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Recent advances in capillary electrochromatography using molecularly imprinted polymers Bogdan-Cezar Iacob, Ede Bodoki*, Radu Oprean Analytical Chemistry Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 4, Louis Pasteur St., 400349, Cluj-Napoca, Romania
Corresponding Author *Ede Bodoki Address: “Iuliu Hatieganu” University of Medicine and Pharmacy, 4, Louis Pasteur St., 400349, ClujNapoca, Romania email: [email protected]. Tel.: +40 264 597 256 Fax: +40 264 597 257
Abbreviations α, separation factor; γ-MAPS, 3-(trimethoxysilyl)propyl methacrylate); AML, amlodipine; C, crosslinking agent; EDMA, ethylene glycol dimethacrylate; HRP, horseradish peroxidase; I, initiator; KET, ketoprofen; LiqCr, liquid crystal; Lyz, lysozyme; M, monomer; MAA, methacrylic acid; MAM, 2methacrylamidopropyl methacrylate; MIP, molecularly imprinted polymer; MSA, methylenesuccinic acid; NAP, naproxen; NIP, non-imprinted polymer; NP, nanoparticle; OFX, ofloxacin; OT, opentubular; Rs, resolution; T, template; TRIM, trimethylolpropane trimethacrylate; ZOP, zopiclone;
Received20-May-2014; Revised: 07-Jul-2014; Accepted: 10-Jul-2014
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400253.
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Keywords capillary electrochromatography, electrophoresis, molecularly imprinted polymers, sample preparation, stationary phases
Total words 8429
Abstract There is an increased and continuous need for developing new methods for the separation and quantification of an increasing number of analytes in the environmental, pharmaceutical, pharmacological and toxicological sciences. CEC is still withholding its popularity, representing a viable alternative to the more conventional techniques (HPLC, GC) due to the numerous advantages, such as, low sample/reagent volumes, high separation efficiencies, hybrid separation principle, etc. One particular promising direction in CEC is the use of molecularly imprinted polymers (MIPs) as stationary phases. They are usually immobilised in the capillary column as a continuous polymeric monolith or as a thin polymer coating attached to the capillary’s inner wall. Another emerging trend is the use of MIPs in the form of nanoparticles (NPs) as a pseudostationary phase. This review discusses the recent developments (2011-2013) in finding the optimal polymerization mixture and the suitable MIP format that should be employed in CEC separations. The most important applications of MIPs in CEC technique are also highlighted.
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I. Introduction One of the biggest challenges for a researcher is to reproduce processes that occur in nature, like translation and transcription of the genetic code, photosynthesis, development of biomimetic materials with molecular recognition properties, etc. A particularly exciting area of biomimetics is Molecular Imprinting which can be defined as an artificial tiny lock for which the assigned key is represented by the target molecule. Molecularly imprinted materials are prepared by assembling around a template molecule by polymerization of functional monomers and a crosslinker. The interactions between the template molecule and the monomer’s functional groups represent the basis of molecular imprinting technique and they must be maintained throughout the polymerization process. The choice of functional monomer is critical for the fabrication of molecularly imprinted polymers (MIPs) in order to provide complementary interactions with the template [1]. Extraction of template leaves behind three-dimensional cavities that are complementary in size and shape to the template. The obtained cavities present a permanent memory for the imprinted specie, allowing the polymer to selectively entrap the imprint molecule from a mixture of closely related compounds. Using this technique, highly selective synthetic receptors with antibody-like recognition characteristics may be obtained and, in principle, they can be applied for molecular structures spanning from ions to biomacromolecules [2-4]. The high chemical and thermal stability, time efficiency, relatively low cost of production, morphology, and good analytical performance of MIPs allowed a rapid growth of their biomedical and biotechnological applications. Their unique selectivity towards a specific target molecule determines a predicted elution order of enantiomers but is disadvantageous when trying to develop a generic stationary phase. They have been used as stationary phases (in liquid chromatography [5], CEC [6], supercritical fluid chromatography [7], thin-layer chromatography [8] and solid-phase extraction [9]), as structures mimicking antibodies [10] and as sensing elements in various devices [11, 12]. Among the mentioned analytical techniques, CEC experienced a significant growth since its This article is protected by copyright. All rights reserved.
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development in 1970s due to its inherent advantages. Furthermore, the small volume of capillaries and therefore the minute amount of needed MIP facilitated the boost of applications using MIP-CEC. Nevertheless, lately a slightly declining trend can be observed in the number of published articles focusing on this topic. This may be due to the persisting shortcomings still associated with this technique. The limited reproducibility of capillary packing or in situ polymerisation, alongside with the lack of commercial availability of a wide selection of CEC columns prevent this technique in becoming a robust alternative for liquid chromatography. Other drawbacks worth mentioning is the need of external pressure to avoid bubble formation during analysis, the incomplete release of template molecule after MIP synthesis causing continuous leakage, or the poor heat dissipation. Nonetheless, recently some new approaches were also reported that may open up new perspectives in the development of CEC methods using MIP. The present review is focusing on new and significant aspects reported in the last 3 years (2011-2013) that may pave the way in alleviating the constraints regarding MIP synthesis and their applications. Recent improvements in the preparation of MIPs in terms of polymerization mixture’s composition and the obtained polymer’s porosity are also discussed. Furthermore, the practical utility and the potential of available MIP-formats are also analysed. Finally, the latest applications of MIPs in CEC concerning sample preparation, chiral analysis and macromolecule analysis are highlighted. Table 1 provides the reader with an overview of all articles covered by this review.
II. MIP-synthesis
II.1. Template /Monomer /Cross-linker ratios Currently, the most widely employed technique in creating MIPs is the non-covalent molecular imprinting approach. The imprinting effect is governed by the formation in the prepolymerization mixture and maintaining throughout the polymerization process of certain This article is protected by copyright. All rights reserved.
