This continuation proposal aims at the design, preparation, and testing of completely new monolithic columns in capillary and sheet 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 life sciences. These new columns will be prepared via a simple free radical polymerization process. The preferred implementation will include preparation of a generic macroporous monolithic polymer with porous properties optimized for the desired application. The pore surface will then be provided with a wide variety of high-density functionalities including hydrophobic, hydrophilic, ionizable, and reactive moieties using UV initiated photografting. This highly versatile approach will also be used to create monoliths with unprecedented sequential and layered surface chemistries. Both preparation steps - polymerization and grafting - are independent, simple, waste-free, as well as less labor intensive, and enable the rapid development of a wide variety of tailor-made monolithic materials. Monolithic capillary columns will be specifically designed for the high throughput and/or multidimensional separations in microHPLC, as well as in capillary and thin layer electrochromatography. Using microHPLC, the separation of difficult protein mixtures will be explored using columns with carefully designed selectivities. Entirely new CEC separation modes enabled by the progress in grafted chemistries will be developed and used for the rapid and efficient separations of proteins and peptides. New approaches to thin layer chromatography and related methods making use of macroporous polymer sheets will be focused on the larger scale and 2-D separation of biomacromolecules and will include both pressure and electrodriven flow. This proposal also targets the development of an enhanced array of monolithic tools available for handling biological samples. Using the benefits of sequential photografting, monolithic devices will be prepared in the tip of electrospray emitters that will integrate sample collection, enzymatic digestion of proteins, and separation of peptides. Direct coupling of this device to mass spectrometer will simplify and accelerate proteomic research. Overall, our targets are to extend the applications of monolithic materials with both enhanced performance and unexpected capabilities in new territories and to demonstrate their vast potential in numerous areas.
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