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 (Ka) and moles of binding sites (mL) 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 Ka accounted for most of the shifts in retention that were seen for L-tryptophan during zonal elution studies. However, mL for this compound was also affected when varying the pH and 1-propanol levels. Changes in Ka 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 mL 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.
Yang
J
J
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Hage
D S
DS
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011485:Protein Binding; D012709:Serum Albumin / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000302:isolation & purification* / Q000378:metabolism
D012709:Serum Albumin; D014364:Tryptophan