Evaluating independent effect of mobile phase components on retention mechanism of ionizable analytes in hydrophilic...

Evaluating independent effect of mobile phase components on retention of analytes would provide robust foundation for managing selectivity in hydrophilic interaction liquid chromatography (HILIC). Since most of the analytes and stationary phases possess weak acid/base properties, mobile phase pH effect is the most important one. This study addresses limitations in current pH variation procedures, summarizes the principles for evaluating the independent effect of pH and proposes the method accounting for different buffer anion affinity in HILIC. Much attention is paid on the shift of dissociation constants and buffer pH in acetonitrile-rich medium. We carefully show how retention trends for the analytes of diverse properties depend on mobile phase pH, counterion concentration, acetonitrile content, and the nature of buffer solution. The valuable tool used to control the ionization state of both stationary phase and solutes was the slope of lgk' = f(lg[C]) dependence. The developed approach was used for the characterization of eremomycin-modified silica and retention behavior of ionizable compounds. Dissociation constants _w^spK_a were evaluated for benzoic acid, xanthine, and cytosine, and conditions for stationary phase charge switching were identified. The registered trends were similar to those obtained for some previously reported stationary phases. These findings offer practical guidance for controlling HILIC separation selectivity for ionizable compounds in multicomponent mixtures.