Chromatographic behavior of ionizable compounds in liquid chromatography. Part 2. Standardization of potentiometric sensors and effect of pH and ionic strength on the retention of analytes using acetonitrile–water mobile phases

Abstract

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 pH S values of NIST buffer solutions and solvatochromic parameters in acetonitrile (ACN)–water. The relationships obtained allow the calculation of standard pH S values in any ACN–water mixture and thus permit correct pH measurement in these media. Through correct pH, p K a 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 p K a 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.