670 SAVILLE : A SCHEME FOR THE COLORIMETRIC DETERMINATION [Vol. 83 A Scheme for the Colorimetric Determination of Microgram Amounts of Thiols BY B. SAVILLE* (Chemical Defence Experimental Establishment, Porton Down, Salisbuvy, Wdts.) A method has been found of converting thiols, and other molecules possessing -SH groups, to their S-nitroso derivatives, which yield an equiva- lent of nitrous acid on mercuric ion-assisted hydrolysis. This nitrous acid is finally used in the formation of a brilliant azo dye from sulphanilamide and N-1-naphthylethylenediamine. The correspondence between dye produced and thiol used has been utilised in the development of a general analytical method. Sensitivity is extremely high, for as little as 2 x lo-* g-equivalents of a thiol in 1 ml of solution can be determined.The accuracy is to within &1 to 2 per cent. for determinations on about 2 x 10-7 g-equivalents of thiol. ALTHOUGH early work112 has shown that thiols can be converted to the corresponding S-nitroso derivatives by means of nitrosyl chloride, etc., little systematic investigation of the more simple chemical properties of these compounds has been reported. It has now been found that the S-nitroso derivatives of some of the more familiar thiols, such as ethanethiol, phenyl- methanethiol, cysteine, thiophenol and thioglycollic acid, undergo facile hydrolysis to nitrous acid in the presence of mercuric, silver or cupric salts. Hence, a solution of S-nitrosocysteine, which can be readily prepared by adding cysteine to excess of sodium nitrite in 0.1 to 1.0 N sulphuric acid, is relatively stable (ti N 50 hours) in the presence of ammonium sulphamate, which is added to remove the excess of free nitrous acid; the reactions are as follows- RSH + HONO =?- RSNO + H,O I Fast NH4S03NH2 NH4+ HS0,- + N, + H,O This indicates that the hydrolysis of the S-nitrosothiol (shown by the broken arrow) is slow in acid solution, in contrast to the corresponding behaviour of alkyl nitrites.On the other hand, if a slight excess of mercuric chloride, mercuric acetate or silver nitrate is added to the solution of the S-nitrosothiol prepared in the way described, there is an immediate liberation of nitrogen and the red colour of the nitroso compound is rapidly destroyed. This extremely rapid hydrolysis can be explained by assuming that those metal cations known to possess high affinities for sulphur can engage in rapid reversible co-ordination with the S-nitrosothiol to form a complex (I), which, owing to the weakened N-S bond, is then highly susceptible to nucleophilic attack by water molecules.The reactions are as follows- ' Hg+ 0 N-S + Hg2+ + N-S+/ I1 I1 I (1) R I R H 0 H 0 \+ I/ /Hg+ \o -N I1 __ :/Hg+ - + 0 - N + S R / H ' 11 \R H H+ + HONO * Present address : The British Rubber Producers' Research Association, 48 Tewin Road, IVelwyn Garden City, Herts.Dec., 19581 OF MICROGRAM AMOUNTS OF THIOLS 67 1 In the experiments described, the liberated nitrous acid immediately reacts with the excess of ammonium sulphamate to form recognisable gaseous nitrogen. However, if sulphanil- amide or some other reactive aromatic amine is mixed with the mercuric salt before addition to the S-nitrosothiol solution, the amine competes favourably with the sulphamate for the nitrous acid equivalent to the S-nitrosothiol and gives a high yield of the corresponding diazonium salt.For sulphanilamide, conditions have been found in which diazonium salt formation is almost exclusive, i.e., reaction ( 2 ) in the following equation is much more rapid than reaction (1)- pNH,IHSO, + rCT2 + HZO NH,SO,NH,/ H+ Hg2+ - - /(I) RSH -+ HONO - RSNO - HONO \(a) Excess of A vNH,,HX\ 4 nitrous acid removed 11[4rN=Nj+X- + 2H,O As the diazonium salt (which is formed in amounts equivalent to the thiol taken) can be made to couple with an amine to yield an intensely coloured azo dye, a potential colorimetric method for the determination of thiols was at once realised.METHOD REAGENTS- of 0.2 to 1.0 N sulphuric acid. Solution A-Mix 1 volume of a 0.01 M aqueous solution of sodium nitrite with 9 volumes Solution B-Prepare a 0.5 per cent. solution of ammonium sulphamate in water. Solution C-Mix 1 volume of a 1.0 per cent. aqueous solution of mercuric chloride with Solution D-Prepare a 0.1 per cent. solution of N-1-naphthylethylenediamine dihydro- This solution can be prepared as required. 