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complexes between the functional monomer and template molecule (Fig. 1). These complexes are formed by non-covalent interactions, such as hydrogen bonding, ion pairing, hydrophobic interactions, dipole-dipole and van der Waals forces. These interactions are weak, but allow a rapid and reversible binding well suited for chromatographic separations [13]. Many parameters have to be assessed in the synthesis of MIPs, since they can influence the morphology, properties and performance of polymers. The prepolymerization mixture is represented by a solvent in which four types of substances are dissolved: one or several functional monomers (M), template(s) (T), initiator (I) and crosslinking (C) agent(s). For the synthesis of organic MIPs, aprotic organic solvents with low polarities represent the appropriate medium for the stabilization of the hydrogen and/or electrostatic bonding between the monomer and template molecule. Highly polar aprotic solvents (e.g., dimethylformamide) tend to interfere with the ionic and van der Waals forces emerging between the imprint molecule and the functional monomer, while protic solvents (methanol, water) competitively interfere the hydrogen bonding interactions [14]. For non-covalent imprinting, the ratios between T, M and C play a crucial role in the imprinting effect. Unfortunately, even nowadays, having many chemometric optimization tools (experimental design) at disposal, the optimal ratios are still achieved empirically following tedious and frequently inefficient protocols by keeping a component constant and varying the other two. Usually, a large excess of functional monomer compared to the template is required during the polymerization to favour the formation of template-monomer assemblies. In fact, the association between the monomer and the template is governed by Le Chatelier’s principle; therefore increasing the concentration of components shifts the equilibrium towards complex formation. Recently, Ye et al. [15] investigated the influence of T/M/C ratio on the size, size distribution and the molecular binding property of resulting MIP-nanoparticles (NPs). They synthesized propranolol-imprinted NPs using precipitation polymerization employing different T/M/C ratios. Using elemental analysis, they observed that the elemental composition of the MIPs and their
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corresponding non-imprinted polymers (NIPs) are almost identical, suggesting that the presence of the template does not influence the ratio of the incorporated monomers. Comparing dynamic light scattering measurements on colloidal dispersions with scanning electron microscopy on dried NPs, it was found that NPs present a larger diameter in a wet state due to swelling. The obtained spherical polymer particles, with a size of several hundred nanometers manifested a good colloidal stability, thus representing a viable option as pseudostationary phases in CEC. Another important conclusion of the study was that in a purely nonimprinting system, the amount of functional monomer (methacrylic acid (MAA)) and the used crosslinking density has little effect on the hydrodynamic size of the synthesized poly(MAA-co- trimethylolpropane trimethacrylate (TRIM))-NPs. In fact, the size of the obtained imprinted NPs is dependent on the template’s polarity and the strength of its molecular interaction with the functional monomer, phenomena previously demonstrated using propranolol as template, which changed the solubility of the growing polymer chains and also affected the rate of particle growth [16]. The highest template binding capacity (~ 170 fmol/mg specifically bound template, value 20 times higher than nonspecific binding) was obtained using a slight excess of template (1–1.3 equivalents) relative to MAA and in the presence of a good crosslinking density. Most often more than one compound of interest needs to be determined in a sample and the single molecule imprinted polymer is not suitable for the analysis of complex mixtures. Furthermore, the fabrication of individual MIPs for each analyte might be severely time-consuming. Recently, Zaidi [17] fabricated inside capillary columns dual-templates (serotonin and histamine) imprinted polymer monolith for their subsequent separation in CEC. In order to achieve best resolutions (Rs) and separation efficiencies different preparation protocols were employed while keeping the molar equivalents of both the templates equal. The influence of two functional monomers, MAA and methylenesuccinic acid (MSA) were evaluated, the latter offering a 2 fold increase in resolution (T/M/C = 1-5-19), as shown in Fig. 2. In the effort of producing a more rigid
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polymer structure able to conserve the imprinted binding sites, the increase of cross-linker ratio did not influence significantly resolution. This enhanced resolution may be explained by the two carboxylate functionalities of MSA, promoting stronger interactions with the templates than MAA, but the degree of monolith uniformity throughout the capillary may also have played its role in case of MSA. Liu et al. [18] observed the same improvement in the imprinting effect using MSA as functional monomer in comparison with MAA. A monolithic polymer with (-)norepinephrine as template was synthesised being employed as CEC stationary phase for the separation of dopamine, (±)-epinephrine, (−)-isoproterenol, (±)-norepinephrine, (±)-octopamine, and (±)-synephrine. In case of MAA, increasing the amount of functional monomer or crosslinker led to a poorer resolution of (±)-norepinephrine. Using MSA as functional monomer, a baseline separation was achieved with a T/M/C molar ratio of 1-9.2-19. In the last decade, molecularly imprinted NPs have gained momentum due to their high surface area and better accessibility to the imprinted cavity, leading to a much faster analyte binding equilibrium [19-21]. The most simple and efficient method in preparing these NPs is the precipitation polymerization method, but in case of some templates difficulties in creating uniform NPs has been reported [22, 23]. As already discussed above, the morphology and size distribution of MIP particles is determined by the nature of imprint molecules added in the polymerization mixture. When Chen and Ye [19] adopted the same polymerization mixture for atrazine as the one with which uniform propranolol-imprinted NPs were obtained, [24] much bigger particles with a very broad size distribution were generated. Interestingly, with the addition of small amount of propranolol (5%, w/w) as an auxiliary template in addition to atrazine, small (147nm) and uniform NPs were achieved. The same improvement was observed when 5% propranolol was added in the prepolymerization mixture for the imprinting of estradiol and theophylline with the reduction of particles sizes from 208 to 99 nm and from 361 to 131 nm, respectively. This significant effect was also reported for
