Microwave Heating in Preparation of Magnetic Molecularly Imprinted

Anal. Chem. 2009, 81, 967–976

Microwave Heating in Preparation of Magnetic Molecularly Imprinted Polymer Beads for Trace Triazines Analysis in Complicated Samples Yi Zhang, Ruijin Liu, Yuling Hu, and Gongke Li*

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School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China The use of microwave heating for preparation in organic synthesis has been accepted as an effective technique in synthesis due to the significant advantages over the conventional method. In the present work, microwave heating was applied to the preparation of atrazine (template molecule) magnetic molecularly imprinted polymer (mag-MIP) beads by suspension polymerization. The term of the polymerization was dramatically shortened by using microwave heating in polymerization, which was less than 1 /10 that by conventional heating. The resultant polymers incorporating molecular recognition and magnetic separation can provide a highly selective material for trace analysis in complicated samples. The magMIP beads were demonstrated with a narrow diameter distribution (80-250 µm) and cross-linking, spherical shape, and porous morphologies and exhibited magnetic property (Ms ) 0.491 emu/g) and thermal stability under 260 °C. An improvement of imprinted efficiency is obtained in comparison to the mag-MIP beads prepared by conventional heating. A method for the determination of triazines in complicated samples by the mag-MIP beads extraction coupled with highperformance liquid chromatography (HPLC) was developed. The results indicated that the mag-MIP beads can be favorably used for the extraction of the triazines in spiked soil, soybean, lettuce, and millet samples. The reused beads displayed a long-term stability after undergoing extraction of 100 times. Sample preparation processes are widely implemented in analytical laboratories for trace analysis in complicated matrixes, necessarily for enhancing selectivity and reducing a potential source of error.1 Rather than being dependent on biomacromolecules recognized roles, attempts have been made to synthesize molecularly imprinted polymers (MIPs) with mimicking of special immunology. The molecularly imprinted technique is to form selective sites in a polymer matrix with the memory of the template, due to shape recognition, hydrogen bonding, and hydrophobic interactions.2,3 With their simplicity of preparation * To whom correspondence should be addressed. Phone: +86-20-84110922. Fax: +86-20-84112245. E-mail: [email protected]. (1) Ridgway, K.; Lalljie, S. P. D.; Smith, R. M. J. Chromatogr., A 2007, 1153, 36–53. (2) Dirion, B.; Cobb, Z.; Schillinger, E.; Andersson, L. I.; Sellergren, B. J. Am. Chem. Soc. 2003, 125, 15101–15109. 10.1021/ac8018262 CCC: $40.75  2009 American Chemical Society Published on Web 01/08/2009

and flexibility of use, MIPs have become a powerful tool used for certain fields.4-8 However, the various applications of MIPs are limited by the tedious synthesis process and laborious after-treatment.9 By far, bulk polymerization has been a most popular method for the preparation of MIPs owing to its simplicity.10 But the aftertreatment processes including crushing, grinding, and sieving to an appropriate particle size for the utilization are wasteful and timeconsuming; only 30-40% of the polymer is recovered as usable material.11-13 Thus, in recent years an interest has been raised in the spherical or particles shape MIPs, including grafting methods, emulsion, precipitation, and suspension polymerization. These methods offer a wide choice to produce spherical or particles shape MIPs, which can sort out some drawbacks associated with bulk polymerization. Grafting method imprinting on the surface of organic polymeric or silica beads is to obtain the desired morphology of the resultant polymer,14-16 but low extraction capacity sometimes limits its application. Emulsion polymerization usually suffers from the remnants of surfactant.17 The precipitation polymerization needs accurate controlling conditions during the free-radical process.18,19 Suspension polymerization is particularly suited to the formation of the porous imprinted polymer beads.20-24 However, this method is not free of some (3) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645–647. (4) Blomgren, A.; Berggren, C.; Holmberg, A.; Larsson, F.; Sellergren, B.; Ensing, K. J. Chromatogr., A 2002, 975, 157–166. (5) Zhu, Q. Z.; Haupt, K.; Knopp, D.; Niessner, R. Anal. Chim. Acta 2002, 468, 217–227. (6) Xie, J.; Zhu, L.; Xu, X. Anal. Chem. 2002, 74, 2352–2360. (7) Zhu, Q. Z.; Degelmann, P.; Niessner, R.; Knopp, D. Environ. Sci. Technol. 2002, 36, 5411–5420. (8) Jodlbauer, J.; Maier, N. M.; Lindner, W. J. Chromatogr., A 2002, 945, 45– 63. (9) Haupt, K. Anal. Chem. 2003, 75, 376A–383A. (10) Wulff, G. Chem. Rev. 2002, 102, 1–27. (11) Bruggemann, O.; Haupt, K.; Ye, L.; Yilmaz, E.; Mosbach, K. J. Chromatogr., A 2000, 889, 15–24. (12) Kempe, H.; Kempe, M. Anal. Chem. 2006, 78, 3659–3666. (13) Mayes, A. G.; Mosbach, K. Anal. Chem. 1996, 68, 3769–3774. (14) Glad, M.; Reinholdsson, P.; Mosbach, K. React. Polym. 1995, 25, 47–54. (15) Norrlrw, O.; Glad, M.; Mosbach, K. J. Chromatogr. 1984, 299, 29–41. (16) Plunkett, S. D.; Arnold, F. H. J. Chromatogr., A 1995, 708, 19–29. (17) Baade, W.; Reichert, K. H. Makromol. Chem., Rapid Commun. 1986, 7, 235–241. (18) Turiel, E.; Tadeo, J. L.; Cormack, P. A. G.; Martin-Esteban, A. Analyst 2005, 130, 1601–1607. (19) Carabias-Martıńez, R.; Rodrı´guez-Gonzalo, E.; Herrero-Hernańdez, E.; DıázGarcıá, M. E. J. Sep. Sci. 2005, 28, 453–461. (20) Tamayo, F. G.; Turiel, E.; Martıń-Esteban, A. J. Chromatogr., A 2007, 1152, 32–40.

