Characterization of Retention Mechanisms in Mixed-Mode HPLC with a Bimodal Reversed-Phase/Cation-Exchange Stationary Phase

Mixed-mode HPLC (MM-HPLC), combining different interactions or retention modes in a single column, can be an interesting alternative to reversed-phase HPLC, notably to achieve the combined retention of polar and non-polar species. In the present fundamental study, we have selected one bimodal stationary phase allowing for both reversed-phase and weak cation-exchange retention modes (Acclaim mixed-mode WCX-1 LC). First, the mobile phase buffer composition (buffer pH ranging from 5 to 7 and concentration ranging from 20 to 100 mM) was explored with a small set of probe compounds (15 acids, bases and neutrals) to ensure adequate retention and peak shapes for the target compounds, and to evaluate the relative contributions of reversed-phase and ion-exchange mechanisms. Second, retention values measured for 63 probe compounds with various proportions of acetonitrile (ranging from 30 to 80%) served to establish linear solvation energy relationships based on (a) the usual and (b) a modified version of the solvation parameter model comprising additional descriptors to take account of interactions with ionizable species to bring some insights into the retention mechanisms. Finally, temperature effects at the low (30%) and high (60%) proportions of acetonitrile were observed between 20 and 40 °C (with 5 °C increments) and Van’t Hoff plots were drawn to measure the changes in interactions energies when the mobile phase composition changed.