Retention of Ionizable Compounds on HPLC. pH Scale in Methanol−Water and the pK and pH Values of Buffers

Abstract

Several properties of methanol−water mixtures (molar volume, molar refractivity, Debye−Hückel parameters, pH scale, HPLC-useful pH range, dissociation constants of acids) are studied. Some macroscopic properties are used to calculate the extent of the methanol−water interaction. Solute properties, such as pKa values, depend on the preferential solvation of the solute by the three solvents that coexist in the mixture (methanol, water, methanol−water aggregrates). Based on this preferential solvation and on the methanol−water interaction, equations that relate the pKa values of acids with the solvent composition are derived. The equations are applied to several acids widely used to prepare HPLC buffers. The pKa of an acid and the pH of a buffered solution at any methanol−water composition can be easily calculated from the proposed equations. The chromatographic retention of any solute can be thus related to the real pH value of the mobile phase used. This procedure is more rigorous and should give better results than the usual method of relating the retention to the pH value of the aqueous buffer before mixing it with methanol.