ACKNOWLEDGMENT
The authors acknowledge the assistance of H. P. House in the preparation of this report. LITERATURE CITED
(1) ‘‘Ayrican Institute of Physics Handbook, Dwight E. Gray, Coordinating Editor, Section 7, pp. 138-9, McGrawHill, New York, 1957. (2) Bankmann, E., Specker, H., 2. anal. Chem. 162, 18 (1958).
(3) Claassen, A., Bastinge, L., Zbid., 160, 403 (1958). (4)Dean, J: A., Burger, J. C., Jr., ANAL. CHEM. 27,1052 (1955). (5) Dean, J. A., Lady, J. H., Zbid., 27, 1533 (1955). (6) Eshelman, H. C., Dean, J. A,, Menis, Q., Rains, T. C., Ibid., 31.183 (1959). (7) Gilbert,‘ P. T:,Jr.,‘Beckman Instruments, Inc., Fullerton, Calif., Bull. 753 (RiIay 1959). (8) Gilbert, P. T. Jr., Pittsburgh Conference on Anaiytical Chemistry and Applied Spectroscopy, March 6, 1958. (9) Hillebrand, W. F., Lundell, G. E. F.,
Bright, H. A, Hoffman, J. I., “Applied Inorganic Analysis,” 2nd ed., pp. 70-2, Wiley New York, 1953. (10) Kehey, M. T., Fkher, D. JTjJones, H. c.,A N A L . CHEM.31,178 (1959). (11) Menis, O., Rains, T. C., Dean, J. A,, Zbid., 31,187(1959). (12)Specker, H,, Boll, W., 2. anal. Chem. 152, 178 (1956). (13) Watanabe, H., Kendall, M. K., Jr., Appl. Spectroscopy 9 , 132 (1955). RECEIVEDfor review April 11, 1960. Accepted September 12, 1360. Division of Analytical Chemistry, 136th Meeting, ACS, Atlantic City, hT, J., September 1959.
oIorimetric Qualitative Test for Glycine R. L. SUSLETT and J.
P. JEWELL
Tennessee Polyfechnic Instifufe, Cookeville, Tenn.
b A color reaction between glycine, ethyl chloroformate, and pyridine was studied as a qualitative test for glycine. Results indicate that the reaction is specific for glycine, pyridine, and the alkyl chloroformates. This method can be adapted to spot testing for glycine with a sensitivity of 1 to 2 pgo The colored compound is stable in some polar solvents,
Hopkins ( I ) reported the formation of a red color in the reaction between pyridine and ethyl chloroformate. However, work in this laboratory has s h o m that this color depends upon the purity of the pyridine and differs from the color obtained when glycine is present. EXPERIMENTAL Reagents.
react with amino acids to produce colored substances v, hich can be used for the identification and determination of these acids. The most widely used reagent in the determination of the amino acids is ninhydrin, although other organic reagents (3) and inorganic complexing reagents (2) have been used. However, none of these reagents are specific for glycine, and the identification of individual amino acids requires previous separation. o-Phthalaldehyde (4) has been found to be specific for glycine. but has never been widely used, This investigation is a study of the color reaction between glycine, pyridine, and ethyl or methyl chloroformate as a specific method of identification of glycine. When glycine is mixed with ethyl chloroformate and pyridine is added dropln-iee, a dark green color is formed which changes t o red on the further addition of pyridine or upon standing. -4 positive test is denoted by obtaining both the green and red colorations. This test combines selectivity, sensitivity, and simplicity. The sensitivity is such that it can be adapted to spot testing for glycine to 1 pg. This test also appears to be specific for pyridine and the alkyl chloroformates. Further work will determine the sensitivity and the selectivity of this reaction as a test for these reagents. AWY REAGENTS
T h e sources of all materials tested and their purification are indicated in Table I. The ethyl chloroformate and glycine were Distillation Products Industries (D. P. I.) White Label, and needed no further purification. The pyridine was obtained from either T h e Matheson Co. or D. P. I., White Label, and was carefully purified b y fractionation. General Procedure. Portions, 0.1 to 0.2 gram, of glycine were moistened with 0.3 t o 0.5 ml. of ethyl chloroformate and mixed thoroughly. Pyridine was added dropwise at room temperature with constant mixing. A dark green color appeared which turns red with further addition of pyridine. T h e red color can be changed to green by the addition of excess of ethyl