DETECTION AND SPECTROPHOTOMETRIC DETERMINATION OF ORGANIC FUNCTIONAL GROUPS ABSTRACT THESIS SUBMITTED FOR THE DEGREE OF Boctor of IN CHEMISTRY BY AHSAN SAEED DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 1087
DETECTION A N D SPECTROPHOTOMETRIC DETERMINATION OF ORGANIC FUNCTIONAL
GROUPS
ABSTRACT
THESIS SUBMITTED FOR THE DEGREE OF
B o c t o r of IN
CHEMISTRY
BY
AHSAN SAEED
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA) 1 0 8 7
ABSTRACT
This thesis comnriscs of five chanters. In first
chapter, a dct.iil'Hl and upto date survey of 1 i t."i at-urf
on the subject has been reviewed. In addition,
studies on thf methods for detection ancS spectrophotomet-
ric determination of organic compounds in pharmaceutical
preparations have been described. The colour reactions
of a nuinbcr oi functional grourjs via spot test hav^ bf en
studied. fhe utility of resin bead techniqu<? has b<'en
discussed tor idi-nt i f ication of different function il qroups.
Resin beads have been utilized in the form of their dual
role i.e. as a detection medium and a medium to catili^' or
to bring about <i chemical reaction. Henc(^, r'»sin :;pot. test
has been ci')f)li<;vi to m-ik. the detection more sen;":it'V - .sn i
free from inter! erinq species. The employmtrnt of './'l' known
instrumental ti?rhnique nam ely spectrophotometry 'M;; V> "n
achieved for detcriain ition of imbst^mces, and v' in
particular.
The ;:ec:ond chapter includes the analysi;; o;' a.- ? bi.r
acid in pharmaceutical pr- paration.":; throuqh i f-;;i.ri ;> •.;!)
detection arni spectrophotometric deter ininatlon . A c^'our
reaction of this compound has been developed with rn-dirltro-
benzene in prfS'-^nc^ o^ sodium hydroxide. The r'" !.•>;) ohf '/s
Beer's law in concentration range O.Ol to 0.2S mq o a: eorbic
3 phenylbuta^ori'^, propyphenazone, codeine and a numb er oL
other excipientr. and can be tolerated upto an anount of
1 mg of each whereas analgin/ oxyphf^nbufcaTion'^, phf^nac^tin,
phenazone saLicylat-^ .nncl n i cot In inter f<>re M i th thf t'-.'it.
The present mt;Lhod has been employed for the detf^rmination
of paracotarnol in various pharmaceutical pr'^parationr,.
The method is reproducible with relative; standard d-.-viation
of 2.35%. The molar absorptivity has been found to be
1.5 X 10^ 1 mole~^cm"^. The Beer's law obeys in the concen-
tration ranq>; of 0.1 - 0,5 nig paracetamol.
In the fourth chapter, an indirect colour r ,T;tion
has been stuaied for detection and determination of novalgin
in tablets. Novalgin is detected by sorption t"chni(Mio and
df?t'..'rrained ;;[)•-ctronhotometrica 1 ly by dt'velopinq th-- colour
with potassium ioJatf?. The detection limit is S u'l. BtM-r's
law is ob(.'y(-d in the concentration rangf^ of ,0.1 t.f) 1.0 mq
per 5 ml.
In th- lust chanter the conditions hav;; b n
for the deter mi rrat Ion of 1-nanhthol through - vr = ' ui'di-
tion, Thf oxidation product is aviolet compound 'isii <r!):;orbs
maximally at 520 nm. nc>-rVs law is obeyed in !"!: • < •-n -n tr it 1 on
range of 0.01 - 0.4 mg of l~nanhthol, Th.'=' m'-thc'i r- :;n-clfic
and has b<'<>n found to bo accuraf^ with standard vi i "n
of 1,66%. A t'.'H t'll. i v;; revK'tlon mechanism h - f. n a
proper,od.
acid. The detection limit is 10 yug. Tho rnothod is
found to be accurate with Q relative standard <i:'via-
tion of 2.12%. The molar absorptivity has been found
to be 1.35 X 10^ 1 mole"^ cm"^, A tentative reaction
mechanism has also been proposed. The method is
selective for ascorbic acid as vitamin B complex does
not give positive test. A negative test was given by-
functional groups such as carbohydrates, carboxylic acids
amino acids, aldehydes, ketones, alcohols, reducing com-
pounds like hydrazine, phenylhydrazine, hydroxylamine, meccap-
toacetic acid and trichloroacetic acid. The determination of
ascorbic acid in the presence of a number 'of foreign substances
has been studied.
In the third chapter spectrophotometric dot'^rmination
of paracetamol has been discussed. For the determination
of paracetamol, tho procedure is as follows. To an aliquot
volume of paracet imol prepared in dioxane ron!:<vninq 0.1 t;p
0.5 mg add 0,3 ml of 5% eerie ammonium nitrate (that is
prepared in 5 M nitric acid) in a 5 ml standard volviin t.-ic
flask. Make the solution upto the mark with dlox in Allow
the reaction mixturt- to stand about 10 minut -r '•o f3.-v lr(t,
yellow colour. Mear.uro the absorbancr:- of ^^sul^^n<T v-iiow
coloured product at 355 nm against a blank solution. i'ho
method has boon found to be vmafloctod by pror.onco i.- nfrin.
DETECTION A N D SPECTROPHOTOMETRIC DETERMINATION OF ORGANIC FUNCTIONAL
GROUPS
THESIS SUBMITTED FOR THE DEGREE OF
H o t t o t of $I|tIosiopI)p IN
CHEMISTRY
BY
AHSAN SAEED
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA) l e s T
il-^f • " Nv ^^
T3419
^^(('c/rf/.j^n^f/t (L f/->rj//f
M.Sc. , Ph.D., C. Chem.. MR/C ( L o n d o n )
P r o f e s s o r o f A n a l y t i c a l C h e m i s t r y
D E P A R T M E N T OF C H E M I S T R Y A L I G A R H M U S L I M U N I V E R S I T Y
ALIGARH-202002. I N D I A
This is to certify that the thesis entitled
"Detection and spectrophotometric determination
of organic functional groups" is the original
work of Mr, ;4isan Saeed and is suitable for
submission for the degree of Doctor of Philosophy
in Chemistry,
(Saidul Zafar^Qureshi) Supervisor i-
ACKNOWLEDGEMENT
It is always a pleasant task to acknowledge
one's debts. I am deeply obliged to Professor Saidul
Zafar Qureshi for his kind and encouraging supervision
which has made these studies possible.
I am grateful to Professor S.M, Osman, Chairiiidn
Department of Chemistry for providing research tdcili-
ties.
I extend my thanks to Dr. Tausiful Hasan for his
help and valuable suggestions rendered to me during the
preparation of this thesis. I- am also thankful to my
colleagues Dr. Nafisur Rehman and Syed Taufiq Ahmad for
their cooperation. I wish to thank all my lab members for
providing me a congenial company and maintaining a cordial
atmosph'?re in the laboratory.
I am also obliged to ny mother for her C"jn::t.int
encouragement and interest in my acedemic ptJLrriui ty.
AHSAN SA.-IKD
C O N T E N T S
Page
CHAPTER - I Introduction
References
1
32
CHAPTER - II Resin bead detection and spoctro-photometric determination of ascorbic acid in pharmaceutical preparations using m-dinitroben-zene.
47
CFiAPTER - III Spectrophotometric determination of paracetamol in tablets using eerie ammonium nitrate.
65
CHAPTER - IV Detection and spectrophotometric determination of novalgin in tablets using potassium iodate.
7S
CHAPTER - V Conditions for 1-naphthol determi-nation through periodate oxidation,
89
LIST OF TABLES
P.iqn
Table - 1 Methods for detection and determination of organic compounds based on functional group.
9 - 1 1
Table - 2 Important terms and their values evalua-ted in the analysis (ascorbic acid deter-mination) .
52
Table - 3 Dptermination of ascorbic acid in pharma- 59 ceutical pre^-parations.
Table - 4 Colour of resin beads on treatment with 60 coloured complex of ascorbic acid.
Table - 5 Determination of paracetamol in pharmaceu- 71 tical preparations.
Table - 6 Important terms and data evaluated in the 72 analysis (paracetamol determination)•
Table - 7 Colour reaction of various drugs v/ith potassium iodate and starch.
80
Table - 8 Detormination of novalqin in pharmaceu-tical preparations.
85
Table - 9 Important data evaluated in thf? analysis (novalqin determination),
86
Table - 10 Important data evaluated in thoanalyni.s 93 (1-napnthol detormination).
LIST OF FTCURES
Paqe
Fig, 1 Absorption spectrum of ascorbic acid 54 m-dinitrobenzene reaction product.
Fig, 2 Effect of sodium hydroxide concentration. 55
Fig, 3 Effect of m-dinitrobenzene concentration. 56
Fig, 4 Calibration curve of ascorbic acid. 57
Fig, 5 Effect of eerie ammonium nitrate 69 concentration,
Fig, 6 Calibration curve of paracetamol. 70
Fig. 7 Absorption spectrum of novalgin - 82 potassium iodate reaction product.
Fig, 8 Calibration curve of novalgin. 84
Fig, 9 Absorption spectrum of l~naphthol " ' 5 potassium periodate reaction product.
Fig. 10 Effect of potassium periodate conc*!ntra- 96 tion.
Fig, 11 Calibration curve of 1-naphthol, 98
CHAPTER - I
I N T R O D U C T I O N
The fundamental importance of analytical chemistry
is shown by the urgent demand of this branch of chemistry.
Our present day knowledge of elements has been made possi-
ble by analysis and separations in all disciplines of
chemistry which are dependent on analytical principles.
Starting discoveries of medicines have been dependent on
accurate analysis. It is possible for experimental research
to ascertain the composition of the product formed. The
methods are accurate, simple, rapid, essential to economic
control of industrial processes.
In analytical chemistry the analysis involving
detection and determination of different organic functional
group containing compounds has been one of the most demanding
problems. The analysis of organic compounds can be made by
using instrumental methods such as IR spectromrstry, uv-visible
spectrometry, mass spectrometry, chromatography, coulomotry,
conductometry, potentiometry, polarography, amprronctry etc.
or by non Instrumental methods as spot-test via colour
reactions.
Spot-test techniquo has been founa to bo inost
versatile and widely used method. Importance of spot
test in qualit.'tive analysis ir> due to the pionooring
efforts of Foiql (l) who has largely devt;loped Lhis
technique. Thc> term snot-test analysis annlics to
sensitive and selective detection based on a chcmic.il
reaction in which a drop of test solution is brot-ght
into contact with suitable reagent on filter pap^^r,
watch glass or porcel<iin plate. It is a v(.>ry simple,
and rapid method. Maximum specificity, sensitivity
and selectivity is attained unly by proper choice of
reaction conditions for analysis and by the use of
masking agents or pH adjustment and 'therefore its use
has been extended to micro or even nanogram detection
of substances.
Resin spot technique has been widely used for the
detection of inorganic ionic species and a few organic
compounds of known colour reaction (2-4). However, resin
beads can also be used to develop new colour reactions as
was described by Qureshi for diphenylamine (5,6), amino
acids (7), ketones (8), thiols (9) and ethylenediaminetetra-
acetic acid in human urine (10). Fujimoto (11) described a
very sensitive test for the detection of fluoride u:;ing ion
exchange resin beads. Bolton (12) has described a method
for the detection of N, S, P and halogens. The sampl'? Ir;
wrapped in a piece of tissue paper, ianited in a flask of
oxygen, the acidic ^jases produced are adsorbed into u
solution of sodium hydroxide (nitric acid for p) ,tnd the
inorganic ions presented in adsorbent are detected by
suitable anion analysis.
