Separation of Proteins with Capillary Electrophoresis in Coated Capillaries with and without Electroosmosis : Studies on Zone Broadening and Analytical Performances

Sammanfattning: Proteins have such structural features that they may interact with different types of surfaces by all possible forces, i.e., electrostatic, hydrogen bonding, hydrophobic. In this thesis two different types of coatings for fused silica capillaries aimed to eliminate such interactions have been studied. The first is a covalent, electroosmosis-free coating with polyacrylamide (PAA) and the second involves a non-covalent coating with the quaternary ammonium compound N, N-didodecyl –N, N- dimethylammonium bromide (DDAB) with a strong anodic electroosmosis. Optimal conditions regarding efficiency and resolution were established by variations of the composition and ionic strengths of buffers at pH below the isoelectric point of the proteins. To achieve high efficiency and resolution the choice of buffer constituents was extremely important. The PAA coating was very stable at neutral and acidic conditions. Ammonium acetate (0.12 M) and ammonium hydroxyacetate (0.15 M) both at pH 4 provided the best separations with plate numbers up to 1 700 000 plate/m that is among the highest reported in the literature. Capillaries coated with DDAB were stable enough to, without recoating, permit consecutive separations of the proteins up to 9 hours (90 injections). High apparent efficiencies (over 1 million plates/m) were achieved with ammonium acetate (0.07 M), ammonium hydroxyacetate (0.08 M) and sodium phosphate (0.1 M) at pH 4. Zone broadening was studied by determination of the variance contributions from all main parameters. Significant variances were contributions from longitudinal diffusion, capillary curvature, injection plug, detector time response and detector slit width while other variances, e.g., variances for Joule heat and vertical sedimentation were negligible. The remaining undetermined variance may have its origin in all types of relatively slow interactions including adsorption onto the capillary surfaces and protein-buffer component interactions. The results indicate that the latter is the main cause to zone broadening in protein separations.