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other β-blockers, like pindolol and atenolol, and was attributed by the authors to the strong hydrogen bond interactions between the isopropylaminopropanediol moiety in the β-blocker’s structure and the used functional monomer (MAA). The effects of polymerization variables (e.g. T/M molar ratio, type and amount of functional and cross-linking monomers) on chiral separation of zopiclone (ZOP) in the preparation of molecularly imprinted NPs were investigated [25]. Even if the T/M and M/C molar ratios were varied on a wide scale (from 1-3 to 1-10) no significant changes on the MIP’s chiral performance were recorded. The positive effect of the optimal polymerisation component ratio was hindered by the agglomeration of the emerging NPs leading to the formation of non-uniform microparticles. Additionally, the adsorption data of the polymer indicated a very wide distribution of pore sizes. The partial substitution of the functional monomer (MAA) with other monomers (acrylamide, 4vinylpyridine or butyl methacrylate) yielded lower column efficiencies and resolutions. The key to a good non-covalent molecular imprinting is a full complexation between the functional monomer and the imprint molecule, which should be maintained throughout the process of polymerization. By adding in the polymerization mixture of a crowding-inducing agent, namely polystyrene dissolved in THF, the complexation is considerable increased [26]. In this case both selectivity and efficiency of the obtained MIP microparticles were improved in comparison with the use of plain ACN as porogen. In the fabrication of S(-)ofloxacin (OFX)-imprinted microparticles, several parameters like the choice of solvents and co-solvents, T/M ratio, type of cross-linking monomers and functional monomer composition were investigated. It was observed that microparticles prepared in polystyrene-THF presented a broader size distribution and lower porosities compared to those prepared in ACN. The optimal T/M ratio was found to be 1-4, offering the best resolution and smallest particle sizes. The type of cross-linking monomer had little influence on the resolution of OFX enantiomers. The addition of a hydrophobic co-monomer (50% butyl
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methacrylate) along MAA led to an increase in separation efficiency, with the baseline separation of OFX enantiomers. As can be seen from the foregoing, even if the formula of MIP preparation is relatively simple, there are many variables needing optimization, like type of functional monomers and crosslinker to be used and the optimum ratio of T/M/C. By combining the functional monomer and the cross-linker into one compound playing both roles, the optimization process is drastically reduced [27]. The functional cross-linker, 2-methacrylamidopropyl methacrylate (MAM), was employed in synthesising a MIP-coating inside a silica capillary column with S-amlodipine (AML), Snaproxen (NAP) or S-ketoprofen (KET) as template molecule for the CEC chiral analysis of the corresponding racemic mixtures. The selected imprint molecule-single cross-linking monomer molar ratio was 1-4 and the porogen used was toluene-isooctane (7/3 (v/v)). The racemic AML, NAP and KET were all resolved employing the same polymerization mixture’s composition with excellent resolutions: 16.1, 1.46 and 4.56, respectively. This approach of using one-monomer in the composition of MIP is not only a simplification improvement, but also offers a higher chiral separation ability and column stability than conventional MAA- ethylene glycol dimethacrylate (EDMA) two-monomer system. In the same trend of MIP synthesis simplification, recently the concept of generalized preparation protocol was introduced, recommending a single polymerization mixture regardless of the nature of imprint molecule [28-31]. In the first reports, the chiral separation capability of the polymerization mixture was demonstrated for a number of acidic analytes, like non-steroidal antiinflammatory drugs, (3-(benzyloxycabonyl)-4-oxazolidine carboxylic acid, OFX, mandelic acid, succinic acid, 2-phenylbutyric acid and camphor [28, 29]. The protocol was further tested for some additional acidic templates, like phenyl carboxylic acids and their derivatives [32] and camphor analogues (10-camphorsulfonic acid, 10-camphorsulfonamide and camphor-p-tosyl hydrazone) [33]. The applicability of the pre-established generalized preparation protocol was expanded to the use of
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neutral templates, like (4S,5R)-4-methyl-5-phenyl-2-oxazolidinone [30]. The open-tubular (OT)-MIP capillary columns employing basic templates were fabricated and evaluated using the preestablished generalized preparation protocol. The basic templates taken into study were: atenolol, sulpiride, methyl benzylamine and (1-naphthyl)-ethylamine [31]. The recommended generalized polymerization mixture consists in the template molecule (0.020 mmol), MAA (0.096 mmol/L), EDMA (0.313 mmol/L), 4-styrene sulfonic acid (2 mg) and AIBN (3.5 mg) dissolved in 1mL 90/10 (v/v) ACN/2-propanol.
II.2. High vs. low crosslinking The standard molecular imprinting procedure involves mixing the template molecule along with the functional monomer to form a complex through covalent or noncovalent interactions over which a large amount of cross-linking agent and a porogen are added. The copolymerization is then started by radical initiation leading to macroporous polymers in which template-monomer complexes are trapped inside. By removing the template molecules after polymerization, welldefined 3D cavities are obtained; the cavities have similar size dimensions and shapes to the template, and functional groups that are complementary to the template molecule or analogue structures. The “molecular memory” of the obtained polymers is “stored” in these cavities, which must be preserved and stabilized by increasing the rigidity of the polymeric network. This is achieved by adding an excess of crosslinking monomer to the polymerization mixture (around 70-95%) [13, 34]. Unlike covalent imprinting where 90-95% of the binding sites are available for template rebinding, in case of non-covalent approach, only 15% of sites can reuptake the template molecule, most probably due to poor site accessibility caused by the shrinkage of the cavities [2, 35]. Therefore, in order to facilitate a fast equilibrium between template release and rebind in the cavity a high flexibility of the polymer is required. These two opposing parameters, rigidity and flexibility of the polymeric network needs to be finely balanced by an optimization step [13].
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An approach of stabilizing binding sites of MIPs was introduced by Mauzac et al. [36-41] relying on the benefits of mesomorphous organization of the liquid crystal (LiqCr) materials. Thus, LiqCr-MIPs with low levels of cross-linking (5-20 mol% of the monomer units) were obtained in which chemical cross-linking was replaced by physical cross-linking, due to the non-covalent reversible interactions between mesogenic moieties. Owing to the low content of cross-linker the LiqCr-MIPs exhibited similar selectivity but much higher loading capacity compared with the classical MIPs [36]. Using the same strategy, Wei et al. [35] reported for the first time a LiqCr-MIP capillary coating (~0.1-0.2 µm thickness) for the chiral separation of AML, NAP and OFX in CEC mode. On the low cross-linked MIP coated capillary separation of racemic AML was achieved in 2.5 minutes with a resolution and selectivity factor of 6.36 and 1.81, respectively. Additionally, no peak tailing for the AML and NAP template enantiomers was observed, providing the better accessibility to the binding sites and/or the improved mass transfer. Using LiqCr-free MIP capillary (80% molar ratio of EDMA) the same separation was accomplished in twice as long time with a 5 times smaller resolution, as shown in Fig. 3.
II.3. Stationary vs. pseudostationary phases Three main types of stationary phase formats are commonly used in CEC: monolithic, particle packed and OT-capillary columns. The alternative is the use of MIP-NPs as pseudostationary phases; however the provided number of interaction sites with the template is lower than in the case of MIP-based stationary phases when a higher amount of polymer is immobilized in the column. The MIP-NPs are most often employed in CEC mode through partial filling technique, being injected prior to the sample, as a plug shorter than the effective length of the capillary. The length of the MIP-NPs plug must be carefully adjusted so that analytes reach the detector before the NPs plug in order to avoid interferences with the employed detection. Therefore, as a rule of thumb the apparent mobility of the analytes should be greater than the one of the NPs.
