Oliver Brüggemann

New configurations and applications of molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) are applicable in a variety of different configurations. For example, bulk polymers imprinted with beta-lactam antibiotics are presented to be used as stationary phases for the chromatographic separation of beta-lactam antibiotics with both aqueous and organic mobile phases. However, in some analytical applications, monosized spherical beads are preferred over the currently used ground bulk polymers. A precipitation polymerization technique allows preparation of monosized spherical imprinted beads with diameters down to 200 nm having excellent recognition properties for different target molecules. Nevertheless, with current imprinting protocols a substantial amount of template has to be used to prepare the polymer. This can be problematic if the template is poorly soluble, expensive or difficult to obtain. It is shown that for analytical applications, the functional monomer:template ratio can be drastically increased without jeopardizing the polymers recognition properties. Furthermore, a substantial reduction of the degree of crosslinking is demonstrated, resulting in much more flexible polymers that are useful for example the preparation of thin imprinted films and membranes for sensors. Apart from analysis, MIPs also are applicable in chemical or enzymatic synthesis. For example, MIPs using the product of an enzyme reaction as template are utilized for assisting the synthetic reaction by continuously removing the product from the bulk solution by complexation. This results in an equilibrium shift towards product formation.

Comparison of polymer coatings of capillaries for capillary electrophoresis with respect to their applicability to molecular imprinting and electrochromatography

Abstract In molecular imprinting (MI), interactive monomers and suitable cross-linking agents are polymerized in the presence of a template. Once the template has been removed, the remaining space acts as a highly specific binding site for the template or analogs thereof, due to the unique three-dimensional arrangement of interaction points. Several steps are involved in producing such a polymer coat inside a capillary electrophoresis capillary. First, the silanization of the inner surface of the capillary with a suitable silane is necessary, to link a monolayer of unsaturated groups suitable for polymerization to the capillary surface. These monomeric groups are then integrated into the three-dimensional polymer coat produced in the next step. MI-capillary coatings ideally are highly porous and of a thickness, δ, which is smaller than the inner radius, r, of the capillary in question. Porous polymer networks can be produced by dispersive polymerization using a suitable solvent (porogen). However, the exact conditions for producing a coating suitable for capillary zone electrophoresis had to be determined experimentally. Seven porogens, namely hexane, toluene, tetrahydrofuran, acetonitrile, CHCl3, dimethyl sulfoxide and dimethylformamide, and two cross-linkers, namely ethyleneglycoldimethacrylate and divinylbenzene, at concentrations of between 5 and 20% (v/v) were investigated. In about 20% of the combinations, a polymer coat of the desired qualities was obtained. The applicability of the MI capillaries to specific separations was demonstrated for the separation of a racemic mixture of S(+)- and R(−)-2-phenylpropionic acid. trans-3(3-Pyridyl)-acrylic acid was used as the interactive monomer in this case.

On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths.

Macroporous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases were synthesized in the confines of 100 microm I.D. fused-silica capillaries via a free radical copolymerization of mono and divinyl monomeric precursors in the presence of porogenic diluents. These columns were used in order to determine their suitability for the reversed-phase separation of small molecules in isocratic nano-LC mode. Carefully designed experiments at varying realized phase ratio by a terminated polymerization reaction, as well as content of organic modifier in the mobile phase, address the most significant parameters affecting the isocratic performance of these monoliths in the separation of small molecules. We show that the performance of methacrylate-based porous polymer monoliths is strongly affected by the retention factor of the analytes separated. A study of the porous and hydrodynamic properties reveals that the actual nature of the partition and adsorption of the small analyte molecules between mobile and stationary (solvated) polymer phases are most crucial for their performance. This is due to a significant gel porosity of the polymeric stationary phase. The gel porosity reflects stagnant mass transfer zones restricting their applicability in the separation of small molecules under conditions of strong retention.

Porous polymer monoliths for small molecule separations: advancements and limitations

Porous polymer monoliths are considered to be one of the major breakthroughs in separation science. These materials are well known to be best suited for the separation of large molecules, specifically proteins, an observation most often explained by convective mass transfer and the absence of small pores in the polymer scaffold. However, this conception is not sufficient to explain the performance of small molecules. This review focuses in particular on the preparation of (macro)porous polymer monoliths by simple free-radical processes and the key events in their formation. There is special focus on the fluid transport properties in the heterogeneous macropore space (flow dispersion) and on the transport of small molecules in the swollen, and sometimes permanently porous, globule-scale polymer matrix. For small molecule applications in liquid chromatography, it is consistently found in the literature that the major limit for the application of macroporous polymer monoliths lies not in the optimization of surface area and/or modification of the material and microscopic morphological properties only, but in the improvement of mass transfer properties. In this review we discuss the effect of resistance to mass transfer arising from the nanoscale gel porosity. Gel porosity induces stagnant mass transfer zones in chromatographic processes, which hamper mass transfer efficiency and have a detrimental effect on macroscopic chromatographic dispersion under equilibrium (isocratic) elution conditions. The inherent inhomogeneity of polymer networks derived from free-radical cross-linking polymerization, and hence the absence of a rigid (meso)porous pore space, represents a major challenge for the preparation of efficient polymeric materials for the separation of small molecules.

