When metal ions with an oxidation number of 3 or 4 are considered, the solution of the various equations requires the application of determinants. These calculations have been made for a number of acetylacetonates (Table 111). On allowing for the intermediates, the maximum change in log K , (f3.78) took place in the case of zinc where the KD,is the lowest (1.8) and pHlla is the second highest (5.30) and the least changes, +0.04 and f0.23, took place in the case of beryllium and uranyl where the K D , values are the highest (41 and 65.7) and the pH1,a among the lowest (0.67 and 1.66). Thus the effect of the presence of charged intermediates is enhanced when KD,is low or the extractions take place at higher p H values. I n addition to the presence of charged chelates, the theoretical calculations will be affected by hydrolysis and changes in the complexity of the molecular species. However in the system studied, the close agreement between the formation constants obtained from extraction and potentiometric studies (Table H I ) , suggests that interference from such causes is negligible. I n the case of zinc(I1)
acetylacetonate, hydrolysis can be ruled out as a factor of any consequence. The low concentrations of metal ions ( l O - a M ) used in this study, along with the fact that the extraction behavior of copper(I1) acetylacetonate has been correctly predicted (Figure 1) from the value of the formation constants obtained independently by potentiometric methods, indicate that increases in molecular complexity, if any, are not significant. ACKNOWLEDGMENT
The authors gratefully acknowledge the financial assistance of the V. S. Atomic Energy Commission. LITERATURE CITED
(1) Allsopp, K. E.,Arthur, T.E.,ASAL. CHEY.23, 1883 (1951). ( 2 ) Biltz, W., Clinch, J. A, 2. anorg. Chem. 40, 218 (1904). (3) Dvrssen. David, Svensk Kern. Tidskr. 68.212 f1'956). (4) Dyrssin, hfargareta, Rec. trav. chiin. 75, 748 (1956). (5)'Eck, C . L. van P. van, Zbid., 72, 529 (1953). (6) Hantzsh, A., Desch, C. H., Ann. Chem. Liebigs 308, 1 (1902) ( i )Hevesy, G. von, Logstromp, 11..Ber. deut. chem. Ges. 59, 1800 (1926). '
.,
(8) Izatt, R. &I., Fernelius, W. C., Block, B. P., J . Phys. Chem. 59, 235 ( 19551. (91 I z a h , R. M., Fernelius, W. C., Haas, C. G., Block, B. P., Zbid., 59,170(1955). (10) Izatt, R. &I., Haas, C. G . , Block, B. P., Fernelius, W. C., Zbid., 58, 1133 (1954). (11) Krishen, Anoop, Ph.D. thesis, University of Pittsburgh, Pittsburgh, Pa., 1957. (12) Krishen, h o o p , Freiser, Henry, ANAL.CHEX 29, 288 (1957). (13) McKaveney, J. P., Freiser, H., Zbid., 29, 290 (1957). (14) Morrison, G. H., Freiser, H., "Solvent Extraction in Analvtical Chemistry,,, Wiley, New York, "1957. (15) Nasanen, Reino, Lumme, Paavo, Mukula, A. L.. Acta Chem. Scand. 5, 1199 (1951). (16) Rydberg, Jan, Zbid., 4, 150 (1950). (17) Rydberg, Jan, Arkiv K e m i 8, 113 (1955).
(iSj z& d., 9, 95 (1955).
(19) Ry dberg, J a n , Svensk Kem. Tidskr. 67, 499 (1956). (20) Steinbach, J. F., Ph.D. thesis, University of Pittsburgh, Pittsburgh, (2
CHEhl. 25, 881 (1 (22) Ibid., 26, 375 (23) Van Uitert, L.,
vania State Colle,,, RECEIVEDfor review August 7, 1958. Accepted December 8, 1958.
Polychromatic Technique for the Identification of Amino Acids on Paper Chromatograms EDWARD D. MOFFAT' and RALPH 1. LMLE Division of Biochemistry, Naval Medical Research Unit No. 4, U. S. Naval Training Cenfer, Great lakes, 111.
