Dựa vào thành phần, tính chất nước thải phát sinh sau quá trình sản xuất công nghiệp và sự tương đồng trong việc áp dụng phương pháp xử lý. Vì thế, có thể phân loại nước thải sản xuất công nghiệp thành các nhóm sau: Nước thải ngành dệt nhuộm, thuộc da, in ấn. Nước thải ngành xi mạ. Nước thải ngành cao su. Nước thải ngành giấy. Nước thải ngành chế biến thực phẩm (bánh, kẹo, nước giải khát, …) Ngành sản xuất bánh, kẹo, bia, nước giải khát, thức ăn đóng hộp, … Ngành chế biến thủy, hải sản. Ngành chế biến hạt: hạt điều, café, … Nước thải ngành chăn nuôi. Nước thải hệ thống xử lý tập trung, khu công nghiệp.
Analytica Chimica Acta 394 (1999) 353±359 Binding study on 5,5-diphenylhydantoin imprinted polymer constructed by utilizing an amide functional group1 Jie Zhoua, Xiwen Heb,*, Yijun Lib a Department of Chemistry, Shandong Agricultural University, Taian, Shandong 271018, China b Department of Chemistry, Nankai University, Tianjin 300071, China Received November 1998; received in revised form 16 March 1999; accepted 20 March 1999 Abstract The molecular imprinting technique was applied for the preparation of a polymer selective for an acidic drug, 5,5diphenylhydantoin in a polar solvent using acrylamide as the hydrogen-bonding functional monomer The binding characteristics of the imprinted polymer were evaluated by batch methods Scatchard analysis showed that two classes of binding sites were formed in the imprinted polymer Their dissociation constants were estimated to be 9.05 mmol/l and 1.87 mmol/l, respectively, by utilizing the model of multiple independent classes of binding sites These results were more reasonable than those obtained by the Scatchard equation Factors that in¯uenced rebinding of the imprinted polymer including pH, template/monomer ratio and functional monomers were explored By contrast, when methacrylic acid was used as functional monomer, the molecular imprinted polymers made in tetrahydrofuran exhibited only very weak binding capacity for the template molecule Finally, the substrate selectivity of imprinted polymer was investigated # 1999 Elsevier Science B.V All rights reserved Keywords: Molecular imprinting; Substrate selectivity; 5,5-diphenylhydantoin; Binding sites; Dissociation constants Introduction The development of synthetic receptors that recognize a target molecule at the molecular level is an important area in chemistry today On the basis of the increasing understanding of supramolecular interactions (hydrogen bonding, ionic interaction, van der Waals interactions, the hydrophobic effect, metal che- *Corresponding author Fax: +86-22-23502458; e-mail: chemczl@sun.nankai.edu.cn Project 29775011 supported by National Natural Science Foundation of China lation, etc.) between substrate±enzyme, antigen±antibody and ligand±receptor, several well-known synthetic recognition systems have been reported [1] and newly synthesized receptors are emerging very rapidly [2] Molecular imprinting is now a well established technique for the preparation of such arti®cial receptors and has recently been reviewed [3±6] The process begins with the desired target molecule called template, which serves two functions The ®rst is as a space-®lling three-dimensional object around which a complementary polymer cavity can be formed The second is to organize complementary interaction between groups on the template and functional mono- 0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V All rights reserved PII: S 0 - ( 9 ) 0 - 354 J Zhou et al / Analytica Chimica Acta 394 (1999) 353±359 mers during polymerization Organization of polymerizable functional monomers by the template can be achieved by either covalent bonds and/or noncovalent forces, that is, supramolecular interactions The polymerization reaction mixture consists of the template, functional monomers and a large excess of crosslinker An appropriate volume of inert solvent and free radical initiator make up the remainder of the polymerization solution Thermal or photochemical initiated polymerization gives a highly cross-linked insoluble network polymer The template can be washed from the polymer matrix, while the functional monomers remain covalently bound to the polymer itself Left in the polymer matrix are three-dimensional cavities that are complementary in both shape and chemical functionality to that of the template The high degree of cross-linking enables the microcavities to maintain their shape after removal of the template, and thus the functional groups are held in an optimal