Determination of sulphur functions with N-iodosuccinimide

00.19-9140/79/1001-0917102.00/0

Jrrlunra. Vol. 26. PP. 9 I7 to 920 0 Pergamon Pms Ltd 1979. Printed in Great Britain

DETERMINATION OF SULPHUR FUNCTIONS N-IODOSUCCINIMIDE

WITH

ASHUTOSHSRIVASTAVA* Department of Chemistry, University of Jabalpur, Jabalpur, India (Received 25

May

1978. Revised 18 Auyusi

1978. Accepted

12 December

1978)

Summary-Titrimetric determination of thioureas, thiols, xanthates and dithiocarbamates with N-iodosuccinimide (NE) is described. The method for xanthate can be applied to carbon disulphide (converted into xanthate with potassium ethoxide). Acidic and non-aqueous solutions of the oxidizing agent are stable. The procedures are rapid and accurate to 0.1% with a precision of 0.2%. Hydrogen sulphide and thiocarbonyl compounds interfere. The behaviour of N-bromosuccinimide and NIS with thiols in aqueous medium is compared. It is shown that iodine is the oxidizing species in both cases. The limitations of iodine as a reagent for thiol determination are discussed. Cysteine, which cannot be determined with iodine, can be determined with NIS. The role of methanol in non-aqueous determination of thiols is discussed. Methanol accelerates the oxidation, which is otherwise slow in acetonitrile medium.

Thioureas are biologically, commercially and medicinally important compounds.1*2 Several approaches for their determination have been reviewed.3-b N-Bromosuccinimide has been found to desulphurize thioureas in presence of potassium bromide.5 As a part of a project on the determination of sulphur-contaming organic compounds through their functional groups, reactions of N-iodosuccinimide (NE) have been investigated and methods evolved for the determination of these sulphur functions. NIS is found to oxidize thioureas readily and quantitatively to forma&dine disulphides according to the general equation. ZRNHC(NH,)=S + (CH2C0)2NI + [RNHC(=NH)S], + (CH,CO),NH

+ HI

In presence of mercuric chloride the following stoichiometry was found. RNHCSNH, + (CH2C0)2NI + H,O--t RNHCONH2 + (CH2C0)2NH + S + HI The wide distribution of the sulphydryl group makes the study of thiols important. The many methods available for the determination of thiols have been reviewed.7-9 Interhalogens,” N-haloamines, N-haloamides,’ ’ periodate,’ 2 chloramine-T and ch10ramine-B’3 have been successfully employed for the assay of thiols. The classical method for the estimation of cysteine by iodine has been improved.14 Recently, thiols in general and cysteine in particular have been estimated with o-diacetoxyiodobenzoate’5*‘6 and ferricyanide.” N-Bromosuccinimide (NBS) in the presence of acid and potassium iodide has been proposed for determination of thiols, including 3-mercaptopropionic and mercaptosuccinic acid. ’ ’ In the presence of acid and potassium iodide * 171. Bhaldarpura, Jabalpur 482002, India.

the reactions of NBS are essentially those of iodine. Danehy and Oester 19s2’ have discussed in detail the limitations of iodine for the determination of such thiols and cysteine. In a systematic study on the determination of sulphydryl compounds, consideration has been given to the behaviour of NIS and NBS, which have similar structures, towards thiols. NIS has aroused a great deal of interest in synthetic organic chemistry. Solutions of NBS are unstable and liberate bromine on keeping, but NIS is very stable in acidic as well as non-aqueous media. Conditions have been found for quantitative oxidation of cysteine to cystine, by varying the pH and iodide concentration. The utility of xanthates has been widely discussed.2’*22 Numerous methods23-2s dealing with the dithiocarbonate functional group are primarily concerned with the determination of a specific alkyl xanthate in the presence of impurities, e.g., sulphides. NIS oxidizes xanthates to dixanthogens. The titration is done in methanol with an acetonitrile solution of NIS or in neutral aqueous medium. The procedure can be applied to carbon disulphide after its conversion into xanthate with potassium ethbxide: CS2 + C2H,0H + C2HSOCSSK Formation of xanthate by this reaction is quantitative provided that significant amounts of water are absent. The analytical importance of dithiocarbamates .has been reviewed.29,30 Various reactions for their detection3’ and determinationj2 have been discussed. Although hypoiodite quantitatively oxidizes dithiocarbamates derived from primary amines, non-stoichiometric results are obtained for those derived from secondary amines. Of the methods proposed, direct titration with iodine33-35 appears to be the best, but in aqueous medium the thiuram disulphide formed hampers the end-point. Linch33 proposed titration 917

