1501
V O L U M E 2 7 , NO. 9, S E P T E M B E R 1 9 5 5 Table 11.
Analyses for Carbon in Macro Range' Using Revised Measuring Unit Carbon Recommended
Sample CaCOa
Carbon Found,
%
%
12.00
h-BS steel 82. 8 samples
2.78
NBS steel 132, 6 samples
0.803
Average
11.97 12.08 11.99, 12.04 12.02 12.05 12.14 11.87 11.98 12.00 1 2 . 0 1 =t0 . 0 5
-4verape
2.79 i0.09
Average
0 . 7 9 3 i 0.005
dioxide were involved, but all permitted varying degrees of loss. The glass wool not only served as a filter to retain the crystals but it also substantially increased the cold surface on which the condensation could occur. Table I1 shows the results obtained on materials containing carbon in the macro range, using the modified measuring unit. ACKNOWLEDGMENT
The author wishes to acknowledge the valuable assistance supplied by W.G. Smiley of the Los Alamos Scientific Laboratory in this application of the capillary trap. Private correspondence and a visit to his laboratory yielded many helpful suggestions. LITERATURE CITED
gave complete recoveries and no carbon dioxide was found to escape into the secondary unit. Other designs tried for the cold trap before adopting the U-bend containing glass wool were a capillary helix coil, a multiple loop coil, and a U-bend containing a fritted-glass filter. These were all improvements over the plain capillary U-bend when large amounts (over 200 y) of carbon
(1) Bennet, E. L., Harley, J. H., and Fowler, R. AI., ANAL. C H E M . , 22, 445 (1950). ( 2 ) Fowler, R. hl., Guldner, W. G.. Bryson, T. C., Hague, J. L., and Schmitt, H. J., Ibid.. 22, 486 (1950). (3) Pepkowitz, L. P., and Rloak, W.D., Ibid., 26, 1022 (1954). (4) Smiley. 1%'. G., Ibid., in pres*. (5) Wooten, L. A., and Guldner, W.G.. IND. EXG.CHEX, - h . t r , . ED., 14, 835 (1942).
RECEIVED for review November
2 , 1954. Accepted April 1, 1955. Based o n urork performed under t h e a u p i c e s of U. S. Atomic Energy Commission.
Detection of Amino Acids on Paper Chromatograms J. A. ClFONELLl and FRED SMITH Department
of Agricultural Biochemistry, University of Minnesota, St. Pad, M i n n .
Amino acids and other amino compounds after being oxidized by periodate may be detected on paper chromatograms by benzidine and by starch-iodide reagents. These methods have enabled certain of the amino acids to be distinguished. Mixtures of tert-amyl alcoholI-propanol-water have been found useful as irrigating solvents for separating amino acids.
N
INHYDRIN is a satisfactory reagent for detecting amino acids on paper chromatograms (5) and the more recent method involving chlorination followed by a starch-iodide spray makes it possible to detect proteins as well as amino acids ( 2 , 8). However, the identification of the amino acids in a mixture still presents considerable difficulty. Since no single solvent mixture permits the separation and identification of all the amino acids on a single one-dimensional chromatogram, recourse has been had to the use of two dimensional chromatography ( S ) , and of specific tests. Thus diazo reagents have been employed for detecting tyrosine and histidine ( 4 , 5, 9); p-aminobenzaldehyde for glucosamine ( 7 ) ; and sodium azide ( I ) , iodoplatinate ( I I ) , and nitroprusside (10) for locating sulfur-contnining amino acids. In an attempt to simplify the identification of amino acids on paper chromatograms the authors have found that certain periodate sprays with or without a subsequent benzidine spray are useful for detecting certain amino acids and groups of amino acids. These new reagents, which have already proved valuable in the paper chromatographic analysis of carbohydrates ( 2 ) are also valuable for the identification of the difficultly separable amino acids such as glycine and serine, threonine and alanine, and valine and methionine on one-dimensional chromatograms. EXPERIMENTAL
Solvents for Irrigating the Chromatograms. >fixtures containing tert-amyl alcohol and 1-propanol were found to be useful
as a second solvent following phenol in two-dimensional chromatographic separation of amino acids. Such solvent mixtures can also be used for one-dimensional chromatograms (eee Table
11). REAGENTS