Another important phenomenon is to combine the
hydrolysis and catalytic reactions of resin beads
with the resin spot techniques. A very interesting
example of this approach is the detection of esters
(13). The mechanism of the method is simple. Ion
exchanger in H"*" form hydrolyses esters effectively
than does an acid (14) and no new ions are introduced
into the solution.
H"** CH2COOC2H5 + H^O > CH3COOH + C2HgOH
Resin spot test has been successfully applied
in microdetection of amides, imides, anilides (15) and
nitriles (16) in which the resin beads play a dual role
i.e. as a reaction medium to concentrate the colour on
the resin surface and secondly as catalytic to facilitate
their acid hydrolysis. The hydrolysis of amides, imides
anilides and nitriles is brought about by H" form cation
exchange resin which catalyses the acid hydrolysis to
corresponding acid and ammonia or aniline. Ammonia gas
and aniline pick-up one proton from resin in H" form and
they are converted to NH "*" and CgH^NH^'''. These ion^; easily
replace H"*" ions from resin and are detected on th'- bead
surface by means of Nensler's reagent or p-dimethylainl no-
benzaldehyde. Reactions are as follows:-
XCONH2 + HOH ^^ > XCOOH + UH^
NH + 2H0H
XCONHCgH^ + HOH
CgH^NH2 + HOH
RH
RH^
RCgHgNH^"^ + OCH N(CH2)2
+ NH.
XCOOH + CgHgNH^
RCgHgNH^
+ » RCgHgNH^tsCH N(CH3)2
Pale yellow resin beads (X = alkyl or aryl groups)
Nitriles can be hydrolysed with dilute sulphuric acid in
the presence of resin beads (H"** form) • The resin spot-test used
for detection of nitriles is not only selective for them but can
be used to differentiate nitriles from unsaturated amides as they
interfere with the test.
Test-I
dil,H,SO. XCN + HOH .. • > XCONH. Heat 2
(X = alkyl or aryl group)
XCOOH + NH. XCOONH 4
RH + XCOONH '4 RNH,
RH"* XCOOH + NH,
XCOOH
positive test with Nesslcr's reagent.
Test-II RH XCN + HOH -Tj— > Hydrolysis does not takp place,
negative test with Messier's reagent.
Qureshi et al. (17) extend the use of ion exchange resins as a
catalyst and as an ion exch mger aimultanooualy for thr> doter-
mination of amides and esters. The acid hydrolynis of
amides and esters is done by using ion exchange rosin
beads in H"*" form. The samole solution is passed through
a specially designed column containing Amberlite IR - 120
resin in H"*" form. The ion exchange column is maintained
at a constant temperature at 80°C during the hydrolysis.
The effluent is recycled three times to ensure a complete
quantitative conversion. The effluent is titrated with a
standard 0,05 N sodium hydroxide solution. The amount of
amide or ester present can be calculated from the corres-
ponding acid produced.
Qureshi et al. (18) developed a simultaneous microgram
detection of nitrogen, sulphur, chlorine and iodine in
organic mixtures using resin spot-test. In another test
(19) they developed ion exchange method for the detection
of aliphatic and aromatic aldehydes. An aqueous solution
of aldehyde is heated with saturated sodium cyanide
containing hydrogen cyanide (HCN) together with a slight
excess of sulphuric acid and cation exchange resin in H"*"
form. This results in the formation of cyanohydrin. The ion
exchange resin in H' form catalyses the hydrolysis of the
cyanohydrin to corresponding carboxylic acid and ammonia.
Ammonium ion is retained by resin beads and is tested by a
drop of Nessler's reagent. The reaction sequenco is given
by 0
R.a R.CHO + CN ^ R.CHCN V • O I
R.CHCN + H^O > R.CFI(0H)CN -h OH
R.CH(OH)CN + H^O + ResH'" > R.CH(0H)C00H + ResNH^
ResNnt + Nessler's reagent (alkaline) > red coloured beads
Sucrose inversion (20), ester hydrolysis (21), benzoin
condensation (22) can also be catalysed by ion exchangers in
the various ionic form. The use of solid ion exchangers as
catalyst has a number of advantages as compared with dissolved
electrolytes.
1, The catalyst can be easily removed from reaction product by filteration or decantation,
2. The purity of product is better sinco side reactions are minimized,
3, The ion exchanger is more selective i.o, it di5tinguishe?s sharply between various reactant molecules and it may be considered to bo hall in the selectivity between dissolved electrolyte and enzymes,
4. No new ions are introduced in the reaction media except the ion which are produced as a result of hydrolysis.
Further the chemical analysis finds a cons Iant
application in the 1 ield of technology, industry, mo'iicine,
agriculture, geology etc.
The constituent to be detected or determined may be
from inorganic or organic substances and the analysis
correspondingly known as inorganic or organic analysis.
Over the last three decades there has been an
increased interest in the field of organic analysis which
may be divided into three groups:-
(1) elemental analysis
(2) functional group analysis and
(3) analysis of compounds individually.
Preference is given for the determination of organic
compounds by functional group analysis (23-25) rather than
to elemental analysis, because the functional group gives
more characteristic information than elements.
Quantitative analysis of organic compounds in traces
is of high importance because this gives the determination
of a component in a sample largely diluted with other mate-
rials. The principal techniques used for this purpose are:
spectroscopic, chromatographic, electrochemical an<i ra'ilo-
tracer technique etc. The sf^ectroscopic techniquf i.nclud<<
nuclear magnetic resonance, ultraviolet, infrared arv i visible
spectrophotometry. The chromatographic methods includt paper,
thin layer, column, electrochromatography and gas chromatogra-
phy. The electrochemical methods mainly used for this purpose
8
are voltametry, conductometry. The radiotracer tochniques
include mainly isotopic dilution and activation analysis.
The use of spectroscopic methods^ however has been
made on the largest scale in organic analysis. Out of
these/ the use of spectrophotometry applied in visible
region is preferred because of its simplicity. This is
very appropriate technique when a colour is formed by a
particular reaction.
The organic compounds may be classified into the
following groups based on different functional groups:
Carboxylic acids, carbonyl compounds, esters, alcohols,
phenols, ethers, amines, amides, nitro compounds,
hydrazines, mercaptans etc. They may also be categorised
on the basis of their natures towards oxidisible and
reducible substances.
The important methods are listed in Table-1 for
the spectrophotometric determination of organic compounds
on the basis of functional group determination.
Ascorbic acid is widely distributed and occurs in
fresh vegetable and fruits, particularly in citrus fruits.
In animals this vitamin occurs in tissues and varioun (jland;
or organs e.g. livor, adrenal glands, thymuij etc., but in
larger quantities it is found in the adrenal cortox. Milk
p. (U o 0 u c 0-1 (U t- CO 0 tH OJ ro in OJ CN CM <N CO ro ro ro ro (t) 'H C OJ O Pi
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12
and blood also contain small quantities of ascorbic acid.
Ascorbic acid is an unsaturated aliphatic acid and its
chemical structure resembles with that of a carbohydrate
' mono saccharide). It was first isolated in crystalline
form by Szent-Gyc^|^i from adrenal cortex and later from the
juice of citrus fruits. It is now easily obtained from
glucose by synthetic method and it was the first vitamin
obtained synthetically. Ascorbic acid shows remarked
reducing property and is oxidised to dehydro-ascorbic.
Ascorbic acid catalyses cellular oxidation-reduction
reaction in the body and due to this oxidation behaviour
Szent-Gyogri suggested that it takes part in the respi-
ratory system. For the estimation of ascorbic acid,
among various available physical and chemical methodr, its
titration with 2,1 - bischlorophenolindophenol is the most
widely used method today.
A number of methods have been described Cor the
detection and determination of ascorbic acid. Ascorbic /
acid has been determined by c<,cx: _dipyrldyl (53, 54). The
formation of violet coiour with 2,6 - dichlorophono1indo-
phenol is used for ascorbic acid determination (55,56,57).
Methylthiazlyl tetrazolium salt (MMT) has been used for
ascorbic acid estimation (58,59,60), A method haa recently been
studied for the flow injection determination of ascorbic acid
(61). 2,6 -Dichlorophenolindophenol has been recommended
13
as a reagont for the detection of ascorbic acid in pharma-
ceuticals. Various compounds present in pharmaceuticals
containing ascorbic acid do not interfere with the test (62).
Mercurous nitrato has been used as a selective lencypnt for
the detection of ascorbic acid (63). The test is not
specific as some o t h e r - u n s a t u r a t e d compounds give the
same colour. Ascorbic acid is detected in fruit juices and
body fluids by impregnating the paper with a solution contai-
ning alkali salt of 2,8 -phosphomolybdic acid, a hydroxy
carboxylic acid and its alkali salt. The test paper changes
its colour on contact with ascorbic acid (64). Nessler's
reagent has been used as a developer for ascorbic acid during
detection by paper chromatography (65)• Ascorbic acid
produces a red coloration when heated with 3% solution of I
p-nitroso - N,N - dimethylalanine in acetic acid (66).
The reaction is not observed for glucose, lactose or water
soluble vitamins. A quantitative method for'the detection
of ascorbic acid in beer has been discussed (67) . A general
test for the detection of the acids have been df^ncribcd by
Qureshi et al. using p-dimethylaminocinnamaldehyde and sodium
nitrite (68). A specific test for the detection of ascorbic
acid is developed by Mueller - Mulot (69). A hot solution of
dimethylformamide acidified with ascorbic acid and Its deri-
vative formed a reddish - purple complex by reaction of
intermediate dehydro-ascorbic acid and glycine. A general
test has been described by Yavor'skii (70) for th'- detection
14
of ascorbic acid, reducing saccharides, adernaline and
some polyphenols. Wawrzyczek proposed a new colour
reaction for ascorbic acid by oxidation with osmium
tetroxide (71), Carbohydrates are found not to inferfere
with the test.
Spectrophotometric methods have proved to be most
useful for the determination of ascorbic acid. Ascorbic
acid can be determined in microgram amount using ammonium
molybdate (72). A rapid and sensitive spectrophotometric
procedure for its determination is based on its interaction
with dimethoxyquinone (73). Reducing substances such as
preservatives, stabilizers, mineral vitamins do not inter-
fere, A method for the ascorbic acid determination is
described by its colour reaction with phenylhydrazine and
hydrochloric acid and measuring the absorbance at 395 nm
(74). Extraction and spectrophotometric determination of
ascorbic acid using 2,6 - dichlorophenolindophenol has been
reported (75) . Another colour reaction ot a s c o r b i c acid ITS
produced by its interaction with diazotized p-aminobenzoic
acid that is used for its spectrophotometric determination (76)
A photometric method for the estimation of ascorbic acid
has been reported (77). Numa (78) investigated a colour
reaction for the determination of ascorbic acid, enayiar;,
protein and amino acids with N-bromosucclnimide, An indirect
spectrophotometric method is described for the determination
15
of ascorbic acid by utilizing a freshly reduced, tervalent
chromium and EDTA. (79) . Another method has been achieved
for its determination in HCl-KCl buffer solution (80).
