Poly(N-alkylpyridinium salt) was synthesized as an ionic polymer phase for HPLC packing material, in which the separation mode can be easily tuned by changing the alkyl side chains through quaternization reactions with various alkyl halides. The prepared ionic polymer phase with methylpyridinium side chains showed good separations of hydrophilic biomolecules in hydrophilic-interaction liquid chromatographic mode, which is different from that with octadecylpyridinium side chains showing better performance in reversed-phase HPLC mode.