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nature drawbacks, such as the tendency of a broad size range from micrometers to millimeter size25 and the disturbance of the dispersing medium.12,26 Thus, the preparation of MIPs with spherical shape in a direct and efficient way would be the best option. Accordingly, the reducing of the possibility of secondary nucleation and the term of nucleation in suspension polymerization would ideally provide a possible way to solve the problem of suspension polymerization. To the best of our knowledge, the majority of MIPs polymerization is dealt with conventional methods, such as conventional heating and UV light. However, the requirement of a long term of reaction time and the ability of poor penetration, the application of conventional methods in the synthetic procedure is necessarily to be improved.27 Accordingly, microwave heating as an alternative choice to replace the conventional method has been proposed.28-32 Synthesis of polymer beads by microwave heating was first used by Murray et al. in 1994,33 which achieved a significantly shortened reaction time and better monodispersity for the resultant polymer. Correa et al. studied the emulsion polymerization in a polar solvent; the reaction can be rapidly and conveniently carried out by microwave heating. It was found that a significant saving of energy and time was possible in the preparation of polystyrene.34 Li et al. investigated emulsion polymerization of styrene and methyl methacrylate. The enhanced conversion and an increasing reaction rate were obtained by pulse microwave heating.35 According to the authors, the physical properties and the mechanism of the resultant polymers were less affected by microwave heating. Despite of the mechanism being still under debate,36-41 the advantages of the application of microwave heating are obvious, such as significant promotion of reaction rate, short term, and high yield.42,43 Until now this technique has been successfully applied for many fields, such as catalysis,44 ring-opening poly(21) Mayes, A. G.; Mosbach, K. Anal. Chem. 1996, 68, 3769–3774. (22) Asier, F.; David, C.; Michael, J. W.; Evgeny, N. V. J. Appl. Polym. Sci. 2000, 77, 1841–1850. (23) Richard, J. A.; Klaus, M. J. Chromatogr., A 1997, 787, 55–66. (24) Lee, Y.; Rho, J.; Jung, B. J. Appl. Polym. Sci. 2003, 89, 2058–2067. (25) Dowding, P. J.; Vincent, B. Colloids Surf., A 2000, 161, 259–269. (26) Ye, L.; Ramstrom, O.; Mosbach, K. Anal. Chem. 1998, 70, 2789–2795. (27) Li, J.; Zhao, J. Q. Polym. Mater. Sci. Eng. 1999, 2, 155–156. (28) Bogdal, D.; Penczek, P.; Pielichowski, J.; Prociak, A. Adv. Polym. Sci. 2003, 163, 51–58. (29) Wiesbrock, F.; Hoogenboom, R.; Abeln, C. H.; Schubert, U. S. Macromol. Rapid Commun. 2004, 25, 1895–1899. (30) Lidstro ¨m, P.; Tierney, J; Wathey, B.; Westman, J. Tetrahedron 2001, 57, 9225–9283. (31) Adams, D. Nature 2003, 421, 571–572. (32) Strauss, C. R.; Trainor, R. W. Aust. J. Chem. 1995, 48 (10), 1665–1692. (33) Murray, M.; Charlesworth, D.; Swires, L.; Riby, P.; Cook, J.; Chowdhry, B. Z.; Snowden, M. J. J. Chem. Soc., Faraday Trans. 1994, 90, 1999–2000. (34) Correa, R.; Gonzalez, G.; Dougar, V. Polymer 1998, 39, 1471–1474. (35) Li, J. A.; Zhu, X. L.; Zhu, J.; Cheng, Z. P. Radiat. Phys. Chem. 2007, 76, 23–26. (36) Berlan, J.; Aiboreau, P.; Lefeuvre, S.; Marchand, C. Tetrahedron Lett. 1991, 32, 2363–2366. (37) Baghurst, D. R.; Mingos, D. M. P. J. Chem. Soc., Chem. Commun. 1992, 9, 674–677. (38) Bram, G.; Loupy, A.; Majdoub, M.; Autierez, E.; Ruiz-Hitzky, E. Tetrahedron 1990, 46, 5167–5176. (39) Wiesbrock, F.; Hoogenboom, R.; Leenen, M. A. M.; Meier, M. A. R.; Schubert, U. S. Macromolecules 2005, 38, 5025–5034. (40) Marand, E.; Baker, K. R.; Graykeal, J. D. Macromolecules 1992, 25, 2243– 2252. (41) Hoogenboom, R.; Wiesbrock, F.; Leenen, M. A. M.; Meier, M.A. R.; Schubert, U. S. J. Comb. Chem. 2005, 7, 10–13.