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The advantages of using MIP-based pseudostationary phases in electrodriven separations, consists in the use of a fresh MIP-NP plug for each analysis, avoiding problems related to strong adsorption of templates during analyses, such as column-ageing and poor reproducibility [42]. Moreover no frits and tedious particle packing protocols are needed. However, there are also some important constraints employing NPs as pseudostationary phases, the most important ones being related with the stability of the NPs colloid and the improved reproducibility ensured by fresh MIPNP plug used in each analysis. One of major factors conditioning the stability of dispersions is particle size, which obviously has to be in the submicrometer-range. Several methods have been developed for the production of MIP-NPs, like: grounding and sieving bulk polymers [43], precipitation polymerization [19, 25, 26, 44], emulsion polymerization [45] and surface templated emulsion polymerization [46]. The simplest, most elegant and widely used approach in preparation of NPs as pseudostationary phases in electrodriven techniques is precipitation polymerization. In this technique a diluted polymerization mixture (identical reaction mixture as in the bulk method, but with a higher volume of porogen) without any other additives is used, resulting in spherical polymer beads with good yields. Due to its versatility, precipitation polymerization leads to an increase in the number of publications focusing on MIPs as pseudostationary phases. This procedure was described for the preparation of MIP-NPs in the enantioseparation of various small drug molecules: ZOP [25, 44], tryptophan [47] and OFX [26]. As previously mentioned, Liu et al. [25] obtained d-ZOP MIP-NPs (~100nm) that formed micrometric agglomerates. These particles ensured a successful separation of ZOP enantiomers within 10 minutes with a resolution and a separation factor (α) of 4.75 and 1.11, respectively. Comparable selectivity with a better Rs reproducibility (RSD below 3%) were observed in case of liquid crystal d-ZOP-MIP-NPs with low crosslinking degree [44]. Adding LiqCrs in the polymerization mixture MIP-NPs with narrower size distribution were obtained with no sign of
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agglomeration between particles (Fig. 4). An even shorter analysis time (below 7 min) with similar selectivity (Rs=4.62, α=1.29) was achieved using a MIP coating grafted on a TRIM core monolith [48]. Unfortunately, the gain in the analysis time is hardly justified by the increased column preparation time: 15 h for TRIM core monolith synthesis and an additional 3 h for MIP coating polymerization. The chiral CEC separation of OFX was achieved by Shi et al. [26] in less than 20 minutes, with Rs and α of 1.53 and 1.07, respectively. As previously mentioned, the pseudostationary phase consisted in MIP-microparticles prepared by the molecular crowding approach. Once again, using MIP grafted poly(TRIM) monolith [48] offered a better resolution (Rs= 4.97) and separation factor (α=2.35), but probably due to the heterogeneity of imprinted sites a broad peak of template enantiomer was observed. The template (S-OFX) peak shape was not improved even when a LiqCr MIP-coating column with low level of crosslinking was employed [35]. Core-shell particles of uniform size (~87 nm) with good colloidal stability in aqueous buffers were achieved by Yue et al. [47] using surface L-tryptophan imprinting on silica NPs. They were employed in CEC as pseudostationary phase for the chiral separation of tryptophan enantiomers (under 10 min, Rs= 2.73) providing symmetrical peak shapes due to the fast mass transfer and good accessibility of the interaction sites. In spite of the highlighted advantages of MIP-based pseudostationary phases (simple, fast and reproducible synthesis, no column ageing and decrease of analytical performances due to strong adsorption, improved mass transfer), most probably due to the more simple analytical method development and optimization, the use of MIP-stationary phases is still more prevalent in the enantioseparation of small drug molecules: AML [27, 35, 48], tyrosine [49], neurotransmitters [18], ractopamine [50], NAP [27, 35], KET [27], atenolol and sulpiride [31]. MIP-based stationary phases, especially monolithic and particle packed columns, tend to be more frequently employed in imprinting large templates (protein recognition), due to their large specific surface area and higher loading capacity in contradistinction to pseudo-stationary phases.
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Thus, a successfully isolation and separation of lysozyme (Lyz) from egg white (Fig. 5) and human serum was achieved on a monolithic stationary phase [51]. A new molecularly imprinted monolithic column was obtained on the surface of a boronicbased polymeric skeleton by polymerization of dopamine using horseradish peroxidase (HRP) as a template [52]. The use of boronate monolithic skeletons permitted the reversible immobilization onto the monolith of the glycoprotein via cyclic boronate esters while the subsequent polymerization of dopamine takes place.
II.4. Monolith // packed // open-tubular capillary columns / chips MIPs were extensively explored as stationary phases in sample treatment, HPLC and CEC. The traditional approach of preparation implied bulk polymerization outside the column resulting blocks of molecularly imprinted materials, that was crushed, grounded and sieved in order to obtain similar sized nano- or micro- particles, followed by their packing into the capillary with the aid of frits. Unfortunately, this method presents many drawbacks. First of all, such preparation of MIP particles is tedious, time consuming and brings low yields. Furthermore, the ground beads are irregular in shape with relatively wide size distribution and increased heterogeneity of the binding sites. Better performances were achieved with improved particle preparation protocols (e.g. suspension or precipitation polymerization method) but upon the packing of capillary the high backpressure remains a problem. Another inconvenience is the need of frit preparation. Ideal frits are tricky to obtain as they should withstand high pressures, to be porous enough and should not be too long or interact with the analytes. Although many of the above mentioned problems are resolved today, the tedious column packing procedure still remains the bottle-neck of such applications and thus the number of publications employing particle-packed columns in CEC presents a noticeable decline.
