Silica-based monolithic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography

This article describes the synthesis, chromatographic characterization, and performance evaluation of analytical (100 x 4.6 mm id) and semipreparative (100 x 10 mm id) monolithic silica columns with mixed-mode RP/weak anion-exchange (RP/WAX) surface modification. The monolithic RP/WAX columns were obtained by immobilization of N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-modified monolithic silica columns (Chromolith) by a radical addition reaction. Their chromatographic characterization by Engelhardt and Tanaka tests revealed slightly lower hydrophobic selectivities than C-8 phases, as well as higher polarity and also improved shape selectivity than RP-18e silica rods. The surface modification enabled separation by both RP and anion-exchange chromatography principles, and thus showed complementary selectivities to the RP-18e monoliths. The mixed-mode monoliths have been tested for the separation of peptides and turned out to be particularly useful for hydrophilic acidic peptides, which are usually insufficiently retained on RP-18e monolithic columns. Compared to a corresponding particulate RP/WAX column (5 microm, 10 nm pore diameter), the analytical RP/WAX monolith caused lower system pressure drops and showed, as expected, higher efficiency (e.g. by a factor of about 2.5 lower C-term for a tetrapeptide). The upscaling from the analytical to semipreparative column dimension was also successful.