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merizations,45 emulsion, and precipitation polymerization.34,46 However, in spite of the success of the proposed approach, further investigation of its application in the preparation of imprinted polymer has not been reported. Hence, we inspirited that microwave heating can be a powerful method to prepare MIPs. Magnetic separation has been proven to be a useful tool in several areas, such as carrier, immobiler, or separator of biomolecules and drugs because of its fast recovery and high efficiency without high cost.47-52 The participation of a magnetic component in the imprinted polymer can build a controllable rebinding process and allow magnetic separation to replace the centrifugation and filtration step in a convenient and economical way. The studies on MIPs with magnetic property have been reported;53-55 however, these MIPs have not been applied to the determination of target analytes in real samples. In the present work, a new approach by microwave heating for the preparation of molecularly imprinted polymer beads in a simple and direct way was proposed. It was based on the suspension polymerization, using iron oxide particles as magnetic cores and methacrylic acid (MAA) as functional monomer. The preparation of mag-MIP beads is performed by the dispersion of prepolymerization solution, which composed the self-assembled mixture of MAA and atrazine in toluene. Then, in the presence of copolymer monomer, cross-linker, and initiator, polymerization was carried out by microwave heating, and the beads prepared by conventional heating were used for the comparison. The characteristics and selectivity of the mag-MIP beads were investigated in detail. A method based on the mag-MIP beads extraction coupled with high-performance liquid chromatography (HPLC) was developed. Also, the performance of the mag-MIP beads for the extraction of triazines in the analysis in the spiked soil, soybean, lettuce, and millet samples was assessed. EXPERIMENTAL SECTION Chemicals. Atrazine, simazine, propazine, simetryn, prometryne, ametryn, and terbutryn standards were kindly provided by Bingzhou Pesticide Plant (Shandong, China). The individual stock (200 mg/L) solutions were prepared in methanol and stored at -18 °C in the dark. Methacrylic acid (MAA) and azo(bis)isobutyronitrile (AIBN) were purchased from Damao Reagent Plant (Tianjin, China). Acrylamide (AA) and 4-vinylpyridine (4VP) were purchased from Sigma-Aldrich (St. Louis, MO). Trim(42) Chen, S. T.; Chiou, S. H.; Wang, K. T. J. Chin. Chem. Soc. 1991, 38, 85– 91. (43) Chia, L. H. L.; Jacob, J.; Boey, F. Y. C. J. Polym. Sci., Part A: Polym. Chem. 1996, 34, 2087–2094. (44) Larhed, M.; Moberg, C.; Hallberg, A. Acc. Chem. Res. 2002, 35, 717–727. (45) Albert, P.; Warth, H.; Mu ¨ lhaupt, R.; Janda, R. Macromol. Chem. Phys. 1996, 197, 1633–1641. (46) An, Z.; Shi, Q.; Tang, W.; Tsung, C. K.; Hawker, C. J.; Stucky, G. D. J. Am. Chem. Soc. 2007, 129, 14493–14499. (47) Haukanes, B. I.; Kvam, C. BioTechniques 1993, 11, 60–63. (48) Safarik, I.; Safarikova, M. J. Chromatogr., B 1999, 722, 33–53. (49) Meldrum, F. C.; Heywood, B. R.; Mann, S. Science 1992, 257, 522–523. (50) Tanaka, T.; Matsunaga, T. Anal. Chem. 2000, 72, 3518–3522. (51) Matsunaga, T.; Kawasaki, M.; Yu, X.; Tsujimura, N.; Nakamura, N. Anal. Chem. 1996, 68, 3551–3554. (52) Kiselev, M. V.; Gladilin, A. K.; Melik-Nubarov, N. S.; Sveshnikov, P. G.; Miethe, P.; Levashov, A. V. Anal. Biochem. 1999, 269, 393–398. (53) Ansell, R. J.; Mosbach, K. Analyst 1998, 123, 1611–1616. (54) Lu, S. L.; Cheng, G. X.; Zhang, H. G.; Pang, X. S. J. Appl. Polym. Sci. 2006, 99, 3241–3250. (55) Lu, S. L.; Cheng, G. X.; Pang, X. S. J. Appl. Polym. Sci. 2006, 99, 2401– 2407.

ethylolpropane trimethacrylate (TRIM) and ethylene glycol dimethacrylate (EGDMA) were purchased from Corel Chemical Plant (Shanghai, China). Styrene (St), pyridine, and N,N-dimethylaniline were purchased from Tianjin Reagent Plant (Tianjin, China). Divinylbenzene (DVB) was purchased from Qunli Reagent Chemical Industry Corporation (Shanghai, China). Ethylene glycol (PEG 6000), oleic acid, and poly(vinyl alcohol) were obtained from Xilong Chemical Plant (Shantou, China). Ferric chloride (FeCl3 · 6H2O) and ferrous sulfate (FeSO4 · 7H2O) were obtained from Shenyang Chemical Industry Corporation (Shenyang, China). The acetonitrile of HPLC grade was purchased from Merck (Darmstadt, Germany). Water was doubly distilled. All other reagents were of analytical grade. All solutions used for HPLC were filtered through a nylon 0.45 µm filter before use. Preparation of Fe3O4 Particles. The procedure to obtain the Fe3O4 particles by coprecipitation was reported in the literatures as follows: a mixture solution containing FeCl3 (1.0 mol/L) and FeSO4 (0.5 mol/L) was prepared with deoxygenated (nitrogen-purged) water.56 NH3 · H2O (28%, weight percent) was then quickly charged into the solution with vigorous agitation; Fe3O4 particles were visually realized by the appearance of a deep brown color of the system. The resultant brown mixture was aged by a MAS-I microwave synthesizer from Sineo Microwave Chemistry Technology Company (Shanghai, China) at 80 °C for 1 h. In all of the above process, nitrogen gas was continuously purged and vigorous agitation was operated. Finally, the precipitate was collected by a magnet and washed three times with 10% acetic acid and distilled water, respectively. The modification of the Fe3O4 particles was preformed with surface modifiers. Briefly, Fe3O4 (2.00 g) and distilled water (30 mL) were, respectively, mixed with PEG (10.0 g), oleic acid (2.0 mL), and poly(vinyl alcohol) (8.0 g) by whisking for 20 min, following sonicating for 30 min until a homogeneously dispersed solution was obtained. Preparation of Imprinted Polymer Beads. Prior to polymerization, prepolymer solutions were prepared by atrazine (1.0 mmol) and MAA (4.0 mmol) dissolved in toluene (37.7 mmol) and were stored in dark for 12 h. The polymerization was carried out in a 300 mL single-necked flask with a vigorous agitation, a condenser coil, and a nitrogen duct, and then was placed in the microwave synthesizer. The prepolymer solutions, PEG-Fe3O4 particles, dispersing solution St (79.6 mmol), cross-linker (TRIM, 8 mmol and DVB, 8 mmol), initiator (AIBN, 0.6 mmol), and dispersing medium (water, 80 mL) were adequately mixed and dispersed by vigorous agitation (600 rpm), bubbled with a nitrogen stream throughout the procedure. Microwave heating was performed for 120 min at 70 °C by raising the temperature in the first 3 min from room temperature. Magnetic nonimprinted polymer (mag-NIP) beads by microwave heating were also prepared as described above but without the addition of template. The preparation of magnetic imprinted polymer beads by conventional heating was obtained with the same recipe except for occurring in an electrothermal water bath at 70 °C for 24 h. Finally, all of the resultant beads were treated with the same processes, collected by magnetic separation, and washed with doubly deionized water in methanol (10%, v/v) for 1 h and then ultrasonically cleaned by 10% (56) Hong, R. Y.; Pan, T. T.; Li, H. Z. J. Magn. Magn. Mater. 2006, 303, 60–68.

