Derived Enzyme Preparations,
148–150Microbiological Attributes Policy, 3Microcrystalline Cellulose, 106–107Milk-Clotting Activity, 917Millon’s Reagent, 967–968Mineral Oil, White, 291–292Minimum Purity Tolerances, 6Mixed Paraffinic Hydrocarbons, 213–214Mixture of 1,2- and 1,3-Benzaldehyde Cyclic
Acetals of Glycerin, 526–527Mixture of Geranial [(E)-3,7-dimethyl-2,6-
octadien-1-al] and Neral [the (Z) isomer],538–539
‘‘mm Hg,’’ Defined, 5Modified Cellulose, 107–108, 225, 290
EC, 158HPMC, 225–227MC, 287–288
Modified Food Starch, 181–183Modified Schiff’s Reagent, 969Molar Solutions, 970Molecular Weight, 892–893Monoammonium L-Glutamate, 292–293
Infrared Spectrum, 774Monoammonium Glutamate Monohydrate,
292–293Monoammonium Glycyrrhizinate, 293Monoammonium Phosphate, 28Mono- and Diglycerides, 293–294Monobasic Calcium Phosphate, 76–77Monobasic Potassium Phosphate, 368Monobasic Potassium Phosphate, 0.2 M, 962Monobasic Sodium Phosphate, 428Mono-tert-butylhydroquinone, 469–471Monocalcium Phosphate, 76–77Monoglyceride Citrate, 294–295Monoglycerides, Acetylated, 12Monoglycerides, Ethoxylated, 155-1561-Monoglycerides, in Fats and Related
Substances, 938Monoglycerides, Total, in Fats and Related
Substances, 938–939Monographs
Codex Specifications, 1Former and Current Titles, xxxivNew, xxxiii
Monoolein, 204–205Monopotassium D-Gluconate, 361Monopotassium L-Glutamate, 295
Infrared Spectrum, 774Monopotassium Glutamate Monohydrate, 295Monopotassium Phosphate, 368Monosodium Dihydrogen Phosphate, 428Monosodium Glutamate, 295–296Monosodium L-Glutamate, 295–296
Infrared Spectrum, 774Monosodium Glutamate Monohydrate, 295–
296Monosodium Phosphate, 428Monostearin, 205–207
Morpholine, 296Infrared Spectrum, 775
MPG, 295MSG, 295–296Murexide Indicator Preparation, 976Mustard Oil, 296–297Myrcene, 600–601Myrcia Oil, 44Myristaldehyde, 600–601Myristic Acid, 297Myristica Oil, 306–307Myristyl Alcohol, 602–603Myrrh Oil, 297–298
Infrared Spectrum, 775
N
1,4-Naphthalenedione, 499–500�-Naphtholbenzein TS, 968Naphthol Green B, 976Naphthol Green TS, 968
* �-Naphthyl Ethyl Ether, 602–603Infrared Spectrum, 775
Natamycin, 298–299National Institute of Standards and
Technology, 5Natural Rubber (Latex Solids), 280Natural Terpene Resin, 471‘‘Negligible,’’ Defined, 5Nerol, 602–603
Infrared Spectrum, 776Nerolidol, 602–603
Infrared Spectrum, 776Nerolin II, 602–603Nerolin Bromelia, 602–603Neryl Acetate, 602–603
Infrared Spectrum, 776Nessler’s Reagent, 968Neutralized Phthalate Buffer, 962Neutral Red, 976Neutral Red TS, 968Neutral Sulfite Method, 930New Monographs, xxxiiiNiacin, 299Niacinamide, 299–300Niacinamide Ascorbate, 300–301Nickel, 301–302
Limit Test, 874Nickel Catalysts, 301–302Nickel Standard Solution TS, 968Nicotinamide, 299–300Nicotinamide Ascorbate, 300–301Nicotinic Acid, 299Niger Gutta, 280Ninhydrin TS, 968Nisin Preparation, 302–303Nispero, 280Nitrate Identification Test, 860Nitre Cake, 406Nitric Acid (reagent), 5, 968Nitric Acid TS, Diluted, 968Nitric Oxide–Nitrogen Dioxide Detector Tube,
977Nitrite Identification Test, 860Nitrogen, 304Nitrogen Determination, 888–889Nitrogen Enriched Air, 304–305Nitrogen Oxide, 305–306Nitrous Oxide, 305–306(E),(E)-2,4-Nonadienal, 602–603
Infrared Spectrum, 777trans,trans-2,4-Nonadienal, 602–603(E),(Z)-2,6-Nonadienal, 602–603
Infrared Spectrum, 777trans,cis-2,6-Nonadienal, 602–603(E),(Z)-2,6-Nonadienol, 604–605
Infrared Spectrum, 777
FCC V Index / pH Determination / 993
trans,cis-2,6-Nonadienol, 604–605�-Nonalactone, 604–605
Infrared Spectrum, 778�-Nonalactone, 604–605
Infrared Spectrum, 778Nonanal, 604–605
Infrared Spectrum, 778Nonanoic Acid, 604–6051-Nonanol, 606–6072-Nonanone, 604–605
Infrared Spectrum, 779Nonbacterial Alpha–Amylase Activity, 900–
901(E)-2-Nonenal, 604–605
Infrared Spectrum, 779trans-2-Nonenal, 604–605(E)-2-Nonen-1-ol, 604–605
Infrared Spectrum, 79trans-2-Nonenol, 604–605cis-6-Nonen-1-ol, 604–605(Z)-6-Nonen-1-ol, 604–605
Infrared Spectrum, 780Nonyl Acetate, 606–607
Infrared Spectrum, 780Nonyl Alcohol, 606–607
Infrared Spectrum, 780Normal Solutions, 970Nutmeg Oil, 306–307
Infrared Spectrum, 781Nutrients, 8
O
(Z),(Z)-9,12-Octadecadienoic Acid, 255–256Octadecanoic Acid, 208, 450(Z)-9-Octadecenoic Acid, 307–308�-Octalactone, 606–607
Infrared Spectrum, 781�-Octalactone, 606–607
Infrared Spectrum, 781Octanal, 606–607
Infrared Spectrum, 782Octanoic Acid, 3071-Octanol, 608–6093-Octanol, 606–6072-Octanone, 594–595(E)-2-Octen-1-al, 606–607
Infrared Spectrum, 782trans-2-Octen-1-al, 606–6071-Octen-3-ol, 606–607
