pH/Organic Solvent Double-Gradient Reversed-Phase HPLC

A new reversed-phase high-performance liquid chromatographic (RP HPLC) procedure has been theoretically and experimentally established. The approach consists of the simultaneous development of a gradient of pH and of the organic modifier in the mobile phase. The proposed theoretical model of the pH/organic solvent double-gradient RP HPLC allows determination of both pK a and the lipophilicity parameter of the ionized and the nonionized form of the analyte and prediction of the retention times at specific separation conditions as well as bandwidth for all analytes. The model provides a rational basis for optimization of separation of ionizable analytes at any given chromatographic mode and analysis conditions. In addition, in the case of pH/organic solvent double-gradient RP HPLC, a compression of analyte peak and its reduced tailing can be expected.