2.2. Preparation methods Several polymerization methods can be used to prepare MIP particles for SPE.

Traditional bulk polymerization

Originally, bulk polymerisation was the first strategy used to synthesize imprinted polymers s. Typically, the reaction mixture of template, functional monomers, cross-linker, initiator, and solvent are added to a test tube, the mixture is purged with nitrogen to remove oxygen, and the test tube is sealed under vacuum. After polymerization under appropriate conditions, the MIP particles can be obtained by grinding, sieving, and repeated flotation in sequence. In this method, the apparatus acquired for synthesis is relatively simple, and the reaction conditions can be easily controlled. Unfortunately, the chromatographic performance of these particles is usually unsatisfactory due to their irregular size and shape. Furthermore, the tedious and timeconsuming process and low yield of MIPs prevent their industrial production and acceptance in analytical laboratories [14]. Gonzalez et al. [55] systemic studied the non-covalent MIP synthesized by bulk polymerization using digoxin as template. These polymers were synthesized under different conditions, i.e., changing the functional monomers employed (methacrylic acid or 2vinylpyridine), the porogenic solvents (acetonitrile or dichloromethane) used, different polymerization and extraction processes were used. The polymerization process was proceeding either under UV light or in a thermostat-controlled water bath. This produced polymers with different structural conformations and characteristics (hardness, porosity, stiffness, loading capacity, strength, etc.). The binding capacity, binding specificity and chemical and thermal capacities of these MIP were found to depend directly on the characteristics of their surface morphology. Molecular imprinting of 2-aminopyridine (2-apy) in bulk polymerizations of acrylic and solgel based polymers has been synthesized by OMahony [56]. Both polymeric systems reveal varying degrees of affinity in rebinding the original template as well as a number of structural analogues. Rebinding was conducted in chloroform, acetonitrile and methanol in order to assess the role of hydrogen bonding in imprinting. The acrylic imprinted polymer retained approximately 50% of the template in rebinding studies in chloroform compared to 100% for the solgel. However, this higher affinity for the solgel was accompanied by a higher degree of non-specific binding. The acrylic polymer exhibited little discrimination between imprinted and reference polymers for 3aminopyridine (3-apy) indicating the high selectivity of the MIP polymer for 2-apy relative to 3apy Suspension polymerization MIPs can also be obtained by suspension polymerisation [36,42,91]. This method proposed by Mayes and Mosbach in 1996 [92] consisted of a heterogeneous polymerisation method suited for the production of spherical beads in a broad size range starting at a few micrometers and reaching up to millimetre size [93]. The organic-based polymerisation mixture is suspended as droplets into an excess of a continuous dispersion phase (water or perfluorocarbon fluids) by agitation (e.g. stirring) in the presence of a stabiliser (suspending agent). The polymerisation mixture contains higher molar concentrations of template and monomers in order to compensate the partial loss of the reagents in the dispersant phase which is

therefore not involved in the polymerization [23]. The polymerisation proceeds in the droplets phase by a free radical mechanism, each droplet acts like a mini bulk reactor. The solubility of both the dispersed droplet phase and the resultant polymer in the dispersion medium is low. The polymer that is produced is soluble in the polymerisation mixture so a gel is formed within the droplet at low conversion rates and this polymerisation method was proposed recently by PerezMoral and Mayes as an easy way to select the optimal compositions of reagents: 36 MIPs were directly prepared in SPE disposable cartridges in order to compare the potential of four different monomers for obtaining a MIP for propanolol and morphine [91]. This method of polymerisationwas also recently compared to the bulk polymerisation by Harvey who developed a MIP for chemical warfare and nuclear signature [10]. Particles obtained by suspension were too small to be used in SPE. However, this approach has been used for developing MIPs for the extraction of triazines [94] and phenobarbital [53]. Recently, two new suspension polymerization techniques based on droplets of prepolymerization mixtures formed in liquid perfluorocarbon [17] or mineral oil (liquid paraffin) [18] have been developed. These liquids, which could not interfere with hydrogen bonding and electrostatic interactions between a template molecule and a functional monomer, were used as a continuous phase instead of water. Both polymerization methods give spherical and polydispersed beads as well as suspension polymerization in water. Furthermore, MIPs for propranolol (PRP) and morphine were directly polymerized using liquid perfluorocarbon as the continuous phase under UV light in SPE cartridge columns, resulting in a rapid, and automatable process that requires no transfer or manipulation of the MIP microspheres [20]. These two new polymerization methods could be good substitutes for a conventional suspension polymerization method for the preparationof molecularly imprinted microspheres. Bovine serum albumin-imprinted polyacrylamide gel beads were synthesized via inverse-phase seed suspension polymerization, using high-density crosslinked gel beads as core, low-density crosslinked polyacrylamide gel as imprinting shell [67]. The selectivity test showed that imprinting gel beads exhibited good recognition for template proteins, as compared to the control protein. The imprinting beads had quick adsorption rate and possessed improved regeneration property in comparison with those prepared directly via inverse-phase suspension polymerization. They consider the formation of multiple hydrogen bonds and complementary shape between the imprinting cavities and the template proteins are the two factors that lead to the imprinting effect. A MIP that uses a stable isotope labeled compound as the template molecule, the so-called IMIP, was developed by suspension polymerization [68]. The selectivity factors of MIP and IMIP for bisphenol A (BPA) were 4.45 and 4.43, respectively. Therefore, IMIP was found to have the same molecular recognition ability as MIP. When MI-SPE with IMIP was used and followed by LCMS in the analysis of river water sample, the detection limit of BPA was 1 ppt with high sensitivity.