ASHUTO~HSRIVASTAVA

918

with iodine in ethanolic solution, the appearance of iodine being taken as the end-point, but the colour is fugitive and over-titration is required for a definite end-point. The method is rapid but furnishes only approximate results. The mercurimetric method36 is excellent only for dithiocarbamates derived from

secondary amines. N-Iodosuccinimide quantitatively oxidizes dithiocarbamates in non-aqueous medium to thiuram disulphides: ZR,NCSSK + (CH,CO),NI --* (R,NCSS-), + (CH2C0)2NK + KI

were added, and the solution titrated with NIS to a blue end-point. Alternatively, a potentiometric titration was done, with an antimony or modified calomel reference electrode and a bright platinum wire as indicator electrode. Xanthates

Carbon

Samples are titrated in methanol solution of the reagent.

with acetonitrile

EXPERIMENTAL Reagents

N-Iodosuccinimide was prepared by the method of Djerassi and Lenk.37 A 0.02M solution was prepared by dissolving the compound in dilute hydrochloric acid or acetonitrile, filtering off any insohrble residue, diluting and standardizing iodimetrically. It was kept in the dark.

disulphide

in aqueous

media

A sample containing 0.1-1.0 meq of carbon disulphide was weighed in a small sealed glass bulb and placed in 0.5-2.0 ml of 10% potassium ethoxide solution and 5 ml of absolute ethanol in a 150-ml conical flask. A sharp rap against the sides of the flask was sufficient to break the bulb and release the sample. The contents were swirled for 2 min. then the flask was cooled in an ice-bath and 30 ml of water and 2-3 drops of phenolphthalein indicator were added. The solution was just acifidied with 1M acetic solution and the potassium ethyl xanthate formed was titrated with NIS as described for xanthates. Thiols

Samples

in aqueous medium

A sample containing 0.1-1.0 meq of the sodium or potassium xanthate was dissolved in 30-50 ml of water, I ml of 1% starch solution was added and the solution was titrated with NIS. The dixanthogen precipitates as it is formed but does not affect the reaction or the end-point. The mixture should be shaken vigorously during the titration.

in non-aqueous

media

Most of the thiols were used as received from Evans Chemetics, New York, and the sodium diethyldithiocarbamate was also a commercial product. Thioureas, xanthates and the other dithiocarbamates were prepared and purified3s by the author. All other chemicals used were of reagent grade.

A sample containing 0.1-1.0 meq of mercapto group was dissolved in 30 ml of methanol containing 50 mg of potassium iodide. The solution was titrated with 0.05N NIS in acetonitrile, the end-point being indicated by the appearance of the colour of iodine or potentiometrically. Enough methanol must be added for it to constitute about 80% of the total solvent at the end-point.

Thioureas

Dithiocarbamates

in aqueous

medium

Samples containing 0.01-0.1 meq of the thiourea group were used. For alkylthioureas the sample was dissolved in water and enough sulphuric or hydrochloric acid was added to make the acid concentration 1N. Aryl derivatives were dissolved in I@-15 ml of 18N sulphuric acid, which was diluted to 3.5N and cooled just before the titration. One ml of 1% starch solution was added as indicator, for visual end-point detection. Potentiometric titrations were done as described for procedure B. Procedure B. An aliquot of aqueous alkylthiourea solution containing about 0.1 mmole of sample was treated with 5 ml of saturated aqueous mercuric chloride solution and 5 ml of chloroform, and titrated with 0.05N N-iodosuccinimide. Appearance of the colour of iodine in the chloroform served to indicate the end-point. For potentiometric titration, no chloroform was added and a modified calomel or antimony electrode was used as reference electrode and a bright platinum wire as indicator electrode. Procedure

L-Cysteine

A.

and a-substituted

D or L-cysteines

A portion of test solution was added to sufficient phosphate buffer to maintain the pH at 7 during the titration and 1 g of potassium iodide was added. The mixture was titrated with NIS until a slight yellowing of the solution occurred. Then 1 ml of starch solution was added, and the titration continued to a blue end-point that persisted for 30 sec. in aqueous medium A sample containing 0.1-1.0 meq of thiol was dissolved in 30-50 ml of water. For water-insoluble thiols 5-10 ml of glacial acetic acid were used to dissolve the sample, and twice as much water was added just before the titraIt&. ‘X%te,ml of 1% starch and 2 ml of 4% acetic ‘acid’

