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ABSTRACT:In this study, the monodisperse-macroporous particles produced by a relatively new polymerization protocol, the so-called, "modified seeded polymerization," were used as column-packing material in the reversed phase chromatography (RPC) of proteins. The particles were synthesized in the form of styrene-divinylbenzene copolymer approximately 7.5 m in size. In the first stage of the synthesis, the monodisperse polystyrene particles 4.4 m in size were obtained by dispersion polymerization and used as the "seed latex." The seed particles were swollen by a low-molecular-weight organic agent and then by a monomer mixture. The monodisperse-macroporous particles were obtained by the polymerization of monomer mixture in the seed particles. In the proposed polymerization protocol, the number of successive swelling stages was reduced with respect to the present techniques by the use of sufficiently large particles with an appropriate average molecular weight as the seed latex. A series of particles with different porosity properties was obtained by varying the monomer/ seed latex ratio. The separation behavior of HPLC columns including the produced particles as packing material was investigated in the RPC mode using a protein mixture including albumin, lysozyme, cytochrome c, and ribonuclease A. The chromatograms were obtained with different flow rates under an acetonitrile-water gradient. The theoretical plate number increased and chromatograms with higher resolutions were obtained with the particles produced by using a lower monomer/seed latex ratio. The separation ability of the column could be protected over a wide range of flow rates (i.e., 0.5-3 mL/min) with most of the materials tested.

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Poly(styrene-co-divinylbenzene)-based monodisperse macroporous particles were obtained by a modified seeded polymerization technique. The monodisperse polystyrene particles, obtained by dispersion polymerization in sufficiently large sizes and with suitable average molecular weights, were directly used as the seed latex in the production of macroporous particles. Therefore, the number of swelling and polymerization stages in the multistage production was reduced. In the first stage, the seed particles were swollen with a diluent, dibutyl phthalate (DBP), and then with a monomer phase including styrene, divinylbenzene, and, as an initiator, benzoyl peroxide. The monodisperse macroporous particles were obtained by the repolymerization of the monomer mixture in the seed particles. The particles, having different porosity characteristics, were synthesized through variations in the dibutyl phthalate/ seed-latex (DBP/SL) ratio. Selected macroporous particle samples were slurry-packed into stainless steel high-performance liquid chromatography columns (300 mm long ϫ 7.8-mm i.d.). The separation of the protein mixture by these columns in the reversed-phase chromatography (RPC) mode was investigated. Liquid chromatograms with high resolutions were obtained under an acetonitrile/water gradient over a wide range of flow rates (i.e., 0.5-3 mL/min), especially for the particles produced with a monomer/seedlatex (M/SL) ratio of 3.0 mL/g. In the RPC experiments, the particles produced with an M/SL ratio of 3.0 mL/g and DBP/SL ratios of 1.0 and 1.5 mL/g exhibited better chromatographic performance than the other samples. The maximum theoretical plate number was 3500 for the particles produced with the M/SL ratio of 3.0 mL/g and DBP/SL ratio of 1.5 mL/g with albumin as the analyte. The size exclusion chromatography (SEC) calibration curves and the back-pressure/flow-rate relationships of the produced columns were also determined. The particles obtained with an M/SL ratio of 3.0 mL/g and a DBP/SL ratio of 1.5 mL/g exhibited the best performance in SEC applications. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: [3685][3686][3687][3688][3689][3690][3691][3692][3693][3694][3695][3696] 2004