Two-step or multistep swelling polymerization is another common approach for preparing MIP beads [26,27]. These methods can produce spherical MIPs of uniform size within the range of 5100 m (Fig. 2B). The spherical particles obtained have separating abilities comparable with those prepared by bulk polymerization [28,29], but with much better column efficiencies and peak shapes. Unfortunately, their chromatographic performance is still unsatisfactory, despite the highly uniform packing. Multi-step swelling and polymerisation basically consists of the swelling of seed particles (polystyrene latex) using a micro-emulsion of a low molecular weight activating solvent (i.e. dibutyl phthalate) in water containing sodium dodecyl sulfate as a stabilizer in presence of the initiator. Once the emulsion droplets have been absorbed into the seed particles, this dispersion is added to a second dispersion containing monomers, cross-linker, porogen, and the template dispersed in water using a polymeric stabilizer such as polyvinyl alcohol. The mixture is stirred for several hours until the droplets are absorbed into the seed particles. Finally, the dispersion is purged with an inert gas and polymerisation is initiated. This methodology has been widely employed by Haginakas group for the synthesis of imprinted beads using different templates. From them, the modification of MIP beads by incorporating a hydrophilic external layer is remarkable. In this way, MIP beads acquire restricted access media (RAM) properties improving the selective extraction of analytes from complex samples such as serum [2931]. Although this method is likely to be one of the most suitable methods for the synthesis of monodisperse imprinted beads with a high yield, it is also one of the more laborious procedures. In addition, since the dispersion phase is water saturated, templatemonomer interactions purely based on hydrogen bonding can be disrupted. Thus, its applicability is limited to templates able to interact with monomers through strong enough electrostatic and hydrophobic interactions. In order to better control the size distribution, the shape of the MIP particles and to decrease the amount of waste material, a polymerisation method based on the swelling of preformed uniform particles was developed. This technique is quite time consuming and sophisticated but it enables the production of mono-dispersed spherical polymer particles over a size range 5100_m. Haginakas group proposed the use of a multi-step swelling approach combining restricted-access media to exclude macromolecules and a MIP to selectively retain target analytes from biological fluids [17,59]. In this approach, the difficulty was to develop a hydrophilic surface that did not modify the selectivity the MIP. A one-step swelling approach was recently proposed by Liu et al. to obtain MIP particles of 7.5_m for the selective extraction of metsulfuron-methyl from drinking water [44]. A two-step swelling approach was used by Kubo et al. [81] to prepare a MIP for domoic acid. Particles of about 5_m were directly packed in a column that was connected to UV detector for the simultaneous clean-up and analysis of this toxin from shellfish extracts. A similar approach was used for the development of a MIP for the on-line selective extraction of 17_-estradiol from river water [38]. 2.1.3 Seed polymerization Seed polymerization, typically multi-step swelling and polymerization, is a method for the preparation of spherical and monodispersed beads [21]. Figure 1 shows a preparation