con®guration for rebinding the template, allowing the receptor to `recognize' the original substrate Thus, many molecularly imprinted polymers (MIPs) have been prepared and utilized mainly as af®nity chromatography media [7±10] MIP-based binding assays have been also developed in which MIPs are successfully used as antibody mimics [11,12] The molecular imprinting technique has been expanded to the ®eld of analytical chemistry of drugs [13,14] The used functional monomer is mostly methacrylic acid (MAA) because the carboxyl group is the most commonly hydrogen-bonding and acidic functional group in molecular imprinting However, the hydrogen-bonding ability of this functional group is not very strong in polar solvents and can only form strong ionic interactions with basic functional groups So it is dif®cult to use MAA in molecular imprinting for acidic drugs insoluble in apolar solvents Here, we ®rst describe the preparation of a synthetic polymer receptor for an effective anticonvulsant acidic drug, 5,5-diphenylhydantoin (DPH) using acrylamide as the hydrogen-bonding functional monomer in a polar solvent, tetrahydrofuran (THF) The imprinting process is shown in Fig The detailed binding mechanism of the MIP is ®rst examined by using the multisite binding model in the molecular imprinting technique, Some factors that in¯uence rebinding of the DPHimprinted polymer including pH, template/monomer ratio and functional monomers are explored Finally, the binding selectivity of the MIP for DPH and its methyl derivatives is also discussed Experimental 2.1 Materials and instruments DPH was obtained from Aldrich Acrylamide, MAA, methanol, acetic acid, acetonitrile, iodomathane, dimethyl sulphate and THF were purchased from Tianjin No Chemical Reagent Factory MAA was puri®ed by distillation prior to use Ethylene glycol dimethacrylate (EGDMA) was prepared from ethylene glycol and methacrylic acid 2,2H -azobisisobutyronitrile (AIBN) was from Nankai University Special Reagent Factory Acetonitrile was chromatographic grade Other chemicals were analytical grade A Shimadsu UV-240 double-beam spectrophotometer, a FT-NMR Model FX90 (JEOL) and a Model SHZ-82 constant temperature bath oscillator (China) were used 2.2 Synthesis of Methyl Derivatives of DPH 3-Methyl-5,5-diphenylhydantoin (MDPH): A ml volume of iodomethane was added with magnetic stirring to a solution of 2.50 g of DPH in 100 ml of 0.011 mol/l NaOH The reaction was allowed to proceed for h at 258C and the white precipitate was collected by ®ltration and recrystallized from EtOH Yield: 67%, mp: 215±2168C (Lit 2178C), H NMR (CDCl3): 3.07 ppm, (N3-CH3); 7.40 ppm (Ar-H); 8.2l ppm (N1-H) 1,3-Dimethyl-5,5-diphenylhydantoin (DMDHP): A 50 ml volume of dimethyl sulphate was added dropwise with magnetic stirring to a solution of 1.25 g of DPH in 250 ml of mol/l NaOH and was prepared according to the procedure of the preparation of MDPH Yield: 73%, mp: 196±1978C (Lit 1978C), H NMR (CDCl3): 2.80 ppm (N1-CH3); 3.12 ppm (N3-CH3); 7.38 (Ar-H) 2.3 Polymer preparation Polymers were prepared using acrylamide or MAA as functional monomer and EGDMA as the crosslinker, DPH as the template The procedure for the J Zhou et al / Analytica Chimica Acta 394 (1999) 353±359 355 Fig Schematic illustration of the preparation of DPH-imprinted polymer synthesis of the standard polymer P2 is as follows DPH (1 mmol) was dissolved in THF (10 ml) in a 50 ml glass ampoule EGDMA (20 mmol), acrylamide (4 mmol) and AIBN (50 mg) were added After nitrogen gas sparged into the solution for min, the ampoule was sealed under vacuum, and the mixture was kept in a shaker bath at 608C for 24 h The resultant bulk rigid polymer was ground to pass through a 75 mm sieve Fine particles were removed by repeated sedimentation in acetone The resulting particles were placed in a home-made extraction apparatus [15] and washed at a ¯ow rate of 1.0 ml/ under continuous stirring condition with 10% acetic acid methanolic solution until the DPH could no longer be detected at 240 nm in the eluent Then the particles were washed with methanol to remove residual acetic acid and dried to constant weight under vacuum at 608C The polymerization conditions for all other materials were shown in Table Table Effect of DPH/monomer molar ratio on affinity of acrylamide MIPa MIPs DPH/monomer molar ratio QÃimp (mmol/g) ÁQ* (mmol/g) P1 P2 P3 1:2 1:4 1:8 18.4 35.0 43.4 5.1 16.6 13.2 a Polymers 20.0 mg, [initial DPH] 1.0 mmol/l, V 2.0 ml, t 258C, Adsorption time 12 h, solvent: acetonitrile, Q* amount