Second Year Analytical Chemistry Lab Manualassets.openstudy.com/updates/attachments/5625a09ce4b...Second Year Analytical Chemistry Lab Manual CHEM 2P42 D2 FALL 2015 ii ANALYTICAL CHEMISTRY

Second Year Analytical Chemistry

Lab Manual

CHEM 2P42 D2

FALL 2015

ii

ANALYTICAL CHEMISTRY

CHEM 2P42

LABORATORY MANUAL

DEPARTMENT OF CHEMISTRY, BROCK UNIVERSITY

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INTRODUCTION

This course introduces students to a variety of basic experimental techniques that have practical

applications in many research and industrial laboratories. The experiments cover the proper handling of

basic laboratory equipment; the collection and analysis of data using the statistical approach, classical

wet techniques such as titrimetry (absolute techniques), and introduces students to the use of modern

instrumental techniques (relative techniques).

Good luck!

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TABLE OF CONTENTS

PAGE INTRODUCTION..........................................................................................................................iii HEALTH & SAFETY To Users of Contact Lenses ............................................................................................................ 1 IN CASE OF EMERGENCY ......................................................................................................... 2 FACULTY OF MATHEMATICS AND SCIENCE SAFETY POLICY ....................................... 3 SAFETY INSTRUCTION MANUAL FOR YEAR II CHEMISTRY STUDENTS ...................... 5

Introduction ................................................................................................................................. 5 Part I. Some Basic Terminology Relating to Fires, Explosions, and Toxicity............................5

A. Fires and Explosions ....................................................................................................... 5 B. Toxicity ........................................................................................................................... 6

Part II. Hazards Associated With Common Inorganic Chemicals .............................................. 6 A. Special Hazards Associated With Common Acids ............................................................ 6 B. Special Hazards Associated with Common Bases ............................................................ 7 C. Hazards Associated with Common Oxidizing Agents...................................................... 7 D. Hazards Associated with Common Reducing Agents........................................................ 7 E. Hazards Associated with Other Inorganic Chemicals ........................................................ 8

Part III. Hazards Associated With Common Organic Chemicals ............................................... 9 Part IV. Spill Cleanup and Waste Disposal .............................................................................. 10 Part V. Proper Storage of Chemicals ........................................................................................ 10 Part VI. Where to Find More Information ................................................................................ 11 Part VII. Penalties for Repeated Violations of Safety Regulations........................................... 11

LABORATORY NOTEBOOOK.................................................................................................. 12 FORMAL LAB REPORT FORMAT ........................................................................................... 13 GENERAL INTRODUCTION TO ANALYTICAL TECHNIQUES AND PROCEDURES...... 14

Analytical Balance .................................................................................................................... 14 Top loading Balances................................................................................................................ 15 Equipment and Procedures Used to Control Adsorbed Moisture in the Samples..................... 16 Drying Procedure ...................................................................................................................... 16 Use of a Desiccator ................................................................................................................... 16 Methods of Filtration................................................................................................................. 18 The Use of Volumetric Apparatus ............................................................................................ 20 Significant Figures .................................................................................................................... 23

INTRODUCTION TO GC AND HPLC ANALYSIS ..................................................................25 Preliminary Lab Exercise – Part 1: Calibration of Eppendorf®Pipette ......................................... 31 Preliminary Lab Exercise -Part 2: Basic Instructions on use of Excel for Mathematical Calculations, Statistical Analysis, Generation of Regression Data and Graphing........................36

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Expt 1. Calibration of Volumetric Glassware, Standardization of 0.1M HCl and Statistical Analysis of Experimental Results ................................................................................................. 42 Expt 2. Determination of Ethanol Content in the Unknown Wine Sample Using Capillary Gas Chromatography............................................................................................................................ 51 Expt 3. Determination of Caffeine in Beverages Using High Performance Liquid Chromatography (HPLC) .............................................................................................................. 62 Expt 4. Potentiometric Acid Base Titration: Analysis of an Unknown Mixture Containing Carbonate and Bicarbonate Species (Automatic Titrator) ............................................................ 72 Expt 5. Determination of Dissolved Oxygen in an Unknown Water Sample (Redox Titration or Winkler Titration) ......................................................................................................................... 80 Expt 6. Determination of Total Inorganic Phosphate in Water (Spectrometric Analysis)............ 86 Expt 7. General Introduction to Ion Selective Electrodes ............................................................. 93 APPENDIX I: Conversion Factors and Physical Constants........................................................ 104 APPENDIX II: Use of Excel for the Generation of a Calibration Curve and Regression Data. 105 APPENDIX III: Notes on Reference Styles ............................................................................... 107 Appendix IV: Useful Web References for Analytical Chemistry .............................................. 109 APPENDIX V: Use of Library Resources for Lab Reports ....................................................... 110

Produced by: Donna Vukmanic and Roger McLaughlin

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Health and Safety

To Users of Contact Lenses:

The increasing use of contact lenses in place of traditional eyeglasses leads to a situation in a chemical

laboratory that is potentially dangerous1. In the event of splashing of a liquid or solution of a chemical into

the eye, both hard and soft contact lenses can act to trap the material in contact with the eye, and so prevent

its removal by irrigation. In addition, soft contact lenses are porous to many chemicals, and can accumulate

material from the atmosphere, bringing it into intimate contact with the eye. In view of these and other

hazards outlined in ref.1, the Department of Chemistry recommends the following policy to users of contact

lenses:

1. That contact lenses not be worn in the laboratory unless there is no alternative, and in this event,

then,

2. That the lenses used should preferably be of the hard type, and that all users of contact lenses in a

laboratory should wear safety goggles, and not safety glasses. The former provide more complete

protection from splashing liquids.

1Chem & Eng. News, 57, (47), 4 (1979).

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IN CASE OF EMERGENCY FIRE

� alert everyone in area and provide assistance where needed � assess:

o if controllable - suffocate or extinguish o if uncontrollable – render area as safe as possible (shut off gas, remove ignition sources

and close door � pull wall alarm or call Campus Police (X3200) or use the closest emergency phone -- give exact

location CLOTHING ON FIRE

� smother flames by rolling on the floor, or wrapping with a fire blanket or douse with water from a safety shower, if available

CHEMICAL SPILL ON BODY

� rinse immediately with lots of water, continue for at least 15 min. � remove contaminated clothing � check Material Safety Data Sheet and get medical attention

CHEMICAL SPILL IN EYE

� flush eye immediately with lots of water while holding eye open, continue for a minimum of 15 minutes

� check Material Safety Data Sheet and get medical attention MEDICAL EMERGENCY

� minor cuts or burns: give first aid (kit in lab) and seek follow-up medical attention � major injury : summon medical help immediately, give any necessary first aid , keep warm and

do not move unless in danger; have one person stay with patient at all times CHEMICAL SPILLS

� contain spill rapidly by dyking with any handy material � warn all those in area � if volatile, extinguish ignition sources and evacuate � if toxic, suppress fumes by covering, and evacuate � check MSDS for clean-up procedures; use kits if available � all materials must be treated as waste until decontaminated.

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FACULTY OF MATHEMATICS AND SCIENCE SAFETY POLICY

All faculty, staff and students working in the Faculty of Mathematics and Science share the responsibility

for protecting from injury those working in the laboratories, others who may be exposed to hazards from the

laboratories and the environment.

General Rules and Procedures

1. Be prepared! Know the safety rules and procedures that apply to the work that is being done.

Determine the potential hazards, appropriate safety precautions and proper waste disposal

techniques before beginning any new operation.

2. Know the location and proper use of emergency equipment (safety showers, eyebaths, fire blankets,

extinguishers, first aid kits etc.) and be familiar with emergency procedures (exits, alarm stations,

evacuation etc.).

3. Safety glasses or goggles must be worn in all areas where chemicals are used, handled or stored, or

where particular eye hazard exists e.g. UV laser light, particulate matter or systems under pressure.

Contact lenses will not be worn in laboratories containing chemicals. (See page 1)

4. Protective clothing must be worn as specified.

5. Open-toe or woven fabric shoes are not to be worn; long hair and loose clothing must be confined;

shorts, cutoffs or miniskirts are not recommended.

6. Horseplay and pranks are expressly forbidden.

7. No eating, drinking, smoking or applying cosmetics is permitted in any laboratory or in any other

chemical storage area.

8. Mouth pipetting is absolutely forbidden.

9. All bottles, flasks & vials containing chemicals (including wastes) must be labelled with

information about contents, concentration, date and individual involved. The only exception is

chemicals which will be consumed in one session by one individual.

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10. Chemicals from unlabelled containers are not to be used and their presence shall be reported to the

demonstrator or faculty supervisor.

11. Suitable warning signs must be put up when hazardous situations are present (e.g. high pressure

experiments, exceedingly toxic experiments, experiments using radioactive substances, or

flammable solvents, or bio-hazardous materials, or involving X-rays or lasers etc.)

12. All work areas must be kept clean and free from obstruction. Access to exits, emergency

equipment, controls and such must never be blocked.

13. Spilled chemicals must be cleaned up immediately and disposed of properly. A separate labeled

container is to be used for broken glassware.

14. Equipment shall be properly maintained, and used only for its designed purpose. Access to

electrical connections and moving parts should be guarded.

15. All accidents must be promptly reported to the supervisor, who will administer first aid and/or

arrange for further medical attention as well as complete an Accident Report.

16. Undergraduate students must not work unsupervised in a laboratory.

17. Laboratories and storerooms must be locked when unattended.

18. Always wash hands and arms with soap and water before leaving the work area. This applies even

if one has been wearing gloves.

19. Proper, responsible, waste disposal practices must be in use at all times.

20. Visitors to laboratories are required to follow these same rules.

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SAFETY INSTRUCTION MANUAL FOR YEAR II CHEMISTRY STU DENTS Introduction Students are expected to know the general rules for working in laboratories, and the basic principles of preventing fires, explosions, cuts, burns, and poisoning. These safety rules may be found in the Handbook for First Year Chemistry Students. This safety instruction manual is intended to increase your knowledge of hazardous chemicals and help you to assume more responsibility for your own safety in the lab. The following topics are covered: 1. Basic definitions related to fires, explosions, and toxicity. 2. Brief details on the hazardous nature of common inorganic chemicals in the second-year labs. 3. Hazardous nature of common organic chemicals in the second-year labs. 4. Principles of spill clean-up and waste disposal. 5. Proper storage of chemicals. 6. Where to get more information. 7. Penalties for repeated violation of safety rules.

THREE RULES FOR SAFETY

1. Know where all the safety equipment is and how to use it. This includes

wearing your safety glasses at all times in the lab!

2. Know what you are doing in the lab. Know the hazardous properties of the chemicals you are going to work with, and how to clean up spills and dispose of wastes.

3. Keep the lab clean and neat. Return bottles to their proper locations on the shelves.

Part I. Some Basic Terminology Relating to Fires, Explosions, and Toxicity A. Fires and Explosions Not all mixtures of flammable vapors and air will burn. The concentration limits within which a fire propagates are called the lower and upper flammable limits (sometimes the explosion limits). For example, the flammable limits of methane are 5-15% by volume in air. Methane-air mixtures containing less than 5% or more than 15% methane will not burn. The flash point is the lowest temperature of a liquid at which its vaporization in air reaches the lower flammable limit. The ignition temperature is the temperature at which combustion can begin. Ether poses serious fire hazard problems because its flammable limit range is large (2-50% in air), its flash point is low (-45° C), and its ignition temperature is low (less than 200° C). The flammable limits, flash point, and ignition temperature for many substances may be found in several places, including Green and Turk Safety in Working with Chemicals.

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B. Toxicity There are two basic types of toxicity: acute and chronic. Acute poisons cause immediate effects; chronic (low dose) poisons only show effects over a long period of time. It is easy to ignore the problem of chronic exposure because nothing happens immediately. However, there are many chemicals where long-term, low-dose exposures cause cancer, sterility, blood diseases, genetic damage, deformed offspring, and other unpleasant consequences. The Chemistry Department tries to avoid undergraduate experiments with extremely toxic chemicals, but many experiments do involve moderately toxic materials. Students should make every effort to avoid exposure to chemicals whose short- and long-range toxicities are proven or suspected. In order for poisoning to occur, the poison must get into the body. This can happen in one of three ways: 1) Oral ingestion 2) Absorption through the skin 3) Inhalation of vapor or dust Oral ingestion can easily be prevented by following well-known rules: no eating or drinking in the lab, no pipetting by mouth, and so on. Absorption through the skin can be avoided by wearing rubber gloves and handling chemicals carefully. This leaves the major source of poisoning: inhalation of dusts and vapors. Students often do not handle toxic materials in the hood. Vapors escape from opened bottles of liquids, and dusts get in the air when bottles of solids are opened. One student recently got a very sore throat when she inhaled NaOH dust when a fresh bottle of NaOH pellets was opened! "Permissible limits" have been established for exposure to certain chemicals. Measures of these limits are the threshold limit value (TLV) and the time-weighted average (TWA). Lists of TLV's or TWA's can be found in several places; the lists are constantly being revised and expanded with new data. Green and Turk, Safety in Working with Chemicals , is a good source of information for some of the common chemicals. The best rule to follow is one of prudence: know which chemicals pose special hazards and avoid unnecessary exposure (work in the hood, wear gloves, etc.), and be cautious when you work with chemicals whose toxicity has not been established. Part II. Hazards Associated With Common Inorganic Chemicals Inorganic chemicals can be divided into 5 groups for the purposes of discussing hazards. These are: Acids Bases Oxidizing Agents Reducing Agents Others A. Special Hazards Associated With Common Acids.

Nitric acid, HNO3

� reacts violently with most organic and inorganic reducing agents. (Do not store acetic acid in the "acids" storage area, as it can react explosively with nitric acid).

� gives off toxic fumes of nitrogen dioxide (wear a breathing mask in cleaning up a major spill). � has the other usual characteristics of strong concentrated acids: produces severe skin and eye burns,

reacts violently with bases. Sulfuric acid, H2SO4

� powerful dehydrating acid, oxidizing agent when hot. � slippery. EXTREME CAUTION is needed when cleaning up a large spill; if you slip and fall in the

acid, you may not be able to get up because of the slipperiness.

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Hydrofluoric acid, HF

� Main problem is that spills on the skin may not produce immediate pain and may go unnoticed until too late. Painful burns may take months to heal; fingers may fall off.

Perchloric acid, HClO4

� powerful oxidant, may explode violently if even a trace of organic compound or reducing agent comes in contact with the concentrated acid. Use only in special fume hoods which can be washed down after use.

� all perchlorates are potential explosives. B. Special Hazards Associated with Common Bases NaOH, KOH alkali metal hydroxides

� pellets and concentrated solutions cause severe skin and eye burns. � may cause violent reactions when in contact with halocarbons (chloroform, methylene chloride, etc.)

Ammonia, NH3

� high levels can cause respiratory paralysis (breathing mask or self-contained air pack is needed for cleaning up a major spill).

Sodium amide, NaNH2

� oxidizes upon exposure to air to form several different explosive compounds. Do not keep partly-used bottles of this material; dispose of the excess after use.

C. Hazards Associated with Common Oxidizing Agents Potassium permanganate, KMnO4 Potassium dichromate, K2Cr2O7 Potassium chlorate, KClO3; potassium iodate, KIO3 Perchlorates, e.g. Mg(ClO4)2 Hydrogen peroxide, H2O2

� all of the above compounds may produce violent or explosive reactions with organic compounds or reducing agents.

� bottles of hydrogen peroxide sometimes blow up because H2O2 decomposes to release oxygen; pressure build-up in the bottle can explode it, breaking other bottles in the process.

� Cr(VI) is a carcinogen (inhaling chromate dusts causes lung cancer) D. Hazards Associated with Common Reducing Agents Metals (elemental K, Na, Mg, Zn, Al, etc.) Hydrides (LiA1H4, NaH, etc.)

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� chief problem is how these things react with other compounds, including water. E.g.

Na

K + H2O ___> H2 gas (forms explosive mix with air) LiAlH 4

Zn + H2O + As2O3 ___> AsH3 (1 mg of arsine contaminates an entire lab)

K (but not Na) forms an explosive superoxide coating on its surface; it is dangerous to keep potassium metal for more than a few months once a container has been opened.

E. Hazards Associated with Other Inorganic Chemicals

• Silver Nitrate, AgNO3 o corrosive to skin and eyes; can cause blindness if solutions splash in the eye and are not

immediately flushed out at the eye-wash station. o long known to form explosive mixtures when dissolved in aqueous ammonia o ethanolic solutions form fulminates, which explode with the touch of a feather when dry

• Beryllium oxide, BeO (and other Be compounds)

o minute amounts of dust, when inhaled, result in incurable (and sometimes fatal) lung disease.

• Liquid bromine, Br2

o skin contact results in long-lasting, painful burns

• Chlorine gas, Cl2 o levels of 50 ppm cause serious injury. For comparison, levels of 50 ppm CO can be tolerated

without ill effect.

• Cyanides, e.g. KCN, NaCN o 0.01 g is the lethal dose o react with acids to produce gaseous HCN

• Mercury and mercury compounds

o most common means of ingestion is by inhalation of mercury vapor or dusts of mercury compounds.

o causes serious damage to kidneys and/or brain ("Mad as a hatter" was the term applied to people who used to make hats - mercury was one of the chemicals used in the felting process). Brain damage is irreversible.

• Hydrogen sulfide, H2S

o more toxic than carbon monoxide. Sense of smell is deadened by exposure to the gas, so you don't know how much you are getting.

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Part III. Hazards Associated With Common Organic Chemicals Most organic chemicals burn in air and react more or less violently with oxidizing agents such as permanganate, nitric acid, and so on. Many are carcinogenic, especially aromatics. The properties of several common organic compounds should be well understood so that you may work safely with them. Chlorinated hydrocarbons (chloroform, carbon tetrachloride, vinyl chloride, etc.)

� can react violently or explosively with concentrated alkali hydroxide and with metals such as Na and K.

� many (not all) are carcinogenic; carcinogenic compounds in this class include chloroform, vinyl chloride, and tetrachlorodibenzodioxin

� not normally fire hazards Aromatic hydrocarbons

� benzene causes bone marrow damage and leukemia � toluene is much less toxic than benzene, and should be used in place of benzene as a solvent when

possible (but watch out if you heat the mixture! Toluene has a higher boiling point than benzene, and a reaction which goes smoothly in refluxing benzene may get violent in refluxing toluene).

� anthracene and higher polynuclear aromatics act as skin photosensitizers, and many are powerful carcinogens

Carbon disulfide, CS2

� a serious fire hazard; ignition temperature is less than 100° C, the flash point is -30 C, and the flammable limits are 1-44% by volume in air.

Amines

� aliphatic amines are generally not a concern � hydrazine and substituted hydrazines cause a variety of effects, including cancer, bone marrow

damage, liver, kidney, and heart damage, and so on � aromatic amines and their derivatives are often carcinogens

Phenol

� serious poisoning or death can result if phenol is spilled on a person, as it is readily absorbed through the skin

Ethers

� fire hazards because of high volatility, wide explosion limits, and low ignition temperature. The surface of a hot plate or heating mantle is hot enough to ignite a diethyl ether-air mixture (no spark or flame needed!). Heat ether on a steam bath.

� explosion hazards due to peroxide formation. All ethers form peroxides; diisopropyl ether is especially prone to peroxide formation. Peroxides frequently concentrate in the cap area, and people have been dismembered while unscrewing the cap of a bottle of diisopropyl ether.

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Part IV. Spill Cleanup and Waste Disposal 1. If the spill is a small one and the chemical is not especially hazardous, wipe it up with paper towels.

Rinse the chemical out of the towels and put the towels into the wastebasket.

2. If the spill is a large one, get people out of the area. Evacuate the lab if necessary.

3. How dangerous is the spill? If fire or explosion hazards are imminent, and it is obvious that you will endanger yourself trying to control the problem, get out of the lab. Set off the fire alarm and YELL LOUDLY FOR HELP.

4. If the spill presents no immediate fire or explosion hazards, get proper clean-up and protection equipment from the spill clean-up box near Chemistry Stores. The box includes face shield, goggles, gloves, rubber boots, breathing masks, dust pan and broom for solid spills, and cat litter for absorbing liquid spills.

5. If the material is a solid and very toxic (e.g. NaCN or As2O3, etc.), shovel the spilled chemical into a clean, dry bottle and LABEL it (e.g. "contaminated NaCN for disposal only.") Make sure the clean-up is thorough.

6. Dust and vapor inhalation is a significant route of poisoning. Wear breathing apparatus when cleaning up a spill of toxic chemical.

7. Acids and bases can be neutralized with sodium bicarbonate, NaHCO3 and then cleaned up with a mop and lots of water.

8. Put ORGANIC WASTES into specially labeled bottles in the labs.

9. Put VERY TOXIC WASTES into separate, labeled bottles if there is no way to safely dispose of them. (But note that many toxic compounds can easily be converted to less toxic materials by simple chemical treatment - e.g., inorganic cyanides can be oxidized to cyanate with hypochlorite bleach in basic solution).

10. Flush small quantities of most INORGANIC WASTES down the drain with lots of water. Part V. Proper Storage of Chemicals Following a few simple rules when you are working with chemicals in the lab can eliminate many problems. 1. Make sure all bottles are labeled properly. Tape labels on if they seem loose.

2. Read the labels, especially if the labels are put on by the manufacturer. Toxicity and flammability

information is often recorded here. Also, note if there is an expiry date or a warning about exposure to moisture or air.

3. Every lab should have storage areas for different classes of chemicals. Chemicals should be returned to their proper area, and put in alphabetical order within that area. In general, labs will have separate cabinets or shelves to store the following types of chemicals:

a. Concentrated acids (but not acetic acid) b. Concentrated base solutions, including ammonia c. Organic solvents (store acetic acid here)

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d. Oxidizing agents e. Reducing agents f. Organic compounds g. Inorganic compounds

4. Keep chemicals away from a strong heat source. 5. Be aware that pressure build-ups can occur in some bottles if the lid is screwed on tightly. (E.g. the

organic waste bottle, bottles of hydrogen peroxide, etc.) If this is a possibility, make sure the lid is placed on loosely.

Part VI. Where to Find More Information Chemistry Stores has several books on safety, including specialized topics such as the handling of radioisotopes, the use of gas cylinders, fire hazard ratings of many chemicals, and so on. The most useful books for an undergraduate student (graduate students too!) are probably the following ones: 1. M. E. Green and A. Turk, Safety in Working with Chemicals. A very readable student guide. 2. G. D. Muir Hazards in the Chemical Laboratory. The "yellow pages" section of this book lists several

hundred chemicals, describes their toxic and explosive hazards, and tells how to dispose of them. 3. M. A. Armour, L. M. Browne, and G. L. Weir, Hazardous Chemicals: Information and Disposal Guide.

Contains tested disposal procedures worked out at the University of Alberta. Part VII. Penalties for Repeated Violations of Safety Regulations Common sense tells you that you will be much safer in the lab if you and your neighbors do your best to

follow the rules above. The rules don't cover every possible situation, so exercise due caution and judgment in your work.

The Chemistry Department does reserve the right to impose the following penalties on a student who

repeatedly violates good safety practices, thus becoming a hazard to self and/or neighbors: 1. The demonstrator may deduct 50% of the student's lab marks if the demonstrator continually has to

remind the student about safety glasses, cleaning up the lab, etc. This is justifiable because following safety rules is a part of lab technique, and failure to show good lab technique is subject to deduction of marks, or

2. The student may be required to write an essay on some aspect of lab safety, topic to be assigned by the instructor, before being allowed to continue work in the lab, or

3. The student may not be allowed to work in the lab at all. These penalties are rarely invoked; however, when they are, any labs missed are counted as zeros

(unexcused, no make-up allowed). Best wishes for a safe, accident-free lab experience.

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LABORATORY NOTEBOOOK Your notebook should approximate as closely as possible the notebook kept in any industrial or research laboratory. Records from a research or industrial laboratory are important legal documents. In the event of a patent dispute, or a particular analysis of a material, the laboratory notebook can be used as evidence of both time and accuracy. The notebook should contain enough information so that another person could reproduce the work and enough raw data to substantiate the conclusions drawn. Notebook Requirements 1. Table of Contents: A page should be left at the beginning of the book for a table of contents. This

page should contain experiment number, title and page numbers where experimental information is found.

