Preparation of an Ion-Exchange Chromatographic Support by A “Grafting From” Strategy Based on Atom Transfer Radical Polymerization Ender Unsal, † Begum Elmas, ‡ Berna C ¸ ag ˇ layan, † Mu 1 rvet Tuncel, § Su 1 leyman Patir, | and Ali Tuncel* ,† Chemical Engineering Department, Chemistry Department, Faculty of Medicine, Anatomy Department, and Faculty of Science and Education, Hacettepe University, Ankara, Turkey A new “grafting from” strategy based on surface-initiated atom transfer radical polymerization (ATRP) was first used for the preparation of a polymer-based ion-exchange support for HPLC. The most important property of the proposed method is to be applicable for the synthesis of any type of ion exchanger in both the strong and the weak forms. Monodisperse, porous poly(glycidyl methacrylate- co-ethylene dimethacrylate), poly(GMA-co-EDM) particles 5.8 μm in size were synthesized by “modified seeded polymerization”. Poly(dihydroxypropyl methacrylate-co- ethylene dimethacrylate), poly(DHPM-co-EDM) particles were then obtained by the acidic hydrolysis of poly(GMA- co-EDM) particles. The ATRP initiator, 3-(2-bromoiso- butyramido)propyl(triethoxy)silane was covalently at- tached onto poly(DHPM-co-EDM) particles via the reaction between triethoxysilane and diol groups. In the next stage, the selected monomer carrying strong cation exchanger groups, 3-sulfopropyl methacrylate (SPM), was polymer- ized on the initiator-immobilized particles via surface- initiated ATRP. The degree of polymerization of SPM (i.e., length of polyionic ligand) on the particles was precisely controlled by adjusting ATRP conditions. Poly(SPM)- grafted poly(DHPM-co-EDM) particles obtained with dif- ferent ATRP formulations were tried as chromatographic packing in the separation of proteins by ion-exchange chromatography. The proteins were successfully sepa- rated with higher column yields with respect to the previously proposed materials. The plate heights between 100 and 150 μm were achieved with the column packed with the particles carrying the shortest poly(SPM) chains. The plate height showed no significant increase with increasing flow rate in the range of 0.5-16 cm/min. Supports in the form of monodisperse polymer particles have attracted significant attention in ion-exchange chromatography. The “activated swelling method” was developed for the synthesis of monodisperse particles in the range of 1-20 μm. 1,2 The monodisperse, porous poly(glycidyl methacrylate-ethylene dimeth- acrylate) (poly(GMA-co-EDM)) particles were produced by “staged shape template polymerization”. 3,4 The particles modified by a pore size-specific functionalization process were used as separation media for the complete separation of complex samples that require a combination of ion exchange with reversed-phase chromatog- raphy. 5,6 Monodisperse poly(glycidyl methacrylate-divinyl- benzene) microspheres carrying strong anion and cation ex- changer groups were used in the separation of macrolide antibiot- ics and proteins by capillary electrochromatography. 7,8 The strong cation-exchange packings based on monodisperse poly(GMA-co- EDM) particles were also used in the separation and purification of combinant human interferon. 9 The ion-exchange polymeric stationary phases presenting amino acids and amine units were prepared by the surface grafting of glycidyl methacrylate onto a silica gel surface and subsequent amination. 10 All of these packings were obtained by the surface derivatization methods involving the use of conventional activation agents or free radical polymerization techniques for the covalent attachment of ion exchanger ligands onto the particles. Atom transfer radical polymerization (ATRP) was proposed as a relatively new technique for the surface derivatization of silica- or polymer-based particles. 11-14 Polymer-grafted silica nano- particles carrying homopolymers, block copolymers, or hyper- branched polymers were synthesized by the surface-initiated ATRP of vinyl- or acrylate-based monomers. 15-20 Poly(divinyl- * Corresponding author. E-mail: atuncel@hacettepe.edu.tr. † Chemical Engineering Department. ‡ Chemistry Department. § Faculty of Medicine, Anatomy Department. | Faculty of Science and Education. (1) Ugelstad, J.; Berge, A.; Ellingsen, T.; Schmid, R.; Nilsen, T. N.; Mørk, P. C.; Stenstad, P.; Hornes, E.; Olsvik, Ø. Prog. Polym. Sci. 1992, 17, 87-161. (2) Ellingsen, T.; Aune, O.; Ugelstad, J.; Hagen, S. J. Chromatogr. 1990, 535, 147-161. (3) Smigol, V.; Svec, F. J. Appl. Polym. Sci. 1992, 46, 1439-1448. (4) Smigol, V.; Svec, F. J. Appl. Polym. Sci. 1993, 48, 2033-2039. (5) Smigol, V.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1994, 66, 4308-4315. (6) Smigol, V.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1994, 66, 2129-2138. (7) Zhang, S.; Huang, X.; Yao, N.; Horvath, C. J. Chromatogr., A 2002, 948, 193-201. (8) Zhang, S.; Zhang, J.; Horvath, C. J. Chromatogr., A 2002, 965, 83-92. (9) Gong, B.; Shen, Y.; Geng, X. J. Liq. Chromatogr. Relat. Technol. 2003, 26, 963-976. (10) Choi, S. H.; Lee, K. P.; Shin, C. H. Macromol. Res. 2005, 13, 39-44. (11) Kickelbick, G.; Paik, H. J.; Matyjaszewski, K. Macromolecules 1999, 32, 2941-2947. (12) Pyun, J.; Jia, S.; Kowalewski, T.; Patterson, G. D.; Matyjaszewski, K. Macromolecules 2003, 36, 5094-5104. (13) Kruk, M.; Dufour, B.; Celer, E. B.; Kowalewski, T.; Jaroniec, M.; Matyjaszewski, K. J. Phys. Chem. B 2005, 109, 9216-9225. (14) Von Werne, T., Patten, T. E. Polym. Prepr., Am. Chem. Soc., Div. Polym. Chem. 1999, 40, 354-355. (15) Shen, Y.; Zhu, S., Zeng, F., Pelton, R. J. Polym. Sci. Pol. Chem. 2001, 39, 1051-1059. Anal. Chem. 2006, 78, 5868-5875 5868 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 10.1021/ac060506l CCC: $33.50 © 2006 American Chemical Society Published on Web 07/13/2006