Chiral Separation Mechanisms in Protein-Based HPLC Columns. 1. Thermodynamic Studies of (R)- and (S)-Warfarin Binding to Immobilized Human Serum Albumin

Bounthon Loun, David S. Hage

Research output: Contribution to journalArticle

240 Scopus citations

Abstract

This work characterizes the thermodynamic processes involved in the binding and separation of (R)- and (S)-warfarin on a high-performance human serum albumin (HSA) column. Frontal analysis was used to determine the strength and degree of binding for each enantiomer. (R)- and (S)-warfarin were found to bind at the same region on HSA; however, (R)-warfarin had a larger number of column binding sites. The number of binding sites for both enantiomers showed a slight increase with temperature. The total changes in free energy for (R)- and (S)-warfarin binding were similar at 37 °C, but the contribution due to entropy was greater for the R-enantiomer. These results suggested that (R)-warfarin was interacting mainly with the binding site interior, while (S)-warfarin interacted more with the site's outer surface. This model was confirmed by examining the retention of (R)- and (S)-warfarin on the HSA column under various pH, ionic strength, and organic modifier conditions. The different changes in entropy for these solutes made it possible to vary their separation by changing column temperature. Both thermodynamic properties and column binding capacities were found to be important in determining the degree of separation obtained for these compounds.

Original languageEnglish (US)
Pages (from-to)3814-3822
Number of pages9
JournalAnalytical chemistry
Volume66
Issue number21
DOIs
StatePublished - Nov 1 1994

ASJC Scopus subject areas

  • Analytical Chemistry

Fingerprint Dive into the research topics of 'Chiral Separation Mechanisms in Protein-Based HPLC Columns. 1. Thermodynamic Studies of (R)- and (S)-Warfarin Binding to Immobilized Human Serum Albumin'. Together they form a unique fingerprint.

  • Cite this