Gas-liquid chromatographic studies on the twenty protein amino acids: A single-column separation

Abstract

The results of earlier investigations by Gehrke et al. toward a single column separation of the twenty protein amino acids showed that polar liquid phases decompose the N-trifluoroacetyl (N-TFA) n-butyl ester derivatives of arginine, histidine, and cystine. Therefore, this study was to evaluate a number of non-polar liquid phases with regard to their ability to separate and reproducibly elute the N-TFA n-butyl ester derivatives. In 1971, a chromatographic system was reported by Gehrke et al. which employed dual columns; one of 0.65% ethylene glycol adipate (stabilized grade EGA) as liquid phase on 80–100 mesh acid-washed Chromosorb W for separation of seventeen amino acid derivatives, and a mixed phase column containing 1.0% (w/w) OV-210 and 2.0% (w/w) OV-17 on Gas-Chrom Q was used for the separation of histidine, arginine, and cystine. Both columns of the system were operated under the same thermal conditions, thus allowing simultaneous operation. The complete separation of the twenty amino acids on a single column is ultimately desirable and would aid considerable with respect to speed and cost; yet a dual-column combination is of significant merit in that additional separations are achieved and valuable cross confirmation is obtained on the amino acids in the sample. A careful study of the separation characteristics of the OV series of siloxane liquid phases demonstrated that none of these phases, singly or in combinations would yield complete and reproducible separation of the protein amino acids on a single column. Studies were then made to determine the optimum initial column temperature and hold time for separation of Ala, Thr, Gly, and Ser, and Phe, Lys, and Tyr by measuring the rates of migration of these derivatives under isothermal conditions on non-polar liquid phases. The optimum separation of all twenty amino acid derivatives was achieved with a 2.5 m × 2 mm I.D. glass column of 10% (w/w) Apiezon M on 80–100 mesh HP Chromosorb W with an initial column temperature of 90° for 6–9 min, then a 6°/min program rate to 260°. Analyses of dipeptides containing histidine resulted in no apparent on-column conversion of diacyl histidine to the monoacyl derivative. Apiezon M is a useful liquid phase for the single column separation of the protein amino acid N-TFA n-butyl esters.