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 aceto nitrile-water mixtures used as mobile phases in reversed-phase liquid chrom atography are discussed. Appropriate buffers of known pH value in acetonitr ile-water mixtures are selected and used to establish the relationship (del ta 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 ac etonitrile-water or water, respectively, as standard state). These delta va lues allow one to convert pH values measured in acetonitrile-water with ele ctrode systems calibrated with aqueous buffers ((s)(w)pH scale) to (s)(s)pH values, which are directly related to the thermodynamic acid-base constant s. This offers an easy way to measure the pH of acetonitrile-water mobile p hases and to relate this pH to the chromatographic retention of acids and b ases through the thermodynamic acid-base constants. The relationships are t ested for the variation of the retention of acids and bases with the pH of the mobile phase at several mobile-phase compositions and favorably compare d with the relationships obtained with the common (w)(w)pH scale (pH measur ed in the aqueous buffer before mixing it with the organic modifier). The u se of the rigorous (s)(s)pH and (s)(w)pH scales allows one to explain the r etention behavior of bases, which in many instances cannot be justified fro m the pH measurement in the ill-founded (w)(w)pH scale.