Gas chromatographic analysis of amino acids as the N-heptafluorobutyryl isobutyl esters.

Abstract

The N-heptafluorobutyryl isobutyl derivatives of proteic amino acids are well resolved by gas chromatography and form the basis of a convenient, rapid assay. The derivatives are prepared by acid-catalyzed esterification at 120 degrees C for 20 min in 3N HCl-isobutanol followed by acylation with heptafluorobutyric anhydride at 150 degrees C for 10 min. The reaction sequence is performed without any transfers or extractions and thus is compatible with microscale analysis. A complete proteic amino acid profile can be completed in less than 20 min by using a packed column or less than 10 min by using a capillary column in combination with an elevated oven temperature program rate. Physiological sample matrixes, which frequently contain a complex mixture of components, and thus require maximum resolution, can be assayed in less than 1 h using a program rate of 4 degrees C/min. A capillary column is recommended for this application. Capillary column chromatography, in combination with a nitrogen-specific detector, is useful for identifying and assaying nonproteic amino acids in physiological sample matrixes. Frequently, a prior cleanup of the sample can be avoided.