Infrared Spectrum, 782cis-3-Octen-1-ol, 606–607(Z)-3-Octen-1-ol, 606–607
Infrared Spectrum, 7831-Octen-3-yl Acetate, 608–609
Infrared Spectrum, 7831-Octen-3-yl Butyrate, 608–609
Infrared Spectrum, 783Octyl Acetate, 608–609
Infrared Spectrum, 7843-Octyl Acetate, 608–609Octyl Alcohol, 608–609
Infrared Spectrum, 784Octyl Formate, 608–609
Infrared Spectrum, 784Octyl Isobutyrate, 608–609Octyl 2-Methylpropanoate, 608–609‘‘Odorless,’’ Defined, 5Oil Content of Synthetic Paraffin, 855–857Oil of Frankincense, 310Oil of Shaddock, 209Oleic Acid, 307–308Oleoresin Angelica Seed, 446, 447Oleoresin Anise, 446, 477Oleoresin Basil, 446, 477Oleoresin Black Pepper, 446, 447Oleoresin Capsicum, 446, 447
Oleoresin Caraway, 446, 447Oleoresin Cardamom, 446, 477Oleoresin Celery, 447Oleoresin Coriander, 447, 448Oleoresin Cubeb, 447, 448Oleoresin Cumin, 447, 448Oleoresin Dillseed, 447, 448Oleoresin Fennel, 447, 448Oleoresin Ginger, 447, 448Oleoresin Hop, 447, 448Oleoresin Laurel Leaf, 447, 448Oleoresin Marjoram Sweet, 447, 448Oleoresin Origanum, 447, 448Oleoresin Paprika, 447, 448Oleoresin Parsley Leaf, 447, 448Oleoresin Parsley Seed, 447, 448Oleoresin Pimenta Berries, 447, 448Oleoresin Rosemary, 447, 448Oleoresin Thyme, 447, 448Oleoresin Turmeric, 447, 448Oleoresins
Color Value, 944Curcumin Content, 944Piperine Content, 944–945Residual Skolvent, 945–946Scoville Heat Units, 946Spice, 446–448Volatile Oil Content, 946–947
Olestra, 308–310Olibanum Oil, 310
Infrared Spectrum, 785Onion Oil, 310
Infrared Spectrum, 785Operating Procedures of the Food Chemicals
Codex, xix–xxivOptical (Specific) Rotation, 844Orange Oil, Bitter, Coldpressed, 310–311
Infrared Spectrum, 785Orange Oil, Coldpressed, 311
Infrared Spectrum, 786Orange Oil, Distilled, 311–312
Infrared Spectrum, 786Origanum Oil, Spanish Type, 312
Infrared Spectrum, 786Origanum Oleoresin, 447, 448Orris Root Oil, 312–313
Infrared Spectrum, 787Orthophenanthroline TS, 968Orthophosphoric Acid, 331–332Oxalic Acid TS, 968Oxalic Acid, 0.1 N, 972Ox Bile Extract, 313Oxidized Hydroxypropyl Starch, 182Oxidized Starch, 1821:8-Oxido-p-menthane, 562–563Oxyethylene Determination, 939–940Oxygen Flask Combustion, 831Oxystearin, 314Ozone, 314–315
P
Packaging and Storage, 8Palmarosa Oil, 315
Infrared Spectrum, 787Palmitic Acid, 315–316Palmitoyl L-Ascorbic Acid, 36–37Palm Kernel Oil (Unhydrogenated), 316Palm Oil (Unhydrogenated), 316–317Pancreatin, 147, 151, 897Pancreatin Activity, 917–919Panthenol, 133–134DL-Panthenol, 317D(+)-Pantothenyl Alcohol, 133–134DL-Pantothenyl Alcohol, 317Papain, 147–148, 898Paprika Oleoresin, 447, 448
Paraffin, Liquid, 291Paraffin, Synthetic, 318
Infrared Spectrum, 787Oil Content, 855–857
Parsley Herb Oil, 318–319Infrared Spectrum, 787
Parsley Leaf Oleoresin, 447, 448Parsley Seed Oil, 319
Infrared Spectrum, 788Parsley Seed Oleoresin, 447, 448Partial Acid Digest of (Source) Protein, 319–
321Partial Enzymatic Digest of (Source) Protein,
319–321Partially Dementholized Mentha Arvensis Oil,
285Partially Hydrogenated Methyl Ester of Rosin,
289–290Partially Hydrolyzed Proteins, 319–321Partially Hydrolyzed (Source) Protein, 319–
321Peach Aldehyde, 626–627Peanut Oil (Unhydrogenated), 321Pectinase, 151, 898Pectins, 321–324PEG, 340–342Pelargonic Aldehyde, 604–605Pendare, 280Pennyroyal Oil, 324–325
Infrared Spectrum, 788�-Pentadecalactone, 608–609
Infrared Spectrum, 789Pentaerythritol Ester of Partally
Hydrogenated Wood Rosin, 325Infrared Spectrum, 789
Pentaerythritol Ester of Wood Rosin, 325Infrared Spectrum, 789
1,2,3,4,5-Pentahydroxypentane, 506–5071,5-Pentanedial, 197–1982,3-Pentanedione, 608–609
Infrared Spectrum, 790Pentanoic Acid, 628–6291-Pentanol, 522–5232-Pentanone, 610–611
Infrared Spectrum, 790Pentapotassium Triphosphate, 372–373Pentasodium Triphosphate, 439–4411-Pentyl Butyrate, 522–5231-Pentyl Formate, 522–523Pentyl Heptanoate, 524–525Pentyl Hexanoate, 576–577Peppermint Oil, 326
Infrared Spectrum, 790Pepsin, 147, 151, 898Pepsin Activity, 920Peptone (Source), 319–321Percentage of Cineole, 931Perchloric Acid, 0.1 N, 972Perchloric Acid, 0.1 N, in Dioxane, 972Perillo, 280Perlite, 326–327Peroxide Identification Test, 860Peroxide Value
in Fats and Related Substances, 940Limit Test, 633
Petitgrain Oil, Paraguay Type, 327Infrared Spectrum, 791
Petrolatum, 327–328Petrolatum, Liquid, 291–292Petrolatum, White, 327–328Petrolatum, Yellow, 327–328Petroleum Jelly, 327–328Petroleum Wax, 329
Infrared Spectrum, 791Petroleum Wax, Synthetic, 329–330
Infrared Spectrum, 792pH Determination, 844–845
994 / �-Phellandrene / Index FCC V
�-Phellandrene, 610–611Infrared Spectrum, 792