2. All pages should be numbered. An important point here is that under no circumstances should

pages be removed from your book. The sequence of pages guarantees the order of the work. 3. All work should be dated. It is usual procedure to date each page of work. 4. The formal part of the report (tables, observations, calculations, etc.) should be placed only on

the right hand pages. Left hand pages can be used for rough work and preliminary calculations such as, the determination of the mass of sodium hydroxide that you would need to make up a 0.10M solution.

5. All experiments should have a purpose, introduction with the balanced equations for all reactions

occurring in the experiment and brief point form procedure. This should be done before the start of the lab period and be no longer than two pages.

6. All the chemicals used in the experiments, their potential hazards and their waste disposal should

be listed. 7. All work in the book should be done in ink. Data tables should be set up ahead of time and should

be filled with the data as obtained. There should be no use of whiteout or similar material. Any mistakes should be neatly crossed out and explanation note added.

8. Examples of all the calculations performed should be shown in the book along with clear

statements of the final results including standard deviation and 95% confidence limits where applicable. The questions found at the end of the experiment should be answered in the discussion section.

9. All graphs should be computer generated and neatly attached in the book. Linear regression

calculations should be performed whenever applicable. 10. All sources used in your write up should be properly referenced. See Appendix III for correct

referencing style. Your notebook mark will be based on the adherence to the requirements above, on the amount of preparation before the lab and on the clarity and accuracy of your observations, results, calculations and conclusions.

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FORMAL LAB REPORT FORMAT

Cover Page contains the title of the experimental investigation, the names of those who performed the

experiment and the name of the author of the report.

Abstract briefly summarizes the purpose of the study, the method or methods used, the results obtained and,

whenever possible, the importance of the results obtained.

Introduction describes the theoretical basis for the method used and for the calculation of parameters

referred to in the Abstract.

Experimental section contains either a full account of the procedure used or a reference to the source of the

experimental procedure that was followed, plus an account of any changes to this experimental procedure

that may have been made. If an instrument is used in the analysis, the instrument name, make and all the

operating parameters are reported. This section also contains sample calculations, tables and graphs. Figures

and Tables should be numbered and titled. Graphs should contain a title, properly labeled x and y-axes (with

units) and clearly marked experimental points. If linear regression was performed on the data, then the slope

and intercept of the line of best fit should be reported.

Discussion section is used to interpret the data and compare it to the reference information. If differences

exist, an attempt should be made to explain why and whether or not they are statistically significant. This

section also contains an evaluation of the method and the possible sources of errors.

References: a correct referencing style is used to indicate all the sources of information used in preparing

the report. A properly referenced lab will allow anyone to immediately check the reference and to discover

whether or not the author has used the reference correctly.

Sample Reports: Available for viewing in R. McLaughlin’s office.

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GENERAL INTRODUCTION TO ANALYTICAL TECHNIQUES AND PROCEDURE S

Analytical Balance

The most important and the most precise instrument available in the analytical chemistry lab is the analytical

balance. It is used to determine the mass of an object. The typical analytical balance has a capacity of 160 to

200 g and a sensitivity of ±0.0001 g.

Rules for Using an Analytical Balance.

• Always use the same balance during the course of an experiment.

• Handle dried object with tweezers or tongs.

• Objects to be weighed must be at room temperature. A warm object tends to form convection

currents inside the balance, which can make mass of an object appear lighter than it actually is.

• Weigh volatile materials in ampoules or tightly sealed bottles.

• Do not overload the balance.

• Always record the mass of an object directly in the notebook.

Weighing Procedure

• Check that the balance pan is clean and that both doors are closed.

• Zero the balance (tare).

• Handle the object to be weighed with tweezers or paper tongs to prevent added weight due to

contamination (i.e. grease/oils and moisture from your skin)

• Open the most convenient door and transfer the object to be weighed onto the center of the pan.

• Close all doors.

• When the scale reading becomes constant record the mass of the sample to four decimal places.

• Remove the object, check that pan is clean, close the door and re-zero the balance.

Procedure for Weighing by Difference

When obtaining the masses of a series of samples the weighing by difference method is usually employed.

• Weigh the weighing bottle plus the bulk of the usually dried and cooled sample using the analytical

balance.

• On the toploading balance, tare the receiving container (usually beaker or an Erlenmeyer flask) and

carefully transfer the approximate amount of individual sample from the weighed bottle into a tarred

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container by gently tapping on the bottle. Note: Avoid any loss of the sample outside the weighing

bottle and the receiving container.

• Re-weigh the weighing bottle and its residual contents on the analytical balance.

• Determine the actual weight of the sample to four decimal places by subtracting the second

weighing from the first.

Top loading Balances

In cases where mass accuracy to four decimal places is not required such as for preparation of solutions that

will be standardized later, a top loading balance is usually used. The typical capacities of the top loading

balance range from 300 g ±0.01g to 2000g ±0.1g.

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Equipment and Procedures Used to Control Adsorbed Moisture in the Samples

Most solid samples used in the analytical laboratory are in the form of fine powders. Since powders have a

large surface area, they tend to adsorb measurable amounts of water from the atmosphere. In cases where a

high degree of accuracy is required in the analysis, the adsorbed water content, which varies from substance

to substance, can significantly affect the final results. To obtain reliable results in the measurement of mass,

a control of the water content of samples is essential. Most often this is accomplished by drying.

Drying Procedure

• Clean and dry a weighing bottle found in your locker.

• Calculate how much sample you will need for the total analysis.

• Transfer slightly more sample than the amount required into weighing bottle. Use the top loading

balance to determine approximate sample weight. Note: transferring excessive amounts of sample

into weighing bottle leads to waste since the excess should not be return to the original stock bottle

due to possible contamination.

• Place an open weighing bottle containing the sample into a 250 mL beaker, placing the ground glass

stopper beside the bottle in the same beaker, and cover the beaker with a ribbed watch glass .

• Dry the sample in the oven at the temperature and for the duration specified in the experiment. It is

standard procedure to dry the samples at 105° C for 1 hour, but there are cases where temperature

should be much higher or much lower. Example of this is disodium salt of EDTA which is very

often used in complexometric titrations. EDTA has two waters of hydration Na2 EDTA . 2H2O and

must be dried at a low enough temperature to remove surface water yet have the two waters of

hydration intact. For this reason, EDTA is dried at 80° C for 1 hour.

• Dried samples must be cooled before weighing. This is done in a dry atmosphere provided by the

desiccator.

Use of a Desiccator

A desiccator provides a dry atmosphere for sample cooling and storage. It consists of a heavy walled glass

base and a top. The contact between the two is ground-glass surface which is kept slightly greased by

applying a small amount of high quality grease. The base contains desiccant, a substance which has a high

capacity for water and effectively absorbs the moisture from the desiccator atmosphere. Most common

desiccants are calcium sulfate (CaSO4) or DRIERITE, calcium chloride (CaCl2 ) and silica gel. You will be

using calcium sulfate as a drying agent. It comes in a regular white or an indicating blue form .

Fill the bottom of the desiccator with white DRIERITE and sprinkle it with a small amount of blue

indicating DRIERITE. Indicating drierite contains a substance which is blue when dry and turns pink in the

presence of moisture. For this reason, periodically check the colour of the indicating desiccant. If the pink

17

colour becomes apparent, empty the spent desiccant into the waste basket and use fresh desiccant to refill.

Place a ceramic desiccator plate, used to support the weighing bottles and crucibles, above the desiccant.

Keep the desiccator covered at all times in order to minimize moisture absorption.

When placing a hot weighing bottle or crucible into the desiccator, wait a short time for the object to cool

somewhat before completely closing the lid. There are two reasons for this cooling period:

1. When the hot object is placed into the covered desiccator, the air around it heats up and expands.

The increase in pressure can pop the lid and break the seal between the top and the base. On many

occasions, desiccator tops have slid off and were broken for this reason.

2. The air around the hot object expands quickly, increasing the pressure even before the lid is put on.

When the air inside the desiccator cools, pressure can be reduced enough to create a partial vacuum

which makes it difficult to remove the top and may cause loss of the sample once the top is

removed due to the sudden rush of air inside. For the same reason, the tops of weighing bottles are

left partially opened on initial cooling.

18

Methods of Filtration

Filtration is a physical method of separating usually a solid material from a liquid or gas substrate in

which it is suspended. The separation is accomplished by passing the mixture through one of many

available types of filters. In the chemistry laboratory, filtration is usually used to separate solid

impurities from a desired liquid or to collect a solid substance from a liquid from which it was

precipitated. There are two general types of filtration: gravity and suction. During gravity filtration

the filtrate or the liquid passes through the filter under the combined forces of gravity and capillary

attraction between the liquid and the funnel. In suction filtration, the receiving flask is evacuated

causing a pressure differential across the filter medium which results in the increase in the rate of

filtration. The choice of the method depends on the nature of the precipitate, the speed required, and

the degree to which one can tolerate the precipitate in the filtrate.

Gravity Filtration

A filter is prepared for use as follows: The dry paper is folded exactly in half and then again so that the two

halves of the first crease do not coincide. When placed in a 60° glass funnel, the filter will not quite fit the

walls at the apex, but will at the top, which permits liquid which has penetrated the filter to run out

unimpeded, making for faster filtration. The corner of the fold on the outside should be torn off so that the

paper will fit the funnel more snugly. The paper is moistened with the solvent to be used, and the top part

pressed against the glass to provide an air seal. The stem of the funnel should fill with solvent, whose weight

provides a gentle suction which increases the rate of filtration considerably. The funnel is placed in a

support and arranged so that the funnel stem touches the side of the receiver. The supernatant liquid is then

slowly poured down a stirring rod into the filter. The precipitate in the bottom of the beaker should be

disturbed as little as possible. The precipitate is then washed by decantation and then the main bulk of the

precipitate is transferred to the filter, using a jet of solvent from a wash bottle, and/or a policeman, as is

necessary. It may be washed further at this time, if necessary. Washing is considerably more efficient if

done with several small volumes rather than one or two large volumes. Sometimes a large surface area for

filtering is desirable so the filter paper is fluted before being put into the funnel.

Fig. 3. Gravity Filtration Setup

19

Vacuum Filtration Using Buchner Funnel

A Buchner funnel has a flat base over which lies the flat piece of filter paper. The Buchner funnel fits into

the filtering flask through a rubber adaptor which ensures a good seal. A hose is attached to the filtering

flask; the other end is attached to the aspirator in the sink. The filter paper is moistened and the aspirator

turned on before filtration is started to prevent filter paper from "floating" when liquid is added. Solution is

poured slowly at first.

Fig. 5. Buchner Funnel Setup Fig. 6. Filtering Crucible Setup

Vacuum Filtration Using Filtering Crucible

Crucible filtration is used in procedures where the accurate weight of the precipitate must be known and

where drying of the precipitate is required. The procedure involves the use of a porous bottom filtering

crucible, filter adaptor, and a filtering flask attached to a water aspirator (Fig. 6). The filter adaptor creates a

tight vacuum seal between the crucible and the filtering flask and in this way facilitates suction of liquid

through the porous crucible bottom. The precipitate is quantitatively collected in the crucible and then dried at

the appropriate temperature until the constant weight of the residue is achieved.

There are two basic types of filtering crucibles. Fritted glass filtering crucibles have an upper temperature

limit of 200°C and are used in filtrations where heating of the precipitates does not exceed 200°C. Gooch

crucibles are usually made of porcelain with perforated bottoms consisting of small circles of glass matting

which tolerate temperatures in excess of 500°C.

Quantitative errors associated with filtration procedure:

• Precipitates left on sides of glassware, stirring rods and filter paper.

• Solvent left on precipitate and filter paper.

• Product left dissolved in solvent because of its solubility.

20

The Use of Volumetric Apparatus

In order to know the exact molarity of a solution, it is necessary to determine the number of moles of the

component of interest and the volume of the solution. The amount of sample is normally determined from

the weight and the volume is usually determined by the use of volumetric glassware. There are three kinds of

volumetric glassware: volumetric flasks are calibrated to hold an accurately known, fixed volume;

volumetric pipettes deliver an accurately known, fixed volume; and burettes deliver an accurately known

volume, but the volume is varied over some range from one application to the next.

Volumetric Flasks

In making up a solution of definite concentration of a pure substance, the substance is accurately weighed

into a beaker, dissolved in a small amount of solvent, and then transferred quantitatively to the flask. The

beaker is then rinsed two or three times with additional solvent which is added to the flask. The dilution

solvent is then added to the flask until it almost reaches the junction neck. The flask is then swirled in order

to mix the solution in order to prevent any expansion or contraction effects due to solvent miscibility after

the final dilution. The final adjustment to the mark is then made dropwise and the solution mixed thoroughly

by inverting the flask at least three times with a simultaneous rotary motion. If the solution sits for any

length of time before use, it should be re-mixed.

Pipettes

Pipettes are used to deliver an accurate volume of liquid. Volumetric or transfer pipettes are used to transfer

a very accurate fixed volume of liquid. These pipettes come in various sizes ranging from 0.5 mL to 200 mL.

Measuring or Mohr pipettes are calibrated, usually in mLs, with 0.1 mL graduations and are capable of

delivering various volumes of liquid up to its capacity. Digital micropipettes are used to deliver small

adjustable or fixed volumes of liquid usually in microliter range.

The following rules ensure proper use:

• When using Volumetric or measuring Pipettes Always use a pipette bulb! Never pipette by

mouth!. Squeeze the air from the bulb by using all four fingers and the palm of your hand. Just

barely slip the bulb onto the blunt end of the pipette--so it is easy to remove quickly. Insert the tip of

the pipette into the liquid, being sure the tip is well below the surface before gradually releasing

your grip on the bulb.

• When the liquid is well above the mark on the pipette stem, slip the bulb off and quickly clamp your

index finger over the opening before the liquid level drops below the etched mark.

• Remove the tip from the solution and carefully wipe any droplets of solution from the outside of the

pipette.

21

• Place the tip of the pipette against the wall of the solution beaker or a waste beaker and slowly

release pressure from your index finger to allow the level to fall until the meniscus is level with the

graduation mark. Once the level has been reached hold your index finger securely to prevent the

level from dropping below the graduation mark.

• Allow the pipette to empty into the desired container. Touch the pipette tip against the wall of the

container for several seconds to drain.

• Most pipettes are marked with either a TD (to deliver) or a TC (to contain), if it is marked with a TD

do not blow out the remaining drop in the tip. The calibration has allowed for some liquid to remain

in the tip.

• Try not to draw liquid into the bulb. If you do, rinse it out carefully with tap water, then deionized

water, and allow it to drain for several minutes.

Burettes

The burette has been calibrated to deliver accurately variable volumes of solution to a maximum of 50 mL

as required in a titration. Graduations to 0.1 mL are marked on the burette wall, but the volume is read by

interpolation to the nearest 0.01 mL.

• The burette is washed with tap water, and then rinsed with 10 mL deionized water. The burette is

then rinsed twice with approximately 10 mL with the solution to be delivered. Each volume of rinse

solution should come in contact with all of the inner surface of the burette by rotation of the burette

while it is held in a nearly horizontal position, and then passed through the burette tip into a

container for waste solutions.

• With the stopcock closed, the burette is filled with solution, using a clean funnel if necessary, to

slightly above the zero mark. Solution is passed through the tip and any air bubbles in the stem are

eliminated by giving the burette a rapid downward shake with the stopcock open. If necessary, the

liquid level in the burette is topped up until it is just below zero.

• A period of 15 seconds is allowed for the meniscus level to settle and the initial volume reading is

made to the nearest 0.01 mL (the initial volume need not be zero exactly--in fact, this is time-

consuming to achieve). In taking a volume reading, the meniscus should be exactly at eye level in

order to avoid error due to parallax.

• A sheet of white paper should be placed below the flask, since such a background aids in

determining the end point. Also, a meniscus reader, which is a card consisting of a solid white

section and a dark coloured section, may be helpful in making volume readings. To use the reader,

the meniscus is viewed at eye level height with the card held behind the burette such that the black-

white interface is lined up exactly with the bottom of the meniscus.

• Keeping the tip of the burette well below the top of the flask containing the sample, the titrant

solution is added from the burette to the sample at a rate which does not exceed that of the

22

dissipation of the colour produced in solution. The rate of addition of titrant solution is controlled

by opening the stopcock with the left hand (for right-handed people) to the desired degree. The

flask containing the sample being titrated is swirled constantly with the right hand (for right-handed

people). Left-handed people should use the left hand to swirl, the right hand to operate the stopcock.

• As the end point approaches, the colour developing in the solution being titrated will persist for a

longer period of time. At this point, the addition of titrant is stopped, the walls of the flask are rinsed

down with a jet of deionized water, and the titrant solution is added one drop at a time with

thorough mixing after each addition until the end point is reached. The volume of titrant solution

remaining in the burette is recorded, and the amount of solution required for the titration is

calculated as the difference between the initial and final volumes.

• Do at least three samples. One strategy is to titrate the first (usually the smallest) sample rapidly,

intentionally over-running the end point. This will give you an idea of about how much titrant you

will need for the other samples.

• Titrate the others quickly to about 1 mL before the expected end point, and then proceed drop wise

to the end-point.

• If the precision is low with the two determinations, or if a gross error occurred (i.e you over/under

ran a sample, or a splash or spill occured), perform another titration so that you have at least two

"true" measurements to work with.

Errors associated with titrations:

• Error in each volume measurement arising from the estimation of the last digit and the accuracy of

burette calibration.

• The appearance of a coloured end point means a slight excess of one of the reactants.

• The non-quantitative transfer of reactants into the flask.

23

Significant Figures

No measuring instrument is completely without error, every measured quantity reflects some degree of

uncertainty. If several measurements are used in a calculation, the accuracy of the final result is limited by

the least reliable measurement. The numerical result must not suggest greater accuracy than the

experimental data warrant.

If a volume is recorded as 23.15 mL, the implication is that the 2, 3 and 1 are known with certainty, and that

the 5 is doubtful. This implies that the exact volume lies between 23.16 mL and 23.14 mL (an uncertainty of

1 part in 2,315). The last written number is assumed to be estimated and uncertain.

Zeroes can be used both to represent a quantity and to fix a decimal point. When used for the latter purpose,

the zero is not considered a significant figure. A zero at the end of a number may or may not be significant.

In the volume, 1,600 mL, both zeros are significant if they are used to indicate measured figures (i.e., the

exact volume between 1,599 mL and 1,601 mL), but not if they are used only to indicate the decimal point.

Such an ambiguity disappears if the quantity is expressed exponentially (see below). If the measurement is

reliable to two significant figures, it would be recorded as 1.6 x 103 mL; but if four figures were significant,

it would be expressed as 1.600 x 103 mL. A zero appearing between other digits is always significant. An

exact number, e.g., 100 cm/meter is considered to have an unlimited number of significant figures.

The following rules governing the handling of significant figures are useful:

Addition and Subtraction The last significant figure in the result is determined by the column containing

the first doubtful figure.

37.25 mL (4 significant figures)

Measured volumes 3.5 mL (2 significant figures)

0.92 mL (2 significant figures)

Calculated volume 41.7 mL (3 significant figures)

The direct result of the addition, 41.67 mL, is incorrect because the second column contains an uncertain

digit, and therefore that column must become the last one in the final result. The number in the uncertain

column is rounded according to the Convention for Rounding Off which is described on the next page.

Multiplication and Division . The number of significant figures in the answer is determined by the quantity

that contains the least number of significant figures.

Measured values 2.024 g/moles/litre (4 significant figures)

x 2.55 litres (3 significant figures)

Calculated value 5.06 g/moles (3 significant figures)

24

The truth of these rules may be proved by identifying the uncertain digits in differently coloured ink

while working through the calculation. The result of any number added to, subtracted from, or

multiplied or divided by an uncertain digit is uncertain.

Convention for Rounding Off:

If digit to be dropped is above 5, round previous digit up. If it is below 5, leave previous digit as is. If the

digit to be dropped is 5 with digits following it, round previous digit up. If it is 5 with no digits following

it, and the previous digit is even, leave previous digit as is. If the previous digit is odd, round the

previous digit up.

Graphing

Graphing allows the determination of an average trend in data measurements, all of which have some error

associated with them and no way to tell which are the most accurate.

Graphical methods are often used in chemistry, as well as many other fields. Most data are subject to easier

interpretation as a result of the visual presentation, but perhaps the most common use is in converting the

dependence of one variable on another by means of a mathematical curve fitting algorithm. In many

instances the dependence between variables is best expressed in terms of a first order or linear relationship.

It is fortunate that the linear relationship is common, as the data can generally be treated with greater

certainty than with higher order (curved) relationships. If, as they often do, the slope and intercept have any

physical significance, graphic methods provide an easy method of effectively averaging data via the familiar

linear equation y = mx + b, where m is the slope and b is the intercept. Additional data can also be extracted

based on how well the individual data points relate to the line of best fit. Many different computer programs

are available today that will perform graphical analysis of the experimental data. In part II of the pre-lab

assignment you will be working with a common computer application; Microsoft Excel, which has very

powerful graphing functions.

Basic rules for graphing:

• Plot the independent variable on the x or horizontal axis (i.e. time, concentration) and the dependent

variable (i.e. absorbance, pH, peak area ) on the y or vertical axis.

• Select proper scale.

• If one of your variables is time, you will always have a starting point at t = 0, on that axis.

• Label the axes properly (quantity plotted and the units used).

• Title your graph. (Be specific.)

• For linear graphs perform linear regression on the data to determine the "line of best fit" and the

equation of a line

• Always include data table used to plot graph.

• Provide the name and the version of the program used in the analysis.

25

INTRODUCTION TO GC AND HPLC ANALYSIS

Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC) are the most popular

and the most efficient forms of quantitative chromatographic separations. In both, the stationary phase is

held in a narrow tube of varying length (length depends on the type and ease of separation), and the mobile

phase is forced through the tube under pressure. The basic process of separation is relatively simple as can

be seen in Fig 1.

Fig. 1 Basic Steps in Chromatographic Separation

In step one, the sample containing a mixture of components is introduced usually through an injection port

into the continually flowing mobile phase. A tight band of solute molecules is formed at the head of the

column. In step two, the sample is carried down the column by the mobile phase. Components in the sample undergo

repeated interactions or partitions between the mobile phase which can be gas or liquid and the stationary

phase which is either liquid or solid. Since each component in the sample interacts in a slightly different

way with the stationary and the mobile phase, the sample components become gradually separated into

bands and emerge from the column in order of increasing interaction with the stationary phase. This means

that the components that have weak interactions with the stationary phase elute first while the components

26

that are strongly retained by the stationary phase elute last. There are two prominent types of interactions

between the solute molecules and the stationary and the mobile phase:

A. Polar interactions arise from the permanent or the induced dipoles. Here polar molecules interact

preferentially with more polar stationary phase. Example of the permanent dipole interaction is

hydrogen bonding. The most popular polar stationary phases are silica based which have a large

number of polar Si−OH groups present on the surface. If a polar molecule such as methanol

(CH3OH) is injected on to the column, it will move slower down the column than the nonpolar

hexane C6H14 because methanol can form hydrogen bonds Si−O---H−O−CH3 with the silica. We

say that methanol is adsorbed onto the polar stationary phase.

B. Dispersion forces give rise to the interaction where the nonpolar molecules interact preferentially

with the nonpolar phases.

The general rule for selecting the stationary phase is LIKE WITH LIKE. It is therefore very important to

know the properties of the solute molecules so that a proper stationary and the mobile phase can be chosen.

In cases where the properties of the sample molecules are not known, one must first determine solubility and

therefore polarity of solutes as well as the boiling point range to be able to choose a proper chromatographic

method.

In step 3, the separated components elute from the column and enter the detector, which sends a response to

a the data processing unit (Computer Application). Results are plotted in the form of detector response

versus the time taken for each component to be eluted. This type of plot is known as a chromatogram. Fig. 2

shows a typical chromatogram. A chromatogram provides a large amount of information which is used to

identify and quantitate individual components as well as to establish and improve the efficiency of the

chromatographic separation.

Fig. 2 Example of a Typical Chromatogram

27

Information Obtained from a Chromatogram.

Retention time (tR) is the time it takes from the sample injection for the analyte peak to reach the detector.

It is the total time the analyte spends in the column and is different for each resolved component. The

retention time consists of two parts: t0 and tR. t0 is the time from sample injection to emergence of an

unretained component (usually air or sample solvent). It is the time the sample spends in the mobile phase.