and xanthates

in non-aqueous

media

A sample containing 0.1-1.0 meq of -CSSK group was dissolved-in 40 ml of methanol; 50 mg of potassium iodide were added and the solution was titrated with 0.05N NIS in acetonitrile from a microburette provided with a guard tube to keep out atmospheric moisture. The end-point was detected by the appearance of a yellow tint or potentiometrically. RESULTS Some typical results are summarized in Tables 1 and 2. Cysteine, methionine, and thiourea interfere seriously in all the procedures, while acetaldehyde (75:1), ally1 alcohol (1OO:l) and diacetone alcohol (4O:l) give results that are 3.5% low, and 4% and 6% high respectively, when present in the molar ratio to determine and that is shown in parentheses. Serine, gfycine, alanine, glutamic acid, valine, glutamine, proline, leucine, threonine urea, acrylonitrile, dimethyldibenzyl sulphide, thiophene and sulphoxide, diphenyl disulphide (OS mmole) do not interfere. Unlike the aqueous solution, the acetonitrile solution of NIS does not liberate molecular iodine on storage (for at least 7 days at 20”). The burette should be .fitted with a guard-tube to protect the oxidant from atmospheric moisture.

Thiols

DlSCU!SSlON

NIS has been reported’* to react stoichiometrically with mercaptosuccinic and o-mercaptobenzoic acids,

Determination Table 1. Determination

of sulphur functions

of thioureas, dithiocarbamates

919

and xanthates with N-iodosuccinimide Purity, %

Present method* Sample

Visual

Thiourea Thioureas N,N’-Dimethyl Butyl Ally1 Isoamyl Phenyl o_Tolyl o-Ethoxylphenyl Na and K dithiocarbamates Dimethyl Di-isopropyl Phenylethyl Diamyl Tetraethyl Na and K xanthates Methyl Isopropyl Ally1 Cyclohexyl Benzyl l

a b c d

Comparison

Potentiometric

Average deviation, %

method

98.5

99.8

99.7”

0.1

97.0 99.6 98.7 96.5 96.4 97.6 98.3

97.1 99.7 98.8 96.5 96.2 97.3 98.2

97.2’ 99.4” 98.9’ 96.2” 96.1b 97.4b 98.5”

0.2 0.3 0.2 0.3 0.2 0.2 0.3

76.6 88.6 83.3 93.8 82.5

76.8 88.4 83.0 93.7 82.6

76.5’ 88.4d 83.1’ 93.9c 82.3d

0.2 0.4 0.3 0.2 0.3

99.8 99.7 99.5 99.4 99.6

99.8 99.6 99.6 99.5 99.7

99.6d 99.8’ 99.4d 99.2’ 99.4d

0.2 0.1 0.2 0.3 0.3

Average of 10 determinations. Sodium hypoiodite3 Iodine titration’ Mercurimetric titration23*‘6 Iodine titration36

these results being compared with those from the iodine titration3’ method, but others have found that neither NBS nor NIS is suitable for determination Table 2. Determination

of these two acids and 3-mercaptopropionic there being a variable positive error; In our opinion,

iodine

is the oxidizing

acid,

species

of thiols with N-iodosuccinimide Purity, % Present method*

Sample L-Cysteine and u-substituted ot-cysteines Ethane thiol Glutathione 2-Mercaptopropionic acid ZDiethylaminoethane thiol hydrochloride Benzyl mercaptan Thioglycollic acid Thiobenzoic acid 2-Mercaptobenzothiozole Methyl 3-mercaptopropionate Benzene thiol ZNaphthalene thiol Glycerol-1-thiol Cyclohexane thiol I-Pentane thiol * Mean of 10 determinations. a o-Diacetoxyiodobenxoate’5 b Iodimetry3’ c Iodimetry43 d Iodine monochloride titration”’ e Mercurimetryz3

Visual

Potentiometric

Comparison method

Average deviation, %

99.1 91.9 .97.9 96.7

99.3 98.0 97.6 96.2

99.4’ 98.1b 97.8’ 96.6b

0.4 . 0.2 0.3 0.2

98.1 96.1 80.5 94.9 97.6 96.3 99.2 98.9 88.1 99.4 88.6

98.5 98.0 80.5 95.2 97.5 96.2 98.8 99.4 80.0 99.5 88.2

98.2d 98.2’ 80.3’ 95.0b 97.5’ 96.0b 99.0’ 99.1’ 79.7” 99.8b 88.4b

0.2 0.3 0.1 0.2 0.5 0.2 0.2 0.3 0.2 0.5 0.2

in

ASHUT~~HSRIVASTAVA

920

NBS titrations in which notassium iodide is added as additional reagent. NBS contains unstably bound bromine and it might be assumed that the actual oxidizing agent is either hypobromous acid formed by hydrolysis of NBS, or Br+, but this is not so because oxidation of thiols with bromine is known to be nonstoichiometric4’ The amino group in 2-mercaptoethylammonium chloride or glutathione remains intact and N-bromosuccinimide attacks only the sulphur atom. Moreover, in the present determinations gly-