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A technique that aroused the interest of scientists in recent years in MIP-CEC field is the use of monolithic stationary phases where no end frits are needed and the imprinted polymer forms a single continuous rod of porous material. In contrast with particle packed capillaries the solid polymeric network of the monolith is abounding with interconnected mesopores. The ratio of (through-pore size)/(polymer surface area) in case of monoliths is much larger, providing a high permeability and column efficiency [53]. In comparison with the OT-format, monoliths possess a much higher loading capacity and large specific surface area. Perhaps the feature that made them so popular among separation scientists is the simplicity of its preparation. They are prepared in a simple, one-step, in situ polymerization stage directly inside the capillary. The polymerization mixture, usually identical with the one used in the traditional approach, is introduced into a pre-treated (silanised) fused-silica capillary up till the detection window. The polymerization is induced by temperature (commonly 50 - 60°C) or UV radiation (generally coupled with low temperatures
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Recent advances in capillary electrochromatography using molecularly imprinted polymers Bogdan-Cezar Iacob, Ede Bodoki*, Radu Oprean Analytical Chemistry Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 4, Louis Pasteur St., 400349, Cluj-Napoca, Romania
Corresponding Author *Ede Bodoki Address: “Iuliu Hatieganu” University of Medicine and Pharmacy, 4, Louis Pasteur St., 400349, ClujNapoca, Romania email: [email protected]. Tel.: +40 264 597 256 Fax: +40 264 597 257
Abbreviations α, separation factor; γ-MAPS, 3-(trimethoxysilyl)propyl methacrylate); AML, amlodipine; C, crosslinking agent; EDMA, ethylene glycol dimethacrylate; HRP, horseradish peroxidase; I, initiator; KET, ketoprofen; LiqCr, liquid crystal; Lyz, lysozyme; M, monomer; MAA, methacrylic acid; MAM, 2methacrylamidopropyl methacrylate; MIP, molecularly imprinted polymer; MSA, methylenesuccinic acid; NAP, naproxen; NIP, non-imprinted polymer; NP, nanoparticle; OFX, ofloxacin; OT, opentubular; Rs, resolution; T, template; TRIM, trimethylolpropane trimethacrylate; ZOP, zopiclone;
Received20-May-2014; Revised: 07-Jul-2014; Accepted: 10-Jul-2014
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400253.
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Keywords capillary electrochromatography, electrophoresis, molecularly imprinted polymers, sample preparation, stationary phases
Total words 8429
Abstract There is an increased and continuous need for developing new methods for the separation and quantification of an increasing number of analytes in the environmental, pharmaceutical, pharmacological and toxicological sciences. CEC is still withholding its popularity, representing a viable alternative to the more conventional techniques (HPLC, GC) due to the numerous advantages, such as, low sample/reagent volumes, high separation efficiencies, hybrid separation principle, etc. One particular promising direction in CEC is the use of molecularly imprinted polymers (MIPs) as stationary phases. They are usually immobilised in the capillary column as a continuous polymeric monolith or as a thin polymer coating attached to the capillary’s inner wall. Another emerging trend is the use of MIPs in the form of nanoparticles (NPs) as a pseudostationary phase. This review discusses the recent developments (2011-2013) in finding the optimal polymerization mixture and the suitable MIP format that should be employed in CEC separations. The most important applications of MIPs in CEC technique are also highlighted.
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I. Introduction One of the biggest challenges for a researcher is to reproduce processes that occur in nature, like translation and transcription of the genetic code, photosynthesis, development of biomimetic materials with molecular recognition properties, etc. A particularly exciting area of biomimetics is Molecular Imprinting which can be defined as an artificial tiny lock for which the assigned key is represented by the target molecule. Molecularly imprinted materials are prepared by assembling around a template molecule by polymerization of functional monomers and a crosslinker. The interactions between the template molecule and the monomer’s functional groups represent the basis of molecular imprinting technique and they must be maintained throughout the polymerization process. The choice of functional monomer is critical for the fabrication of molecularly imprinted polymers (MIPs) in order to provide complementary interactions with the template [1]. Extraction of template leaves behind three-dimensional cavities that are complementary in size and shape to the template. The obtained cavities present a permanent memory for the imprinted specie, allowing the polymer to selectively entrap the imprint molecule from a mixture of closely related compounds. Using this technique, highly selective synthetic receptors with antibody-like recognition characteristics may be obtained and, in principle, they can be applied for molecular structures spanning from ions to biomacromolecules [2-4]. The high chemical and thermal stability, time efficiency, relatively low cost of production, morphology, and good analytical performance of MIPs allowed a rapid growth of their biomedical and biotechnological applications. Their unique selectivity towards a specific target molecule determines a predicted elution order of enantiomers but is disadvantageous when trying to develop a generic stationary phase. They have been used as stationary phases (in liquid chromatography [5], CEC [6], supercritical fluid chromatography [7], thin-layer chromatography [8] and solid-phase extraction [9]), as structures mimicking antibodies [10] and as sensing elements in various devices [11, 12]. Among the mentioned analytical techniques, CEC experienced a significant growth since its This article is protected by copyright. All rights reserved.
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development in 1970s due to its inherent advantages. Furthermore, the small volume of capillaries and therefore the minute amount of needed MIP facilitated the boost of applications using MIP-CEC. Nevertheless, lately a slightly declining trend can be observed in the number of published articles focusing on this topic. This may be due to the persisting shortcomings still associated with this technique. The limited reproducibility of capillary packing or in situ polymerisation, alongside with the lack of commercial availability of a wide selection of CEC columns prevent this technique in becoming a robust alternative for liquid chromatography. Other drawbacks worth mentioning is the need of external pressure to avoid bubble formation during analysis, the incomplete release of template molecule after MIP synthesis causing continuous leakage, or the poor heat dissipation. Nonetheless, recently some new approaches were also reported that may open up new perspectives in the development of CEC methods using MIP. The present review is focusing on new and significant aspects reported in the last 3 years (2011-2013) that may pave the way in alleviating the constraints regarding MIP synthesis and their applications. Recent improvements in the preparation of MIPs in terms of polymerization mixture’s composition and the obtained polymer’s porosity are also discussed. Furthermore, the practical utility and the potential of available MIP-formats are also analysed. Finally, the latest applications of MIPs in CEC concerning sample preparation, chiral analysis and macromolecule analysis are highlighted. Table 1 provides the reader with an overview of all articles covered by this review.
II. MIP-synthesis
II.1. Template /Monomer /Cross-linker ratios Currently, the most widely employed technique in creating MIPs is the non-covalent molecular imprinting approach. The imprinting effect is governed by the formation in the prepolymerization mixture and maintaining throughout the polymerization process of certain This article is protected by copyright. All rights reserved.
