In this paper we report a study on the mechanism of the enantiomeric separation of unmodified D,L-amino acids in RP-HPLC by copper(II) complexes of two tetradentate diaminodiamido ligands, (S,S)-N,N'-bis(phenylalanyl)ethanediamine (PheNN-2) and (S,S)-N,N'-bis(methylphenylalanyl)ethanediamine (Me2PheNN-2), added to the eluent. The aim is to investigate whether and how a copper(II) complex with no free equatorial positions can perform chiral discrimination of bidentate analytes such as unmodified amino acids. The problem is approached in a systematic way by: (a) varying the different chromatographic parameters (pH, selector concentration, eluent polarity); (b) performing chiral separation with the selector adsorbed on the stationary phase; (c) studying the ternary complex formation of these ligands with D- and L-amino acids in solution by glass electrode potentiometry and electrospray ionization MS. All the experimental data are consistent with a mechanism of chiral recognition, based on ligand exchange, which involves as selectors the species [Cu2L2H(-2)]2+ and [CuLH(-2)] and proceeds by displacement of two binding sites from the equatorial positions, giving rise to the ternary species [CuLA]+ and [CuLH(-1) A]. The most important factor responsible for chiral discrimination seems to be the affinity of the diastereomeric ternary complexes for the stationary phase since no enantioselectivity is observed in solution.