CONTINUOUS BEDS FOR STANDARD AND MICRO HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Conventional high-performance liquid chromatographic columns are built up of small, uniform beads. The preparation of the columns involves many expensive and cumbersome steps. This paper describes a simpler method, which is not based on the use of preformed beads. The chromatographic bed consists of a compressed gel plug with intercommunicating "channels", the "walls" of which are impermeable to proteins. The gel bed is formed in one step: a monomer solution. including ligands, is polymerized in the chromatographic tube under such conditions that the polymer chains aggregate into bundles, for instance, by hydrophobic interaction [the voids ("channels") thus created between the polymer bundles, are large enough to permit passage of eluent]. The gel plug is then compressed 10-15 fold, which decreases the average diameter of the "channels". The gel bed formed shows a resolution that, at constant gradient volume, is independent of or increases with an increase in flow-rate, a property that it shares with a compressed. non-porous agarose bed. The potential of a continuous gel bed has been previously illustrated by a cation-exchange chromatography experiment. This paper gives details of the preparation of this cation-exchanger. as well as of beds for anion-exchange and hydrophobic-interaction chromatography. The usefulness of the beds is demonstrated by the separation of model proteins on columns with the diameters of 6 and 0.3 mm. For the micro column both on- and off-tube detection were used. In the latter procedure the protein zones were transferred by a buffer flow to a conventional UV detector as they leave the gel bed. and then to a fraction collector. This detection technique has the advantage that any detector and any flow-cell for high-performance liquid chromatography can be used. The sample becomes diluted, but the resolution and the sensitivity are about the same as those obtained with standard micro-flow-cells (the same technique has been used successfully for micro-preparative high-performance capillary electrophoresis). A new method of preparing salt gradients for micro columns is presented.