A modified ninhydrin reagent for the photometric determination of amino acids and related compounds.

The photometric ninhydrin method described previously (1) for use in the chromatography of amino acids has been the subject of further study during the development of the Dowex 50-X4 procedure (2). The adjustment of the pH of the effluent fractions prior to analysis required by the earlier method has been largely eliminated by increasing the strength of the buffer in the reagent 5-fold. A second modification involves the elimination of the stannous chloride which was previously added to form reduced ninhydrin (hydrindantin) in the reagent solution. Hydrindantin itself is now added directly, and this change avoids the precipitation of tin salts which occurs when the previous reagent is used to analyze samples of the phosphate buffers frequently employed in the chromatography of proteins (3). The effective concentration of hydrindantin has been raised to 0.3 per cent. As a result, the antioxidant effect of 1 ml. of the modified reagent is sufficient to prevent interference from the dissolved oxygen in a 2 ml. sample submitted to analysis. With 1 ml. fractions, 0.5 ml. of the ninhydrin reagent is adequate. The quantity of reagent required is thus reduced to one-half or one-quarter the amount formerly recommended (1). Troll and Cannan (4) have recently described conditions under which the yield of blue color (diketohydrindylidenediketohydrindamine) in the reaction of ninhydrin with amino acids is raised to 100 per cent of theory in most cases. For this purpose, the reaction is performed in a predominantly organic solvent (phenol-pyridine-alcohol) having a maximal water content of about 20 per cent. The method, when applicable, has the fundamental advantage of giving maximal color yields. The present procedure, in which the reproducible color yield from leucine is 95 per cent of theory, appears to be more convenient for the analysis of the aqueous effluent fractions obtained in ion exchange chromatography.