Phenethyl Acetate, 610–611Infrared Spectrum, 792
2-Phenethyl Acetate, 610–611Phenethyl Alcohol, 610–611
Infrared Spectrum, 7932-Phenethyl Alcohol, 610–611Phenethyl Isobutyrate, 610–611
Infrared Spectrum, 793Phenethyl Isovalerate, 610–611
Infrared Spectrum, 7932-Phenethyl 2-Methylbutyrate, 610–611Phenethyl Phenylacetate, 610–611
Infrared Spectrum, 794Phenethyl Salicylate, 610–611
Infrared Spectrum, 794Phenolphthalein, 976Phenolphthalein Paper, 977Phenolphthalein TS, 968Phenol Red, 976Phenol Red TS, 968Phenols
Essential Oils and Flavors, 931–932Free, 932Qualitative Testing for, 634
Phenolsulfonphthalein TS, 968Phenoxyethyl Isobutyrate, 612–613
Infrared Spectrum, 794Phenylacetaldehyde, 612–613
Infrared Spectrum, 795Phenylacetaldehyde Dimethyl Acetal, 612–
613Infrared Spectrum, 795
Phenylacetic Acid, 612–613Infrared Spectrum, 795
DL-Phenylalanine, 330Infrared Spectrum, 796
L-Phenylalanine, 330–331Infrared Spectrum, 796
Phenyl Carbinol, 526–527�-Phenyl Ethyl Acetate, 598–5992-Phenylethyl Alcohol, 610–611Phenylethyl Anthranilate, 612–613
Infrared Spectrum, 796Phenylethyl Butyrate, 612–613
Infrared Spectrum, 797* Phenyl Ethyl Cinnamate, 612–613
Infrared Spectrum, 797* Phenyl Ethyl Propionate, 614–615
Infrared Spectrum, 797p-Phenylphenol TS, 9683-Phenyl-1-propanol, 614–615
Infrared Spectrum, 7983-Phenylpropenoic Acid, 536–5372-Phenylpropionaldehyde, 614–615
Infrared Spectrum, 7983-Phenylpropionaldehyde, 614–615
Infrared Spectrum, 7982-Phenylpropionaldehyde Dimethyl Acetal,
614–615Infrared Spectrum, 799
3-Phenylpropyl Acetate, 614–615Infrared Spectrum, 799
Phenylpropyl Alcohol, 614–615Phenylpropyl Aldehyde, 614–615PHMO, 280–284Phosphate Buffer,Phosphated Distarch Phosphate, 183Phosphate Identification Test, 860Phosphate Standard Solution, 963Phospholipase A2, 147, 151, 898Phospholipase A2 Activity, 920–921Phosphoric Acid, 331–332Phosphoric Acid (reagent), 5, 968Phosphorus Limit Test, 874–875Phosphotungstic Acid TS, 968Phylloquinone, 499–500
Physical Tests and Determinations, 834–858Physicochemical Properties of Substances,
841–854Phytase, 151, 898Phytase Activity, 921–922Phytase (Aspergillus niger var.), 150, 898Phytonadione, 499–500Picric Acid TS, 968Pimaricin, 298–299Pimenta Berries Oil, 332–333Pimenta Berries Oleoresin, 447, 448Pimenta Leaf Oil, 332
Infrared Spectrum, 799Pimenta Oil, 332–333
Infrared Spectrum, 800Pimento Leaf Oil, 332Pimento Oil, 332–333�-Pinene, 614–615�-Pinene, 616–6172-Pinene, 614–615l-�-Pinene, 614–615Pine Needle Oil, 179–180, 333Pine Needle Oil, Dwarf, 333
Infrared Spectrum, 800Pine Needle Oil, Scotch Type, 333–334
Infrared Spectrum, 800Piperidine, 616–617
Infrared Spectrum, 801Piperine Content, 944–945Piperonal, 616–617
Infrared Spectrum, 801Piperonyl Aldehyde, 616–617Plant Proteolytic Activity, 922–923Platinum–Cobalt CS, 846Policies and Guidelines, 1–4
Added Substances Policy, 2Arsenic Specifications Policy, xiv, xv, 2FCC Substances Containing Sulfiting
Agents, 2Flavor Chemicals Policy, 1–2Fluoride Limits Guidelines, xiv, 2General Policy, xiv–xv, 1–2Labeling Policy, 2–3Heavy Metals Limits Policy, xiv, xv, 3Mg/Kg and Percent Policy, 4Microbiological Attributes Policy, 3
Poloxamer 331, 334–335Poloxamer 407, 335–336Polydextrose, 336–339Polydextrose Solution, 339Polydimethylsiloxane, 141Polyethylene, 339–340
Infrared Spectrum, 801Polyethylene Glycols, 340–342Polyglucitol, 221–222Polyglycerate, 155–156Polyglycerol Esters of Fatty Acids, 343Polyglycerol Esters of Interesterified
Ricinoleic Acid, 343–344Polyglycerol Polyricinoleate, 343–344Polyglycerol Polyricinoleic Acid, 343–344Polyisobutylene, 344–345
Infrared Spectrum, 802Poly[1-(2-oxo-1-pyrrolidinyl)ethylene], 351–
352Poly(oxy-1,2-ethanediyl) Derivative, 346–349Polyoxyethylene (20) Mono- and Diglycerides
of Fatty Acids, 155–156Polyoxyethylene (20) Sorbitan Monolaurate,
346–347Polyoxyethylene (20) Sorbitan Monooleate,
349Polyoxyethylene (20) Sorbitan Monostearate,
347–348Polyoxyethylene (20) Sorbitan Tristearate, 348Polypropylene Glycol, 345–346Polysorbate 20, 346–347Polysorbate 60, 347–348
Polysorbate 65, 348Polysorbate 80, 349Polyvinyl Acetate, 349–350