method of MIP microspheres by multi-step swelling and polymerization, where uniformly-sized polystyrene seed particles are utilized as the shape template. The seed particles were swollen by microemulsion droplets containing an activating solvent (i.e., dibutylphthalate) and further swollen by prepolymerization mixtures (porogen, template molecule, functional monomer, cross-linker, and initiator). Completion of swelling followed by thermal or photo polymerization resulted in the formation of monodispersed MIPs. However, interactions between a template molecule and a functional monomer could be interfered since water is used as a continuous phase. MIPs for bisphenol A (BPA) [22, 23] were prepared using 4-vinylpyridine (4-VPY) or 2-vinylpyridine as a functional monomer and applied for SPE of BPA in river water samples. The advantages of multi-step swelling and polymerization techniques are easy to modify the surface of prepared MIPs. Two surface modification methods have been tried: one is an addition method and the other is a dispersion method [24]. The former was performed by the addition of polar monomers such as a mixture of glycerol monomethacrylate (GMMA) and glycerol dimethacrylate (GDMA) after initiation of the polymerization step in the multi-step swelling and polymerization method, while the latter was performed by the dispersion of MIPs in a solvent including a mixture of GMMA and GDMA or methacrylic acid 3-sulfopropyl (MAS). i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 1550 J. Haginaka J. Sep. Sci. 2009, 32, 1548 1565 Using an addition method, restricted access media (RAM)-MIPs for (S)-naproxen [25, 26], whose surfaces are modified with GMMA-co-GDMA polymers, were prepared and applied for direct serum injection assays of ibuprofen. Using a dispersion method, an MIP for 4,4-methylenebisphenol modified with MAS, which is an ionic monomer, was prepared and utilized for ultra-trace assays of BPA in river and lake water samples [24, 27]. The MIP modified with MAS could effectively remove the interferences (negatively charged ones such as water soluble oligomers of humic materials) in those samples,

compared with that modified with a mixture of GMMA and GDMA. 5.2. Multi-step Swelling Polymerization In recent years, much effort has been dedicated to developing alternative methods to prepare imprinted stationary phases, which are superior in terms of efficiency and mass transfer properties. Int. J. Mol. Sci. 2006, 7 1 6 8 Micrometer-sized spherical imprinted polymers with narrow size distribution have been prepared through several techniques [57-59]. Uniformed spherical particles have been obtained by using multi-step swelling method [60-63]. Particles can be prepared directly in the form of spherical beads of controlled diameter. Beads synthesized in this way can be rendered magnetic through inclusion of iron oxide particles. Although particles obtained using this technique are comparatively monodisperse in size and shape and well suited for chromatographic applications, however, fairly complicated procedures and reaction conditions are required, and the aqueous suspensions used in this technique could interfere with the imprinting and thus lead to a decrease in selectivity. The requirement for aqueous emulsions can interfere with the imprinting process and the selectivity of these particles is still not completely satisfactory. Uniformly sized MIP for d-chlorpheniramine (CP) and -brompheniramine (BP) prepared by a multi-step swelling polymerization method and evaluated using a mixture of phosphate buffer and acetonitrile as mobile phase [64]. CP and BP enantiomers were retained the most as a monovalent cation on MAA-co-EDMA polymers and a divalent cation on TFMAA-co-EDMA polymers. Ion exchange and hydrophobic interactions could mainly work for the retention and enantioseparation of CP and BP on both MAA-co-EDMA and TFMAA-co-EDMA polymers in hydro-organic mobile phases.

Dispersion polymerization can produce MIP beads of nanometer size [30]. The main advantage of this method is that it can be used for water-soluble templates.
2.1.4 Dispersion/precipitation polymerization
In dispersion polymerization, primary particles swell in a polymerization medium and the polymerization proceeds in the particles, resulting in the formation of spherical beads, while in precipitation polymerization,

primary particles do not swell in a polymerization medium and the polymerization takes place in the medium, leading to irregularly shaped, and polydispersed particles [28]. However, there is no sharp distinction between dispersion and precipitation polymerization methods [28, 29]. A crucial difference between the bulk polymerization and dispersion/precipitation polymerization techniques is a volume of a polymerization medium used. The latter requires larger volumes of the mediumthan the former. The first application of MISPE was reported in 1994 by Selleregren. An MIP for pentamidine was prepared by using methacrylic acid (MAA) as a functional monomer in situ in the chromatographic column by dispersion polymerization [30, 31]. The resultant polymers gave agglomerates of micron-sized globular particles with micro- or meso-pores. The selective enrichment of pentamidine, which has two amino groups, in urine samples was attained with the absorption and desorption of pentamidine by changing mobile phase pH. In general, precipitation polymerization gave MIP aggregates or microspheres of one or a few hundred nanometers in diameter [32]. Recently, it was found that matching the solubility parameter of the developing polymer network to that of the porogenic solvent(s) is particularly important in precipitation polymerization. An MIP for theophylline was prepared by the copolymerization of MAA and divinylbenzene (DVB) in a mixture of ACN and toluene, resulting in spherical and monodispersed beads of ca. 4 lm id [32]. Similarly, the MIP microspheres for thiabendazole [33] and (S)-nicotine [34] (for both ca. 3.5 lm id) were prepared by using MAA and DVB as a functional monomer and cross-linker, respectively, in a mixture of ACN and toluene. The former MIP was applied for the determination of thiabendazole in fruits [33], and the latter was applied for the selective removal of nicotine from cigarette smoke extracts [34]. An MIP prepared for isoproturon (one of phenylurea herbicides) gave aggregates of nano-particles of different sizes [35]. However, the method gave high yields for the MIP preparation. The obtained MIP could be employed in SPE of phenylurea herbicides from corn sample extracts.

