Influence of the mobile phase and molecular structure parameters on the retention behavior of protonated basic solutes in chaotropic chromatography

Abstract In this study, we present novel insights into the pH-dependent retention behavior of protonated basic solutes in chaotropic chromatography. To this end, two sets of experiments were performed to distinguish between mobile phase pH and ionic strength effects. In the first set, the ionic strength ( I ) was varied with the concentration of NaPF 6 and additives that adjusted the mobile phase pH, while in the second set, I was kept constant by adding the appropriate amount of NaCl. In each set, the retention behavior of 13 analytes was qualitatively examined in 21 chromatographic systems, which were defined by the NaPF 6 concentration in their aqueous phases (1–50 mM) and the pH of their mobile phases (2, 3 or 4); the acetonitrile content was fixed at 40%. The addition of NaCl significantly reduced the differences among retention factors at studied pH values due to the effect of the Na + ions on PF 6 − adsorption to the stationary phase and the magnitude of the consequential development of the surface potential. A quantitative description of the observed phenomenon was obtained by an extended thermodynamic approach. The contribution of ion-pair formation in the stationary phase to the retention of the solutes was confirmed across models at the studied pH values in the set with varying I. In the systems with a constant I , the shielding effect of the Na + ions on the surface charge lowered the attractive surface potential and diminished the aforementioned interactions and hence the effect of the mobile phase pH on analyte retention. Eventually, we developed a readily interpretable empirical retention model that simultaneously takes into account analyte molecular structures and the most relevant chromatographic factors. Its coefficients have clear physical meaning, and owing to its good predictive capabilities, the model could be successfully used to clarify the contributions of analyte molecular structures and chromatographic factors to the specific processes underlying separation in chaotropic chromatography.