Determination of Organic Disulfides Reduction with Sodium Borohydride C. R. STAHL and SIDNEY SlGGlA Central Research laborafory, General Aniline und Film Corp., Easton, Pa.
,A method is presented for the determination of organic disulfides by reduction to the mercaptan with sodium borohydride and aluminum chloride and titration of the mercaptan with silver nitrate.
B
oxidation (6) and reduction (3-5) methods for the determination of disulfides have been reported. The oxidation method is more precise and accurate for the assay of pure disulfide samples but is subject to interference from dialkyl sulfides, mercap tans, and other easily oxidized compounds. Disulfides cannot be determined by oxidation in the presence of sulfides, and results are not accurate in the presence of mercaptans. Sulfides and mercaptans do not interfere with the reduction methods for disulfides, but in the methods reported low results are obtained because of incomplete reduction or because the time necessary for the reduction is too long to be practical. Kolthoff, May, Morgan, Laitinen, and O'Brien (6) and Earle (3) report an accuracy only within 5%. Harnish and Tarbell (4) obtained quantitative results for diphenyl disulfide, but the reduction required 16 hours. Because of its freedom from interferences, reduction is the better means of determining disulfides, if quantitative reduction can be accomplished in a reasonable length of time. After various means of reducing disulfides had been considered, metal hydrides seemed to be the most promising reducing agents to investigate, as it had been observed that when reductions with hydrides are quantitative they proceed in a short time. Also, the hydrides would present no problem in the analysis of the mercaptan formed on reduction once they were decomposed. Lithium aluminum hydride was first tried, but high results were obtained. This appeared to be due to reduction of part of the disulfide to hydrogen sulfide, since on titration of the reduced samples with silver nitrate two potential breaks were observed. These corresponded to those obtained by titrating mixtures of hydrogen sulfide and OTH
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ANALYTICAL CHEMISTRY
mercaptans, and no silver nitrate was consumed in blank titrations of the decomposed reagent. Reduction with sodium borohydride was attempted when it appeared unlikely that conditions could be adjusted to eliminate the difficulties observed in using lithium aluminum hydride. Brown and Rao found that in the prrsence of aluminum chloride a diethylene glycol dimethyl ether solution of sodium borohydride (2) reduced diphenyl disulfide. An estimated reaction completeness of about 90% is reported. A method for quantitatively determining disulfides was developed with this reduction as a basis. After reduction the excess hydride is decomposed, and the mercaptan obtained is titrated potentiometrically in an aqueous solution of sodium and ammonium hydroxides with silver nitrate. The reducing agent will interfere with the titration unless it is completely decomposed. The decomposition of the hydride presented a problem in the development of the method, because sodium borohydride can readily be decomposed only by the addition of acid nhich causes a rapid evolution of gas and loss of mercaptan. Because sodium borohydride and aluminum chloride apparently form aluminum borohydride ( I , 2 ) to some extent, it seemed probable that the addition of dilute sodium hydroxide would destroy most of the hydride in this case, and no mercaptan would be lost from the basic solution; however, the decomposition with sodium hydroxide nTas found to be vigorous and mercaptan n-as lost unless the deconiposition was carried out in a flask submerged in an ice bath and equipped with a long coiled condenser. In some cases a small amount of sodium borohydride remained; hon-ever. it R 3s possible to decompose this with acid without loss of mercaptan, since little gas was evolved. The procedure finally adopted !vas to add sodium hydroxide and then nitric acid t o ensure complete decomposition. REAGENTS
Sodium borohydride, 2 grams dissolved in 100 ml. of diethylene glycol dimethyl ether (Ansul Chemical Go., Marinette, Wis.).
Anhydrous aluminum chloride. Sodium hydroxide, 1N. Sodium hydroGde, 6 N . Yitric acid, 3M. Concentrated ammonium hydroxide. Standard O.1N silver nitrate. PROCEDURE
A sample containing approximately 0.001 mole of disulfide is accurately weighed and placed in a 150-ml. roundbottonied flask containing 15 ml. of sodium borohydride solution and approximately 0.5 gram of aluminum chloride. The flask is immediately attached to a coil condenser 40 em. long, and the reduction is allowed to proceed a t room temperature for 0.5 hour. The order of addition of reagents and sample to the flask is unimportant except in the case of methyl disulfide. When the
Table 1. Determination of Disulfide by Sodium Borohydride Reduction Compo I 1n d
Phenyl disulfide"
Methyl disulfide"
%-Butyldisulfide
Isoamyl disulfide
Disulfide Found, c/o r7
99 2 99 3 99.8 99.6 99.4 98 9 99.0 99.2
98.7 98.7 98.6 99.1 95.5 95.7 96.3 96.6 95.9 95.4
a Sulfur values obtained on these samples. Phenyl disulfide found = 29.52y0, theory = 29.37%. Methyl disulfide found = 67.80%, theory = 68.08%. Ethyl disulfide found = 50.63%,
theory = 52.46y0.
