Chiral separation mechanisms in protein-based HPLC columns. 2. Kinetic studies of (R) - and (S)-Warfarin binding to immobilized human serum albumin

This work used plate height measurements to investigate the kinetics of (R)- and (S)-warfarin binding to an immobilized HSA column. The dissociation rate constants for (R)- and (S)-warfarin on this column increased from 0.06 to 1.9s^-1 and from 0.06 to 0.36s^-1 between 4 and 45 °C. The corresponding association rate constants increased from 2.4 × 10⁴ to 3.2 × 10⁵ M^-1 s^-1 for (R)-warfarin and from 4.4 × 10⁴ to 7.2 × 10⁴ M^-1 s^-1 for (S)-warfarin over the same temperature range. From the dissociation data, it was found that an increase in temperature led to a large decrease in the plate height due to sationary phase mass transfer for both enantiomers. Further studies indicated that (R)- and (S)-warfarin had similar activation emergies for their binding to HSA. For (R)-warfarin, most of this energy requirement was due to the change in enthalpy of the system, while for (S)-warfarin, it was mainly due to the change in entropy. All of these results agree with an earlier model, in which (R)- and (S)-warfarin were proposed to interact with regions on the interior and exterior of HSA, respectively. In addition, these results offer a number of useful insights into the mechanisms of protein-based chiral seprations.