The adjusted retention time t' R is the time from the unretained peak to the component peak maximum. It is

the time that the sample spends in the stationary phase (refer to Fig. 2)

0' ttt RR −=

Peak width, w, is a measure of the length of time taken by the sample to pass the detector. It is a measure

of how much the sample has spread or has diffused as it travelled down the column.

Partition Ratio or Capacity Ratio k' is one of the most used parameters in column chromatography. It

describes the migration rates of the solute on the column and is a measure of how well sample molecules are

retained by the column. k' is obtained by dividing the time the component spends in the stationary phase by

the time the component spends in the mobile phase.

0

''

t

tk R= or

0

0'

t

ttk R −

=

k' should be different for each component in the analysis. Ideally k' should be in the range between 2 to 20.

If it is less than one, the elution is too fast and determination of retention time is difficult. If it is over 20, the

solute is retained too long on the column, resulting in band broadening and therefore poor separation

efficiency. In gas chromatography, k' can be varied by changing the temperature and the column packing. k'

is usually lowered by increasing the column temperature. In liquid chromatography, k' can be varied by

changing the composition of the mobile phase and the type of the stationary phase.

Resolution - R measures how well two closely eluting peaks are separated or resolved. R is equal to ∆t, the

time between peaks, divided by the average peak width and can be obtained from a chromatogram.

)(5.0 21

12

ww

ttR RR

−−

=

∆∆∆∆t, w1 and w2 all have the same units in the same units making R unitless.

28

Types of Chromatographic Results

1. Qualitative Qualitative analysis is used to confirm the presence or the absence of a particular component. In a fixed

chromatographic system (constant flow, temperature, column, injection volume and rate of injection),

retention time, tR is a constant for a particular solute and it is therefore possible to identify the separated

components of a complex mixture by comparing their retention times with those of a pure standard. There

are two ways of identification when using a non specific, or universal detector.

a) Comparison to a Pure Standard. Separate chromatograms are obtained for the sample and the

standard under the identical chromatographic conditions and then the retention time of the sample

peak is compared to the retention time of the standard. In this way, absence of the particular

component can be positively confirmed by the absence of a peak at a particular tR, but if the

component is present, the positive identification of the unknown sample requires a second more

specific method.

b) Standard Addition Method. First, the chromatogram of the sample is obtained and the retention

times are recorded, then the pure component (standard) that is being identified is added to the

sample and the sample is analyzed again. If the identification is positive, the peak height of the

component in question should increase by the amount of the standard added.

The major drawback in the qualitative analysis is the need to anticipate the presence of the specific

compounds in the sample and the requirement to have available the pure reference standards. The second

drawback is the requirement for the second method of identification. However, this drawback can be

eliminated by coupling the chromatographic column directly to a specific detector such as an infrared

detector (LC-IR) or mass spectrometer (GC-MS, LC-MS), both of which can provide a positive

identification of the components and in most instances quantitate the results.

29

2. Quantitative

In quantitative analysis, one measures either the height or the area of the analyte peak. However, it is not

enough to just obtain the height or the area of the analyte peak since this value by itself tells us nothing. To

be meaningful, this value must be compared to the peak height or the area obtained for a known quantity of

the same substance (standard).

Peak height measurements are obtained by connecting the baselines on the two sides of the peak by a

straight line and measuring the perpendicular distance from this line to the peak maximum. Peak height

measurements can be of high precision and accuracy if factors such as: temperature, flow rate and rate and

volume of sample injection are kept constant for the duration of sample and standard analyses. Advantages

of this method are ease of measurement and since only one measurement is involved smaller measurement

error is introduced. For narrow reproducible peaks, peak height measurement can produce more accurate

determination than peak area.

Peak area measurements are independent of peak broadening effects and are therefore more widely used in

quantitative analyses. For symmetric peaks, area is calculated as follows:

A = peak height x peak width at half height or A = peak height x 1/2 peak width

Computers or integrators are most often used in determination of peak areas. Computers use change of slope

from zero to positive value as a start of peak integration and a change of slope from negative to zero value as

an end of integration. It is therefore essential that proper slope threshold is defined in this type of analysis.

Standard Calibration Plot: A series of standard solutions similar in the composition to that of an unknown

are prepared. Ideally the concentration of the standards used should bracket that of the unknown sample.

The chromatograms of the standards are obtained and the peak heights or the peak areas are plotted as a

function of concentration. A plot should be a straight line passing through the origin. Peak height or area of

the sample of interest is then measured and the sample concentration is interpolated from the graph. The

most significant source of error in this method is the uncertainty associated with injecting a reproducible

volume of sample and standards, where volume can be as small as 0.1 µL.

30

Internal Standard: This procedure involves addition of a known quantity of internal standard into each

standard and sample flask, obtaining a chromatogram of each solution and preparing a calibration graph by

plotting the ratio of analyte peak area or height to the internal standard peak area or height against the

standard concentrations. The analyte concentration in the sample is then determined from the calibration

graph. The internal standard must possess the properties similar to those of the analyte such as boiling point,

polarity and structure in order to behave in a similar manner during analysis. The internal standard peaks

must be well separated for accurate measurements but must appear close enough to the analyte peak in order

to experience similar line broadening. Because uncertainties introduced by the sample injection and any

losses during extraction, preconcentration and sample preparation are compensated for, this method of

measurement achieves the highest precision in chromatographic determinations.

31

Preliminary Lab Exercise – Part 1 Calibration of Eppendorf®Pipette

Pipettes allow transfer of accurately known volume of solution from one container to another. Volumetric or transfer pipettes deliver a single fixed volume of liquid.

Measuring pipettes are calibrated in convenient units and permit delivery of variable volumes of

liquid up to a maximum capacity.

Digital pipettes deliver adjustable microliter volumes of liquid. In recent years digital pipettes have

become the most widely used mode of accurate liquid transfer. There are several manufacturers of

digital pipettes. In this laboratory you will be using Eppendorf digital pipettes.

Digital pipettes operate on the principle that a known and adjustable volume of air is displaced from

a disposable tip by depressing a button on the top of the pipette to a first stop. Digital pipettes are

easy to use and deliver consistently reproducible, and when calibrated, highly accurate volumes of

solution. Due to the mechanical nature of these pipettes, the accuracy may change over time due

to the mechanical component wear or to misuse by the operator. In order to have confidence in

the accuracy of the volume delivered, digital pipettes should be calibrated on regular basis.

In this lab you will learn correct procedures for operating and calibrating of Eppendorf pipettes. Operation of Eppendorf Pipettes Volume setting: The volume is adjusted continuously by turning the setting ring. The digits of the

volume display should be read from top to bottom.

Pipette tips: Pipette becomes a functional unit only when a suitable tip is attached. The color of

the control button of the pipette matches the color of the tip or the tip rack.

Filling (Aspirating):

1. Securely attach appropriate pipette tip.

2. Press control button down to first stop (measuring stroke).

3. Hold pipette vertically and immerse tip approximately 3 mm into the liquid.

4. Let control button glide back slowly, wait 3-5 sec. for the liquid to enter the tip. Be careful to

not let the tip come out of the liquid during this step, or liquid can enter the air displacement

cylinder which can cause permanent damage to the pipette.

32

5. Slide the tip out of the liquid along the inside of the vessel. Wipe off any droplets with lint-free

tissue. Ensure that no liquid is aspirated out of the tip.

Note: if the tip is removed from the liquid too quickly, coaxial forces may push liquid out of the tip.

This may result in the pipetted volume being too low.

Warning!! Never lay the pipette down with liquid in the tip as liquid can flow into the pipette

Emptying (Dispensing):

1. Hold the tip at an angle against the inside of the vessel.

2. Press control button slowly down to the first stop (measuring stroke) and wait until no more

liquid is emptied.

3. Press button down to second stop (blow-out) to empty tip completely.

4. Hold down control button. Slide tip out along the inside of the vessel.

5. Let control button glide back.

6. If you have completed all dispensing of the liquid eject the tip by pressing the lateral tip

ejector button.

Calibration

1. Firmly attach an appropriately sized pipette tip onto the pipette.

2. Adjust to the desired pipette volume. Calibration should be carried out at three volumes:

nominal, 50% and the smallest volume. For example, for 1000 µL pipette, calibration

should be done at 1000, 500 and 100 µL (see Table 1 below for the calibration volumes for

each pipette).

3. Place a receiving vessel onto the pan of the Analytical Balance. Close any open doors and

Zero the balance. (Wait until balance stabilizes)

4. Aspirate and dispense three volumes of distilled water into a waste beaker.

5. Open the top sliding door on the analytical balance. Aspirate the volume of distilled water into

the pipette. Carefully insert the pipette from the top of the balance and dispense the test volume

slowly and uniformly up to the first stop and wait for 1-3 sec.

6. Press the control button to the second stop and dispense any liquid remaining in the tip.

7. Hold down the tip and gently touch it against the weighing vessel.

33

8. Remove the pipette from the balance and close the sliding glass door. Record the weight

after the display has come to a standstill.

9. Perform 10 measurements at each assigned volume. Calculate the inaccuracy and the

imprecision. (See equations below.)

10. Enter the calculated values on the sheet provided. Compare your values to those provided by

the manufacturer. See Table 1.

Note: In order to achieve good accuracy and precision for volumes less than or equal to 10 µL, a

new tip should be used each time (do not pre-rinse) and the sample should be dispensed into a

weighing vessel that already contains a volume of the liquid (i.e. 1.0 mL).

Table 1. Technical data for Eppendorf® Series 2100 Pipette

Model Color of Increment Volume Systematic Error Random Error Button µµµµL µµµµL ( Inaccuracy) (Imprecision;

CV) 0.5 - 10 µL light gray 0.01 1 ± 2.5% ≤1.8% 5 ± 1.5% ≤0.8% 10 ± 1.0% ≤0.4% 10 - 100 µL yellow 0.1 10 ± 3.0% ≤ 1.0% 50 ± 1.0% ≤ 0.3% 100 ± 0.8% ≤ 0.2% 100 - 1000 µL blue 1.0 100 ± 3.0% ≤ 0.6% 500 ± 1.0% ≤ 0.2% 1000 ± 0.6% ≤ 0.2% 500 - 5,000 µL violet 5.0 500 ± 2.4% ≤ 0.60% 2500 ± 1.2% ≤ 0.25% 5000 ± 0.6% ≤ 0.15% Liquid : bidistilled water, degassed Room temperature: 20 -25 oC Number of determinations: 10 Table 2. Factor Z (µµµµL/mg) as a function of temperature and air pressure: Temperature oC Pressure (mbar)960 Pressure (mbar) 1013 Z Z 21.0 1.0030 1.0031 21.5 1.0031 1.0032 22.0 1.0032 1.0033 22.5 1.0033 1.0034 23.0 1.0035 1.0035 23.5 1.0036 1.0036 24.0 1.0037 1.0038

34

For an assigned pipette enter your experimental data and complete the following questions on

the tear out sheet provided. Show all sample calculation on the back of the sheet.

1. Convert to actual volume (VI) dispensed by multiplying each weighing by a correction factor Z

(µL/mg). See Table 2 above.

(V i) (µL) = M (mg) • Z (µL/mg)

2. Calculate the mean actual volume delivered (V ):

(V )(µL) =( )n

Vi∑ where n = number of measurements

3. Calculate the standard deviation (s) for the actual volume delivered:

(s) = 2

1)(

1

1xx

n

n

ii −∑•

− =

4. Calculate % Inaccuracy (d): (d ) = nominal

nominal

x

xx − • 100

5. Calculate the Coefficient of variation or Imprecision (CV):

(CV) (%) = V

s • 100

6. Calculate the difference between maximum and minimum value or Range (R):

R(µL) = Vmaximum - Vminimum

7. Compare your results to manufacturer’s specifications in Table 1.

8. Discuss sources of error in the calibration procedure.

35

Micro-pipette Assigned ________ uL Name:____________________________

Volume measured ______ µµµµL Measured Mass Volume Delivered ( ix - x )2 mg µµµµL 1. 2. 3. 4. 5. 6. 7. 8. 9. Random Error Systematic Error 10. Imprecision % Innaccuracy Range Mean Volume SD CV (100%) d R Report: _______ ___ ________ ________ ________ ________

Volume measured ______ µµµµL Measured Mass Volume Delivered ( ix - x )2 mg µµµµL 1. 2. 3. 4. 5. 6. 7. 8. 9. Random Error Systematic Error 10. Imprecision % Innaccuracy Range Mean Volume SD CV (100%) d R Report: _ _________ ________ ________ ________ ________

Volume measured ______ µµµµL Measured Mass Volume Delivered ( ix - x )2 mg µµµµL 1. 2. 3. 4. 5. 6. 7. 8. 9. Random Error Systematic Error 10. Imprecision % Innaccuracy Range Mean Volume SD CV (100%) d R Report: _______ ___ ________ ________ ________ ________

36

Preliminary Lab Exercise -Part 2 Basic Instructions on use of Excel for Mathematical Calculations, Statistical Analysis,

Generation of Regression Data and Graphing Spreadsheet applications are an extremely useful tool for carrying out a variety of computational tasks in analytical chemistry. There are a variety of spreadsheet programs available, such as Microsoft Excel, Quattro Pro, Lotus 1-2-3 etc. You will only be introduced to the basics of Excel in this course; however, you may use any available spreadsheet. The following steps are a general outline and not intended to be a complete step by step procedure. Your own personal experience and understanding of spreadsheets may be of assistance, and if you have none this should provide you with a relatively simple introduction. Your Analytical text also has a quite useful section on the use of Excel. Excel can be used to analyze experimental data by performing a regression analysis (linear) to obtain the slope, intercept, and the equation of the line. It can also generate a graphical representation of the experimental data with a line of best fit, and the above parameters can be displayed on the graph. Further uses of Excel are to create a calibration curve; to compute mean; standard deviation (SD); pooled standard deviation (PSD); relative standard deviation (RSD); and coefficient of variation (CV). Calibration Curve for Two Dimensional Data: The Least-Squares Method Calibration Curves are used in many analytical methods. For example, a measured quantity y (dependent variable) is plotted as a function of the known concentration x (independent variable) for a series of standards. By plotting the measured value (y) for an unknown sample, its concentration can be extrapolated based on the corresponding value of x. In this exercise you will construct a calibration curve for determination of caffeine in an unknown caffeine sample. The ordinate (the dependant variable) equals the area under the chromatographic peak for a series of caffeine standards, and abscissa (the independent variable) equals the concentration of the caffeine standards in mg/L. It is often easy to identify that a linear relationship exists between a dependent variable y and an independent variable x, which is represented by equation (1):

Y = mx + b (1) Where m is the slope and b is the y-intercept (where x = 0) Due to the indeterminate errors introduced during the analytical process, not all data points will lie exactly on the straight line. The analyst must therefore draw a line of best fit through the points. The most widely used and least biased approach is the statistical method of regression analysis, which also allows determination of uncertainties associated with the results. For a two-dimensional data set, a simple regression method of least squares is used.

Use of Excel functions to calculate slope, intercept, and equation of the line or to determine the concentration of an unknown from a calibration plot

For calibration calculations data is normally entered into two separate columns. The top of one column should be labeled for the ‘x’ values (this is a usually the independent variable such as time, speed or

37

standard concentration). The second column is then labeled for the ‘y’ values (this is normally the dependent variable, or what you measure, i.e. response from the measurement such as Absorbance, peak area, current etc.). Each value entered has its own cell address based on the spreadsheet array e.g. C5, C6, C7 etc… indicating column C row 5, then 6, then 7 etc…Cell addresses are used in equations, or functions where they represent the specific values in the cell. For example if you want to add the values of three cells in cells A3 to A5, you could type in cell A6 “=A3+A4+A5” or you could insert the function “=Sum(A3:A5)” Note: Cell addresses can be inserted into functions using mouse clicks for individual cells, or by selecting a range of cells i.e. highlighting cells A3 to A5 while the curser is in the brackets of the =Sum( ) function. For calibration data where we have ‘x’ values in one column, and ‘y’ values in another, we can readily determine the values for the slope and y-intercept for a line of best fit using functions that are built into Excel. Once we have established these values, the linear equation can then be arranged to solve for sample values by entering the dependant or measured ‘y’ values into the expression and solving for ‘x’. The program function LINEST allows for the calculation of the slope (m) of the line for the data entered. The format of this function is as follows: =LINEST(cell range for y, cell range for x) The program will return the slope of the line in the cell where you entered this information. Use the program function INTERCEPT to obtain the intercept of the line for the data entered. Follow the format: =Intercept (cell range for y, cell range for x) The program will return the intercept of the line in the cell where you entered this information. Note for advanced users: The Linest function can provide a matrix of values that includes the slope, intercept, the standard deviations associated with each, and the correlation coefficient for the line of best fit. For further information consult the least squares section of your textbook. In order to determine sample ‘x’ values the regression equation must be rearranged to x = (y-b)/m. For example you should have a cell that contains information resembling: =(A10-F8)/G8 where A10 is the ‘y’ data measurement for the unknown, F8 is the cell containing the intercept value and G8 is the cell containing the slope value. The formula will return the ‘x’ value, which is the concentration of the unknown. A graphical display can be made for your data by highlighting the cells that contain the calibration values for ‘x’ and ‘y’, and then selecting the chart function from the toolbar, or from the Insert Menu, and selecting the X-Y Scatter chart type. On the chart you can add the line of best fit (a.k.a. the trend line), and the corresponding equation can be displayed. In the exercise that follows you will be using the above functions and features of Excel.

38

Exercise #1a: Performing a Regression Analysis and Determining the Concentration of an Unknown

Sample

Launch the Excel application and set up a spreadsheet as shown below. The standard concentration values (x) are in cells A2 to A5, with corresponding Peak Areas (y) in Cells B2 to B5.

1) Use both methods to calculate the slope and intercept:

Method 1 - calculate the slope (m) and the intercept (b) for the above data as follows: click on cell C2 and in

the formula bar type =LINEST(B2:B5,A2:A5); click on cell D2 and in the formula bar type =INTERCEPT

(B2:B5, A2:A5); the calculated values will be the slope and the intercept.

Method 2 - click on cell C3, then select Insert ⇒Function from the menu (or select f(x) from the tool bar if

it is visible). Select Statistical as function category, then LINEST as a function name. Next, you will be

asked to enter cell ranges for y and x. You can type in the values i.e. B2:B5 for the y-values, or you can use

the mouse and select the cell range on the spreadsheet. On the bottom of the box you will find a function

definition which can be helpful for some functions. Click o.k. when finished. The value for the slope will

appear in C3. It should be the same as in C2. Follow a similar procedure for the intercept in cell D3 (note

that other functions can also be done using both methods).

2) Cells B8 to B10 contain the data collected for three replicate samples. In order to determine the

concentrations in mg/L for these samples the rearranged linear equation x =(y-b)/m must be entered into the

corresponding cells in the A column. Click on cell A8 and in the formula bar type = (B8-D$2)/C$2 which is

equal to rearranged formula.

(Note: The $ in front of the number indicates a constant, in this case the slope and intercept are constants.)

Click on A8 then pull down to highlight cells A9 and A10, select Edit ⇒Fill ⇒down from the menu (or

Ctrl+D for Windows based systems). This will enter formulas and calculate concentrations for the two

39

remaining samples. Note that in the formula for A9 the value for ‘y’ is automatically adjusted for the

corresponding cell address B9, but the cell addresses for the slope and intercept stay the same, as they were

defined as constants.

Alternate way, highlight cell A8, select Edit ⇒Copy, or in the tool bar click on Copy (two page icon).

highlight the remaining cells A9 and A10 and select Edit ⇒Paste or click on Paste in the tool bar

(Clipboard icon).

3) Calculate the Mean sample concentration; In cell A12 enter the column label ‘Mean’ then click on cell

A13 and in formula bar enter = SUM(A8:A10)/3, or type =AVERAGE(A8:A10), or select Insert ⇒Formula

and Select AVERAGE from the list, then insert the cell range.

4) Calculate the Standard Deviation (SD or s); click on cell B12 and enter the Label ‘STDEV’, click on cell

B13 and in the formula bar enter =STDEV(A8:A10), or use the insert function method. This will calculate

the standard deviation for the three sample concentration. Next, ensure that you report correct number of

significant figures for Cells A13 and B13, using one or two significant figures for the SD. (The Mean should

not have more significant figures then is indicated by standard deviation. For example if the mean value

reads 25.6778 and the SD is 0.246, then the result should be reported as 25.68 ± 0.25 or 25.7 ± 0.2. If your

result indicates that the number of decimal places needs to be adjusted then select Format ⇒Cells

⇒Number and select the appropriate number of decimal places for the cell, or click on the tool bar icons

that show a small arrow beside decimal zeroes to increase or decrease the number of decimal places

displayed.

*NOTE: When reporting your experimental results you must always report the correct number of significant

figures in your mean, SD and 95% CI. Marks will be deducted if incorrect significant figures are reported.

5) Calculate coefficient of variation (CV) for the results. CV = x

sx 100%

Exercise 1b: Using the Charting Features of Excel to Generate a Calibration Curve and to Perform a

Regression Analysis

An alternate way of analyzing experimental data is to create a chart (graph) for the data and to perform an

automatic linear regression on the graph. In this part of the exercise you will be plotting two sets of data on

the same graph, and then you will use the built in functions to add the lines of best fit, the equations for the

line, and the correlation coefficients, R2 (this indicates how well the data fits the line with values that range

from 0 to 1, when R2 equals exactly 1 it indicates a perfect fit. In most cases the value will be less than 1).

The displayed equations will then be used to calculate values for unknown samples in a spreadsheet.

40

Data Set 1 Standard Concentration (mg/L) Peak Areas (au) 1 2 3

5.332 10.233 15.399

1023 2103 3022

Sample A 1 2 3

1434 1465 1398

Data Set 2 Standard Concentration (µµµµg/L) Peak Areas (au) 1 2 3

1010.456 5040.214 10050.155

125.645 653.115 1303.321

Sample B 1 2 3

621.340 634.118 626.654

1) In your file from exercise 1A (called a workbook) select Insert ⇒Worksheet from the menu.

Enter the data for the two sets above in a neat and organized manner. You might have noticed that the

concentration values are not the same for the two data sets. For Data Set 2 add an additional column labeled

Concentration (mg/L), and convert the concentration values to mg/L using a mathematical function ( i.e. if

you want a value in cell C15 to be ten times greater than in cell B15 then you can enter =B15*10 into cell

C15, for subsequent operations that are the same in the column below the cell, i.e. C16 etc…, use the Edit

⇒Fill ⇒Down menu).

2) Highlight the x,y data for Data Set 1, then select Insert ⇒Chart, or click on the chart icon from the

toolbar. Select XY Scatter with a sub-type without lines, then click on next.

3) To add a second data set to the chart select the series tab at the top of the window, and then select Add.

Series 2 should then be displayed below Series 1 (For Mac Users: To add a second data set Select Chart

⇒Source Data then select Add under the series box). Set the curser into the x values mailbox, and then

select the concentration values from the worksheet (in mg/L) by selecting the cells. Set the curser into the y

values mailbox (delete any text already there), and then repeat as per the x values. You can also add names

41

to the series which will be displayed in the legend should you decide to add one.You should now see the two

data sets on the chart. Click on Next

4) Add an appropriate title, and axis labels, and click Next. For Mac users titles and axis labels are added

using chart tools and selecting the layout tab.

5) Select New Sheet for the chart location and select Finish. For Mac users select Chart ⇒Move Chart

⇒New Sheet from the menu.

6) Create a line of best fit by selecting Chart ⇒ Add Trendline from the menu. For Mac users, you might

have to select the series by clicking on a data point before going to the Chart menu. Select the desired series

and Linear for the type of fit, then select the options tab and select the Display Equation on Chart and

Display R2 on Chart buttons. Then click OK. Repeat for the remaining series.

7) Use the slope and intercept values displayed on the chart to calculate the concentrations for the unknown

samples on the worksheet, then calculate the Mean and Standard Deviation for the two samples with the

correct significant figures.

Report: When you have completed this exercise print out the entire workbook and hand it into the

appropriate report box labeled with your demonstrator’s name.

42

EXPERIMENT 1

Calibration of Volumetric Glassware, Standardization of 0.1M HCl and Statistical Analysis of

Experimental Results (Read only – complete analysis questions 2-6)

The accuracy of analytical results depends on four factors: proper use of equipment, good

technique, knowledge of the chemistry of the experiment, and the correct calculation and reporting of the

results. In this experiment you will examine the importance of all four factors.

