Retention of ionizable compounds on HPLC. 6. pH measurements with the glass electrode in methanol–water mixtures

The relationship, δ values, between the two rigorous pH scales, sspH (pH measured in a methanol–water mixture and referred to the same mixture as standard state) and swpH (pH measured in a methanol–water mixture but referred to water as standard state), in several methanol–water mixtures was determined (δ=swpH−sspH). δ values were measured using a combined glass electrode and a wide set of buffer solutions. The results are consistent with those obtained with the hydrogen electrode. This confirms the aptness of the glass electrode to achieve rigorous pH measurements in methanol–water mixtures. An equation that relates δ and composition of methanol–water mixtures, and allows δ computation at any composition by interpolation, is proposed. Therefore, sspH can be achieved from the experimental swpH value and δ at any mobile phase composition. sspH (or swpH) values are related to the chromatographic retention of ionizable compounds through their thermodynamic acid–base constants in the methanol–water mixture used as mobile phase. These relationships were tested for the retention variation of several acids and bases with the pH of the mobile phase. Therefore, the optimization of the mobile phase acidity for any analyte can be easily reached avoiding the disturbances observed when wwpH is used.