Role of binding capacity versus binding strength in the separation of chiral compounds on protein-based high-performance liquid chromatography columns - Interactions of D- and L-tryptophan with human serum albumin

Frontal analysis was used to examine changes in the association constant (K-a) and moles of binding sites (m(L)) for D-and L-tryptophan on an immobilized HSA column under various elution conditions. Both enantiomers had single-site interactions under all conditions tested. At pH 7.0 and 25 degrees C, the strength of L-tryptophan/HSA binding was determined mostly by the change in enthalpy of the system, while D-tryptophan/HSA binding was dominated by the change in entropy. The interactions of L-tryptophan with HSA showed a large change when varying the temperature, pH, ionic strength or 1-propanol content of the mobile phase. In each case, changes in K-a accounted for most of the shifts in retention that were seen for L-tryptophan during zonal elution studies. However, m(L) for this compound was also affected when varying the pH and 1-propanol levels. Changes in K-a were responsible for most of the shifts in D-tryptophan retention that were seen when adjusting the mobile phase pH or ionic strength. In addition, the value of m(L) for D-tryptophan was affected by pH, temperature and 1-propanol levels. It was concluded that varying such chromatographic conditions can alter either the binding strength or number of binding sites for solutes injected onto immobilized protein columns.