In the first part you will learn how to use and calibrate the analytical chemist's most important tools:

the analytical balance, the burette, the volumetric flask and the volumetric pipette. (Note: before coming to

the lab please read the section in your lab manual "General Introduction to Analytical Techniques and

Procedures" (p 23) which outlines the correct procedure steps for use of the above equipment.

The technique of calibration involves weighing an amount of water delivered by the burette or

contained by the volumetric flask or the volumetric pipette, and converting the weight to volume by making

use of the known density of water at a given temperature. More detailed discussion of calibration techniques

can be found in your analytical text1.2 You should be aware of the effects of temperature on solution

concentrations. Volumes of aqueous solutions increase upon heating and a student who makes up a standard

solution on a cold day is frequently astonished to see the solution well above the calibration mark on the

volumetric flask when the lab warms up. The capacity of volumetric glassware changes very little with

temperature (over a few degrees range), so it is not difficult to calculate the solution concentration at a

temperature different from the temperature at which the solution was made, provided both temperatures are

known. As a rule of thumb, the concentration decreases by about 0.1% (relative) for each 4° C increase in

temperature between 15 and 30° C. This change will not drastically affect an analyst's results except in the

most accurate work.

Safety Considerations

There are no harmful chemicals in this experiment unless you have to use strong acids or bases to

clean your glassware. Concentrated acids and bases cause severe skin burns and eye damage. If you should

spill one of these solutions on yourself, wash it off immediately. Use the safety shower if the spill is large.

You shouldn't get anything in your eyes because you wear safety glasses, but if anything does splash in your

eye, use the eyewash fountain. You will be taken to the hospital so that a doctor can determine the extent of

injury.

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 778-811. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 25-33, 213-290. McGraw-Hill Higher Education Inc., 2000.

43

Waste Disposal of Concentrated Acids and Bases

Dilute concentrated acid or base by slowly pouring it into a large container of water. BE CAREFUL!

This is a strongly exothermic reaction. DO NOT pour water into concentrated acid or base and DO NOT

MIX large quantities of concentrated acids and bases. Neutralize the diluted solution, use pH paper to check

that solution is in the 6-8 pH range, then pour slowly down the sink with lots of water.

Experimental Procedure

Part I: Preliminaries to Calibration of Glassware

All volumetric glassware must be cleaned so that the liquid flows smoothly down the inside walls

without beading up. Ordinary soap solution is often sufficient. If there is a lot of beading, the glassware may

contain a film of grease, which should be removed with a small quantity of cleaning solution found in the

fume hood. (Cleaning solution contains concentrated sulfuric acid, H2SO4, and should be handled and

disposed of in the same manner as concentrated acid). The cleaning fluids should be rinsed out of the

glassware with tap water but the final rinsing should be done with the deionized water.

Obtain from your locker a 500 mL volumetric flask, a 25 mL volumetric pipette, a 50 mL

Erlenmeyer flask and a 1000 mL beaker, and from the rack found at the back right hand side of the lab a 50

mL burette. Fill the clean 1000 mL beaker with distilled water and allow the water to equilibrate to room

temperature. Record the temperature of the water several times while cleaning the remaining glassware to

establish constant temperature. Remeasure the temperature just before performing individual calibrations. A

list of conversion factors used to convert the mass of water at temperature T to the corresponding volume at

the same temperature will be posted in the lab and can also be found in Ref.1 (pp.807).

Clean the remaining glassware as described above. Place your name and date at the top of a clean

burette using the tape provided. This will ensure that you are the only person using the burette for the

duration of this course. It is usually not necessary to dry volumetric glassware, but for calibration purposes,

a 500 mL volumetric flask should be dried. Drying should not be done in the oven since higher temperatures

may deform the glass and therefore change the volume. If the flask is rinsed with ethanol (CH3CH2OH) and

inverted on the bench it will dry much quicker since ethanol evaporates faster than water.

Calibration of a 25 mL Pipette

Review the pipetting technique outlined on page 29. Dry the outside of a 50 mL Erlenmeyer flask

and obtain a rubber stopper that fits it. Weigh the flask and the stopper on the analytical balance and record

the weight in your notebook to four decimal places. Use the cleaned 25 mL volumetric pipette to transfer 25

mol of the temperature-equilibrated water into weighed flask and reweigh the flask. Empty the water from

the flask, dry out the mouth, and reinsert the rubber stopper. Weigh the flask and repeat the sequence of

pipetting and weighing. Repeat this procedure at least three times. Ensure that the outside of the flask and

the rubber stopper stay dry. Record the water temperature. Convert the weights to volumes. The spread on

your results should not exceed 0.02 mL. Larger spreads are due to incorrect technique or to dirty pipettes.

44

Example of lab notebook entry:

Calibration of a 25 mL volumetric pipette

temperature of water 23.0°C

weight of flask + water + stopper 75.0524 g

weight of flask + stopper 50.0121 g

weight of water 25.0403 g

conversion factor at temp. of 23° C 1.0035 mL/g

volume of water at 23°C = 25.0403 g X 1.0035 mL/g

= 25.1279 mL

Therefore, the true volume of the 25 mL volumetric pipette is 25.13 mL.

Calibration of a 500 mL Volumetric Flask

The volumetric flask must be clean and dry. Weigh the 500 mL volumetric flask on a top-loading

balance with a capacity of at least 1000 g ± .00g. Fill the flask to the calibration mark with the temperature-

equilibrated water, then re weigh the flask. Convert the weight of the water to the volume by using the table

of water densities provided in the lab.

Example of lab notebook entry:

Calibration of a 500 mL volumetric flask

water temperature 23.0°C

weight of flask + water 753.05 g

weight of flask 254.00 g

weight of water 499.05 g

conversion factor at temp. of 23° C 1.0035 mL/g

volume of water at 23°C = 499.01 g X 1.0035 mL/g

= 500.7565 mL

Therefore, the true volume of 500 mL flask is 500.76 mL.

Data Analysis

1. Calculate the volume corrections for the two pieces of glassware that you have calibrated.

2. List several sources of weighing error.

3. Report the capacity and the precision of the analytical balance in the analytical lab.

4. Describe the difference between volumetric or transfer pipette and measuring or graduated pipette.

What are the typical tolerances for each? (Ref 1)

5. Use the information from the Material Safety Data Sheets located at the back of the lab and the safety

literature available online, or in the Library to list the hazards, the proper handling and the waste

disposal of the following chemicals: CH3OH, H2SO4, H2O2 and NaCN. Be brief.

45

6. Draw a plan of the analytical lab and clearly mark the locations of the following safety equipment:

safety shower, fire blanket, eye wash, fire extinguishers, lab exits, fume hoods, flammable storage, acid

storage, oxidizing storage shelf, first aid kit, spill clean-up kit, fire alarm and Material Safety Data

Sheets.

Part II: Standardization of 0.1 M HCl and Statistical Analysis of the Experimental Data

Introduction

Any experiment involving the quantitation of data must inherently contain error. Accordingly, we

must learn how to deal with that error, minimize it where possible, and express it properly. To that end, we

employ the science of statistics. The fact is that, without statistical analysis of raw data, we would have

difficulty in determining the degree to which our experimental data can be trusted. Statistical analysis does

not produce new results. It simply helps us to interpret the results honestly.

A stock solution of ~0.1 M HCl has been prepared for use in this experiment as well as in one in the

future. For the purpose of demonstrating the statistics of experimental error, you will determine the

concentration of this stock solution by simple titrations of a known quantity of basic material with the acid.

Each student will perform four titrations. The results from each student will be pooled into a single large

data set. Each student must have the complete data set to work on for their lab report.

The raw data will be analyzed statistically and a value for the concentration of the standard HCl

solution, along with the estimated error in that value, will be reported.

Statistical Evaluation of Results

The statistical evaluation takes place in several steps. Terms will be defined as necessary in this

portion, however, bear in mind that the major portion of the details has been omitted. You should refer to

your text1 (pp. 11-20 and 21-44) on statistics for clarification and elaboration. There are two types of errors

to be encountered in experimentation: determinate and indeterminate.

Determinate or systematic errors are those which can be identified as arising from a particular

source, often affecting all of the measurements in the same way. For example, if you are measuring time,

and your watch is 3 seconds fast, then all measurements will be 3 seconds fast. This is an instrument error.

Similarly, there are errors in the calibration of everything from glassware to sophisticated electronic

equipment. There may also be method errors, which arise because the method presumes ideal behavior,

while the reality is non-ideal behavior. The titration process requires that you titrate the weighed amount of

base plus the five drops of indicator, which you have added. This error is intrinsic to the method. Finally,

there may be human error, due to lack of skill (i.e. incorrect reading of volume in a burette), oversight, or

simple carelessness.

Indeterminate or random errors arise from minute variations in each experiment which are not

reproducible. Examples are the minute fluctuations in the power supplied on the standard wall socket power

outlets, or the variations in room temperature and pressure during the course of an afternoon. These

46

variations impose a small, non-reproducible uncertainty in measurements, and result in a small scatter of

data about the true, error-free value. The spread of results is usually a Gaussian (bell-shaped) curve. It is the

random or indeterminate error of the experimental results that will be determined using statistical analysis.

Common Calculations Carried Out in Statistical Analysis of Analytical Results

1. Determination of whether any of the data should be rejected from the set, on the grounds that they fall

outside the 95% confidence interval. This is also known as the Q-test, where Q is the rejection quotient:

Qexp = w

xx nq−

xq = the experimental value which is being called into question

xn = the experimental value which is nearest in magnitude to xq

w = spread or range of the entire data set (including the questionable data)

The value Qexp is compared to the value Qcrit which has been tabulated for the desired confidence

interval and number of measurements. See for example Table 4-4, (p 58) in your text1. The

experimental value is rejected if its quotient, Q exp, is greater than the critical quotient Qcrit.

Your first rough value may fall outside the confidence limits. This would be the result of the method

(rapid titration), and its rejection does not pose any problem. If you are careful with the remaining

titrations, they should be fairly reproducible.

2. Calculation of the sample mean (equation 2), the sample standard deviation (equation 3) and the

confidence interval (equation 4) on your own data:

Sample Mean (−x ) =

∑i=1

N (xi)

N (2)

Sample Standard Deviation s =

∑i=1

N

(xi − −x)

2

N − 1 (3)

Confidence Interval = −x ±

t.sN

(4)

−x = mean or average value of the HCl concentration.

47

N = the number of measurements.

t = the appropriate t-value for the Student's t-test, for the number of measurements you have averaged

(N) and the desired confidence interval (95%). For 95 % CI student t is 4.30 for 2 degrees of freedom

and 3.18 for three degrees of freedom. For more detailed explanation and more t values see Table 4-2,

(p. 50), Ref1, and read the section on the t-test.

s = the standard deviation of your results, given by equation 3.

3. Calculation of pooled standard deviation sp :

Spooled =

∑i=1

N1

(xi − −

x1)2 + ∑

j=1

N2

(xj − −

x2)2 . . .

N1 + N2 + N3 . . . − Ns (5)

where N1 = the number of measurements in the data set of student number 1, etc., and

−x 1 = the mean of the data in set number one

Ns = the total number of data sets which are being pooled

The purpose of pooling data is the same as the repetition of each titration many times, that is, to

increase the reliability of the results.

Note: When you do these calculations be aware of round-off errors and acceptable numbers of

significant digits. Never round a standard deviation until the end. It is incorrect to report more

significant digits than the experiment warrants and you will lose marks accordingly.

Experimental Procedure

Materials

Indicators: Methyl Orange (Already prepared in dropper bottles)

Titrant: ~ 0.1M HCl

Primary Standard: Sodium Carbonate- Na2CO3 F.W. 105.989 g/mol

Gassware: 250 mL Erlenmeyer flasks, 50 mL burette, burette holder, analytical balance, 250

mL beaker, spatula,

1. Clean and dry on the outside, three 250 mL Erlenmeyer flasks.

2. Obtain a weighing bottle containing previously dried primary standard sodium carbonate from the

desiccator found on the back counter in the lab.

3. Check that the pan on the analytical balance is clean and that the doors are closed. Next, place the dry

Erlenmeyer flask in the center of the pan, close the door and press re-zero on the balance bar. Balance

should read 0.0000. The fluctuations in the last decimal point should not be more than ± .0001g. Open

48

the balance door and, using a spatula, carefully transfer between 0.21 and 0.22 g of Na2CO3 (e.g.

0.2123 g). Close the door, wait until balance reading stops fluctuating, then record in your notebook the

mass of Na2CO3 to four decimal places. Remove the flask, rezero the balance, check that the pan is

clean (use the brush provided if necessary) and close the door. Use the same balance for all the

weighings.

4. Add 25 mL of deionized water to the flask and dissolve the sample by swirling the flask.

5. After the sample has dissolved completely add 5 drops of methyl orange indicator solution and swirl the

flask to mix the indicator in the solution.

6. Rinse the calibrated burette with small amount of 0.1 M HCl solution. Then fill the burette to

somewhere between 0.00 and 0.50 mL mark using the burette funnel. Check that no air bubbles are

present in the tip of the burette. Allow 30 seconds for liquid to drain, then take initial reading to the

nearest 0.01 mL.

7. Do a quick "rough" titration to establish the approximate endpoint volume. The endpoint is indicated by

the solution color change from yellow to orange. The rough titration will enable you to do the rest of the

titrations more efficiently. If the "rough" endpoint volume is about 40 mL, then you can safely add ~36

mL of titrant quickly, for each subsequent titration, and then go slowly for the last few mLs to establish

the endpoint as precisely as possible. You may have to perform a Q-test in order to determine if the

rough titration should be included in your results.

8. Repeat the titration three more times. For each titration record the initial and the final burette readings

to the nearest 0.01 mL. Record also the color changes observed during the titration process.

9. Perform a blank titration by titrating 50 mL of 0.05M NaCl (provided) instead of the carbonate standard.

Subtract the blank volume from the titration volume.

10. Calculate the concentration of the HCl solution for each titrations. You will need to consider the

following information: Carbonate ion can be titrated to two equivalence points.

1st equivalence point (pH~8.4): CO32- + H3O+ HCO3− + H2O (5)

2nd equivalence point (pH ~4.0): HCO3- + H3O+ H2CO3 + H2O CO2 (g) + 2 H2O (6)

When titrating to the second equivalence point equations 5 and 6 indicate that two moles of H+ ions

reacts with one mole of CO32- ions.

Molarity = no. moles of solute

no. L solution

[HCl] =

g of Na2CO3 x 1 mol Na2CO3

105.989 g Na2CO3 x

2 mol of HCl 1 mol Na2CO3

Volume of HCl (L)

49

11. Calculate the mean, the standard deviation and the 95% confidence interval for the HCl concentration.

Enter your results for the individual titrations, the mean and the standard deviation in the pooled data

table set up on the lab computer. Completed tables will be posted on the Sakai for further analysis.

Data Analysis

1. Use the pooled data column to determine the pooled mean and the pooled standard deviation for HCl

concentration.

2. Compare your mean and SD values to the pooled values. Explain the difference.

3. How would you determine the population mean and the population standard deviation for this

experiment?

Note: A true statistical sampling requires many data points, and errors inevitably arise from under

sampling. You may recall that Gallop polls generally sample about 1000 persons and describe the results as

being accurate within 4%, 19 times out of twenty, that is, a standard deviation of 4% with a 95% confidence

interval.

50

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Experimental Results : Concentration of 0.1M HCL Replicate # 1 ___________ Replicate # 2 ___________ Replicate # 3 ___________ Replicate #4 ____________ Sample Mean ___________ Pooled Mean ___________ Sample SD ___________ Pooled SD ___________ Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations (show example of all the calculations involved in data analysis):

51

EXPERIMENT 2

Determination of Ethanol Content in the Unknown Wine Sample Using Capillary Gas

Chromatography1,2

Introduction

Gas Chromatography (GC) is one of the most widely used analytical separation techniques. It is

used in both, qualitative and quantitative applications, but most applications are of quantitative nature.

The GC separation uses a long tubular column that contains a stationary phase, which is a thin film of

high boiling organic liquid, coated either on the walls of the column or on a small size tightly packed

solid particles. The moving phase is always an inert, low molecular weight gas such as helium. The

sample, injected onto the column, spends some of the time in the stationary phase and some in the mobile

phase. The sample moves along the column only when it is in the moving phase. If two types of

molecules, such as ethanol and n-propanol, are applied onto the column, they will spend different

amounts of time in the moving phase and will move down the column at different rates becoming

completely separated in the process. The separated components enter the detector. Each component of

the sample can be identified by its characteristic retention time, on a particular column at a particular

carrier gas flow and column temperature. Quantitative results are obtained by comparing the detector

response of the particular component in the sample to the detector response of a component in a series of

standard solutions.

Boiling points of the analytes have the greatest affect on the separation efficiency. Low boiling

components are eluted first and high boiling components come out last. For this reason, it is essential that

the column temperature during the analysis is carefully monitored and is reproducible. Separation of the

components with the similar boiling points is achieved by selection of appropriate stationary phase. For

the separation of highly polar compounds, a polar stationary phases such as polyethylene glycols are

used, while for the separation of non-polar compounds, a non-polar stationary phases such as dimethyl

polysiloxanes are used. Since most analytes exhibit some polar character a number of stationary phases

of varying polarities exist. These can range from 95% dimethyl - 5% phenyl polysiloxane to 65%

diphenyl-35% dimethyl polysiloxane or 50% cyanopropyl-50% phenyl-methyl polysiloxane. Most GC

stationary phases are organic in nature. However, when analyzing gases or aqueous samples, a solid

stationary phases consisting of small particles of highly porous polymers such as Porapak or natural

zeolite material are often used. Here separation is based on the molecular size. Smaller molecules travel

faster down the column and are eluted first while larger molecules are eluted last.

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 686-700. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 544-577. McGraw-Hill Higher Education Inc., 2000.

52

Samples analyzed by GC could be gases, liquids and solids. They could be inorganic or organic in

nature. The only requirement is that the analytes be sufficiently volatile to be carried through the

column. The rule of thumb is that analytes that can generate a vapor pressure of a few torr when heated

to the column operating temperature, will pass through the GC system.

The basic components of a gas chromatograph are shown in Fig. 1.

Fig.1 Basic Components of a Gas Chromatograph

1. Cylinder of carrier gas (most often used gases are He and H2).

Carrier gas should be inert or non-reactive in order not to change the analyte. It should also be pure and

dry in order not to introduce impurities, which can contaminate the column and the detector.

2.Two-stage pressure regulator provides a constant gas flow rate, which is essential for achievement of

reproducible retention times.

3. Sample Injector - The aim of the injection is to introduce the sample as a sharp band into a heated

carrier gas stream. For that reason, the injector zone is heated 25-40°C higher than the column

temperature to insure rapid volatilization of the sample. The sample is usually introduced into the

injector using a 1 or 10 µL syringe. A long syringe needle pierces the flexible septum, which closes

tightly around the needle and prevents any backward leakage of the sample. The heated block rapidly

vaporizes the sample and the carrier gas sweeps the vapor through the column.

4. Thermostated Column Oven provides a circulating air bath for the column. The temperature in the

oven is controlled by the thermocouples to within 0.1° C of the set value. Oven temperature can also

be programmed to increase from 1 to 40° C per minute in the range from 30 to 300° C.

5. Column is the heart of the chromatographic system. It determines the efficiency and selectivity that can

be achieved in the separation. There are two basic types of columns, packed and open tubular columns.

53

Packed columns are usually 6 to 10 feet in length with 1/8" to 1/4" outside diameter. Open tubular or

capillary columns are the most widely used type of columns today. These columns contain no support

material, instead the thin film of liquid uniformly coated on the walls of the column serves as the

stationary phase. Capillary columns range in length from 10-100 meters with internal diameter ranging

from 0.1 to 0.53 mm as compared to packed columns that are usually 2 to 4 meters in length and 2 to 4

mm in diameter. Capillary columns are much more efficient and can separate much larger numbers of

components.

6. Detector - The elution of the analyte from the GC column is monitored by the detector. It is the

performance of the detector that determines the sensitivity of the analysis. The detector produces an

electrical output that is proportional to the amount of analyte being eluted. The response to various

compounds may differ. Various kinds of detectors are used in the GC, depending on the type of the

analyte and the detection limit required. The most widely used detectors are Thermal Conductivity

Detector (TCD), known as a universal detector, and Flame Ionization Detector (FID), a highly sensitive

detector used for detection of most organic compounds.

7. Flow meter measures carrier gas flow through the column. If the Electronic Pneumatic Control (EPC)

is installed in the GC it will automatically measure and control all the flow rates as entered through the

chromatography station.

8. Recorder typically a computer terminal and printer. It records the detector output as a continuous trace

of the signal strength against time to produce a chromatogram.

In this experiment you will be determining ethanol content in the unknown wine samples using a

capillary GC and an internal standard method. The quantity of ethanol present in the wine has to be

monitored carefully since there are legal limits that determine the taxation levied on a particular wine.

A 0.1% error can result in a significant monetary loss or gain for the producer. The Internal Standard

method involves addition of a known quantity of internal standard (ie.1-propanol) into each standard

and sample flask. A gas chromatogram of each solution is then obtained and a calibration graph is

prepared from standard solutions by plotting the ratio of the ethanol peak area to the internal standard

peak area versus the standard ethanol concentrations. A linear regression is run to obtain the equation

of the line for the standard curve. The ethanol concentration in the sample is then determined by

obtaining the peak area ratio of the ethanol and the internal standard present in the sample, substituting

it for y in the equation of the line for the standard curve and solving for x (ethanol concentration). The

internal standard must possess properties similar to those of the analyte such as boiling point, polarity

and structure in order to behave in a similar manner during analysis. The internal standard peaks must

be well separated for accurate area measurements but must appear close enough to the analyte peak in

order to experience similar line broadening. Because uncertainties introduced by the sample injection

and any losses during extraction, pre-concentration and sample preparation are compensated for, this

54

method of analysis achieves much greater precision especially for volatile samples than the external

standard method.

Experimental Procedure

Reagents: Ethanol, 1-propanol, distilled water, unknown wine sample.

Preparation of Ethanol/Propanol Standards

You need to prepare 5 standard solutions. Solution #1 will contain only 1% ethanol, while solution #2

will contain only 1% of 1-propanol. The chromatograms of these two standards will be used to

establish the specific retention times for ethanol and 1-propanol under experimental conditions. The

established retention times will then be used to positively identify ethanol and 1-propanol

chromatographic peaks in the entire analysis. Solutions #3, #4 and # 5 will be the three ethanol

standards used in preparing a calibration plot. Each standard solution will contain 1% of 1-propanol as

internal standard.

NOTE: Students working in pairs need to prepare and analyze only one set of standards,

however each student needs to obtain and analyze different unknown wine sample.

Standard Preparation: clean with soap and rinse with distilled water five 100 mL volumetric flasks.

Label flasks as follows: #1-1% ethanol, #2-1% 1-propanol, #3- 0.5% Ethanol STD, #4- 1.0%Ethanol

STD and #5-1.5% Ethanol STD. To each of the 5 flasks add approximately 50 mL of distilled water. To

prepare #1-1.0 % ethanol solution use 1000 µL Eppendorf pipette to transfer 1000 µL of absolute

ethanol into 100 mL volumetric flask then dilute to the mark with distilled water. To prepare #2-1% 1-

propanol solution use1000 µL Eppendorf pipette to transfer 1000 µL of 1-propanol to flask #2, then fill

to the mark with distilled water. To prepare #3-0.5% ethanol standard, transfer 500 µL of ethanol and

1000 µL of 1-propanol to flask #3 and fill to mark with distilled water. Follow similar procedure for

flasks #4 and #5 by adding 1000µL and 1500 µL of ethanol to respective flasks along with 1000 µL of

1-propanol to each flask. (NOTE: You should check calibration of Eppendorf pipette using procedure

outlined in Preliminary Lab Exercise before starting preparation of standards. Make sure to use

different tips for ethanol and 1-propanol and remember that these two chemicals have 100% purity and

are quite volatile so that high degree of care and accuracy is required when pipetting. This is

especially true since you are transferring small volumes. Loss of one drop (~ 10 µL ) may result in

greater than 1% error depending on the volume being transferred. In order to minimize error, use the

same pipette and the same tip for all ethanol standards. For 1-propanol use the same pipette and the

tip for standards and the unknown samples.)

