Comparative evaluation of bonded-silica reversed-phase columns for high-performance liquid chromatography using strongly basic compounds and alternative organic modifiers buffered at acid pH

The performance of eight silica-based RP-HPLC columns evaluated previously with relatively high-pK(a) bases using isoeluotropic mixtures of either methanol, acetonitrile or tetrahydrofuran in combination with phosphate buffer pH 7.0, was further examined with the same solutes and mobile phases buffered at pH 3.0. Differences in column performance dependent on choice of organic modifier were shown to be considerably reduced at acid pH, although tetrahydrofuran again gave significantly better performance than acetonitrile or methanol. While differences between columns were less apparent at pH 3.0, some columns again clearly performed better than others. Furthermore, the ranking of columns according to average asymmetry of the set of basic compounds was found to vary somewhat with pH; thus column evaluation should be performed preferentially at the pH of intended use. The dependency of column performance on choice of solute was reduced, but still evident at pH 3.0. All solutes except pyridine gave superior results at acid pH. Solutes which were the most challenging probes at pH 7.0 were not necessarily the most difficult at pH 3.0.