Precipitation polymerization is another simple approach to prepare MIP beads, which are usually of nanometers size (Fig. 2C) and used in capillary electrochromatography [31] and immunoassay [32]. Recently, a new precipitation polymerization method was developed for the one-step preparation of monodisperse and spherical MIP particles, about 5 m in diameter [33] (Fig. 2D), which can be applied to HPLC and SPE separation. The advantages of this method include that it requires no surfactant and only minimal optimization of polymerization conditions, while producing high-quality products in good yield in one step. Unfortunately, in the MIP beads prepared by the above methods the binding sites are inside their network, causing a slow mass transfer of target molecules. Precipitation polymerisation [28] has been proposed as a simple and easy strategy for the obtainment of MIP beads quickly in

high yield. This method consists on carrying out the polymerisation in a large amount of a suitable solvent and has been successfully employed for the preparation of imprinted microspheres to be used in competitive radioassays [32], capillary electrophoresis [33] and SPE [34]. However, this methodology seems to be not generally applicable to any polymerisation mixture. Several examples can be found in the literature describing the obtainment of agglomerates instead of independent beads which has been attributed to the influence of the different template used. However, it is important to stress that the compatibility between the cross-linker and the used porogen is of vital importance for the success of this methodology. In any case, just the simplicity of the experimental procedure used and the elimination of crushing and sieving steps justify the use of this methodology for the preparation of MIPs as shown in recent papers for the MISPE of triazines [35] and phenylurea [36] herbicides from vegetables and bisphenol A from biologic One of them is the polymerisation by precipitation [89,90] that takes place in the presence of a larger amount of porogen than that typically used in the bulk method (2 to 10 times higher). As the polymerisation proceeds, the growing polymers chains become insoluble in the liquid phase and they precipitate. Micro- and nanospheres can be generated when accurate control of the parameters governing the precipitation polymerisation is achieved (i.e. polymerisation temperature, cross-linker . . .). Turiel et al. [89] obtained MIP particles close to 3.5_mthat were packed in a column directly connected to a fluorescence detector for the determination of thiabendazole in fruit extracts.

5.4. Precipitation Polymerization MIP microspherical shapes with more uniform size can be obtained by the method of precipitation polymerization, which offers a higher active surface area by manipulating its compositions. As regards precipitation polymerization, this technique involves coagulation of nano-gel beads followed by ordered particle growth due to capture of oligomers from surrounding solution [69-70]. In this manner, near-monodispersed spherical beads can be prepared, and size and porosity can be fine-tuned thereby changing the polymerization conditions. This technique has been reported in MIP-based competition assays [71-72] and capillary electrochromatography [73-74], but only recently works have been published, in which it is clearly shown that precipitation polymerization can be a potentially fruitful technique for preparing chromatography-grade molecularly imprinted beads [75]. Recently, precipitation polymerization has been employed for the production of molecularly imprinted microspheres by Ye and Puoci et al [76-78]. Despite the higher yields, a large amount of template molecules is needed for the preparation process, because of the high dilution factor. The feasibility of preparing highly selective morphine imprinting polymer particles using precipitation

polymerization is demonstrated by Ho et al. [79]. Since the template, morphine hydrochloride, is dilute in the polymerization solution, it is assumed that the pH effect can be neglected in the system. The MIP prepared by precipitation polymerization gave uniform particles, and this proved to be a feasible method for fabricating MIP. By controlling the separation point during the cross-linking polymerization process, starting with a dilute monomer solution, uniform molecular imprinted microspheres were obtained in good yield. Compared with the traditional method, these particles exhibit greater recognition binding ability in the sensing of morphine without a time-consuming process of grinding and sieving. The rebinding of MIP by the precipitation polymerization in a solution containing morphine exhibited better performance than that of NMIP. Baggiani [80] prepare near monodispersed polystyrene beads by precipitation polymerization in acetonitrile, and these polymeric beads cheap and easy-to-make, represent a convenient alternative to the expensive chromatography-grade silica in the iniferter-mediated grafting of MIP. The chromatographic behaviour of a column packed with these imprinted beads was compared with another column packed with irregular particles obtained by grinding of a bulk pyrimethanilimprinted polymer. These results are consistent with an influence of the polymerization method on the morphology of the resulting polymer and not on the molecular recognition properties due to the molecular imprinting process. Int. J. Mol. Sci. 2006, 7 1 7 0

Recently, a surface-initiated polymerization method was developed for the preparation of an MIP film grafted on the surface of beads [34]. The process was carried out by initially grafting the initiator to the surface of spherical particles, and then carrying out the polymerization reaction of monomers on the surface of the supports, causing the formation of grafted MIP films. Due to its controllable film thickness, relatively fast mass transfer and regular size, this MIP film grafted on the surface of beads will draw much attention in MISPE.

