V O L U M E 28, N O . 4, A P R I L 1 9 5 6
501
in the solid state. As stated previously, 8-quinolinol begins to eublime a t 85’ C. However, when held as a molecule of solvation, it sublimes a t 230” C. The results seem to indicate that the lattice forces holding this extra molecule of 8-quinolinol must be of a greater order of magnitude than 1 kcal. per mole. The normal chelate was stable up to 380” C., where it decomposed to the oxide level, U30a, beginning a t 450” C. This is certainly in disagreement with Duvnl (6) who did not obtain an oxide level even at 9 4 i ” C. Although it is possible to prepare the normal chelate of uranium and 8-quinolinol by thermal decomposition, it is not possible to remove the extra solvate molecule of the 5,7-dihalo-&quinolinol chelates by heating. There was no evidence from the pyrolysis curves for the existence of the normal chelates in this series of compounds. The 5,7-dihalo chelates were even less stable thermally than the 8-quinolinol chelate; thus, there is no advantage in using them for the determination of uranium. It is difficult to prevent coprecipitation in the chelates, as is shown by the decomposition of the uranium 5,7-dibromo-8-quinolinol chelate in Figure 3. The excess reagent began to sublime a t about 200’ C. going directly to the chelate, U01(CPH1Br2K0)2.C oH4Br2NOH.
LITERATURE CITED
(1) Berg, R., “Die Analytische Verwenclung von o-Oxychinolin (‘Oxin’)und seiner Derivate,” 2nd ed., F. Enke, Stuttgart, 1938. (2) Borrell, II.,Pbris, R., And. Chim. Acta 4, 267 (1950). (3) Classen, A , , Visser, J., Rec. trau. chim. 6 5 , 211 (1946). (4) Dupuis, T., D u d , C., Anal. Chiin. Acta 3, 589 (1940). (5) Duval, C., Ibid., 3, 335 (1949). (6) Fleck, H. R., Analyst 62, 378 (1937). (7) Frere, J. F., J. Am. Chetn. SOC.55, 43653 (1933). (S) Got& H., Sci. Repts. TBhoku Imp. Univ., First Ser. 26, 391 (1937). (9) Hecht, F.. Ehrmann, FV., 2. anal. Chem. 100, 98 (1935): (10) Hecht, F., Reich-Rohrwig, TI7., illonatsh. 53, 596 (1929). (11) Moeller, T., Ramaniah, JI. IT., J . Am. Chem. SOC.75, 3946 (1953). (12) Zbid., 76, 2022 (1954). (13) Ibid., p. 5251. (14) Ibid., p. 6030. (15) Moeller, T., II‘ilkins, D. H., Inorg. Suntheses 4, 2G7 (1953). 23, 757 (1951). (16) Pokras, L., Bernays, P. bl., ANAL.CHEM. (17) Pokras, L., Bernays, P. AI., J . Am. Chein. SOC.73, 7 (1951). (18) Pokras, L., Kilpatrick, hl., Bernays, P. M., Ibid., 75, 1254 (1953). (19) Wendlandt, W. FV., ANAL.CHEM.27, 1277 (1955).
.
RECEIVED for review November 15, 1955.
Accepted December 30, 1955
Color Reaction of Amino Acids with Alloxan, Isatin, and Ninhydrin in Circular Paper Chromatograpby ABRAHAM SAIFER and IRWIN ORESKES Isaac Albert Research Institute and Department
of Physical Chemistry, Jewish Chronic Disease Hospital, Brooklyn 3, N. Y.
Color reactions of 51 amino acids and their sensitivities were determined as spot tests on filter paper with alloxan, isatin, and ninhydrin. R f values for these amino acids were obtained with the circular chromatographic method using phenol- and butanol-acetic acid as developing solvents. Alloxan gave stable and uniform colors for most amino acids and was a more sensitive reagent than ninhydrin for some of them. For aniino acids with similar R , values, simultaneous use of these reagents applied to sectors from one chroniatograni often permits the ‘‘differential” determination of a single amino acid in a mixture. Isatin is the most useful for this purpose because of the wide variety of its color reactions and lack of reactivity with many amino acids. The nature of these color reactions for amino acids and their possible application to amino acid niixtures are discussed.
