Capillary electrochromatography of proteins and peptides with porous-layer open-tubular columns

Capillary electrochromatography (CEC) of basic proteins and peptides was carried out with porous-layer open-tubular (PLOT) columns which had a functionalized rugulose polymeric porous layer grafted to the innerwall of 20 microm I.D. fused-silica capillaries. The porous layer was highly crosslinked and prepared by in situ polymerization of vinylbenzyl chloride and divinylbenzene in the presence of 2-octanol as a porogen inside a pretreated fused-silica capillary. The chloromethyl functions at the surface of the porous polymeric support layer were reacted with N,N-dimethyldodecylamine to obtain a positively charged chromatographic surface with fixed C12 alkyl chains. A mixture of lysozyme, cytochrome c, ribonuclease A and alpha-chymotrypsinogen A was separated isocratically by counterdirectional CEC with hydro-organic mobile phases containing acetonitrile and phosphate buffer, pH 2.5. The overall migration behavior of the four proteins was the result of an interplay of chromatographic retention and electrophoretic migration, and was different from that observed in capillary zone electrophoresis or in reversed-phase chromatography under similar conditions. The separation of three basic peptides by CEC also exhibited the same behavior. The stability of the PLOT column was tested by measuring electroosmotic mobility during continual use.