A colorimetric method for ascorbic acid determination is 3+ 2+ based on reduction of Fe to Fe by quinone group and
2 + subsequent formation of pink colour complex between Fe
and bipyridine (81). An intense violet complex formed
between equimolar mixture of cacotheline and ascorbic acid
is used for its spectrophotometric estimation (82). A new
diazo method for the determination of ascorbic acid in blood
plasma is based on colour reaction between zinc chloride
double salt of diazotized 4 - nitroaniline - 2, 5 - dimethoxy
aniline and ascorbic acid (83), A spectrophotometric method
for the determination of small amount of ascorbic acid in
pharmaceutical preparations is reported by Karas-Gasparce and
Hankogi (84). In this method the compounds are reacted with
potassium ferrocyanide at pH 3.5 in the presence of 1,10 -phenanthroline. A quantitative method for determination of 1 ascorbic acid, involving the formation of blue colour with
phosphomolybdic acid in sulphuric acid medium is described
by Godyyatskii et al (85). Ascorbic acid has been determined
colorimetrically in fruit juices, fresh vegetables and phar-
maceutical preparations by reaction with diazotized 4-chloro-
2-nitroaniline (86 ) .
16
A potentiometric method for its determination is
based on potentiometric titration with 2,5-dichloroin-
dophenol (87) . Paper chromatography" has been used for
the detection and determination of ascorbic acid. Spots
are detected by spraying a solution of sodium salt of
0.03% 2,6-dichloroindophenol. After cutting out the spot
the concentrated acid is determined by 2*4-dinitrophenyl-
hydrazine (88). An iodometric method for the determination
of acid has also been described (89). A complexometric
determination of ascorbic acid is described (90). The amount
of acid in the sample is determined by reaction with mercuric
chloride. The excess of mercuric chloride in the filtrate is
determined by complex formation using EDTA and Eriochrome
Black-T as the indicator. A number of methods are also
available in the literature for ascorbic acid determination
(91-99).
In the third decade of the ninteenth century the high
cost and scarity of qxiinine motivated people to search tor
synthetic antipyretics. As a result, a large number compounds
are introduced into medicine. They have ability to produce
antipyretic and analgesic effects. Analgesics and antipyr^^tics
are drugs which not only relie/e pain but they reduce elevated
body temperature too. Analgesic and antipyretics can be
classified as:-
(I) The salicylates. (Il) The para-aminophenol derivatives.
17
(III) The pyrazolone derivatives.
(IV) Propionic acid derivatives.
Paracetamol is the derivative of para-aminophenol.
This exerts analgesic and antipyretic effects like salicy-
lates. Paracetamol is a slightly more potent antipyretic
than phenacetin and is equianalgesic with aspirin. It can
be used in liquid dosage form in children. Novalgin has
potent analgesic and antipyretic action but no uricosuric
effect. It does not offer any distinct advantage over
aspirin except it can be injected. The toxic effects are
similar to those of phenylbutazone. Hence, it is risky
to use this drug in a routine fashion.
Paracetamol has been determined in tablets by a
colorimetric method based on the nitration of the drug and
treatment of resulting nitro compounds with dimedone (III)
and triethylamine-> to form a coloured complex. Boar's law
is obeyed in the concentration range 0.03 - 0.1 mq/ml (100)..
However, oxyphenbutazone interferes with the test. A buffered
solution of Folin and Ciocalteau is a colorimetric reagent
for the spectrophotometric determination of paracetamol in
analgesic preparations. Ascorbic acid also reacts with the
reagent and can be deactivated by oxidation with hiydrogen
peroxide (101). Another colorimetric method for the estima-
tion of paracetamol has been reported. The mtrthod in based
18
on reaction with ferric hydroxamate resulting in a reddish
violet coloured product (102). A simple colorimctric
method estimates paracetamol that is based on the hydrolysis
ot complex with P-dimethylaminobenzaldehyde (103). A rapid
automatic colorimetric method for the determination of para-
cetamol has been developed (104). It is based on its reaction
with acidic hypochloride followed by coupling with alkaline
phenol resulting in the tormation of blue indophenol. Para-
cetamol can be determined in combination, colorimetrically
by adding the solution of HCl and NaCLo at pH 3.4 (105).
A method for the quantitative determination of paracetamol
as 2-nitro-4-acetamidophenol formed by reaction with nitrous
acid has been developed (l06). Phenacetine causes only
negligible interference. However, salicylamide forms a
chromophore which interferes in the determination of parace-
tamol. A method for the its determination in syrup and in
combination with other drugs i-i tablet form has been studied
(197). Spi'c rophotometric mf.'thod for the detorminotion of
compound after separ ition by butanol extraction and dircct
ultrabiolet spectrophotometry was reported, after trf;atnv nt
with O.IN sodium hydroxide (108), The ultraviolc^t spoctro-
photometric method has been developed for paracemamol determi-
nation after separation by means of column chromatography(109).
Codeine phosphate, phenylephedrine hydrochloride nnd pyrilenine
maleate can also be determined by using same mothofl. An
independent method for paracetamol makes use of acotylnalicylic
19
-u't'l, I I I .iiiii Mpl iMir.iln" In I ot (">U(|(.r
(110). Th<; t.iblot in dlsr.olv.-d in mothanol, flltorod
to remove the insoluble excipients and divided in two
portions. First portion is used for ultraviolet spectro-
photometry of paracetamol and salicylamide, second portion
is analyzed for aspirin by titration, Semimicro analysis
of paracetamol in blulk and in tablets, based on oxidation
of paracetamol with Ce^"^ is described (ill) • A colorimetric
method for the determination of paracetamol has been reported
by Ninomiya (112). The method is based on the oxidation of
drug with ferricyanide in sodium hydroxide in ice cold
conditions. A blue coloured product is obtained on addition
of phenol to the reaction mixture. The formation of mono-
nitro derivatives ot paracetamol was studied for its
estimation (113). Two simple and rapid methods are described
for the identification of paracetamol (114). In the first
method, a yellow colour is obtained with sodium nitrite in
acidic medium, which changes to orange after alkalization, in
the second method the paracetamol is hydrolysed with acid to
give a violet colour with ammonium hydroxide. The sensitivity
of the method (I) is 10 ^g/ml, of (ll) is 0.5 /ig/ml. Parace-
tamol, chloropromazine hydrochloride and tetracycline were
separated and identified by thin layer chromatograDhy on silica
gel containing Jluorescein and observing the spot the ultravio-
let light (115). A dark brown spot of paracetamol is observed
20
when ethyl methyl ketone is used a developer. Chioropromazine
and tetracycline give zero R^ value. Paracetamol is identified
by thin layer chromatograp'iy on silica gel G or Fuller's earth
plates using a 90:8:2 butanol-water-ammonia solvent system and
Drazendorff's reagent for detection (116). A differential non-
aqueous titration procedure has been described for the determi-
nation of a mixture of paracetamol and salicylamide (117). In
another method a mixture of paracetamol and salicylamide is
dissolved in dimethylformamide and has been determined poten-
tiometrically using 0-. IN lithium hydroxide as a titrant (118).
Gas chromatographic technique has been used for the
determination of paracetamol in plasma, and urine (119-120),
Nitrometric titration of paracetamol has been reported. In
this method the determination is carried out via hydrolysis
with hydrochloric acid, followed by nitrimetric titration of
resulting p-aminophenol using a mixture of tropeolln and
methylene blue as an indiettor and KBr as the catalyst (12]).
A simple colorim^'trlc method for the detenninvit Lon o? parace-
tamol in biological specimen has been describf'd (122). The
method is based on the measurement of intensity of th'» crimson
colour produced when paracetamol is treated with 10% aqueous
sodium hydroxic No interference is caused by the comnounds
such as phenacetin, asnirin, caffeine, oxyphenbutazone which
are present in the various analgesic formulations of paracetamol,
A new screening method for its determination in blood serum has
21
been studied (123). The method is well suited for use in
emergency situations.
An automated colorimetric method has been used for its
determination by mean of its reaction with Folin and Ciocal-
teau's reagent at pH 8 (124), Inamdar et al. report the esti-
mation of paracetamol (125). If analgin is present in the
sample it must be removed by extraction with anhydrous acetone.
A method for quantitative determination of paracetamol^ tropa,
brucine, caffeince etc. by means of ion exchange and spectro-
photometry is described. It is based on the adsorption of
acidic and basic substances on ion exchange resin followed by
the elimination of interfering products by washing or back wash
procedure and elution of pure drug. The resins used are Dowex
50-X-2 and 1-x-l (126). The reaction of paracetamol with nitrous
acid to give 2-nitro-4-acetamidophenol is used for easy colori-
metric determination of paracetamol (127). A method for its
determination in several forms by ion exchange and non-aquoous
titration hay boon reporte:d by Hent (128). KoL;hy haa developed
a chromatographic method for the simultaneous determination of
aspirin, coffeine and paracetamol (129),
A method has been established for the estimation of
novalgin in pharmaceutical preparations with p-dirnethylamino-
benzaldehyde (1j,0). Paracetamol can also be determined by this
method. However the presence of chloronheninamine maloate,
caffeine, and diazepam do not affect the determination. In
another method novalgin has been determined spectrophotornetrically
22
by its rcdction wiLh CUCI2.2H2O at pH 4,6 and the absorbance
of resulting colourt?d product measured at 340 and 408 nm
(131). However, aminophenazone and phenazone interfere with
the test. An ultraviolet spectrophotometric method is repor-
ted for its analysis (132). It has also been determined by
differential spectrophotometric method (133). A method for
the simultaneous estimation of novalgin and pyramidon is
discribed (134). It is noted that pyramidon is readily soluble
in chloroform while novalgin is relatively insoluble. To a
solution of novalgin in ehtanol and pyramidon in chloroform, /
ferric chloride and oC / c< - bipyridyl are added and the
resulting red complex is measured spectrophotometrically,
Novalgin has been determined on the capability of forming a
coloured compound with bromophenol blue (II) which is extracted
into chloroform and the absorbance measured at 414 nm (135).
Novalgin can also be determined in powder form, tablets and
medicated drops. A photoelectro colorimetric technique is
described for quantitative determination of novalgin (136),
The method is basci on its reaction with p-benzoqu!nono in
acetic acid. The method has also been utilized for the determi-
nation of novalgin in i^s mixtures with caffeine, amidopyrine
and phenacetine. In another method it has been identified and
determined spectrophotometrically either in pure form or in a
mixture by its reaction with acetic acid, phenol, ammonia buffer
and potassium ferricyanide (137). A red coloured complex of
novalgin and lumogallion is utilized for its spectrophotometric
23
estimation (138). A rapid qualitative test for the detection
of pyramidon-novalgin mixture has been described by Labanov
(139). The filter paper to which analgin or its mixture is
applied turned to purple with boiling hydrogen peroxide.
Another qualitative test for its detection is based on its
interaction with 1ml of 1% Mn (NO^)2#1 drop of 1% NaOH and few
crystals of organic acid to give an intense blue colour (140),
Gallic acid has been reported as a specific reagent for novalgin
detection (141). Identification and spectrophotometric deter-
mination of novalgin with sodium 1,2 - naphthaquinone -4- sul-
fonate is reported by Maggiorelli and Conti (142). The reaction
can not be used in the presence of primary amines but ant^pyrine
does not interfere. Flame photometric methods have also been used
for the quantitative estimation of novalgin (143). A rapid,
characteristic and specific method for novalgin detection has
been described (144). In this method dilute sulphuric acid (3-5 4
drops) is heated on a watch glass and then, a drop of schiff's
reagent and 1 drop of the test solution are added, a violet
colour appears immediately.
