Boctor ofvolume of paracet imo l prepare id n dioxane ron!:

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

^ 4 > U \ .

IW 0) 0 c f-H o

-o <k (1) -n 0) C 0 (U t/1 QJ «H L •d fO u (U 'O XJ 0 M 0) 0 M o tn u M 4J -d n r-f % 0 0 0) o 4J % d) 0) M C u Q) c c Cn o> 0 rH 0) Q) c c A; (!) 0) rH

0 o O 0 0 QJ 0 OI CN >H o O

u o + tu

•H ft) 'D C ' D 0 •H TO •H -P r) CN tH 0 M 0 0 0 o C -H rH t C

a o M x: + 0 m o •H 0 C 0 •H o •H -p JC 0 ID (0 x: •p +J 0 ft •H M OJ 0 0 c 0 rH rH 4J •p x: u rH 0) o iH 0 0 0 (U 1 'd to 0 +J •H 0 w M rH 4J n 0 0 to X} (0 Q) + OJ to C •H to (D td n 0 Xi c 0 6 X) C •H •H tt) •H 0) Xi -H rH •H to E •H X e u c to (U M > Q +> o •H to to -H M c 0 1 oi O p E +> c 6 Di C u c •H 3 p 0 0 •H 0 • G (t) M •H •H TA N •rH E M U 0 x: u •H M -a TS -H 03 M 0 x> 0 U 0 , u •d c OJ 0 0 0 •H (U M 1 0 •H OJ 0 0 to to to Q u ra 2: <30 to Q ^ to •H •p u Q) +J 0) !>i TS M to M 01 W w CO 0 TS XJ a ip g C c C 0 !0 to

B 0 d 0 'd XJ to 0 o 0 0 0) •H -H •a & 0< 9< to to 10 0) u 0 0 5 E E g <D 0) -d ro <0 ^ 0 0 0 0 •d 4J +J •H

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1 6 R^ 0) <v (U 0) 0 U -H 0 jt: M M X M JH 0 0 Xi sz x: X! r H U RH 0 (0 (0 0 to to rH u c 04 (14 a. a* 0 < CU to JQ u u XJ U u 0) 04 U u b

(0 X! H (0 to u Oi < U to u

0) u c a» M (U U-J (U a;

u p 0) 0 I-H ,-H 0 -r) 0 •H (U m u > a

•d Q) c •H -P C o u I Q) rH 03

10

VD ro r-ro

CO m cr> ro

d) Qi

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wt 0 U (0 c . r J M P 0 fO I . 0 0 p. tj 0 r

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t + P, (0 0) rH OJ rH IH CM 0 l - i X3 x: ^ x: V 'd 0 ) r ) U IX) U ( 0 >1 •p + V -p > 1 + a) + ( 0 x :

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(0 U N o r} P. COO) COO) Q) • H (0 u 0 0 •H XI -P •H Xi +J M l-l p in M U iH fO 0 i H ( 0 + O T) •H Q> +J C> (fl 0 m CO C 11 dj u P o a; - x: x: •H c c in c in c a U H •H •H 0 + o 0 + o IP 0 0 0 1—1 T) nj tJ t3 •r-l U C • H l-f (0 1 0 cu - P 0 (U -J-J •H x: T ) C n n Ai < < • H T l fO • H ' O f d u u > i J C 0 r-I % 1 (U 1 -H (U CO n: p c n jz <c OJ s X x : 5 s X x :

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5

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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),

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4. H, Kakihana, Y, Mori and M, Yamaski, Nippon Kagaku Zasshi 907 (1954).

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33

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34

28. Ibid, p. 107 (1955).

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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 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



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)


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)


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).

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