Solute attributes and molecular interactions contributing to retention on a fluorinated high-performance liquid chromatography stationary phase

by 1964- Bell, David S.

iii The structural attributes and molecular interactions contributing to “U-shape” retention on pentafluorophenylpropyl (PFPP) HPLC stationary phases are systematically investigated. Only basic analytes exhibit retention that increases with the acetonitrile content in mixtures of acetonitrile and aqueous ammonium acetate, with some basic analytes not eluting at all from PFPP columns using 100% acetonitrile. U-shaped retention as a function of mobile phase acetonitrile content was more dramatic on a PFPP column relative to C18. Retention of the quaternary ammonium salt bretylium on these stationary phases and on the same bare silica support showed minimal influence of ionexchange mechanisms on the C18 phase, however a significant influence of ion-exchange mechanisms was observed for both PFPP and bare silica. The retention of bretylium on PFPP was only slightly less than on bare silica. These findings suggest ion-exchange mechanisms dominate retention of basic analytes in the high acetonitrile realm on PFPP. The PFPP stationary phase exhibits a substantial increase in effects of ionized surface silanol groups compared to the alkyl phase despite similar surface coverage. Retention of some basic analytes on a PFPP phase was enhanced relative to retention on silica alone, and implicates other dispersive interactions that might be exploited for selectivity different from either alkyl phases or silica alone. NMR spectroscopy is demonstrated to be a rapid and useful technique for the determining pKa values of solutes in HPLC mobile phases. The variation of chemical shift data for protons in the vicinity of a basic nitrogen atom as a function of the medium pH can be related through the Henderson-Hasselbalch equation to estimate analyte pKa values. The use of a pH scale based on the measurement following addition of organic component ( s w pH) is shown to reflect the true thermodynamic reality of the environment about the analyte as compared to the pH scale based on the purely aqueous component. The present study demonstrates that one can not assume that bases are protonated in high acetonitrile content even if the aqueous pH is adjusted to less than two pKa units from the literature pKa value as is commonly practiced. The pKa values for the basic analytes used in this study were shown to decrease by approximately one pKa unit in approximately 90 v/v% acetonitrile from their aqueous value. Where ion-exchange mechanisms are present, improved prediction and manipulation of HPLC selectivity results from more accurate knowledge of the analyte degree of dissociation values. NMR spectroscopy provides a non-invasive and direct measure of the equilibria and its dependence on the pH of the media. Knowledge of such equilibria allows one to further explore the fundamental mechanisms of retention in chromatographic processes. Fluorinated, silica-based stationary phases are becoming increasingly popular alternatives to traditional alkyl phases owing to their differential selectivity and retention for a variety of analyte classes. In this report, the ion-exchange mechanisms characteristic of a fluorinated phase are exploited to rapidly develop separation conditions for ephedrine alkaloids and synephrine using a mobile phase compatible with mass spectrometry. A linear relationship of basic analyte retention with the reciprocal of ammonium acetate concentration is first established. This linear relationship can then be used to optimize retention and selectivity in just two experiments. The relationship of retention with temperature is also explored. Greater retention with increasing temperature is demonstrated on the fluorinated phase at high percentages of organic iv v modifier, which is in contrast to behavior observed in typical reversed-phase separations. The unexpected observation is explicated based on the reduction in solvent solvating power with increasing temperature. As solvation power of the mobile phase decreases, decreased solvation of both mobile phase and ionized surface groups of the stationary phase leads to stronger interactions between analyte and stationary phase. Both mobile phase ion concentration and temperature are shown to be powerful tools for the manipulation of analyte retention and selectivity.