Continuous beds for microchromatography: cation-exchange chromatography.

Abstract

Microcolumns (i.d. 10-320 microns) for cation-exchange chromatography can be prepared simply by polymerization of an aqueous solution of appropriate monomers, including the desired ligand, directly in the chromatographic tube (fused-silica tubing) in the presence of salt. The beds thus prepared are in the form of rods traversed by channels through which the eluent can pass. The walls of the channels are composed of very small particles and are impermeable to peptides and proteins, which is important for rapid mass transfer and thus for high resolution at high flow rates. The bed becomes attached covalently to the tube wall during synthesis. A complicated column tube design with a supporting frit 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 continuous bed column with an inner diameter as small as 10 microns is easily obtained. The resolution, binding capacity, and flow rate (i.e., run time at a given pressure) can be varied by changing the composition of the monomer solution. One can thus tailor the beds to each separation problem. The chromatographic properties of the microcolumns are demonstrated by separations of model proteins.