of adsorbed DPH 356 J Zhou et al / Analytica Chimica Acta 394 (1999) 353±359 2.4 Binding experiments The sized and washed polymer particles (20.0 mg) were placed in a 10 ml conical ¯ask and mixed with 2.0 ml of a known concentration of DPH acetonitrile solution The conical ¯ask was oscillated in a constant temperature bath oscillator at 258C for 12 h The mixture was transferred into a centrifuge tube and centrifuged at 4000 rpm for The concentration of free DPH in the solutions was determined by measuring the absorbance at 240 nm The amount of DPH bound to the polymer Q was calculated by subtracting the concentration of free DPH from the initial DPH concentration We de®ne the imprinting factor as ÁQ Qimp À Qnon, where Qimp and Qnon are the amounts of bound DPH on the imprinted and nonimprinted polymers Thus, Q is a measure of the af®nity of polymers for DPH, while ÁQ is a measure of the effect of the imprinting process The average data of triplicate independent results were used for the following discussion Results and discussion 3.1 Template/monomer molar ratio The molecular imprinting of DPH using acrylamide as functional monomer was performed essentially by a well-known procedure described previously [16] Because different template molecules have different functional groups and different degrees of functionalization [17], a constant ratio of functional monomer to template is required to obtain a high-af®nity MIP for a particular template Thus, in order to ®nd the optimum conditions for DPH template, we synthesized imprinting polymers P1, P2 and P3 at a constant template/ cross-linker ratio (1 : 20) and corresponding nonimprinting polymers The value of Q of the polymers for DPH was determined by the equilibrium binding method and ÁQ was obtained (Table 1) Table shows that Q increases with the acrylamide content and ÁQ of P2 is the highest among those of P1, P2, and P3 This is that the more selective binding sites and more nonselective adsorption in the imprinted polymers are produced as the acrylamide content increases The selective binding sites increases faster when the DPH/monomer molar ratios change from : to : So ÁQ is enhanced While the DPH/monomer molar ratios vary from : to : 8, on the contrary, the nonselective adsorption increases faster and ÁQ decreases Based on this, we have chosen P2 as a standard DPH-MIP to investigate the binding characteristics of the acrylamide imprinting polymer for DPH 3.2 Effect of functional monomer MAA is known as a common functional monomer and has been extensively used in the preparation of MIPs [18] We replaced acrylamide with MAA to prepare DPH-MIP in polar solvent(THF) with the same template/monomer/cross-linker molar ratio as P2 Q and ÁQ of the polymer for DPH were 7.1 mmol/ g and 4.5 mmol/g under the same conditions The properties of the polymer were vastly inferior to those of P2 This result was in agreement with that of MIPs made in a polar solvent using carboxylic functional monomers and print molecules [19] Theoretically, the difference between amide and carboxylic group MIPs can be explained (i) The dielectric constant and dipole moment of the amide group is higher than that of the carboxylic group For example, acetic acid has a dielectric constant of 6.20 and a dipole moment of 1.70 D, while for acetamide these values are 67.6 and 3.76 D [20] (ii) In a peptide, the amide oxygen has 0.42e (e 1.602 Â 10À19 C) negative charge and the hydrogen 0.20e positive charge [21] These constants indicate that the amide functional group may be capable of forming stronger hydrogen bonds than the carboxylic group in polar solvents or water In addition, DPH is a weakly acidic drug, it can not form ionic interactions with MAA So the acrylamide imprinting polymer yields much more highly selective binding for DPH than the MAA imprinting polymer prepared in the polar solvent THF 3.3 Determination of binding parameters of DPHimprinted polymer In the binding study of MIPs, it has been found that two classes of binding sites often existed [18] The binding parameters of MIPs were mainly estimated by Scatchard analysis [18] At ®rst, we investigated the binding performance of P2 The equilibrium binding experiments were carried out by varying the concen- J Zhou et al / Analytica Chimica Acta 394 (1999) 353±359 357 in the determination of lower af®nity binding parameters So the binding parameter values obtained by Scatchard analysis are less accurate in these systems In order to overcome the insuf®ciency of Scatchard