Characterization of the binding and chiral separation of d- and l-tryptophan on a high-performance immobilized human serum albumin column

Abstract

High-performance affinity chromatography was used to study the separation and binding of d- and l-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both d- and l-tryptophan were binding to single but distinct sites on HSA. l-Tryptophan bound to the indole site of HSA. d-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of d- and l-tryptophan at pH 7.4 and 25°C were 0.4·10 4 and 2.7·10 4 M −1, respectively. The value of Δ G for these sites ranged from −5.2 to −5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of d- and l-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of d- and l-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.