Enantiomeric mixtures of alanine, serine, threonine, valine, methionine, leucine and norleucine were resolved in ligand exchange reversed phase HPLC (reproducibly), by using L-proline, L-hydroxyproline or N,N-dimethyl-L-phenylalanine (2 mmol L-1) and Cu(CH3COO)2 (1 mmol L-1) in water or in water/methanol. The latter mobile phase greatly decreased the retention time of the more hydrophobic α-amino acids, preserving enantioseparation. pH must be high enough to allow the presence of free –NH2 groups in order to make the complexation with Cu(II) easier. The more restricted conformation of L-proline and L-hydroxyproline led to lower enantioseparations. The ligand exchange formation of pseudo-homochiral and pseudo-heterochiral complexes, thermodynamically and kinetically controlled, plays a fundamental role for the desired enantiomeric chromatographic separation. This simple and inexpensive methodology can be used routinely by any laboratory involved in α-amino acid synthesis.
α-amino acids; HPLC; pseudo-homochiral; pseudo-heterochiral