In order to reduce the analysis time, start injecting the first standards as soon as they are

prepared. Carry out sample preparation while analyzing the standard solutions.

55

Sample Preparation: Clean with soap and rinse with distilled water three 50 mL volumetric flasks.

Add 10-15 mL of distilled water to each. Next, use 5000µL Eppendorf pipette (calibrate pipette first) to

transfer quantitatively 5 mL of unknown wine sample and 1000 µL pipette to transfer 500 µL of 1-

propanol (internal standard). Fill to the mark with distilled water. Prepare samples in triplicate. Shake

each flasks well to insure homogeneous concentrations.

Make 0.4 µL injections for each standard and sample. (You will be shown correct injection

procedure by the demonstrator.)

Instrumental Parameters

An HP-6890 Capillary Gas Chromatograph will be used in the experiment. This instrument consists of

Electronic Pneumatic Controlled (EPC) split injector, thermostated column oven, column, Flame

Ionization Detector (FID) and Agilent ChemStation used for instrument control and data acquisition

and processing. The column used is an HP-5 30m x 0.32 mm I.D; the stationary phase is 5% phenyl-

95% dimethyl polysiloxane; film thickness is 1µm. This is a relatively non-polar stationary phase and is

commonly used to separate wide range of organic compounds. High purity helium gas, which is inert

and non-toxic, is used as the carrier gas and also as a make-up gas for FID detector. High purity

hydrogen and air are used as fuel and oxidant respectively for the FID detector. The injector, oven and

detector temperatures are adjusted separately and can range from ambient to 250°

List of Parameters:

Agilent-6890 Gas Chromatograph; Agilent-5 30 m x 0.32 mm ID column; Agilent FID (Flame

Ionization Detector) detector; Dell computer with Agilent ChemStation software; 10 µL Syringe; high

purity gases: He, H2, and air; Column flow rate: 1.5 mL/min; Split Ratio: 50:1 (1/50 of the injected

sample goes to the column while the rest is flushed out). Note: when toxic compounds are being

analyzed split vent must be vented properly.

GC Temperature Settings:

Initial column temperature: 70O C Injector Temperature: 200O C

Initial Time: 0 Detector Temperature: 250O C

Rate: 5O C/ min Signal 1 ON

Final Temperature: 95O C/ min H2 flow 40 mL/min

Final Time: 0 Air flow 450 mL/min

Makeup flow (He) 45 mL/min

56

GC Operating Procedure

1. Turn on helium, hydrogen and air flow by turning main valve on the cylinder one complete turn. Do not

touch any of the regulator valves. They have been adjusted when the initial flow rate was set. The flow

rate is controlled from the chromatography station using Electronic Pneumatic Control (EPC). In most

cases there is no need to measure the gas flow using the bubble meter.

2. Turn on the GC instrument by depressing the square switch found on the front bottom left hand side.

3. Next, load the ChemStation Program as follows: Select: Start > Programs>AgilentChemstation

>Instrument 1 Online.

4. It will take few minutes for the ChemStation program to be loaded and to take control of the GC

instrument. You will see on the GC display window…loading of method parameters. If Chem 2P42

method was the last method used, the parameters from that method will be loaded and flame in the FID

detector will be automatically ignited.

5. Check that CHEM2P4212M method is loaded. Then check that the correct parameters are loaded. This

can be done two ways. One way is from the front panel of the Gas Chromatograph. This is done by

depressing black buttons that indicate various GC functions. For example, to check the temperature

settings, press OVEN. You will see: Temp.70, Initial Time 0:00. Press down arrow on the pad to see

additional settings; Rate: 5 deg/min; Final Temp. 1-120 oC; Final Time 1- 0:00 min. If you need to

change a parameter, you will have to do that from the ChemStation program since GC is now under

computer control.

Second way: Once the Agilent ChemStation is loaded, the Instrument 1 (online): Method and Run

Control screen should be visible and CHEM2P4212.M method should be selected. A diagram of

instrument components is visible on the screen. Click on a square button marked S/S to check inlet

settings. Instrument Inlet (6890) will appear and inlet button should be highlighted. Split-Splitless

Inlet parameters will be displayed. For Chem2P4207 method parameters should be: Mode: Split, Gas:

He, Heater: 200oC, Pressure: 8~10 psi, Total flow: 70-100 mL, Split Ratio: 50:1, Split flow ~90

mL/min, Gas Saver 20.0 mL/min at 2 min. If any of the parameters need to be changed it is done from

this panel.

Once the parameter is changed Apply button needs to be selected for changes to be stored. To check

the settings for other GC components use the same screen and click on the component of interest. For

example, by selecting Detectors, all the detector operating parameters will appear: Heater 250oC, H2

flow 40 mL/min, Air flow 450 mL/min , Makeup flow (He) 45 mL min. Flame should be ON.

IMPORTANT NOTE: none of these parameters should be changed before checking with the

demonstrator!!!!

GC SHOULD BE TURNED ON BEFORE STARTING STANDARD AND SAMPLE

PREPARATION. This will allow all the GC components to reach the desired temperatures before

starting with the injections.

57

Injecting a sample and obtaining a chromatogram:

1. Select RunControl form the toolbar, then Sample Info. Sample Info window will appear and

following information should be entered before first sample is injected: Operator names; Path:

Chem 32\1\DATA\; Subdirectory: CHEM2P42; Manual injection; (correct path, subdirectory

and injection mode are usually already selected). Signal 1 Filename must be changed for each

injection in order not to overwrite previous data. File name should contain student initials, date

of analysis and injection number. For example if Mark and Emily are making first injection on

September 25, 2007 the file name would be ME2509071. For injection two, 1 should be changed to

2 and so on. Other information to be entered is Sample name, Injection volume (0.4 µµµµL) , Internal

Standard amount (1) and a comment indicating more precisely what is being injected.

2. Select-Run Method at the bottom of the screen. The main screen appears and Ready Status is

highlighted in green.

3. Obtain a 10 µL syringe. Rinse the syringe two times with distilled water, then two times with the

sample being analyzed. If bubbles appear, pump the plunger several times then take up

approximately 2µL of the sample. Carefully reduce the volume in the syringe so that only 0.4 µL

remains, wipe the needle with the Kimwipe to remove any sample on the outside, being careful that

no sample from the syringe is removed. Insert the needle all the way into the injector, depress the

plunger and as quickly as possible depress the start button on the GC. (You will be shown the

correct injection procedure by the demonstrator.)

On the screen green light will change to blue and Run in progress will appear.

4. For all standards and samples except for standards one and two, two peaks should appear with

retention times between 2 and 3 minutes. Let the GC run until temperature reaches ~ 95oC, then

select RunControl and Stop Run. Chromatogram will appear on the screen and will be printed on

the printer. Each printout will contain: sample information entered, sample chromatogram and a

table listing peak retention times, peak areas, type of integration and area % for each peak. You will

be using retention times for peak identification and peak area (NOT % area) for the

quantitative analysis.

5. After all the runs have been completed, increase the column temperature to 200oC to remove any

residual water and other higher boiling compounds from the column.

6. Turning off the GC: Before leaving the lab you must turn off the GC. First reduce the column

temperature to 50oC (this can be done on the ChemStation or on the GC panel). After column

temperature has reached 50oC turn off the three gases at the main valve on the cylinders.

Check that the flame is out then depress the square switch in front of GC. You can now exit

the ChemStation program.

58

Data Analysis

Most chromatographic runs can be thought of as having two distinct phases: Data Acquisition and Data

Analysis. Data Acquisition is the part of a run where analytical data is collected and stored in a file. You

access Data Acquisition parameters from the Run Control, Instrument and Method menus. Data Analysis is

the part of a run where the data file is processed (integrated) and the results reported in the desired format.

In the first part you have obtained a printed copy of a Chromatogram and an Area Percent report for each

sample analyzed. A Percent report is based on peak-area data from the integration results providing

information about the relative sizes of all the integrated peaks. This type of report is un-calibrated so it

cannot provide quantitative results. It can, however, be used to develop the calibration tables required for

External Standard, Normalized %, and Internal Standard reports. You will use the Area Percent reports from

the ethanol standards to prepare Internal Standard Calibration Plots using two different procedures.

Editing acquired chromatograms: To edit the chromatogram in any way, you must be in Data Analysis

screen. From View select Data Analysis or click on Data Analysis Box at the bottom left hand side of the

screen. To load the chromatogram of interest-select - File >Load Signal >CHEM32 > 1 >Data > CHEM

2P42. In the CHEM2P42 folder a list of file names will be displayed on the left hand side. Select the file of

interest and click OK. An integrated chromatogram will be loaded. It can now be edited, i.e. re-integrated,

expanded, printed, etc..

Preparation of Calibration Curve and Determination of % Ethanol Using the Internal Standard

Calibration Procedure and ChemStation Method

The first step in the procedure is preparation of the Calibration Table. For each ethanol standard following

information must be entered into the Calibration Table: retention times, area responses and the exact

concentrations of ethanol and internal standard (1-propanol). This is accomplished by loading Area Percent

Report for standard one, entering relevant information into the calibration table (retention times and areas

are entered directly by the computer while exact concentration of the standard must be entered by the

operator). Lowest standard is designated as level one, second highest as level two and third as level three.

Multilevel calibration can compensate for detector non-linearity when enough properly spaced calibration

points are included (at least one calibration point should have concentration higher that the highest expected

sample concentration and the lowest standard should have concentration lower than the lowest expected

concentration in the sample). The presence of the internal standard reduces errors due to the loss of sample

during injection. ChemStation provides several different calibration curve fits. You will be using linear

curve.

ChemStation Method

1. From the RunControl screen load the calibration method: File>Load>Method (CHEM2P4207C)

2. From View on the main toolbar select Data Analysis.

3. Load the data file for the 0.5% Ethanol standard: File>Load Signal (i.e. ME2509073)

4. Click on the Calibration Task button to select the calibration tool set.

59

5. Next create a new calibration table as follows:

a) From the toolbar select Calibration>New Calibration Table

b) Select Automatic Setup for Level 1

c) Specify the concentration of the calibration standard. (You can overwrite this value in the

calibration table.) Then select OK to display the calibration table.

6. Identify ethanol and 1-propanol peaks in the table by the retention times that were established from

chromatograms of standards one and two. For each peak of interest in the Calibration Table (ethanol

and 1-propanol), select the line containing the peak and perform the following steps:

a) Type the name of the compound in the Compound column.

b) Type the amount for the compound in the Amt column if necessary.

c) If this compound is an internal standard, select the ISTD box (1-propanol).

d) If the peak is a reference peak, select the Ref box (1-propanol).

e) To continue to the next compound, choose Enter button on the dialog box or press the down

arrow on your keyboard.

e) Select OK when the table is complete to accept the edits and exit the table.

7. Load the data file for the next calibration level (1% ethanol): File>load signal>file name.

8. To add a new level: select>Calibration >Add Level (the level number is automatically

incremented). Enter the standard concentration for this level then select OK . Enter the relevant

information for standard two into the calibration table and click OK .

9. Repeat Step 7 and 8 for the third level.

10. Select OK when the table is complete to accept the edits and exit the table.

11. To print the calibration table and curves select >File > Print >Calibtable and Curves. Your

printout will show the calibration table you defined and the calibration curve.

11. To determine % ethanol in the unknown sample: Load Chromatogram for the first sample

replicate: File>load signal>file name. Check that the chromatogram is correctly integrated, then

select>Report>Print Report . Report will appear on the screen and will be printed on the printer.

The table at the bottom of the page indicates Retention times, Amounts and Compound Names.

You can read % Ethanol from the table. (This value does not take into account any dilution steps.)

The Internal Standard Calibration Method Using Least-Squares 1,2

1. Your standard solutions consist of three components: water, ethanol and 1-propanol. Since FID

detector does not detect water, you should observe two major peaks on each chromatogram. Use the

information from injections of vial #1(% ethanol) and #2 (1% 1-propanol) to determine the retention

times of ethanol and 1-propanol. Use the retention time information to identify ethanol and 1-

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 60-64. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 105-110, 115-129. McGraw-Hill Higher Education Inc., 2000.

60

propanol peaks on the standard and sample chromatograms. (Hint: peaks should increase as %

ethanol increases while 1-propanol peaks should remain relatively constant.)

2. Use Excel spreadsheet to perform linear regression on your data. First construct a data table

consisting of % ethanol, retention times and peak areas of ethanol and 1-propanol for each standard.

Use a spreadsheet to determine the ratio of ethanol area count to 1-propanol area count for each

standard. Use the format established in Preliminary Exercise lab to perform linear regression on the

data and obtain the slope (m) and intercept (b). Use the % ethanol as x-values and area ratios of

ethanol to 1-propanol are y-values. Construct a calibration plot for visual presentation. Next, enter

data for the three samples and determine ethanol/1-propanol peak area ratios. For each sample,

substitute sample area ratio for Y in the Y= mx + b equation, where m is the slope and b is the

intercept of the calibration plot above. Solve the equation for x to determine % ethanol in each

prepared sample. Calculate mean, SD and 95% CI for the unknown wine sample. (Remember to

account for the dilution.) Summarize the information from all the chromatographic runs in a table.

Table should include the following columns: peak identity (ie.5% ethanol standard), retention times,

concentration, peak area counts, peak area ratios and final sample ethanol concentration.

Questions

1. Report the mean % ethanol in the unknown sample along with standard deviation and 95% CI.

2. Discuss all possible sources or error in the experiment.

3. Discuss the importance of retention time in the chromatographic analysis. What were the retention times

for the two analytes? Use the physical and structural properties of the two compounds to explain why they

eluted in this particular order.

4. What are the advantages of the internal standard method? How is it different from the simple external

standard method?

5. Choose one of the chromatograms and the information found in the Introduction to GC and HPLC Analysis section (pg 42) to calculate the adjusted retention times (tR') and the capacity ratios

(k') for ethanol and 1-porpanol peaks. Calculate the resolution (Rs ) between the peaks. Use the

value of 0.25 min for to.

6. What is the difference between isothermal and temperature programmed analysis? Explain why is

the temperature control important in the GC analysis?

7. Briefly describe operation of a Flame Ionization Detector (FID). Discuss its advantages and disadvantages

in terms of its sensitivity, selectivity, linear range and ease of use. Although water constitutes 98% of the

sample why does water peak not appear on your chromatograms?

61

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of the Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Unknown Sample Number : __________________________________ Experimental Results : % Ethanol –Least Square Method Replicate # 1 ___________ Replicate # 2 ___________ Replicate # 3 ___________ Sample Mean ___________ Sample SD ___________ Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations (show example of all the calculations involved in data analysis):

62

EXPERIMENT 3

Determination of Caffeine in Beverages Using High Performance Liquid Chromatography

(HPLC)1,2

Introduction

HPLC separations are based on the distribution of the analyte between a liquid mobile phase and

either a solid, liquid or bonded stationary phase. Liquid chromatographic method is used for analysis of non-

volatile liquids (b.p. higher than 300°C), solids and labile compounds (compounds that would decompose at

temperatures required for GC analysis).

Basic components of high performance liquid chromatograph are illustrated in Fig. 1.

1

2 34

5 6

Fig.1 Basic Components of HPLC System

1. Degassed mobile phase reservoir. In HPLC, the liquid mobile phase composition is one of the

principle factors affecting separations. We can vary mobile phase composition relatively easily by

combining solvents that have different polarities but that are mutually miscible. In this way, a wide

range of stationary phase-mobile phase combinations can be achieved.

2. High performance pumping system. In order to achieve highly efficient separation, high performance

columns are packed tightly with very small size particles (3-10 µm in diameter). This tight packing

produces very high back pressure when the liquid mobile phase is passed through the column. For that

reason, the HPLC system employs highly efficient high pressure pumps capable of pumping at back

pressures up to 200 atmospheres. The major requirement of the pumping system is that it be pulse free

so that the sample bands being separated on the column are not disturbed. The most widely employed

pulseless pumps are reciprocating and syringe types.

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 701-720. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 578-593. McGraw-Hill Higher Education Inc., 2000.

63

3. Sample injector allows a sample in solution to be applied onto the column as a tight band. This is done

by injecting a known volume of sample solution into a continuously flowing mobile phase under

pressure.

4. Columns. As in GC, the HPLC column is the heart of the instrument. This is the part where the actual

separation takes place. The analytical HPLC columns range in length from 3 to 30 cm, depending on the

stationary phase particle size and the resolution requirement, and are usually 4.6 mm in internal

diameter. They are made of thick walled stainless steel which can withstand high pressures and are

resistant to corrosion. Columns are classified by the type of stationary phase they contain and by the

type of separation that they are used for. Two basic types of columns are:

4.1. Polar silica columns, used for normal-phase or adsorption chromatography. The separation occurs

between the polar stationary phase, usually silica and the nonpolar organic mobile phase. The major

separation mechanism is adsorption, which involves interaction of the polar groups of the analyte

with the polar groups of the stationary phase. In this process, the molecules of the mobile phase and

the solute are in competition for the discrete adsorption sites on the surface of the stationary phase.

In this mode, the elution power of the mobile phase increases with its polarity. Hydrocarbons such

as hexane are the weakest eluents and elution strength increases through dichloromethane

(CH2Cl2), tetrahydrofuran (THF), acetonitrile (CH3CN), and methanol (CH3OH). Usually mixtures

of these solvents are used to achieve the desired separations.

4.2. Nonpolar stationary phase is usually prepared by bonding alkyl substituents to a silica surface (Fig.

2). The most popular nonpolar stationary phase is C18 or octadecyl silica (ODS) which has 18 carbon

alkyl chains attached to silica backbone. The method of separation is known as reversed-phase or

partition chromatography . The reversed-phase title came from the fact that this type of separation

was introduced later than normal phase separation and most of the solute interactions occur in

reverse order to that of the normal phase. In reversed phase chromatography, the driving force behind

the retention mechanism is not the favorable interaction of solute with the stationary phase, but the

effect of the polar mobile phase solvent which forces the nonpolar part of the solute molecule into the

hydrocarbonateous nonpolar stationary phase (hydrophobic interactions).While in normal phase, the

driving force is the favorable interaction of solute with stationary phase; in the reverse phase, we say

that solute is partitioned between the polar mobile phase and the nonpolar stationary phase. The

degree of partitioning is determined by the hydrophobic interactions of the analyte with the mobile

phase. Hydrophobic interactions and therefore analyte retention increase as the polarity of the

analytes decrease and as the polarity of the mobile phase increases. Water is the weakest mobile

phase and gives the greatest retention for the nonpolar organic molecules. The elution strength is

increased by the addition of the organic solvents such as methanol or acetonitrile. These solvents are

also known as the organic modifiers because they act to decrease or modify polarity of the aqueous

64

mobile phase. Acetonitrile and methanol are popular modifier solvents because they have low

viscosity, are readily available with excellent purity, are miscible with water in all proportions and do

not absorb UV light in the region where most other more complex organic molecules do. The samples

analyzed by this method should be non-ionic, of medium to low polarity and be soluble in weekly

polar to polar solvents. You will be using this type of separation in your HPLC experiment.

Fig. 2: Preparation of Bonded Stationary Phase

5. Detectors. Separated analyte bands leave the column and are carried in the mobile phase to the

detector. The detector must be able to measure quantitatively some property of the tight band of analyte

molecules. The most popular HPLC detectors are ultraviolet-visible (UV-VIS) spectrophotometers and

the refractive index detector. Detection by a UV-VIS spectrophotometer is based on the quantitative

absorption of ultraviolet or visible light by the sample molecules. It is useful for organic molecules

which possess conjugated double bonds or functional groups with C =O. It is not very useful for the

detection of aliphatic hydrocarbons since they tend to have very weak or no absorption in this region.

When a UV-VIS detector is not suitable, the refractive index detector is used. It measures the changes in

the refractive index of the eluent that are caused by the elution of different solute bands. The refractive

index detector is known as a universal or general detector since it responds to all the solutes.

Caffeine is one of the most widely consumed drugs in the world. It is found in a wide variety of

consumer products such as coffee, tea, cola beverages, pharmaceuticals, and many others. In recent

years, caffeine has been linked to many human health problems such as heart attacks and birth defects.

For these reasons, there exists a substantial interest in the levels of caffeine present in the commercially

available products. High Performance Liquid Chromatography provides a simple, fast and accurate

determination of caffeine in beverages.

Si

O

OH

O

Si OH

O

Cl Si

CH3

CH3

(CH2)n-CH3+ 2

Si

O

O

O

Si O

O

Si

CH3

CH3

(CH2)n-CH3

Si

CH3

CH3

(CH2)n-CH3

65

Experimental Procedure

In order to shorten the analysis time students should work in pairs to prepare and analyze only one set of

caffeine standards required for the preparation of calibration curve. However, each student should

analyze individual unknown sample.

Apparatus and Materials

1. Liquid Chromatograph: Agilent Technologies 1200 Series Quaternary Pump, Inline Degasser,

Thermostated Column Compartment, Autosampler, Diode Array Detector, Agilent ChemStation

Operating Software and Dell computer.

2. Column: HP 150 X 4.6 mm; Zorbax 5µ ODS 2 (C18) analytical column; flow - 1.5 mL/min.

5. Reagents: HPLC grade Methanol; distilled water; caffeine standard; sample of tea, coffee or cola

Preparation of Standards

Weigh approximately 0.7 g of caffeine standard and dry at 110°C for 1 hour. To prepare 1000 ppm

caffeine stock solution, weighing on the analytical balance to four decimal places ~0.5 g of dried caffeine

standard, transfer quantitatively into a 500 mL volumetric flask, add ~100 mL of distilled water and swirl

the flask until the caffeine is completely dissolved, then dilute to volume with distilled water. You will now

have 1000 ppm* (see Note 1) stock solution. To prepare 100 ppm standard, pipette 25 mL (volumetric

pipette) of 1000 ppm stock solution into a 250 mL volumetric flask and dilute to volume with distilled

water.

Preparation of 10, 30 & 50 ppm Working Standards. Pipette 10, 30 & 50 mL of 100 ppm caffeine

standard into separate 100 mL volumetric flasks and dilute to volume with distilled water.

Preparation of Samples

Obtain sample of pop, tea or coffee from your demonstrator. The initial sample preparation will

depend on the type of sample you obtained.

1. Preparation of Coffee Sample: Weigh on the analytical balance ~ 2 g of coffee sample, transfer the

sample to a 400 mL beaker and dissolve completely in 200 mL of boiling distilled water. When cooled,

transfer the solution to a 250 mL volumetric flask and dilute to volume with distilled water. This

represents an average coffee serving.

2. Preparation of Tea Sample: Place a single tea bag into a 600 mL beaker then add ~ 400 mL of boiling

distilled water. Leave the bag in boiling water for 2 minutes. Stir occasionally. Remove the tea bag and

let the solution cool, then quantitatively transfer the solution to a 500 mL volumetric flask and dilute to

volume with distilled water.

3. Preparation of Cola Sample: Transfer contents of the pop can into a clean and dry 500 mL filtering

flask, place a magnetic stirring bar into the flask and degas the sample using the suction setup found

next to the HPLC instrument.

66

From this point on, samples are treated in the same way.

Filter each beverage through a coarse filter paper, then transfer three 10 mL aliquots of the filtered beverage

into three 100 mL volumetric flasks and dilute to volume.

Final filtration of samples and standards: In order to ensure complete removal of particles from the

solutions to be injected on to the column (presence of particles may lead to clogging and degradation of

column efficiency) obtain six clean screw cap vials if working alone or 9 vials if working with a partner and

filter approximately 1 mL of each of the prepared solutions (3 standards and 3 or 6 samples) using 0.45µm

syringe filters.

Note 1. Vials will be provided by the demonstrator. Major damage can be done to the auto-sampler if

wrong vials are use. Only special vials can be used in the auto sampler.

Note 2. Concentrations for very dilute solutions are often expressed in weights rather than molarities. Since

the density of water is close to one (1mL of water weighs 1g ), the concentration terms (µg g-1 or mg kg

-1) and

(µg ml-1 or mg L-1) are interchangeable for aqueous solutions. This allows the concentrations of aqueous

solutions to be expressed in units of parts per million (ppm) (i.e. 1 µg ml-1 equals 1 µg per 106 µg).

Warning!! The ppm concentration units can only be used with accuracy for dilute aqueous solutions. A

hybrid unit, (µg ml-1) is used when organic solvents are used in the analytical process.

