High-performance ligand-exchange chromatography of α-amino acid enantiomers

Abstract

Three packings with the following surface composition were synthesized on LiChrosorb Si 100, d p  10 μm: [Cu(II) 3-( L-prolyl)propyl] + (packing I), [Cu(II) 3-( L-hydroxyprolyl)propyl] + (packing II) and [Cu(II) 3-( L-hydroxyprolyl)propyl] + with remaining excess 3-(iodopropyl) groups (packing III). The separation of α-amino acid enantiomers was studied in eluents containing a constant concentration of copper(II) acetate (10 −4 M). The pH of the buffered eluent was varied in the range 4–6 and the ammonium acetate concentration was varied from 0.001 to 0.1 M, and these were established as dominating parameters in controlling the retention and enantioselectivity. In contrast, the addition of an organic solvent such as methanol, acetonitrile or tetrahydrofuran gave only minor changes. The same was observed for the changes in k′ and α on increasing the methanol content up to 30% (v/v) and by varying the column temperature between 298 and 323°K. The order of elution of α-amino acid enantiomers was generally found to be L- < D-; there were, however, a series of exceptions, depending on the type of packing and the eluent composition. Enantioselectivity can be understood in terms of complexation of the enantiomer to a trans-bis(amino acidato)—copper complex and additional interaction through complexation of free ligand sites, hydrophobic interaction between the radical R and the n-propyl spacer of the bonded ligand, hydrogen bonding, etc. The columns permitted the separation of six racemic α-amino acids into the corresponding enantiomers, using common high-performance liquid chromatographic equipment with a UV photometer (254 nm).