T
WO general approaches have been tried for the complete
qualitative identification of amino acids in complex mixtures using paper chromatography. The first approach aims a t complete resolution of amino acids into bands or spots containing but one component. A single, generally applicable coloring reagent would then suffice for the identification of all the amino acids present. However, complete resolution is difficult to achieve in practice. An alternative approach is the use of specific or selective coloring reagents to identify a single amino acid in a band or spot containing several components. Attempts to obtain more complete separation of the individual amino acids in a mixture have led to such developments as: ( a ) increased path of travel of components by using larger paper sheets (48) or ascending-descending chromatography (6, 48); ( b ) the use of buffered filter paper (26, 30); (c) the use of multiple solvent sys-
tems (14, 19, 21, 30, 37-39); (d) multiple development techniques (11, 23); and ( e ) elution from one chromatogram followed by rerunning the mixture on a second chromatogram employing a different solvent system (IS). It is the opinion of Block, Durrum, and Zweig (6) that many of these inovations represent no major improvement over the two-dimensional, two-solvent technique originally described in the classical paper by Consden, Gordon, and Martin (9). Indeed, many of these modifications have made the procedure more costly, complex, and laborious. One exception to this general trend appears to be the increasing popularity of the circular chromatographic method proposed by Brown (8) and Rutter (41, 42). In this method the substances to be analyzed are resolved into circular bands instead of spots. Recent work by LeStrange (25), as well as previous studies by hluller and Clcgg (SS), have confirmed the simplicity of the method, as well as the facts that separation occurs more r a p idly, and the definition between components is superior to that obtained with the descending technique. Careful experimental studies of some of the physical factors which influence circular paper chromatography were made by Saifer and Oreskes (43). These studies have led Block and others (6) to conclude that circular paper chromatography is a more rapid and a simpler method than the more conventional techniques. In addition to the advantages stated above, circular paper chromatography lends itself most readily t o the use of several different color reagents, because a large number of sectors can be cut from a single chromatogram (41, 4 2 ) . Such an approach permits the identification of the separate amino acids even where complrte resolution has not occurred (17). The use of such diffeIentia1 reagents which give colors with one or several amino acids has often been cited in the literature (4, 6, 17, 18, 48). Although ninhydrin comes closest to the ideal coloring reagent because of its reactivity and sensitivity for most amino acids, even it has certain drawbacks, such as the faint yellow colors obtained with proline and hydroxyproline (9, BS). For
502
ANALYTICAL CHEMISTRY
this reason isatin was proposed as an additional color reagent, because it gives intense bluish colors with these two imino acids (a). Subsequent studies (44) showed that a number of other amino acids also react with this reagent, thus enabling it to be used for their identification. In a recent publication (34) a scheme was proposed for the complete identification of the amino acids of protein hydrolyzates, except for separation of the leucines. This system employs two solvents (phenol-water and butanol-acetic acid) and two coloring reagents (ninhydrin and isatin). The’present paper deals with an extension of this system to other amino acids besides those previously discussed (34). It also makes use of alloxan as a useful reagent for amino acids in paper chromatography to supplement the ninhydrin and isatin reactions. Although the reactivity of alloxan with amino acids in general has been noted in the literature (1, 10, 82, 27), only one previous application to the field of paper chromatography of amino acids and related compounds was found (40). I t is believed that the data presented here will be useful in identifying the less common amino acids such as may be found in urine, plant and tissue extracts, protein hydrolyzate?, and the like (7, 16, 52).
