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 m M sodium borate (pH 9.2), 150 m M sodium dodecyl sulfate (SDS) and 40 m M γ‐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 m M sodium phosphate (pH 6.8) or a 5 m M sodium borate buffer (pH 9.2), each of which contained 25 m M 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 m M 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 n M level, which was about 100 × more sensitive than UV absorption at 200 nm. Analyte concentrations as low as 3 × 10 −8 M ( DL ‐Val) could be derivatized with (+)‐FLEC.