| For capillary electrochromatography (CEC) to become a practical analytical method for the separation of biopolymers, further progress is needed in understanding the separation mechanism and in developing especially designed columns to meet the unique requirements arising from the combined separation powers of chromatographic retention and electrosmotic flow (EOF). In addition to providing retentive sites for the sample components, the stationary phase in CEC is also involved in generating EOF. This makes the column the ‘heart’ of the CEC system.; In this study, several novel polymeric stationary phases for CEC of biopolymers were designed and prepared. They include highly crosslinked porous methacrylate and styrenic monoliths as well as micron-sized, monodisperse methacrylate microspheres. The chromatographic surfaces of the stationary phases were conveniently functionalized to carry both retentive and ionic sites. In order to separate positively charged biomolecules, several types of the sorbents were functionalized to have quaternary ammonium or tertiary amine groups at the surfaces that are also positively charged at low pH to generate EOF. The electrostatic repulsion between the surface and the sample components diminishes the electrostatic binding and the long hydrocarbonaceous chains grafted on the surface function as the chromatographic retentive sites. Meanwhile, novel anionic- and cationic-exchangers were also prepared for the CEC separation of biomolecules when the retention mode is based mainly on electrostatic interactions between the migrants and the sorbents.; Several important applications of these stationary phases are illustrated. The capillary elecrochromatographic behaviors of the sorbents for the separation of proteins, peptides, nucleotides and macrolide antibiotics were tested. The efficiencies of the columns were exceptionally high compared to that of high performance liquid chromatography (HPLC). This was attributed to the success in designing the stationary phases and the use of electrosmotic flow.; Studies were also carried out to investigate the influence of temperature and mobile phase composition on the retention and selectivity of CEC separations. The results suggest that the separation is governed by a dual mechanism that involves the complex interplay between selective chromatographic retention and differential electrophoretic migration. It also shows that the major advantage of our CEC system is that biomolecules may be separated in the isocratic elution mode with the resolution approaching that achieved in gradient HPLC.; The studies of the effect of temperature on CEC separations also demonstrate the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. This again clearly proves the successful design of the stationary phase and the potentially important role of CEC in the separation of biopolymers. |
