"Effect of temperature on the chromatographic retention of ionizable compounds. Modelling retention of pharmaceuticals as a function of eluent pH and column temperature in RPLC

GAGLIARDI, L. G.; C. B. CASTELLS*; RÀFOLS, C.; ROSÉS, M.; BOSCH, E.

Ghent, Belgica

Congreso; International Symposium on HPLC Separations and Related Techniques. HPLC'2007; 2007

Comite Organizador

We propose a general simple equation for accurately predicting the retention factors of ionizable compounds upon simultaneous changes in mobile phase pH and column temperature at a given hydroorganic solvent composition. Only four independent experiments provide the input data: retention factors measured in two pH buffered mobile phases at extreme acidic and basic pH values (e.g. at least ±2 pH units far from the analyte pKa) and at two column temperatures. The equations, derived from the basic thermodynamics of the acid-base equilibria, additionally require the knowledge of the solute pKa and enthalpies of acid-base dissociation of both, the solute and the buffer components in the hydroorganic solvent mixture. The quite good performance of the predictive model is corroborated with the comparison between theoretical and experimental retention factors of several weak acids and bases of important pharmacological activity (profens, b-blockers and alkaloids), in mobile phases containing different buffer solutions prepared in 25% (w/w) acetonitrile in water and at several temperatures. Our experimental results show that the retention factor significantly varies from one to another buffer (e.g., tris buffer compared to phosphate buffer). And, as it is expected, at mobile phase pH close to the pKa of the analyte and for buffer components which have an enthalpy of dissociation differing from that of the analyte, a special dependence of the retention on temperature would be conceived. Finally, we found no correlation between the mobile phase pH and the retention factor of aminoalcohols (b-blockers and alkaloids) in tris buffer. Instead, the nature of the tris buffer mainly controls the retention of these analytes, based on another retention mechanism besides hydrophobicity.