Amino acid analysis by capillary gas chromatography

Two developments have enabled major advancements in the use of capillary gas chromatography (GC), the result being its much more widespread use in investigations on a broad range of chemical and biological problems. The 2 technological developments were the introduction of fused silica capillary columns and the development of immobilized stationary phases for capillary GC columns. Because fused silica columns with immobilized stationary phases of varying polarities are offered by numerous vendors of chromatographic equipment, they have become widely used for many analytical tasks. We conducted a study to compare the effectiveness of commercially available fused silica capillary columns with the classical ion-exchange method in the separation and quantitation of amino acids. We selected the N-trifluoroacetyl (TFA) n-butyl and the N-heptafluorobutyryl (HFB) isobutyl ester derivatives for this study because of the extensive research and application of these derivatives during the past 20 years. The amino acid content of hydrolysates of 5 materials was measured: ribonuclease, beta-lactoglobulin, lysozyme, soybean meal, and a commercial poultry feed. Single 6N HCl hydrolysates of each material were prepared to minimize sample preparation differences, and 3 independent analyses of each hydrolysate were made by each of 3 techniques: the N-TFA n-butyl and N-HFB isobutyl ester methods using capillary gas chromatography and the ion-exchange chromatographic method using a Beckman 121 M amino acid analyzer. Our results clearly demonstrate that capillary GC analysis of amino acids using fused silica bonded-phase columns provides data with good precision and in general excellent agreement with ion-exchange analyses.