Common concentration expressions:

All solutions Aqueous solutions only

1 mg solute/1L of solution mg/L or µg/mL ppm (1 mg solute/106 mg solution)

1 µg solute/1L of solution µg/L or ng/mL ppb (1µg solute/109 µg solution)

1 ng solute/1L of solution ng/L or pg/mL ppt (1 ng solute/1012 ng solution)

HPLC Operating Procedure

1. Check that solvent reservoirs A( water) and B(methanol) contain at least 700 mL of liquid each.

2. Turn on 1) Pump, 2) Degasser, 3) Injector, 4) Column Compartment and 4) Diode Array Detector by

depressing a switch on the front left hand side of each component.(Most of the time instrument will

be already on.)

NOTE 3. Instrument components should always be Turned On before the ChemStation Program.

Also, ChemStation program should always be Turned Off before instrument components are

turned off.

3. Next, load the ChemStation Program as follows: Select: Start > Programs>AgilentChemstation

>Instrument 1 Online.

67

4 It will take few minutes for the ChemStation program to be loaded and to take control of the HPLC

instrument. If Chem 2P42 method was the last method used, the parameters from that method will be

loaded.

5. Once the Agilent ChemStation is loaded, the Instrument 1 (online): Method and Run Control screen

should be visible and CHEM2P4209.M method should be selected. If not, from Methods menu select

Chem2P4209.M method. A diagram of instrument components is visible on the screen. At this point you

can set parameter and turn on different components of the instrument.

1) Click on the Injector (syringe) than select “Set up Injector” -5 µL should be selected- if

not enter 5 µµµµL in the injection volume box, select Injection with needle wash and the

Position of the wash vial as 100. (Note: Always check that wash vial contains

methanol.)

2) Click on the Pump icon –Select: “Set up Pump” then check that following parameters are

selected: flow should be 1.5 mL/min; Stop Time: 8 min; Solvent Table: A-60% water, B-

40% methanol; Timetable: Time-0.00, % B-40%, %C-0, %D-0%, Flow-1.5 mL, Max

Pressure-350.

3) Click on DAD (Diode Array Detector)-under Signals- Select B-254 nm, BW 16, Ref 360,

BW 100; select UV and Vis Lamps, then OK to exit the setup.

Next, click on a square box at the bottom RHS of DAD. Select YES to question-Turn on the

lamps of the DAD Module?

4. Next, Prime the Pump (you will be shown this step by the demonstrator if it is required).

To prime the pump, first open the black knob located in the center of the pumping unit.

(This allows solvent and any trapped air to bypass the column and the detector and to drain

directly to the waste container.) Next, click on Pump icon and select “ Control->Turn the

Pump On->OK”. Pump should have been in the Standby position previously. When the

pump starts pumping, check that the column pressure is close to zero (2-3 bar), then select

Pump->Set up Pump->and change flow to 5 mL/min-OK.

(Warning: Increasing the flow to 5 mL if the purge valve is not completely

opened ( pressure across the column is much greater than zero) may result in

damage to the HPLC system.) Run PURGE for 2 to 3 minutes or until all the air

bubbles are removed from the lines. Next, click on Pump icon again->Set up Pump->and

change flow back to 1.5 mL/min->OK. Only after you are sure that flow has

changed to 1.5 mL should you close the purge valve. (You should see 1.5 mL

displayed on the green pump icon on screen before closing the purge valve.) The continuous

stream of solvent should now be pumped through the injector, column and the detector.

Pressure should increase to ~ 170 bar. Continue pumping for 10 minutes to equilibrate the

column.

68

5. Click on Column icon, then select “Set Up Column Thermostat” –> 30oC -> OK. This

will keep the column at 30oC during the analysis.

Running a Sample

7. Open the auto-sampler door and carefully place the sample vials in the tray with position one being at the

bottom left corner of the tray and position 10 at the back left corner. Make sure that you know exactly

what samples are placed in what positions since you will have to enter the vial position when

running the sample.

8. Select RunControl from the toolbar, then Sample Info. Sample Info window will appear and following

information should be entered before first sample is injected: Operator names; under Data Files:

Path:C:\Chem32\1\DATA\; Subdirectory: CHEM2P42; manual; under Sample Parameters: file name

(Filename must be changed for each injection in order not to overwrite the previous data!)

File name should contain student initials, date of analysis and injection number. For example if Jack and

Mary are making first injection on September 15, 2009 the file name would be JM15090901.D. For

injection two, 01 should be changed to 02 and so on).

Location: Vial position (must enter correct vial position for each sample).

Other information to be entered is Sample name, Sample amount(5 ), and a comment indicating more

precisely what is being injected.

Select Run Method at the bottom of the screen. At this point the green light on the screen will change to

blue and Run in progress will appear. Auto-sampler will proceed to inject the sample (Warning!! DO

NOT DISTURB INJECTOR WHEN IT IS IN THE PROCESS OF I NJECTING A SAMPLE.

DISTURBANCE MAY ALTER ARM POSITION AND RESULT IN SE ROUS DAMAGE TO THE

INJECTOR.) Injector will take few minutes to pick up and inject the sample, then rinse the syringe.

9. An active chromatogram will appear on the screen where you can follow the progress of the analysis. The

analysis time is set to 8 minutes. You should not stop the run until all the components present in the

sample have been eluted since they will show up in the following analysis. If you need to stop the run for

some reason, you can click on STOP button or under Run Control select-Stop the run.

10. While the Instrument is completing the run, record all the actual operating parameters such as: pump

pressure, column temperature, flow rate, solvent composition etc.

11. After the run is completed chromatogram will appear on the screen and be printed on the printer. Each

printout will contain: sample file name and any information entered, sample chromatogram and a table

listing peak retention times, peak areas, peak area integration results and area % for each peak. You will

be using retention times in minutes for peak identification and peak area for the quantitative

analysis.

69

12. If for some reason chromatogram needs to be reintegrated, select Data Analysis function on the bottom

left of the screen. If the current chromatogram does not appear on the screen, scroll through the file name

window and select file of interest. Next, click on the Integration icon,-> manual integration icon. Use a

mouse to carefully draw a baseline from the start to the end of the peak of interest. This will generate new

peak area. (Make sure that integration baselines are consistent between all the samples and

standards.) When you are satisfied with the integration, select Report from the menu bar than Specify

Repot. Check that in the destination box at the bottom left all three destinations: printer, screen and file

are selected. If you do not want to print to the printer, deselect the printer. Next, from Report select

Print Report . Report will now be printed to the printer.

13. To analyze another sample, first select Method and Run Control on the bottom left hand side. From the

menu bar select Run Control then Sample Info. On sample info screen change file name, vial location

and detailed sample description, ensure that sample is placed in the correct position in the auto-sampler

and select Run Method.

14. Proceed by injecting the first standard in triplicate to establish injection reproducibility. Then perform

single injections for the second and third standard and the samples.

Instrument Shutdown:

1. Select Pump icon->Set up Pump->change Solvent B to 100%->OK. (Green arrow will be shown

from B solvent bottle only.) When pressure across the column is reduced from ~ 180 bar to stable

pressure ~ 75 bar, select Pump icon->Control ->Standby. (Pump will be left in Standby mode for the

next lab period.)

2. Next, select Column->Set up Thermostat->Not controlled->OK .

3. Click on DAD->Control ->Lamps->UV-off ->Vis-off->OK .

4. To turn off the light in the auto-sampler compartment click on Injector->deselect Illumination.

NOTE: To completely shut down the instrument (This should be done only by the

demonstrator!!) Chromatography Station software must be exited first!!! Select File->Exit-

> Close Instrument 1 Online (YES)-> Close pump etc. (YES). Only when the Chromatography

Station is off, the power buttons on the five instrument modules could be turned off.

Data Analysis

1. Identify the caffeine peaks in the sample chromatograms by comparison to the retention times of the

standard caffeine peaks.

2. Prepare caffeine calibrations plot by plotting the area count for the three standard caffeine peaks vs

caffeine concentration (ppm). (Use the actual concentrations of your standards based on the exact

weight of the caffeine used in preparing the caffeine stock solution. Use 0.0 as one point on the plot.)

Perform linear regression to obtain the slope and the intercept. Use the equation of the line to determine

70

caffeine concentration for each of the three sample replicates. (Use the format established in the

Preliminary Lab Exercise.

3. At this point you have obtained the caffeine concentrations for the three diluted samples. By considering

the dilution steps (10 mL of the original sample was diluted to 100 mL), calculate in ppm the caffeine

concentration in the original sample. Report the mean, SD and 95% CI values for the caffeine

concentration in your unknown sample.

4. List all the possible sources of error in the analysis. Why is the use of external standard calibration

method acceptable for determination of caffeine by HPLC but not for determination of ethanol by GC?

5. Calculate how many milligrams of caffeine will be consumed by drinking an average serving, where a

serving is 250 mL of coffee, or tea or one can of pop (355 mL).

6. Discuss the requirements of the reversed-phase HPLC process in terms of column, mobile phase and

types of samples being analyzed. Calculate R, k’, N and H for one chromatographic run. See

Introduction to Chromatography (pg 42) at the front of the manual or your text for the appropriate

formulas.

7. Discuss the differences between isocratic and gradient elution. Which method did you use?

8. What type of detector did you use in this experiment? Why is this detector suitable for the detection of

caffeine but not for the detection of sucrose?

71

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of the Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Unknown Sample Number : __________________________________ Experimental Results : Concentration of Caffeine in mg/L Replicate # 1 ___________ Replicate # 2 ___________ Replicate # 3 ___________

Sample Mean __________ Amount of Caffeine in one cup of coffee or tea (250 mL)

Sample SD ___________ or in 355 mL can of pop. Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations ( show example of all the calculations involved in data analysis):

72

EXPERIMENT 4

Potentiometric Acid Base Titration: Analysis of an Unknown Mixture Containing Carbonate

and Bicarbonate Species (Automatic Titrator)

Introduction

In this experiment you will determine the composition of a pair of substances, sodium carbonate

(Na2CO3) and sodium hydrogen carbonate (NaHCO3) by acid-base titration of a single sample to two

successive end points. The usual method involves use of a burette and two indicators, one for each end

point. In this experiment the automatic titrator equipped with a pH combination electrode will be used to

determine the two endpoints by automated potentiometric titration. Potentiometric titration allows

construction of the titration curves which lead to more accurate determinations of end points or equivalence

points.

Na2CO3 is a base that produces two equivalence points when titrated against acid:

CO3 2- + H3O

+ HCO3- + H2O pHeq.pt. = 8.4 (1)

HCO3- + H3O

+ H2CO3 + H2O CO2 + 2H2O pHeq.pt. = 4.0 (2)

HCO3- comes from two sources in the titration: As an original quantity provided in the unknown sample

and as a product of the reaction between the CO32- and HCl. For a successful analysis, it is important to

know the exact amount of HCO3- from each source. This information is easily obtained from the titration

data. At the first end point (~pH 8.4), 100 % of the original CO32- has reacted but none of the original

HCO3-. From the volume of HCl required to reach the first end point, one can calculate the amount of

CO32- in the sample and therefore the amount of HCO3

- generated. Titrating to the second end point at pH

4.0 will reveal the total amount of HCO3- in the sample.

The amount of HCO3- in the unknown sample can then be calculated as follows:

[HCO3- ]orig = [HCO3

- ]total - [CO32- ]orig

samp samp samp (3)

It is also possible to estimate the ionization constants for carbonic acid (H2CO3) from the titration

curve. The expressions for K1 and K2 are:

K1 = [H+] [HCO3- ] / [H2CO3 ] (4)

K2 = [H+] [CO32-

] / [HCO3- ] (5)

73

The hydrogen ion concentration is obtained from the titration curve using the relationship:

pH = -log[H+] (6)

pH is measured and [H+] is calculated. The carbonate/bicarbonate and bicarbonate/carbonic acid ratios are

also obtained from the titration curve.

Potentiometric Measurement1,2

The pH value of a sample can be readily determined by the use of pH paper or the acid-base

indicators. However, the most accurate pH measurements are performed potentiometrically. The

potentiometric method uses a pH meter and two electrode system. The two electrodes, indicating and

reference, are immersed in the solution of interest, and a very small voltage, produced in the electrode

circuit which possesses extremely high resistance, is measured accurately by the pH meter. The voltage is

related in a very precise way to the pH of the solution. The measured voltage can be expressed by the

simplified Nernst equation:

E = Eo - 2.303

F RT (pH) (7)

where E is the measured voltage; Eo is the total of all constant voltages in the measuring system, R is the

Gas Law constant; T is the temperature in °K; and F is Faraday's constant.

The electrode system consists of a pH sensing glass electrode and a constant voltage reference

electrode. Figures 1a and 1b illustrate basic construction of the two electrodes. The sensing glass electrode

consists of a nonconducting glass or polymer body to which is attached a sealed bulb of special conductive

glass, also known as a pH sensing membrane. The body contains a buffered electrolyte which has fixed pH

and ionic concentration and in which is immersed an internal reference element composed of Ag/AgCl. The

electrode works on the principle that at pH 7.0 the sum of voltages on the inner surface of the glass

membrane is approximately equal to the voltages on the outer surface of the glass membrane (in the

solution). The voltage potential at this point is zero. As the [H+] of the sample solution increases or

decreases, the change in the potential across the glass membrane is measured and related to the pH. The

reference electrode consists of a glass or polymer body; an electrolyte filling solution; a reference element, usually calomel, Hg2Cl2 or Ag/AgCl, and a porous junction which allows a small flow of the electrolyte to

complete the electrical circuit. Two major functions of a reference electrode are to complete the electrical

measuring circuit and to provide a stable reference potential. Today the use of two individual electrodes for

routine measurements of pH is being replaced by a single combination electrode (also used in this

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 386-430. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 273-293, 462-479. McGraw-Hill Higher Education Inc., 2000.

74

experiment). The combination electrode contains both the pH-sensing and the reference electrodes as

shown in Fig.2. The pH-sensing electrode is located in the center with the pH-sensing membrane being

exposed at the probe tip. The surrounding jacket contains the reference element, the reference electrolyte

and the porous junctions through which contact is made with the sample solution.

Buffer Solutions

The most important property of pH buffer solutions is their ability to resist pH change upon dilution or

acid/base contamination (detailed discussion of buffer behavior can be found in most analytical texts).

Buffers have found many uses in analytical chemistry. In this experiment, buffers are used to standardize the

pH meter/electrode system by immersing the electrodes into buffer solutions of known pH value and

75

adjusting the pH meter to display these values. The three buffers that are encountered most often are: pH

4.0 buffer (can be prepared by adding 50 mL of 0.1M potassium hydrogen phthalate and 8.2 mL of 0.1M

HCl to 100 mL flask and diluting with deionized water to the mark); pH 7.0 buffer (can be prepared by

adding 50 mL of 0.1M potassium dihydrogen phosphate and 29.1 mL of 0.1M NaOH to 100 mL flask and

diluting with deionized water to the mark); pH 10.0 buffer (can be prepared by adding 4.6 mL of 0.1M HCl

and 50 mL of 0.025M borax to 100 mL flask and diluting with deionized water to the mark). In most

instances buffer salts are purchased prepackaged and buffer solutions are prepared by diluting contents of

the package with deionized water to specified volume Experimental Procedure (You will be using 0.1 M HCl standardized in Experiment 1 and will therefore carry out only Part II of this experiment)

Safety Considerations No special considerations in this experiment.

Waste Disposal Solutions can go down the sink with lots of water.

Equipment G20 Mettler Titrator equipped with pH combination electrode.

Apparatus: weighing bottle, 1 L volumetric flask, 10 mL graduated cylinder, 8 x 100 mL plastic titration

cups, 1 x 10 mL volumetric pipette, box of Kimwipes.

Reagents Sodium Carbonate (Na2CO3) - primary standard F.W. 105.99 g/mol

Concentrated Hydrochloric Acid (HCl)Concentration = 12 moles/L

pH 4.0 buffer

pH 7.0 buffer

Part I: Preparation and Standardization of 0.1M HCl Using G20 Mettler Titrator

Drying of Primary Standard Na2CO3: Transfer approximately 0.5 g of primary standard Na2CO3

(Analar grade) into a clean and dry weighing bottle. Let the standard dry at 110ο C for 45 minutes then cool

in desiccator for 30 minutes before weighing.

Preparation of 0.1M Hydrochloric Acid: Clean 1L volumetric flask and fill half full with deionized water.

Add about 8 mL of concentrated HCl to the flask. Mix the contents by swirling the flask several times. Fill

the flask to volume with deionized water and mix thoroughly by inverting the flask several times.

Calibration of a pH Sensing Combination Electrode: The pH electrode is calibrated with two buffers.

The pairs of buffer solutions that could be used are: pH 4 and pH 7, pH 4 and pH 10, pH 7 and pH 10. The

order of buffer solutions in the measurement is immaterial.

1. Rinse the electrode along with the stirrer and the titrant delivery tube by immersing them into a

sample cup filled with deionized water. Gently tap dry the components to remove any excess water

using Kimwipes. Pour approximately 50 mL of the pH 7 buffer into a plastic sampling cup and secure

76

the cup to the titration head. Make sure that both, the glass membrane and the contact point of the

reference electrode (frit, taper) are immersed.

From the home screen press the CALIB shortcut icon. The method for the standardization of the pH

electrode will be loaded. Press the START soft key and you will be prompted to enter sample 1. Press

o.k.

2. After the potential of the first buffer has been determined you will be prompted to add sample 2.

Remove the cup containing the pH 7.0 buffer and rinse the electrode as described above. Transfer

approximately 50 mL of pH 10.0 buffer into the second sampling cup and attach to the titration head.

Press o.k. The measurement is made as before. The electrode parameters are automatically calculated,

stored and printed. Press

Standardization of 0.1M HCl: The titrant concentration (HCl concentration in this experiment) is

determined by a special titration called a Titer determination. To perform this titration we make use of a

standard substance (primary standard in this case sodium carbonate). The titrant concentration is stored in

the G20 as a Titer value

1. Check that the 1L titrant reservoir (brown bottle) contains sufficient 0.1 M HCl (at least ¼ full). If you

need to add acid carefully unscrew the reservoir and fill it using the stock acid solution provided.

2. Attach an empty plastic cup to the titration head by pressing the cup against the head and tightening

the gray screw (turn to the right but do not over tighten). Fill the burette with the titrant by pressing

the Rinse Burette shortcut icon on the home screen.

3. Prepare three standards by weighing out on the analytical balance (to four decimal places) (0.08 - 0.09

g) of Na2CO3. Make the weighing directly into clean and dry plastic titration cups by placing a cup

onto the balance pan and rezeroing the balance, then transferring carefully with a clean spatula

approximately 0.09g of Na2CO3 directly into the cup. Record the exact weights in your notebook.

Remove the cup from the balance and add approximately 50 mL of deionized water to the sample cup,

repeat for the other two standards

4. Rinse the electrode with the deionized water as described in step 1, then connect the first sample cup

to the titration head as described in step 2.

5. Press the Titer shortcut icon from the home screen, and the press the start key. You will be prompted

to enter the number of standards (3) and the mass of sodium carbonate for each sample cup.

6. When the titer determination is complete rinse and connect the next sample cup until and proceed as

prompted until all three standards have been run. The instrument will print out a summary of the

standardization and will give a titer value for the 0.1 M HCl.

77

Part II: Sample Analysis

1. Quantitatively transfer the unknown sample into a 100 mL volumetric flask (use the powder funnel for

the transfer), dissolve the sample in approximately 50 mL of deionized water by swirling the flask,

then dilute to the mark. Using 10 mL volumetric pipette, transfer 10 mL of the prepared sample

solution to the clean sample cup. Fill the cup to 60 mL mark with deionized water. Rinse the

electrodes in deionized water and tap dry with Kimwipe. Attach the sample cup to the titration head

by adjusting the gray screw.

2. From the home screen press the EXP 4 shotcut icon to load the method. Press the start key and enter

the sample number into the Sample ID. The instrument will then perform the titration and a summary

will be printed that includes the titration curve and the first derivative plot, plus two results R1 and R2

in mmol., which is the mmol of acid used to reach equivalent points 1 and 2.

3. Repeat the steps above for each sample.

Data Analysis

1. For each titration you will obtain a measured value table and two graphs. The measured value table

consists of the following : volume of HCl in mL, ∆V, pH, ∆pH and derivative ∆pH/∆V. The first

graph is a plot of pH vs mL of 0.1 M HCl added. The steepest parts of the plot indicate the two

equivalence points (indicated by the dotted lines). The second graph is a first derivative plot of

∆pH/∆mL vs mL of 0.1 M HCl added. The two equivalence points are determined from the two

maxima and the volumes of 0.1 M added to the equivalence points are recorded. The results are

calculated according to the following formula:

R1 = Q1

R2 = Q2

Where Q is the number of mmol of titrant that has been delivered. You will have to calculate a third

result R3 which is:

R3 = Q2 – Q1

This takes into account the mmol of the original carbonate that was converted into bicarbonate after

the first equivalence point.

Use the results obtained from R1 and R3 (in millimoles) to calculate the milligrams of sodium

carbonate and sodium bicarbonate present in the original sample. Remember that you used 10 mL

aliquot in the titration out of the total sample volume of 100 mL.

2. Report the mean, the standard deviation and the 95% confidence interval of each carbonate species.

3. The results for the two equivalence points are reported in mL of titrant added and in millimoles. For

one run only, calculate the concentrations of Na2CO3 and NaHCO3 in the 100 mL volumetric flask in

mmol/L and ppm .

78

4. Briefly describe the electrode system used in the experiment.

5. Write balanced equations for the reactions occurring at the two end points.

6. Calculate the dissociation constants K1and K2 for carbonic acid (H2CO3) at half-neutralization

point and compare to literature values.1,2

7. Sketch a typical two end point titration curve for the titration of carbonate/bicarbonate mixture with

0.1M HCl. Explain what reactions are occurring at pH 10,8, 6 and 3 along the titration curve. Give

appropriate equations.

8. Draw and label components of a G20 Mettler Titrator.

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp 426. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pg 310. McGraw-Hill Higher Education Inc., 2000.

79

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of the Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Unknown Sample Number : __________________________________ Experimental Results : Amount of Na2CO3 (mg) Amount of NaHCO3 (mg) Replicate # 1 ___________ Replicate # 1 ___________ Replicate # 2 ___________ Replicate # 2 ___________ Replicate # 3 ___________ Replicate # 3 ___________ Sample Mean ___________ Sample Mean ___________ Sample SD ___________ Sample SD ___________ Sample 95% CI ___________ Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations ( show example of all the calculations involved in data analysis):

80

EXPERIMENT 5

Determination of Dissolved Oxygen in an Unknown Water Sample (Redox Titration or Winkler Titration)

Introduction

Dissolved oxygen (DO) is essential for survival of aquatic life. For adequate game fish population

the dissolved oxygen content should be in the 8 to 15 mg/L range. The water bodies with high content of

organic pollutants, which come from sewage, dead vegetation, industrial discharge etc., tend to have an

insufficient amount of dissolved oxygen to support the normal levels of fish and other water dwelling

organisms. The main cause for low oxygen levels in the polluted waters is the presence of microorganisms

that readily oxidize organic pollutants by aerobic metabolism and in the process consume dissolved oxygen.

For this reason, an important test in determining the water quality of our lakes and rivers is the measurement

of dissolved oxygen (DO).

Usually two tests are carried out when determining water quality. One test determines the straight

dissolved oxygen content (DO), while the second test determines the amount of oxygen needed by the

microorganisms to remove the organic pollutants. We say that the second test determines the biological

oxygen demand or BOD. The DO test is usually performed immediately after sampling. For the BOD test,

the duplicate to the DO sample is incubated for approximately 5 days to give microorganisms a sufficient

amount of time to break down organic pollutants. The amount of oxygen remaining after incubation is

measured using the same test as for DO. The Biological Oxygen Demand is the difference in oxygen content

before and after incubation.

In this experiment, you will be determining the amount of dissolved oxygen (DO) present in the

water sample. The determination consists of a redox titration (Winkler Method) which involves a series of

oxidation-reduction reactions. Reaction steps: Step 1-Manganese (II) precipitates in a highly basic solution to give manganese (II) hydroxide (1).

Mn2+ + 2OH- Mn(OH)2(s) (1)

Step 2- Dissolved oxygen present in the sample oxidizes manganese (II) hydroxide to manganese (III)

hydroxide (2).