A simplified polychromatic technique was developed to study the amino acid composition of protein hydrolyzates; the previous two-dimensional chromatographic systems were not entirely satisfactory, because of the time consumed and the inherent variables encountered in determining the reproducibility of Rl values. Twenty common, naturally occurring amino acids in amounts as low as 1.2 +y were completely differentiated after 6 hours of resolution when sprayed with ninhydrin-cupric nitrate (N-CN) and were readily identified by their characteristic color complexes. This method was equally satisfactory in the detection of single amino acids or mixtures on a unidimensional chromatogram in n-butyl alcohol-acetic acid-water (4: 1 :5 ratio) solvent system, although some possessed only slightly different R,'s-i.e., leucine-isoleucine. 926
ANALYTICAL CHEMISTRY
P
chromatographic techniques have contributed much toward simplifying the analysis of numerous multicomponent systems, such as mixtures of sugars, amino acids, and inorganic ions (4). Many methods for the identification of amino acids on a paper strip chromatogram are satisfactory. However, this report presents a simplified procedure that was developed in these laboratories. Investigations have recently been conducted to find a selective indicator to identify amino acids. Many indicators containing ninhydrin have been used, but with the exception of the yellow color of proline, they do not differentiate shades of red, violet, and purple. The excellent procedure of Hardy et al. was not applicable to the present problem because of its limited application (identification of 11 commonly occurring amino acids), such as the differentiation of leucine from isoAPER
leucine ( G ) . I n the quest for a polychromatic method for the identification of amino acids, released from a protein moiety, the following indicators were employed: 0.25% w./v. ninhydrin in acetone ( l 7 ) , 0.3% ninhydrin in 95% ethyl alcohol ( l a ) , 0.201, ninhydrin in water-saturated n-butyl alcohol ( 6 ) , 4% ninhydrin in pyridine (I@, and the method reported by Levy in 1953 ( 8 ) . S o n e of these reagents proved to be as selective t o color formation as the reagent described, which is a modification of the ninhydrin-copper complex method cniployed by Levy and Chung
(5). EXPERIMENTAL
TKO standard amino acid mixtures containing 10 amino acids in each were 1
Present address, Grove Laboratories,
I X ~ C8877 . ~ 1,adue Rd., Clayton 24, Mo.
prepared. Group I contained 50 lug. of the amino acid in 5 ml. of 0.28N hydrochloric acid. Group I1 contained 50 mg. of the amino acid in 5 ml. of 0.32N hydrochloric acid. Group I 1. Lysine 2. Aspartic acid
3. Glycine 4. Threonine 5 . Proline 1. 2. 3. 4. 5.
6. Valinc
7 . Trytophail 8. Phenylalanine 9. J,eucine
10. Isoleucine
Group I1 Glutamic acid 6. Arginine Alanine 7 . Cystine Tyrosine 8. Cysteine Histidine 9. Asparitginc Methionine 10. Serine
The ninhydrin-cupric nitrate indicator (N-CN) consisted of two solutions, I and 11, combined in a ratio of 25 to 1.5 just before using. Solution I contained 0.27, ninhydrin (anhydrous 1,2,3-triketohydrindene) in 50 ml. of absolute ethyl alcohol, 10 nil. of glacial acetic acid, and 2 ml. of 2,4,6-collidine. Solution I1 was a 1% solution of cupric nitrate trihydrate (analytical reagent llallinckrodt Chemical Works) in absolute alcohol. Ten microliter aliquots of thc individual amino acids as the hj-drochlorides in Groups 1 and 2 and a n equal mixture of the tn-0 groups u-ere placed on Whatman No. 1 filter paper. The strips were placed in a glass chromatographic chamber containing n-butyl alcohol-acetic acid water (4: 1: 5 ) as the solvent (11). Using descending chromatography the strips were developed for 6 to 7 hours, the time needed for the solvent front t o migrate 32 to 35 em. during each run. The strips \\ ere then removed, dried in a n oven for 5 niiiiutes a t 104" to 110" C., sprayed with the N-CN indicator, and placed in a n oven for 1.5 to 2 minutes a t 105' C. RESULTS
The resulting color formations on a white background arc uniquc for each amino acid studied, n-ith the cqc>ption of glutamic acid and alanine, which both yielded purplc spots, but of different intensity-i.e., alanine is much darker if equal concentrations are present. Arginine and valinc also form a purple spot, but thcir rates of flow are so dissimilar that identification is very readily achieved. Table I describes the color of thc amino acid spots formed after using this indicator, immcdiatcly upon removal from the drying oven. Illthin 10 to 15 minutes the bright blue ring of tryptophan disappears. K i t h fcw other exceptions, the remaining amino acids are color stable for about 30 minutes; then slight changes occur, such as a pink ring forming around lysine and serine. After these changcs in color occur and if the strips are kept in the dark, the color remains stable for 1 to 2 months, other than slight fading. The back-
Table 1.