EXPERIMENTAL
Reagents. NINHYDRIN, reagent grade, 0.25% in reagent grade acetone. This reagent remains stable over a period of several months. ISATIN, reagent grade, 0.2% in reagent grade acetone containing 4% acetic acid. This reagent is also stable for several months. A slight precipitate n-hich forms occasionally should be filtered off prior to use. ALLOXAX,reagent grade, 0.25% in reagent grade acetone. When the solution is first made, any undissolved material should be filtered off. This reagent is relatively unstable; it should be made fresh meekly and kept refrigerated. ISOPROPYL ALCOHOL, reagent grade. Procedure for Determining Reactivity and Sensitivity of Various Amino Acids. Solutions of 5 mmoles per liter of the various amino acids shown in Table I were prepared by disso!ving the pure amino acid in the appropriate volume of 10% isopropyl alcohol. Whatman No. 1 filter paper was used, and 20 cu. mm. of each amino acid were spread uniformly over an area within a penciled circle 1 em. in diameter. This operation Tvas performed on three separate sheets of filter paper, one for each coloring reagent to be tested. Each sheet was dipped into one of the coloring reagents, air dried, and then heated a t about 100” C. for 10 minutes. If the reaction mas positive, the above procedure was repeated with smaller aliquots of the same solution-e&, 15, 10, and 5 cu. mm. At this point, if the reaction was still positive, a 1 t o 5 dilution of the amino acid was made, and the procedure again repeated. This was continued until no perceptible reaction was noted. Table I. Circular Paper Chromatographic R / Values The next higher concentration was then taken as the [Phenol-water (0.1% ammonia) and butanol-acetic acid-water (40 to 10 to EO) used as developins solvents. Minimum detectable quantities glven for various amino acids with ninhydrin, isatin, and alloxan color reactions Perlimit of sensitivity for the formed on Whatman No. 1 paper.] particular amino acid with Eensitivity Limits R/ X 100 each coloring reagent. These .___ Alloxan Phenol-Hx0 ButanolNinhydrin Isatin values, as well as the colors Amino Adda 0.1% NH,’ HAC-H~O (>olor y Color y Color o b t a i n e d , a r e g i v e n in Table I. 0.4 P L 5 1. Alanine 64 40 0.2 Pll. 0.4 R-P N.R. 48 0.4 P11. 2. .&Alanine 72 D e t e r m i n a t i o n of Rf 0.4 P 0.1 B 75 50 3. u-Aminobutyric acid 0.1 P11. Values by Circular Paper 0 . 5 R-P 0.5 Pu. 84 . 54 4. 7-Aminobutyric acid 0.5 Ll,. Pu. Chromatography. The Rj 0.2 P B 10 58 2.6 PU. 5. 8-Aminoisobutyric acid 82 1.0 P 2.0 L 25 0.2 Pu. 86 0. Arginine values for the various amino 1 . 3 R-P 24 0.7 Gy.-Pu. N.R. 7. Asparagine 50 acids listed in Table I were B-G 0.7 P 3 0.3 P 1. 24 31 8. Aspartic acid determined with the circular 0.4 P L 13 22 0.3 P 1. 71 9. Canavanine 0.7 P B 20 B 88 17 Carnosine 5.7 chromatographic technique, 10. 0.8 P 3.5 L 0.9 P u . 70 25 11. Citrulline using phenol-water (0.1% 0.2 P 1.9 B-Gy. 0.4 Pu. 15 12. Cystathionine 36 ammonia) and butanol-acetic 0.8 P 0.8 L 15 18 0.6 L.Pu. 13. Cysteic acid 0.8 T-P 3.6 B-G 13 14. Cysteine 39 2.7 Lt. T acid-water (40 to 10 to 50) 1.2 B-Gy. b 0.8 P-Pu. 18 18 0.5 B-Pu. 15. Cyatine , as the developing solvents, 0.3 Br. 0.2 Br. 35 16. 