This continuation proposal aims at the design, preparation, and testing of completely new monolithic columns in capillary and thin layer formats that will provide unmatched potential for the study, isolation, separation, detection, and identification of biologically active molecules and enable to fully exploit the rich potential of studies in the life sciences. The new monolithic columns and layers will be prepared via a simple free radical polymerization process initiated both thermally and photochemically. One of our main targets will be design and development of macroporous monolithic column suitable for the separation of small molecules and stable in the entire pH range using a sacrificial scaffold approach. Another target will involve novel narrow bore open tubular capillary columns with inner walls covered with a thin layer of porous monolithic polymer. The pore surface of the capillary monolithic columns will then be used directly for high efficiency separations, or modified through UV initiated photografting to incorporate a wide variety of high-density, tailor-made functionalities including hydrophobic, ionizable, and reactive groups. These capillary columns will be specifically designed to achieve the very high efficiency separations of difficult protein and peptide mixtures in nano LC mode. Finally, new approaches to thin layer chromatography and related methods making use of macroporous polymer thin layers will focus on new, extremely thin layers for the 1-D and 2-D separations of biomacromolecules such as peptides and proteins and their direct detection using MALDI-TOF MS. The mobile phase will be driven by capillary forces, pressure, and/or electroosmotic flow. In order to achieve MALDI detection of midsize and large molecules without the need for a low molecular weight matrix, this proposal also targets the development of thin layers including specifically designed monomers carrying functionalities enhancing desorption and ionization of biological samples. These monolithic plates will be prepared using both copolymerization and photografting techniques. Direct use of these plates in mass spectrometer will simplify and accelerate proteomic research. Overall, our targets are to extend the applications of monolithic materials providing them with both enhanced performance and unexpected capabilities in new applications and to demonstrate their vast potential in various areas. This proposed research focuses on the development of monolithic polymers that will serve as separation media, adsorbents, and supports providing real benefits to both the scientists who perform ever more demanding separations with ever smaller amounts of complex samples as well as the engineers who design advanced separation units. Our results can also be used for the engineering of new separation and detection devices suitable for separation of very complex samples such as proteomes, in biochemical assays, and in early diagnostics of diseases with direct impact on affordable health care.
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