Enantiomeric separation of amino acids using micellar electrokinetic chromatography after pre-column derivatization with the chiral reagent 1-(9-fluorenyl)-ethyl chloroformate.

Direct enantiomeric separations of some racemic amino acids derivatized with 9-fluorenylmethyl chloroformate were obtained using cyclodextrin-modified micellar electrokinetic chromatography (CD/MEKC) with a buffer made up of 5 mM sodium borate (pH 9.2), 150 mM sodium dodecyl sulfate (SDS) and 40 mM gamma-CD. Alternatively, enantiomeric separations were also achieved indirectly using MEKC after pre-column derivatization with (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC). Using either a 10 mM sodium phosphate (pH 6.8) or a 5 mM sodium borate buffer (pH 9.2), each of which contained 25 mM SDS and 10-15% of acetonitrile, FLEC-derivatized serine, alanine, valine, methionine, leucine, phenylalanine, tryptophan, and their diastereomeric pairs were all separated: the L-isomers migrated faster than the corresponding D-isomers. However, when (-)-FLEC was used for derivatization, the D-isomers migrated faster than the corresponding L-isomers. Also, the diastereomers of aspartic acid, glutamic acid, and proline were resolved using a 10 mM sodium citrate buffer (pH 4.4). Using KrF (248 nm) laser-induced fluorescence, the detection limit of (+)-FLEC derivatized DL-amino acids was obtained at the nM level, which was about 100 x more sensitive than UV absorption at 200 nm. Analyte concentrations as low as 3 x 10(-8) M (DL-Val) could be derivatized with (+)-FLEC.