Infrared Spectrum, 802Polyvinylpolypyrrolidone, 350Polyvinylpyrrolidone, 351–352Pork Collagen, 352–354Potassium Acetate TS, 968Potassium Acid Phthalate, 0.1 N, 972Potassium Acid Tartrate, 354Potassium Alginate, 354–355Potassium Alum, 22Potassium Benzoate, 355Potassium Bicarbonate, 355–356Potassium Biphosphate, 368Potassium Biphthalate, 0.2 M, 962Potassium Bitartrate, 354Potassium Bromate, 356Potassium Carbonate, 356–357Potassium Carbonate Solution, 357Potassium Chloride, 357–359Potassium Chloride, 0.2 M, 962Potassium Chromate TS, 968Potassium Citrate, 359–360Potassium Dichromate TS, 968Potassium Dichromate, 0.1 N, 972Potassium Dihydrogen Phosphate, 368Potassium Ferricyanide TS, 968Potassium Ferrocyanide TS, 968Potassium Gibberellate, 360–361Potassium Gluconate, 361Potassium Glutamate, 295Potassium Glycerophosphate, 361–362Potassium Hydroxide, 362Potassium Hydroxide, 0.5 N, Alcoholic, 973Potassium Hydroxide Solution, 362–363Potassium Hydroxide TS, 968Potassium Hydroxide TS, Alcoholic, 968Potassium Hydroxide, 1 N, 972–973Potassium Identification Test, 860Potassium Iodate, 363Potassium Iodate, 0.05 M, 973Potassium Iodide, 364Potassium Iodide TS, 968Potassium Kurrol’s Salt, 369–370Potassium Lactate Solution, 364–366Potassium Metabisulfite, 366Potassium Metaphosphate, 366Potassium Nitrate, 366–367Potassium Nitrite, 367Potassium Permanganate TS, 968Potassium Permanganate, 0.1 N, 973Potassium Phosphate, Dibasic, 367–368Potassium Phosphate, Monobasic, 368Potassium Phosphate, Monobasic, 0.2 M, 962Potassium Phosphate, Tribasic, 368–369Potassium Polymetaphosphate, 369–370Potassium Polyphosphate, 369–370Potassium Pyroantimonate TS, 968Potassium Pyrophosphate, 370–371Potassium Pyrosulfite, 366Potassium Salt, 371Potassium Sodium Tartrate, 430Potassium Sorbate, 371Potassium Sulfate, 371Potassium Sulfate TS, 968Potassium Sulfite, 371Potassium Triphosphate, 372–373Potassium Tripolyphosphate, 372–373Povidone, 351–352Precipitated Calcium Phosphate, 77Precision of Analytical Methods, xxv–xxviPressure Measurements, 5Product Security, 8L-Proline, 373–374
Infrared Spectrum, 802Propane, 374–3751,2-Propanediol, 376
FCC V Index / Sodium L-Ascorbate / 995
1,2,3-Propanetriol, 204–2051,2,3-Propanetriol Octadecanoate, 205–2071,2,3-Propane Tristearoyl Ester, 2082-Propanol, 235n-Propanol, 616–6172-Propanone, 101-Propene-1,2,3-tricarboxylic Acid, 16p-Propenylanisole, 524–525Propenylguaethol, 616–617
Infrared Spectrum, 8034-Propenyl Veratrole, 596–597Propionaldehyde, 616–617
Infrared Spectrum, 803Propionic Acid, 375–376Proprietary Information, xxiiiPropyl Acetate, 616–617
Infrared Spectrum, 803n-Propyl Acetate, 616–617Propyl Alcohol, 616–617
Infrared Spectrum, 804p-Propyl Anisole, 616–617
Infrared Spectrum, 804Propylene Chlorohydrin Determination, 953–
954Propylene Glycol, 376Propylene Glycol Alginate, 376–377Propylene Glycol Ether of Methylcellulose,
225–227Propylene Glycol Lactostearate, 241–243Propylene Glycol Mono- and Diesters, 377–
378Propylene Glycol Mono- and Diesters of Fatty
Acids, 377–378Propylene Glycol Monostearate, 377–378
* Propyl Formate, 618–619Infrared Spectrum, 804
Propyl Gallate, 378Propyl p-Hydroxybenzoate, 379
* Propyl Mercaptan, 618–619Infrared Spectrum, 805
Propylparaben, 379Propyl Propionate, 618–619
Infrared Spectrum, 805n-Propyl Propionate, 618–619Protease, 151, 898Protease (Aspergillus niger var.), 150Protease (Aspergillus oryzae var.), 150Proteolytic Activity
Bacterial (PC), 923–924Fungal (HUT), 924–925Fungal (SAP), 925–926Plant, 922–923
Pteroylglutamic Acid, 180–181Public Access and Participation, xxi–xxiiiPullulanase, 151, 898Pullulanase Activity, 926–927Purified Oxgall, 313PVP, 351–352PVPP, 3503-Pyridinecarboxylic Acid, 299Pyridoxine Hydrochloride, 379–380Pyridoxol Hydrochloride, 379–380Pyromucic Aldehyde, 562–563Pyrrole, 618–619
Infrared Spectrum, 805l-2-Pyrrolidinecarboxylic Acid, 373–374
Q
Qualitative Test for Phenols Using FerricChloride, 634
Quantitation, Limit of, xxviiQuimociac TS, 968–969Quinaldine Red, 969Quinaldine Red TS, 969Quinine Hydrochloride, 380–381Quinine Sulfate, 381–382
Quinones, in Chewing Gum Base, 893–894
R
Racemic Pantothenyl Alcohol, 317Range of Analytical Method, xxviiRapeseed Oil, Low Erucic, 86–88Rapeseed Oil, Fully Hydrogenated, 382Rapeseed Oil, Superglycerinated, 382–383Readily Carbonizable Substances, 845–846Reagents
Hazardous or Toxic, 5Readily Carbonizable Substances, 845–846Solutions, 963–976Specifications, 5
Red Litmus Paper, 977Reduced Iron, 231–232Reduced Lactose Whey, 503Reduced Minerals Whey, 503–504Reducing Sugars Assay, 954–955Reference Standards, 5Refined Bleached Shellac, 398Refined Menhaden Oil, 284–285Refined Microcrystalline Wax, 329Refined Paraffin Wax, 329Refractive Index, 517, 846Regular Bleached Shellac, 397–398Reichert-Meissl Value, 940–941Rennet, 898
Bovine, 147, 151Calf, 147, 151Microbial (Endothia parasitica), 150Microbial (nonpathogenic strain of Bacillus
cereus), 150Microbial (Rhizomucor (Mucor) sp.), 150
