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The different procedures used in HPLC to measure the pH of a mobile phase are evaluated in terms of the rigorous IUPAC definition of pH. The three procedures evaluated are as follows: measurement of the pH of the aqueous HPLC buffer before mixing it with the organic modifier, measurement of the pH of the HPLC buffer after mixing it with the organic modifier using a pH electrode system calibrated with aqueous buffers, and measurement of the pH of the HPLC buffer after mixing it with the organic modifier but calibrating the electrode system with reference buffers prepared in the same mixed solvent used as mobile phase. Following IUPAC definitions and recommendations, the three pH values can be related with the pH scales: w(w)pH, s(w)pH, and s(s)pH, respectively. The relationships between these three pH scales are also presented. The retention of several compounds with acid/base behavior in a C-18 and a polymeric column with buffered methanol/water as mobile phase is related to the mobile phase pH value measured in the three pH scales. It is demonstrated that the s(w)pH and s(s)pH scales give better relationships than the w(w)pH scale (pH measured in the aqueous buffer before mixing it with the organic modifier), commonly used on HPLC. The s(w)pH scale is specially recommended because of its simplicity of measurement: the pH is measured after mixing the aqueous buffer with the organic modifier, but the pH calibration is performed with the common aqueous reference buffers.

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A new model that relates the retention of a weak acid in HPLC columns with the pH and ionic strength of the mobile phase is derived and tested for different benzoic acids in methanol-water mobile phases. The proposed model uses the pH value in the mobile phase instead of the pH value in water, takes into account the effect of the activity coefficients, and considers different holdup times for the neutral and ionic species. The dependence of the holdup time of the ionic species on the mobile phase properties (pH, solvent composition, and ionic strength) is evaluated. It is demonstrated that the holdup time of the neutral species does not depend on the mobile phase properties, but the holdup time of the ionic species depends on the particular buffer used. The proposed equations can be combined with previously derived equations that relate the retention with the solvent composition of the mobile phase to establish a general model that relates the retention of the solute with the significant mobile phase properties:  composition, pH, and ionic strength.

The relationships between retention and mobile-phase pH in gradient elution are studied for acids and bases. The apparent pH shift caused by the increasing amount of acetonitrile and methanol has been determined starting from a wide range of pH values. It is shown that good relationships between the retention of ionizable compounds and the pH of the aqueous buffer can be established if the same type of buffer (ammonium acetate in this work) is used for all pH points. Equations are proposed to fit the gradient retention data to the pH of the aqueous buffer. The proposed equation gives an account of the relative variation of the pKa of the compound in the reference to the variation of the pH of the buffer as both parameters change during gradient elution.