sodium borohydride solution and the aluminum chloride are mixed, a rapid reaction takes place, and methyl disulfide is lost if it is added to the solution before the aluminum chloride. I n t h e determination of methyl disulfide the sodium borohydride solution and the aluminum chloride are mixed and allowed to stand in an ice bath for a few minutes while the samde is being weighed. After reduction is complete, the flask is submereed in an ice bath, and 5 ml. of 1N sGdium hydroxide 'are added through the condenser. The sodium hydroxide is added a few drops a t a time until the initial vigorous reaction subsides, and then the remainder is added, and the condenser is rinsed with a few milliliters of distilled water. The solution is allowed to stand for 2 or 3 minutes and 10 ml. of 3144 nitric acid are added. The ice bath is removed, and 10 ml. of 6N sodium hydroxide are added after about 2 minutes. I n determining methyl disulfide it is better to allow the solution to stand 5 minutes in the ice bath after adding the nitric acid and to add the sodium hydroxide before removing the ice bath. u
The condenser is rinsed with a few milliliters of distilled water, and the flask is removed. The contents of the flask are rinsed into a 400-ml. beaker, and 10 ml. of concentrated ammonium hydroxide are added. Using a pH meter equipped with silver and calomel electrodes, the solution is titrated potentiometrically with standard 0.1N silver nitrate solution. The break occurs between approximately -325 and -175 mv., although it varies somewhat for the different mercaptans being titrated. Per cent disulfide is calculated in the following manner:
with the procedure. n'hen sulfitlrs are treated in the same manner used to determine disulfides, no silver nitrate is consumed on titration. In determining the disulfide content of a sample which contains mercaptan, the mercaptan must be determined on an unreduced sample and subtracted from the value obtained after reduction. The mercaptan is unchanged by reduction and titrates Ivith the reduced disulfide. LITERATURE CITED
Brown, H. C., Rao, B. C., J . dm. Chern. SOC.77, 3164 (1955).
Per cent disulfide = milliliters X N of AgKOa X molecular weight X 100 weight of sample x 2000
Zbid., 78, 2582 (1956). Earle, T.E., ASAL. CHEhf. 2 5 , 769-71 (1953). Harnish. D. P., Tarbell, D. S., Ibid., 21,968-9 (i959). ' Kolthoff, I. M., May, D. R.,, Morgan, P.. Laitinen. H. A.. O'Brien. A. S.. EKG.CHEJI.,'ANAL. f i ~ 18; . 442-4 (;1946). Siggia, S., .Edsberg, - - - R.. ANAL CHEX. 20, 938-Y (1948).
RESULTS AND DISCUSSION
Results obtained for the six disulfides used to test the procedure are given in Table I. The precision and accuracy of the method are within &l%. Mercaptans and sulfides do not interfere
RECEIVED for review June 13, 1956. Accepted September 23. 1956.
Spectrophotometric Method for Estimation of Menadione VANAMALA SATHE, J. B. DAVE, and C. V. RAMAKRISHNAN Department o f Biochemistry, M. S. University o f Baroda, Baroda, India
b During investigations on the biosynthesis of Menadione in molds, it was found that the spectrophotometric methods described b y Novelli and b y Reddy and Srinivasan could not b e used to determine micro quantities of Menadione because of incomplete separation of the two layers and the instability of the color formed. The improvements on the method of Reddy and Srinivasan reported here make i t possible to use it for the estimation of 2-methyl-1 ,4naphthoquinone.
R
EDDY and Srinivasan ( 2 ) experienced great difficulty in adapting the technique of Kovelli (1) to estimate Menadione, because even the slightest excess of ammonia resulted in a reddish yellow precipitate which masked the green color. During the course of the present investigations, it was observed that on standing the layers do not separate clearly and the intensity of the color a t the junction of the two layers increases, with the result that the intensity of the color
is not uniform throughout the layer. Hence, a modification of the method was proposed to estimate Menadione in the range of 2 to 300 y ( 3 ) . APPARATUS AND REAGENTS
Menadione. A 0.1% solution was prepared by dissolving 0.1 gram of 2-methyl-] .d-naphthoquinone in 100 ml. of ethyl alcohol. 2,4-Dinitrophenylhydrazine. A saturated solution of 2,4-dinitroplienylhydrazine (Merck) in 2N hydrochloric acid was used.
ethyl alcohol, and 0.1 ml. of a saturated solution of 2,4-dinitrophenylhydrazine in 2N hydrochloric acid was added. This solution was incubated a t 35" C. for 10 minutes and then cooled to room temperature. After incubation, 0.3 ml. of 20% sodium carbonate was added, and the mixture was shaken well until the green color appeared. Three milliliters of amyl alcohol, 1 ml. of ethyl alco-
Table I. Effect of Concentration of Menadione on Color Development"
Sodium Carbonate Solution. A 20% sodium carbonate (British Drug Houses) solution was used. Alcohols. %-Amyl alcohol (BIerck) and ethyl alcohol were distilled before use. Spectrophotometer. A Beckman Model DU spectrophotometer with a 1-cm. light path was used. PROCEDURE
One tenth milliliter of Menadione solution was shaken up with 0.5 ml. of
Quantity of Menadione, Y
Ahsorbance (A635 MF)
0 (blank) 5 10 20 30 50 100 200 300
0.006 0.030 0.054 0.102 0.150 0.246 0.486 0.966 1.446
Experiment carried out at 30" C. Absorbance not corrected for blank. ~
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VOL. 29, NO. 1, JANUARY 1957
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