Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive.

Abstract

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.