3 4-Dihydroxyphenylalanine 38 4.9 Br. exactly as described in pre2,2 T-P 2.0 G 70 65 2.0 GY. 17. 3:5-Diiodotyrosine 0.8 T 2.5 T 14 47 1.3 T vious publications (34, 4 3 ) . 18. Djenkolic acid 0.2 P 1.6 B-G 65 0.2 k.-Pu. 19. Ethionine 87 The Ry values, as shown in 0.8 P 4.0 L 0.3 FU. 36 20. Glutamic acid 35 T a b l e I, r e p r e s e n t t h e 3.0 L t . P N.R. 35 21. Glutamine 33 1.5 E U. 4 P Gy.-T 21 5 20 0.1 F u. 22. Glutathione (oxidized) average values for a t least six 1.4 P 6.2 Pu. 12 23. Glutathione (reduced) 17’ 1.5 E’U. separate runs for each de0.4 P Lt. L 4 31 24. Glycine 50 0.2 I’U. veloping solvent. I n most 1.0 P 23 2.0 B-Gy. 80 0.4 c:y.-pu. 25. Histidine 0.3 P 1.0 B-G 44 24 26. Homocysteine 0.3 ’Ir-Pu. instances ninhydrin was used P 0.2 P 3.8 46 I9 15.0 f : U . 27. Homocysteine thiolactone as the coloring reagent t o 0.8 P P 10 64 0.04 1 u. 38 28. Homoserine show the exact location of 0.4 P-L 16 0.8 29. Hydroxylysine 70 0.2 I’U. N.R. 1.3 33 0.6 ‘r 30. Hydroxyproline 71 the circular band in relation 0.7 P L 10 86 73 0.1 I’U. 31. Isoleucine to the solvent front. The R, 0.2 Br. 14 0.4 T 35 32. Lanthionine 0.2 I h . values for each chromato0.7 P L 10 86 73 0.1 I’U. 33. Leucine 0.5 P 2.0 Br. 22 86 gram were calculated in the 34. Lysine 0.1 l’u. 0.8 P 0.7 B-G 55 80 35. Methionine 0.1 :?U. manner previously described 0.4 P 4 35 0.08 3 1 . 70 36. Methionine sulfone GY. (43). I n other cases, where 0.4 P 4 B-G 36 82 0.08 PU. 37. Methionine. sulf oxide 0.6 P 2.0 B-G 28 92 38. Methylhistidine 0.04 IGY.-PU. ninhydrin was less sensitive 0.7 P 2.0 B 0.1 ?U* 76 87 39. Norleucine or reacted poorly, alloxan or 0.6 B 83 71 2.0 B 0.1 eu. 40. Norvaline isatin was used. I n every 0.6 P 0.3 Pu. 19 83 41. Ornithine 0.3 Pu. 0.8 P 0.7 66 B-G 86 0.7 Pu. 42. Phenylalanine case care was taken to reP 10 0.6 Y-P 77 57 43. Phenylserine 0.04 L move even faint traces of the N.R. 92 44 0.2 B 44. Proline 0.5 Y developing solvents, either 31 3 0.5 P Lt. L 0.2 Pu. 43 45. Eerine 0.6 P 23 52 0.6 Lt. Pu. N.R. 46. Taurine by acetone washing in the 0.4 P 25 1.9 B-Gy. 47. Thiolhistidine ’ 37 0.8 Y-Br. case of phenol, or by air dry0.6 P Lt. L 36 55 3 0.1 Pu. 48. Threonine ing with a stream of warm 1.0 P 47 0.8 B-G 49. Tryptophan 74 0.2 Pu. 0.9 B-G 2.0 T 45 64 0.2 Br. 50. Tyrosine air in the case of the butanol10 0.6 P L 62 80 51. Valine 0.1 Pu. acetic acid. This was necessary to prevent any interferLegend for Table: B = Blue. Br. = Brown. D = Dark. G = Green; Gy. = Gray; L = Lavender; Lt. = Light; P Pink; Pu. = Puiple; R Red: T = Tan; k = Yellow; N.R. = No reaction. ence with thesubsequent color reactions and to make shades a The following substances were tested and gave weak or no reactions with all three color reagents: sarcosine of color obtained on the (alloxan faint reaction) betaine guanidine and glycocyamine. actual chromatogram comb This’ value for cystihe is sodewhat low& than t h a t previously reportzd with isatin (44). c This material gave rather diffuse bands with this solvent, E O that the Rf value is only an approximation. parable with those obtained with the spotting technique. C
PGy.
