Evaluation of new mixed-mode UHPLC stationary phases and the importance of stationary phase choice when using low ionic-strength mobile phase additives.

In this study, the selectivity, retention properties, peak shape and loading capacity for bases were practically evaluated using two UHPLC mixed-mode hybrid CSH stationary phases modified by C18 or Phenyl group. The data were compared with the data obtained on other UHPLC hybrid stationary phases (BEH C18, BEH C8, BEH Phenyl and BEH Shield RP18) at both basic and acidic conditions using conventional HPLC buffers (50mM ammonium formate/acetate) as well as low ionic-strength additives such as, e.g. 0.1-0.01% formic/acetic acid and 1mM solution of ammonium formate/acetate, which are widely used in LC-MS applications. Ten pharmaceutically important compounds encompassing acids, bases and neutral were included into the study. Due to properties of CSH sorbent (which possess positively charged surface besides RP group), much improved peak shapes and weaker retention was obtained for bases even at very low concentration of acidic additives. Such conditions are ideally suited for LC-MS analysis of bases, where typical RP chromatographic separation (retention and good selectivity at basic pH) and LS-MS conditions (efficient ionization at acidic pH) are not in agreement. On the other hand, acids were more strongly retained and for some compounds the peak shape was influenced negatively due to ion-exchange mechanism. Further, the behavior of acidic, basic and neutral solutes is discussed using various additives at both basic and acidic pH for all above stated columns. The robustness of retention times after pH change from basic to acidic was also evaluated. The new CSH stationary phases represent an interesting selectivity tool preferably for separation of basic compounds.