Aqueous size exclusion chromatography in semimicro and micro-columns by newly synthesized monodisperse macroporous hydrophilic beads as a stationary phase

A new class of monodisperse macroporous beads in the hydrophilic form were synthesized by seeded microsuspension copolymerization of two acrylic crosslinking agents, glycerol dimethacrylate (GDMA) and glycerol-1,3-diglycerolate diacrylate (GDGDA). The monodisperse porous poly(glycerol dimethacrylate-co-glycerol-1,3-diglycerolate diacrylate), poly(GDMA-co-GDGDA) beads were highly hydrophilic in nature due to hydroxyl functionality resulting from both crosslinking agents. The beads with different particle sizes between 4.5 and 6.7 μm and with different porous properties were obtained by changing the seed latex to monomer ratio. The bead size decreased, the average pore size increased and the specific surface area decreased with increasing seed latex to monomer ratio. Poly(GDMA-co-GDGDA) beads were slurry packed in microbore and semimicro-HPLC columns and successfully used as a stationary phase in aqueous size exclusion chromatography (SEC) mode in a micro-liquid chromatography system. The aqueous SEC runs were performed by using dextran standards in the molecular weight range of 1000–670,000 Da. SEC calibration curves exhibiting linearity in a wider range of molecular weight were obtained with the semi-micro and micro-HPLC columns packed with the poly(GDMA-co-GDGDA) beads synthesized with the seed latex to monomer ratios of 0.038 and 0.058 g/mL. The dextran standards could be eluted in an analysis time shorter than 2 min using micro or semi-micro columns packed with poly(GDMA-co-GDGDA) beads as stationary medium. These packings are suitable for molecular weight determination between 5 × 103 and 5 × 105 Da in the aqueous medium by using mobile phase flow rates in the range of 25–250 μL/min. The average molecular weight determinations of different water soluble polymers, an ionic polymer, poly(2-dimethylaminoethyl methacrylate), a zwitterionic polymer, poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), and a non-ionic polymer, poly(vinyl alcohol) were performed on a semimicro-column packed with the poly(GDMA-co-GDGDA) beads. Satisfactory results were obtained in the molecular weight determination of hydrophilic polymers by aqueous SEC.

► Highly hydrophilic, monosized macroporous beads were synthesized.
► The beads were successfully used as stationary phase in aqueous SEC in micro-HPLC.
► The molecular weights of different water soluble polymers were determined by aqueous SEC.