Uniformly Sized Polyninylphenol) Porous Beads: A Versatile HPLC Separation Medium Allowing Fast Switching Between Size-Exclusion, Normal Phase, and Reversed Phase Chromatography

Uniformly sized, 10 um porous poly(4-fert-butoxycarbonyloxystyrene-co-divinylbenzene) beads with controlled porosity were prepared by polymerization of uniform size droplets consisting of small polymeric particles that were used as shape templates and enlarged with monomers and porogens. The fert-butoxycarbonyl groups were removed by a thermal treatment that affords the desired poly(4-hydroxystyrene-co-divinylbenzene) separation medium. The pore size of the beads does not change when immersed in THF as the beads are additionally crosslinked upon heating in the deprotection step. The beads were tested in size-exclusion, normal phase, and reversed phase chromatography. The size-exclusion calibration curve is almost linear in the range of molecular weights from 100 to about 480 000. Columns packed with the polymeric phenol beads can be switched between normal and reversed-phase chromatography simply by changing the solvent without removing from the chromatograph and their chromatographic properties do not change even after several hundred injections in different chromatographic modes including also multiple washing with aqueous sodium hydroxide. The retentions in separations of series of homologous alkylbenzenes, N-substituted, and ring substituted anilines were found to be linear functions of the number of carbon atoms, while interactions with the phenolic hydroxyls increased the retention times of N-alkylanilines. Separation of a protein mixture using a column packed with the beads has also been demonstrated and very high protein recoveries have been obtained.