(v/v) acetic acid in methanol and methanol for 30 min, respectively, until no leakage and no residue of polymerization was observed. Morphology Observation. The morphological evaluation was examined by an Olympus conventional heating-20 transmission electron microscope from Shimadzu (Tokyo, Japan) and scanning electron micrography (SEM) with a Philips XL-30 scanning electron microscope from Philips (Eindhoven, Netherlands). The particle size distribution was tested by a Malvern MasterSizer 2000 particle size analyzer from Malvern (Malvern, Britain). The infrared absorption spectrum was obtained with the KBr method by a Shimadzu IR-prespige-21 FT-IR spectrometer from Shimadzu (Tokyo, Japan). The thermogravimetric analysis was performed in a Netzsch STA-409 PC thermogravimetric analyzer from Netzsch (Bavaria, Germany). The magnetic properties of particles and polymeric beads were measured using a SQUID-based magnetometer form Quantum Design (San Diego, CA). Extraction Performance. The beads were screened and selected at the range from 120 to 150 µm. Before each use, the recycled beads were sonicated in methanol for 20 min and revived at 120 °C for 12 h. A known mass beads were immersed in a 3.0 mL standard or extraction solution, which was added to a 30 mL vial under a reciprocating shaking-table at room temperature. After being incubated for 30 min, the beads were magnetically separated, cleared up with 3 mL of n-hexane to reduce nonspecific adsorption, and then eluted in 1.0 mL of desorption solvent. The analytes elution was dealt with a nitrogen drying step and redissolved in 100 µL of methanol; the desorption solvent was placed into a 200 µL conical insert tube in a glass vial for automatic syringe. All chromatographic measurements were performed using a Shimadzu LC-2010 system for reversed-phase chromatography that consisted of a SCL system controller. An autosampler equipped with a UV-vis detector was used for the analysis of the samples. A C18 column 250 mm × 4.6 mm i.d., 5 µm from Dikma (Beijing, China) was used as the analytical column. A 7.5 mm C18 security guard column from Phenomenex (Torrance, CA) was attached to the analytical column. The mobile phase was acetonitrile/water (30:70, v/v) at a flow rate of 1.0 mL/min. Triazines were monitored at 225 nm. Analysis of Triazine in Spiked Samples. To remove water, the samples (soybean, millet, lettuce, and soil) were dried at 60 °C for 12 h in the vacuum oven. The samples were ground and then sieved with a mesh gauge. The spiked samples were prepared with the mixture of 1.0 g of soybean, corn, soil, and lettuce powder with the particle size of 0.198-0.246 mm. The extraction of triazines from the spiked samples was performed by the MARS-X microwave oven from CEM experimenting company (Matthews, NC) with the power of 300 W. An amount of 30.0 mL of acetonitrile was used as extraction solvent for spiked samples at 60 °C. Soybean, millet, and lettuce were extracted for 10 min except 20 min for soil. The extraction solutions were concentrated under vacuum distillation until the solvent was removed and then dissolved in 10 mL of n-hexane. The spiked concentrations for each triazine were set with three levels of 5.0, 15.0, and 30.0 µg/L, respectively. The procedures of the samples extraction by mag-MIP or mag-NIP beads were the same as those for the standard solutions. The reproducibility (between-assay precision) was based on three separate runs. Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Figure 1. Schematic representation of PEG modification of iron oxide particles (a) and atrazine mag-MIP beads preparation (b).

RESULT AND DISCUSSION Preparation of Mag-MIP Beads by Microwave Heating. This work focused on the application of microwave heating in the preparation of atrazine magnetic molecularly imprinted polymer beads. With optimized parameters, it was expected the resultant beads could possess a homogeneous morphology, highly selective recognition, and magnetic property for the extraction of the triazines from complicated samples. However, it has to be mentioned herein, microwave energy generally gets through the reaction matrix, but without dissipation, when the low polararity or low dielectric of polymeric monomers is used; therefore, it is difficult to apply microwave heating in this condition. However, the way by addition of iron oxide to improve microwave absorption capability was proposed by Wang and Shen in 1997,57 and the same effect was found with graphite and metal powder in the polymeric matrix.58,59 Consequently, in this work, Fe3O4 particles have the double function of both enhancing microwave absorption for the reactant and acting as magnetic cores of the resultant beads. Initially, Fe3O4 particles were produced using coprecipitation, following aging by microwave heating at 80 °C for 1 h. The average diameter of the Fe3O4 particles, measured by transmission electron microscope, was obtained at the range from 30 to 50 nm, and aggregation of these particles was not found. It seems clear that the aging procedure using microwave heating allows the obtainment of highly aged efficiency, as compared with the particles obtained by conventional heating that required 3 days. It has been proved by Hong et al.56 After the aging process, different surface modifiers were tested for modification of the Fe3O4 particles, which is a crucial procedure for further encapsulation. Poly(vinyl alcohol), oleic acid, and PEG were mixed with Fe3O4 particles and dispersed in water under ultrasonication, respectively. The resultant beads showed (57) Wang, Z.; Shen, J. R. Chin. Polym. Bull. 1997, 2, 113–117. (58) Inkpen, S. C.; Melcher, J. R. Polym. Eng. Sci. 1985, 25, 289–294. (59) Baziard, Y.; Gourdence, A. Eur. Polym. J. 1988, 24, 873–880.

