CHARACTERIZATION OF THE BINDING AND CHIRAL SEPARATION OF D-TRYPTOPHAN AND L-TRYPTOPHAN ON A HIGH-PERFORMANCE IMMOBILIZED HUMAN SERUM-ALBUMIN COLUMN

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-degrees-C were 0.4.10(4) and 2.7 - 10(4) M-1, respectively. The value of DELTAG 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.