This continuation proposal is aimed at the design, preparation, and testing of "molded" monolithic separation media in entirely new shapes and formats that complement current bead-based packing materials and provide new and unmatched capabilities for the study, detection, isolation, and separation of biologically active molecules. These new separation media are based on a continuous body of macroporous polymer molded directly into the housing of the separation device rather than made of small beads mechanically packed into the column. The "molded" media offer many advantages such as ease of preparation and handling, compactness, enhanced reproducibility, versatility of separation chemistries, and capability of incorporate gradients of chemistry. The molded media are prepared from a single phase allowing the use of many monomers that cannot be used for the preparation of conventional beads. In contrast to packed columns, the preparation of monoliths is simple, waste-free, and less labor intensive, leading to a low unit cost per device. Dramatic improvements in the column characteristics are expected to originate from the redesigned porous structures with both a large pore volume and a high connectivity of pores within the monolithic structure. The versatility of separation chemistries and the overall binding capacities will be further enhanced by the grafting of active chains within the pores of the monoliths using in situ "living" polymerization techniques. These new molded monoliths will, in addition to remarkably enhanced mass transfer through the built-in transport pores, include smaller pores with improved connectivity and afford separation devices such as columns and capillaries with substantially increased efficiencies. Overall, our targets are to significantly enhance the array of analytical and diagnostic tools available for handling biological samples. The new media developed within the framework of this project will contribute to both better performance and new capabilities in areas such as high- performance liquid chromatography, capillary electrochromatography, and microchannel "lab-on-chip" separations. The unequaled versatility of the molded systems and their unique features make them ideal for the development of a broad range of products specifically designed for the high throughput separation of biopolymers using capillary electrochromatography and laboratory-on-chip formats in unprecedented separation modes.