Retention of ionizable compounds on HPLC.: 5.: pH scales and the retention of acids and bases with acetonitrile-water mobile phases

The pH calibration procedures that lead to the different pH scales in acetonitrile-water mixtures used as mobile phases in reversed-phase liquid chromatography are discussed. Appropriate buffers of known pH value in acetonitrile-water mixtures are selected and used to establish the relationship (delta values) between the two rigorous acetonitrile-water pH scales: (s)(s)pH and (s)(w)pH (pH measured in acetonitrile-water mixtures and referred to acetonitrile-water or water, respectively, as standard state). These delta values allow one to convert pH values measured in acetonitrile-water with electrode systems calibrated with aqueous buffers ((s)(w)pH scale) to (s)(s)pH values, which are directly related to the thermodynamic acid-base constants. This offers an easy way to measure the pH of acetonitrile-water mobile phases and to relate this pH to the chromatographic retention of acids and bases through the thermodynamic acid-base constants. The relationships are tested for the variation of the retention of acids and bases with the pH of the mobile phase at several mobile-phase compositions and favorably compared with the relationships obtained with the common (w)(w)pH scale (pH measured in the aqueous buffer before mixing it with the organic modifier). The use of the rigorous (s)(s)pH and (s)(w)pH scales allows one to explain the retention behavior of bases, which in many instances cannot be justified from the pH measurement in the ill-founded (w)(w)pH scale.