4 Mn(OH)2(s) + O2 + 2 H2O 4 Mn(OH)3(s) (2) Step 3- When the mixture is acidified manganese (III) is reduced to manganese (II) and iodide is oxidized to iodine (3).

2 Mn(OH)3(s) + 2I- + 6 H3O+ I2 + 12 H2O + 2 Mn

2+ (3)

81

Iodine is only slightly soluble in water, but in the presence of excess iodide, it forms soluble triiodide

complex (4).

I2 + I- I3- (4)

iodine iodide triiodide

Step 4-Generated triiodide is then titrated with a standardized sodium thiosulfate (Na2S2O3) solution (5).

2 S2O32- + I3- 3 I- + S4O62- (5)

Starch is used as an indicator since it forms an intense blue complex with a small amount of iodine/triiodide.

In a colourless solution, it is possible to see the blue colour in a solution with [I3- ] of 5x10-6 M. The end

point of the titration is signified by a sudden disappearance of the blue colour. Experimental Procedure I. Preparation and Standardization of 0.02M Sodium Thiosulfate Apparatus Volumetric flask 500, 250 mL Cylinder, graduated 25 mL Erlenmeyer flask 3 x 250 mL Burette 50 mL Reagents Potassium Iodate (KIO3) FW 214.00 Sodium Thiosulfate Pentahydrate(Na2S2O3.5H2O) FW 248.17 Potassium Iodide (KI) FW 166.00 Hydrochloric Acid 1M Starch Indicator

Preparation of 0.02 M Sodium Thiosulfate Solution Boil 1 L of deionized water for at least 5 minutes to remove CO2. Let the water cool, then to about

300 mL of cooled water add 3.0 g of Na2S2O3.5H2O. Stir until dissolution is complete, then add 0.4 g of

NaOH and dilute to the mark in a 500 mL volumetric flask.

* Sodium thiosulfate solutions are light sensitive and are subject to slow bacterial decomposition. For this

reason, solution should be stored in a tightly closed brown bottle and used within several weeks after

standardization. For longer storage periods, a small amount of bacterial inhibitor such as HgI2 is added.

82

Preparation of Starch Indicator (this solution will be prepared for you)

Make a paste by grinding 2 g of soluble starch and 10 mg HgI2 in 30 mL of H2O. Add this mixture to

1L of boiling water, and continue heating until clear. Cool, and store in stoppered bottles. *HgI2 is added to

inhibit bacterial growth. If starch is used immediately after preparation, it is not necessary to add HgI2.

Standardization of 0.02 M Sodium Thiosulfate Solution

Weigh out on the analytical balance approximately 200 mg of dried potassium iodate (primary

standard), transfer the reagent to 250 mL volumetric flask and dissolve in approximately 100 mL of water.

After dissolution is complete, dilute to the mark. Use cooled freshly boiled water for dilution. Use 25 mL

volumetric pipette to transfer 25 mL of this solution into a 250 mL Erlenmeyer flask. Add 0.5 g of

potassium iodide (KI) and 10 mL of 1.0 M HCl to the flask and titrate immediately with thiosulfate solution

until the colour of the solution becomes pale yellow. At this point add 100 mL of boiled deionized water, 2

mL of starch indicator, and titrate to the first disappearance of the blue colour. Perform triplicate analyses.

Calculate the molarity of the thiosulfate solution. Example of calculations:

IO3- + 5I- + 6H+ 3I2 + 3H2O (6)

I2+ I- I3- (7)

2 S2O32- + I3- S4O62- + 3 I- (8)

From equations 6 to 8, we see that one mole of iodate (IO3- ) consumes 6 moles of thiosulfate (S2O32- ).

Therefore, we can calculate molarity of Na2S2O3 as follows:

Concentration of Na2S2O3(moles/L) = moles of Na2S2O3

Vol. Na2S2O3 (9)

Taking into account that only 25 mL out of 250 mL of iodate solution was used in the titration, following

expression is obtained for the concentration of sodium thiosulfate solution:

[Na2S2O3 ] (moles/L) = Wt. of KIO3 (g) x 1mole KIO3

214.00 g KIO3 (10)

x 25 mL250 mL x

6 moles Na2S2O31 mole KIO3

x 1

Vol. Na2S2O3 (L)

83

II. Determination of Dissolved Oxygen in Water Samples Sample Collection

Samples are collected in a clean 300 mL glass bottles. Water is allowed to overflow the bottle and tight

fitting stoppers are used to insure that no air bubbles remain trapped. Analysis for DO should be carried out

as quickly as possible after sampling in order to reduce the biological activity which reduces the actual

amount of DO. The dissolved oxygen content will change with depth, turbulence, temperature, organic

impurities and other factors. For that reason, several samples should be taken at different locations, times

and depths. You will receive for analysis three water samples in 300 mL ground glass stoppered bottles. Apparatus Sample bottles 300 mL Cylinders, graduated 10 mL Erlenmeyer flask 3 x 500 mL Burette 50 mL Volumetric Flask 100 mL Reagents Sodium Thiosulfate (Na2S2O3) 0.02 N Manganese (II) Sulfate (MnSO4.4H2O) FW 223.06 Sodium Hydroxide (NaOH) FW 40.00 Potassium Iodide (KI) FW 166.00 Concentrated Sulfuric Acid 18 M H2SO4 Starch Indicator

Preparation of Special Reagents 1. Prepare 10 mL of 2 M manganese (II) sulfate by adding 4.8 g MnSO4.4H2O to 10 mL of boiled

deionized water. This solution must be freshly prepared.

2. Prepare the alkaline iodide reagent by dissolving 5 g of NaOH and 1.4 g of KI in 10 mL of deionized

water. * Small amount of sodium azide can be added to alkaline iodide solution to suppress

interferences from any nitrate present.

Sample Treatment

Step 1- Remove the stopper from the 300 mL sample flask and carefully add 2 mL of 2 M manganese (II)

sulfate from 10 mL graduated cylinder by letting the solution run down the side of the bottle. Restopper the

bottle over the sink since some of the solution will overflow. Make sure that no air bubbles are trapped

inside. Invert the bottle several times.

Step 2- Add 2 mL of alkaline iodide solution in the same manner as above. Again stopper the bottle, check

for air bubbles, and mix the solution by inversion. At this stage, a flocculent precipitate will appear. It will

be orange-brown if oxygen is present or white if oxygen is absent.

84

Step 3- Let the solution settle for few minutes, then remove the stopper and add 2 mL of concentrated

sulfuric acid. Re-stopper and mix by inversion until the precipitate dissolves and the yellowish-brown colour

due to liberated iodine appears.

Titration

Use 100 mL volumetric pipette to transfer 200 mL of the prepared sample into 500 mL Erlenmeyer flask.

Fill 50 mL burette with standardized sodium thiosulfate solution and titrate the sample until mixture turns

pale yellow. At this point, add 2 mL of starch solution and titrate to the first disappearance of the blue

colour. Record the volume of titrant used.

Data Analysis 1. Calculate the concentration of Na2S2O3 in moles/L and determine the mean, standard deviation and

95% confidence interval.

2. The net balanced equation (9) indicates that 4 moles of Na2S2O3 are used for each mole of O2 present

in the sample. Use this information to calculate the moles of dissolved oxygen present in the 200 mL

sample.

8 OH- + O2 + 12 H3O+ + 4 S2O32- 22 H2O + 2 S4O62- (9)

3. Calculate the concentration of dissolved oxygen in the water sample in mg/L. Report the mean, standard deviation and 95% confidence interval for your results.

4. Discuss the sources of error in this experiment.

5. Discuss physical changes (i.e. change in colour, precipitate formation, etc.) that occur in the sample

solution as different reagents (MnSO4, NaOH, KI, H2SO4) are added. Provide appropriate equations for

the reactions.

6. How would you go about determining BOD?

85

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of the Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Unknown Sample Number : __________________________________ Experimental Results : Concentration of DO in mg/L Concentration of Na2S2O3 mol/L _________ Replicate # 1 ___________ Replicate # 1 ___________ Replicate # 2 ___________ Replicate # 2 ___________ Replicate # 3 ___________ Replicate # 3 ___________ Sample Mean ___________ Sample Mean ___________ Sample SD ___________ Sample SD ___________ Sample 95% CI ___________ Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations (show example of all the calculations involved in data analysis):

86

EXPERIMENT 6

Determination of Total Inorganic Phosphate in Water (Spectrometric Analysis)1,2,3

Introduction

Phosphorus is present in natural waters and wastewater almost exclusively in the form of

phosphates. Phosphates can be classified as orthophosphates (1), condensed phosphates (2) and organically

bound phosphates (3).

Na+

OH

O

Na+ -

O

O Na- +Na+

-

O−P−OH

O−Na+

O

-

Na+- Na+O P_ −O− P− OO−P

O O O

O-Na+ O- Na+

−O-Na+

R−O−P−O−R'

OH

O

R & R = organicgroups

O−P−O−Na+

O −P− OH-

(1) orthophosphates (2) condensed phosphate- triphosphate (3) organically bound phosphate

Certain amount of phosphate is essential for most plant and animal growth, but excessive amount of

phosphates present in the water acts as a nutrient and stimulates abnormally rapid growth of plants and

algae. When these plants die, oxygen is needed in the decay process. If large amount of the decaying matter

is present in the water, the biological oxygen demand (BOD) may deplete the level of dissolved oxygen in

the water to an extent where aquatic life can no longer be supported. This process is known as eutropication.

Phosphates may enter water from household and industrial waste, agricultural run-off or may be

added to the water in municipal and industrial water treatment processes to control corrosion. Most

detergents contain some form of phosphate. Phosphates can functions as water softeners (they complex

Ca2+ and Mg2+ ions in hard water), as dirt dispersers and as fillers. Many laundry detergents used to

contain up to 40% sodium triphosphate (Na5P3O10). After it was recognized that the excessive amounts of

phosphates in the water bodies leads to eutropication the phosphate content of the detergents was

substantially reduced.

In this experiment concentration of the orthophosphate in the unknown water sample will be

determined using spectrometric analysis. The determination of phosphate involves a reaction of

orthophosphate with ammonium molybdate in acid solution to form a yellow-coloured phosphomolybdate

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 557-587. Philadelphia: Saunders College Publishing, 1996.

2 Hach Water Analysis Handbook, Hach Co., Loveland, Colorado, 1989, p. 379. 3 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 395, 656-657. McGraw-Hill Higher Education Inc., 2000.

87

complex (H3PMo12O40)(4). The phosphomolybdate complex is then reduced by ascorbic acid, giving a

characteristic molybdenum blue species (5). The actual structure of the molybdenum blue species is not

known with certainty.

12 H2MoO4(aq) + H3PO4 (aq) H3P(Mo12O40) + 12 H2O (l) yellow species (4)

H3P(Mo12O40)(aq) + C6H8O6 molybdenum blue species (5)

Since the above reaction occurs only with orthophosphate, the polyphosphates present in the sample must be

first converted to orthophosphate by acid hydrolysis (6).

Na 4P2O7 + 2 H2 SO4 + H2O 2 H3PO 4 + 4 Na+ + 2 SO42- (6)

The intensity of the blue colour is measured with a spectrophotometer. The spectrometric

measurements in the dilute solutions obey Beer-Lambert law (7) which states that the amount of light

absorbed by a solution at a particular wavelength is directly proportional to the concentration of the

absorbing species.

A = εbc (7)

A = absorbance ε = molar absorptivity coefficient b = path length c = concentration

You will be using the Genesis 5 microprocessor-controlled UV/Visible Spectrophotometer designed

for quantitative spectrophotometric measurements. It performs absorbance, % transmittance and

concentration measurements within the wavelength range of 200 to 1100 nanometers. It is a split-beam

instrument equipped with two detectors, as shown in Figure 1. One detector is used for sample

measurements and the other for reference measurements. It has two light sources, deuterium lamp is used as

UV source and tungsten lamp as visible source. The capability of the instrument is enhanced by the use of

Application and Memory SoftCards. The Application SoftCard contains the software used for: Survey

Scans, Simple Kinetics, Linear and segmented (non-linear) standard curves and Selected data manipulations.

The standard curve program that will be used in this experiment has the ability to construct a standard

calibration curve and to determine the concentration of the sample from the curve. Instrument takes

absorbance measurements of the blank and the standards and plots them against the standard concentrations.

The sample absorbance is then measured and the concentration is determined from the standard curve.

88

Experimental Procedure

Equipment: Thermo Scientific Genesis 10S UV-Vis Spectrophotometer

Apparatus: 7 x 50 mL, 1 x 250 mL and 1 x 1000 mL volumetric flasks; 1, 2, 5 and 25 mL volumetric

pipettes; 3 x 50 mL Erlenmeyer flasks; 1 weighing bottle; 3 gravity funnels

Reagents: -Potassium Dihydrogen Phosphate (KH2PO4) Primary standard FW 136.09

-Ammonium Molybdate ((NH4)6Mo7O24.4H2O) 17.5 g in 500 mL of 5M H2SO4 (Prepared )

-5% Ascorbic Acid (C6H8O6) Prepare fresh by dissolving 1.0 g in 20 mL of deionized water.

-2.5 M H2SO4 and 5M NaOH

Drying of Primary Standard: Transfer approximately 0.7 g of Potassium dihydrogen phosphate into a

clean and dry weighing bottle and dry for 45 minutes at 110ο C. This should be done as soon as you enter

89

the lab. Cool the sample in the desiccator for 15 minutes before weighing. (Note: Check in the desiccator if

there is any previously dried standard available.)

Cleaning of Glassware: Clean all the glassware using phosphate free soap and rinse in deionized water.

Sample Collection and Preparation for Analysis: Water samples for analysis should be collected in clean,

glass or plastic bottles which were previously washed with a phosphate free detergent and rinsed with dilute

HCl and deionized water. Sample bottles should be filled to the top and tightly stoppered. If prolonged

storage is required, 5 mL of chloroform should be added per each liter of water to prevent phosphate loss by

microbiological activity.

Pipette three 25 mL aliquots of the unknown water sample into three 50 mL volumetric flasks. At this

point stopper the flasks and set aside for further analysis.

Preparation of 25 ppm Phosphate Stock Solution: Weigh to four decimal places approximately 0.36 g of

dried phosphate standard and transfer quantitatively into 1 L volumetric flask. Dissolve the sample in

deionized water and dilute to 1L. Using volumetric pipette transfer 10 mL of the prepared standard into 100

mL volumetric flask and dilute to volume with deionized water. At this point calculate the exact

concentration of phosphate ion (PO43-)) in mg/L.( NOTE: You used potassium dihydrogen phosphate as

your primary standard but are asked to calculate the concentration of the phosphate ion in the stock

solution.)

Preparation of 5% Ascorbic Acid Reagent: Into 50 mL beaker weigh to two decimal places 1 g of

ascorbic acid. Add 20 mL of deionized water and stir the solution until dissolution is complete. This

solution should be prepared the same day it is used.

Molybdate Reagent: In the fume hood transfer 10 mL of the prepared molybdate reagent into a clean 50

mL beaker. (Caution! This reagent contains 5 M sulfuric acid.)

Preparation of the Calibration Curve: Clean and label four 50 mL volumetric flasks. Fill the first flask

half full with deionized water. This will serve as a blank. Transfer 1, 2 and 5 mL of the 25 ppm phosphate

standard into flasks 2, 3 and 4 respectively. Fill each flask half full with deionized water. Since the colour

development is time dependent, careful attention must be paid to the time elapsed from reagent addition to

the actual measurement being taken. The measurements should be taken as close to 15 minute development

time as possible. Using micro-pipette transfer 1mL of the molybdate reagent, then 1mL of the ascorbic acid

reagent to the flask designated as the blank. Start the timer. After 1 minute has elapsed transfer 1mL of the

90

molybdate reagent, then 1mL of the ascorbic acid reagent to the flask designated STD #1. Add reagents to

standards two and three at one minute intervals. Fill the flasks to volume with deionized water, shake well

and let the colour develop for 15 minutes. When preparing a calibration plot the actual standard

concentrations must be entered through the keyboard. While waiting for colour to develop (~ 10 min),

calculate actual standard concentrations as ppm of PO43- and then set up the instrument

parameters.

Instrument Setup

1. Press the power switch to ON (back of the instrument) to turn on the instrument.

2. Wait for the initialization sequence to be completed and check to see that the instrument is set to

STANDARD CURVE mode by looking at the top left of the display, and that the test name is PHOS. If

the instrument is set-up properly proceed to Standardizing the Spectrometer.

3. If the instrument is in standard curve mode, but the test method is not PHOS then press the Stored Test

soft key and load the method PHOS.

4. If the instrument is not in Standard Curve mode then press the TEST button and use the up/down arrow

keys to select Standard Curve. Once chosen, check to see that the test PHOS has been loaded, if not

press the Stored Test soft key and load the method PHOS.

Standardizing the Spectrophotometer

After a minimum of 15 minutes from the time of reagent addition fill a plastic sample cuvettes to ~ 0.5 cm

from the top with the blank solution. Open up the sample compartment and insert the cuvette into cell holder

B, making sure that the transparent sides are in the light path. Repeat the above procedure for standards 1 –

3, placing them into cell holders 1 – 3 respectively.

Press the Run Standards soft key, the instrument will prompt you to enter the concentrations of the three

standards. Once complete follow the prompts to run the standards.

Once complete the absorbance values for the standards will be displayed. Press the Standard Curve soft key

to display the curve, and then use the soft key to print it.

Sample Analysis after Reaction with Molybdate and Ascorbic Acid: To the three volumetric flasks

containing earlier prepared samples add 1mL of molybdate reagent and 1mL of ascorbic acid reagent.

Immediately after addition start the timer. Dilute the samples to volume with deionized water and mix

thoroughly. Let the colour develop for 15 minutes.

When the samples are ready to be analyzed remove the standards from the cell holders, but leave the blank

in place. Place the samples in cell holders 1 – 3. Press the Run Samples soft key. The instrument will then

measure the absorbances of the three samples, and the repective concentration values will be displayed.

Print your results using the print key.

91

When all samples have been analyzed shut down the instrument using the power switch.

Data Analysis

1. Your printed data should include a Standard Curve Plot as well as a Standard Data Table which includes:

standard number, absorbance, entered concentration, calculated concentration from the regression curve

and the difference in actual and calculated concentration. Also included in the table is the slope,

intercept, correlation coefficient and the standard deviation of the curve. You should also have a printout

of Unknown Measurement Table which includes: unknown number, date and time the measurement is

taken, observation wavelength, sample absorbance and calculated concentration. Since your sample was

prepared in triplicate you should have three absorbance values and three concentration values.

a) Use the concentration values obtained from the instrument for the three sample replicates to

calculate the concentration of PO43- (in mg/L) in the original water sample. Remember that you diluted

your sample. Report the mean, SD and 95% CI for your result. Include printed data output.

b) Use the absorbance values and the calculated concentrations for the three standards and any

graphing program capable of regression analysis to construct a manual calibration curve. Include 0,0 as

a data point. Perform linear regression on the curve to obtain slope and intercept, then use equation of

the line to determine concentrations of the three replicates in mg/L.

Report the mean, SD and 95% CI for your analysis. Include you plot.

c) Discuss the importance of Beer-Lambert law in quantitative spectrometric measurements.

Compare the results from the two calibration procedures. Are there any differences? Explain why of

why not.

2. Calculate the concentration (in ppm) of elemental phosphorous in the water sample. Report mean, SD

and 95% CI.

3. Comment on the sensitivity and the reproducibility of the method.

4. Give the equations for the reactions responsible for the development of the molybdenum blue species.

5. How would you go about determining the concentration of total inorganic phosphate in the unknown

water sample?

6. What is achieved by the process of hydrolysis? Give appropriate equation.

7. Briefly describe operation of the Spectronic Genesis 5 spectrophotometer.

92

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of the Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Unknown Sample Number : __________________________________ Experimental Results : Instrument Calibration Results Linear Regression Results Replicate # 1 ___________ Replicate # 1 ___________ Replicate # 2 ___________ Replicate # 2 ___________ Replicate # 3 ___________ Replicate # 3 ___________ Sample Mean ___________ Sample Mean ___________ Sample SD ___________ Sample SD ___________ Sample 95% CI ___________ Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations (show example of all the calculations involved in data analysis):

93

EXPERIMENT 7

General Introduction to Ion Selective Electrodes12

Introduction

In recent years, ion-selective electrodes (ISE) have become an important analytical tool for many

chemists. They have found wide use in the analysis of drinking water and wastewater, in industrial process

control measurements, in quality control and in many areas of research. For example: ammonia electrodes

are used to determine ammonia in air and stack gases, beer, biological samples, boiler feed water, fertilizers,

and fish tanks; calcium electrodes are used to determine calcium in blood, feed, plant tissue, milk, soils,

fertilizers, and water; fluoride electrodes are used to determine fluoride in bone, carbonated beverages,

stack gases, coal, teeth, toothpaste, dental plaque, cryolite, drinking water, glass, household products,

phosphate rock, feeds and fluorospar. Ion-selective electrodes are available for most major elements and

ions as well as many gases. Reference 1 summarizes construction and applications of various ion-selective

electrodes.

Following is a brief review of theory of potentiometry, the concepts of ionic strength and activities

in ionic solutions and the preparation and the importance of total ionic strength adjusting buffer (TISAB) in

specific-ion measurements.

Theory

The concentration of ions or gases in solution may be determined by measuring the potential

difference which develops when the reference electrode and the sensing electrode are immersed in the

measuring solution. Ion-selective electrode is a sensing electrode which has a high degree of specificity for a

single ion or a class of ions. The reference electrode has a fixed potential held constant by being immersed

in an electrolyte (salt solution) of constant composition. When the two electrodes are immersed into a

measuring solution, the electrolyte surrounding a fixed potential reference electrode contacts the sample

through a porous membrane allowing current to flow between the electrodes (Figure 1). The voltage that

develops between the two electrodes is measured by a high impedance voltmeter (commonly known as a pH

meter). The meter also serves to complete the measuring circuit as shown in Figure 1.

1 D. A. Skoog, D. H. West, and F. J. Holler, Analytical Chemistry, 7th ed., pp. 151-158; 386-411; 412-423. Philadelphia:Saunders College Publishing, 1996. 2 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 172, 475-481. McGraw-Hill Higher Education Inc., 2000

94

The voltage is related in a very precise way to the concentration of the reactive species. As the

concentration of the reactive species varies, so does the voltage measured between the two electrodes. The

electrode response can be expressed by a linear Nernst equation (1):

E = Eo + 2.3RT

nF log ai (1)

where E = measured voltage; Eo = total of all constant voltages in the measuring cell at 25oC (called

standard potential); R = gas constant; n = number of electrons lost or gained in the electrode reaction; ai =

activity of the ion being measured. Another way of expressing equation (1) is:

E = Eo + S log ai (2)

where S is the electrode slope. The slope S is defined as the change in the electrode output voltage resulting

from a decade change in the activity of the ion to which it responds. At 25oC, the ideal slope for the ISE

with n = 1 is -59.16 mV. The equation (2) then becomes :

E = Eo - 59.16 log ai (3)

95

Activity ai is defined as the effective concentration of a free ion i in solution. It is related to the electrical

force each ion in solution exerts with respect to every other ion. The relation between activity and

concentration is given by the following expression:

ai = γi Ci (4)

where ai = activity; γi = single ion activity coefficient and Ci = ion concentration. In very dilute solutions,

where the single ion activity coefficient γi approaches unity, the activity and the concentration can be

considered equivalent and can be used interchangeably. However, in most chemical solutions in which

specific ion measurements are made, γi may vary significantly from unity. In these cases, the activity and the

concentration of the measured ion become different as can be seen from equation (4). The activity

coefficient γi is a function of the total quantity of all ions present in solution, known as the ionic strength of

the solution µ and is expressed by (5):3

µ = (1/2) Σi Ciz2i (5)

where zi is the charge on an ion. The activity coefficient γi in a solution of a specific ionic strength can be

calculated from the Debye-Huckel equation (6):

- log γi = 0.51 zi2 µ

1 + 0.33αi µ (6)

where αi = effective diameter of the hydrated ion i in angstroms. (Table of activity coefficients and αi

values for some common ions can be found in Ref.1, pp 154.)

In addition to the activity coefficient, the observed ion activity ai also depends on the ions tendency

to associate with other ions to form undissociated species such as weak acids, weak bases, insoluble

precipitates and complex molecules or ions. Most of these reactions tend to be pH dependent. It is therefore

important that all measurements be carried out at optimum and constant pH.

