Protein separation and analysis by a high performance capillary isoelectric focusing system
Abstract
This thesis reports separations of proteins by a capillary isoelectric focusing (CIEF) system with deactivated fused silica capillaries. Deactivation was achieved by adsorbing either a surfactant or hydrophilic polymer to alkylsilane derivatized capillaries. The coating reduced electro-osmotic flow (EOF) 20 to 30 fold in comparison to underivatized capillaries. Although EOF was reduced sufficiently to allow focusing to permit separations to be completed before proteins were swept through the capillary, there was adequate flow to obviate the need for a separate mobilization step. This reduces the complexity of CIEF and increases reproducibility. Based on resolution of hemoglobin variants, proteins that varied 0.03 pH units in isoelectric point were resolvable. This is equivalent to the highest resolution achieved in conventional slab and tube gel isoelectric focusing. Another novel approach in CIEF mobilization was the use of pumping-capillary electroosmotic flow mobilization (PCEOF). The resolution of separation from PCEOF is as good as that from the common salt mobilization. The different magnitudes of EOF in coated capillaries can be used for the control of analysis time and resolution without compromising separations. Rapid resolutions of Hb Al indicated that CIEF has potential to become a routine clinical monitoring tool. CIEF will become a powerful analytical tool in the fast growing field of biotechnology.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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