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 mum in size. In the first stage of the synthesis, the monodisperse polystyrene particles 4.4 mum 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. (C) 2004 Wiley Periodicals, Inc.