Recently, we have developed a reversed-phase chiral HPLC method for a basic pharmceutical compound with a primary amine on one of three stereogenic centers. The method provided baseline resolution of five stereoisomers, including the API, undesired enantiomer and three potential diastereomers on a tris (3,5-dimethylphenylcarbamate) cellulose chiral stationary phase (Chiralcel OD-RH). The chiral separation was found highly sensitive to mobile phase pH. Optimal operational pH encompassed a range of 5.3 - 5.8 using citrate buffer. Interestingly, the van't Hoff plot of retention (log k) against the reciprocal of column temperature (1/T) showed a non-linear relationship, while the enantioselectivity plot, i.e. log a vs. 1/T, remained linear.Non-linear van't Hoff behavior is commonly seen in reversed-phase chromatography, especially in chiral separations. It may be due to phase-ratio change, or changes in transfer enthalpy or enthopy [1]. For ionizable compounds, buffer composition, pKa and ionization enthalpy may play significant roles in the observed phenomenon [2]. In this presentation, chiral method development strategy, pH effect, and the selection of mobile phase buffer system will be discussed. More importantly, the efforts to address non-linear van't Hoff behavior of chiral analytes will be outlined and results will be presented. References:T.L. Chester, J. W. Coym, J. Chromatogr. A 1003 (2003) 101�111.S. Heinisch, J.L. Rocca, J. Chromatogr. A 1216 (2009) 642�658.

Study of nonlinear van't Hoff behavior in reversed-phase chiral separation

248th National Meeting (2014)

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