chloroformate. Amino acids other than glycine and related compounds were tested. The results are given in Table 1. Dilute aqueous solutions of glycine were tested. A positive test was obtained if the quantity of the solution tested was less than 0.1 ml., or, if the molar ratio of ethyl chloroformate to water is greater than one. The effect of temperature and the order of addition of the reagents, glycine, pyridine, and ethyl chloroformate, are given in Table 11. I n order to determine the specificity of the reagents, alkyl chloroformates, pyridine, and compounds similar to these were substituted for the reagents in various combinations. With respect to reagents substituted for ethyl chloroformate, methyl chloroformate (D. P. I.,
practical) only gave a positive test. Acetyl chloride, benzoyl chloride, thionyl chloride (E. I?. I., White Label), and carbobenzoxy chloride (K &K Lab., Inc.) gave negative tests. The reagents substituted for pyridine in the test for glycine all gave a negative test. The ones used in the experiment were diethylamine, ethylenediamine, 8-quinolinol, 2- (redistilled), 3-, and 4picoline, and triethylamine (D. P. I., White Label) ; morpholine and quinoline (D. P. I., practical); diphenylamine (D. P. I., technical); pyridine (moisture present) ; and ammonium hydroxide. Diethylamine and diphenylamine produce a red coIor only. The product formed by the reaction of glycine, ethyl chloroformate, and pyridine was soluble in polar solvents. The solubility of this colored product in various solvents is given in Table 111. Pyridine and ethyl chloroformate were reacted under the same conditions as described b y Hopkins (1). Some commercial grades of pyridine gave a pale pink color, but when purified by careful distillation, no color was observed. Spot Test Procedures. A solution containing 0.1000 gram per liter of glycine r a s prepared, and then diluted to contain 10-5 gram per ml, Portions of this solutior, ranging from 0.1 to 1 ml. were pipetted onto a spot plate. The spot plate was placed in an ovcn at 135’ to 140’ C. and allowed to dry for 30 to 40 minutes. The plate was removed from the oven, and, while hot, ethyl chloroformate and pyridine were added alternately and dropwise. The colors produced were either green or red depending on whether ethyl chloroformate or pyridine was in excess. It was found that 0.1 ml. of the solut: or 10-8 gram of glycine gave a faint red or green color, while 2 x 10-6 gram of glycine gave a distinct coloration. The intensity of the coloration increased with an increase in concentration of glycine. The same results were obtained when the spot plate was allowed VOL. 32, NO. 13, DECEMBER 1960
a
1841
Table 1. Results of Color Test with Compounds Substituted for Glycine
COMPOUNDS GIVINGPOSITIVE TEs?rs Acetylglycinea Glycylglycine hydroGlycine hydrochlo- chlorideb ride* Hydrolyzed alGlycine (recryst. buminb from ethyl Hydrolyzed gelatin a1cohol)a-* (23.6% g1ycine)b Glycine (recryst. Sodium glycinateb from water)aJ COXPOUNDS GIVINGNEGATIVE TEST& D&ct-A1aninea &Histidine'" 6-Alaninea Hydroxyprolined Albumin. L-( - )Leucine. Zhin0-2-(hyDbIsoleucinea droxymethy1)-1,- nL-Lysme mono3-propanedioP hydrochloride" nL-2-Aminohexanoic Methyl glycinate acid0 hydrochlorideb Anthranilic acida DLhfethionined L-Ar inine hydro- hlonochloroacetic chforidee acida DL-Aspartic acid0 Morpholinef Beneamidef 2-Xaphthylamineh CarbobenzoxynL-fl-Phenylalanine0 glycineb N-Phen ylalaninea Creatinine. N-Phen yiglycine" L-( )Cysteine 2-Picoline? (rehydrochlorideo distilled) L-(- )Cystines LProiined Diethylaminea Sarcosine' Diethylanilinea nL-Serined sym-Diketopipera- Taurineo zineb DL-Thre0ninea.o Diphenylamine! Triethylamine0 N-E thylanilinea L-Tryptophane Gelatink L-Tyrosinel G( )Glutamic m-ValineO acida a D. P. I. (White Label). Prepared,in this laboratory. Fisher Scientific Co. Kutritionai Biochemicals Corp. e Mann Research Laboratory. f D. P. I. (Prartical). 0 California Foundation for Biochemical Research. J. T. Baker Chemical Co. K &: K Laboratories, Inc. i D. P. I. (Technical). E. H. Sargent and Co. (U. S.P.). General Biochemicals, Inc. Matheson Coleman and Bell.