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a good magnetic property and stability when PEG-Fe3O4 suspension was participated in MIPs polymerization, which was in full agreement with those obtained in previous studies on the proteins immobilization with magnetic particles.60,61 It could be attributed to the side chain of PEG to physically trapping Fe3O4 particles, which is illustrated in Figure 1a. Atrazine was used as the template molecule to demonstrate the imprinted principle.62,63 As can be illustrated in Figure 1b, MAA and atrazine were self-assembled through the hydrogenbonded interaction. After PEG-Fe3O4 suspension, cross-linker, copolymer monomers, initiator, and water were added and mixed and polymerization was generated by microwave heating. After polymerization, atrazine was removed from the resultant beads by ultrasonic cleaning, leaving cavities in the polymeric matrix that were complementary to the template. The specific interactions and the morphology of the beads were markedly influenced by monomer, cross-linker, solvent, and reaction time, which were necessarily optimized to obtain an appropriate recipe. Initially, the recognized capability was extremely dependent on the functional monomer; thus, AA, MAA, and 4-VP were tested with the same amount (4.0 mmol) for monomer selection. Similarly produced yields were obtained by using the mentioned monomers, but the best homogeneous morphology and specific selectivity of the resultant beads were achieved by choosing MAA. To avoid leakages of Fe3O4 particles and fragility of the resultant beads, styrene was utilized as a copolymer monomer, which was attributed to its unsaturated bonds to form the cross-linking main chain within the polymeric network. The amount of styrene was carried out at the 79.6 mmol level, which was (60) Suzuki, M.; Shinkai, M.; Kamihira, M.; Kobayashi, T. Biotechnol. Appl. Biochem. 1995, 21, 335–345. (61) Yoshimoto, T.; Takeshi, M.; Mihama, T.; Takahashi, K.; Saito, Y.; Tamaura, Y.; Inada, Y. Biochem. Biophys. Res. Commun. 1987, 145, 908–914. (62) Hidalgo, C.; Sancho, J. V.; Hernańdez, F. Anal. Chim. Acta 1997, 338, 223–229. (63) Pacakova, V.; Stulik, K.; Prikoda, M. J. Chromatogr. 1988, 442, 147–155.

Figure 2. Micrographs from a scanning electron microscope of mag-MIP beads at different magnifications. The inset of panel c shows the image of cross section (scale bar ) 10 µm), and panel d was after the use of more than 100 times.

proved to shift the equilibrium of the reaction toward the direction of the complex formation side, as published elsewhere.64 One of the parameters that can affect the degree of crosslinking of the polymeric network is cross-linkers. Thus, TRIM, DVB, EGDMA, and the two of the three compounds (1:1, molar ratio) were used as cross-linkers in the mag-MIP beads preparation. The results indicated that the polymer beads prepared with TRIM/DVB (1:1, molar ratio) had better uniformity and spherical shape structure than either EGDMA, TRIM, and the mentioned mixed cross-linkers did. It is well-known that solvent of the prepolymerization step affects the morphology and recognition capability of MIPs. A lowpolarity solvent was commonly chosen to preserve the interaction between monomer and template during the prepolymerization procedure. In this case, different solvents, such as acetone, ethanol, tetrahydrofuran (THF), and toluene were tested as prepolymerization solvent. It was concluded that polymerization did not take place with acetone, and a nonuniform morphological (64) Matsui, J.; Goji, S.; Murashima, T.; Miyoshi, D.; Komai, S.; Shigeyasu, A.; Kushida, T.; Miyazawa, T.; Yamada, T.; Tamaki, K.; Sugimoto, N. Anal. Chem. 2007, 79, 1749–1757.

and floppy structure was obtained when ethanol or THF were used. Homogeneous and dense structure can be reproducibly prepared with toluene as prepolymerization solvent. Thus, toluene was chosen as optimum prepolymerization solvent for the preparation of mag-MIP beads. The effect of microwave heating conditions was optimized, since it controlled the degree of the polymeric reaction. Thus, the beads were prepared with different polymerization times (40, 60, 90, 120, and 180 min) and temperatures (60 and 70 °C). At polymerization times of 40, 60, and 90 min, the resultant beads had low yields, likely because polymerization was insufficient yet. At polymerization time of 180 min at 70 °C, a broad size distribution was obtained, which could be attributed to a secondary polymerization. The size distribution of the beads produced at 60 °C was not significantly different from the beads obtained at 70 °C; however, its extraction yields for atrazine standard solution were lower, likely because of incomplete cross-linking. Thus, a polymerization time of 120 min at 70 °C was chosen for the preparation of the mag-MIP beads. Mag-NIP beads were also fabricated in the similar manner by using microwave heating, which did not contain atrazine. The magMIP beads prepared by conventional heating were carried out in Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Figure 3. Thermogravimetric analysis of mag-MIP beads prepared by microwave heating (MH, line a) and that prepared by conventional heating (CH, line b).

