## 3 Results and Discussion  
### Preparation and Characterization of Imprinted Polymers  
This study aimed to prepare an imprinted material which could selectivity bind CS and its structural analogues in aqueous media. The template (CS), which has amide, hydroxyl and amino groups, can form abundant hydrogen or ionic bonds with the carboxyl group of MAA. Besides, it has been reported that HEMA, as a hydrophilic monomer, could improve the hydrophilicity of the exposed surface of MIPs which will enhance specific recognition under aqueous medium [28]. Keeping this in mind, MAA and HEMA were used as co-monomers to synthesize selective MIPs. Generally, in precipitation polymerization, polymers are formed in an appropriate solvent in which polymerization system is highly diluted, that is monomers/porogen (_w/v_) less than 5%. Compared with traditional bulk polymerization, imprinted particles (beads or agglomerates) prepared by this method usually hold more homogeneous binding sites, thus exhibiting excellent performance [29]. However, in order to obtain high quality polymers with stable pore structures and keep their average diameters to 1 \(\upmu\)m, the porogenic solvent(s) should be screened according to the solubility parameter of the forming polymer network. From this point, the presence of DVB in the mixture of ACN/toluene (75/25, _v/v_) contributes to generate monodisperse polymer microspheres with average diameters of \(\sim\)5 \(\upmu\)m [30; 31]. Accordingly, the challenge of polymer preparation was that CS is neither soluble in non-polar solvents (i.e., toluene) nor in some polar solvents (MeOH, ACN and dimethyl formamide, etc.). The key was to improve the solubility of CS in the reaction system, while circumventing the participation of water because it would significantly suppress recognition of imprinted polymers. In this case, several combinations of MeOH, ACN and toluene as porogens were preliminary screened. Practically, high proportions of ACN or toluene produced colloidal suspension of polymers on account of extremely poor solubility of CS in these porogens. Considering the solubility of CS and trying to keep mentioned porogen requirements in precipitation polymerization, mixtures of ACN, toluene and MeOH were assayed as porogen. From these experiments, it was concluded that the use of a high amount of MeOH in ACN:Toluene could effectively dissolve CS with the participation of little DMSO after sonication and finally a mixture of MeOH:ACN:Toluene (75:18.75:6.25, _v/v/v_) was chosen as optimum. However, once DVB was added as a cross-linker, it affected CS solubility and further made the polymerization mixture turbid. Increasing the amount of MeOH would negatively affect CS:MAA interactions based on hydrogen bonding and thus such porogen modification was discarded. Alternatively, mixtures of DVB and EGDMA were tested assuming that the presence of EGDMA would increase the overall polarity of the polymerization mixture and thus favoring CS solubility. After some experiments, it was concluded that a mixture of DVB:EGDMA (16/84, _v/v_) allowed the complete solubilization of CS in the polymerization mixture and was subjected to polymerization. Figure 2 shows the SEM images of the MIP and NIP particles. It could be seen that agglomerates, instead of independent spherical particles, were obtained. Such agglomerates, for both MIP and NIP, were constituted by beads with particle size lower than 1 \(\upmu\)m, which can be attributed to the great difference between the polymerization conditions used in this study and those recommended in conventional precipitation polymerization. Specific surface area of both MIP and NIP, measured by nitrogen sorption porosimetry, were as low as 10 m\({}^{2}\)-g\({}^{-1}\), which is directly related with the low amount of toluene used in the polymerization mixture [32].