Evaluation of affinity microcolumns containing human serum albumin for rapid analysis of drug-protein binding

Michelle J. Yoo, John E. Schiel, David S. Hage

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


This study examined the use of affinity microcolumns as tools for the rapid analysis and high-throughput screening of drug-protein binding. The protein used was immobilized human serum albumin (HSA) and the model analytes were warfarin and l-tryptophan, two solutes often used as site-specific probes for drug binding to Sudlow sites I and II of HSA, respectively. The use of HSA microcolumns in binding studies was examined by using both zonal elution and frontal analysis formats. The zonal elution studies were conducted by injecting the probe compounds onto HSA microcolumns of varying lengths while measuring the resulting retention factors, plate heights and peak asymmetries. A decrease in the retention factor was noted when moving from longer to shorter column lengths while using a constant amount of injected solute. However, this change could be corrected, in part, by determining the relative retention factor of a solute versus a reference compound injected onto the same microcolumn. The plate height values were relatively consistent for all column lengths and gave an expected increase at higher linear velocities. The peak asymmetries were similar for all columns up to 1 mL/min but shifted to larger values at higher flow rates and when using short microcolumns (e.g., 1 mm length). The association equilibrium constants and number of binding sites estimated by frontal analysis for warfarin with HSA were consistent at the various column sizes that were tested and gave good agreement with previous literature values. These results confirmed affinity microcolumns provide comparable results to those obtained with longer columns and can be used in the rapid analysis of drug-protein binding and in the high-throughput screening of such interactions.

Original languageEnglish (US)
Pages (from-to)1707-1713
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Issue number20
StatePublished - Jun 15 2010


  • Affinity microcolumn
  • Drug-protein binding
  • Frontal affinity chromatography
  • Frontal analysis
  • High-performance affinity chromatography
  • High-throughput screening
  • Human serum albumin
  • Tryptophan
  • Warfarin
  • Zonal elution

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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