Requirements for Listing Substances in theFood Chemicals Codex, xx
Residual Blank Titration, 4, 852–853Residual Solvent in Oleoresins, 945–946Residual Styrene in Chewing Gum Base, 894–
895Residue on Evaporation, 634, 932Residue on Ignition (Sulfated Ash), 857–858Retardation Factor (Rf), 834All-trans-Retinol, 494–496Revising Specifications, Procedure for, xxiii–
xxivRhodinol, 618–619
Infrared Spectrum, 806Rhodinyl Acetate, 618–619
Infrared Spectrum, 806Rhodinyl Formate, 618–619
Infrared Spectrum, 806Riboflavin, 383–384Riboflavin 5′-Phosphate Ester Monosodium
Salt, 384–386Riboflavin 5′-Phosphate Ester Monosodium
Salt, Dihydrate, 384–386Riboflavin 5′-Phosphate Sodium, 384–386Rice Bran Wax, 386
Infrared Spectrum, 807Ricinus Oil, 105Ring-and-Ball Method, 948–950Robustness of Analytical Method, xxviiiRochelle Salt, 430Rose Geranium Oil, Algerian Type, 192Rosemary Oil, 386–387
Infrared Spectrum, 807Rosemary Oleoresin, 447, 448Rose Oil, 387
Infrared Spectrum, 807Rosidinha, 280Rosins and Related Substances, Tests and
Assays, 947–950Rotation, Optical (Specific), 844Rubber, Butadiene-Styrene, 54–57Rubber, Butyl, 233–234
Rubber, Natural, 280Rue Oil, 387–388
Infrared Spectrum, 808Ruggedness of Analytical Method, xxviiiRum Ether, So-Called, 558–559
S
Saccharin, 388–389Safflower Oil (Unhydrogenated), 389–390Sage Oil, Dalmatian Type, 390
Infrared Spectrum, 808Sage Oil, Spanish Type, 390–391
Infrared Spectrum, 808SAIB, 456–457Salatrim, 391–395
Infrared Spectrum, 809* Salicylaldehyde, 618–619
Infrared Spectrum, 809SALP, 403Salt, 407–410Sandalwood Oil, East Indian Type, 395
Infrared Spectrum, 809Sandalwood Oil, West Indian Type, 30–31Santalol, 620–621
Infrared Spectrum, 810Santalyl Acetate, 620–621
Infrared Spectrum, 810Saponification Value
Essential Oils and Flavors, 931Fats and Related Substances, 941
Savory Oil (Summer Variety), 395–396Infrared Spectrum, 810
Schiff’s Reagent, Modified, 969Scoville Heat Units, 946Seignette Salt, 430Selenium Limit Test, 875–876DL-Serine, 396
Infrared Spectrum, 811L-Serine, 396–397
Infrared Spectrum, 811Shea Butter, 397Sheanut Oil, Refined, 397Shellac, Bleached, 397–399Shellac, Bleached, Wax-Free, 398Short- and Long-Chain Acyl Triglyceride
Molecules, 391–395Sieve Analysis of Granular Metal Powders,
858Significant Figures, 5-6Silica, Diatomaceous, 137–138Silica, Synthetic Amorphous, 398–399Silicon Dioxide, 398–399Silver Diethyldithiocarbamate Solution, 861–
862Silver Nitrate, 0.1 N, 973Silver Nitrate, Ammoniacal, TS, 964Silver Nitrate TS, 969Slaked Lime, 67–68Smectite, 45–46Soap, 942Soda Alum, 22–23Soda Ash, 407Sodium Acetate, 400Sodium Acetate, 0.1 N, 973Sodium Acid Pyrophosphate, 400–401Sodium Acid Sulfate, 406Sodium Acid Sulfite, 406Sodium Alginate, 401Sodium Alum, 22–23Sodium Aluminosilicate, 401–403Sodium Aluminum Phosphate, Acidic, 403Sodium Aluminum Phosphate, Basic, 403–
404Sodium Arsenite, 0.05 N, 973Sodium Ascorbate, 404–405Sodium L-Ascorbate, 404–405
996 / Sodium Benzoate / Index FCC V
Sodium Benzoate, 405Sodium o-Benzosulfimide, 432–433Sodium Bicarbonate, 405Sodium Biphosphate, 428Sodium Bisulfate, 406Sodium Bisulfite, 406Sodium Bisulfite TS, 969Sodium Bitartrate TS, 969Sodium Borate TS, 969Sodium Carbonate, 407Sodium Carbonate TS, 969Sodium Carboxymethylcellulose, 107–108Sodium Chloride, 407–410Sodium Chlorite Solutions, Acidified, 15–16Sodium Choleate, 313Sodium Citrate, 410–411Sodium Cobaltinitrite TS, 969Sodium Dehydroacetate, 411Sodium Diacetate, 411–412Sodium Erythorbate, 412Sodium Ferric Pyrophosphate, 412–414Sodium Ferrocyanide, 414–415Sodium Fluoride TS, 969Sodium Gluconate, 415–416Sodium D-Gluconate, 415–416Sodium Glutamate, 295–296Sodium Hexametaphosphate, 429–430Sodium Hydrogen Carbonate, 405Sodium Hydrogen Diacetate, 411–412Sodium Hydrogen Sulfite, 406Sodium Hydroxide, 416Sodium Hydroxide, 0.5 N, Alcoholic, 973Sodium Hydroxide Solutions, 416–417Sodium Hydroxide TS, 969Sodium Hydroxide 0.2 M, 962Sodium Hydroxide, 1 N, 973Sodium 3-(1-Hydroxyethylidene)-6-methyl-
1,2-pyran-2,4(3H)-dione, 411Sodium Hypophosphite, 417–418Sodium Hyposulfite, 438–439Sodium Identification Test, 860Sodium Indigotindisulfonate TS, 969Sodium Iron Pyrophosphate, 412–414Sodium Lactate Solution, 418–419Sodium Lauryl Sulfate, 419–420Sodium Lignosulfonate, 420–421Sodium Magnesium Aluminosilicate, 421–
423Sodium Metabisulfite, 423–424Sodium Metaphosphate, Insoluble, 424Sodium Metasilicate, 424–425Sodium Methoxide, 425–426Sodium Methoxide, 0.1 N, in Pyridine, 973–