analysis, we ®rst treated our observations according to the model of many independent classes of binding sites [22] Because of the presence of two classes of binding sites in P2, the equilibrium binding equation can be written as: Q Fig Scatchard plots to estimate the binding nature of P2 Q is the amount of DPH bound to 20.0 mg of P2 tration of DPH from 50 mmol/l to 4.0 mmol/l in acetonitrile in the presence of a ®xed amount of P2 The obtained data were plotted according to the Scatchard equation [18] to estimate the binding parameters of P2 As shown in Fig 2, the Scatchard plot was not linear indicating that the binding sites in P2 are heterogeneous in respect to the af®nity for DPH Because there are two distinct sections within the plot which can be regarded as straight lines It reveals that two classes of binding sites were produced in P2 The equilibrium dissociation constant Kd1 and the apparent maximum number Qmax1 of the higher af®nity binding sites can be calculated to be 2.l mmol/l and 17.2 mmol/g of dry polymer from the slope and the intercept of its Scatchard plot By the same treatment, Kd2 and Qmax2 of the lower af®nity bonding sites were found to be 1.56 mmol/l and 104.0 mmol/g However, Scatchard analysis does not consider the contribution of the lower af®nity binding sites to the binding capacity of MIPs at low concentrations of substrates in the determination of higher af®nity binding parameters Similarly, at a high concentration of substrates, binding capacity of higher af®nity binding sites is ignored Qmax1 DPH Qmax2 DPH Kd1 DPH Kd2 DPH (1) where Q is the amount of DPH bound to P2 [DPH] is the free concentration in solution, Qmax1 and Qmax2 are the appearent maximum numbers of the higher and lower af®nity binding sites, respectively, and Kd1 and Kd2 are the equilibrium dissociation constants of the binding sites Using the binding parameter values obtained by Scatchard analysis as a set of initial parameter estimates, we ®tted the experimental points by Eq (1) The obtained ®tting curve is in good agreement with the experimental points as shown in Fig Kd1, Kd2, Qmax1 and Qmax2 obtained by the ®nal parameter estimates were 9.05 mmol/l, 1.87 mmol/l, l0.0 mmol/g, and 94.6 mmol/g, respectively Because the treatment is not limited by the substrate concentration used in the experiments, we think that the values obtained by the model are more reasonable 3.4 Effect of pH on characters of DPH-imprinted polymer Since biological recognition mainly occurs in aqueous buffer systems and is a function of pH, it is quite important to make MIPs capable of recognition in water in order to mimic biomolecules Unlike the carboxylic group the amide group is not ionizable, which could be advantageous for molecular recognition in water Using KH2PO4±K2HPO4 (aq)/acetonitrile (3/7) as an aqueous buffer solvent system, a correlation between binding and pH of adsorbed solution is seen in Fig for the DPH-imprinted polymer The pH was altered by adjusting the balance of mono- and dibasic phosphate salts(or adding HCl to KH2PO4 solution for pH values lower than 4.2), while the total concentration of phosphate salts was held constant at 0.05 mol/l 358 J Zhou et al / Analytica Chimica Acta 394 (1999) 353±359 Fig The fitting curve obtained by Eq (1) Q is the amount of DPH bound to 20.0 mg of P2 The binding of DPH on the DPH-imprinted polymer is strongly in¯uenced by the pH in the solutions as shown in Fig Enhanced binding was obtained in the pH 4.7±6.9 range This can be accounted for by the protonation of the amide group and ionization of the N-3 nitrogen atom of the DPH molecule When the pH is lower than 4.7, the amide group of P2 can bind a proton to form a positively charged group When the pH is higher than 6.9, a proton of N-3 in DPH molecule can be lost to form a negatively charged DPH molecule These results lead to the weak selective hydrogen-bonding interaction between DPH and P2 and they lower the binding capacity of P2 for DPH In particular, this is more obvious in basic solutions This suggests that the binding of the DPH-imprinted polymer for DPH may be controlled by hydrogenbonding interaction, which plays a crucial role in biological recognition systems and in determining the structure of protein and nucleic acids In looking at the nonimprinted polymer, its binding capacity for DPH is distinctly lower than P2 and there is little dependence of binding on pH The reason for this may be that, although the optimum binding conditions of the polymer can be controlled