El ideal del análisis de nivel neutro 1. Nattiez y Roy

Polymer synthesis methods to prepare MIPs have been well reviewed (Yan and Row, 2006). Some of the important available polymerization methods include bulk,

dispersion/precipitation, multi–step swelling, suspension, surface and in-situ polymerization.

2.6.2.1.3.1. Bulk polymerization

Most molecular imprints have been synthesized by bulk polymerization where the functional monomers have been prearranged around a template molecule in an organic solvent and copolymerized with an excess of crosslinkers in the presence of free radical initiator induced by either thermal or photochemical methods. The resulting block polymer is then crushed to the desired particle size and the template is washed. The polymers thus prepared are irregular in shape with a wide range in particle size distribution and have been often utilized as solid phase extraction material due to good specificity in binding (Al-Kindy et al., 2000; Holthoff and Bright, 2007; Petcu et al., 2009). However, some studies have identified disadvantages of bulk

polymerization, including a complicated synthesis process, low recognition binding sites within the polymer matrix and poor binding kinetics for the template molecules (Pichon and Chapuis-Hugon, 2008; Beltran et al., 2010).

2.6.2.1.3.2. Precipitation polymerization

Precipitation polymerization is preferred over bulk polymerization to synthesize regularly shaped and monodispersed particles of polymers that have better binding properties.

Basically, the synthetic process starts with very dilute monomers that are allowed to propagate in solution to reach a critical mass and then precipitated from the solution by decreasing the solubility using a large amount of crosslinkers. The polymer beads are separated by

centrifugation and then washed. This technique offers an efficient methodology for the

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synthesis of MIP beads and has been applied in the preparation of MIP nanospheres for capillary electrochromatography (Nilsson et al., 2004; Turiel and Martin-Esteban, 2005; Priego-Capote et al., 2008). However, the limitation of this approach is that the monomers should be soluble in the solvent while the resulting beads should be insoluble and the beads may aggregate resulting in inferior mass transfer properties. To overcome the problems of aggregation, some have used multi-step swelling and polymerization that also produced spherical particles (Beltran et al., 2010; Gokmen and Du Prez, 2012).

2.6.2.1.3.3. Multi-step swelling and polymerization

In this approach, uniformly sized seed particles are suspended in water and allowed to swell to a certain pre-determined range (usually 5-10 µm) by several additions of appropriate organic solvents. The components involved in the production of MIP are then added to the solution during the swelling stage and polymerization is initiated (Beltran et al., 2010). A uniformly sized molecularly imprinted polymer prepared by multi-step swelling and polymerization using methacrylic acid for d-chlorpheniramine showed superior selectivity towards template compared to non-imprinted polymer and slight recognition for its structurally related compounds (Haginaka and Kagawa, 2002). In another study, methacrylic acid-based MIP non-covalently prepared by a two-step swelling technique using isomers of diaminonaphthalene or a chiral amide derived from (S)-α-methylbenzylamine as the template molecule have been shown to enhance chiral recognition (Hosoya et al., 1996). However, even though polymer particles are relatively monodispersed in size and shape and appropriate for chromatographic applications, fairly complicated procedures and reaction conditions are required. Additionally, the requirement for aqueous suspensions (emulsion) used in this technique could interfere with the imprinting leading to a decrease in selectivity.

2.6.2.1.3.4. Suspension polymerization

A different approach to preparing spherical particles is to use suspension

polymerization. In this method, all the components are dissolved in organic solvent and later transferred to a larger volume of immiscible solvent. The mixture is stirred vigorously to

facilitate droplet formation and then subjected to polymerization induction. Imprinted polymers synthesized using acrylamide monomers for bovine serum albumin using this technique

exhibited good recognition for template proteins as compared to the control protein and also showed better adsorption kinetics of MIP (Pang et al., 2005). Using this two-phase system,

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regular MIP microspheres have been synthesized that exhibited an excellent chromatographic performance with superior selectivity, even at high flow rates (Mayes and Mosbach, 1996;

Zhang et al., 2003). Unfortunately, this approach uses immiscible liquids (e.g., perfluorocarbons) instead of water as the continuous phase to avoid the detrimental effect of water on the non-covalent complex between monomers and imprint molecule thereby imposing limits on the applicability and practicality of this method.

2.6.2.1.3.5. Surface imprinting

Surface imprinting is another polymerization technique aimed at delivering spherical particles with an imprinted layer grafted on the surface (Pichon and Chapuis-Hugon, 2008;

Beltran et al., 2010). This approach is well suited to make the coating on chromatography-grade porous silica or spherical polymers (Vasapollo et al., 2011). Here, all the components involved in the polymerization process are absorbed into the beads or silica particles and then polymerized.

The silica particles or the beads are then etched away to reveal the final product that retains the shape of the original bead or silica particle (Komiyama et al., 2003; Tan and Tong, 2007). This surface imprinting technique was used to prepare metal-ion imprinted membranes by water-in-oil emulsion polymerization (Araki et al., 2005). Superior chiral selectivity towards the original template was observed with surface-initiated radical polymerization of ultra-thin MIP films grafted on gold-coated quartz crystal (Piacham et al., 2005). Despite their good recognition properties in aqueous media, these membranes were not stable in an organic environment.

2.6.2.1.3.6. In-situ polymerization

In-situ polymerization uses interpenetration of polymer networks by crosslinking and are commonly used to synthesize MIP membranes. The in-situ polymerization technique was first used for the preparation of molecularly imprinted monoliths (Matsui et al., 1993).

Template, initiator, monomer and crosslinker are dissolved in porogenic solvents and poured into a stainless steel column and polymerized. The template and porogenic solvent are removed by thorough washing with acidic methanol solvent to form a monolithic molecularly imprinted polymer. This technique has advantages including ease of preparation, high reproducibility, high selectivity and sensitivity, and rapid mass transport. Their great porosity, and hence good permeability, and high surface area are well suited for both small molecules and large

biopolymers. In recent years, monolithic molecularly imprinted stationary phases have become popular in chromatographic stationary phase preparations (Liu et al., 2005). However, some

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conflicting results were also reported showing low membrane permeability (Sergeyeva et al., 2007; Vasapollo et al., 2011) which was suggested to be an obstacle for application in separation technology.