tine, alanine and serine do not interfere. This further indicates that bromine is not the oxidizing species in N-bromosuccinimide titrations. Danehv19,20 studied thiol-iodine reactions and * suggested that a carboxyl group on a carbon atom /I to the thiol group causes an intramolecular displacement reaction and that a five-membered cyclic intermediate is formed. This intermediate can un-

dergo further oxidation to sulphenic acid and higher oxidation states instead of forming the disulphide, which vitiates the titration. On the other hand, thiols such as 2-mercaptopropionic, mercaptoacetic and mand p-mercaptobenzoic acids without a /I-carboxyl group give the correct stoichiometry. Finally gluta-

thione shows minimal tendency to be over-oxidized because the mercapto group is too remote from the carboxyl groups. Although in acetonitrile or glacial acetic acid, either

alone or mixed, the reactions of thiols with N-iodcsuccinimide are slow and iodine appears during the course of titration, in methanol either alone or mixed with acetonitrile (about 80% methanol at the end-point), the reactions are instantaneous and the end-points accurate. The function of the methanol is to deprotonate the complex. MeCN R

RSH+&_____-_ -2

S-1

4. B. Singh and B. C. Verma,

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1965,

24, 536. 5.

R. D. Tiwari and U. C. Pandey,

Analyst, 1969, 94, 813. 6. .I. H. Karchmer, The Analytical Chemistry of Suijiir and its Compounds, Part II, 668. Wiley-Interscience,

New York, 1972. 7. J. H. Karchmer, op. cit., Part I. 8. P. C. Jocelyn, Biochemistry of the -SH Group, Academic Press, New York, 1972. 9. M. R. F. Ashworth, The Determination of Sulphur-conraining Groups, Vol. II, pp. l-55. Academic Press, New York, 1976. 10. A. Srivastava and S. Bose, J. Indian Chem. Sot.. 1974. 51, 736. II. Idem, ibid., 1975, 52, 217. 12. ldem. ibid.. 1975. 52. 214. 13. ldem, Cur;. Sci. India, 1977, 46, 562. 14. A. Srivastava, Z. Anal. Chem., 1977, 287, 316. 15. A. Srivastava and S. Bose, Talanta, 1977, 24, 517. 16. A. Srivastava, K. K. Verma, J. Ahmed and S. Bose. J. Indian 17.

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1971, C6, 159.

21. S. R. Rae, Xanthates and Related Comoounds. Dekker. New York, 1971. 22. A. Granville, N. P. Finkelstein and S. A. Allison, Trans. fnstn. Min. Metall., 1971. Cl. 81. 23. R. J. Magee, CRC Crit. Rev. Anal. Chem., 1973, 1, 355. 24. R. C. Paul, K. Singh, R. K. Chauhan and R. Prakash, Mikrochim.

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=[RS,j=

RSI + I-

HI + &OH RSI + RSH = xiii

RSSR+ HI

This scheme has the merit of suggesting that if it is the basicity of methanol which induces the reaction chain, then a more basic species should fulfil this function even more effectively. It may be mentioned here that pyridine is used as catalyst in the titration of thiois with iodine in benzene medium.44 Acknowledgements-Sincere thanks are due to Dr. S. Bose and Dr. B. P. Sinha for vahiable suggestions. REFERENCES I. D. Schroeder, Chem. Reo., 1955, 55, 181. 2. J. A. Vinson, Anal. Chem., 1969, 41, 1661. 3. P. C. Gupta, Z. Anal. Chem., 1963, 1%. 412.

31. F. J. Welcher, Organic Analytical Reagents, Vol. IV, Chap. IV. Van Nostrand, Princeton, 1948. 32. G. D. Thorn and R. A. Ludwigs, The Dithiocarhamates and Related Compounds, Elsevier, Amsterdam, 1962. 33. A. L. Linch, Anal. Chem., 1951, 23, 293. Acta, 1957, 844. 34. H. Roth and W. Beek, Mikrochim. 35. A. Stevenson, J. Sci. Food Agr., 1964, IS, 505. 36. F. P: Przybylowicz and L. B. Rogers, Anal. Chim. Acta. 37. 38. 39.

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