5.5. Surface Imprinting Polymerization Surface grafting of MIP layers onto preformed beads has been recently proposed as attractive and apparently general techniques to obtain chromatography-grade imprinted materials. In this method, thin imprinted layers have been successfully used as coatings on chromatography-grade porous silica using several techniques to restrain the radical polymerization at the surface of the beads [81-82]. An imprinted layer selective to specific molecules on the surface of widely used polymers without affecting the bulk features was prepared by Sreenivasan [83]. The methodology is simple and modified

surface could be used in applications as diverse as separation, sensing, medical uses, etc. Say et al. [84] prepared phosphorotriesterase mimic surface imprinted polymeric microbeads using MAH Cu(II) as a new metalchelating monomer. The paraoxon hydrolytic activity results showed that hydrolytic activity of PIBs was higher than NIBs. The preparation of the polymer is simple, inexpensive and results demonstrated that the catalytic activity of microbeads has decreased by only 17% after several uses. The surface imprinting technique utilizing water-in-oil (W/O) emulsions was applied to the preparation of a metal ion-imprinted membrane by Araki [85]. The zinc(II) ionimprinted membrane was successfully prepared by emulsion polymerization with 1,12-dodecanediol-O,O-diphenylphosphonic acid (functional host molecule), l-glutamic acid dioleylester ribitol (emulsion stabilizer), and divinylbenzene (polymer matrix-forming monomer). To obtain flexible and mechanically stable membranes for practical applications, the polymerization was conducted in the presence of acrylonitrile-butadiene rubber and hydrophilized poly(tetrafluoroethylene) membranes. The use of acrylonitrile-butadiene rubber and a porous solid support in the polymer matrix resulted in improved flexibility and mechanical strength of the imprinted membrane. The permeation mechanism of the metal ions was considered to be hopping of metal ions on the binding sites in the membranes. Piacham et al. [86] prepare ultra-thin MIP films using surface initiated radical polymerization. Polymer films are directly formed on gold-coated quartz crystal resonator, which offers easy monitoring of polymer growth. With this approach its easy control the thickness of the MIP film to be below 50 nm, where the selective recognition of target analytes can be easily detected by the underlying quartz crystal resonator. When used in a flow injection analysis system, the assembled QCM sensor generated a large frequency change (>30 Hz) upon encountering a small amount of analyte (0.19 mM). The sensor had a very short response time (<1 min), and displayed certain chiral selectivity towards the original template, (S)-propranolol at a concentration higher than 0.38mM in aqueous solution
In addition, in situ polymerization is a very simple and easy method for preparing MIPs for HPLC or SPE separation [29,35,36]. In a typical process, the reaction mixture containing the template molecules, functional monomers, cross-linking agents, porogenic agents and initiators are poured into a stainlesssteel tube, sealed at one end, and degassed ultrasonically. the other end is sealed and the reaction is allowed to process by heating polymerization. After the removal of template molecules,

