| Scope and method of study. The broad objective of this research entailed an integrated approach to furthering the advancement of CEC by providing (i) improved design of stationary phase for use in reversed-phase CEC (RP-CEC) for the separation of complex biomolecules (e.g., peptides, proteins and glyconjugates), (ii) ensuring a relatively strong EOF without the presence of fixed charges on the stationary phase surface but still achieve fast analysis and good chromatographic selectivity, we developed and evaluated a neutral C17-monolithic stationary phase devoid of fixed charges and proved effective in RP-CEC and (iii) the development of biospecific (affinity) interactions in microcolumns, e.g., affinity CEC and affinity nano-LC based on poly(glycidylmethacrylate) polymers with immobilized lectins for the isolation and separation of minute amounts of biopolymers. We also introduced the serial use of lectin affinity nano-LC and RP-CEC for the sequential capturing and separation of glycopeptides.; Findings and conclusions. The overall investigation described in this dissertation has contributed to enlarging the scope of CEC by demonstrating its capabilities in areas that were not exploited previously. We introduced a novel neutral PEDAS monolith that belongs to the stratified type of stationary phases whereby the surface top layer is essentially the non-polar C17 ligands for chromatographic partitioning/adsorption of solutes by a reversed phase retention mechanism and the surface sub-layer contains polar functional groups for ions binding which impart the monolith with a given zeta potential to generate the EOF. On the other hand, lectin affinity CEC provided the simultaneous capturing and separation of different glycoconjugates due to differences in charge-to-mass ratio, thus emerging as a very suitable microcolumn separation technique for nano-glycomics/proteomics. This investigation has demonstrated for the first time the coupling of lectin capillary columns in series or sequential with RP-CEC for enhanced separation by LAC using the CEC modality. The novel stationary phases developed within the framework of this investigation will not only facilitate the solution of many important analytical separation problems in the life sciences, but also contribute to new concepts that will open the way to increasing the utility of the technique in the future. |
