Monosized-porous and hydrophilic polymer microspheres with reactive chlorine functionality were first obtained in the form of poly(3-chloro-2-hydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(HPMA-Cl-co-EDMA) copolymer by a new seeded polymerization. A macromolecular ligand, polyethylenimine (PEI) was covalently attached onto the poly(HPMA-Cl-co-EDMA) microspheres via a single stage reaction between 3-chloro-2-hydroxypropyl and amine groups. PEI attached-poly(HPMA-Cl-co-EDMA) microspheres were slurry packed into the microbore columns and used as stationary phase in hydrophilic interaction chromatography (HILIC) mode. The separation of various compounds, nucleosides, polar organics and peptides was successfully performed. The results showed that hydrophilic poly(HPMA-Cl-co-EDMA) microspheres are a new, alternative starting material for the synthesis of stationary media with different functionalities for various liquid chromatographic applications. (C) 2013 Elsevier B.V. All rights reserved.