Benzidine Reagent A. Mix equal volumes of 0.1M benzidine in ethyl alcohol and 0.8N hydrochloric acid. Benzidine Reagent B. Mix 10 volumes of 0.1V benzidine in 50% ethyl alcohol with 1 volume of 0.2N hydrochloric acid and add 2 to 3 volumes of acetone t o dissolve the benzidine hydrochloride. Neutral Periodate. Saturated aqueous solution of potassium metaperiodate. hlkaline Sodium Iodide. A 4% aqueous sodium iodide solution saturated with sodium bicarbonate. Starch, 0.1% aqueous solution. Sodium Iodide, 5 % aqueous solution. Glycine, 5% aqueous solution. PROCEDURE AND R E S U L T S
Treatment of Chromatograms with Periodate. The chromatograms were freed from volatile solvents by allowing them t o stand in air. When phenol was used it was removed by extraction with either ether or acetone-ether. The papers were then sprayed with the saturated aqueous potassium metaperiodate solution and allowed to stand for 1 to 2 minutes. Procedure 1. Detection of Amino Acids with Benzidine Reagent A. When the periodate treated paper is sprayed with Reagent A it gives within 1 minute a blue color with glycine and a yellow color with methionine on an almost colorless background. With insufficient hydrochloric acid in the reagent, the background will turn blue, while too much acid reduces the sensitivity of the reagent. Maximum sensitivity for all reactive amino acids is obtained by spraying the chromatogram lightly with Reagent A and, after the blue spots appear, respraying with Reagent A; this inteneifies the spots due to the sulfur-containing amino acids and tryptophan. The blue spots generally fade under this treatment, but
1592
ANALYTICAL CHEMISTRY
they can usually he made to reappear by respraying the chromatogram with potassium metaperiodate. The more basic amino acids appear as blue spots, while the sulfur-containing amino acids and tryptophan appear yellow or yellow-brown (see Table I group 1.4).
Table I.
Detection of Amino Compounds on Paper Chromatogramsa Groiin I Group Group IAby I B by Procedure Procedure 1 2 Blue Blue YellowC ColorlessC -1
Compound Alanine Arginine b Cysteineb Cysteic acid Cystine Yellow Glucosamineb Blue Glycine Blue Blue Histidine b Hydroxyproline Blue Lysine Blue Yellod Methionine Serine Threonine YellowTryptophan 6
___
hrnwn -
Colorless Colorless
Colorless Colorless Colorless Colorless
Group I1 Procedure 3Before After starch 5taicli
Grouri 111 Procedure 4
Light brown Dark brown Dark brown Dark brown
Blue Blue Blue Blue
Colorless Colorless Colorless Colorless
Dark brown
Blue
Colorless Colorless Colorless Colorless Colorlea8
Valine Blue a Blank spaces in table indicate compound not detectable. b Applied as hydrochloride. c Colorless spot detected b y Procedure 2 corresponds in R / value t o colorless area within yellow trailing spot produced by Procedure 1. d Colorless center can generally be detected within yellow ring. ' Dissolved in 0.1N HC1.
Cysteine hydrochloride gives a long, trailing, rapidly darkening yellow spot usually containing a colorless area when the chromatogram is developed with such solvents as phenol saturated with water, tert-amyl alcohol-acetic acid-water, tert-amyl alcohol1-propanol-water or methyl Cellosolve-water. The colorless area is in the center of the yellow trailing spot when tert-amyl alcohol-1-propanol-water is used as the irrigating solvent, but when phenol saturated with water is used the colorless area is a t the lower edge of the trailing $pot. The colorless area is best seen if the chromatogram is given an additional spraying with glycine solution, since this produces a blue background (see later). It is possible to detect the amino acids of groups IA and I B on the same chromatogram by spraying the periodate treated chromatogram first with the benzidine Reagent A, to detect the amino acids in group IA, rand then, within 5 minutes, with the 5% glycine solution whereupon colorless spots due to the amino acids of groups I B soon rappear on a blue background. I n this manner, pairs of overlapping spots due to amino acids of group IA and I B such as glycine-serine, alanine-threonine, and valine-methionine can be differentiated. Concentrations of 10 y of amino acids were detected in this way on one-dimensional chromatograms that had been irrigated with methyl Cellosolvewater (9 to 1). Procedure 2. Detection of Amino Acids with Benzidine Reagent B. The periodate treated paper is sprayed lightly with reagent