In this study several properties of acetonitrile-water (ACN-water) mixtures were examined which have an influence on the liquid chromatography (LC) behavior of ionizable compounds, such as Debye-Hückel parameters, pH scale, LC-useful pH range and solvatochromic parameters. Factor analysis techniques were applied to the correlation between standard pHs values of NIST buffer solutions and solvatochromic parameters in acetonitrile (ACN)-water. The relationships obtained allow the calculation of standard pHS values in any ACN-water mixture and thus permit correct pH measurement in these media. Through correct pH, pKa and activity coefficient values, a model describing the effect of pH on retention in LC is established and tested for several diuretic acids and peptide ampholytes using an octadecylsilica column and ACN-water mixtures of 7, 30, 35, 40, 45 and 50% (v/v) ACN as mobile phases. The proposed model uses the pH values in ACN-water mobile phases instead of in water, and takes into account the effect of activity coefficients. The usefulness of the proposed equations is twofold: they permit the determination of pKa of analytes in the hydroorganic media used as mobile phases, and also permit the prediction of the optimum pH for the separation methodologies using a minimum number of measurements. The proposed equations can be combined with the previously derived equation, which relates the retention to the composition of the mobile phase, in order to establish a general model relating elution behavior of the solutes with the significant mobile phase properties: composition, pH and ionic strength.