Gas-Liquid Chromatography of Histidine, Arginine, and Cystine. Interactions with the Liquid and Solid Support Phases

The interactions of the N-trifluoroacetyl n-butyl ester derivatives (TAB) of histidine, arginine, and cystine with the liquid phase, ethylene glycol adipate (EGA), and/or solid support were investigated. Glutamic acid was used as internal standard to determine the relative weight response (RWR) values of the three amino acids. Changes in the RWR values served as an index of substrate or solid support interaction. Essentially nonpolar Gas-Chrom Q and more reactive Chromosorb W AW were coated with a nonpolar OV-101 polymer and tested to determine whether the solid support contributed to the breakdown of the TAB derivatives of these three amino acids. Then a bilayer column was prepared by coating OV- 101/Gas-Chrom Q with polar EGA to determine the interaction of the EGA polymer with the TAB derivatives of histidine, arginine, and cystine. It was found that the polar substrate, EGA, was primarily responsible for the breakdown of histidine. The decomposition of arginine and cystine was caused mainly by the solid support (Chromosorb W AW), and the more active support significantly increased the degree of breakdown. As the loading of the EGA polymer on the Gas-Chrom Q of the polymer bilayer was decreased, an increased histidine response was observed while the arginine and cystine response remained unaffected. Also, the histidine response was substrate contact time and temperature dependent and increased significantly on a bilayer column with decreased residence times; whereas, the arginine and cystine response decreased, again indicating complex temperature dependent interactions with the EGA-polymer and/or solid support phase. Nickel and glass tubing filled with the EGA bilayer packing were compared in the chromatography of the TAB derivatives. The nickel tubing completely destroyed the derivatives of histidine, arginine, and cystine. For the quantitation of all 20 protein amino acids by gas-liquid chromatography (GLC) the two-column system introduced by Gehrke and Kuo, et al. (33) still remains a valuable method.