Monodispersed, molecularly imprinted polymers as affinity-based chromatography media

This review article deals with preparation methods for spherical and monodispersed molecularly imprinted polymers (MIPs) in micrometer sizes. Those methods include suspension polymerization in water, liquid perfluorocarbon and mineral oil, seed polymerization and dispersion/precipitation polymerization. The other methods are the use of beaded materials such as a spherical silica or organic polymer for grafting MIP phases onto the surfaces of porous materials or filling the pores of silica with MIPs followed by dissolution of the silica. Furthermore, applications of MIP microspheres as affinity-based chromatography media, HPLC stationary phases and solid-phase extraction media, will be discussed for pharmaceutical, biomedical and environmental analysis.     

Study on the recognition of templates and their analogues on molecularly imprinted polymer using computational and conformational analysis approaches

A simplified computational model was proposed to simulate the synthesis of molecularly imprinted polymers (MIP), removal of template and recognition of the template and its analogues by MIP. The MIPs with nicotinamide and iso-nicotinamide as templates were prepared using methacrylic acid as functional monomer. Based on our computational model, the interaction energies between the monomer and the template or its analogues were calculated, which were well correlated with the retention factors and imprinting factors obtained on HPLC columns packed with the corresponding MIP particles. The imprinting effects of the template and its analogues were also investigated from the viewpoint of conformational analysis. The computational data were successfully used to predict the chromatographic behaviour of some chemicals in separation on HPLC columns. We believe that the computational method will find application in designing monomers for MIP synthesis and in studying recognition of templates and their analogues on MIP.     

Synthesis and evaluation of molecularly imprinted polymers for enalapril and lisinopril, two synthetic peptide anti-hypertensive drugs

Molecularly imprinted polymers (MIPs) for the recognition of enalapril and lisinopril were prepared using 4-vinylpyridine as the functional monomer. Following thermal polymerisation the resulting materials were crushed, ground and sieved. First generation MIPs were produced in protic polar porogenic solvents (mixture of methanol (MeOH) and acetonitrile (ACN)). These MIPs were used and validated as sorbents for solid phase extraction and binding assays. Second generation MIPs were produced with polar aprotic porogenic solvent (DMSO). These polymers were packed in HPLC columns in order to investigate their molecular recognition properties in a dynamic mode. The study of the mobile phase composition included two major parameters: organic modifier content and pH value. Retention factors illustrate selective binding of the template from the imprinted polymers, compared to structurally related compounds.     

Novel biphasic separations utilising highly selective molecularly imprinted polymers as biorecognition solvent extraction agents

Molecularly imprinted polymers (MIPs) represent a class of artificial receptors that promise an environmentally robust alternative to naturally occurring biorecognition elements of biosensing devices and systems. However, in general, the performance of conventional MIPs in aqueous environments is poor. In the study reported here, this limitation has been addressed by the novel application of MIPs as a solvent extraction solid phase in a biphasic solvent system. This paper describes a previously unreported use of MIPs as solvent extraction reagents, their successful application to aqueous sample media and the opportunities for utilisation of this unique system in novel biosensing and separation procedures. This study demonstrates the development of a novel biphasic solvent system utilising MIP in the extracting phase to enhance both efficiency and selectivity of a simple two phase liquid extraction. Monodisperse propranolol imprinted polymer microspheres [p(divinylbenzene-co-methacrylic acid)] were prepared by precipitation polymerisation. Initially, the affinity of the polymers for (R,S)-propranolol was assessed by established techniques whereby the MIP demonstrated greater affinity for the template than did the non-imprinted control polymer (NIP). Importantly, MIP performance was also assessed using the novel dual solvent system. The depletion of (R,S)-propranolol from the aqueous phase into the polymer containing organic phase was determined. When compared to control extractions containing no polymer the presence of MIP in the extracting solvent phase resulted in an increased extraction of (R,S)-propranolol from the aqueous phase. Importantly, this extraction was significantly greater in the presence of MIP when compared to NIP. This unique principle generates opportunities for MIP based extractions and chemical enrichments in industrial applications, offering commercial, ecological and practical advantages to traditional solvent extraction techniques. The technique is readily transferable to analytical microsystems utilising MIP recognition elements generating promising opportunities for MIP based sensing of aqueous sample media.     

