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V O L U M E 2 7 , NO. 12, D E C E M B E R 1 9 5 5 of phosphoric acid eliminates the interference of aluminuni(II1) during the distillation of fluorine with perchloric acid ( I , 2, 6). LITERATURE CITED
(1) Brunisholz, G., and Jlichod, J., HeZv. Chim. Acta, 37, 874 (1954).
(2) Grimaldi, F. S.,Ingram, Blanche, and Cuttitta, Frank, ANAL. CHEW,27, 918 (1955).
(3) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A,, Hoffman, J. I., "Applied Inorganic Analysis," 2nd ed., p. 860, Wiley, New York, 1953. ENG.CHEM.,ANAL.ED., 1, (4) Schrenk, W. T., and Ode, W. H., IND. 201 (1929). (5) Shell, H. R., and Craig, R. L., ANAL.CHEM.,26, 996 (1954). (6) Shell, H. R., and Craig, R. L., U. S. Bur. Mines, Pittsburgh, Pa., Rept. Invest., in press (1955). RECEIVED for review June 14, 1955.
-4ccepted August 8, 1955.
Determination of Neutral Equivalents by Titration in Alcohol ERIC ELLENBOGEN' and ERWIN BRAND2 Department o f Biochemistry, Columbia University College o f Physicians and Surgeons, N e w York,
5-eutral equivalents of amino acids, peptides, and peptide derivatives can be determined on 2- to 10-micromole samples by titration in alcohol or aqueous alcohol. By means of the proper indicator mixtures, sharp end points are obtained. .4lthough the method is useful only for virtually pure compounds and not applicable to mixtures, it is precise enough to serve as adjunct to the methods usually employed in establishing the chemical purity of these substances.
I
N ORDER to study t,he kinetics of enzymic proteolysis, methods have been devised to determine amino acids by titration in alcohol ( I ) using thyniolphthalein as indicator. The success of such methods is impaired by absorption of at'mospheric carbon dioxide; t'he end point (colorless to light blue) is often masked by turbidity due to precipitation of enzyme and buffer salts, and comparison with a color standard at the end point is difficult. These disadvantages prompted a reinvestigation of the procedure, and resulted in the development of a method which is suitable for the titration of carboxyl groups and acid bound by the amino group of amino acids, peptides, and their derivatives. .4tmospheric carbon dioxide is excluded and indicator mixtures, rather than single indicators, produce sharp end points even in slightly turbid or colored solutions. Forty-five indicat,ors and indicator mixtures have been studied in order t o find some whose color change is sharp enough to be applicable to titration of weak acid and basic groups. The following groups have been titrated so far under the conditions described: carboxyl, cu-aniino.X, &amino.X, e-amino.X, imidazol. X , phenol, and sulfonate (.S st.ands for bound HC1, HBr, HI, HzS04,etc.). This method is not suitable for the determination of guanido.X, however. The sharpest end points were obtained by the following indicator mixtures: (-1)3 nil. of 0.1% phenosafranine in 40% ethyl alcohol plus 4 ml. of 0.1% m-cresol purple in 95% ethyl alcohol; (B) 3 ml. of 0.1% ethylbis-(2,4dinitrophenyl)-acetate in methanol and 1 ml. of 0.1% phenol red in 95% ethyl alcohol; (C) 3 ml. of 0.1% ethylbis-(2,4dinitrophenyl)-acetate in methanol and 1 nil. of 0.1% m-cresol purple in 95% ethyl alcohol; (D) equal parts of 0.1% solutions of o-cresolphthalein and aminoazotoluene in 95% ethyl alcohol; ( E ) equal parts of 0.1% solutions of tropaolin 000 and o-cresolphthalein in 95% et'hyl alcohol; (F) equal parts of 0.1% phenosafranine in 40yc ethyl alcohol and 0.1% thymol blue in 95% ethyl alcohol; and ( G ) equal parts of 0.1% ethylbis-(2,4dinitrophenyl)-acetate in methanol and 0.1% cresol red in 95yc ethyl alcohol. The proper choice of indicator mixture will often depend upon an individual's color perception, and for t,his reapon, mixture -1was employed as most suitable in this study. 1 Present address, Departnient of Biochemistry and Nutrition, Graduate School of Public Health, Csiversity of Pittsburgh, Pittsburgh 13, P a . U.S. Public Health Service Postdoctorate Research Fellow, 1949-51. 2 Deceased.