In practical applications it is difficult to determine single ion activity coefficient. For this reason, it

is common practice to raise the ionic strength of both standards and samples to high, and essentially

constant, levels. This is usually accomplished by the addition of a total ionic strength adjusting buffer

(TISAB). At constant ionic strength, the Nernst equation (2) may be rewritten to describe electrode response

to the concentration C of the measured species (7):

E = Eo + S log C (7)

3 D. Harvey, Modern Analytical Chemistry, 1st ed., pp 172. McGraw-Hill Higher Education Inc., 2000

96

Most measurements by ion-selective electrodes are performed at constant ionic strength and the unknown

concentrations are usually determined from the standard calibration curve.

Standard calibration curve is prepared by immersing a suitable sensing and reference electrodes in a series

of solutions of known concentration, and recording the resulting voltages. The measured voltage in

millivolts is plotted (on linear scale) against concentration (on log scale) on semilogarithmic graph paper.

The straight line drawn through the points gives a calibration curve. Any number of sample concentrations

can be determined from the calibration curve by immersing the sensing and the reference electrodes into the

sample solutions at adjusted ionic strength to that of the standard solutions, recording the voltage, and

reading corresponding concentration from the graph. Newer, microprocessor based instruments, such as the

Accumet 25 pH/Ion Meter have the capability to construct internal calibration curve and to give direct ion

concentration readings for the unknown solutions. In-depth discussion of activities and activity coefficients

is found in Ref.1, pp 151-158. More detailed discussion of potentiometric measurements and ion-selective

electrodes is found in Ref.1, pp 386-411; 412-423.

Fig.2. Typical Calibration Curves for Selective-Ion Measurements

Molarity

≅ 56 mv

10 fold change

10-4 10-3 10-2 10-1 10-5

300

260

220

180

140

100

90

97

Determination of Fluoride in Drinking Water or Toot hpaste

(Ion-Selective Electrode Method)4

Introduction

In this experiment, you will be asked to determine the concentration of fluoride in either an

unknown toothpaste sample or in an unknown sample of drinking water. Since the fluoride concentrations

are different in the two types of samples, a different set of calibration standards as well as sample

preparation procedures are required for the two analyses. Follow procedure steps 1, 2, and 4 for both

experiments, but choose procedure 3A if determining fluoride in toothpaste or 3B if determining fluoride in

drinking water.

Construction and Operation of Fluoride Electrode

The Fluoride electrode is an example of a solid state electrode. Its sensor is a lanthanum fluoride

(LaF3) crystal doped with europium fluoride (EuF2) and bonded into an epoxy body. When the fluoride

electrode is immersed in the solution containing fluoride ions, a potential proportional to fluoride

concentration develops across the LaF3 crystal. This potential is measured against a reference electrode and

is related to fluoride concentration by measuring potentials of solutions with known fluoride concentrations

.

Fluoride concentrations from 10-6 M to saturation can be measured directly, provided that the ionic strength

and the pH are adjusted and complexing agents for fluoride are essentially absent. This is achieved by the

addition of TISAB to the sample solution. TISAB raises the ionic strength of the measuring solution to

essentially constant levels, it buffers the solution to pH 5, and provides sodium citrate ion which

preferentially complexes with polyvalent cations such as aluminum and iron (III), which would otherwise

form stable complexes with fluoride ions. To accurately measure the fluoride concentration with an

electrode, the fluoride must exist free, in an uncomplexed state. Most anions including Cl-, Br-, SO2-4 , NO-

3 ,

do not interfere with fluoride ion measurements; however, hydroxide ion (OH-) does. In acid solutions,

hydrogen ion complexes with fluoride to form HF and HF2- complexes. Buffering the solution at pH 5

removes both hydroxide ion and hydrogen ion interferences.

Experimental Procedure

Equipment

Fisher Model 25 pH/Ion Meter

Fisher 13-620-523 Fluoride Ion Selective Electrode

Fisher 13-620-47 Double Junction Reference Electrode

4 T. S. Light and C. C. Cappuccino, J. CHEM. EDUC., 52, 247 (1975).

98

Apparatus

3 x 200 mL tall form beakers

5 x 150 mL plastic beakers

1 x 1000 mL, 1 x 500 mL and 6 x 100 mL volumetric flasks

1, 10 and 25 mL volumetric pipettes

5 magnetic stirring bars

Chemicals

Sodium Fluoride (NaF)-Primary Standard FW 41.99

Sodium Chloride (NaCl) FW 58.44

Sodium Acetate (CH3CO2Na) FW 82.03

Glacial Acetic Acid (CH3CO2H), d = 1.049g/mL FW 60.05

Trisodium Citrate Dihydrate(Na3C6H5O7.2H2O) FW 294.10

1. Preparation of Total Ionic Strength Adjusting Buffer (TISAB)

To a 500 mL volumetric flask containing 250 mL of deionized water add: 7.50 g glacial acetic acid,

29.22 g sodium chloride, 30.76 g sodium acetate and 0.15 g (0.001M) sodium citrate dihydrate. Dissolve

the salts by swirling the flask, then fill the flask to the mark with deionized water. Mix well, then check the

pH using a pH meter.

Operation and Standardization of a pH Meter

1. Before starting the pH measurement you should have the following items on hand: pH meter,

combination electrode, three 50 mL beakers , pH 4.00 or 4.01 and pH 7.00 or6.86 buffer solutions, box

of Kimwipes, wash bottle and a waste beaker.

2. Set FUNCTION selector on the pH meter to STANDBY and connect line cord to an electrical outlet.

3. Place a combination electrode into the electrode support holder, then connect electrode cable leads into

REF and INPUT jacks found at the back of the pH meter. Secure the connection by tightening the

knurled locking screw.

4. Prior to making the actual pH measurement, the pH meter must be standardized to compensate for the

difference in the zero potential of the electrode system. This is accomplished by the use of buffer

solutions of known pH values.

i) Set the FUNCTION selector to STANDBY position.

ii) Set the SLOPE control at 100%.

99

iii) Rinse the electrode by immersing into a beaker of deionized water and gently dry with a

Kimwipe.

(iv) Immerse the electrode into the pH 6.86 buffer solution.

(v) Check the temperature of the buffer and adjust the TEMPERATURE control to the actual buffer

temperature.

(vi) Set the FUNCTION selector to the pH position.

(vii) Adjust the STANDARDIZE control until the digital display indicates the pH of the buffer

solution. NOTE: determine the exact pH of the buffer solution from a table of buffer pH versus

temperature.

(viii) Set the FUNCTION selector to STANDBY position.

(ix) Remove the electrode system from the buffer solution and rinse with deionized water.

(x) Immerse the electrode system into pH 4.01 buffer solution; wait until the pH reading stabilizes,

then adjust the SLOPE control until the digital display indicates the pH of the second buffer.

(xi) Set the FUNCTION selection to STANDBY position, remove the electrode system from the

buffer solution and rinse with deionized water.

5. Transfer 50 mL of TISAB into a clean and dry beaker, immerse the electrode and gently swirl the

solution, set the FUNCTION selection to pH and read the pH value. The pH should be in the pH 5 to

5.5 range. If it is not, adjust pH using acetic acid or sodium acetate.

2. Preparation of 1000 mg/L Standard Fluoride Solution

From the desiccator next to the drying oven, transfer ~ 2.5 g of sodium fluoride, which has been dried

for two hours at 110oC, into a clean and dry weighing bottle. Weigh on the analytical balance to four

decimal places approximately 2.2 g of NaF and transfer quantitatively into 1 L volumetric flask. Add

approximately 300 mL of deionized water, swirl until solid dissolves, then dilute to mark.

Preparation of 100 mg/L Fluoride Standard

Using volumetric pipette, transfer 10 mL of 1000 mg/L stock standard into 100 mL volumetric flask,

dilute to the mark with deionized water, stopper the flask, then invert several times to mix well. (NOTE: Do

not store standard fluoride solutions for prolonged periods in glass containers since fluoride ions react with

glass. For longer storage periods, fluoride solutions should be stored in plastic containers.)

3A. Experiment I- Determination of Fluoride in Drin king Water

Preparation of 0.20 and 2.0 mg/L Working Fluoride Standards for Water Analysis

Using a volumetric pipette, transfer 10 mL of a 100 mg/L fluoride standard into 100 mL volumetric

flask, then dilute to volume with deionized water to prepare 10 mg/L fluoride standard. Next, transfer 2 mL

100

and 20 mL of the 10 mg/L working standard to two 100 mL volumetric flasks. Add 50 mL of TISAB buffer

to each and dilute to the mark with deionized water. Standards are now ready for measurement.

Note: **Before proceeding to standard measurements you need to calculate the exact fluoride ion

concentration in mg/L.

Preparation of Water Sample

Using a volumetric pipette, transfer three 50 mL aliquots of the unknown water sample into three 100 mL

volumetric flasks. Fill the flasks to volume with TISAB buffer. Mix thoroughly then empty entire content of

the flask into a beaker, stir and measure the fluoride concentration.

3B. Experiment II-Determination of Fluoride in Toothpaste

Preparation of 1 & 10 mg/L Working Fluoride Standards

Use volumetric pipettes to transfer 1 mL of 100 mg/L fluoride standard into one 100 mL volumetric

flask and 10 mL into second 100 mL volumetric flask. Use a 50 mL volumetric pipette to transfer 50 mL of

the TISAB ionic strength adjusting buffer to the same two flasks. Dilute to volume with deionized water.

Mix well by shaking the flasks. Standards are now ready for measurement.

Preparation of Toothpaste Samples

Clean and dry three 200 mL tall form beakers. Obtain an unknown toothpaste sample from the

demonstrator. Transfer into each of the three beakers approximately 200 mg of toothpaste using tarring

procedure on the analytical balance. (Hint- insert the tube from the top of the analytical balance, squeeze out

approximately 0.5 cm of toothpaste, cut the toothpaste with a spatula tip and let fall into the beaker.)

Record the weight to four decimal places. To each beaker add 50 mL of TISAB buffer and a glass stirring

rod and place on the preheated hot plate. Boil the solutions while stirring for two minutes making sure that

all the toothpaste has dissolved and that solutions do not boil over. Let the solutions cool, then use gravity

funnels to transfer solutions into 100 mL volumetric flasks. Dilute to the mark with deionized water and mix

well by inverting stoppered flasks several times.

4. Calibration of Ion Meter and Measurement of Sample Concentration

1. Check that the fluoride selective ion electrode and the double junction reference electrode are properly

connected to channel A. Check that the temperature probe is connected. Remove plastic cap from

fluoride electrode, adjust the electrodes in a holder so that they are at approximately the same level,

then immerse in a beaker of deionized water to rinse.

101

2. From the main screen, press the CHANNEL key until display indicates channel A.

3. Press the ION key to select the ion measurement.

4. Press the STANDARDIZATION key. The menu of standardization options appears.

5. Press the 2 key to select CLEAR EXISTING STANDARDS.

6. The meter returns to the main screen, but with all ion standardization points cleared from the memory.

Press the STANDARDIZATION key again.

7. Press the 1 key to select UPDATE OR ADD A STANDARD.

8. The SELECT ION UNITS menu appears. Press the 5 key to select units in mg/L. Then press the

ENTER key to implement the selection.

9. STD CONC. entry screen appears. Key in the concentration (mg/L) to three significant figures for the

lowest fluoride standard. Press ENTER key.

10. The PREPARE BUFFER/STANDARD screen appears. Remove the electrodes from the deionized

water and pat dry with a Kimwipe. Transfer the standard with lower fluoride concentration into a clean

150 mL plastic beaker, add 1" magnetic stirring bar and stir solution at medium speed on the stirring

plate provided. Immerse the electrodes into the standard solution making sure that no bubbles are

present on the tips of the electrodes and that the electrodes are not touching the stirring bar.

11. Press the ENTER key to measure the first standard. Record the millivolt reading when reading

stabilizes. (If you wait long enough, the meter will auto-enter the electrode signal but you may miss the

millivolt reading.)

12. Press the STANDARDIZATION key again.

13. Press the 1 key to select UPDATE OR ADD A STANDARD.

14. Key and enter the concentration of the higher standard.

15. Transfer the higher standard into a 150 mL plastic beaker and add 1" stirring bar. Rinse electrodes in a

beaker of deionized water, dry with a Kimwipe and place into the stirred standard solution. Press the

ENTER key to measure the second standard. Record the millivolt reading when stable, then press the

ENTER key. Check that the millivolt difference (slope) between the two readings is in -57 to -59

millivolt range. If it is not repeat steps 5-15 or see the demonstrator.

16. At this point, a two point calibration curve is stored in memory and you may proceed to measure the

fluoride concentrations of the unknown samples.

17. Transfer the first unknown solution into a 150 mL plastic beaker, add 1" stirring bar and stir the

solution at the same speed as for standards. Rinse and dry the electrodes, then immerse into the sample

solution. Press the MEASURE key. The instrument will display the fluoride concentration in the

sample in mg/L. Wait until the reading stabilizes before recording the concentration. In some cases,

especially in very dilute solutions, it may take 30 seconds to 1 minute before the reading stabilizes.

18. Repeat step 17 for unknown solutions 2 and 3.

102

5. Data Analysis

1. A)For the unknown water samples calculate the concentrations of F- and NaF in mg/L. (Remember the

dilution factor). Report the mean, the standard deviation and the 95% CI for the determined

concentrations.

OR

B)For the unknown toothpaste sample calculate the % F- and NaF. Report the mean, SD and 95% CI for

the results.

2. What are the sources of error in the analysis?

3. Calculate the ionic strength µ of the TISAB buffer that you prepared. (Remember that acetic acid is a

weak acid.)

4. Briefly explain why the use of TISAB in the analysis allows the measurement of fluoride ion

concentration and not activity as indicated by equation (1).

5. Briefly explain operation of the fluoride ion selective electrode.

103

Chem 2P42 Quantitative Report Form

Student Name : _________________________________________________ Date : ____________________ Lab Section : _______________ Title of the Experiment : _______________________________________________ Type of Analysis: ______________________________________________________ Unknown Sample Number : __________________________________ Experimental Results : Unknown Fluoride Concentration Standard Measurements (mV) Replicate # 1 ___________ 0.2 mg/L Standard ___________ Replicate # 2 ___________ 2.0 mg/L Standard ___________ Replicate # 3 ___________ Actual Standard Concentrations (mg/L) Sample Mean ___________ ___________ Sample SD ___________ ___________ Sample 95% CI ___________ Comments : _____________________________________________________ _________________________________________________________________ _________________________________________________________________ Sample Calculations (show example of all the calculations involved in data analysis):

104

APPENDIX I

Conversion Factors and Physical Constants

Velocity of light (in a vacuum) c 2.9979 x 1010 cm/s

Angstrom unit °A 10-8 cm, 10-10 m

micron µ 10-4 cm, 10-6 m

millimicron mµ 10-7 cm, 10-9 m nanometer nm

atomic mass unit amu 1.6606 x 10-24 g

Avogadro number NA 6.023 x 1023 particles/mol gas constant R 8.3145 J/mol K 1.987 cal/mol K 0.08206 L atm/mol K molar volume (STP) Vm 22.414 L/mol

joule J 107 erg thermochemical calorie cal 4.184 joules (heat required to raise temperature 0.04129 L atm

of 1 g H2O from 14.5°C to 15.5°C 2.612 x 1019 eV Standard pressure P 760 mm Hg

1.013 x 105 pascals 760 torr 1 atm

Absolute 0 To -273.16°C

Standard temperature T 273.16oK or 0.0oC π 3.1416 e 2.7183 ln x 2.303 log x Approximations: 1 mL = 20 drops; 1 dropperful = 10 drops

105

APPENDIX II Use of Excel for the Generation of a Calibration Curve and Regression Data

The following steps are a general outline and not intended to be a complete step by step procedure. Your

own experience and understanding of spreadsheets may be of assistance, and if you have none this should

provide you with a relatively easy introduction.

Ultimate goal: To use the data generated from a calibration to calculate the concentration of an ‘unknown’

when the measurement is made in the same manner as the standards.

The formula will return the x value, which is the concentration of the unknown.

Load the program. Enter the data from the standards in two columns. Label the top of one column as ‘x’

(standard concentration, with the appropriate units). Label the second column ‘y’ (response from the

measurement). Each value entered has its own cell address, e.g. C5, C6, C7 etc… indicating column C row

5, then 6, then 7 etc… In other locations on the spreadsheet it is helpful to put in statements or comments

which help identify various symbols, definitions or formulas which will be used in the spreadsheet.

In this case we are making use of the regression line equation; y = mx + b

Where y is the measured response

X is the concentration (with appropriate units)

m is the slope of the calibration line

b is the intercept value on the y axis, when x = 0

use the program function LINEST to obtain the slope of the line for the data entered. Follow the format:

=LINEST(cell range for y, cell range for x, True, False)

The program will return the slope of the line in the cell where you entered this information. Identify the cell

as ‘m’

Use the program function INTERCEPT to obtain the intercept of the line for the data entered.

Follow the format:

=Intercept (cell range for y, cell range for x)

The program will return the intercept of the line in the cell where you entered this information. Identify the

cell as ‘b’

106

Rewrite the regression equation as x = (y-b)/m. Enter the data for the unknown measured. You should have

y values with no corresponding x value. To calculate the x value, use the right hand side of the above

expression and enter the formula using the Cell addresses in place of the latter or actual numbers.

For example you should have a cell that contains information resembling:

+(+A5-F8)/G8

where A5 is the y data measurement for the unknown, F8 is the cell containing the intercept value and G8 is

the cell containing the slope value.

Graphical Display:

Using the graphing capabilities of Excel, you can graph your data to see what your data looks like. It is also

possible to draw the regression line from your data. There may be number of ways of doing this. One is

described below

1. Enter x,y data into Excel spreadsheet.

2. Highlight x,y data.

3. Click on chart icon.

4. Select xy scatter. Choose sub-type-one without lines. Then click on Next. If you need to plot several lines

on one graph select Series. Click on Add Series. In the Series box you should see Series 2 highlighted.

Click on mailbox in the y-values box. Mailbox will appear on your work sheet. Select the y-values you

want to include in second plot and click on the mailbox. Repeat for x values (x values can be same as for

series one or they can be different.) You should see second set of data points. Click on Next.

5. Enter title, labels for X and Y-axis and units. Remove grid lines and add legend if desired. Then click on

Next to select chart location (choose: as new sheet or as object in). Click on Finish.

6. To remove gray background – double click on graph background, choose white square from plot area

then click OK.

7. From Chart in toolbar select Add Trendline , then select Linear . Click on the series that you want to

perform regression on. Click on Options-then Display equation on chart and R squared. Do not select

Set intercept. Then click OK. Repeat for the remaining series.

8. Use slope and intercept from the equation of the line and the experimentally obtained y-value to calculate

concentration in the unknown sample.

107

APPENDIX III

Notes on Reference Styles

References should be given in a standard format: Journals

Spector, A., John, K.: "Effects of Free Fatty Acid on the Fluorescence of Bovine Serum Albumin"

Arch. Biochem. Biophys.127(1) 65-71 (1968) {QP 501 A77}

Author; last name, initial Article Name in quotes

Journal Name in Italics or underlined

Volume Number in BoldIssue Number in brackets page range

date of journalcall number

Often, the journal name has been condensed, as is common in the scientific literature. A partial list of contractions:

Arch. Biochem. Biophys. Archives of Biochemistry and Biophysics Biochim. Biophys. Acta BBA } Biochimica Biophysica Acta Biochem. J. Biochemical Journal Chem. Phys. Let. Chemical Physics Letters J. Am. Chem. Soc. Journal of the American Chemical Society J. Chem. Ed. Journal of Chemical Education Phys. Chem. Let. Physical Chemistry Letters

For a complete list see: CAS Directory Books

Sjöholm, I.: "Binding of drugs to Human Serum Albumin" inAlbumin: Structure,

Biosynthesis, Function, (Peters, T., Sjöholm, I., Eds.) Pergamon Press: Toronto (1978).

Author; last name, initial.Section/Chapter Title in Quotes (if applicable)

Book Title in Italics

Editors, followed by “Ed.” or “Eds.” Company city date inbrackets

Note: if book is part of a series, the series name follows the Editors in Italics (if the series as a whole has an editor, then that editor follows the series name)

108

World Wide Web All Web sources should contain the following basic information: Author’s name (last name first), Document Title, Date of Internet Publication, Date of access and <URL>. Example: (Book) Harnack, Andrew and Kleppinger Eugene. Preface. Online! A reference Guide to Using Internet Sources. Boston: Bedford/St. Martin’s. 2000. 5 Jan. 2000. <Http://www.bedfordstmartins.com/online>. For more details see one of the many style books in the library. In particular see: VCH Style to Scientific Writing.

109

Appendix IV

Useful Web References for Analytical Chemistry

1. Significant Figures: http://slc.umd.umich.edu/slconline/SIGF/page0.html 2. Analytical Instruments and Spectroscopic Concepts (adopted from-Dr. Thomas G. Ghosteen's

Home Page-Houston State University (videos and written material): http://www.shsu.edu/~chm_tgc/primers/primers.html

http://www.shsu.edu/~chm_tgc/sounds/sound.html 3. Good Analytical Site: http://www.anachem.umu.se/jumpstation.htm 4. Chromatographic Separations (Supelco Site):

http://ull.chemistry.uakron.edu/chemsep/index.html 5. LC/GC Chromatography Journal: http://www.chromatographyonline.com/lcgc/ 6. This site provides a general treatment of chromatography: http://ull.chemistry.uakron.edu/analytical/24_Chromatography.pdf 7. Good beginner's guide to high performance liquid chromatography (HPLC): http://hplc.chem.shu.edu/HPLC/index.html 8. Chemistry Based Videos: http://www.shsu.edu/%7Echm_tgc/sounds/sound.html (Updated August 2011)

110

APPENDIX V

Use of Library Resources for Lab Reports

The following is a general procedure for finding relevant material at Brock library. In addition to the widely used Google search engine (www.google.com) students may find journal articles via database search or search the library for available textbooks related to the subject of interest.

How to search for JOURNAL ARTICLES

1. Go to www.brocku.ca/library 2. Click on DATABASES 3. Select subject area (e.g. Analytical Chemistry) 4. Select desired database under the Title. Among the popular ones are: Scholarsportal,

Academic Search Premier, Web of Science. 5. Enter search terms (e.g. Gas Chromatography) and select journal category (e.g. chemistry).

Submit for search. 6. Once the search is complete, you may select Article Full Text PDF and view the article

online or save it to the disk. 7. If the article is not available in full text, you may find it in the original journal in the

following way: • Write down the journal Name, Volume # and Publication date. If the name of the

journal is abbreviated use Periodical Title Abbreviations available at the library (Call Number: PE 1693 A567 )

• Go to www.brocku.ca/library • Select RESEARCH on the top grey menu bar • Click on CATALOGUE • Select JOURNAL TITLE from the drop-down menu • Type in the name of the journal into the Title Search (e.g. Journal of Chemical

Education) • Click on search results and it will tell you if the journal is available at Brock library,

either electronically or in the stacks

• If the journal is not available at Brock library, it may be requested via INTERLIBRARY LOAN (RACER) (see link on library main page)

How to search for BOOKS

1. Go to www.brocku.ca/library 2. Select RESEARCH on the top grey menu bar 3. Click on CATALOGUE 4. Click on SUBJECT or KeyWORDS 5. Enter keywords (e.g. protein purification) and click search.

111

Click on search results and write down the Call Number. How to search for a Reference if you know its CALL NUMBER.

1. If a reference provided at the end of the experiment has a Call Number (e.g. QD 453 M 826 1965):

2. Go to www.brocku.ca/library 3. Select RESEARCH on the top grey menu bar 4. Click on CATALOGUE 5. Click on LC Call # (this stands for Library of Congress Call Number) 6. Type in the Call Number and click search.

As a Final Note…

General RULE OF THUMB for using the library: most of the science text books are located on the 5th floor of the library, while most of the scientific journals are on the 6th floor.

USEFUL WEBSITES for finding out more about the chemicals used in the lab: � Chem Finder

<http://chemfinder.cambridgesoft.com/> � Chemical Safety Database Searcher <http://ptcl.chem.ox.ac.uk/MSDS/msds-

searcher.html>