-
-
503
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 RESULTS
In agreement with Rao and Giri (36) it \.\’asfound that butanolacetic acid-mater is a most effective solvent for the well-defined separation of amino acids. It was also confirmed that circular paper chromatography gives somelyhat higher R , values with this solvent than are obtained by other techniques (46). While there are some differences between the R, values for this solvent as reported in Table I and those found by Rao and Giii (36), the sequence of the various amino acids is viitually the same. The widely used phenol-water developing solvent gives more diffuse bands than dors the butanol-acetic acid-water system (56). However, phenol-water is most useful because, in the presence of 0.1% ammonia as an additional phase, it separates into pure bands those commonly occurring amino acids not resolved by the butanol solvent (34). The E , values given in Table I for phenol are in bettei agreement with those reported (0)for the descending trchnique, than for the circular method (36). I t has been reported (9) that the use of an ammonia atmosphere in the phcnol runs results in an improved separation of the basic and acidic amino acids. This would explain the wide divergencc bctveen the results for these amino acids reported by Rao and Giri (36) and in the present study. Rao and Giri (36) and other investigators (0, 55) have tended to minimize the importance of exact R j values and consider them as a “mere indication of the relative positions of the acids with respect to each other.” Admitting that the order of separation of amino acids is of prime importance, i t has been demonstrated that thr circular chromatographic method, as modified for a closed system, permits one to obtain aveiage Rj values nhich are reproducible to about k 0 . 0 2 unit (34). This makes such R, data useful for identification purposes. Although slight differences in these R, values may be obtained from one laboratory to another because of differences in paper, reagents, equipment, and temperature, consistent Rj values within the experimental error can be obtained in any one laboratory under controlled experiniental conditions. One might expect to obtain separations of aniino acid mistures whose R, values differ by *0.04 unit for a particular solvent. However, this cannot be predicted in advance without experimental trial, because other substances present may exert an effect on the mobility of a compound (41). In certain instances i t has been found possible to identify amino acids whose l2, values differ by only 0.01 or 0.02 unit, as in the case of lysine, arginine, and histidine (34). Identification is based on their distinctive color reactions n i t h isatin and ninhydrin, rather than 011 the use of a multiplicity of solvents which increase the complexity of the method. Such identification is also obtained in the case of overlapping amino acids where one of the mixtures gives a distinctive color reaction with a differential reagent (44). The limits of sensitivity of the ninhydrin reaction for some amino acids using tm-0-dimensional chromatography has been reported by Dent (I$), Auclair (S), and Berry ( 4 ) . The results obtained in the present study with this reagent, as shown in Table I, compare most closely 75-ith those obtained by the latter t v o authors. I n general, the results tend to be slightly lower than those reported by Auclair (3). However, in the case of certain amino acids such as arginine, tryptophan, threonine, etc., which are partially destroyed by phenol, either during the developing or drying process (14, 15), considerably loner values nere obtained. This is due to the fact that sensitivity limits were determined as spot tests on filter paper and not on chromatograms. The useful sensitivity limits on actual chromatograms would probably be about two to three times greater than those reported in Table I. In the case of circular paper chromatography, larger amounts of the amino acids are required for visual detection with incremhg R, values. T h k in turn is dependent on the solvent system used. It should, perhaps, again be stressed that successful application of the color reactions described here to actual chromatograms requires complete
