Determination of monoamino monocarboxylic acids by quantitative paper chromatography.

Many methods for chromatographic separation of amino acids are now available, but their subsequent determination, generally employing ninhydrin, often leaves much to be desired. It was felt, therefore, that there was a need for a simple method of analysis based on the convenient paper technique and employing some means of determination less fickle than ninhydrin colorimetry. The method now presented involves an initial one-dimensional separation of amino acids in considerable quantity, using bands rather than spots. The problem of accurate marking of these bands, with little space between them and often having irregular fronts, has been solved by spraying with an indicator solution of bromothymol blue, formaldehyde and alkali, in ethanol (Kemble & Macpherson, 1952). This restricts the range ofsolvents to those neutral in character, but several such are known to give useful separations of amino acids. Methods of determination were then investigated. Measurement of carbon dioxide following oxidation with ninhydrin is a most specific and accurate method (Van Slyke, Hamilton, McFadyen & Dillon, 1941), but note was taken, in the same paper, of the use of chloramine-T for decarboxylation at low temperatures. Cohen (1940) has measured the evolution of carbon dioxide by chloramine-T in a Warburg respirometer. As numerous determinations can be performed simultaneously, this apparatus was adopted for the present work. A 9 in. wide strip of Whatman 3MM paper will separate at least 2 mg. of each amino acid, sufficient to give a maximum reading on the manometer. Recoveries of carbon dioxide from amino acids obtained by Krebs (quoted by Cohen, 1940) are significantly higher than the more nearly theoretical values reported by Van Slyke et al. (1941). Preliminary work showed that yields of carbon dioxide following direct oxidation with chloramine-T were in accord with the results of Krebs, while yields obtained from a sprayed chromatogram agreed with those of Van Slyke. This difference was found to be due to the suppression by the formaldehyde of the indicator spray of interference due to ammonia, and all recovery factors (moles C02/mole amino acid) have been determined in the presence of a standard amount of formaldehyde. Work has been confined in the present instance to the monoamino monocarboxylic acids, which can be isolated readily by ionophoresis in a three-compartment cell.