974Sodium Methoxide, 0.02 N, in Toluene, 974Sodium Methylate, 425–426Sodium Monohydrogendicarbonate, 434Sodium Nitrate, 426Sodium Nitrite, 427Sodium Nitroferricyanide TS, 969Sodium Phosphate, Dibasic, 427Sodium Phosphate, Monobasic, 428Sodium Phosphate, Tribasic, 428–429Sodium Phosphate TS, 969Sodium Polyphosphates, Glassy, 429–430Sodium Potassium Tartrate, 430Sodium Potassium Tripolyphosphate, 430–
431Sodium Propanoate, 430–431Sodium Propionate, 430–431Sodium Pyrophosphate, 431–432Sodium Pyrosulfite, 423–424Sodium Saccharin, 432–433Sodium Sesquicarbonate, 434Sodium Silicoaluminate, 401–403Sodium Stearoyl Lactylate, 434–436Sodium Stearyl Fumarate, 436–437Sodium Sulfate, 437
Sodium Sulfide TS, 969Sodium Sulfite, 437–438Sodium Tartrate, 438Sodium Tetraphenylborate TS, 969Sodium Tetrapolyphosphate, 429–430Sodium Thiosulfate, 438–439Sodium Thiosulfate TS, 969Sodium Thiosulfate, 0.1 N, 974Sodium Trimetaphosphate, 439Sodium Triphosphate, 439–441Sodium Tripolyphosphate, 439–441Softening Point of Rosins and Related
SubstancesDrop Method, 947–948Ring-and-Ball Method, 948–950
Solidification Point, 846–848Solin Oil, 441Solubility in Alcohol, 932Solubility Specifications and Statements, 7
for Flavor Chemicals, 517Soluble Saccharin, 432–433Solutions, 6
Colorimetric (CS), 962Standard Buffer, 962Standard, for the Preparation of Controls
and Standards, 963Test, and Other Reagents, 963–970Volumetric, 6, 970–974
Sorbic Acid, 441–442Sorbitan, Monododecanoate, 346–347Sorbitan, Monooctadecanoate, 347–348Sorbitan, Mono-9-octadecenoate, 349Sorbitan Monostearate, 442–443D-Sorbite, 443–444Sorbitol, 443–444D-Sorbitol, 443–444Sorbitol Solution, 444Soybean Oil (Unhydrogenated), 444–445Soy Protein Concentrate, 445Spearmint Oil, 446
Infrared Spectrum, 811Specifications and Statements, 7–8
Data Requirements, xxi, xxiv–xxviiiDevelopment of, xiv–xv, xx–xxiiiFlavor Chemicals, 517International Harmonization of, xvRevision of, xxiii–xxiv
Specific Gravity, 6Flavor Chemicals, 517
Specificity of Analytical Methods, xxviSpecific Rotation, 844Spice Oleoresins, 446–448
Angelica Seed, 446, 447Anise, 446, 447Basil, 446, 447Black Pepper, 446, 447Capsicum, 446, 447Caraway, 446, 447Cardamom, 446, 447Celery, 447Coriander, 447, 448Cubeb, 447, 448Cumin, 447, 448Dillseed, 447, 448Fennel, 447, 448Ginger, 447, 448Hop, 447, 448Laurel Leaf, 447, 448Marjoram Sweet, 447, 448Origanum, 447, 448Paprika, 447, 448Parsley Leaf, 447, 448Parsley Seed, 447, 448Pimenta Berries, 447, 448Rosemary, 447, 448Thyme, 447, 448Turmeric, 447, 448
Spike Lavender Oil, 448–449Infrared Spectrum, 812
Stability of Fats and Related Substances, 942Standard Buffer Solutions, 962Standard Solutions for the Preparation of
Controls and Standards, 963Stannous Chloride, 449Stannous Chloride TS, 969Starch Acetate, 182Starch, Acid-Modified, 181–182Starch Aluminum Octenyl Succinate, 182Starch, Bleached, 182Starch Esters, 182–183Starch, Gelatinized, 181–182Starch Hydrolysate, Hydrogenated, 221–222Starch, Hydroxypropyl, 182, 183Starch Iodate Paper, 977Starch Iodide Paper, 977Starch Iodide Paste TS, 969Starch Octenyl Succinate, 182Starch, Oxidized, 182Starch Phosphate, 182Starch Sodium Octenyl Succinate, 182Starch Sodium Succinate, 183Starch, Thin-Boiling, 181–182Starch TS, 969Starter Distillate, 449–450Stearic Acid, 450Stearin, 208Stearyl Monoglyceridyl Citrate, 450–451Sterculia Gum, 237Storage Specifications and Statements, 8Strawberry Aldehyde, 558–559Stronger Ammonia TS, 964Styrene
Bound, in Chewing Gum Base, 892Residual, in Chewing Gum Base, 894–895
Submission and Development ofSpecifications, xx–xxiv
Succinic Acid, 452Succinylated Monoglycerides, 452–453Sucralose, 453–455Sucroesters, 457–458Sucrose, 455–456Sucrose Acetate Isobutyrate, 456–457Sucrose Fatty Acid Esters, 457–458Sucrose, Refractive Index Scale, 960–961Sugar, 455–456Sugar Beet Fiber, 458–460Sugar Beet Pulp, 458–460Sugar, Invert, 229Sugar Syrup, Invert, 229Sulfanilic Acid TS, 969Sulfated Ash, 857–858Sulfate Identification Test, 860Sulfate Limit Test, 863Sulfite Identification Test, 860Sulfiting Agents Policy, 2Sulfur Determination, 889–891Sulfur Dioxide, 460–461Sulfur Dioxide Detector Tube, 977Sulfur Dioxide Determination, 955–956Sulfuric Acid, 461–462Sulfuric Acid, Alcoholic, 0.5 N, 974Sulfuric Acid, Alcoholic, 5 N, 974Sulfuric Acid (reagent), 5, 845, 969Sulfuric Acid Table, 848Sulfuric Acid TS, 969Sulfuric Acid TS, Diluted, 969Sulfuric Acid, 1 N, 974Sulfurol, 600–601Summer Savory Oil, 395–396Sunflower Oil (Unhydrogenated), 462–463
* Sunset Yellow, 463–464Sunset Yellow FCF, 166–167, 463Superglycerinated Fully Hydrogenated
