The complete gas—liquid chromatographic separation of the twenty protien amino acids

Abstract

The complete gas—liquid chromatographic separation of the twenty protein amino acids is presented. In our previous publications, we reported on in effective chromatographic separation of seventeen N-trifluoroacetyl n-butyl esters of the amino acids on a packing composed of stabilized grade EGA and acid-washed Chromosorb W (heated at 140° for 12 h). A new column packing has been found to replace the OV-17; it is composed of a mixed phase of OV-17 and OV-210. This is a superior packing and shows quantitative elution, and highly efficient and complete separation of histidine, internal standard tranexamic acid, lysine, arginine, tryptophan, n-butyl stearate (I.S.), and cystine. No longer is it necessary to make a separate “subtraction-calculation” for histidine. With these two packings, EGA and mixed siloxane phases, one can now simultaneously analyze and separate the 20 protein amino acids in 30 min or less with automatic electronic integration of all peaks. A second internal standard (tranexamic acid) is introduced which contains both -NH 2 and -COOH functional groups. It can be used to follow the performance of ion-exchange cleanup, thereby increasing the reliability of analysis of complex biological materials. The use of two columns for separation of the twenty amino acids has important advantages over a single column system with respect to resolution, cross confirmation, and identification of the eluted compounds.