B. Colorless spots on a blue background appear a t the location of a-hydroxyamino acids, glucosamine, tryptophan, cysteine, and methionine (see Table I, group IB). h o c e d u r e 3. Detection of Amino Acids with Neutral Periodate and Sodium Iodide. The chromatogram, after being lightly sprayed with a saturated solution of potassium metaperiodate, is warmed for 20 to 30 minutes a t 50" to 60' C. Upon spraying with 5'70 aqueous sodium iodide the amino acids are located by the formation of brown spots which turn blue when the paper is sprayed with starch solution (see Group 11, Table I ) (6). The warming of the chromatogram prior to spraying with sodium iodide appearg to be necessary to produce maximum color stability of the spots. Cysteic acid may be distinguished from other amino acids by the formation of a blue color upon spraying the periodate treated chromatograms with the starch solution
Procedure 4. Detection of Amino Acids with Alkaline Periodate and Sodium Iodide. After the irrigating solvent has been removed, the chromatogram is sprayed with the sodirim iodidesodium bicarbonate solution and then with starch. After drying in the air, the paper is light'ly sprayed with potassium metaperiodate. Cystine, histidine, and cysteic acid are revealed by thr immediate formation of brown spots (the color disappears with excessive spraying). .4fter a fe\v minutes a number of the other amino compounds listed in Table I appear as colorless spots on a blue background. With this reagent reducing sugars will also form colorless spots ( 2 ) ,hut they may be distinguished from the colorless spots due to
amino compounds by the fact that they turn brown on being heated for 15 to 20 minutes a t 110" C. whereas the colorless spots due to thP amino acids remain unchanged. In this test tryptophan develops a slight color (Table I. group 111).
'Table 11. R f Values of Amino Compounds Using Mixtures of tert-Amyl Alcohol (A), 1-Propanol (B), and Water (C). R/ Values Using Solvent Mixture .4, 4; B , 1: -4 4 . B 0.8: Compound C , 1 vol. 6, i.s h i . .$lanine 0 24 0 26 hrginineb 0 os 0 13 Aspartic acid 0 04 0 11 Cysteic acid 0 10 0 12 Cysteine b 0 27= Cystine 0 04 0 os Glucosamine b 0 23 0 27 Glycine 0 16 0 20 Histidineb 0 10 0 14 0 24 Hydroxyproline 0 24 0 62 Leucine 0 57 0 06 Lysine 0 10 Methionine 0 46 0 43 Proline 0 27 0 31 Serine 0 16 0 20 n 21 Threonine 0 24 0 44 Tryptophand 0 42 0 43 Valine 0 38 a Ascending technique u s i d ; coinpounds detected by ninhydrin unless vtated otherwise. Applied as hydrochloride. Cysteine hydrochloride not easily detected by ninhydrin. H f vgue refers t o colorless spot detected by Procedure 1 followed bv treatment mth 5% glycine. .i 1 % solution in 0.1.V HC1.
Since this work was completed, a similar procedure using periodate followed by a starch-iodide buffered spray reagent has been published for detecting periodate oxidizable compounds including amino acids (6). The above procedures have enabled the authors to show that in the hydrolyzate of a purified preparation of yeast invertase the following amino acids are present: alanine, aspartic acid, cysteic acid, glycine, glutamic acid, leucine, lysine, serine, threonine, tyrosine, valine, and one as yet unidentified. LITERATURE C I T E D
Chargaff, E., Levine, C., and Green, C., J . Bid. Chem., 175, 67 (1948). Cifonelli, J. A., and Smith, F., .%XAL. CHEM.,26, 1132 (1954). Consden, R., Gordon, A. H., and Martin, A. J. P., Riochem. J., 38, 224 (1944). Dent. C. E.. Ibid.. 41. 240 (1947). DeVay, J. E., Chang, 'W. H., and Hossfeld, R. L., J . Am. Chem. SOC..73. 4977 (1981). Mitchell, H. K., and ketmenberg, 11. L., Ibid., 76, 4187 (1954). Partridge, S. M., Biochem. J., 42, 238 (1948). Rydon, H. N., and Smith, P. W. G.. Yuture, 169, 922 (1952). Sanger, F., and Tuppy, H.. Riochevz. ,J., 49, 463 (1951). Toennies, G.. and Kolb, J. J., . % s k i , . CHEM..23; 823 (1951). Winepard, H. M., Toennies, G., and Block. R. J.. Science, 108. 506(1948).
RECEIVED for review October 18, 1954. Accepted April 19, 1955. This paper (No. 3230, Scientific Journal Series, hlinnesota Agricultural Experiment Station) forms part of a thesis submitted by J. A. Cifonelli t o the graduate school of t h e University of Minnesota in partial fulfillment of t h e requirements for t h e Ph.D. degree. 1952.
