Qualitative Determination of Amino Acids in Protein Hydrolyzates by Circular Paper Chromatography

Color Reaction oK Amino Acids with Alloxan, Isatin, and Ninhydrin in Circular Paper Chromatography

“…The widely used phenol-water developing solvent gives more diffuse bands than does the butanol-acetic acid-water system (36). 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 Rf values given in Table I for phenol are in better agreement with those reported (9) for the descending technique, than for the circular method (36).…”

“…One might expect to obtain separations of amino acid mixtures 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 it has been found possible to identify amino acids whose Rf 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 with isatin and ninhydrin, rather than on the use of a multiplicity of solvents which increase the complexity of the method.…”

“…Because of these characteristics, isatin is a most useful color reagent in helping to differentiate certain amino acids in mixtures with similar Rf values. Some examples of its use have been cited in previous publications (34,44)• Other interesting findings from the present data include: (a) the large difference in sensitivity obtained with isatin between the 3-and Y-aminobutyric acids with similar Rf 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, 3,4dihydroxyphenylalanine, 3,5-diiodotyrosine, «-and -aminobutyric acids, ornithine, and phenylalanine. It is, therefore, 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.…”

“…Although ninhydrin and isatin are among the two most widely used color reactions for amino acids (9,34,48), the reaction which takes place wras originally investigated by Strecker (47) for alloxan. Subsequent work has shown that these three substances and others possessing the group -CO(CH=CH)"CO-react in a fairly general manner with «-amino acids having the structure RCH (NIIalCOOH.…”

“…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 (1,10,22,27), only one previous application to the field of paper chromatography of amino acids and related compounds was found (40).…”