Capillary electrochromatography with monolithic-silica columns. II. Preparation of amphiphilic silica monoliths having surface-bound cationic octadecyl moieties and their chromatographic characterization and application to the separation of proteins and other neutral and charged species

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Three different synthetic routes have been introduced and evaluated for the preparation of amphiphilic silica-based monoliths possessing surface-bound octadecyl ligands and positively charged groups. The amphiphilic silica monoliths (designated as cationic C18-monoliths) have been designed for use in reversed-phase capillary electrochromatography (RP-CEC) with hydro-organic mobile phases. These amphiphilic stationary phases yielded anodic electroosmotic flow (EOF) over a wide range of mobile phase pH. The magnitude of EOF remained constant up to pH 4.0 and then decreased at pH > 4.0 due to the ionization of silanol groups and the subsequent decrease in the net positive surface charge density of the amphiphilic monoliths. The cationic C18-monoliths exhibited reversed-phase chromatography (RPC) behavior toward non-polar solutes (e.g., alkyl benzenes), which parallels that observed with octadecyl-silica (ODS) monoliths. On the other hand, the amphiphilic stationary phases exhibited both non-polar and polar interactions toward slightly polar solutes such as anilines and PTH-amino acids. CEC retention factor k* and velocity factor k*e, which reflects the contribution of the electrophoretic mobility, were evaluated for charged solutes such as anilines and proteins.