Determination of D- and L-Amino Acids by Ion Exchange Chromatography as L-D and L-L Dipeptides

Abstract

Abstract A procedure for the precise determination of the d and l isomers in a given sample of an amino acid has been based upon chromatographic separation of the diastereoisomeric dipeptides obtained by derivatization with an l-amino acid N-carboxyanhydride. The conditions for the preparation of dipeptides developed by Hirschmann and associates make the derivatization step simple and rapid; the coupling proceeds at pH 10.4 in 2 min in about 90% yield. For each of 21 individual amino acids conditions of elution have been found for the separation of the resulting pairs of l-d and l-l dipeptides by ion exchange chromatography on an amino acid analyzer. One part of d-amino acid can be detected in the presence of 1000 parts of the l isomer with 2-µmole samples. With a mixture of amino acids, a given component can be isolated chromatographically for subsequent determination of the d to l ratio or chromatographic separation of the dipeptides prepared by derivatization of the mixture can be undertaken. If the mixture is a hydrolysate of a peptide or a protein, the degree of racemization occurring during acid hydrolysis must also be considered. For this purpose hydrolysates of synthetic bradykinin and of pancreatic ribonuclease have been examined. The amounts of several d-amino acids (0.2 to 4.4%) determined in the hydrolysates were not significantly higher than those found in control experiments when the pure l-amino acids or l-l dipeptides were subjected to the hydrolytic conditions.