the column of MIP monolith can be connected directly to the HPLC system for on-line SPE or analysis of target molecule 2.4. In situ polymerisation methods The previously reported methods are used for the synthesis of MIP particles for SPE. Other authors proposed the direct synthesis of an imprinted polymer prepared by electrochemical polymerisation of pyrrole (Py) onto a stainless-steel frit to be coupled on-line [51] or to prepare a thin-layer MIP by rinsing a commercial membrane of Nylon-6 by the polymerisation mixture before initiating the polymerisation [84]. Polymerisation inside the pores of preformed silica-beads was also proposed for synthesizing a MIP used as a LC stationary phase for the direct analysis of phenylureas from vegetable sample extracts [95]. The monolith obtained by bulk polymerisation can also be directly synthesized in situ into a capillary. This approach has been largely reported by Schweitz et al. who developed MIP monoliths for capillary electrochromatography (CEC) to combine the intrinsic high efficiency and enhanced flow dynamics of the CEC with the high selectivity of the MIP for the separation of enantiomers [96,97]. MIP monoliths are covalently anchored to the inner wall of fused silica capillaries so that the use of retaining frits, whose fabrication is very difficult [98], is avoided. Briefly, a typical procedure starts by the activation of the inner surface of the silica capillary using 3-(trimethoxysilyl)propyl methacrylate for a better attachment of the monolith [99]. The capillary is then filled with the polymerisation mixture and the polymerisation is carried out by placing the capillary under a UV source or in a heating bath. When the polymerisation is achieved, the capillary is flushed with a range of solvents to remove remaining reagents and to equilibrate it for later use. This approach enables the preparation of a MIP monolith within 3 h. The success of the synthesis relies on the presence of macropores to provide good flow-through properties and on the simultaneous generation of the selective binding sites [96]. Different parameters control both the pore distribution and the recognition properties, thus a careful optimisation based on chemometric concepts seems interesting. Currently, research focuses on the use ofMIPmonoliths as SPE sorbents. It has to be mentioned that there is a great attention to test and to optimize the MIP monolith format [100]. The future trend in the molecular imprinting field moves towards the introduction of MIP into miniaturized devices [101,102]. There is no doubt that in situ polymerisation is particularly suited for miniaturized devices. However, this approach can also be helpful on a larger scale: an in situ polymerisation was carried out in a stainless-steel column 50mm4mm [16,34] by thermal initiation to be directly used as a pre-column coupled on-line with LC. In this application, the flow-through property was control by optimizing the composition of the porogen, i.e. the amount of toluene in dodecanol [16].

2 Type of molecularly imprinted polymers

There are two types of MIPs: molecularly imprinted particle and monolith. Molecularly imprinted particles are generally used for sample preparation. They are prepared by bulk, suspension, seed, and dispersion/precipitation polymerization methods. The other polymerization methods include grafting or coating of thin films of MIP phases on beaded materials and a molecularly imprinting solgel technique.

2.1 Molecularly imprinted particle

2.1.5 Other polymerization methods

An MIP for sulfamethazine was synthesized on the surface of silica gels using a molecular imprinting solgel technique. First, sulfamethazine was complexed with 3aminopropyltriethoxysilane to form the functional silica gel compounds. Next, functional silica gel compounds of sulfamethazine were integrated with the silica gels into a polymer of sulfamethazine [39]. The prepared MIP was applied for the determination of trace levels of sulfonamides in pork and chicken samples [39].

2.2 Molecularly imprinted monolith

Molecularly imprinted monoliths were divided into two categories: one is monolith materials prepared in a column or capillary column. The other is SPME fiber monoliths. Molecularly imprinted monolith materials for BPA [40], sulfamethoxazole [41], and theophyllin [42] were prepared in a stainless steel column in situ polymerization. Those monolith materials were successfully applied for the determination of BPA and other phenolic compounds in river water samples, sulfamethoxazole in pharmaceutical tablets, and caffeine and theophyllin in green tea, respectively. Furthermore, molecularly imprinted monolith materials were synthesized by two steps in a fused-silica capillary column: a trimethylolpropane trimethacrylate (TRIM) core material was prepared in a 100 lm id capillary and then surface of the core materials was grafted with thin layers of polymers imprinted with local anesthetics (bupivacaine, mepivacaine, and (S)-ropivacaine) using MAA and EDMA as the functional monomer and cross-linker, respectively [43]. However, the molecularly imprinted monolith materials have not been applied to real samples yet. Previously, SPME based on MIPs were carried out using silica fibers coated with an imprinted layer, which are unstable under basic conditions [4446]. SPME fiber monoliths imprinted with diacetylmorphine were prepared by copolymerization of MAA and EDMA. The fiber is stable and flexible enough to repeat SPME of a target compound from aqueous samples [47]. Furthermore, SPME fiber monoliths imprinted with propazine were prepared using fused-silica capillaries as molds with silica being etched away after polymerization. The imprinted SPME fibers showed selectivity for triazines and were successfully applied for triazines in environmental and food samples [48].