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complexes between the functional monomer and template molecule (Fig. 1). These complexes are formed by non-covalent interactions, such as hydrogen bonding, ion pairing, hydrophobic interactions, dipole-dipole and van der Waals forces. These interactions are weak, but allow a rapid and reversible binding well suited for chromatographic separations [13]. Many parameters have to be assessed in the synthesis of MIPs, since they can influence the morphology, properties and performance of polymers. The prepolymerization mixture is represented by a solvent in which four types of substances are dissolved: one or several functional monomers (M), template(s) (T), initiator (I) and crosslinking (C) agent(s). For the synthesis of organic MIPs, aprotic organic solvents with low polarities represent the appropriate medium for the stabilization of the hydrogen and/or electrostatic bonding between the monomer and template molecule. Highly polar aprotic solvents (e.g., dimethylformamide) tend to interfere with the ionic and van der Waals forces emerging between the imprint molecule and the functional monomer, while protic solvents (methanol, water) competitively interfere the hydrogen bonding interactions [14]. For non-covalent imprinting, the ratios between T, M and C play a crucial role in the imprinting effect. Unfortunately, even nowadays, having many chemometric optimization tools (experimental design) at disposal, the optimal ratios are still achieved empirically following tedious and frequently inefficient protocols by keeping a component constant and varying the other two. Usually, a large excess of functional monomer compared to the template is required during the polymerization to favour the formation of template-monomer assemblies. In fact, the association between the monomer and the template is governed by Le Chatelier’s principle; therefore increasing the concentration of components shifts the equilibrium towards complex formation. Recently, Ye et al. [15] investigated the influence of T/M/C ratio on the size, size distribution and the molecular binding property of resulting MIP-nanoparticles (NPs). They synthesized propranolol-imprinted NPs using precipitation polymerization employing different T/M/C ratios. Using elemental analysis, they observed that the elemental composition of the MIPs and their
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corresponding non-imprinted polymers (NIPs) are almost identical, suggesting that the presence of the template does not influence the ratio of the incorporated monomers. Comparing dynamic light scattering measurements on colloidal dispersions with scanning electron microscopy on dried NPs, it was found that NPs present a larger diameter in a wet state due to swelling. The obtained spherical polymer particles, with a size of several hundred nanometers manifested a good colloidal stability, thus representing a viable option as pseudostationary phases in CEC. Another important conclusion of the study was that in a purely nonimprinting system, the amount of functional monomer (methacrylic acid (MAA)) and the used crosslinking density has little effect on the hydrodynamic size of the synthesized poly(MAA-co- trimethylolpropane trimethacrylate (TRIM))-NPs. In fact, the size of the obtained imprinted NPs is dependent on the template’s polarity and the strength of its molecular interaction with the functional monomer, phenomena previously demonstrated using propranolol as template, which changed the solubility of the growing polymer chains and also affected the rate of particle growth [16]. The highest template binding capacity (~ 170 fmol/mg specifically bound template, value 20 times higher than nonspecific binding) was obtained using a slight excess of template (1–1.3 equivalents) relative to MAA and in the presence of a good crosslinking density. Most often more than one compound of interest needs to be determined in a sample and the single molecule imprinted polymer is not suitable for the analysis of complex mixtures. Furthermore, the fabrication of individual MIPs for each analyte might be severely time-consuming. Recently, Zaidi [17] fabricated inside capillary columns dual-templates (serotonin and histamine) imprinted polymer monolith for their subsequent separation in CEC. In order to achieve best resolutions (Rs) and separation efficiencies different preparation protocols were employed while keeping the molar equivalents of both the templates equal. The influence of two functional monomers, MAA and methylenesuccinic acid (MSA) were evaluated, the latter offering a 2 fold increase in resolution (T/M/C = 1-5-19), as shown in Fig. 2. In the effort of producing a more rigid
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polymer structure able to conserve the imprinted binding sites, the increase of cross-linker ratio did not influence significantly resolution. This enhanced resolution may be explained by the two carboxylate functionalities of MSA, promoting stronger interactions with the templates than MAA, but the degree of monolith uniformity throughout the capillary may also have played its role in case of MSA. Liu et al. [18] observed the same improvement in the imprinting effect using MSA as functional monomer in comparison with MAA. A monolithic polymer with (-)norepinephrine as template was synthesised being employed as CEC stationary phase for the separation of dopamine, (±)-epinephrine, (−)-isoproterenol, (±)-norepinephrine, (±)-octopamine, and (±)-synephrine. In case of MAA, increasing the amount of functional monomer or crosslinker led to a poorer resolution of (±)-norepinephrine. Using MSA as functional monomer, a baseline separation was achieved with a T/M/C molar ratio of 1-9.2-19. In the last decade, molecularly imprinted NPs have gained momentum due to their high surface area and better accessibility to the imprinted cavity, leading to a much faster analyte binding equilibrium [19-21]. The most simple and efficient method in preparing these NPs is the precipitation polymerization method, but in case of some templates difficulties in creating uniform NPs has been reported [22, 23]. As already discussed above, the morphology and size distribution of MIP particles is determined by the nature of imprint molecules added in the polymerization mixture. When Chen and Ye [19] adopted the same polymerization mixture for atrazine as the one with which uniform propranolol-imprinted NPs were obtained, [24] much bigger particles with a very broad size distribution were generated. Interestingly, with the addition of small amount of propranolol (5%, w/w) as an auxiliary template in addition to atrazine, small (147nm) and uniform NPs were achieved. The same improvement was observed when 5% propranolol was added in the prepolymerization mixture for the imprinting of estradiol and theophylline with the reduction of particles sizes from 208 to 99 nm and from 361 to 131 nm, respectively. This significant effect was also reported for