the same recipe, such as functional monomer, cross-linker, and prepolymerization solvent, except for a polymerization time of 24 h. Characteristics of Mag-MIP Beads. The morphology of magMIP beads with optimum factors prepared by microwave heating was observed by SEM. As can be observed in Figure 2a, the wellshaped beads with diameter distribution from 80 to 250 µm were achieved. The majority of the beads were spherical, and the surface of beads was porous and rough (Figure 2b), which is suitable for rebinding or releasing the target molecules from the mag-MIP beads. From the inset in Figure 2c, the cross section of the bead was found with a compact inner structure. The thickness of the porous structure located on the bead was less than 5 µm, which can be suitable for the molecular transfer, leading to reach extraction equilibrium within a short time. The resultant beads have a core-shell structure in theory, but in reality, however, the diameter of the Fe3O4 particles is only 30-50 nm as cores, which is too small to be observed by SEM on the same magnifications versus resultant MIPs with 80-250 µm. No significant differences of the morphology of the mag-MIP beads were observed after undergoing more than 100 times extraction procedures (Figure 2, parts c and d). The mag-NIP beads prepared with the same recipe had similar morphological structure and size distribution. Figure 3 shows the thermogravimetric analysis (TGA) of the beads prepared by microwave heating and compared with that by conventional heating. There was no dramatic weight loss below the temperature of 260 °C. Significant mass loss began from 260 to 590 °C. The remaining mass was attributed to the thermal resistance of Fe3O4 particles, and the quantity of Fe3O4 particles in the beads was 0.45% (microwave heating) and 7.5% (conventional heating), respectively. Figure 4 shows the magnetic hysteresis loops analysis of the beads. It is seen that there is a similar general shape of the three curves, being symmetrical about the origin, which illustrated a response to an external magnetic field but without magnetization. This result agreed with the experiments of thermogravimetric analysis. The mag-MIP beads by microwave heating achieved a saturation magnetization value of 0.491 emu/g, and it was 0.423 emu/g of mag-NIP beads with parallel manner, respectively. The 972


Figure 4. Magnetization curve of mag-NIP beads (curve a, S saturation magnetization is 0.423 emu/g), mag-MIP beads (curve b, S saturation magnetization is 0.491 emu/g) prepared by microwave heating, and mag-MIP beads (curve c, S saturation magnetization is 5.133 emu/g) prepared by conventional heating.

saturation value of the beads obtained by conventional heating is higher (5.113 emu/g), because of increasing average diameter. It seems clear that a larger average diameter of the beads allows more encapsulation of Fe3O4 particles. However, the mag-MIP beads with less magnetite encapsulation also possess enough magnetic response to meet the need of magnetic separation, as can be observed in Figure 5. Figure 5 shows the magnetic separation process of the magMIP beads. Initially, the mag-MIP beads were incubated in the standard solution. By reciprocated oscillation, the beads were fully dispersed for absorption. After a certain time, a magnet was adjoined to the vessel bottom to attract the beads. Following the desorption process, the beads were redispersed in desorption solvent, and then the elution was assessed by HPLC after magnetic separation. The infrared spectra of the mag-NIP and mag-MIP beads prepared by microwave heating were measured, respectively. The main functional groups of the predicted structure can be observed with corresponding infrared absorption peaks. An absorption band at 536 cm-1 of the mag-MIP beads corresponded to the Fe-O bond for Fe3O4 particles, which was also obtained for magNIP. A broad absorption band at 3420 cm-1 of the mag-MIP corresponded to the stretching vibration of O-H bonds to the hydroxyl groups for MAA molecules (monomer); so did the mag-NIP beads. The typical bands of mag-MIP beads were at 3030 and 2920 cm-1, and mag-NIP beads are similarly at 3030 and 2930 cm-1 due to the C-H aromatic stretching vibrations of styrene units. Other absorption bands, such as 1740 cm-1 (stretching vibration of CdO bonds on carbonyl groups), 1580 cm-1 (stretching vibration of residual vinylic CdC bonds), 1490 and 1450 cm-1 (stretching vibration of CdH bonds of phenyl), matched just minor peaks for mag-NIP beads. The solvent-resistant experiment indicated that the mag-MIP beads prepared with microwave heating remained with good surface quality and no desquamating or cracking in these solvents, such as methanol, acetonitrile, 10% (v/v) acetic acid in methanol, or 10% (v/v) acetic acid in acetonitrile.

Figure 5. Photos of immersion, dispersion, and magnetic separation of mag-MIP beads. Table 1. Extraction Yields and Imprinting Efficiency of the Imprinted Polymer Beads beads prepared by microwave heating extraction yield (pmol) compd

MIP

NIP

imprinting efficiencya

atrazine simazine simetryn ametryn propazine terbutryn prometryne

137 126 121 117 107 98 86

29 28 30 31 29 33 32

4.7 4.5 4.0 3.8 3.7 3.0 2.7

beads prepared by conventional heating extraction yield (pmol) MIP

NIP

imprinting efficiency

71 65 60 68 46 43 36

26 23 25 28 29 30 24

2.7 2.8 2.4 2.4 1.6 1.4 1.5

a The imprinted efficiency was calculated from extraction yield of mag-MIP (QMIP) and that of mag-NIP (QNIP) beads: imprinting efficiency ) QMIP/QNIP. In formula, Qpmol was the analysis of pmol for the extraction unit volume.

Figure 6. Particles size analysis of mag-MIP beads prepared by microwave heating (MH) and by conventional heating (CH). The vertical axis is the percentage of the size range of the total volume.

From the mentioned characteristic analysis, it could be concluded that no significant differences were observed between the mag-MIP and mag-NIP beads. The template molecules, added into the polymerization solution, only changed the arrangement of the monomer in the polymer network, but without participating in MIPs polymerization. Evaluation of Mag-MIP Beads. As can be observed in Figure 6, mag-MIP beads prepared by microwave heating had a narrower size distribution, with the majority particles size of 100-200 µm, being comparable to the beads obtained with a broad size distribution (100-1200 µm) of the mag-MIP beads prepared by conventional heating. The poor penetration in the polymerization procedure sometimes takes place with conventional heating, which does potentially lead to further polymerization between primary and ultimate production and yields resultant polymer with irregular shape and broad beads size distribution. The results are also agreement with those observed from the SEM (Figure 2) and particles size analysis (Figure 6). The similar phenomena were also reported in the published articles.65,66 The comparison of the

extraction capability of the beads prepared by microwave heating and conventional heating was studied by the simultaneous analysis of triazines in standard solution, which is illustrated in Table 1. There was no obvious difference of the extraction yields between the two mag-NIP beads prepared by microwave heating or conventional heating, because of the same mechanism of mainly nonspecific adsorption. The imprinting efficiency of the mag-MIP beads (microwave heating) was obtained at the range of 2.7-4.7, which was enhanced than the range of 1.4-2.8 for the mag-MIP beads prepared by conventional heating. In this sense, the improvement of imprinting efficiency was likely to occur, whereas the binding sites on the polymeric network were better formed in polymerization by microwave heating. The interaction of the noncovalent binding system mainly bases on the hydrogen bond between template and monomer, which is easily interrupted in principle when polar solvent is presented in imprinted polymerization. However, the interaction was obtained in this experiment while water was used as dispersing medium. It was possibly ascribed to the accelerated reaching of polymerization temperature of the matrix by microwave heating, so the binding sites can be preserved in a very short time. Extraction Capability of Mag-MIP Beads. Figure 7 shows the extraction yield curves of the mag-MIP and mag-NIP beads

(65) He, W. D.; Pan, C. Y.; Lu, T. J. Appl. Polym. Sci. 2001, 80, 2455–2459.