Colors of Amino Acids after Use of N-CN Indicator"
Compound Cystine
Color Gray Reddish brown, pink ring forms upon standing Light brown p-ith dark brown ring, inside a yellow ring 2%ine Golden Asparagine Arginine Dark purple Greenish brown, red ring forms upon standing Serine Light blue (if removed from the oven too soon, aspartic acid \ d l be iispartic acid bright green) Orange brown with bright orange ring Glycine Greenish brown, changes t o purplish brov n upon standing Threonine Purple, fades slightly upon standing Glutamic acid Blanine Dark purple Light green mith yellow ring Proline Cysteine Gray Tyrosine Light brou-n Valine Purple Grayish purple n-ith yellow ring Methionine Brown a i t h bright blue ring (ring fades rapidly) Tryptophan Isoleucine Light blue Phenylalanine Greenish yellow Light purple with yellow ring Leucine a hmino acids listed in order of separation after resolution in n-butyl alcohol-acetic acid-m-ater (4: 1 : 5 ) ratio on Whatman KO. 1 filter paper. Table
II.
Evaluation of Chromatograms Developed with Different Solvents and Sprayed with the N-CN Color Reagent
Grading.
Solvent
1. n-Butyl alcohol-pyridine-xater, 3 : 2 : 1.5 (16) 2. n-Butyl alcohol-pyridine-water, 4 : 5 : 1 (9) 3. n-Butyl alcohol-water, 85: 15 (14) 4. %-Butyl alcohol-saturated n-ith 2 5 ",OW (7) 5 . n-Butyl alcohol-dioxane, 4:1, saturated nith n-atcr (3) 6. i 0 q ethyl alcohol ( 1 0 ) 7. Pyridine-water, 65 :35 (2). 8. in-Cresol saturated nith aater ( 5 ) 9. 2,4-Lutidine-collidine-water 1 : 1 :2 ( 1 ) 10. tert-Butyl alcohol-ethyl methyl ketone-dieth~-lam~i~e-~s-atcr, 40: 40: 20:4 (18) a Method of grading ranges from 1 to 4+ and is derived by comparison with cohol-acetie acid-water (4:1: 5 ) which is arbitrarily :issigned a 4+ value. b
3+ 2+ 4+ 2+ 3+ 3+ 2+ 1+ 3+ 2+b
butyl al-
Leaves dark background.
