Application of Cinchona alkaloid-based zwitterionic chiral stationary phases in supercritical fluid chromatography for the enantioseparation of Nα-protected proteinogenic amino acids

Abstract

Stereoselective supercritical fluid chromatographic separations of the enantiomers of a large set of Nα-Fmoc proteinogenic amino acids were carried out on the recently developed Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+)™ and ZWIX(-)™ with protic solvents as co-solvents. The effects of the mobile phase composition, the natures and concentrations of the acid or base additives, the co- and counter-ions and temperature on the separations were investigated. The retention time in most cases slightly increased, while the separation factor decreased with increasing temperature. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van't Hoff plots derived from the lnα vs 1/T curves in the studied temperature range (20-60°C). The values of the thermodynamic parameters depended on the natures of the selectors and the structures of the analytes. On both ZWIX(+)™ and ZWIX(-)™ columns, enthalpically-driven separations were observed. The elution sequence was determined in all cases and was observed to be opposite on ZWIX(+)™ and ZWIX(-)™ which acted for the presented applications as chiral anion exchanger.

Keywords: Enantiomer separation; N(α)-Fmoc-protected proteinogenic amino acids; Supercritical fluid chromatography; Temperature effect; Zwitterionic chiral stationary phases.