Investigation of Warfarin–Human Serum Albumin Binding by Ultrafast Affinity Extraction Using Single- and Dual-Column Affinity Microcolumn Systems

Authors

• Samiul Alim, University of Nebraska-LincolnFollow

First Advisor

David S. Hage

Committee Members

Rebecca Lai, Martha Morton

Date of this Version

6-2026

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Chemistry

Under the supervision of Professor David S. Hage

Lincoln, Nebraska, June 2026

Comments

Copyright 2026, Samiul Alim. Used by permission

Abstract

This dissertation examined the interaction of warfarin with human serum albumin (HSA) by using affinity chromatography, with particular emphasis on ultrafast affinity extraction (UAE) and affinity microcolumns. HSA affinity supports were prepared by immobilizing HSA onto diol-bonded silica via Schiff base chemistry and used to prepare affinity microcolumns for chromatographic studies under physiological-like conditions at pH 7.4 and 37.0 °C. UAE studies were performed to investigate the effects of residence time, flow rate, and column configuration on the measured apparent free fraction of warfarin and on the calculated apparent binding parameters. The results showed that the measured apparent free fraction decreased with increasing flow rate, consistent with the expected behavior of UAE, in which shorter residence times reduced dissociation of a protein-bound drug during passage through the affinity microcolumn. However, the measured binding parameters were influenced by the column dimensions, flow-rate range, and system geometry. Shorter affinity microcolumns produced more physically reasonable free fraction measurements than longer microcolumns and gave apparent association constants that were closer to previously reported literature values for warfarin–HSA binding. A dual-column UAE system consisting of two HSA microcolumns connected in series was also examined to determine the effect of segmented column architecture and column order on the measured response. Although both dual-column arrangements showed the expected UAE behavior, differences were observed in the free fractions and apparent association constants obtained for the two configurations. These results suggested that transport-related effects, including partial re-equilibration occurring within the intercolumn connector, contributed to the measured response. Overall, this work demonstrated that affinity microcolumns and UAE are useful tools for studying drug–protein interactions while also showing that the measured apparent binding parameters can be influenced by the residence time, column format, and transport-related effects within the chromatographic system.

Advisor: David S. Hage