Resolution of Optical Isomers as the Mixed Chelate Copper(III) Complexes by Reversed Phase Chromatography

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Abstract
Highly selective separation of D and L amino acids can be effected by reversed phase chromatography of mixed chelate complexes of the analyte acids with equimolar concentrations of Cu(II) and an optically active second amino acid in the mobile phase. The stabilities of the formed diastereomeric ternary complexes will determine the resolution of enantiomers. By this approach, amino acids were resolved as the dansyl and O-phthalaldehyde (OPA) derivatives and imino acids were separated without derivatization. Resolution of D and L dansyl amino acids was accomplished as the mixed complexes of Cu(II) with L-proline, L-arginine, L-histidine and L-histidine methyl ester as the second amino acids. Among the chiral ligands we studied, L-histidine methyl ester is unique in that it possesses both achiral selectivity for the dansyl amino acids and chiral selectivity for the respective D and L enantiomers. With a mobile phase gradient of acetonitrile in a buffer containing Cu(II) L-histidine methyl ester complex, we devised a stereoselective procedure for the analysis of D and L amino acid enantiomers, achieving the separation that the current amino acid analyzer failed. The mixed chelate metal complexation approach was recently extended to resolve OPA derivatives. The free amino acid was reacted with OPA in the presence of a chiral sulfhydryl reagent, N-acetyl-L-cysteine (NAC). HPLC of the derivatives was then performed on a reversed phase column, with a mobile phase containing Cu(II) L-proline, using fluorescence detection and resolved the optical isomers of the common primary amino acids. The same approach was also used to resolve compounds containing amine and sulfhydryl groups. The simultaneous detection and resolution of D and L imino acids is a more complicated problem. The reason is that the nitrogen of the imino acids must be derivatized in order to be detected whereas both the carboxyl and the imino groups must be free to allow complexation for chiral separation. A procedure for the resolution of D and L isomers of pipecolic acid and proline was devised by complexing the analytes with Cu(II)-L-aspartame and detecting the complexes at UV 235 nm. The urine concentration of D and L pipecolic acid was also measured this way for patients with disorders of lysine metabolism.