SYNTHESIS AND CHARACTERIZATION OF NEW ZIRCONIA-BASED POLYMERIC CATION-EXCHANGE STATIONARY PHASES FOR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHYOF PROTEINS

Ion-exchange chromatography is a major method used for large-scale pro tein separations, New zirconia-based polymeric cation-exchange HPLC st ationary phases have been developed for protein separations, Two route s were employed for the synthesis. In one method, polyethyleneimine (P EI) was adsorbed onto porous zirconia particles and cross-linked with 1,4-butanediol diglycidyl ether (BUDGE). Succinic anhydride was then r eacted with the remaining primary and secondary amine groups on PEI to afford anionic functionalities. The second method utilizes poly(acryl ic acid) anhydride as both the crosslinker and the stationary phase. T he resulting stationary phases act to separate proteins by a weak cati on-exchange mechanism with a slight contribution to retention from hyd rophobic interactions, In the presence of 20 mM phosphate buffer, Lewi s acid/base interactions between the zirconia support and the proteins , which can significantly broaden the peaks, are sufficiently suppress ed. The effects of ionic strength, mobile phase pH, and salt type are discussed. Protein mass recovery and loading capacity for protein sepa rations on these phases have been evaluated. These weak cation-exchang e stationary phases exhibit good stability under normal separation con ditions for months and are stable in alkaline solution up to pH 10. In contrast to zirconia supports modified with small anionic species, th ese new phases have no limitation on the type of salt used as the elue nt, and they exhibit unique selectivities, Therefore, they offer inter esting alternatives for protein separations. To our knowledge, us work represents the first successful example of protein separations using porous zirconia-based polymeric phases under normal chromatographic co nditions, which will definitely help make zirconia-based supports more useful for bioseparation.