(66) Bao, J. J.; Zhang, A. M. Polym. Mater. Sci. Eng. 2002, 2, 78–81.


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Figure 7. Extraction yield curves of atrazine mag-MIP (a) and magNIP (b) beads to atrazine in n-hexane of 0.5-50 µg/L.

prepared by microwave heating. It was investigated with a series of atrazine standard solutions of 0.100-200.0 µg/L in n-hexane. The experiment revealed that the extraction yield continuously increased along with the increase of concentration in range of 0.500-50.00 µg/L for mag-MIP and reached equilibrium as the concentration was up to 50.00 µg/L. The extraction capacities with the mag-MIP beads were 220 pmol for atrazine and only 43 pmol obtained by mag-NIP beads. Although the physical property of mag-MIP was close to that of mag-NIP beads, the inherently different arrangement of functional monomer caused selectivity to associate absorption with mag-MIP beads. A good reproducibility with RSD of 5.6% was obtained for extraction of 15.00 µg/L atrazine standard solution by six batch mag-MIP beads. The adsorption and desorption kinetics of the beads was investigated with 15.00 µg/L atrazine standard solution. The extraction yields exhibited a significantly increase as prolonged extraction time. A time of 30 min was obtained to reach absorption equilibrium for the mag-MIP beads, which was likely ascribed to the highly cross-linked and porous structure of the beads. During the desorption process, the porous structure of mag-MIP beads had a positive effect on the desorption kinetics. A majority of the rebinding atrazine can be desorbed within 10 min, and the equilibrium time was about 20 min. Consequently, a desorption time of 20 min was chosen as optimum for the process. Figure 8 shows that the extract capabilities of mag-MIP beads for seven triazines and reference molecule (N,N-dimethylaniline and pyridine) were investigated using standard solution at 15.00 µg/L level. It was obvious that extraction yields of the mag-MIP beads for seven triazines were obtained at the range of 86-137 pmol, which was much higher than the range from 36-71 pmol of the mag-NIP beads. The interaction between mag-MIP beads and template can perform selective extraction of atrazine and its analogues, which have triazine ring structure and identical secondary amino groups. The degree of molecular analogy to the template is relative to the extraction yields of the mag-MIP beads. The extraction yields of mag-MIP beads for the reference molecules (N,N-dimethylaniline and pyridine), which are nitrogenous compounds but lack analogous structure with the template, are performed 40.0 and 11.4 pmol, respectively. This result indicated that the interaction is not only based on hydrogen 974


Figure 8. Extraction yields of atrazine, simazine, propazine, simetryn, prometryne, ametryn, terbutryn, N,N-dimethylaniline, and pyridine with mag-MIP and mag-NIP beads at the 15.0 µg/L level.

bonding between polymeric matrix and template but also the complement to template in size and shape. The degree of molecular analogy to the template is relative to the extraction yield of mag-MIP beads. There is not an obvious difference among the extraction yields of the mag-NIP beads for seven triazines and reference molecules, which likely depended on the same mechanism of nonspecific absorption. In order to further investigate the competitive recognized coefficients of the beads, N,N-dimethylaniline (nitrogenous branched chain) or pyridine (nitrogenous heterocyclic) as reference molecules but without structural similarity to the template were chosen to measure in the mixed solution of atrazine, N,Ndimethylaniline, and pyridine. The distribution coefficient (Kd),

Table 2. Binding Constants of the Mag-MIP and Mag-NIP Beads C0 (µg/L)

Cfinal (µg/L)

Kd (mg/L)

k

N,NN,NKd2 (N,NdimethyldimethylKd1 dimethylKd3 samples atrazine aniline pyridine atrazine aniline pyridine (atrazine) aniline) (pyridine) MIP NIP

15.0 15.0

15.0 15.0

15.0 15.0

5.6 ± 0.1 13.0 ± 0.1 14.7 ± 0.1 144.0 ± 2.2 12.8 ± 0.5 12.8 ± 0.1 13.0 ± 0.1 14.2 ± 0.1 14.7 ± 0.8 13.3 ± 1.1

K1

k′

K2

5.6 ± 0.2 11.3 ± 0.3 25.7 ± 0.7 5.2 ± 0.2 1.1 ± 0.1 2.8 ± 0.1

K′1

K′2

10.3 ± 0.6 9.2 ± 0.1

a Kd, distribution coefficient; Kd ) (C0-Cfinal)/(Cfinal) × (solution volume [mL]/absorbent mass [g]), where C0 and Cfinal represent the initial and final concentrations, respectively; k, selectivity coefficient, k ) Kd1/Kd2; k1 ) Kd1/Kd2, k2 ) Kd1/Kd3; k′, relative selectivity coefficient, k′ ) KMIP/KNIP, k′1 ) K1MIP/K1NIP, k′2 ) K2MIP/K2NIP.

