Chiral separation of selected proline derivatives using a polysaccharide-type stationary phase by supercritical fluid chromatography and comparison with high-performance liquid chromatography

Chiral separation of selected proline derivatives was conducted on a polysaccharide-type chiral stationary phase (CSP) using supercritical fluid chromatography (SFC). Effect of both modifier percentage and column temperature was studied. As modifier content decreased, the retention and selectivity increased. However, retention was decreased as temperature increased from 30 °C to 40 °C. In comparison to high-performance liquid chromatography (HPLC), better resolution was achieved for Boc-2-methylproline using SFC along with a shorter run time. Selectivity is impacted less by change of ethanol content in the mobile phase under SFC conditions than with HPLC. Chiral recognition mechanism under SFC and HPLC conditions was also examined. With SFC, the contribution from hydrogen bonding is most likely a dominant interaction for retention rather than chiral recognition. This is supported by the limited thermodynamic data and the elution order change for Boc-proline between SFC and HPLC. Linearity, injection repeatability and limit of quantitation were determined for Boc-2-methylproline.