Capillary gas chromatographic analysis of protein amino acids as their N(O,S)-isobutoxycarbonyl methyl ester derivatives

Abstract

A simple and rapid method for the determination of the 22 protein amino acids by capillary gas chromatography is described. The amino acids were converted into their N(O,S)-isobutoxycarbonyl (isoBOC) methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. Arginine was converted into ornithine by arginase treatment prior to the isoBOC reaction. The N(O,S)-isobutoxycarbonylation of amino acids with isobutyl chloroformate was completed within 15 s by sonication in aqueous alkaline media. All of the derivatives were quantitatively resolved as single symmetrical peaks within 9 min by GC on a single column. The detection limits of amino acids were 0.2-4.0 ng per injection, and the calibration curves were linear in the range 0.2-50 micrograms for each amino acid. This method was successfully applied to protein hydrolysate samples. The analytical results of amino acid compositions of several proteins are presented.