5.6. Monolithic Imprinted Polymerization Monolithic molecularly imprinted technology combined the advantage of monolithic column and molecular imprinted technology, which was prepared by a simple, one-step, in-situ, free-radical polymerization molding process directly within a chromatographic column without the tedious Int. J. Mol. Sci. 2006, 7 1 7 1 procedures of grinding, sieving, and column packing. Monolithic MIP is expected to improve the separation and enable direct analysis with high-speed and high performance after insitu polymerization. Matsui et al. [87-88] first used the in-situ polymerization technique for preparation of molecularly imprinted monoliths. Template, functional monomer, cross-linker and initiator were dissolved in mixture porogenic solvents (cyclohexanol and 1-dodecanol) and the mixture was degassed and poured into a stainless steel column. After polymerization, the template and porogenic solvents were removed by exhaustive washing with methanol-acetic acid. The monolithic molecularly imprinted technology has attracted significant interest because of their ease of preparation, high reproducibility, high selectivity and sensitivity, and rapid mass transport. Furthermore, the preparation of this type of MIP is more cost-efficient, because the amount of template molecules required is much lower. Moreover, their greater porosity, and hence good permeability, and high surface area are well suited for both small molecules and large biopolymers. Monolithic molecularly imprinted stationary phases have become a rapidly expanding field in chromatographic stationary phase preparation in recent years [89]. The proportion of mixture composition and polymerization temperature defines the monolithic structure and separation characteristic without further processing. The key to successful column preparation of MIP monolith is both choice of the composition of the pre-polymerization mixture and porogenic solvents and careful timing of the polymerization reaction. In order to compare the different polymerization methods, Mayes et al. [90] prepared three types of MIP by bulk, multistepswelling and grafting methods when some -blockers were used as the template molecules. In that study, ground monolithic imprinted polymer was thought to be the best all-round performer for enantiomeric separations of drugs by HPLC. In-situ polymerization has similar recognition ability and possesses the advantages of a one-step preparation procedure and high yield. Matsui and Huang [91-93] prepared a set of monolithic molecularly imprinted polymers with cinchonine and amino acid derivatives as the

template molecules. Separation of the corresponding enantiomers was achieved but the separation mechanism was not mentioned. In recent years, the uses of monolithic media for superior chromatographic separation in high-performance liquid chromatography and capillary electrochromatography have attracted considerable attention [94-102]. Imprinted monolithic membranes by polymerizing mixtures of methacrylic acid and dimethacrylate crosslinkers within microporous support of filtered paper is described by Kieczynski [103]. These membranes are selectively permeable for template molecules but transport of others species is mostly limited. The measured transport stereoselectivity varied from 1.1 to 3.7 depending on the system used and the presence of cinchonine in the monomer mixture made membrane more permeable while cinchonidine reduced its permeability. Optimization of the imprinted polymer membrane in terms of the number of binding sites and their selectivity for the template enantiomer ought to be connected with optimization of membrane porosity and template interactions with functional groups. When EDMA and EDMA:MAA monolithic membranes behave in the predicable way, i.e. transport of cinchona alkaloids follows membrane templating, TEGDMA-family membranes show abnormal preference to transport cinchonidine more effectively.

5. Preparation Methods of MIP 5.1. . . 2.3. MIPs Physical Forms and Methods MIPs can be prepared in a variety of physical forms, using different method, depending on their final application [9,20]. The conventional approach is to synthesize MIPs in bulk, then grind the resulting polymer and successively sieve the particles into the desired size ranges according to the specific application. This method is the most popular since it is simple; nevertheless, crushing, grinding and sieving to obtain the appropriate particle sizes is tedious and time-consuming and often produces particles that are irregular in size and shape. In addition, some interaction sites are destroyed during grinding, reducing the MIP loading capacity and, since only a portion of the original polymer is used, this method suffers from high consumption of the template molecules. Most imprinting

publications are still based on use of this method although the scarce control of the MIP physical form and difficulties in scaling up MIP production are important drawbacks. In order to overcome these problems, alternative methods to prepare novel MIP formats, such as MIP beads, membranes, in situ prepared monoliths, surface imprinting, molecularly imprinted monolayers have been developed in recent years [9]. Regular beads can instead be obtained using precipitation polymerization method. This technique allows the formation of imprinted beads with the same reaction mixture used in the bulk method except for the presence of a higher amount of porogen. Polymer chains will continue to grow, precipitating only when become large enough to be insoluble in the reaction mixture. Then the polymer beads are easily recovered by washing and centrifugation operations. This technique is easy, less time consuming than bulk polymerization and provides regular beads in good yields. The particles diameters decrease, increasing the porogen volume probably due to the formation of less oligomers and nuclei under diluted conditions and with an increase of flow ability of the polymer, thus less monomers and cross-linkers diffuse to the surface and polymerize into the small particles. In a recent work [53], a feasible defined control of MIP size from nanoparticles to microbeads that can be utilized in different applications by changing the reaction conditions was obtained. The change of particle size, while maintaining excellent recognition property, was achieved by varying the ratio of two different cross-linking monomers (DVB and TRIM) in similar precipitation polymerization conditions. Polymers in the range of 130 nm to 2.4 m covering all pore sizes typically obtained with precipitation technique were prepared. Mosbach and Mayes [55] prepared spherical beads in liquid perfluorocarbon by suspension imprinting polymerization in the presence of a stabilizer and a surfactant. More recently Zourob et al. [56], using a polycarbonate-based spiral microflow reactor, demonstrated that in mineral oil, which is less expensive than perfluorocarbon, it is not necessary to add any stabilizer or surfactant to produce monodisperse MIP beads.

