Photopolymerized monolithic capillary columns for rapid micro high-performance liquid chromatographic separation of proteins

The preparation of monolithic poly(butyl methacrylate–co-ethylene dimethacrylate) capillary columns using photoinitiated in situ polymerization within 200 μm i.d. capillaries and their application for μHPLC separations of proteins have been studied. The low resistance to flow characteristic of monolithic columns, enabled the use of very high flow rates of up to 100 μL/min representing a flow velocity of 87 mm/s. Very good separations of a model protein mixture consisting of ribonuclease A, cytochrome c, myoglobin, and ovalbumin was achieved in less than 40 s using a very simple single step gradient of the mobile phase. Interestingly, no effect of the pore size on the separations of proteins was observed for these monolithic columns within the size range of 0.66–2.2 μm. The monolithic μHPLC columns are found very robust and no changes in the long term separation performance and back pressure were observed.