Rapeseed Oil, 382–383Superglycerinated Rapeseed Oil, 382–383
FCC V Index / Tunu (Tuno) / 997
Sweet Basil Oil, 43–44Sweet Orange Oil, 311Sweetwood Bark Oil, 102–103Symbols and Abbreviations, 6–7Synthetic Amorphous Silica, 398–399Synthetic Magnesium Silicate, 263–265Synthetic Paraffin, 318
Infrared Spectrum, 787Oil Content of, 855–857
Synthetic Terpene Resin, 471–472Synthetic Wax, 329–330
T
Talc, 464Tallow, 464–465Tangerine Oil, Coldpressed, 465–466
Infrared Spectrum, 812Tangerine Oil, Expressed, 465–466Tannic Acid, 466–467Tannic Acid TS, 969Tared Container, 6Tarragon Oil, 467
Infrared Spectrum, 813Tartaric Acid, 467–468L(+)-Tartaric Acid, 467–468Tartrate Identification Test, 860
* Tartrazine, 165–166, 468–469TBHQ, 469–471Temperatures, 6Terpene Resin
Natural, 471Synthetic, 471–472
�-Terpinene, 620–621�-Terpinene, 620–621Terpinen-4-ol, 620–621�-Terpineol, 620–621
Infrared Spectrum, 813Terpinyl Acetate, 622–623
Infrared Spectrum, 813Terpinyl Propionate, 622–623
Infrared Spectrum, 814Test Methods for Flavor Chemicals
Acidity Determination by IodometricMethod, 632
Acid Value, 634Alcohol Content of Ethyl Oxyhydrate, 631–
632Aldehyde and Ketone Assays, 630–631Aldehydes–Hydroxylamine Method, 630–
631Aldehydes–Hydroxylamine/Tert-Butyl
Alcohol Method, 630Gas Chromatography, 630, 635–636Ketones–Hydroxylamine Method, 631Limit Test for Antioxidants in Ethyl
Acrylate, 632–633Limit Test for Hydrocarbons in Eugenol,
633Limit Test for Hydrocyanic Acid in
Benzaldehyde, 633Limit Test for Lead, 633Limit Test for Methyl Compounds in Ethyl
Acetate, 633Limit Test for Peroxide Value, 633Limit Test for Readily Carbonizable
Substances in Ethyl Acetate, 633Limit Test for Readily Oxidizable
Substances in dl-Menthol, 633–634Limit Test for Reducing Substances, 634Qualitative Test for Phenols Using Ferric
Chloride, 634Residue on Evaporation, 634Titrimetric Procedures, 631
Tests and Assays. See Assays and Tests* �-Tetradecalactone, 622–623
Infrared Spectrum, 814
Tetradecanal, 600–601Tetradecanoic Acid, 2971-Tetradecanol, 602–603Tetradecyl Alcohol, 602–603Tetrahydrofurfuryl Alcohol, 622–623
Infrared Spectrum, 814Tetrahydrogeraniol, 548–549Tetrahydrolinalool, 622–623Tetrahydro-2H-1,4-oxazine, 2962,3,5,6-Tetramethylpyrazine, 622–623
Infrared Spectrum, 815Tetrapotassium Pyrophosphate, 370–371Tetrasodium Diphosphate, 431–432Tetrasodium Pyrophosphate, 431–432Thermometers, 831–832Thiamine Chloride, 472–473Thiamine Hydrochloride, 472–473Thiamine Mononitrate, 473–474Thiamine Nitrate, 473–474Thibetolide, 608–609Thin-Boiling Starch, 181–182Thin-Layer Chromatography, 835–836Thiobismethane, 548–549Thiosulfate Identification Test, 860–861Thorium Nitrate Colorimetric Method, 864–
865Thorium Nitrate, 0.1 M, 974L-Threonine, 474
Infrared Spectrum, 815Thuja Oil, 105–106Thyme Oil, 474–475
Infrared Spectrum, 815Thyme Oleoresin, 447, 448Thymol, 622–623
Infrared Spectrum, 816Thymol Blue, 976Thymol Blue TS, 970Thymolphthalein, 976Thymolphthalein TS, 970Tight Container, 8Time Limits, 6Tin Dichloride, 449Titanium Dioxide, 475–478Titrimetric Assays, 631, 851–854All-rac-�-Tocopherol, 478–479DL-�-Tocopherol, 478D-�-Tocopherol Concentrate, 479–480RRR-�-Tocopherol Concentrate, 479–480Tocopherols Concentrate, Mixed, 480–481RRR-Tocopherols Concentrate, Mixed,
480–481All-rac-�-Tocopheryl Acetate, 483D-�-Tocopheryl Acetate, 482–483DL-�-Tocopheryl Acetate, 483RRR-�-Tocopheryl Acetate, 482–483D-�-Tocopheryl Acetate Concentrate, 483–484RRR-�-Tocopheryl Acetate Concentrate,
483–484D-�-Tocopheryl Acetate Preparation, 483–484D-�-Tocopheryl Acid Succinate, 484–486RRR-�-Tocopheryl Acid Succinate, 484–
486Tolerances, 5–6
weights and balances, 833p-Tolualdehyde, 624–625Tolualdehyde, Mixed Isomers, 624–625
Infrared Spectrum, 816Toluene Distillation Method for Water, 853–
854�-Toluic Acid, 612–613�-Toluic Aldehyde, 612–613p-Tolyl Acetate, 540–541Tolyl Acetate, So-Called, 590–591p-Tolyl Aldehyde, 624–625Tolyl Aldehyde, mixed isomers, 624–625p-Tolyl Isobutyrate, 624–625
Infrared Spectrum, 816Torula Yeast, 508–510
Total Alcohols, 932Total Ash, 854–855Total Color, 882–884Total Monoglycerides, 938–939Total Solids,
Glucose Syrup (Corn Syrup), 957–958High-Fructose Corn Syrup Solids, 958Invert Sugar, 960–961Liquid Fructose, 958Maltodextrin, 959Total Unsaturation, 895–896
Trace Impurities, 1–2, 6Tragacanth, 486Tragacanth Gum, 486‘‘Transfer,’’ Defined, 4
* Transglutaminase, 150, 151Transglutaminase Activity, 927–928Transglutaminase (Streptoverticillium
Mobaraense var.), 150, 151* Trehalose, 486–487
Triacetin, 487–488Infrared Spectrum, 817
