Gas-liquid chromatography of protein amino acids separation factors

Abstract

This research reports on chromatographic and instrumental studies which will permit one to accurately, rapidly, and routinely analyze the twenty natural protein amino acids in biological substances. A number of factors influencing the performance of the chromatographic system were evaluated, and the separation characteristics of various polyesters of neopentyl glycol were evaluated. Also, an evaluation was made of support materials which had undergone various heat treatments. It was shown that the optimum chromatographic performance for neopentyl glycol polyesters was observed at a carbon chain length of ten (neopentyl glycol sebacate). The separation ability of ethylene glycol adipate as a liquid phase was found to be superior to neopentyl glycol sebacate, consistent, and reproducible with respect to time and temperature. Arginine, histidine, and cystine were not reproducibly eluted from this column. This is a result of interaction between the substrate phase and the amino acid derivative. However, seventeen of the amino acid derivatives were well separated and quantitatively eluted in 33 min from columns containing 0.325 w/w % ethylene glycol adipate coated on 80/100 mesh acid-washed heat-treated Chromosorb G. In general, the retention temperatures for the amino acids were lower and a significant improvement in resolution was noted when columns were prepared with Chromosorb G which had been treated at 450° to 600° for 15 h. For the analysis of arginine, histidine and cystine, columns containing 1.5 w/w % OV-17 coated on high performance 80/100 mesh Chromosorb G were used, Di-acyl histidine was converted to the mono-acyl derivative by injection of n-butanol immediately after injection of the sample. The di-acyl derivative of histidine does not interfere with any of the amino acids on the EGA column. A dual column chromatographic system of ethylene glycol adipate and OV-17 as the liquid phases is described which quantitatively separates that protein amino acids in 55 min. The quantitative gas-liquid chromatographic analysis of the amino acids in ribonuclease is reported.