Conformational changes in polysaccharide-based chiral selectors induced by mobile phase composition: Effects on enantioselective retention and enantiomer elution order reversal

Despite having identical physicochemical properties, chiral molecules require effective separation techniques due to their distinct pharmacological effects. Polysaccharide-based chiral stationary phases (CSPs) are widely used for chiral separations in liquid chromatography; however, the mechanisms of chiral recognition are not well understood. This research explored the adsorption, retention, and chiral recognition mechanisms of three amylose-based CSPs: Chiralpak ID, IF, and IG. The effect of mobile phase composition on enantioselective retention was examined using four acyloin-type chiral solutes in normal-phase mode. For pantolactone (PL) and methyl mandelate (MM), reversals in enantiomer elution order were observed with ID and IG sorbents, respectively, at 2 vol.% isopropanol (iPrOH). As the iPrOH concentration increased, the adsorption of MM enantiomers reached an energetic barrier at this concentration, causing discontinuities in the enthalpy-entropy compensation. Conversely, while the reversal behavior of PL was also attributed to conformational changes in the ID polymer, it did not encounter an energetic barrier and thus remained in line with the enthalpy-entropy compensation. For the IF sorbent, no significant changes in enantioselective retention or enthalpic curves were noted. Nevertheless, a reversal was observed for benzoin (B) enantiomers on the IF sorbent at 10 vol.% iPrOH. It was postulated that the IF sorbent contains two chiral sites with opposing recognition abilities, and their relative contributions to the apparent enantioselectivity of B are influenced by the iPrOH concentration. These findings highlight the importance of conformational changes in chiral selectors, driven by mobile phase composition, in chiral recognition mechanisms. Understanding these effects is crucial for developing predictive models of chiral retention and enhancing optimization of chiral separation processes.