ground becomes a yelloir ish green after 5 or 6 days and the intensity of this color increases with time. The sensitivity of this indicator was determined by diluting the 1% standard amino acid solution to 0.50, 0.25, 0.13, 0.06, and 0.03%, placing from 2- to 10pl. aliquots of the various concentrations on a strip, and chromatographing them. Asparagine, proline, and phenylalanine could be detected by position alone with 2 111. of a 0.06% concentration. However, as a very faint yelloTT- color was produced which was not typical of the original 1% solutions, these amino acids could be identified by a characteristic color a t this level only with a n 8 p1. application of 0.06% concentration. The remaining amino acids could be identified b y color alone a t the 2 pl. t o 0.06% level, but below this concentration, the colors were atypical of the 1% solutions, fading to yellow as t h e concentration was decreased. The effect of developing solvents other than the n-butyl alcohol-acetic acidn-ater system on the selective color formation of the K-CN indicator was studied in the same manner. The results using various developing solvents were graded by comparing them with
the la-butyl alcohol-acetic acid-n-ater system (Table 11). DISCUSSION
The great advantage of paper chromatography lies in its simplicity and the relatively small expense of the equipment. Paper chromatography can be further simplified for the detection of amino acids b y the polychromatic method. This technique virtually eliminates the time-consuming two-dimensional chromatography for the study of complex mixtures and provides a sensitive indicator for the identification of amino acids in concentrations as low as 1.2 y. dlthough the quantities of amino acids required to give a visible spot on the chromatogram are dependent on a number of factors, the X-CK color reagent is equally satisfactory for the differentiation of 20 different amino acids either individually or in a mixture a t levels of 0.06%. Although as noted in Table I both cystine and cysteine appear as gray spots when sprayed with ninhydrincupric nitrate (N-CK), there is no difficulty in identifying them, because of the marked differences in rate of flow. Lysine, histidine, and asparagine VOL. 31, NO. 5, M A Y 1959
927
(and the other combinations with the one exception of cystine-cysteine) in a similar mixture of 20 amino acids appear frequently as homogeneous spots, when chromatographed by conventional techniques. However, this method indicates that there are sufficient differences in their R, values within a given spot to warrant the selective identification of each of the amino acids by their color complexes. This technique has been employed routinely in these laboratories for the identification of amino acids in hydrolyzates of the beta hemolytic streptococci fraction. It was of help in distinguishing between the troublesome leucine and isoleucine in amino acid mixtures. Maximum resolution is the most outstanding feature of two-dimensional chromatography. However, this degree of resolution is not required when the polychromatic procedure is employed, even though some of the amino acids possess relatively similar R, values. I n unidimensional chromatography lysine, histidine, and asparagine butyl alcohol-acetic acid-water, 4:1: 5 ratio), when sprayed with ninhydrin, appear as a homogeneous substance. Each amino acid, however, can be readily distinguished after a minimum of 6 hours of resolution when sprayed with N-CN. Other combinations of amino acids selected on the basis of the similarity of R, values, such as cystinecysteine, glycine-aspartic acid, arginine-
serine, threonine-glutamic acid-alanine, tyrosine-valine-methionine, tryptophanphenylalanine, and isoleucine-leucine also were studied to determine if they could likewise be detected after a minimum of 6 hours of resolution. These mixtures of amino acids would also appear as homogeneous mixtures and give singular spots when treated with the ninhydrin reagents. However, when treated as outlined, each amino acid could be characterized by its color complex. A single application provides a far superior chromatogram than using t F o sprays to effect only semipolychromatic results. Perhaps the most outstanding advantage of this method is that the interfering factors such as abnormalities occurring as a result of ionizable substances, cation concentration, the rate of movement of solvent, and temperature, are of no major significance; in two-dimensional chromatography they would be of considerable importance in the reproducibility of RI values for the identification of amino acids.