removal of phenol by washing with organic solvents prior to the heating step (14, 16). This is most readily performed where small circles, or strips, of filter paper are used for the chromatogr :tphy . I n general, ninhydrin is the most sensitive of the three amino acid reagents investigated. It gives purple or bluish colors with almost all amino acids except proline and hydroxyproline (yellow); 3,4-diliydrox~~plienylalanine, tyrosine, lanthionine, thiolhistidine, cysteine, and djenkolic acid (brown or tan); and 3,5-diiodotyrosine (gray). Alloxan, whose structure resembles ninhydrin closely, also gives brownish colors with the same acids listed above, except for proline and hydroxyproline, with which it gives no color. Pinkish colors are obtained with practically all the other amino acids tested. Alloxan should prove to be a most useful new color reagent for the detection of small amounts of certain amino acids. Its reactivity with such acids as p-aminoisobutyric acid, carnosine, cysteine, cystathionine, and homocysteine thiolactone is far superior to that obtained with either ninhydrin or isatin. I n addition, the colors obtained on paper chromatograms are surprisingly uniform and stable over long periods of time with practically all the commonly occurring amino acids; this is in contrast to the more rapidly fading ninhydrin colors. I n general, its sensitivity for most amino acids is only slightly less than is found for ninhydrin, and is much superior to isatin. Isatin differs from either ninhydrin or alloxan in giving a wider variety of colors with amino acids (Table I). It gives no discernible color reaction with a number of ninhydrin-reacting substances such as asparagine, taurine, glutamine, and p-alanine. I n addition, it gives very weak reactions Iyith a number of other amino acids such as glycine, homoserine, glutamic acid, canavanine, phenylserinr, valine, leucines, carnosine, m i n e , threonine, and alanine. These amino acids give colors which do not intensify much with increasing concentration and which tend to fade rather rapidly. Because of these characteristics, isatin is a most useful color reagent in helping to differentiate certain amino acids in mixtures with similar R, values. Some examples of its use have been cited in previous publications (S4, 44). Other interesting findings from the present data include: ( a ) the large difference in sensitivity obtained with isatin between the p- and yaminobutyric acids with similar R, values; ( b ) the distinct blue color of norleucine with isatin, while leucine and isoleucine give faint levender colors; (c) the use of isatin to distinguish norvaline (blue) from valine (lavender); ( d ) the fact that isatin is equal to or greater in its sensitivity than either ninhydrin or alloxan for the following amino acids: proline, 3 , 4 dih3‘droxyphenylalaIiine, 3,5-diiodotyrosine, a- and 7-aminobutyric acids, ornithine, and phenyldanine, I t is, thereforc7 to be expected that depending on the complexity and content of the amino acid system to be analyzed, one or more of these color reagents can be used to obtain identifications even where complete chromatographic resolution has not been achieved, DISCUSSION
Although ninhydrin and isatin are among the two most widely used co!or reactions for amino acids (9, 5’4,48),the reaction which takes place was originally investigated by Strecker (47) for alloxan. Subsequent work has shown that these three substances react and others possessing the group -CO (CH=CH)XOin a fairly general manner with a-amino acids having the structure RCH(NI3,)COOH. I n each instance, the reactive amino acid is degraded to the corresponding aldehyde having one less carbon atom (45),with carbon dioxide and ammonia being liberated during the reaction. Although liberation of both carbon dioxide (60) and ammonia (28), as well as aldehyde formation (51), has been used for the quantitative determination of LYamino acids, this discussion is concerned only with the nature of the colored substances formed in these reactions. Of these three
504 reagents, the mechanism of the reaction of ninhydrin with c’amino acids has been recently studied by Moore and Stein ( S I ) and MacFadyen and Fowler (29). The purple color (Ruhcniann’s purple) is attributed to the anion of diketohydrindaminediketohydrinylidene. Under the usual experimental conditiona , such as those employed here, both the molar color yields ( S I , 32) and the spectral curves (IS) differ within a series of amino acidr. However, under controlled conditions of pH and temperature, and in the presence of organic solvents having maximal water content, it is possible to obtain purple color yields which approach 100% of theory (IS,49). These conditions do not apply to the reaction of ninhydrin with the imino acids proline and hydroxyproline. With these acids, although carbon dioxide is evolved, deamination does not occur and the acid residues condense di. rectly with the ninhydrin to form the yellow pigments (49). According to Abderhalden ( I ) , the pink-colored component:i formed by the interaction of alloxan with amino acids are 0.’ unknown constitution. However, because of the similarities in the structure and reactivities of alloxan and ninhydrin, thej‘ are believed to be of a composition similar to Ruhemann’a purple (10). This viewpoint is supported by the present exper. imental data in that all amino acids which gave purple colon with ninhydrin gave pink colors with alloxan, and all those \\-hich gave other shades with one reagent also did so with the other. There are, however, marked differences in their sensitivities which may be due to steric factors or side reactions. A mechanism for the Strecker reaction of 0-amino acids with isatin, similar to that for ninhydrin and alloxan, has been proposed by Schonberg and others (46) in which the colored product formed is isatide. Again, as in the case of ninhydrin, the hluecolored products formed with the imino acids are believed to be of an entirely different structure than the other amino acids-Le., pyrrole blues (20). Because of the somewhat different structure of isatin, as compared to the other two reagents, its reaction n-ith various amino acids mould not be expected to parallel their reactions. HoxT-ever, on the basis of such a mechanism, it would still be difficult to explain the wide variety of colors obtained nith this reagent which make it most useful in paper chroniatography. In addition to the possibility of side reactions and steric factors accounting for some of these color differences, they may represent various oxidative stages of the products formed during the reaction (24). This n*ould account for the weak colors formed and the relative ease with which they fade from the paper chromatograms. Because reagents described are general reagents for the CYamino acid grouping, the authors have preferred the terms “selective” or “differential” reagent to the more commonly used “specific” reagent. This latter term is more applicable to reagents such as the phenol reagent for tyrosine or the Sakaguchi reaction for arginine, although it is also used loosely to apply to reagents such as platinic iodide or phenol-hypochlorite which react with a number of amino acids (4, 6). Such specific reagents are readily applied t o small sectors from the circular chromatograms to supplement the differential reagents discussed here. While not so convenient, specific reagents are useful in helping to confirm the presence or absence of a particular amino acid in a band containing a mixture. Many of the sensitivity data reported here for amino acid reactions with isatin and alloxan on paper, and some of them for ninhydrin, are new to the literature, These data should be useful to investigators engaged in paper chromatography of amino acids. They also hold promise for the development of new methods for the quantitative determination of such acids as norleucine, norvaline, the prolines, etc., as well as for peptides and proteins. Work in this direction is a t present in progress in this laboratory.
ANALYTICAL CHEMISTRY ACKNOWLEDGMENT
The authors wish to acknowledge the aid and encouragement given them by Bruno W. Volk, Director of Laboratories, who permitted this work to be performed under Grant B-285 of the U. S. Public Health Service, and to Lillian Salowitz for the typing and editing of the manuscript. The authors also gratefully acknowledge the gift of a number of amino acids included in this study from Louis B. Rockland, U. S.Department of Agriculture Laboratory, Pasadena, Calif., and from Alfred Deutsch, California F o u n d d o n for Biochemical Research, Los Angeles, Calif. LITERATURE CITED
Abderhalden, R., Z. physiol. Chem. 252, 81 (1938). Acher, R., Fromageot, C., Jutisz, M.,Biochent. et Biophys. Acta 5, 81 (1950). Auclair, J. L., Dubreuil, R., Can. J . Zool. 30, 109 (1952). Berry, H. IC., others, Univ. Texas Publ., No. 5109, 22 ( R h y 1951). Block, R. J., AKAL.CHEM.22, 1337 (1950). Block, R. J., Durrum, E. L., Zweig, G., “A Afanual of Paper Chromatography and Paper Electrophoresis,” Academic Press, New York, 1955. Brims, C., Fromageot, C., Adrances in Protein Chem. 8 , 1 (1953). Brown, W., Nature 143, 377 (1939). Consden, R., Gordon, A. H., Martin, A. J. P., Biochem. J. 38, 224 (1944). Copley, G. N., Analust 66, 492 (1941). Csoban, G., Magyar Kem. Folydirat 56, 449 (1950). Dent, C. E., Biochem. J . 43, 169 (1948). Fitapatrick, W.H., Science 109, 469 (1949). Fowden, L., Biochent. J . (London) 50, 355 (1052). Fomden, L., Penney, J. R., Nature 165, 846 (1950). Fromageot, C., Jutisa, hl., Ann. Rev. Biochem. 22, 629 (1953). Giri, I
