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.
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
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J
Yang
D S
DS
Hage
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