Table 111. Solubility of Colored Product in Different Solvents Insoluble Soluble Very Soluble Acetone Ammonium hydroxide Carbitol Benzene Ethyl alcohol Cellosolve Butyl ether Isoamyl alcohol Cyclohexanol Carbon tetrachloride Isopropyl alcohol Diethylene glycol Chloroform Methanol Dimethylformamide Diethylamine Water Ethylene chlorohydrin 2-Picoline Ethylene glycol Trichloroethylene Triethylamine
A contained 0.1000 gram of glycine in addition to the above amino acids. Solutions A and B were tested on a spot plate as indicated above. A positive
test for glycine in solution A was obtained if as much as 2 x gram of glycine were present. A negative test was obtained for solution B regardless of the quantity used. DISCUSSION OF RESULTS
The results given in Table I indicate that this test is specific for glycine and some of its simple derivatives such as sodium glycinate, glycine hydrochloride, glycylglycine hydrochloride, and acetylglycine. The properties of these compounds are such that they could readily form glycine in the presence of the reagents and conditions used for the test. Under the usual conditions carbobenzoxyglycine gives a negative test. However, if carbobenzoxj-glycine is heated with pyridine prior to the addition of ethyl chloroformate, a positive test is obtained, which is probably due to the decomposition of carbobenzoxyglycine. The glycine-containing proteins, when hydrolyzed, gave a positive test. The alkyl chloroformates were the only compounds that gave a positive test with glycine and pyridine. This test was also specific for pyridine. Although some of the pyridine substitutes gave a red coloration, no green to cool before the addition of ethyl color was obtained with or without the chloroformate and pyridine. presence of glycine. I n order to determine the interference The spot tests indicate that 1 wg. of other amino acids, two solutions, A of glycine can be detected or, if other and B, were prepared. Each solution amino acids are present, the sensitivity contained 0,1000 gram per liter of the may be of the order of 2 pg., depending following amino acids: L-( -)leucine, DL-p-phenylalanine, ~~-2-aniinohexanoic upon the concentration of the other amino acid?. acid, B-alanine, m-lysine hydrochloride, As indicated in Table 11, a positive DL-a-alanine, DL-isoleucine, L-( -)cystine, and L-(+)glutamic acid. Solution test was not obtained when glycine
+
10
+
Table II.
Order of Addition of Reagents and Effect of Temperature
1st Addition
2nd Addition Glycine Ethyl chloroformate Glycine Pyridine Ethyl chloroformate Pyridine Pyridine Ethyl chloroformate Glycine Ethyl chloroformate Glycine Ethyl chloroformate Glycine Ethyl chloroformate a White solid produced.
1842
B
ANALYTICAL CHEMISTRY
Tempera3rd Addition ture Results Pyridine Room Pos. Ethyl chloroformate Room POS. Glycine Room Neg.a Glycine Room Neg.0 Pyridine 15'@. ru'eg." Pyridine 0" C. Neg." Pyridine -15" C. n'eg.0
was added to a mixture of pyridine and ethyl chloroformate. However, a positive test was obtained regardless of the order of addition of pyridine and ethyl chloroformate to glycine. The reaction that produces the colored compound does not occur or is very slow a t temperatures below 15" C. (Table 11). The reagents, glycine and ethyl chloroformate, needed no purification, but some grades of unpurified pyridine required purification. Upon distillation the color-forming impurity in the pyridine remained in the residue. Infrared curves of the unpurified pyridine and the distillate were identical, which indicated that the concentration of the impurities was very small. These curves were obtained in an Infracord 137. The red compound produced by glycine, ethyl chloroformate, and pyridine fades rapidly in the presence of water. I t s stability in other solvents is such that i t could be adapted t o quanticative colorimetric determinations. Work on this is now in progress. Esters, polyamides, nitrogen-substituted glycines, and all amino acids other than glycine gave negative tests. This evidence indicates that a -CQOH, -a-NHz, and -CH&OO(two alpha hydrogens) must be available to obtain a positive test. The negative tests obtained in reactions with substituted pyridines and other amines indicate that pyridine is not just a hydrogen chloride acceptor, but probably enters into the structure of the colored materials. The fact that only the alkyl chloroformates gave positive tests suggests that the colored product also contains some part of the alkyl chloroformate molecule in its structure. Work is now in progress to determine the structure of the colored compound. LITERATURE CITED
(1) Hopkins, Thomas, J . Chem. SOC.117,
278 (1920). (2) Ley, H. Arends, B., 2. physiol. Chem. 193, 131 (1930). (3) Meneie, Calvin, ANAL. CHEM. 28, 1321 (1956). (4) Zimmerman, Walther, Z . physiol. Chem. 189, 4 (1930). RECEIVEDfor review May 23, 1960. Accepted Sugust 22, 1960.
