CONTINUOUS BEDS FOR MICROCHROMATOGRAPHY - CATION-EXCHANGE CHROMATOGRAPHY

Microcolumns (i.d. 10-320 mu m) for cation-exchange chromatography can be prepared simply by polymerization of an aqueous solution of approp riate monomers, including the desired ligand, directly in the chromato graphic tube (fused-silica tubing) in the presence of salt. The beds t hus prepared are in the form of rods traversed by channels through whi ch the eluent can pass. The walls of the channels are composed of very small particles and are impermeable to peptides and proteins, which i s important for rapid mass transfer add thus for high resolution at hi gh flow rates. The bed becomes attached covalently to the tube wall du ring synthesis. A complicated column tube design with a supporting fri t at the bottom is thus eliminated. The absence of a frit reduces the flow resistance and facilitates interfacing to mass spectrometers. The covalent linkage of the bed to the tube wall also serves to suppress the zone-broadening ''wall effect.'' A homogeneous ''packing'' of a co ntinuous bed column with an inner diameter as small as 10 mu m is easi ly obtained. The resolution, binding capacity, and flow rate (iie., ru n time at a given pressure) can be varied by changing the composition of the monomer solution. One can thus tailor the beds to each separati on problem. The chromatographic properties of the microcolumns are dem onstrated by separations of model proteins. (C) 1994 Academic Press, I nc.