Research Article Received: 4 March 2012 Revised: 5 May 2012 Accepted: 9 May 2012 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jsfa.5757 Application of molecularly imprinted hydrogel for the preparation of lactose-free milk Farzin Hadizadeh, a Maryam Hassanpour Moghadam b and Seyed Ahmad Mohajeri b* Abstract BACKGROUND: A variety of lactose imprinted hydrogels were prepared and their binding properties were studied in comparison with blank non-imprinted hydrogel. Methacrylamide and ethylene glycol dimethacrylate were used as functional monomer and cross-linker, respectively. Dimethylsulfoxide was also applied as polymerisation solvent. RESULTS: Different template/monomer ratios were studied and the optimised imprinted hydrogel (MIP 2 ), with a lactose/methacrylamide ratio of 1 : 8, was selected in a rebinding test. In Scatchard analysis of MIP 2 -lactose interactions, the dissociation constant and maximum binding sites were 0.33 mmol L -1 and 67.76 μmol g -1 hydrogel, respectively. The selectivity of MIP 2 for lactose in aqueous media was also evaluated in comparison with different mono- and disaccharides. The data showed that the affinity of MIP 2 for lactose is significantly higher than other saccharides. The imprinted hydrogel was finally used as a sorbent for separation of lactose from milk. CONCLUSIONS: The results indicated that MIP 2 , as an optimised imprinted hydrogel, can effectively bind lactose and decrease its concentration in milk. c  2012 Society of Chemical Industry Keywords: lactose; milk; molecularly imprinted hydrogel; molecular recognition; selectivity INTRODUCTION The preparation of specific binding sites via the molecular imprinting technique has become the main objective of many studies and major interest of some researchers in recent years. 1–5 Molecularly imprinted polymers (MIPs) are synthesised by radical polymerisation of monomers in the presence of a suitable template molecule. The functional monomers are arranged around a template compound and are then fixed in a spatial arrangement with a cross-linker monomer. 6–8 The MIPs could be applied as the sorbent in solid-phase extraction, 1–3,9–11 as the stationary phase in high-performance liquid chromatography, 12,13 in biosensors 14,15 and as new drug delivery systems. 16–18 Covalent and non-covalent methods are two main strategies for preparation of MIPs. 19 The non-covalent method is much more flexible and simpler in preparation. In addition, MIPs prepared by the non-covalent imprinting method show much faster rebinding kinetics than those prepared by the covalent approach. 20 Thus, the non- covalent method is more suitable for analytical applications such as their use in solid-phase extraction and chromatography methods. 20 Lactose is an important disaccharide which is found in milk with 3.8–5.3% concentration. 21 In the small intestine, lactose is cleaved to its two submits by lactase. 22,23 Lactose intolerance (lactase deficiency) is an inability to digest and absorb the lactose sugar in milk. It is caused by a lack or low concentration of the enzyme lactase in the digestive system. 22,24,25 The primary symptoms of lactose intolerance are abdominal pain, abdominal bloating, diarrhoea and nausea. Approximately 65% of the human population has a reduced ability to digest lactose after infancy. 26 Thus, separation of lactose from milk is very important. In this work, we prepared a series of lactose imprinted hydrogels using methacrylamide (MAAM) and ethylene glycol dimethacrylate (EGDMA) as monomers and dimethyl sulfoxide (DMSO) as polymerisation solvent. Binding properties of hydrogels were studied in water and the selectivity of the optimised MIP was evaluated. Finally, we applied the optimised imprinted hydrogel for separation of lactose from milk. MATERIALS AND METHODS Materials Methacrylamide (MAAM), ethylene glycol dimethacrylate (EGDMA), 3,5-dinitrosalicylic acid (DNSA) and sodium potassium tartrate were purchased from Sigma-Aldrich (Steinheim, Germany); glucose, fructose, maltose, sucrose, lactose, zinc acetate, dodeca- tungstophosphoric acid and glacial acetic acid were obtained ∗ Correspondence to: Seyed Ahmad Mohajeri, Department of Pharmacodynam- ics, Pharmaceutical Research Center, School of pharmacy, Mashhad University of Medical Sciences, P.O. Box 91967-73117, Mashhad, Iran. E-mail: mohajeria@mums.ac.ir a Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran J Sci Food Agric (2012) www.soci.org c  2012 Society of Chemical Industry