Another method is described for the determination of
novalgin by potentiometric titration or titration v/ith crystal
violet as indicator in acetone. Tho titration In carrle<3 out
by O.IN HCl in acetic acid (145). Chloramine has been usoci as
a titrant for the determination of novalgin. In thin method
novalgin is dissolved in 30 ml water, 10 ml of 8% aqucious HCl
24
and 5 ml of 10% KI aro added and the reaction mixture is
titrated with O.IN chloramine to a blue colour of the
solution (146). Another method involves the vanadomotric
determination of novalgin. In this method 5 ml of O.OIM
aqueous novalgin solution is added to 19.5 ml sulphuric
acid (1:1) and 20 ml of 0.02N ammonium vanadate. After 30
minutes the excess of reagent is titrated with 0.02N Mohr's
salt by using 3 drops of 0.2% aqueous sodium phenylanthranilate
as indicator (147). Novalgin can be determined volumetrically
with barium chloride. The SO^ group present in the novalgin is
oxidised to SO^ and is determined by titration with barium chlo-
ride (148). Juan and Rosa have demonstrated a qualitative test
for the group present in novalgin and they have recommended colo-
rimetric method for the determination of novalgin (149),
Medicinally used methanesulfonic acid derivatives as
novalgin and melubrine are determined by iodometric mothod
based on the fact in moderately alkaline solution they can be
split into sulfite and glycolic acid by potassium c-yani<if!. The
sulfite is then titrated with iodine solution to starch iodide
end point in sulphuric acid of moderate concentration (150).
A number of methods based on different technique for the
determination of novalgin are also available in tho litt»rature
(151-158).
25
Some industrial wastes usually contain phenols and
their derivatives, and their detection and determination
occupy an important place in analytical chemistry. There-
fore, a sensitive and rapid method for microgram detection
and determination is needed. Phenolic compounds have been
detected by colour formation with chlorotoludine (159).
This reaction is the modification of Greig and Leaback
reaction used for detection of amino and imino compounds
(160). The aqueous or methanolic alkaline sample is
treated with potassium chlorate and saturated o-toludine
solution in 2% acetic acid. The contents are allowed to
react 30 minutes with aqueous 1% potassium iodide. The
resulting colours are identified by thin layer chromatogra-
phy and the R^ values are recorded. Phenols are determined
spectrophotometrically with 4-aminoantipyrine in the presence
of potassium ferricyanide used as a oxidising agent (161-163)«
A spectrophotometric method for the determination of 1-naphthol
in the presence of 2-naphthol has been described (164). The
method is based on the faster rate of diazotizatlon of 1-naph-
thol than 2-naphthol. A spot test has been developed by Kagaku
(165) for the detection of phenols in which indophonol reacts
with p-aminophenol. A drop of sample solution and a drop of 2%
sodium hydroxide are placed in a micro test tube. If necessary,
1-2 drops of ethyl alcohol is added to complete the dissolution.
To this solution, 0.5 ml p-aminophenol reagent is added and the
mixture is shaken to develop a characteristic blue, green or
26
1>H[1.>1« i-iildui, 'Pli<! m<<t ho>1 1!. .'itiiia i t i Vf. to dl- <inii irlhydrlr
ph<>noly, 0-, m-, <md p-isornnrs qivG a different colour whern-
a i p-crt;:!ol and hydt oc}uinoni- qivt; a nogatlvu toot. A apectro-
photomntrlc mothod for thn dotrrmination of phonol has been
given, which is based on the addition of three moles of
N-bromosuccinimide in methyl alcohol at O^C (1966) and phloro-
glucinol takes up seven bromine atom's with ring opening and can
be reduced with the atoms of iodine. 1-Naphthol can be deter-
mined only in 1% hydrobromide. Another colour reaction of
phenolic compounds is obtained by the interaction of phospho-
molybdic acid reagent (167), Phenol, pyrocatechol, resorcinol
and hydroquinone give colour with maximum absorption at wave-
length 700 nm.
Di and trihydroxy phenols are the chief constituents of
many plants and their identification and determination helps
in establishing the composition of several natural products.
Most phenols are known to give colour reaction with many organic
and inorganic reagents and several spectrophotometric and color-
imetric methods for the determination of simple and o-substi-
tuted phenols have been developed (168-171).
A new colour reaction of titanium with ortho and meta-
phenol, resorcinol, phloroglucinol, 1 and 2-naphthol, thymol,
galic acid and chromotropic acid in presence of sulphuric acid
has been described (172). The effects of sulphuric acid concen-
tration, rea '?nt ration, dehydratinq agents and colour formation
27
in hydrochlorir acid has bocn studied. Smith et al. (173)
have mentioned twenty three reagents for the detection of
phenols. A spectrophotometric determination of phenols
with 3-methyl-l,2-benz0thia20lin0ne hydrazone and cerium
ammoniiim sulfate has been studied (174), The photometric
determination is based on colour formation with nitrous acid
followed by ammonium hydroxide. This gives phenolate ion
which depends on the formation of 2- or 4-nitrophenol,
2/4-dinitrophenol or picric acid (175). LegT^di (176) has
described a method for the detection and spectrophotometric
determination of naphthols, eresols and polyphenols in the
presence of phenol. In this method various phenols react
with sodium nitrite to give coloured products in very dilute
solutions. The reaction shows different reaction rates,
differences in the colour of products and their acidity.
Selectivity is improved by changing the concentration of
sulphuric acid (177).
An amperomotric titration procedure has been usod to
determine the phenol contents in epoxyresins (178). In this
procedure, the bromine titrant is generated from acidic KBr-
KBrO^. A combined separation procedure using liquid chroma-
tography on silica gel and high perform;ince liquid chromato-
graphy followed by ultraviolet spectroscopy has been used to
determine alkylphenols in coal-derivatives solvents (179).
The formation of complijx between phenols and p-ni tronniline.
28
sulphanll lc acid, 4-aniLnoanttpyt inc or 3-mc>thy U)''nv,(jthi.i7.o-
1 J,no-2-yl hyUi n; ' . I ti<' I n t h " l i o n I ri <»! d d d in I n n I I o n ' > | m o i K )
and polyhydroxy phenols in water (180), The concentration
of complex is measured spectrophotometrically. A gravime-
tric method, which is regarded as being more useful than
bromination on nitration method for the determination of
phenols involves the formation of addition compound between
penols and chromic anhydride (181), By use of optimum
reaction conditions and a calibration curve, the results
are within 2% relative error. The use of an internal
standard, o-ethylphenol is recommended for gas chromatographic
determination of phenol in urine (182). The trace level phenols
in water have been determined by high pressure liquid chroma-
tography with standard deviation ol 0»3-1.0% (183).
It has been found that sulfite and sulfur dioxide inter-
fere with colour development in the aminoantipyrine spectro-«
phtometric determination of phenols (184). The modifications in
the procedure which result in improvement oi. the detection of
phenols by using 4-aminoantipyrine continue to be used and
studied. In one report 2 2 monohydric phenols are determined
by high performance liquid chromatography after deriva-tization
with 4-aminoantipyrine, The method is applicable to the deter-
mination of phenols at the ppm levels in water (185), In a
study of the apparent recoveries of 35 phenols in 4-aminoanti-
29
pyrine method, the recoveries ranged from 0 to 100% (186).
Some investigators found an even wider range of recoveries
(100%) when they use ultraviolet spectrophotometric method.
Ultraviolet spectra of phenolate ion is rs.'porte d to provide
a simple, reliable technique for phenol determination. When
the pH of a phenol solution is raised from 7 to 12, the
difference in absorptivity at 291 nm is a reliable measure
of phenol concentration (187). An extraction spectrophoto-
metric method is described for determination of 1-naphthol
and 2-naphthol in coal tar by colours after coupling with
diazotized p-nitroaniline (188). A colour developed between
titanium sulfate and o-phenylphenol, m-aminophenol, o-amino-
phenol, p-aminophenol, p-bromophenol, p-hydroxybenzoic acid,
1-naphthol, 2-naphthol, pyrorallol, resorcinol is used for
spiTtrophotom^^tric an-ilyr.is a^tf^r separation from inter'f>r ing
materials by steam ciisti 11 ation or by extraction (189). The
small amount of naphthols have oeen determined b^ luminescence
method (190). Another spectrophotometric method for the deter-
mination of 1-naphthol is based on their reaction with sodium
sulphate and p-N, N-dimethylphenylenediamine at pH 7.8 (191).
The so formed indaniline deriv.->tive are extracted into n-butanol
and the absorbance of organic layer was measured. Fluorometric
analysis has been recommended for determination of a r' -"n of
1-naphthol (192), Two tests have been given by Leqradi (193-194)
The first test differentiates phenols derivatives by their
indicator action <-ind th ^ acid base character of their coupling
30
products with diazotized sulfanilic acid. Nitro phenols
behave in the same faishon. This test can be used to
differentiate 1-naphthol from 2-naphthol.
Phenol produces a characteristic colour with an alco-
holic solution of xynthydrol (195) in the presence of hydro-
chloric acid. Though the test is sensitive, the derivatives
of phenols containing acid, basic, halo and nitro groups gave
a negative test. A number of common phenols oxidised with
hydrogen peroxide in dilute sodium bicarbonate solution contai-
ning some copper sulfate,producing dilferent colour were found
to be useful for their characterization upon addition of few
drops of hydroxylamine (196). A spot test of phenols is
available by indophenol method (197) based on their reaction
with 1,3-aminophenol in basic medium producing a characteristic
blue, green or purple colour. The most widely used method for
determining phenols in aqueous scimple is 4-aminophenazone method,
whorn phenol in m.tdc to react with hcxacyanofnrrato ( i n ) and
4-aminophenazone to form a red dye (198-199). The test is .
sensitive, rapid and selective towards these phenols having
free hydrogen electron at free position, however, inapnlicable
for p-substituted phenols.
Qureshi and coworkers have developed the ion exchange
method for the detection and spectrophotometric determination
of phenols (200-201).
31
This thesis describes the develooment of new colour
reactions and the establishment of procedures for detection
and determination of ascorbic acid, paracetamol, novalgin
and 1-naphthol, Ascorbic acid has been found to intnract
with m-dinitrobenzene in presence of sodium hydroxide to
produce a violet colour. The product is sorbed by anion
exchange resin and has been used for resin bead detection of
ascorbic acid in pharmaceutical preparations. The colour
reaction obeys Beer's law and hence has been studied spectro-
phtometrically for its determination. It has been observed
that paracetamol produces a yellow colour with eerie ammonium
nitrate that has been employed for paracetamol determination
in tablets. The iodate oxidation of novalgin has been studied
for its analysis in tablets. The detection has been achieved
by adsorption of reddish yellow product by starch. The reaction
is found to obey Beer's law ana therefore, used for its aeter-
mination. During the studies on the oxidation of' 1-naphtViol
it has been found that a violet product is obtained by its
interaction with potassium periodate in presence ol nodium
tungstate. The conditions have been set for determining
1-naphthol through oxidation. 2-Naphthol has not bor-n found to
undergo this oxidation.