Polystyrene/Divinylbenzene Based Monolithic and Encapsulated Capillary Columns for the Analysis of Nucleic Acids by High‐Performance Liquid Chromatography‐Electrospray Ionisation Mass Spectrometry

Monolithic capillary columns are prepared by copolymerization of styrene and divinylbenzene, encapsulated capillary columns by immobilizing silica particles with different pore sizes inside a 200 μm i.d. fused silica capillary by encapsulation of the derivatized silica sorbent in a poly(styrene/divinylbenzene) (PS/DVB) matrix. Both allow the rapid and highly efficient separation of single‐ and double‐stranded DNA by ion‐pair reversed‐phase high‐performance liquid chromatography (IP‐RP‐HPLC). The high resolving power of monolithic and encapsulated capillary columns can be utilized for mutation screening in polymerase chain reaction (PCR) amplified polymorphic loci by denaturing HPLC (DHPLC). Recognition of mutations is based on the separation of homo‐ and heteroduplex species by IP‐RP‐HPLC under denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Separations can be readily hyphenated to electrospray ionization‐mass spectrometry.     

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC–diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 μg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.     

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%.     

Binding site characteristics of 17beta-estradiol imprinted polymers

The variety of applications utilizing molecularly imprinted polymers (MIPs) requires synthetic strategies yielding different MIP formats including films, irregular particles, or spheres, along with precise knowledge on the specific material characteristics, such as binding capacity and binding efficiency of these materials. In response to this demand, MIPs are prepared in different formats by variation of the polymerization methodology. It is commonly agreed that micro- and sub-microspheres are particularly advantageous MIP formats, due to their monodispersity and facile synthesis procedures in contrast to conventional imprinted polymers prepared by bulk polymerization. However, the differences in actual rebinding characteristics of different MIP formats based on molecular interactions under a variety of binding/rebinding conditions have not been studied in detail to date. Consequently, the present work details an analytical strategy generically applicable to MIP systems for rebinding studies including equilibrium binding, non-equilibrium binding, and release experiments enabling more profound understanding on the molecular interactions between the imprinted materials and the template molecules. In this study, three MIP formats were considered for the same template molecule, 17beta-estradiol: irregularly shaped particulate polymers prepared by bulk polymerization and grinding, microspheres, and sub-microspheres. The latter two formats were synthesized via precipitation polymerization using different processing strategies. The morphologies and porosities of the resulting imprinted materials were characterized by scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET) analysis, respectively. The obtained results indicate that microspheres prepared by precipitation polymerization provide superior rebinding properties during equilibrium binding in contrast to bulk polymers and sub-microspheres, and that the rebinding properties are different during equilibrium binding versus non-equilibrium binding. The median binding affinity constant determined during non-equilibrium rebinding is higher than the values obtained from equilibrium rebinding. Furthermore, the binding site distribution appears more homogeneous thief derived from non-equilibrium rebinding, as reflected in a heterogeneity index of m=0.725. Moreover, it is hypothesized that the specific interactions between template and monomers are related to the porosity of the imprinted polymers, which implies that the amount of binding sites and the pore sized distribution of the imprinted materials are a critical factor in achieving the desired MIP performance in various analytical applications. The BET results indicate that particles prepared with lower cross-linker-to-template ratio have a reduced surface area. Furthermore, it can be expected that there are less specific binding sites available at particles with reduced surface area and pore volume given similar distribution of the binding sites, as confirmed by the equilibrium binding isotherm studies. The pore size distribution results reveal that control of the pore size in the range of 100-180 A is essential to obtain the desired retention properties and Gaussian peak shape during HPLC analysis of small molecules.     

Direct rapid synthesis of MIP beads in SPE cartridges

Selecting optimal compositions for non-covalent molecularly imprinted polymers (MIPs) and screening for appropriate rebinding conditions necessitates synthesising a large number of polymers. This is extremely labour-intensive and usually results in very limited "optimisation" in studies of MIPs. Here, a new method is proposed for rapid synthesis of MIPs in a beaded form that can be used directly in many different performance evaluation studies. The method is based on synthesis of spherical particles by suspension polymerisation in liquid fluorocarbon [Mayes, A., Mosbach, K., 1996. Molecularly imprinted polymer beads: suspension polymerisation using a liquid perfluorocarbon as the dispersing phase. Anal. Chem. 68, 3769-3774]. The polymers were directly polymerised under UV light in solid phase extraction (SPE) cartridges, then washed and extracted in the same cartridges where they had been synthesised, resulting in a rapid and automatable process that requires no transfer or manipulation of the polymer particles. The particles were similar in terms of size, morphology and functional performance to particles obtained by suspension polymerisation in fluorocarbon solvent using a conventional reactor. In this initial study, 36 polymers were synthesised to study the effect of a variation in the type and amount of four different functional monomers, methacrylic acid (MAA), acrylic acid (AA), hydroxyethyl methacrylate (HEMA) and 2-vinylpyridine (2-VPy), for the imprinting of propranolol and morphine. The performance of polymers synthesised using MAA was as expected, but those synthesised with AA as functional monomer showed more surprising rebinding properties as a function of monomer to cross-linker ratios, demonstrating the potential value of pragmatic synthesis and screening approaches to polymer optimisation.     

