A comparative study of factors affecting chiral separations under reversed-phase conditions using cellulosic and Pirkle-type chiral selectors.

Limited studies have been conducted in reversed-phase chiral chromatography despite the relevance to water soluble chiral analytes. Systematic investigation and interpretation of factors affecting chiral separation in reversed-phase LC is crucial to efficient chiral method development. Two of the most useful chiral stationary phases (CSPs), a cellulosic (Chiralcel OJ-R) and a Pirkle-type (Chirex 3005), were chosen as the chiral selectors in this study to systematically investigate the factors affecting chiral separation of a series of Fmoc-amino acids. The effects of temperature, pH, buffer concentration, buffer type and organic modifiers on retention and chiral selectivity were compared between the cellulosic and the Pirkle-type selectors. Temperature affected retention and selectivity, as predicted by thermodynamics, using both types of chiral selectors. On the cellulosic CSP, buffer pH was the only factor which caused a significant change in chiral selectivity, while several factors, including buffer concentration and organic modifier type, affected chiral selectivity on the Pirkle-type CSP. The results also indicated that the cellulosic CSP had a retention mechanism analogous to conventional reversed-phase LC, however, the Pirkle-type selector appeared to show a mixed mode retention mechanism for these analytes.