REFERENCES 32
1. F, Feigl, "Spot test in Inorganic Chemistry", 5th Edn,, Elsevier, Amsterdam, p. 103 (1958).
2. M. Fujimoto, Bull, Chem. Soc, Japan, 30, 93 (1957),
3. M. Fujimoto, Ibid, 283 (1957).
4. H, Kakihana, Y, Mori and M, Yamaski, Nippon Kagaku Zasshi 907 (1954).
5. M, Qureshi, S.Z. Qureshi, Anal, Chem. 1956 (1966).
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34
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CPiAPTER - II
RESIN BEAD DETECTION AND SPECTROPHOTOMETRIC DETERMINATION OF ASCORBIC ACID IN PHARMACEUTICAL PREPARATIONS USING m-DINITR0BEN2ENE.
47
INTRODUCTION
Ascorbic acid contains an ene-diol functional qroup
and has antiscorbutic property. Its detection and deter-
mination are therefore, very important in pharmaceutical
preparations. A review that has recently been published
describes the studies on detection, and determination of
ascorbic acid (l), A new colorimetric technique for its
determination has been reported using Folin-phenol reagent
(2). A method has been developed for spectrophotometric
determination of ascorbic acid in pharmaceutical preparation
using Chloranil (3). Ferrozene has been used as a colori-
metric reagent for ascorbic acid and the method was compared
with that of 2,4-dinitrophenylhydra5!ine (4), The advantage
of ferrozene method includes its speed, ease and sensitivity.
A new water soluble ferroin type chromogen of supe^rior sensi-
tivity has been reported for ascorbic acid determination(5).
Some variants of method for ascorbic acid determination with
2,4-dinitrophenylhydrazine have also been studied (6). The
ascorbic acid content of commercial samples has b e n nnalyned
spectrophotometrical ly after a coupling reaction v it h diazo-
tised sulphanilic acid (7), A comparative study of . ^ 'l-dini-
trophenylhydrazine and phosphotungstic acid method'-, for
spectrophotometric determination of ascorbic acid h.i: .ilso
been made (8). It has been determined spectrophotomf'trical ly
by reaction with 2,5-diphenyl-3-thiazolyltetrazolium chloride
(9). A recent paper describes the determination in pharma-
4 8
ceuticdl products (10). Tbi; colourler.s si Ivor q-l.itln
complex has been quantitatively reduced to y 1 ow silver
solution by ascorbic acid which was used for its determi-
nation (11). A spectrophotometric method based on oxi-
dation with bromine has been developed for acid determina-
tion (12). The colour reaction of ascorbic acid with ferri-
cinixim trichloroacetate (II) has been used for extraction
spectrophotometry (13). A number of spectrophotometric
methods are also available in literature (14-16).
In our studies ascorbic acid has been found to
interact with m-dinitrobenzene to produce a violet product
in alkaline medium. The product is sorbed by Amberlite
I.R.A. 400, an anion exchange resin. The colour reaction
has been stuaied for the resin bead detection and spectro-
photometric determination of ascorbic acid in pharmaceutical
preparations.
49
EXPERIMENTAL
Apparatus. A Bausch and Lomb Spectronic - 20 (U3A) vias
used for absorptlometric measurements.
Reagents. All chemicals used were of analyticcO. grade.
Test solution. A 0.1% (w/v) ascorbic acid solution was
prepared in conductivity water. This solution was prepared
fresh daily. The analyzed tablets were purchased locally.
Their solution were also prepared immediately before use.
Reagent and ion exchange resin. m-Dinitrobenzene solution
0.2% (w/v) was prepared by dissolving Oo2 g. in 100 ml of
formaldehyde.
A IM sodium hydroxide solution was prepared in conduc-
tivity water. An anion exchange resin, Amberlite r.R.A.- 400
was converted into OH^form.
Detection of ascorbic acid using resin beadss
Procedure: Place about 4-6 ion exchange r^sinr. in OH" form
in the depression of white spot plate and dry thi'in \)y blotting
with filter paper. Place 2-3 drops of test solution with few
drops of reagent followed by 1-2 drops of socHurn hydroxide.
The resin beads turn violet that indicate a po;i L L i v - c"5t,
Spectrophotometric determination of ascorbic acid:
Procedure: To an aliquot volume of ascorbic acid cont.ilnin-f
10-250 /ug, add 1.25 ml of m-dinitrobenzene and 1,5 ml of sodium
hydroxide in 5 ml standard flask. Allow tho reaction mixture
50
to stand about 10 minutes to develop a violet coloured
product. Make up the solution upto the mark with formal-
dehyde. Measure the absorbance at 540 nm againtit a blank
solution at room temperature(25 + 10°C).
Determination of ascorbic acid in pharmaceutical preparation:
Procedure : Stir finely ground tablet with about 30 ml of
conductivity water. Allow the mixture to stand for 15 minutes.
Filter the residual solids on Whatman no, 42 and wash with
water. Make the filtrate upto the 50 ml in volumetric flask.
In case of ampoules the solution is prepared directly by
taking an appropriate volume and diluting to a desired concen-
tration of ascorbic acid. Follow the recommended procedure for
spectrophotometric determination,
51
RESULTS
A number of other organic compounds with dirffrrnt
functional groups were tested and found that only ascorbic
acid gives a characteristic violet colour, both in solution
and in resin phase. The detection limit was 10 /ig. Some
important terms and their values evaluated in the present
analysis are presented in Table -2.
The ion exchange test was performed in the presence of a
large amount of foreign substances and found that no inter-
ference was obtained. They are listed below.
Carbohydrates : fructose, glucose, sucrose, xylose, arabinose,
rhamnose, and lactose.
Carboxylic acids : acetic acid, formic acid, tartaric acid,
phthalic acid, pyroqallic acid, oxalic acid, citric acid,
malic acid, adipic acid.
Amino acids : cystfino, '7lyclnc, and glutamic arid, m-'thiionin'',
leucir. , lysine, tryntophan .
Aldehydes : crotonaldehyde, anisaldehyde, cinnaina] dr hyd- ,
benzaldehyde, acetaldehyde, formaldehyde, salicylaldohydo.
Ketones : diethyl ketone, methyl propyl k o t o n a c : t jnh ;n'5ne,
bensophenono.
Alcohols i methinol, r-thanol, 1-prop.^nol, 1-butanol.
52
Table - 2 Important terms and their values evaluated in the analysis.
Term Value
Dilution limit 1 2 100
Molar absorptivity 1.35 X 10- - 1 - 1 1 mole cm
Standard deviation 4.25 jug (v/ith 200 /ug of ascorbic acid)
Relative standard deviation 2.125%
Correlation Coefficient 0.95
Confidence Limit 200.83 + 3.04 yug (at 95% confidence level
53
Reducing compounds : hydrazine, phenylhydraz.1 nc, hydro-
xylamine, mercaptoacetic acid, trichloroacetic acid.
Determination of ascorbic acid :
Absorption spectrum : The absorption spectrum of violet
coloured product obtained by interaction of ascorbic acid
with m-dinitrobenzene was prepared (Fig, 1), The optimum
wavelength was jifound to be 540 nm.
Optimum condition : The optimum conditions for the formation
of violet coloration were studied and maintained throughout
the work.
There was no effect of time on the stability of product
upto 45 minutes. However a slight decrease in absorption was
recorded after it. Therefore, it was recomrnendcKi that the
absorption should be measured within this time.
The effect of reagent concentration was studied by adding
different volume of m-dinitrobenzene solution to a constant
volvime of ascorbic acid and was found that 1,25 ml of 0,2%(w/v),
m-dinitrobenzene is acceptable for the determination of ascorbic
acid (Fig. 2). It was also observed that 1.5 ml ot sodium hy-
droxide solution is optimum volume for determination (Fiq,3).
Confirmatory with Beer's Law : Beer's law is obr-yed in the
concentration ranq'^ of 10 to 250 jaq of ascorbic! acid for mono-
chromatic radiation. Results are shown in Fig, 4,
Study of precision : To tost the reproducibility o' the method,
ten replicate determinations of 200 ^g ascorbic acid were done.
54
<u c; <u N d <0 JD O
o L_ o UD C
"O 1 1 E 1
O 1
"O KD u Ln ro
U XI l_ o
o c o (NJ c Ln cz n)
sz H— O cn c: E <u o l_
C30 <u > U O rt) OJ D a.-a in o
Q. o Zj C CL o o tn XI <
u ro a;
an iT
9 D u e q j o s q V
55
0-4
0-3-
o c 03 n L -o U) n <
o-zA
oi-J
0-23 0-50 0-75 1 0
ML of 1 M NaOH
F i g •• 2 - E f f ee t of NaO H c o n c e n t r a t i on
56
0-25 0 -50 0-75 1 0 1-25 1-50
Ml of 0-2 7o m - d i n i t r o b e n z e n e
F i g : 3 - E f f e c t of m - d i n i t r obe n z e n e c o n c e n t r a t i o n
57
o c fT3 JQ O CO n <
0. 5
0 4-
0 - 3 -
130 160 190 2 2 0 250
A m o u n t , u g / 5 m l
Fig: 4 - C a l l b r a t i o n c u r v e of a s c o r b i c a c i d
58
The results are given in Table-2,
Study of charge on complex s The charge on tho complex was
determined by adding two type of resins :
(a) Amberlite I.R.A, 400^ anion exchange rosin and;
(b) Amberlite I.R. 120, cation exchange resin.
The anion exchange resin beads turned violet/ as they are
exchanged by negatively charged complex. Results arc given
in Table-4,
Effect of foreign substances in ascorbic acid determination :
In order to study the applicability of the method, the
determination of ascorbic acid was studied in the presence of
foreign substinces. The amount at which they were tolerated
are given in the paranthoses.
Leucine (4 mg), resorcinol (3 mg), citric acid (6 mg),
sucrose (7 mg), acetoni tri L o (l.S mg), accton«' (3.'i rr><7) , -
1,4-dioxane (8.25 mg) , formamide (6 mg), beniscnc (0-4 62 :ng),
aniline (0,5 mg).
The results of ascorbic acid determination in diff'-Tont
pharmaceutical pr^:pardtions are given in Tablc-3.
59
Table - 3 Determination of ascorbic acid in Pharmaceutical Preparations.
,, , amount in roq
nominal found % error
Cilin (IDPL) tablet 250 247 - 1.20
Suckcee (Glaxo) tablet 250 255 + 2.00
Chewcee (Lederle) tablet 250 252 + 0.80
Redoxon (Roche) injection 250 245 - 2.00 Becozyme C forte (Roche) tablet 250 255 + 2.00 (B Complex plus C)
o X (u rH a e o u Xi 0) u o >H o o x: -(J •H
c (U (0 0) u 4-> c o V) T) (d 0) XI c •H • tn fO 0) -H M U (0 m o u •H M X! P M o o rH U O n U to
I
(U r H Xi ft) EH
(H
O r H o u c
•H
<u C m 5
c •H m 0) cn
w (0 Q) .Q c: •H tn 0) u MH o M o
rH o u
M cl o r H o u 0)
r H o •H >
o o I
CC H
o r H r H (U >1 4J JG Di -H
QJ tr C fO x: u o
o QJ -!J x: •rl
60
61
DISCUSSION
The present colour reaction is based on the general
roactlon Involvlnq a compound thnh contain.-j an aromatic
ring with two or three electron attracting groupa in tho
meta positions to interact with an anion suitably activated
to give highly coloured complex (17). The colour produced in
alkaline solution is due to the interaction of ascorbic acid
with ra-dinitrobenr-.ene. On the basis of above studies a
tentative reaction mechanism has been proposed (scheme~l),
Ascorbic acid possesses acidic and reducing characters since
it contains enediol functional group. Both the hydroxyl groups
attached to -C = C- interact with m-dinitrobenzene at position 5.
