Reverse-Phase Liquid Chromatography Of Small Molecules Using Silica Colloidal Crystals

Khanina, Natalya. M.S., Purdue University, December 2014. Reverse-Phased Liquid Chromatography of Small Molecules using Silica Colloidal Crystals. Major Professor: Mary J. Wirth. Capillaries packed with a silica colloidal crystal (SCC) of 470 nm nonporous particles have recently been shown to offer significant improvements in the peak width, resolution, and speed of protein analysis due to the reduction of the A and C term broadening contributions of the Van Deemter equation. While protein separations using this technology have shown vast improvements, small molecules remain largely unexplored. As the SCC allows for an approximately diffusion limited system, extremely high plate numbers are expected for small molecule separations. The research presented herein studies the feasibility of pressure-driven small molecule separation using SCCs and commercial instrumentation. Studies are performed both in capillaries and stainless steel columns. Separating small molecules presents a unique challenge due to their size and high diffusion coefficients. For capillary SCC separations, these properties are shown here to exacerbate problems in the nano-ultra high performance liquid chromatography (UHPLC) commercial instrumentation, such as extra-column band broadening and adherence to tubing walls. While this is less of a problem for gradient separations, isocratic separations are considerably affected. Thus, the full potential of these capillary