Photometric ninhydrin method for use in the chromatography of amino acids.

For the investigations on the chromatographic separation of amino acids outlined in the preceding communication (1)) it was necessary to have available a suitable quantitative method for the determination of the concentration of amino acids in the effluent from the column. For this purpose, the method should be sufficiently general to include the determination of most of the amino acids and peptides likely to be encountered in protein hydrolysates or other material of biological origin. The method should have as high a sensitivity as possible to permit the determination of low concentrations of amino acids in the effluent from the chromatogram. Also the laboratory procedure should be fairly simple to permit the method to be applied conveniently to large numbers of effluent samples. It appeared probable that a photometric method would best fulfil these requirements. The two calorimetric methods of this type which had received the most study were the procedures based on the use of /3-naphthoquinonesulfonic acid and ninhydrin (triketohydrindene hydrate) as reagents. For reasons which will be described, the ninhydrin reaction was selected for further investigation. The color reaction between CY-NH2 acids and ninhydrin has been studied extensively in the past. It has been established that colored compounds are formed not only with amino acids, but also with peptides, proteins, and other classes of substances possessing free amino groups. The reaction is known to be extremely sensitive for qualitative work. In earlier attempts to render the color reaction quantitative (2-S), however, it has been found that the color yield per microgram of amino acid decreased markedly as the concentration of amino acid was reduced. In addition, the results have not been reproducible. In the present investigations, it has been observed that, when the color development is carried out in tubes exposed to the air, these difficulties appear to result primarily from the influence of dissolved oxygen. Improved results can be obtained when the reaction is performed in tubes evacuated to 20 mm. Under these conditions, the relationship between color yield and amino acid concentration is more nearly linear, although the deviations are still marked. By the addition of a strong reducing agent directly to the reaction medium, however, the oxidative side reaction has been eliminated. In preliminary experiments