SEPARATION OF PRECOLUMN-LABELED D- AND L-AMINO-ACIDS BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY WITH UV AND FLUORESCENCE DETECTION

Micellar electrokinetic chromatography (MEKC) was examined for the sep aration of labelled D- and L-amino acids to permit rapid screening of protein amino acid enantiomers in microchemical analytical work. Preco lumn chiral derivatization was performed using 2,3,4,6-tetra-O-acetyl- 1-thio-beta-D-glucopyranose (OPA/TATG) reagent and the diastereomers f ormed were detected by UV or fluorescence detection. Optimization of s eparation buffer pH, ionic strength and surfactant concentration was c arried out and was focused on the effective separation window availabl e for the resolution of the amino acid derivatives. The effects of add ed organic modifiers, methanol, acetonitrile and tetrahydrofuran, on t he relative retention of the derivatives were characterized for the pu rpose of fine tuning the separation selectivity. The resolution of the derivatives of the D- and L-forms of each protein amino acid was very high (mean value of R(S) = 14.3, range 0.8-28), except for aspartic a cid and glutamic acid, whose enantiomers could not be resolved at the alkaline pH studied. A separation of 34 D,L-amino acids in less than 5 min, is demonstrated with only a few peaks co-eluting.