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other β-blockers, like pindolol and atenolol, and was attributed by the authors to the strong hydrogen bond interactions between the isopropylaminopropanediol moiety in the β-blocker’s structure and the used functional monomer (MAA). The effects of polymerization variables (e.g. T/M molar ratio, type and amount of functional and cross-linking monomers) on chiral separation of zopiclone (ZOP) in the preparation of molecularly imprinted NPs were investigated [25]. Even if the T/M and M/C molar ratios were varied on a wide scale (from 1-3 to 1-10) no significant changes on the MIP’s chiral performance were recorded. The positive effect of the optimal polymerisation component ratio was hindered by the agglomeration of the emerging NPs leading to the formation of non-uniform microparticles. Additionally, the adsorption data of the polymer indicated a very wide distribution of pore sizes. The partial substitution of the functional monomer (MAA) with other monomers (acrylamide, 4vinylpyridine or butyl methacrylate) yielded lower column efficiencies and resolutions. The key to a good non-covalent molecular imprinting is a full complexation between the functional monomer and the imprint molecule, which should be maintained throughout the process of polymerization. By adding in the polymerization mixture of a crowding-inducing agent, namely polystyrene dissolved in THF, the complexation is considerable increased [26]. In this case both selectivity and efficiency of the obtained MIP microparticles were improved in comparison with the use of plain ACN as porogen. In the fabrication of S(-)ofloxacin (OFX)-imprinted microparticles, several parameters like the choice of solvents and co-solvents, T/M ratio, type of cross-linking monomers and functional monomer composition were investigated. It was observed that microparticles prepared in polystyrene-THF presented a broader size distribution and lower porosities compared to those prepared in ACN. The optimal T/M ratio was found to be 1-4, offering the best resolution and smallest particle sizes. The type of cross-linking monomer had little influence on the resolution of OFX enantiomers. The addition of a hydrophobic co-monomer (50% butyl
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methacrylate) along MAA led to an increase in separation efficiency, with the baseline separation of OFX enantiomers. As can be seen from the foregoing, even if the formula of MIP preparation is relatively simple, there are many variables needing optimization, like type of functional monomers and crosslinker to be used and the optimum ratio of T/M/C. By combining the functional monomer and the cross-linker into one compound playing both roles, the optimization process is drastically reduced [27]. The functional cross-linker, 2-methacrylamidopropyl methacrylate (MAM), was employed in synthesising a MIP-coating inside a silica capillary column with S-amlodipine (AML), Snaproxen (NAP) or S-ketoprofen (KET) as template molecule for the CEC chiral analysis of the corresponding racemic mixtures. The selected imprint molecule-single cross-linking monomer molar ratio was 1-4 and the porogen used was toluene-isooctane (7/3 (v/v)). The racemic AML, NAP and KET were all resolved employing the same polymerization mixture’s composition with excellent resolutions: 16.1, 1.46 and 4.56, respectively. This approach of using one-monomer in the composition of MIP is not only a simplification improvement, but also offers a higher chiral separation ability and column stability than conventional MAA- ethylene glycol dimethacrylate (EDMA) two-monomer system. In the same trend of MIP synthesis simplification, recently the concept of generalized preparation protocol was introduced, recommending a single polymerization mixture regardless of the nature of imprint molecule [28-31]. In the first reports, the chiral separation capability of the polymerization mixture was demonstrated for a number of acidic analytes, like non-steroidal antiinflammatory drugs, (3-(benzyloxycabonyl)-4-oxazolidine carboxylic acid, OFX, mandelic acid, succinic acid, 2-phenylbutyric acid and camphor [28, 29]. The protocol was further tested for some additional acidic templates, like phenyl carboxylic acids and their derivatives [32] and camphor analogues (10-camphorsulfonic acid, 10-camphorsulfonamide and camphor-p-tosyl hydrazone) [33]. The applicability of the pre-established generalized preparation protocol was expanded to the use of
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neutral templates, like (4S,5R)-4-methyl-5-phenyl-2-oxazolidinone [30]. The open-tubular (OT)-MIP capillary columns employing basic templates were fabricated and evaluated using the preestablished generalized preparation protocol. The basic templates taken into study were: atenolol, sulpiride, methyl benzylamine and (1-naphthyl)-ethylamine [31]. The recommended generalized polymerization mixture consists in the template molecule (0.020 mmol), MAA (0.096 mmol/L), EDMA (0.313 mmol/L), 4-styrene sulfonic acid (2 mg) and AIBN (3.5 mg) dissolved in 1mL 90/10 (v/v) ACN/2-propanol.
II.2. High vs. low crosslinking The standard molecular imprinting procedure involves mixing the template molecule along with the functional monomer to form a complex through covalent or noncovalent interactions over which a large amount of cross-linking agent and a porogen are added. The copolymerization is then started by radical initiation leading to macroporous polymers in which template-monomer complexes are trapped inside. By removing the template molecules after polymerization, welldefined 3D cavities are obtained; the cavities have similar size dimensions and shapes to the template, and functional groups that are complementary to the template molecule or analogue structures. The “molecular memory” of the obtained polymers is “stored” in these cavities, which must be preserved and stabilized by increasing the rigidity of the polymeric network. This is achieved by adding an excess of crosslinking monomer to the polymerization mixture (around 70-95%) [13, 34]. Unlike covalent imprinting where 90-95% of the binding sites are available for template rebinding, in case of non-covalent approach, only 15% of sites can reuptake the template molecule, most probably due to poor site accessibility caused by the shrinkage of the cavities [2, 35]. Therefore, in order to facilitate a fast equilibrium between template release and rebind in the cavity a high flexibility of the polymer is required. These two opposing parameters, rigidity and flexibility of the polymeric network needs to be finely balanced by an optimization step [13].
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An approach of stabilizing binding sites of MIPs was introduced by Mauzac et al. [36-41] relying on the benefits of mesomorphous organization of the liquid crystal (LiqCr) materials. Thus, LiqCr-MIPs with low levels of cross-linking (5-20 mol% of the monomer units) were obtained in which chemical cross-linking was replaced by physical cross-linking, due to the non-covalent reversible interactions between mesogenic moieties. Owing to the low content of cross-linker the LiqCr-MIPs exhibited similar selectivity but much higher loading capacity compared with the classical MIPs [36]. Using the same strategy, Wei et al. [35] reported for the first time a LiqCr-MIP capillary coating (~0.1-0.2 µm thickness) for the chiral separation of AML, NAP and OFX in CEC mode. On the low cross-linked MIP coated capillary separation of racemic AML was achieved in 2.5 minutes with a resolution and selectivity factor of 6.36 and 1.81, respectively. Additionally, no peak tailing for the AML and NAP template enantiomers was observed, providing the better accessibility to the binding sites and/or the improved mass transfer. Using LiqCr-free MIP capillary (80% molar ratio of EDMA) the same separation was accomplished in twice as long time with a 5 times smaller resolution, as shown in Fig. 3.
II.3. Stationary vs. pseudostationary phases Three main types of stationary phase formats are commonly used in CEC: monolithic, particle packed and OT-capillary columns. The alternative is the use of MIP-NPs as pseudostationary phases; however the provided number of interaction sites with the template is lower than in the case of MIP-based stationary phases when a higher amount of polymer is immobilized in the column. The MIP-NPs are most often employed in CEC mode through partial filling technique, being injected prior to the sample, as a plug shorter than the effective length of the capillary. The length of the MIP-NPs plug must be carefully adjusted so that analytes reach the detector before the NPs plug in order to avoid interferences with the employed detection. Therefore, as a rule of thumb the apparent mobility of the analytes should be greater than the one of the NPs.