4 volumes of a 3.4 per cent. solution of sulphanilamide in 0.4 N hydrochloric acid. chloride in 0.4N hydrochloric acid. PROCEDURE- To 5 ml of solution A in a 25-ml calibrated flask, add 1 ml of a solution of the thiol (0*00002 to 0.0005 M ) in water or aqueous ethanol.Set aside for & to 5 minutes, according to the nature of the thiol, and then add 1 ml of solution B. Insert the stopper, and shake well for a few seconds to ensure complete removal of excess of nitrous acid. After 1 to 2 minutes, rapidly add 10ml of solution C (to hydrolyse the S-nitrosothiol and form the diazonium salt), and then make up to the mark with solution D. Colour development is rapid and is usually complete in 3 to 5 minutes with no further change in intensity over a prolonged period. After 10 minutes, measure the coloured solution against an appropriate blank solution with a Spekker absorptiometer and Ilford No. 605 yellow-green filters. (It is advisable to cover the cell containing the blank solution with a cover-glass to prevent access of atmos- pheric nitrogen oxides.) Calibration graphs relating absorptiometer reading to thiol concentration can be plotted.These graphs are linear over a wide range of readings and are extremely reproducible. This solution must be freshly prepared each day. DISCUSSION OF THE METHOD ACCURACY- The accuracy of the method depends on the concentration of thiol being analysed. For 1 ml of 0.00002 M thiol, the accuracy is to within + l o per cent., whereas, for 0*0001 and 0.0004M solutions, the respective accuracies are to within i 2 to 3 per cent. and + 1 to 2 per cent. INTERFERENCES, SPECIFICATION AND SCOPE- As far as I am aware, the method is of general applicability to all thiols and has been shown to be specific for thiols in the presence of substances that might be thought, on a rational basis, to interfere. Hence, small amounts of dialkylamines, which could form nitros- amines as a potential source of nitrous acid, do not interfere.This may be because the rate of nitrosation of secondary aliphatic amines is low in acid solution3 and the rate of hydrolysis672 GAGE: THE DETERMINATION OF TRACES OF LEAD [Vol. 83 of secondary nitrosamines is high. In general, modifications will need to be introduced to compensate for interfering substances that either rapidly destroy nitrous acid or form coloured nitroso derivatives. As the rate of S-nitrosation of cysteine is several orders greater than the rate of de-amination of simple amino acids, cysteine can be determined without inter- ference from extremely large excesses of amino acids in protein hydrolysates. The method may therefore be of immediate importance to 'biochemists. A convenient standard thiol solution can be prepared by allowing the corresponding isothiuronium salt to undergo quantitative hydrolysis in dilute sodium hydroxide solution containing about 0.05 per cent. of alkali cyanide to prevent oxidation of the thiol. The reaction is as follows- NH \ // HO- + C-S-R- H'O-C + RSH (urea) I thank R. Bond (vacation student from the University of Glasgow, 1956), H. F. Liddell and Miss P. M. Smyth for assistance and advice during this work. REFERENCES 1. 2. 3. Tasker, H. S., and Jones, H. O., J . Chem. SOC., 1909, 95, 1917. Rheinboldt, H., Ber., 1926, 59, 1311; 1927, 60, 184. Taylor, T. W. J., J . Chem. SOL, 1928, 1897. Received May 29th, 1958