A molecularly imprinted polymer on indium tin oxide and silicon

Molecular imprinting is a technique for creating artificial receptor sites in a polymer. Molecularly imprinted polymers (MIPs) are produced by forming a polymer around a molecule that is used as the template. Upon removal of the template, molecular holes remain which are specific in shape and size to the target molecule. In this research, a MIP was formed for theophylline using a copolymer of methacrylic acid and ethylene glycol dimethacrylate. The theophylline MIP was formed on two platforms: indium tin oxide (ITO) and silicon, which were used as the working electrode for cyclic voltammetry measurements. The presence of theophylline was measured using cyclic voltammetry and corresponded to the peak current on the cyclic voltammograms. The results of this research agreed with previous results of MIPs immobilized on an ITO platform. The peak currents of the MIP in the presence and absence of theophylline were compared to the blank polymer for each platform. The ratio of peak currents on ITO increased by a factor of 9.5 for the MIP compared to the non-imprinted polymer. Similarly, the ratio of peak currents on silicon increased by a factor of 6 compared to the non-imprinted polymer. This research demonstrated a procedure for evaluating a MIP layer on two different platforms.     

Molecular imprinting and solid phase extraction of flavonoid compounds

Molecularly imprinted polymers (MIPs) for quercetin have been successfully prepared by a thermal polymerization method using 4-vinylpyridine (4-VP) and ethylene glycol dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The obtained molecularly imprinted polymers were evaluated by HPLC using organic eluents, with respect to their selective recognition properties for quercetin and related compounds of the flavonoid class. Two equivalent control polymers, a blank polymer and a polymer imprinted with a structural analogous template, were synthesized, in order to confirm the obtained results. Furthermore, preliminary experiments confirm the applicability of the prepared MIPs for solid phase extraction (SPE), as rapid and facile clean-up of wine samples for HPLC analysis is an envisaged field of application. The successful preparation of molecularly imprinted polymers for flavones provides an innovative opportunity for the development of advanced separation materials, with applications in the field of wine and fermentation analysis.     

Evaluation of water-compatible molecularly imprinted polymers as solid-phase extraction sorbents for the selective extraction of sildenafil and its desmethyl metabolite from plasma samples

The evaluation of molecularly imprinted polymers (MIPs) as selective sorbents for the solid-phase extraction of sildenafil and its principal metabolite, desmethylsildenafil, was investigated. Two MIPs were synthesised using structural analogues of sildenafil as templates, and a comparison of the performance of the two MIP sorbents in organic and aqueous media was performed. Additionally, the feasibility of applying molecularly imprinted solid-phase extraction (MISPE) to the clean-up of plasma samples containing sildenafil and desmethylsildenafil was investigated. A preliminary, quantitative MISPE for the determination of both compounds in plasma was also performed. The results showed that the MIPs used for the selective extraction of sildenafil gave better compound recovery when aqueous samples were used in comparison to organic-based samples. A preliminary, quantitative MISPE of sildenafil and desmethylsildenafil indicated that the imprinted materials could be used successfully as SPE sorbents for sample pre-treatment for the determination of sildenafil, and related compounds, in plasma.     

Molecularly imprinted polymer using-p-hydroxybenzoic acid, p-hydroxyphenylacetic acid and p-hydroxyphenylpropionic acid as templates.

Molecularly imprinted polymers (MIPs) using p-hydroxybenzoic acid (p-HB), p-hydroxyphenylacetic acid (p-HPA) and p-hydroxyphenylpropionic acid (p-HPPA) as templates were synthesized. The performance of the templates and their analogues on polymer-based high performance liquid chromatography (HPLC) columns was studied. The imprinting effect of the MIP using p-HB as template is more obvious than that of MIP using either p-HPA or p-HPPA as template, and the mixture of p-HB and p-HPA can be well separated on the MIP using p-HB as template, but not on the blank. Interestingly, the recognition of MIP (p-HB as the template) to p-HB showed a synergistic effect. The retention factor of p-HB is not the sum of those of phenol and benzoic acid. We also found that the imprinting effect decreased when increasing the concentration of acetic acid in mobile phase. The possible reason is that acetic acid molecules occupied the binding sites of the polymer, thereby decreasing the concentration of binding sites. Furthermore, polymers, which showed specificity to 3,4-dihydroxybenzoic acid, can be prepared with p-HB as template. It is thus possible to synthesize a specific polymer for a compound that is either expensive or unstable by using a structurally similar compound as template.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

