Chiral ion-exchange chromatography. Correlation between solute retention and a theoretical ion-exchange model using...

Mobile phase effects were studied in the separation of D- and L-phenylalanine anilide (D,L-PA) on an imprinted chiral stationary phase (CSP). Using an aqueous-organic mobile phase, an improved column performance was seen, reflected in a two-fold decrease in the reduced plate height and an almost doubling of the resolution as compared to when a pure organic mobile phase was used. A strong dependence of retention (k') and enantiomer selectivity (alpha) on mobile phase pH was observed. k' reached a maximum at a pH close to the pKa value of the solute and alpha was high at low pH value but decreased when pH exceeded the solute pKa. Potentiometric titration data allowed estimation of the state of protonation of both the carboxylic acid containing CSP and the amino group containing solutes. The data are analyzed using a simple cation-exchange model to allow simulation of the retention as a function of mobile phase pH. The close agreement between the simulated and experimental curves for retention versus pH suggests that a simple cation-exchange mechanism controls the retention in this system. Moreover, the slightly lower average pKa of the imprinted polymer compared to that of a corresponding blank polymer explains the high selectivity seen at low pH values. Based on these findings, a model describing the events controlling binding and selectivity as a function of pH is proposed.