High-Performance Liquid Chromatography of Complex Mixtures Using Monodisperse Dual-Chemistry Polymer Beads Prepared by a Pore-Size-Specific Functionalization Process. A Single Column Combination of Hydrophobic Interaction and Reversed-Phase Chromatography

A novel separation medium for HPLC combining hydrophobic interaction and reversed-phase separation modes in a single column has been prepared from monodisperse 10-μm poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads using a pore-size-specific functionalization process. In this approach, the large pores of each bead were provided with phenyl groups interspersed among hydrophilic functionalities while a much higher surface concentration of hydrophobic phenyl groups was introduced into the small pores. Due to the size-specific character of the modification process, no protein interaction with any highly hydrophobic surface was observed during chromatography. The beads were used for the separation of samples containing both proteins and small hydrocarbon or drug molecules. A plot of log k′ against salt concentration in the mobile phase clearly documents the clean hydrophobic interaction mechanism of protein separation and the absence of charged groups while the linear plot of log k′ against acetonitrile concentration for numerous compounds demonstrates the reversed-phase separation ability. No decrease of the efficiency of the test column (23 000 plates/m) was observed in long-term experiments during which more than 1000 injections and many changes between the modes were performed.