Construction Of Capillary Electrophoresis-Based Two-Dimensional Separation System

Complex protein/peptide samples are beyond the capacity of 1D separationtechniques. 2D-PAGE,a widely used 2D separation technique in proteomics hasinherent limitations,therefore new developments in the area of multidimensionalprotein/peptide separation involving at rapid pace, high-throughput play a key role inproteomics research. The key problem in constructing a 2D separation system lies inthe construction of a reliable interface to switch the effluents from thefirst-dimension column to the second one. Although various switching valves andstorage loops employed as interfaces for LC/LC have been developed, it is still ahard nut to fabricate a satisfactory interface for a 2D-CE system. The object of thisdissertation is to develop interfaces for on-line CE-based 2D separation systems aswell as the related technologies for the analysis of complex protein samples.Commercial micro-dialysis hollow fiber as the interface has been employed inconstructing 2D-CE systems, however, the significant dead volume would be usuallyintroduced in 2D systems due to the dimensions of the capillary unmatched to thoseof the commercial fiber. In this work, a simple and fast technique for preparingmicro-dialysis hollow fibers with desired dimensions was introduced, and the2D-CIEF-CNGSE system for proteins has been developed using the self-madehollow fiber. The feasibility and the application of the combined CIEF-CNGSEseparation technique were demonstrated by examining each technique independentlyfor the separation of Haemoglobin and mixtures ofproteins excretingfrom lung cancercells of rats. The 2D separation strategy was found to greatly increase the resolvingpower and overall peak capacity over those obtained for either dimension alone.The significant dead volume could not be avoided in all the present 2D-CEsystems, whether by an interface of cross, valve/loop or dialysis hollow fiber, whichdefinitely decreases the resolution of 2D separations. Here, we presented theconstruction and evaluation of a simple porous junction with no dead volume bysimply etching a short segment on a capillary. A 2D-CIEF-CZE method using thisnovel junction was developed for the separations of protein mixtures. A largeimprovement was made in preparation of a good conductive interface by the etchedporous junction, which is particularly well suited for an on-line coupling CE-basedmultiple dimensional separation system. To reduce the dispersion of the focused protein zones in CIEF and mobilize thefocused zones to detect during the performing a two-step CIEF, a simple open tubularelectroosmotic pump (OTEOP), operating in a high electric field, was connected toCIEF capillary and isolated from other components by an on-column etched porouselectrical junction that permitted the pumped fluid hydrodynamically to mobilize thefocused zones in CIEF capillary. The output flow rate of the OTEOP is controlled bythe applied voltage, which in turn controls the mobilization rate accordingly. Coaxial sheath flow interface is the basis of most commercial instruments incoupling CE on-line with MS. However,sheath flow suffers in sensitivity largelyfrom the relatively high sheath flow compared the flow from the CE capillary,resulting in not only a large dilution of the eluting analytes but also in hindereddesorption of ions. In this work, a rugged and simple sheathless etched porousjunction has been designed to couple CE on-line with MS where the separationcolumn, a porous electrical junction, and the electrospray tip were integrated on asingle piece of a fused-silica capillary. The new interface employed in theCE/ESI-MS was systemically investigated and was used for the analyses of complexprotein samples. The sheathless interface is particularly well suited for the detectionof low levels of proteins and peptides because of the compatible flow rates of eachtechnique.