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The advantages of using MIP-based pseudostationary phases in electrodriven separations, consists in the use of a fresh MIP-NP plug for each analysis, avoiding problems related to strong adsorption of templates during analyses, such as column-ageing and poor reproducibility [42]. Moreover no frits and tedious particle packing protocols are needed. However, there are also some important constraints employing NPs as pseudostationary phases, the most important ones being related with the stability of the NPs colloid and the improved reproducibility ensured by fresh MIPNP plug used in each analysis. One of major factors conditioning the stability of dispersions is particle size, which obviously has to be in the submicrometer-range. Several methods have been developed for the production of MIP-NPs, like: grounding and sieving bulk polymers [43], precipitation polymerization [19, 25, 26, 44], emulsion polymerization [45] and surface templated emulsion polymerization [46]. The simplest, most elegant and widely used approach in preparation of NPs as pseudostationary phases in electrodriven techniques is precipitation polymerization. In this technique a diluted polymerization mixture (identical reaction mixture as in the bulk method, but with a higher volume of porogen) without any other additives is used, resulting in spherical polymer beads with good yields. Due to its versatility, precipitation polymerization leads to an increase in the number of publications focusing on MIPs as pseudostationary phases. This procedure was described for the preparation of MIP-NPs in the enantioseparation of various small drug molecules: ZOP [25, 44], tryptophan [47] and OFX [26]. As previously mentioned, Liu et al. [25] obtained d-ZOP MIP-NPs (~100nm) that formed micrometric agglomerates. These particles ensured a successful separation of ZOP enantiomers within 10 minutes with a resolution and a separation factor (α) of 4.75 and 1.11, respectively. Comparable selectivity with a better Rs reproducibility (RSD below 3%) were observed in case of liquid crystal d-ZOP-MIP-NPs with low crosslinking degree [44]. Adding LiqCrs in the polymerization mixture MIP-NPs with narrower size distribution were obtained with no sign of
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agglomeration between particles (Fig. 4). An even shorter analysis time (below 7 min) with similar selectivity (Rs=4.62, α=1.29) was achieved using a MIP coating grafted on a TRIM core monolith [48]. Unfortunately, the gain in the analysis time is hardly justified by the increased column preparation time: 15 h for TRIM core monolith synthesis and an additional 3 h for MIP coating polymerization. The chiral CEC separation of OFX was achieved by Shi et al. [26] in less than 20 minutes, with Rs and α of 1.53 and 1.07, respectively. As previously mentioned, the pseudostationary phase consisted in MIP-microparticles prepared by the molecular crowding approach. Once again, using MIP grafted poly(TRIM) monolith [48] offered a better resolution (Rs= 4.97) and separation factor (α=2.35), but probably due to the heterogeneity of imprinted sites a broad peak of template enantiomer was observed. The template (S-OFX) peak shape was not improved even when a LiqCr MIP-coating column with low level of crosslinking was employed [35]. Core-shell particles of uniform size (~87 nm) with good colloidal stability in aqueous buffers were achieved by Yue et al. [47] using surface L-tryptophan imprinting on silica NPs. They were employed in CEC as pseudostationary phase for the chiral separation of tryptophan enantiomers (under 10 min, Rs= 2.73) providing symmetrical peak shapes due to the fast mass transfer and good accessibility of the interaction sites. In spite of the highlighted advantages of MIP-based pseudostationary phases (simple, fast and reproducible synthesis, no column ageing and decrease of analytical performances due to strong adsorption, improved mass transfer), most probably due to the more simple analytical method development and optimization, the use of MIP-stationary phases is still more prevalent in the enantioseparation of small drug molecules: AML [27, 35, 48], tyrosine [49], neurotransmitters [18], ractopamine [50], NAP [27, 35], KET [27], atenolol and sulpiride [31]. MIP-based stationary phases, especially monolithic and particle packed columns, tend to be more frequently employed in imprinting large templates (protein recognition), due to their large specific surface area and higher loading capacity in contradistinction to pseudo-stationary phases.
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Thus, a successfully isolation and separation of lysozyme (Lyz) from egg white (Fig. 5) and human serum was achieved on a monolithic stationary phase [51]. A new molecularly imprinted monolithic column was obtained on the surface of a boronicbased polymeric skeleton by polymerization of dopamine using horseradish peroxidase (HRP) as a template [52]. The use of boronate monolithic skeletons permitted the reversible immobilization onto the monolith of the glycoprotein via cyclic boronate esters while the subsequent polymerization of dopamine takes place.
II.4. Monolith // packed // open-tubular capillary columns / chips MIPs were extensively explored as stationary phases in sample treatment, HPLC and CEC. The traditional approach of preparation implied bulk polymerization outside the column resulting blocks of molecularly imprinted materials, that was crushed, grounded and sieved in order to obtain similar sized nano- or micro- particles, followed by their packing into the capillary with the aid of frits. Unfortunately, this method presents many drawbacks. First of all, such preparation of MIP particles is tedious, time consuming and brings low yields. Furthermore, the ground beads are irregular in shape with relatively wide size distribution and increased heterogeneity of the binding sites. Better performances were achieved with improved particle preparation protocols (e.g. suspension or precipitation polymerization method) but upon the packing of capillary the high backpressure remains a problem. Another inconvenience is the need of frit preparation. Ideal frits are tricky to obtain as they should withstand high pressures, to be porous enough and should not be too long or interact with the analytes. Although many of the above mentioned problems are resolved today, the tedious column packing procedure still remains the bottle-neck of such applications and thus the number of publications employing particle-packed columns in CEC presents a noticeable decline.
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A technique that aroused the interest of scientists in recent years in MIP-CEC field is the use of monolithic stationary phases where no end frits are needed and the imprinted polymer forms a single continuous rod of porous material. In contrast with particle packed capillaries the solid polymeric network of the monolith is abounding with interconnected mesopores. The ratio of (through-pore size)/(polymer surface area) in case of monoliths is much larger, providing a high permeability and column efficiency [53]. In comparison with the OT-format, monoliths possess a much higher loading capacity and large specific surface area. Perhaps the feature that made them so popular among separation scientists is the simplicity of its preparation. They are prepared in a simple, one-step, in situ polymerization stage directly inside the capillary. The polymerization mixture, usually identical with the one used in the traditional approach, is introduced into a pre-treated (silanised) fused-silica capillary up till the detection window. The polymerization is induced by temperature (commonly 50 - 60°C) or UV radiation (generally coupled with low temperatures
