Development of a new strategy for the modification of monolithic stationary phases used in capillary electrochromatography

摘要

In the past two decades, growing research and commercial activity in the fields of biotechnology and medicine has spawned the development of minore efficient separation methods capable of handling increasingly complex and smaller samples. In this new research playpen, capillary electrochromatography (CEC), combining both electrophoretic and chromatographic separation mechanisms, has the potential of playing an important role. Nonetheless, the development and optimization of specialized electrochromatographic stationary phases capable of providing high separation efficiencies for specific applications, such as proteomics, is first required. This Ph.D. research thesis reports on the development of multifunctional monolithic stationary phases employed principally in capillary electrochromatography. In order to enable the fabrication of such multifunctional monoliths, a simple and novel copolymer grafting followed by functionalization strategy for the adjustment of a monolith's properties was developed. The developed methodology was compared to the classical approach based on adjustment of individual polymerization conditions for the fine-tuning of monolithic support. The grafting approach enabled the efficient introduction of functional moieties on an existing monolithic stationary phase while retaining physical and morphological properties critical to its electrochromatographic properties. The classical approach would have required reoptimization of the polymerization conditions. To demonstrate that the developed technique allows easy fabrication of a multitask monolithic support, selected proteomics applications were developed. One of these applications was the development of proteolytic microreactors fabricated via direct and linker-mediated immobilization of trypsin, a proteolytic enzyme, on a reactive monolith. The monolith was made reactive by photografting glycidyl moieties. The proteolytic reactors exhibited high proteolytic efficiency, reproducibility and stability. In addition to the enzymatic capacities the monolith preserved its electrochromatographic properties which could enable its implementation for on-line high-throughput digestion of minute amounts of proteins. The same photografting methodology was used to produce boronate-functionalized monoliths for the (electro)chromatographic differentiation of glycosylated substrates from their non-glycosylated counterparts. Spectroscopic analyses clearly showed the efficient immobilization of boronate and resulted in efficient separation of glycosylated and non-glycosylated proteins. Finally, an atomic force microscopy (AFM) characterization technique able to provide physical information in wetted and dry conditions was developed in order to address the general lack of information on structural and morphological properties of monolithic stationary phases in their working (electro)chromatographic conditions.