The H^ ions produced from ascorbic acid combine with OH of
sodium hydroxide to form water. Since ascorbic acid contains
two identical enolic groups, addition takes place at these two
positions of ascorbic acid through S-position of two m-dinitro-
benzene molecules. Both the nitroaromatic nuclei ar" ictivated
and become n<'gativ<:-ly charged ana thr-n bond form<ition v/lth oxygen
of enolic group occurs with the removal of Tho nr;gative
charge on the product is confirmed by the exchange of colour
on an anion exchange rosin in OH form.
62
nj 2: CO + o CO X CJ OJ o
X 0 ra z: CJ CO + 0 2:
Csl o z: -c TD
CU E cu JH o U1
W // CJ
u fU u JD i_ O U t/1 no
63
R E F E R E N C E S
1. W. Geottel and H. Hallstein, Prax. Naturwiss Chem. 29, 295 (1980).
2. S.K. Jagota, S.K. Danl, Anal. Biochem. ITJ , 178 (1982).
3. N.K. Pandey, Anal. Chem, 763 (1982).
4 . E.L. M C G O W H / M . G . Rusnak, C . M . Lewis and J.A. Tlllotson, Anal. Biochem. 119, 55 (1982).
5o A.A. Schilt and M.R. DiTusa, Talanta, 129 (1982).
6. R.R. Juna, M.C. Dora and P.A, Jorge, Rev. Cubana Farmj, 16, 82 (1982).
7. M.A. Dexheimtr and C.A. Perazzolo, Quim. Nova 4, 40 (1981).
8. A Castelli, G.E. Martorana, A.M. Frasca and K, Meucci, Acta Vitdminol Enzymol 3, 103, (1981).
9. P.W. ALexandrova and A. Nejtscheva, Mikfochim, Acta 1, 387 (1982).
10, M.E. Abdel Hamid, M.H. Barary, E.M. Hassan and M.A.
Elsayed, Analyst 110, 831 (1985).
11, T. Pal and D.S. Maity, Anal. Lett. 18, 1131 (198S).
12, M.A. Abdel-Slam, M.E. Abdel-Hamid, M.A. Els^iy^d, Sci. Pharm. 11 (1982).
13, M.M. Aly, M.K. Hassan and ^.M. Souliman, J. Choni. Techno!. Biotechnol 30, 435 (1980).
64
14. F, Lagaro, A, Rios, M.D. Luque deCastro and M.Valcarel, Analyst 111, 167 (1986).
15. I.Z. Al-Zamil, M.A. Al-Hajjaji and S.M, Sultan, Orient. J. Chom. 2, 6 (1986).
16. D. Nagahama, Rinsho Byori 31' 331 (1983).
17. E. Sawlcki, Photometric Organic Analysis, Part I, P. 577, Wiley-Interscience, London (1970).
CHAPTER - III
SPECTROPHOTOMETRIC DETERMINATION OF PARACETAMOL IN TABLETS USING CERIC AMMONIUM NITRATE.
65
INTRODUCTION
Paracetdmol or acetaminophen is N-acetyl d'-rivative
of 4-aminophenol, It. is conamonly used as an antipyretic
and analgesic drug. The detection of paracetamol has
been achieved by thin layer chromatography and its combi-
nation with adsorbent resin utilized for extraction of
this compound from the samples Cl). It has been determined
spectrophotometrically by oxidation v/ith iodylbenzene in
acetone to produce yellov/ orange N-acetyl-1,4-benzoquino-
neimine (2). A simple spectrophotoraetric method for the
analysis of a mixture of paracetamol, salicylamide and
codeine phosphate in tablets is based on pH induced diffe-
rential spectral changes of their nitroso derivatives (3)«.
The ultraviolet spectrophotometry has been employed for its
determination in tablets and capsules (4-6), Paracetamol
has been determined through nitrosation and 'subsequent
chelation with cobalt (II) and copper (II) (7). Di, ? ' n^nt
approaches for its determination based on couplino vrith
diazotized 2-nitrodniline have been described (n). Acid
hydrolysis of paracetamol followed by reaction with .ilkaline
sodium nitroprusside has been employed fur itu d' t • r:-.in ition
(9)„
In our studies paracetamol has been found to interact
with eerie ammonium nitrate giving a yellow product which absorbs at 355 nm. The use of eerie ammonium nitrate as a
spectrophotonietric roagent has been made for determining
paracetamol.
66 EXPERIMENTAL
Apparatus t A l.nir.rh .irn) liornb nrx-ctr on ic-2() (U..">.A.)
was lot vibLiorbviacc ia«.u;uc'om<'nt.
Reagents : All chemicals used were of reagent grade.
Test solution : A 0,1% paracetamol (Sigma Chemicals)
solution was prepared in dioxane.
The analyzed cablets were purchased locally.
Reagents : A 5% eerie ammonium nitrate solution was
prepared in 5M nitric acid,
Spectrophotometric determination of paracetamol :
Procedure : To an aliquot volume of paracetamol prepared
in dioxane containing 0,1 to 0,5 mg^ add 0,3 ml of eerie
ammonium nitrate in a 5 ml standard volumetric flask.
Make the solution upto the mark with dioxane. Allow the
reaction mixture to stand about ten minutes to develop a
yellow colour. Measure the absorbance at nra ciq iinst
a blank solution.
Determination of paracetamol in pharmaceutical preparations
Procedure : Dissolve the finely ground tablet in 30 ml of
dioxane. Allow the mixture to stand for 15 miniiten and
filter the residual solids on Whatman no. 42. Mai: ' the
filtrate upto 50 ml in a volumetric flask. Fol loi; the
recommended procedure for spectrophotometric d.->tormination.
67
RESULTS
A number of organic compounds v/ere tested rmd found
that paracetdmol gives a characteristic colour.
Other organic compounds with different functional
groups are found to give the negative test. They are
listed below,
Amino acids : histidine, aspartic acid, glutamic acid,
leucin , lysine, glycine, tryptophan, asparagine,
arginine, 1-alanine, tyrosine.
Acids : acetic, formic, oxalic, citric, malic, adipic,
propionic, tartaric, pyruvic.
Sugars : glucose, fruc-ose, rhamnose, sucrose, maltose,
arabinose, xylose.
Aldehydes : acctaldehyde, benzaldehyde, crotonaldohyde,
anisaldehyde.
Ketones : acetone, ehtyl methyl ketone, diethyl ketone,
methyl propyl ketone, cyclopentanone.
Amines : ethyl, methyl, butyl, propyl, diethyl, triethyl,
Alcohols : tithanol, methanol, propanol, but inol.
Other compounds : nicotine, nicotinamide, acetaraiof,
vitamin B complex and acetylsalicylic acid.
68
Determination of paracetamol : The abr.ocptIon 3o<'ctrum
of yellow coloured product obtained by interaction of
paracetamol with eerie amrnoniurn nitrat-' was pr'-p.irod.
The optimum wavelength was found to be 355 nm.
In order to set the optimum conditions for the
determination, the effect of different volumes of eerie
ammonium nitrate was studied. It was found that 0,3 ml
of 5% eerie ammonium nitrate is optimum volume (Fig» 5)•
Confirmatory with Beer's Law : Beer's law holds good in
the concentration range of 0.1 to 0,5 mg paracetamol for
monochromatic radiation as shown in Fig, 6.
Study of precision : To test the reproducibility of
method ten replicate determinations of r>,2 mg particetamol
were done. The rt-sults are presented in Table-G.
Study of interferences : An interference study in the
determination of 0.2 mg paracetamol was made. It v/ -T found
that aspirin, cod(3ine sulphate, ph(!nylbutazon'^, (i i '••,'->pam,
propyphenazone, aininophenazone and nicotinamld-• could b«
tolerated upto an amount of 1 mg of each. An,iLnjn, o;<yphen-
butazone, phenacetin, phenazone salicylate ani nlcotinf^
interfered in the determination.
The determination of paracetamol was dont.' in vnrious
pharmaceutical preparations .;m cloying tho prt— -nt -thod,
the results are shown in Table-5. Some important data
evaluated in the pre:5f?nt analysis are given in r.'bl —
69
1 1 ! 0 05 0 1 0 15 0 20 0-25 0-30 0 35
Ml of 57o C e r i c a m m o n i u m n i t r a t e
F i g : 5 - E f f ( 2 c t of Cen ' c a m m o n i u m
n i t r a t e c o n c e n t r a t i o n
7(
0 5
0.1 0-2 0 -3 0 - 4
A m o u n t , m g / 5 n n l
Fig 6 - C a l i b r a t i o n c u r v e of
p a r a c e t a m o l
0 5
71
Table - 5 Determination of paracetamol in pharmaceutical preparations
Sample No. Samples Amount in mq ^ k e n found*^ % error
1 Proxyvon (Ciba-Geigy) 400 403 + 0,75
2 Anafebrin (Themis) 250 255 + 2.00
3 Aristopyrin (Aristo) 250 253 + 1.20
4 Metacin (Themis) 500 493 ~ 1,40
^Average of three determinations
72
Table ~ 6 Important terms and data evaluated in the analysis
Terra Value
Dilution limit 1 s 1 0 0 0
Molar absorptivity 1,5 X 10.5 1 mole"^cm"^
Standard deviation 4.71 Aig (with 200 yug of paracetamol)
Relative standard deviation 2.35%
Correlation coei ficient 0.99
Confidence limit 200' + S.S'/c^at 95% conf Idf-nr • Icvr^l) , ,
73
DISCUSSION
Ceric ammonium nitra :e has been used as an oxidant
in organic analysis. On the basis of its oxidising pro-
perty* a tentative reaction mechanism between paracetamol
and ceric ammonium nitrate has been proposed with the
formation of a yellow coloured guinone.
18
NHCOCH O
+ l8(NH4)2Ce(N03)^= 18 . | + IBCH^COOH + 15N2 + 18Ce (N03)3
OH O + SSNH^NG^ + 2HN0, + 4N0
paracetamol p-benzoquinone + 4N0 + 8H2O
Amann et al. under taken the study to test the application
of cerium (IV) oxidation to a wide range of pharmaci^utical
substances (10)® The solution of ceric ammonium nitrate
has been prepared in nitric acid to check it irom
precipitation.
74
REFERENCES
1. L. Eva, Pharmazie, 40, 358 (1985).
2. K.K. Verma and A.Jain, Talanta, 32, 238 (1985).
3. A.A. Kheir, S.F, Belal and A. Shanwani, J, Assoc. Off. Anal. Chem,, 1048 (1985).
4. P. Guoguang, Yaowu Fenxi Zazhi, 5, 58 (1985).
5. W, Changlin and Y, Guang, Yaowu Fenxi Zazhi 5, 52 (1985).
6. S.M, Hassan and S.A.M. Shaaban, J. Pharm. Belg., 258 (1983).
7. S.F. Belal, E.M. Abdel-Hady, A.El-Waliely and H. Abdine, Analyst, 104, 919 (1979).
8. S.F. Belal, E.M. Abdel-Hady, A.El-Waliely and H. Abdine, J. Phar.n. Sci., 750 (1979).
9. N.M. Sanghavi and S.P. Kulkarni, Indian J. Pharm. Sci., 42, 10 (1980).
10. G. Amann and G. Gubitz, Anal. Chim. Acta, 116, 119 (1980).
CHAPTER - IV
DETECTION AND SPECTROPHOTOMiCTRIC DETER-MINATION OF NOVALGIN IN TABLETS USING POTASSIUM lODATE,
75
INTRODUCTION
Novalgin or dipyrone is the sodium salt of
(2,S-dihydro-l,5-dimethyl-3-oxo-2-phenyl-lH Pyrazol-
4-yl) ethylamino methane sulphonic acid. It is a
commonly used analgesic drug. It has been analysed
in tablets and injections using high performance
liquid chromatography on a reverse phase column and
ultravilet detection (l). The spectrophotometric
determination of novalgin has been achieved by reaction
with cerium (IV) measuring cerium (III) formed with
arsenazo (III) (2). Antipyrine and pyramidon can also
be determined by this method, A coulometric method
for novalgin determination in tablets has also been
reported (3). It has been determined spectrophotome-
tricdlly by reaction with N-bromosuccinimide in acid media *
and measurement of absorbance of resulting colour-^d solu-
tion at 290-450 nm (4), However, antipyrine and amido-
pyrine also give the positive reaction. A number of
spectrophotometric methods for novalgin and other anal-
gesics have also been reported using potassium ferrocy-
anide (5)/ sodium nitrite (6), 4-dimethylaminobenzalde-
hyde (7), bromophenol blue (8) and potassium aurichloride
(9) reagents.