selectivity coefficient (k), and relative selectivity coefficient (k′) of the beads prepared by microwave heating were evaluated herein. Table 2 shows the measured values of the three parameters for the tested compounds for the beads. The distribution coefficient (Kd) is defined as the ratio of the concentrations of the solute at the two phases under equilibrium state, suggesting the adsorbed capability of a substance, and we obtained 144.0 ± 2.2 mL/g of atrazine for mag-MIP beads, compared to 14.7 ± 0.8 mL/g for mag-NIP beads. The selectivity

coefficient (k) is defined as the ratio of the Kd values of the two competitive solutes and indicates the difference of two substances adsorbed by the polymer beads. The relative selectivity coefficient (k′) is defined as the ratio of the k values of the two competitive solutes.67,68 The k value of the mag-MIP beads (11.3 ± 0.3 or 25.7 ± 0.7) is 10.3-fold (9.2-fold) of that for the mag-NIP beads (1.1 ± 0.1 or 2.8 ± 0.1) for N,N-dimethylaniline and pyridine, respectively. It is evident that the mag-MIP beads had a high selectivity for atrazine over the reference compounds

Figure 9. Chromatograms of 15.00 µg/L triazines mixed standard solution, 15.00 µg/L triazines spiked solutions of soil, soybean, millet, and lettuce samples, and determination of triazines in spiked sample solutions with mag-MIP and mag-NIP beads: (a) triazines-spiked extraction solutions of sample, (b) spiked sample solution extracted with the mag-MIP beads, (c) spiked sample solution extracted with mag-NIP beads, and (d) triazines mixed standard solution; (1) simazine, (2) simetryn, (3) atrazine, (4) ametryn, (5) prometryne, (6) propazine, (7) terbutryn; injection volume of 20 µL. Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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of N,N-dimethylaniline or pyridine, depending upon the imprinted effect in the matrix. Application of Mag-MIP Beads Extraction Coupled with HPLC. It is necessary to optimize conditions in order to extract as much analytes as possible. An oscillation speed of 100 period/ min and n-hexane were the optimum conditions of extraction solvent with mag-MIP beads. Under the optimized conditions, the linearity of the mag-MIP beads extraction coupled with the HPLC method was investigated with a serial of atrazine, simazine, simetryn, ametryn, propazine, terbutryn, and prometryne mixed standard solutions. The results indicated that good linearity was achieved in the range of 5.00-50.00 µg/L for atrazine, simazine, simetryn, and prometryne and 5.00-30.0, 7.00-45.0, and 10.00-35.0 µg/L were obtained for ametryn, propazine, and terbutryn, respectively. The correlation coefficient was at the range of 0.9768-0.9957. The detection limits for seven triazines were in the range of 2.10-8.17 µg/L. The precision of the method was investigated with 15.00 µg/L triazines mixed standard solution, and the RSD was varied from 2.4% to 10.2%. The aim of this work was to provide a simple procedure by using microwave heating in the preparation of the mag-MIP beads, which can be applied in determination of trace analytes from complicated samples. Accordingly, soybean, millet, and lettuce samples were treated as described in the Experimental Section, and samples extraction were spiked with a mixture of triazines at three levels (5.00, 15.00, and 30.00 µg/L). Mag-MIP beads were subjected to extracted procedures of the mag-MIP beads coupled with HPLC. The mag-NIP beads extraction and direct injection analysis were used for comparison. As can be observed in Figure 9, the sensitivities of seven triazines analysis in four spiked samples were greatly enhanced with the proposed mag-MIP beads extracted procedure (Figure 9b), which cannot be totally detected by direct injection analysis (Figure 9a) or by mag-NIP beads extraction (Figure 9c). A high degree of selectivity is obtained by the proposed mag-MIP beads extracted procedure (Figure 9b), which would be an extremely difficult determination without the proposed sample preparation process. It was obvious that the baseline obtained for the analysis extracts by mag-MIP beads was

as clean as that shown in Figure 9d for the standard solution, likely owing to the interaction of imprinted recognition to the analytes. The recoveries of the spiked soil, soybean, millet, and lettuce samples were 71.6-126.7%, 72.1-120.2%, 78.9-119.9%, and 79.6-120.5%; the RSDs were 4.5-12.8%, 3.0-10.0%, 1.8-8.2%, and 2.5-10.5%, respectively, except for propazine and terbutryn in spiked samples at 5.0 g/L level could not be determined quantitatively.

(67) Han, D. M.; Fang, G. Z.; Yan, X. P. J. Chromatogr., A 2005, 1100, 131– 136. (68) Luo, W.; Zhu, L. H.; Yu, C.; Tang, H. Q.; Yu, H. X.; Li, X.; Zhang, X. Anal. Chim. Acta 2008, 618, 147–156.

Received for review August 31, 2008. Accepted December 15, 2008.

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CONCLUSION In this work, a microwave heating technique for the preparation of magnetically imprinted polymer beads was proposed. The resultant mag-MIP beads exhibited good characteristics, such as narrow size distribution, uniform morphology, and superior selectivity for triazines, and gave rather higher imprinted efficiency factors. The term of polymerization was dramatically shortened as compared to that by conventional heating. It is concluded that microwave heating is a powerful technique to prepare mag-MIP beads in this simple and efficient manner. Magnetic separation has been used in various fields to replace filtration and centrifuge steps, but rarely coupled with the molecular imprinted technique. The produced mag-MIP beads with magnetic property and large specific surface area could be flexibly dispersed and attracted by simple treatments, which could be suitable to the sample preparation for trace analysis in complicated matrixes. Consequently, the analytical method based on mag-MIP beads extraction coupled with HPLC was successfully used for triazines analysis in spiked samples. Finally, the application of the microwave heating technique in specific imprinted substances, which can be applied to the online HPLC or GC apparatus, is still at a further investigation. In a sense, the preparation of imprinted polymer by the microwave heating technique could be a promising tool used in the artificialantibody synthetic fields. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China under Grant Nos. 20775095, 20705042, and 20375050 and by the Guangdong Provincial Natural Science Foundation of China under Grant No. 06023094.

AC8018262

Microwave Heating in Preparation of Magnetic Molecularly Imprinted

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