The objectives of this proposal are to design, prepare and test an entirely new form of separation media for use in the separation and isolation of biological molecules. The new separation media are based on a continuous body of porous polymer molded directly into the housing of a chromatography column rather than made of small particles mechanically packed into the column. The new separation media offer many advantages in ease of preparation, reproducibility, versatility of separation chemistry, ability to incorporate gradients of chemistry, compactness, adaptability to microscale diagnostics, preparative separations, and bioreactors. The preparation of the continuous media is simple, amounting to a plain bulk polymerization in the presence of porogenic substances, rather than the more tedious and demanding techniques used to prepare monodispersed particulate polymer media. As a result, many monomers which cannot be used readily in the preparation of particulate media, including many hydrophilic or water soluble monomers that are particularly well-suited for the separation of biopolymers, can be used directly. The versatility of the separation chemistry is further enhanced by the ability to mix several chemistries in a single continuous medium. This can be achieved with monomers having complementary chemistries, or through the creation of concentration or reactivity gradients within the column housing during preparation of the media, or even by the combination of several chemistries in series within a single column. The ease of preparation is an important advantage as it removes the vagaries of packing which are known to affect greatly the efficiency of packed columns. In addition the continuous media have no interparticular void volumes which are responsible for mass transfer in packed particulate columns and account for ca. 40% of total column volume. Instead, the continuous media contain an interconnected array of pores including large flow-through, or perfusion pores. Total perfusion by the mobile phase means that the flow of the substances to be separated is through the porous medium itself, rather than around the packed particles. Our targets will be to gain an understanding and control the factors that are essential for the reproducible preparation of continuous porous separation media. Emphasis will be placed on efficiency and versatility of separation chemistry for application to biological systems such as proteins, drugs, or metabolites.
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