In recent years molecularly imprinted polymer membranes have attracted significant interest. Porous free-standing molecularly imprinted polymer membranes were synthesized by the method of in situ polymerization using the principle of synthesis of interpenetrating polymer networks and tested Int. J. Mol. Sci. 2011, 12 5918 in solid-phase extraction of triazine herbicides from aqueous solutions. Addition of oligourethane acrylate provided formation of the highly cross-linked MIP in the form of a free-standing 60 m thick flexible membrane [57]. However, for separation purposes low membrane permeability was still the main obstacle for their application in separation processing. Yang et al. [58] prepared molecularly imprinted nanotubes supported by a porous alumina membrane. The imprinted nanotubes could be used directly for the separation of biomolecules, without the alumina support being removed. Monolithic MIPs have also been prepared by a simple, one-step, in situ freeradical polymerization process directly within the confines of a chromatographic column without the need of grinding, sieving and column packing. Surface grafting of MIP layers onto preformed beads has also been proposed as a technique to obtain chromatography-grade imprinted polymers. In this method, thin imprinted layers have been used as coatings on chromatography-grade porous silica or spherical polymers using several techniques to retain the radical polymerization at the surface of the beads. Molecularly imprinted monolayers have also been prepared for sensor applications. Lahav et al. produced recognition sites for a naphthacenequinone derivative in a thiol monolayer on gold electrodes through a photochemical imprinting method [59]. The group of Piletsky and Turner developed a MIP sensor for domoic acid (DA) based on amino-substituted methacrylate crosslinked with ethylene glycol dimethacrylate with a direct photografting of their polymer onto a sensitive gold layer suitable for Surface Plasmon Resonance (SPR) detection [60].
MIPs can be prepared in a variety of physical forms to suit the final application desired (Table 1). The conventional

approach is to synthesize the MIP in bulk, grind the resulting polymer, and sieve the resulting particles into the desired size ranges [4244]. This method is by far the most popular, since it is simple but the crushing, grinding and sieving to obtain the appropriate particle size is tedious and time-consuming and often produces particles that are irregular in size and shape. Some interaction sites are destroyed during grinding, and this reduces the chromatographic performance and the MIP loading capacity. Moreover, since only a portion of the original polymer is used as packing material, this method suffers from high consumption of the template molecules. In order to overcome these problems, much effort has been dedicated in recent years to developing alternative methods to prepare MIPs which are superior in terms of efficiency and mass transfer properties [45, 46]. Uniform spherical particles have been obtained by using multi-step swelling methods [4749], in which particles can be prepared directly in the form of spherical beads of controlled diameter. The particles obtained using this technique are comparatively monodisperse in size and shape and well suited for chromatographic applications. However, fairly complicated procedures and reaction conditions are required, and the aqueous suspensions used in this technique may interfere with the imprinting and thus lead to a decrease in selectivity. Suspension polymerization [5052] and precipitation polymerization [5356] are two other methods for the preparation of imprinted supports that yield aggregates of spherical particles, and if the system is sufficiently dilute, uniformly sized microspheres. In this way nearmonodispersed spherical beads can be prepared, and size and porosity can be fine-tuned by changing the polymerization conditions. To avoid interference in multi-step swelling, perfluorocarbon solvents have been applied for suspension polymerization to avoid the influence of water. Although regular molecularly imprinted microspheres have been prepared and excellent chromatographic performance obtained, the specialized fluorinated surfactant limits the applicability and practicality of this method. Surface grafting of MIP layers onto preformed beads has been recently proposed as an attractive and apparently general technique to obtain chromatography-

grade imprinted materials [5760]. In this method, thin imprinted layers have been successfully used as coatings on chromatography-grade porous silica or spherical polymers using several techniques to retain the radical polymerization at the surface of the beads. Monolithic MIP technology, as a novel method for the preparation of chromatographic stationary phases, combines the advantages of molecular imprinting and monolithic column technology [6163]. Monolithic MIPs areprepared by a simple, one-step, in-situ, free-radical polymerization moulding process directly within the confines of a chromatographic column without the need of grinding, sieving, and column packing. The proportion of mixture composition and polymerization conditions defines the monolithic structure and separation characteristics without further processing [64]. This method has attracted significant interest because of its ease of preparation, high reproducibility, high selectivity and sensitivity, and rapid mass transfer. Furthermore, the preparation of this type of MIP is more costeffective, because the amount of template molecules required is much lower. The use of monolithic MIPs has become a rapidly expanding field in stationary phase preparation in recent years [65