In our studies novalgin has been found to interact
with potassium iodate in presence of hydrochloric acid
76
yielding a yellowish red coloration. Thn product is
sorbed by starch producing a blue colour. This test
has been employ^jd for the detection of novalgin in
tablets. The formation of a yellowish red solution
by interaction of novalgin and potassium iodate in
presence of hydrochloric acid has been studied for
the determination of drug.
77
EXPERIMENTAL
Apparatus : A Bausch and Lomb Spectronic-20 tUSA)
was used for absorbance measurement.
Reagents : All chemicals used were of analytical grade.
Test solution : A 0,5% (w/v) novalgin was prepared in
distilled ethanol. The analyzed tablets were purchased
locally.
Reagents : A 0.01 M potassium iodate solution was
prepared in conductivity water.
A 0.1 M hydrochloric acid solution was prepared in
conductivity water.
Starch grannules were used as a self detector.
Detection of novalgin in adsorbent phase :
Procedure s Take small amount of starch <jr<mnul'^r. or
coarsely ground wheat in a depression of v/hit-a 3oot plate.
Add one drop ot novalgin follov^jd by tv/o to tVir"n drop::
of potassium iodate and hydrochloric acid r^Tipnctivdy.
The starch grannules turn blue within 2 minut-fn ' n i i f stina
the presence of novalgin.
Spectrophotometric determination of novalgin :
Procedure : To an aliquot volume of no.aloin c?ontai.ning
0.1 to 1 mg, added 1 ml of potassium iodata -'ol lovfod by
1 ml of hydrochloric acid 'Wahdardtj^oiuin<-tric
78
flask. Allow the reaction mixture to stand for about
5 minutes to develop a •'^ellowish red colour. Make the
solution upto the iwarlc with water. Measure' the absor—
bance at 460 nm against water.
Detection of novalgin in pharmaceutical preparations :
Procedure : Stir finely ground tablet with about 30 ml
of distilled ethanol. Allow the mixture to stand for 10
minutes. Filter the residual solid on Whatman no. 42,
Make the filtrate upto 50 ml in volumetric flask and
follow the recommended procedure for the detection.
Determination of novalgin in pharmaceutical preparations:
Procedure : Dissolve the finely ground tablet in 30 ml
of distilled ethanol. Allow the mixture to stand for
10 minutes and filter the residual solid on Whatman no,42,
Make the filtrate upto the 50 ml in a volumetric flask
and follow the recommended procedure for spectrophotometric
determination.
79
RESULTS
A number of organic compounds were tested and
found that novalgin gives a characteristic colour in
adsorbent phase. The detection limit was 5 ug.
Many drugs (Table-7) and a number of compounds
containing different groups were found to give a
negative test.
Amino acids : histidino/ aspartic aci<i, glutamic acid,
leucin , lysine, glycine, tryptophan, asparagine,
arginine, 1-alanine, ^ -alanine, tyrosine.
Acids : acotic, formic, oxalic, citric, malic, adipic,
propionic, tartaric, pyruvic.
Sugars : glucose, fructose, rhamnose, sucrose, maltose
arabinose, xylose.
Aldehydes : acetaldehyde, benzaldehyde, crotcnaldehyde,
anisaldehyde, koton-'>5.
Ketones : .ic>'ton", ethyl m>'thyl kotonf>, diothyl k'H.on' ,
methyl propyl ketone, cyclopentanone.
Amines : ethyl, methyl, butyl, propyl, diethyl, trifthyl
Alcohols : ethanol, methanol, propanol, butanol.
Other compounds : nicotine, nicotinamide, ac^tanilidf^
vitamin B complex, and acetylsalicylic acid.
80
Table-7 Colour reaction of various drugs with potassium iodate and starch
Drug Trade name Reaction
Dipyrone
Dipyrone
Dipyrone
Dipyrone
Dipyrone
Dipyrone
Aspirin
Aspirin
Aspirin
Aspirin
Paracetamol
Codeine Sulphate
Oxyphenbutazone
Propyphenazone
Ascorbic acid
Phenylbutazone
Phenasone Salicylate
Aspirin Phenacetin Caffeine
Diazepam
- Indicates no reaction
+ Indicates that colour develops
Baralgan (IDPL) Blue +
Spasmizol (IDPL) Blue +
Algesin-o (Alembic) Blue +
Ginox (Averest Chem.Lab) Blue +
Maxigon (Unichem) Blue +
Spazmolysin(Standard Blue + Pharm)
Mejoral (CFL)
Disprin (Reckitt & Colman)
Micropyrin (Nicholas) -
Tuxyne (Griffon)
Proxyvon (Ciba-geigy) Blue •}*
Codeine Sulphate . -(Indo Pharm)
Tendril (Ranbaxy)
Cibalgin (Ciba-Geigy)
Sukcee (IDPL) Blue +
Phenylbutazone (A.B.M. Research Lab)
Cosavil (Hoechst)
APC (Boots)
Calmed (IDPL)
81
The following compounds were found to interfere
with the test.
Methionine, serine, cysteine, cystine, ascorbic
acid, aminophenazone and acetaminophen.
Determination of novalgin :
AbsoiTption spectrum : The absorption spectrum of
yellowish-red coloured product obtained by interaction
of novalgin with potassium iodate i-/as prepared. The
optimum wavelength was found to be 460 nm. The visible
spectrum of novalgin-potassium iodate is shown in Fig,7.
Optimum condition » The optimum conditions for the
formation of yellowish-red coloration were studied and
maintained throughout the work.
There was no effect of time on the stability of
product upto 30 minutes. However, a slight decrease in
absorption was recorded after it, Therefpre, it v/as
recommended that the absorption should be measured v/ithin
this time.
The effect of reagent concentration v/as studied
by adding different volumes of O.OIM potassium iodate; to
0,5 mg of novalgin. The absorbance were 0.05, 0.14, 0.7,8,
0.31, 0,33, 0.33, 0.33, and 0.33 with 0.32, 0.34, 0.36, 0.40,
0.48, 0.50, 0.60 and 0.70 ml of potassium iodate. It is
clear from these data that 0.5 mg of novaLqin (1.6 /imole)
needs 0.48 ml of O.OIM potassium iodate (i.e. 4.8 /umole) .
82
c o o •w o o to OJ 0) L_
(L> ro "O o t
£ •D 'in in ro » 1
o o rsj a. Ln 1
«
o
c o
i t i o-g
o -a u < Q.
cn •
L L ro o
oa o o
9DUBqjosq\/
83
However, the uue of higher volume kept the absorb nee
constant. 1.0 mg novalgin will, therefort-, need 0,96
ml of O.OIM potassium iodate that contains 9,6 /umole.
1.0 ml of O.OIM potdsylum iodate haa, therr-tore, been
used for 1,0 mg of novalgin that is the upper concen-
tration limit of method. It is also observed that 1.0 ml
of hydrochloric acid is the optimum volume for determination,
Confirmatory with Beer's law : Beer's law holds good
in the concentration range of 0,1 to 1,0 mg of novalgin
for monochromatic radiation as shown in Fig, 8.
Study of precision : To test the reproducibility of
method ten replicate determinations of 0,2 mg novalgin
were done. The results are presented in Table-9,
Study of interferences : An interference study in the
determination of 0,2 mg novalgin was done. It was found
that aspirin, codeine sulphate, phenylbutai:one, diazepam,
propyphenazone, aminophenazonu, nicotinamide, nicotine,
oxyphenbutazon J, phenacetin, phenazonc salicylate' could
be tolerated up to an amount of 1 mcj of each.
Tho determination of novalgin v/nr don'^ in v h iou::
pharmaceutical proparationr, .'mployinq ' he rirc.-: mi' inf^thod.
Other substances which can be tolerated <iro linf: d in u
footnote to Table-8. Some important data evaluated in the
present analysis are given in Table-9.
84
A m o u n t , mg / 5 ml
Fig : 8 - C a l i b r a t i o n c u r v e of nova lg i n
85
Table-8 Determination of novalgin in pharmaceutical preparations
Sample No. Sample Amount In mq taken found^ % error
1 Baralgan (IDPL) 500 505 + 1.0
2 Spamizol (IDPL) 500 504 + 0.8
3 Algesin-0 (Alembic) 300 293 - 2.3
4 Ginox (Averest Chem. Lab)
500 510 + 2.0
5 Maxigon (Unichem.) 500 495 - 1.0
6 Spasmolysin (Standard Pharm.)
500 514 + 2.8
^Average of three determinations
Other substances present include the following :
(1) p-piperidinoethoxy-o-carboethoxybenzoph(7none hydrochloride (50 mg) / diphcjnylpipfjridinouthyl acetamide brom-o-methylate (0.1 mq),
(2) Phenobarbitone (10 mg), Homatropin [nethylbromide (2.5 mg),
(3) Oxyphenbutazone (100 mg),
(4) Oxyphenbutazone (100 mg),
(5) p-piperidinocthoxy-o-carbemcthoxyb<'nzopVi(^nonf' hydrochloride (5 mg), dipheylpip<'rj <iino thy L brom-o-methylatG (0.1 mg), hydroxyr.ino hydro-chloride (7.5 mg),
(6) Dicyclomine hydrochloride (10 mg), diazepam (2 mg).
86
Table-9 Important data evaluated in the analysis
Term Value
Molar absorptivity 4 X 10^ 1 mole'^cm"^
Standard deviation 3.36 Aig (with 200 /ag of novalgin)
Relative standard deviation 1.68%
Correlation coofficiont 0.99
Confidenc(> limit 199.9 + 2.S3 Mn (at 95% confidencc level)
87
DISCUSSION
The oxidation of organic compounds of pharma-
ceutical interest by potassium iodate has bc^en studied
by many workers (10-11). On the basis of these studies
a tentative reaction mechanism between novalgin and
potassium iodate has been proposed. Potassium iodate
interacts with drug in presence of hydrochloric acid
to liberate iodine.
HO2SCH2
+ eKio^ + 6HCI =
novalgin
0 2
CHO 2 I +4HC00H
+ N2 + + 6H^0 +
i '"i-Ji H"
The liberation of iodine is supported by thf> nroduction
of blue coloration with starch.