by the pH of the external solvent system, the selective binding is controlled by the imprinting process The selectivity of the MIPs is due to the shape-selective cavity built into the polymer matrix and the preorganization of functional groups complementary to the template molecule 3.5 Substrate-selectivity of DPH-imprinted polymer Fig Effect of pH on the binding of P2 in buffer solvent system Solid square: DPH-imprinted polymer (P2); open square: nonimprinted polymer The substrate selectivity of P2 was studied using DPH and its methyl derivatives, MDPH and DMDPH, as substrates in acetontrile and an aqueous buffer solvent system, KH2PO4±K2HPO4 (pH 6.9)/acetonitrile (3/7) Their amounts bound to P2 and nonimprinted polymer were determined by batch methods (Table 2) Table shows that the amount of DPH bound to P2 was to times more than for a nonimprinted polymer in both acetonitrile and aqueous buffer solvent system This indicates that the binding ability is introduced into the polymer by the molecular imprinting technique This template effect induced a good binding performance for DPH As can be seen, P2 showed very low af®nity to the DPH methyl derivatives MDPH and DMDPH The cause for this can be that, J Zhou et al / Analytica Chimica Acta 394 (1999) 353±359 Table Binding amounts (mmol/g) of tested substrates on P2 and Pnon by batch methoda Substrates DPH MDPH DMDPH Acetonitrile KH2PO4±K2HPO4/MeCN P2 Pnon P2 Pnon 51.3 9.0 2.4 18.4 7.9 6.3 44.5 5.2 3.2 12.5 4.8 3.8 a Polymer 20.0 mg, [initial substrate] 2.0 mmol/l, V 2.0 ml, adsorption time 12 h, t 258C in spite of their similar structures to DPH, MDPH has an active hydrogen on N-1, which can form a hydrogen bond as a proton donor whereas DMDPH has not the active hydrogen in its molecule In addition, the molecular structures of MDPH and DMDPH can not be complementary to the shape of the cavities in P2 which is disadvantageous to the molecules entering the cavities This can result in the formation of very weak or no hydrogen-bonding interactions between the DPH methyl derivatives and the amide groups of P2 In conclusion, good hydrogen-bonding speci®c recognition sites for acidic drugs can be created within the synthetic polymer P2 in polar solvents using an amide functional group This study may further enlarge the application of molecularly imprinted polymers to the separation and determination of trace drugs Acknowledgements The authors are grateful to National Natural Science Foundation of China for ®nancial support 359 References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] G Wenz, Angew Chem Int Ed Engl 33 (1994) 803 M Famulok, J Am Chem Soc 116 (1994) 1698 G Wulff, Trends in Biotechnol 11 (1993) 85 S Vidyasankar, F.H Arnold, Curr Opin Biotechnol (1995) 218 G Wulff, Angew Chem Int Ed Engl 34 (1995) 1812 J Zhou, X.W He, J Instr Anal 17 (1998) 87 I.A Nicholls, A RamstroÈm, K Mosbach, J Chromatogr A 691 (1995) 349 O RamstroÈm, I.A Nicholls, K Mosbach, Tetrahedron: Asymmetry (1994) 649 L Fischer, R MuÈller, B Ekberg, K Mosbach, J Am Chem Soc 113 (1991) 167 J Matsui, O Doblhoffer-Dier, T Takeuchi, Chem Lett (1995) 489 G Vlatakis, L.I Andersson, R MuÈller, K Mosbach, Nature 361 (1993) 645 M.T Muldoon, L.H Stanker, J Agric Food Chem 43 (1995) 1424 J Mathew-Krotz, K.J Shea, J Am Chem Soc 118 (1996) 8154 M Senholdt, M Siemann, K Mosbach, L.I Andersson, Anal Lett 30 (1997) 1809 G Wulff, W Vesper, R Grobe-Linsler, A Sarhan, Makromol Chem 178 (1977) 2799 M Kempe, K Mosbach, J Chromatogr 664 (1994) 276 M Kempe, K Mosbach, J Chromatogr A 691 (1995) 317 J Matsui, Y Miyoshi, O Doblhoff-Dier, T Takeuchi, Anal Chem 67 (1995) 4404 O RamstroÈm, L.I Andersson, K Mosbach, J Org Chem 58 (1993) 7562 D.R Lide, CRC Handbook of Chemistry and Physics, CRC Press, Boca Raton, 1994, pp 6±155, pp 9±42 T.E Creiqhton, Protein's Structure and Molecular Properties, W.H Freem, New York, 1993, p 45 A.F Heny, Anal Biochem 48 (1972) 317 ... recrystallized from EtOH Yield: 67%, mp: 215±2168C (Lit 2178C), H NMR (CDCl3): 3.07 ppm, (N3-CH3); 7.40 ppm (Ar-H); 8.2l ppm (N1-H) 1,3-Dimethyl-5,5-diphenylhydantoin (DMDHP): A 50 ml volume of dimethyl... the preparation of MDPH Yield: 73%, mp: 196±1978C (Lit 1978C), H NMR (CDCl3): 2.80 ppm (N1-CH3); 3.12 ppm (N3-CH3); 7.38 (Ar-H) 2.3 Polymer preparation Polymers were prepared using acrylamide... (1993) 7562 D.R Lide, CRC Handbook of Chemistry and Physics, CRC Press, Boca Raton, 1994, pp 6±155, pp 9±42 T.E Creiqhton, Protein's Structure and Molecular Properties, W.H Freem, New York, 1993,