N. Y.
Table I. Neutral Equivalents of Amino Acids and Peptides and Their Derivatives (Mixture A as indicator)
Compound Glvcine L-Tyrosine L-Tyrosine amide Diiodo-L-tyrosine L-Aspartic acid L-Lysine.HCI L-Arginine.HC1 Taurine L-Glutamic acid gaiiirna ethyl ester.HC1 .4cetyl-~-alanine Benzoyl-L-lysine methyl ester. HC1 Tricarbobenzoxyl ysyll gsine (LD)
Carbobenzoxyglycylalanylalanine (Ln) Dicarbobenzoxyalanyllgsine (nL) 8-Carbobenzoxy-L-ornithine --&L..l "'C"")' c:, ster.HC1 Lysyllysineamide.3HCl (LLj Alanylalanine ( L D j .4lanyllysine.HCI (nL) Lysyllysine.2HCl.HzO f L L j Lysyllysyllysine.3HCl i m L j
-
h-eutral Equivalent Calculated Found 75.0.5 74.7 181.09 179.8 180.09 1 8 1 . 2 216.51 211.6 66.53 67 1 91.6 91.29 210 69 209.3 125 14 126.4
Groups Titrated 1iCOOH) l(CO0H) l(OH) . 2(COOH, OH) 2(COOH) 2iCOOH), HCIj l(CQ0H) l(S0aH)
R,+ covery,
%
100.5 100.7 99.4 100.9 99.2 99.7 100.6 99.8
2(COOH, HC1) l(CO0H)
105 32
l(HC1j
300.77
301 0
99.9
l(C0OH)
677.4
677 5
100.0
l(C0OH)
351 3
330 3
100.3
l(C0OH)
486 44
487.0
99.8
l(HC1) 3(HC1) l(C0OH) 2(COOH, HCIj 3(COOH, HCI) 4(COOH, HCI)
316.i7 127.55 16O,l8 126.87 121.77 128.22
311 9 127.6 161.0 127.4 120.6 129.1
101.5 100.0 99.5 99.6 100.9 99.3
105 8 131.13 131 7
99.5 99.6
One nlilliliter of solution containing 2 to 10 micromoles of substance is placed in a 25-m1. Erlenmeyer flask. Ten milliliters of absolute ethyl alcohol are added, followed by 5 drops of indicator, the flask is placed under manifold, and oil-pumped nitrogen is bubbled through the solution for about 3 minutes. The flask is then transferred t o the titration rack. h buret, calibrated in 0.01 ml., is lowered into the flask, a bubbling tube is inserted, and nitrogen is passed through the solution during the titration in order t o exclude atmospheric carbon dioxide. The buret is connected by means of a three-way stopper to a reservoir of 0.01.Y alcoholic potassium hydroxide. This is prepared by placing 0.6 gram of potassium hydroxide pellets into 100 ml. of absolute ethyl alcohol and stirring by means of a stream of dry nitrogen until dissolved. The solution is allowed to stand a t room temperature for 2 days under nitrogen in order to allow all carbonate t o settle and is then syphoned off under nitrogen through a porous plate and diluted with 9 volumes of absolute ethyl alcohol. The titer of the base is determined by titrating against either standard potassium acid phthalate or 0.01.V hydrochloric acid, using the miaed indicator, and correcting for the indicator blank. Optimum results are obtained by chosing conditions such that the total volume of titrant does not exceed 5 ml. I n Table I are listed compounds representative of the different types. LITERATURE CITED
(1) Bergmann, M., and Hoffman, K., J . B i d . Chem., 130,81 (1939). RECEIVEDf o r review December 3 , 1954. Accepted September 21, 1955, Division of Biological Chemistry 117th Meeting, ACS, Philadelphia, Pa., 4 p r i l 1950.
