In this study, 2-hydroxyethyl methacrylate (HEMA) was used as functional monomer and diethylene glycol diacrylate (DEGDA) and polyethylene glycol (200) diacrylate (PEG(200)DA) were used as crosslinking agents to imprint D(+)glucose. D(+)glucose imprinted polymers were prepared in the presence of dimethyl sulfoxide (DMSO) /isopropyl alcohol (IPA) (3/1, v/v) at room temperature, in the air by radiation-induced polymerization/ crosslinking. The control polymers were synthesized by the same procedure in the absence of D(+)glucose. In order to evaluate the recognition and separation properties of the imprinted system high performance liquid chromatography (HPLC) experiments were carried out where β (-) lactose, D(+)glucose and glycerol were used as analytes. To increase the affinity of the template to the stationary phase polarity of the mobile phase was decreased by the addition of acetonitrile into water. Optimum composition of acetonitrile/water (1/5 v/v) was determined according to the swelling experiments. The sizes of the cavities in the polymeric networks were determined by positron annihilation lifetime spectroscopy (PALS). The average radii of cavities were found as 0.254 and 0.279 nm for freeze-dried imprinted polymers prepared by using PEG(200)DA after swollen in water and acetonitrile/water mixture (1/5 by volume), respectively.