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 s a m d e 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. I n 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). Zbid., 78, 2582 (1956). Earle, T.E., ASAL. CHEhf. 2 5 , 769-71 (1953). Ha rnish. 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.
Per cent disulfide = milliliters X N of AgKOa X molecular weight X 100 weight of sample x 2000 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
20, 938-Y (1948).
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
Quantity of Menadione,
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.
Y
PROCEDURE
One tenth milliliter of Menadione solution was shaken up with 0.5 ml. of
0 (blank) 5
10 20 30
Ahsorbance (A635 MF) 0.006 0.030 0.054
0.102 0.150 50 0.246 100 0.486 200 0.966 300 1.446 Experiment carried out at 30" C. Absorbance not corrected for blank. ~
~
VOL. 29, NO. 1, JANUARY 1957
155
Table
II.
Effect of Incubation Time on Color Development"
Time, hlinutes
Absorbance Sample Blank Net 0 0 004 0 004 0 000 5 0 240 0 004 0 236 10 0 540 0 006 0 534 20 0 538 0 006 0 532 Xeasurements made at 635 mw
Table V. Effect of Relative Amounts of Amyl Alcohol and Ethyl Alcohol on Color Development"
Amyl Ethyl Alcohol, Alcohol, Absorbance hI1. MI. Sample Blank Net 4.0 .. 0.520 0.006 0.514 3.0 1 . 0 0.540 0.006 0.534 2.0 2 . 0 0.480 0.018 0.462 1.0 3 . 0 0.300 0.088 0.212 Measurements made a t 635 mp. Table VI.
Table 111. Effect of Temperature on Color Development"
TpPf C. 26 30 35 40
a
Absorbance Sample Blank Net 0 260 0 002 0 258 0 493 0 005 0 488 0 540 0 006 0 534 0 638 0 004 0 634 60 0 650 0 004 0 646 90 0 919 0 004 0 915 Measurements made at 635 mp.
Table IV. Effect of Concentration of Sodium Carbonate on Color Development"
Sodium Carbonate, Absorbance hII. Sample Blank Net 0.2 0.423 0.004 0.419 0.3 0.540 0.006 0.534 0.4 0.520 0.008 0,512 0.5 0.441 0.008 0.433 a Measurements made at 635 mp.
hol, and 1 ml. of distilled water were added, and the solution was shaken thoroughly, and then allowed t o stand for 5 minutes to permit the salts to settle. The amyl alcohol layer was removed, and the color was read at 636 mp in a Beckman spectrophotometer
F. WITTER
Nenadione Added,
hIenadiSbsorbone Reance Recovered, covery, (3635)" -, 76 5 0.027 5.0 100.0 10 0.053 9.9 99.0 50 0.268 49.6 99.2 100 0.532 99.6 99.6 a Corrected for blank. Blank run on mold extract alone (3635 mp for blank was 0.002 n-hich remained constant in all cases).
2,4-Dinitrophenylhydrazine on Color Development"
2.CDinitrophenylhydrazine, Absorbance hI1. Sample Blank Set 0 08 0 250 0 004 0 246 0 10 0 540 0 006 0 534 0 15 0 540 0 005 0 535 0 20 0 548 0 004 0 544 a Measurements made at 635 mp.
against a blank containing all reagents except Menadione.
ratio of the concentrations of amy1 alcohol and ethyl alcohol, and the concentration of 2,4-dinitrophenylhydrazine. The addition of more than 1 nil. of water made the solution very turbid. Table VI1 presents the percentage recovery of different amounts of Mensdione added to the mold extract. I t shows that the method is useful in estimating the amounts of hIenadione in mold extracts. CONCLUSION
RESULTS
Tables I and I1 show that the rate of color formation depends on the concentration of Xenadione and the period of incubation. Table I11 shows the effect of temperature on the formation of the color complex. Even though the intensity of the color increased with temperature of incubation, it was observed that the color formed a t 35" C. was stable for 24 hours; the color that developed at higher temperatures was more intense at the junction and erratic results mere obtained. Tables IV, V, and VI show that the rate of color formation depends on the concentration of sodium carbonate, the
Spot Test for 3-Hydroxy ROBERT
Effect of Amount of
Table VII. Recovery of Menadione Added to Mold Extract
The results obtained in these experiments show that this method can be used to estimate Menadione in the range of 2 to 300 y. It remains to be seen whether this method will accurately measure natural vitamin K (K, and K2) in plant or animal tissues. LITERATURE CITED
(1) Sovelli, A., Science 93,358 (1941). (2) Reddy, D. V. S., Srinivasan, V., Current Sci. (India) 17. 22-3 11948). (3) Sathe, Vanamala; Dave, J.' B., 'Ramakrishnan, C. V., Nature 177, 276 (1956).
RECEIVED for review December 13, 1955. Accepted June 25, 1956.
Steroids
and SHELLEY STONE
Department of Biochemistry, School o f Medicine und Dentisfry, University of Rochester, Rochesfer 20, N. Y.
b Steroids
containing the 3-hydroxy-
A-5 groups give a characteristic pink test when the paper is heated a t 80" C. for 8 minutes after spraying with phenol and perchloric and molybdic acids. The presence of a keto group or a double bond in position 7 interferes with the test. Fructose and its derivatives give a purple color.
156
ANALYTICAL CHEMISTRY
S
specific for the various functional groups of the steroids are needed. It has been found that steroids containing the 3-hydroxyl A-5 groups form a characteristic pink color when heated a t 80' C. on paper that has been sprayed with phenol and then with perchloric and molybdic acids. The steroids can be detected a t concentraPOT TESTS
tions as low as 5 y per 10 pl. (The spot area is 1.8 sq. cm.) EXPERIMENTAL
Reagents. C.P. phenol is saturated with water at room temperature. This reagent is used for about 2 weeks and is kept in the dark. The Hanes-Isher-