The starch contains amylose which given ,1 blu-^ colour
with iodine. The coloured nroduct iv, an vid;:<!i ion comnlex
of iodine and amylose. The intense blue colour form d is
due to the occlusion like arrangement of tri1od:de ions in
spiral amylose chains, which contain:; c<-] , 1 ycor.ido
bonds.
88
REFERENCES
1. N.H. Eddinf, F, Bressollo, B. Mandrou and H.Fabre., Analyst, 107, 67 (1982).
2. F. Buhl and U. Hachula, Chem. Anal. 395 (1981).
3. N. Kosta, V. Ksenija and M. Mirjana. Acta Pharm, Jugosl. 177 (1984). .
4. N.V, Pathak and I.C. Shukla, J, Indian Chem. Soc. 206 (1983).
5. P. George, Indian J. Pharm. 3_6, 14 (1974).
6. H.-Abdine, A.S, Sophi and G.I. Morcas Magdi, Pharm. Sci 1834 (1973) .
7. R. Bontemps, J. Parmentier and M. Diesse, J. Pharm. 23, 222 (1968).
8. D.M. Singhal, Indian Drugs Pharm. Ind. 37 (1976)
9. N. Shiritsu and Yakugakuku, Konbyu Nome, 1 (1965).
10. G. Cavaszutti, L. Giigliavdi, A. Axnuto, Pr<)tili and V.J. Zagarese, J. Chromatog., 26 3, S28 (1933).
11. K. Macek and I.M. Haia, in G. Fisher (i-:riitor), Handbuch der papierchromatographio, :ian<i I, V -rlag-Jena, Jena, (1958).
CHAPTER - V
CONDITIONS FOR 1-NAPHTHOL DETERMINATION THROUGH PERIODATE OXIDATION.
89
INTRODUCTION
1-Naphthol is industrially an important comoound
as it is foiind in coal tar, hair dyes etc. A methods
for its specific determination are available. It has
recently been determined using diazotized anthranilic acid
as a novel chromogen (l). The method is not specific for
1-naphthol as 2-naphthol also undergoes the same reaction.
1-Naphthol in aqueous solutions has been determined by
extraction spectrophotometry (2), A recent method describes
the use of crystal violet hexachloroantimonate solution as
a colorimetric reagent for its determination (3). However,
a number of compounds interfere with this method. 1-Naphthol
has been determined in air and water samples spectrophoto-
metrically by the Berthelot reaction using ammonium chloride
as a reagent and sodium tungstate as a catalyst.(4). The use
of diazotiaed sul fonyl acid has also been report f'u for its
determination (5). It has Vjccn dctormlnod l-iy i t cito-
metric meaGuroment at 510 nm after oxidation v;itfj tu:inioniurn vanadate in presence of hydrochloric acid (6). Gouluin
cuprobromide has been used for its spectrophoto;n->tric deter-
mination in pr<;-3(:?nco of 2-naphthol at microrjr am 1- v. (7) but
the colour fades after five minutes. Another m ;':::ho<i i:; based
on the fact that 1-naphthol is diazotize(i factor than 2-
naphthol (8). A direct fluorometric method tor 1-nanhthol
determination ha::; also been reported (9). Most of those
90
methods havu two limitationi; (a) thtj reaction:; involved
are not specific for 1-naphthol as many compounds give
the same test and (b) the recommended procedures are
complicated.
In this study we describe a colour reaction of
1-naphthol with potassium periodate in the presence of
sodium tungstate. The periodic acid oxidation has
earlier been used for determining a number of phenols (10)
However, the oxidation product was yellow and tht test
was general for all phenols.
91
EXPERIMENTAL
Apparatus : A Bausch and Lomb Spectronic-20 (USA) was
used for absorptiometric measurements.
Reagents : All the chemicals used were of analytical grade.
Test solution : A 0,1% (w/v) l-naphthol solution was
prepared in distilled etnanol. This solution wan prepared
fresh daily.
Reagent : A 1 X 10"^M potassium periodate and 0,5M sodium
tungstate solution were prepared in conductivity water.
Spectrophotometric determination of 1-naphthol :
Procedure : To an aliquot volume of 1-naphthol containing
O.Ol to 0,40 mg, add 1 ml of soaium tungstate and 0,28 ml of
potassium periodate in 5 ml standard flask. Allow the
reaction mixture to stand for about 20-25 minutes to develop
a violet colour. Make the solution upto the mark v/ith form-
amide. Mccisure th<' absorbanco at 520 nm again?;? hl.mk
solution.
92
A number of other organic compounds W < T O
and found that only 1-naphthol gives a charactiT istic colour.
Some important terms and data evaluated in th' present
analysis are presented in the Table - 10,
Other organic compounds with different functional
groups are found to give the negative test. Thny are
listed below.
Phenols : 2-n iphthol, m-cresol, orcinol, phl oroglucinol,
3-nitrophenol, p-nitrophenol, 4-acetaminophenol, picric acid
and 8-hydroxyquinoline, catechol, phenol, salicylic acid,
quinol and resorcinol.
Carbohydrates : fructose, glucose, sucrose, xylose, arabinose,
rhamnose and lactose.
Amino acids : methionine, cysteine, glycine,, leucir.,
lysine, glutamic acid, asparagine, arganine anH threonine.
Aldehydes : crotonaldehyde, aniraIdehydo, cinnamald'-hyde,
benzaldehyde, ac^taldehyde, formaldehyde and nalicyl-ildehyde.
Ketones : diethyl ketone, methyl propyl ketone;, acetonhenone
and benzophenone.
Alcohols : methanol, ethanol, 1-propanol, l-butanol,
Pyrogallol was found to intorfero with th- tost.
Absorption spectrum : The absorption spoctrurn o! violf.>t
product obtained by interaction of potas.'jium p T i o d 'to v/.i;-
93
Table-lO Important data evaluated in the analysi;
Term Valuf
Standard deviation 1.66 ^g
Relative standard deviation 1.66% (with 100 jag of 1-naphthol)
Confidence limit 99.76 + 1.25 lag (at 95% confidencn level)
Correlation coefficient
Molar absorptivity
0.99
3.1 X 10^ 1
94
prepared. The optimum wavolength was found to be 520 nm
(as shown in Pig. 9 )•
Optimum condition : The optimum conditions for thr>
formation of a violet coloration were studied and maintained
throughout the experiments.
There was no effect of time on the stability of the
product upto one hour. However slight decrease in absorp-
tion and a light turbidity was noted after it. Therefore,
it was recommended that the absorption should be measured
within this time.
The effect of reagent concentration was studied by
adding different volumes of potassium periodate to 0,1 mg of
1-naphthol : the absorbanceawere: 0,04, 0.09, 0,16, 0,21,
0.21, 0,21, 0,21, 0.21 for 0.2, 0.4, 0.6, 0.7, 0,8, 1.0, 1.2
and 1.4 ml of potassium periodate. It is clear from these
data that 0.7 ml of O.OOIM potassium periodate is needed for
0.1 mg of 1-naphthol. Therefore, 2.8 ml rf-aq- nt has been
recommended for 0,4 mg of l-nanhthol that is the upper
concentration limit of method (Fig. 10 ).
It was also observed that 1 ml of O.IM r;od?ii:n
tungstate is nut t icient to catalyse the r^acc.! on rrixLuro.
Higher volume of sodium tungstate produces th'- turbidity.
Confirmatory with Beer's Law : Beer's law obeyoii in the
concentration range of 0,01-0,4 mg of 1-naphthol for
95
0 25
CJ u c 03 n
o U) n <
0 2-
0-1 5 -
O'l-
0 0 b -
AOO A 8 0 5 60 6 AO 7 20
W a v e l e n g t h ( n m )
F i g : 9 - A b s o r p t i o n s p e c t r u m of 1 _ n a p h t h o l - p o t a s s i u m
p e r i o d a t e r e a c t i o n p r o d u c t
96
Cb) o C
fO JD O U) n <
0 25
0 2
0 1 5 -
0 1 -
0.0 5-
0 2 0 4 0 6 0 8 1-0 1 2 1 A
Ml of 0 001 M P o t a s s i u m per ioda tc
Fig:10 - E f f c c t of p o t a s s i u m p e r i o d a t e
c o n c e n t r a t i o n
97
monochromatic radiation (as shown in Pig. n ) .
Study of precision : To test the reproducibility of method,
ten replicate determinations of O.lO mg of 1-naphthol were
done. The rt^sults are given in Table-10.
Effect of foreign substances in 1-naphthcl determination :
In order to study the applicability of the method, the
determination of 1-naphthol was studied in the presence of
foreign substances. The amount at which they were tolerated
are given in the parantheses.
2-naphtol (1.00 mg), citric acid (l.OO mg), glycine
(1,5 mg), maltose (2.0 mg)/ acetonitrile (1.5 mq), acetone
(3.9 rag), 1,4-dioxane (3.25 mg), 4-acetaminophenol (1.00 mg),
benzanilide (3.2 mg), acetaldehyde (3.9 mg), thiourea (1.2 mg)
98
100 200 3 0 0
A m o u n t M g / 5 m l F ig 11 - Cal i b r a t i on c u r v e of
1- n a p h t h o l
4 0 0
99
DISCUSSION
Periodate oxidation of some phenols has bK>on
studied by Sklarz (ll). A tentative reaction mochanism
between periodate and 1-naphthol has been propoi>ed. It
is oxidised by periodate to compound (11),
+ 2 HIO + 2 HIO^ + 2 H2O
The oxidation proceeds through 4»4 -di-l-naphthol (l)
as an intermediate
TungstatG ion iacksthe oxidising power (12). It Ccatalyat-s
periodate oxidation of 1-naphthol resulting the loination
of oxidation compound (II). The support of this mechanism
is obtained from the work of Edwards and Canhaw (13) who
obtained a violet compound (ll) by oxidation of 4,4 -di-l-
naphthol (l) with lead tetracetate.
100
REFERENCES
1. D, Arain and W.A, Bashir, Microchem. J., 33(1)^ 78-80 (1986).
2. Ya. I. Korenman, E.M. Tishchenlco and G.S. Lineva, Zh. Anal. Khim., ^ ( 7 ) , 1288-91 (1982).
3. G.m, Sergeev, I.M. Korenman and S.V, Tikhomirova, Zh. Anal. Khim., 40(1), 154-7 (1985).
4. R. Tao and fc. Fan, Fenxi Huaxue, 1(6), 463-5 (1983)
5. Ya. I.Korenman, R.N. Bortnikova, V.M.Bolotov, S.N. Taldykina, N.N. Sel'man Bhchuk and Tischenko, Zh. Anal. Khim., ^ (9), 1803-9 (1980).
6. J.P. Rawat and P. Bhattacharji, Talanta, 26, 283-284 (1979).
7. S. Sass, J.J. Kaufman and Kierman, Anal. Chem., 29, 143 (1957).
8. J.S. Sarson, W. Seaman and J.T. Wood, Anal.. Chem., 27, 21 (1955).
9. M.J. Larkin and M.J. Day, Anal. Chim. AcL>!, l')8, . 426-7 (1979).
10. J.P. Rawat and K.P. Singh Muktawat, Microchnra. J., ^ ( 3 ) , 289-96 (1984).
11 . B. Sklarz, Quart Rev., 21(1), 3 (1967).
12. F.A. Cotton and Wilkinson, *Mvanco Incirqanic Chemistry", Inters-ience publishtjr, p. 7B3 (1064).
13. J.D. Edwards and J.L. Cashaw, J. Am. Ch<Mn. Sor., 76, 6141 (1954).