High-performance liquid chromatography of proteins on compressed, non-porous agarose beads. I. Hydrophobic-interaction chromatography.

Macroporous agarose beads were converted into non-porous beads by shrinkage and cross-linking in organic solvents. These beads could be used for high-performance hydrophobic-interaction chromatography without derivatization with non-polar ligands, because the 1,4-butanediol diglycidyl ether, used as cross-linker, gives relatively hydrophobic bridges. The resolution for compressed columns packed with these beads was determined as a function of gradient time at constant flow-rate, flow-rate at constant gradient volume and flow-rate at constant gradient time and as a function of loading capacity. Interestingly, the resolution is virtually independent of flow-rate at constant gradient volume even when the column is packed with relatively large beads (diameter 30 microns). The beads have the advantage of being stable up to pH 14.