Elimination of Losses in the Quantitative Estimation of Amino-Acid by Paper Chromatography

Abstract

SEVERAL methods^1–6 have been published for the quantitative estimation of amino-acids after separation by filter-paper chromatography. In general, these methods have only been applied to simple amino-acid mixtures, which can be resolved on a one-dimensional chromatogram. In investigating the possibility of quantitative estimation of amino-acids in complex mixtures, we have examined the accuracy of most of these methods. The most sensitive and accurate method in our hands is a colorimetric ninhydrin method based on that of Moore and Stein⁷. Applying the method directly to pieces of filter paper, there is a high and variable ‘amino-acid’ value, due mainly to the presence of absorbed ammonia. The colour response per mole of ammonia is about the same as that of an amino-acid. By treatment of the paper with dilute alkali immediately before the determination, the absorbed ammonia is removed, the ‘paper blank’ becomes very low and constant, and the colour response from a standard amount of an amino-acid added to small squares of filter paper is identical with that produced from the same amount of amino-acid in the absence of paper, provided the appropriate paper blank correction is applied.