Triatomic Oxygen, 314–315Tribehenoyl-sn-glycerol, 202–203Tributyrin, 624–625
Infrared Spectrum, 817Tricalcium Phosphate, 77Trichloroethene, 488–489Trichloroethylene, 488–4891,1,2-Trichloroethylene, 488–4894,1′,6′-Trichlorogalactosucrose, 453–455
* 2-Tridecanone, 624–625Infrared Spectrum, 817
2-Tridecenal, 624–625Infrared Spectrum, 818
Tridocosanoyl-sn-glycerol, 202–203Triethanolamine, 0.5 N, 974Triethyl Citrate, 4893,7,12-Trihydroxycholanic Acid, 1122,4�,7-Trihydroxy-1-methyl-8-methylenegibb-
3-ene-1,10-dicarboxylic Acid-1,4�-lactone, 193
Triketohydrindene Hydrate TS, 970Trimagnesium Phosphate, 263Trimethylamine, 624–6254-Trimethylamino-3-hydroxybutyrate, 100–1012,6,6-Trimethylbicyclo[3.1.1]hept-2-ene, 614–
6154(2,6,6-Trimethyl-1-cyclohexenyl)-3-butene-2-
one, 576–5774(2,6,6-Trimethyl-2-cyclohexenyl)-3-butene-2-
one, 574–5753,7,11-Trimethyl-1,6,10-dodecatrien-3-ol,
602–6033,7,11-Trimethyl-2,6,10-dodecatrien-1-ol,
562–5633,5,5-Trimethyl Hexanal, 624–6252,4,5-Trimethyl �-3-Oxazoline, 626–627
Infrared Spectrum, 8182,3,5-Trimethylpyrazine, 627
Infrared Spectrum, 8181,3,7-Trimethylxanthine, 59Trinitrophenol TS, 970Triphosphate, 439–441Tripotassium Citrate, 359–360Tripotassium Phosphate, 368–369Tripropionin, 566–567Trisodium Citrate, 410Trisodium Dipotassium Tripolyphosphate, 430Trisodium Phosphate, 428–429Tristearin, 208Trypsin, 147, 151, 898Trypsin Activity, 928DL-Tryptophan, 489–490
Infrared Spectrum, 819L-Tryptophan, 490
Infrared Spectrum, 819Tunu (Tuno), 280
998 / Turmeric Oleoresin / Index FCC V
Turmeric Oleoresin, 447, 448L-Tyrosine, 490–491
Infrared Spectrum, 819
U
Ultraviolet Absorbance of Citrus Oils, 932–933
Uncombined Ingredients and Products of SideReactions, 884–886
�-Undecalactone, 626–627Infrared Spectrum, 820
�-Undecalactone, 626–627Infrared Spectrum, 820
Undecanal, 626–627Infrared Spectrum, 820
2-Undecanone, 626–627Infrared Spectrum, 821
1,3,5-Undecatriene, 626–627Infrared Spectrum, 821
10-Undecenal, 626–627Infrared Spectrum, 821
Undecen-10-al, 626–627(E)-2-Undecenol, 626–627
Infrared Spectrum, 822Undecyl Alcohol, 626–627
Infrared Spectrum, 822n-Undecyl Aldehyde, 626–627United States Pharmacopeia, 5Unmodified Food Starch, 183–184Unsaponifiable Matter, 942–943Urea, 491
V
‘‘Vacuum,’’ Defined, 6Valeraldehyde, 628–629
Infrared Spectrum, 822Valeric Acid, 628–629
Infrared Spectrum, 823�-Valerolactone, 628–629
Infrared Spectrum, 823‘‘Validation,’’ Defined, xxvValidation of Codex Methods, xxiv–xxviiiL-Valine, 492
Infrared Spectrum, 823
Vanillin, 628–629Infrared Spectrum, 824
Vegetable Oil, Brominated, 53–54* Vegetable Oil Phytosterol Esters, 492–493
Infrared Spectrum, 824Venezuelan Chicle, 280Veratraldehyde, 628–629Veratryl Aldehyde, 628–6291-Vinyl-2-pyrrolidone Crosslinked Insoluble
Polymer, 350Viscosity Tests, 848–851
Cellulose Gum, 850–851Dimethylpolysiloxane, 848–849Methylcellulose, 849–850Rosins and Related Substances, 950
Vital Wheat Gluten, 500Vitamin A, 494–496Vitamin B1, 472–474Vitamin B1 Hydrochloride, 471–473Vitamin B1 Mononitrate, 473–474Vitamin B2, 383–384Vitamin B6, 379–380Vitamin B6 Hydrochloride, 379–380Vitamin B12, 496–497Vitamin C, 36Vitamin C Sodium, 404–405Vitamin D, 497–499Vitamin D2, 497–498Vitamin D3, 498–499Vitamin E Acetate, 483Vitamin K, 499–500Volatile Acidity, 943Volatile Oil Content
Essential Oils and Flavors, 933Oleoresins, 946–947
Volumetric Apparatus, 832–833Volumetric Solutions, 6, 970–974
W
Water and Loss on Drying, 4, 6Water, Carbon Dioxide-Free, 5Water Determination, 851–854Water for Solutions, 6Water (reagent), 5Water Vapor Detector Tube, 977Weighing Practices, 6Weights and Balances, 833Weights and Measures, Symbols, and
Abbreviations, 6–7
Well-Closed Container, 8Wheat Gluten, 500
* Wheat Protein Isolate, 500–501Whey, 501–502Whey Protein Concentrate, 502
* Whey Protein Isolate, 502–503Whey, Reduced Lactose, 503Whey, Reduced Minerals, 503–504White Cedar Leaf Oil, 105–106White Mineral Oil, 291–292White Petrolatum, 327–328White Shellac, 397–398White Wax, 44–45Wine Yeast Oil, 120Wintergreen Oil, 504
Infrared Spectrum, 824Wool Fat, 245
X
Xanthan Gum, 504–505Xylenol Orange, 976Xylenol Orange TS, 970Xylitol, 506–507Xylose Isomerase, 151
Y
Yam Flour, 238–239Yeast, Autolyzed, 507–508Yeast, Dried, 508–510Yeast Extract, 510–511Yellow Petrolatum, 327–328Yellow Prussiate of Soda, 414–415Yellow Wax, 45
Z
Zein, 511Zinc Gluconate, 511–512Zinc Identification Test, 861Zinc Oxide, 512–513Zinc Sulfate, 513Zinc Sulfate, 0.05 M, 974Zingerone, 628–629
Infrared Spectrum, 825
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