Hydrophobic-interaction chromatography of proteins on continuous beds derivatized with isopropyl groups

A new support is described for fast, high-resolution hydrophobic-interaction chromatography of proteins based on a continuous bed containing isopropyl groups. The bed is prepared by a simple procedure: a mixture of appropriate acrylamide monomers, including the monomer with the isopropyl group, is polymerized directly in the chromatographic tube or, alternatively, in a beaker for columns with diameters greater than 1-2 mm. The concentrations of the monomers were adjusted for optimum resolution and low flow resistance. Both standard (6 mm I.D.) and microbore columns (0.015-0.320 mm I.D.) have been employed successfully for the separation of seven model proteins. A linear negative gradient of ammonium sulfate (2.2-0 M) in phosphate buffer was used for the elution. These columns share with all continuous bed columns the properties that the resolution is roughly independent of the flow-rate, that the gel is sufficiently rigid to give high flow-rates at moderate pressures and that the efficiency for the separation of proteins is high and independent of the bed height for beds longer than 4-5 cm. The residence time of the proteins on the column has no observable influence on the appearance of the chromatograms.