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 1.9 s - 1 and from 0.06 to 0.36 s - 1 between 4 and 45 °C. The corresponding association rate constants increased from 2.4 × 10 4 to 3.2 × 10 5 M - 1 s - 1 for ( R )-warfarin and from 4.4 × 10 4 to 7.2 × 10 4 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 stationary phase mass transfer for both enantiomers. Further studies indicated that ( R )- and ( S )-warfarin had similar activation energies 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 separations.