HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF COMPLEX-MIXTURES USING MONODISPERSE DUAL-CHEMISTRY POLYMER BEADS PREPARED BY A PORE-SIZE-SPECIFICFUNCTIONALIZATION PROCESS - A SINGLE-COLUMN COMBINATION OF HYDROPHOBIC INTERACTION AND REVERSED-PHASE CHROMATOGRAPHY

A novel separation medium for HPLC combining hydrophobic interaction a nd reversed-phase separation modes in a single column has been prepare d from monodisperse 10-mu m poly(glycidyl methacrylate-co-ethylene dim ethacrylate) beads using a pore-size-specific functionalization proces s. In this approach, the large pores of each bead were provided with p henyl groups interspersed among hydrophilic functionalities while a mu ch higher surface concentration of hydrophobic phenyl groups was intro duced into the small pores. Due to the size-specific character of the modification process, no protein interaction with any highly hydrophob ic surface was observed during chromatography. The beads were used for the separation of samples containing both proteins and small hydrocar bon or drug molecules. A plot of log k' against salt concentration in the mobile phase clearly documents the clean hydrophobic interaction m echanism of protein separation and the absence of charged groups while the linear plot of log k' against acetonitrile concentration for nume rous compounds demonstrates the reversed-phase separation ability. No decrease of the efficiency of the test column (23 000 plates/m) was ob served in long-term experiments during which more than 1000 injections and many changes between the modes were performed.