A Technique for the Determination of Enantiomeric Amino Acids in Biological Samples

Abstract

Racemic amino acids can be separated into their enantiomers by means of gas-liquid chromatography. The most applied technique, today, is the conversion of chiral compounds into diastereoisomers with optically active reagents and subsequent chromatography on conventional optically inactive stationary phases. In previous studies it has been realized that this technique is associated with various problems. We studied the use of optically active stationary phases for separating enantiomers directly via a diastereo- isomeric association complex. The optically active stationary phases employed are N- and C-terminal substituted dipeptides of the type N-trifluoroacetyl-dipeptide-cyclohexyl esters and have been synthesised by the I-hydroxibenztriazole dicyclohexylcarbodiimide method.

The quality of these phases with respect to separation factors, resolution factors, and thermodynamical properties have been evaluated. All synthetic phases show excellent properties; however, when attempting separation of mixtures of naturally occurring amino acids extensive overlap in the elution diagram was detected. Only one phase — N-TFA-L-α-amino-n-butyryl-L-α-amino butyric acid cyclohexyl ester — gave complete resolution of the naturally occurring amino acids alanine, valine, glycine, threonine, leucine, isoleucine, serine and proline on a 400 ft x 0.02 in capillary column. Less volatile amino acids such as aspartic acid, phenylalanine, methionine, glutamic acid, tyrosine, arginine, and tryptophan can be resolved at a 100 ft × 0.02 in column.