General method allowing the use of 100% aqueous loading conditions in reversed-phase liquid chromatography

Reversed-phase HPLC purification of peptides, using n-alkyl modified spherical silica, has become a widely used technique within the pharmaceutical industry. One drawback of these materials is the necessity of having at least 5% organic modifier in the mobile phase, in order to avoid de-wetting of the porous stationary phase. For some preparative reversed-phase separations, it is an advantage if the feed solution can be loaded onto the column under 100% aqueous conditions. This study describes the use of post-column pressure control to avoid de-wetting of regular reversed-phase stationary phases when operated under 100% aqueous conditions. The applicability of post-column pressure control as a means of maintaining the column fully wetted is demonstrated with various buffers and with packing materials having different alkyl-chain lengths. Two peptides, insulin and oxytocin, in overloaded quantities, were loaded under 100% aqueous conditions onto a regular C8 column, and then eluted by a acetonitrile gradient following standard procedures. The retention volume and the peak shape showed that the separation was satisfactory, and proved that post-column pressure control can be used to overcome wettability problems, which are otherwise often observed for reversed-phase packing materials with high ligand density.     

HPLC-based bioseparations using molecularly imprinted polymers

HPLC-based separations of amino acids and peptides, nucleotide bases, drugs, sugars and steroids using molecularly imprinted polymers (MIPs) have been reviewed in this article. The molecular recognition mechanisms of the template molecules on the MIPs in organic and aqueous eluents were discussed. Furthermore, new polymerization methods suitable for preparations of HPLC columns and packing materials using molecular imprinting techniques, and their applications to HPLC-based separations are also dealt with.     

Molecularly imprinted polymer formats for capillary electrochromatography

The research aimed towards the adaptation of molecularly imprinted polymers (MIPs) to the capillary format and the use of these highly selective matrices for capillary electrochromatography (CEC) is reviewed in this article. The MIP is prepared by incorporation of a template molecule into a polymerization protocol. After polymerization and extraction of the template from the resulting polymer a highly selective material with recognition cavities complementary to the template in size, shape and chemical functionality is obtained. MIPs have been used as recognition elements in several different analytical techniques. In combination with CEC a novel separation system with a unique selectivity towards a predetermined target (the template) is achieved. The merge of molecular imprinting technology (MIT) and CEC have introduced several interesting polymer formats, due to the adaptation of the MIP to the miniaturized capillary format. The polymer formats can be classified according to their preparation protocols and appearance into three conceptually different categories, i.e. the monolith, the coating and the nanoparticles. The preparation protocols, characteristics and applications of these formats will be discussed.     

Separation and screening of compounds of biological origin using molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) represent a new class of materials possessing high selectivity and affinity for the target molecule. Since their discovery in 1972, molecularly imprinted polymers have attracted considerable interest from bio- and chemical laboratories to pharmaceutical institutes. They have been utilized as sensors, catalysts, sorbents for solid-phase extraction, stationary phase for liquid chromatography, mimics of enzymes, receptors and antibodies. Among which, the application of molecularly imprinted polymers for molecular recognition-based separation and screening of compounds has undergone rapid extension and received much attention in recent years. This article mainly focuses on the separation and screening of certain pharmacophoric compounds of interests from biological origin using molecular imprinting technology. Examples of extraction and recognition of active components as anti-tumors or anti-Hepatitis C virus inhibitors from Chinese traditional herbs using molecularly imprinting technology are particularized in this article. Comparison between the screening effect based on MIPs and that based on antibodies is also represented. Consequently the merits and demerits of these two technologies are highlighted.     

Separation of ursodeoxycholic acid from its isomeric mixture using core–shell molecular imprinting polymer

In order to separate ursodeoxycholic acid (UDCA) from its isomeric mixture, the molecular imprinting polymers (MIPs) were synthesized by using core–shell emulsion polymerization. In the porous imprinting polymer, ursodeoxycholic acid was used as imprinting molecule, acrylamide (AM) and α-methacrylic acid (MAA) were functional monomers, and CaCO₃ was used for the porogen in the polymerization to obtain large pore. Characterization of the MIP structure with IR spectra demonstrated the expected MIPs. Through adsorption and selectivity assays, AM as the functional monomer showed better separation efficiency than MAA, and nonspecific and specific adsorption capacities of MIP with AM were 43.52 and 13.93 mg/g, respectively. The separation factor of MIP with AM for UDCA was 2.20. Furthermore, MIP with AM could be applied to separate UDCA from the isomeric mixture by column chromatography successfully.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u = 4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u = 8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.