The molded macroporous poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate) rods produced by a facile molding process were polymerized in situ within a tubular mold, chromatographic column (4.6×100 mm) by free radical polymerization. It was complemented by epoxy derivatized monolithic column and chemical modification of the epoxide groups with the sulphuric acid. By variation of the polymerization conditions, such as the ratio of the monomers, the porogen (pore generating material), and the temperature, the pore size could be varied, so the retention time of the samples may be adjusted. For the mixture of caffeine and tryptophan in the prepared monolithic column, the influences of polymerization material compositions to the efficiency, selectivity, and resolution of the monolithic column were investigated.