(2) Bentley, H. R., Whitehead, J. K., Bzochem. J . 46, 341 (1950). (3) Block R. J., Bolling, Diana, “Amino Acid ,$omposition of Proteins and Foods, p. 576, Charles C Thomas, Springfield, Ill., 1951. (4) Block, R. J., Durrum, E. L., Zweig, Gunter, “Manual of Paper Chromatog-
raphy and Paper Electrophoresis,” Academic Press, New York, 1955. (5) Consden, R., Gordon, A. H., Martin, A. J. P., Biochem. J . 38,224 (1944). (6) Hardy, T. L., Holland, D. O., Nayler, J. H. C., ANAL.CHEM.27,971 (1955). (7) Hird, F. J. R., Trikojus, V. M., Australian J . Sci. 10,185 (1948). (8) Levy, A. L., Chung, D., ANAL. CHEM.25,396 (1953). Miettinen, J. K.. Suomen Kemistilehti (9) ~,
26,49 (1953). ’ (10) Miller, H., Kraemer, D. M., ANAL. CHEM.24,1571 (1952). (11) Partridge, S. M., Biochem. J . 42, 238 (1948). (12) Patton, A. R., Chism, P., AKAL. CHEM.23,1683 (1951). (13) Redfield, R. R., Biochim. et Biophys. Acta 10, 344 (1953). (14) Redfield, R. R., Barron, E. S. G., Arch. Biochem. Biophys. 35, 443 (1952). (15) Rockland, L. B., Blatt, J. L., Dunn, M. S., ANAL.CHEM.23, 1142 (1951). (16) Smith, P. B., Pollard, A. L., J . Bacteriol. 63, 129 (1952). (17) Toennies, G., Kolb, J. J., ANAL. CHEM.23,723 (1951).
ACKNOWLEDGMENT
The authors express their appreciation to Jack H. Davis and Irwin Schultz, M C USNR, for helpful suggestions in the preparation of this manuscript. LITERATURE CITED
(1) Auclair, J. L., Dubreuil, Robert, Can. J . Zool. 30, 109 (1952).
RECEIVEDfor review April 9, 1958. Accepted November 14, 1958. From Research Project NM 52 06 04.4, Bureau of Medicine and Surgery, Navy Department, Washington 25, D. C. The views and opinions expressed herein are those of the authors and do not necessarily reflect those of the Navy Department or the Naval Service a t large.
Titrimetric Determination of AI kyl Mercaptan-Dial kyl Sulfide and AI kyl Mercaptan-AI kyl Disulfide Mixtures BRUNO JASELSKIS Department o f Chemistry, University o f Michigan, Ann Arbor, Mich.
Mixtures of n-alkyl mercaptans and dialkyl sulfides can b e determined titrimetrically using two aliquots. The first aliquot is titrated for mercaptan with iodine, and the second, after treatment with basic acrylonitrile, is acidified and titrated with bromatebromide solution. The amount of dialkyl sulfide is found by the difference. The results agree to better than 1%. Mixtures of n-alkyl mercaptans and alkyl disulfides are determined iodimetrically. Mercaptan alone is titrated first, and then the total mercaptan is titrated after the reduction of alkyl disulfide with zinc in an acetic-hydrochloric acid-alcohol mixture. Alkyl
928
ANALYTICAL CHEMISTRY
disulfides with higher molecular weights than propyl disulfide are recovered quantitatively.
A
for determination of mercaptans (thi01s) have been developed using iodine ( 7 ) or silver nitrate (3, 8, 11). The determination of alkyl sulfides by bromate-bromide standard solutions has been described b y Siggia and Edsberg (10). However, samples containing a mixture of alkyl sulfide and alkyl mercaptan in excess of 10% cannot be analyzed satisfactorily, because of the slow and incomplete oxidation of CCURATE TITRIMETRIC METHODS
mercaptan and alkyl sulfide Kith bromine and the interference of alcohol in exhaustive oxidation. This difficulty can be overcome by using the addition reaction of acrylonitrile to alkyl mercaptan to yield sulfide, which is readily oxidized to sulfoxide. The reactions of acrylonitrile and alkyl mercaptans have been studied b y Earle (2) and Hurd and Gershbein (4, and the determination of acrylonitrile and alpha-beta-unsaturated compounds has been described b y Beesing et al. ( 1 ) . Mixtures of alkyl mercaptans and alkyl disulfides in the absence of alcohol have been determined by bromine oxidation as described by Siggia and Eds-
