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

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233 Citations (Scopus)

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

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Warfarin
Serum Albumin
Thermodynamics
Enantiomers
Proteins
Binding Sites
Entropy
Ionic strength
Free energy
Thermodynamic properties
Temperature

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

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