Towards porous polymer monoliths for the efficient, retention-independent performance in the isocratic separation of small molecules by means of nano-liquid chromatography

We have investigated the free-radical copolymerization dynamics of styrene and divinylbenzene in the presence of micro- and macro-porogenic diluents in 100 μm I.D. sized molds under conditions of slow thermal initiation leading to (macro)porous poly(styrene-co-divinylbenzene) monolithic scaffolds. These specifically designed experiments allowed the quantitative determination of monomer specific conversion against polymerization time to derive the porous polymer scaffold composition at each desirable copolymerization stage after phase separation. This was carried out over a time scale of 3h up to 48 h polymerization time, enabling the efficient and repeatable termination of the polymerization reactions. In parallel, the porous and hydrodynamic properties of the derived monolithic columns were thoroughly studied in isocratic nano-LC mode for the reversed-phase separation of a homologous series of small retained molecules. At the optimized initiator concentration, polymerization temperature and time, the macroporous poly(styrene-co-divinylbenzene) monoliths show a permanent mesoporous pore space, which was readily observable by electron microscopy and indicated by nitrogen adsorption experiments. Under these conditions, we consistently find a polymer scaffold composition which suggests a high degree of cross-linking and thus minimum amount of gel porosity. These columns reveal a retention-insensitive plate height in the separation of small retained molecules which only slightly decreases at increased linear mobile phase velocity. As the polymerization progresses, a build-up of less-densely cross-linked material occurs, which is directly reflected in the observed consistent increase in retention and plate heights. This leads to a significant deterioration in overall isocratic separation performance. The decrease in performance is ascribed in particular to the increased mass transfer resistance governing the monoliths performance over the whole linear chromatographic flow velocity range at polymerization times significantly higher than that of phase separation. The performance of the optimized monoliths only becomes limited by fluid dispersion due to the poorly structured macroporous pore space.

Polyphosphazenes: Multifunctional, Biodegradable Vehicles for Drug and Gene Delivery

Poly[(organo)phosphazenes] are a unique class of extremely versatile polymers with a range of applications including tissue engineering and drug delivery, as hydrogels, shape memory polymers and as stimuli responsive materials. This review aims to divulge the basic principles of designing polyphosphazenes for drug and gene delivery and portray the huge potential of these extremely versatile materials for such applications. Polyphosphazenes offer a number of distinct advantages as carriers for bioconjugates; alongside their completely degradable backbone, to non-toxic degradation products, they possess an inherently and uniquely high functionality and, thanks to recent advances in their polymer chemistry, can be prepared with controlled molecular weights and narrow polydispersities, as well as self-assembled supra-molecular structures. Importantly, the rate of degradation/hydrolysis of the polymers can be carefully tuned to suit the desired application. In this review we detail the recent developments in the chemistry of polyphosphazenes, relevant to drug and gene delivery and describe recent investigations into their application in this field.

Catalytically active polymers obtained by molecular imprinting and their application in chemical reaction engineering.

Molecular imprinting is a way of creating polymers bearing artificial receptors. It allows the fabrication of highly selective plastics by polymerizing monomers in the presence of a template. This technique primarily had been developed for the generation of biomimetic materials to be used in chromatographic separation, in extraction approaches and in sensors and assays. Beyond these applications, in the past few years molecular imprinting has become a tool for producing new kinds of catalysts. For catalytic applications, the template must be chosen, so that it is structurally comparable with the transition state (a transition state analogue, TSA) of a reaction, or with the product or substrate. The advantage of using these polymeric catalysts is obvious: the backbone withstands more aggressive conditions than a bio material could ever survive. Results are presented showing the applicability of a molecularly imprinted catalyst in different kinds of chemical reactors. It is demonstrated that the catalysts can be utilized not only in batch but also in continuously driven reactors and that their performance can be improved by means of chemical reaction engineering.

Analysis of amatoxins α-amanitin and β-amanitin in toadstool extracts and body fluids by capillary zone electrophoresis with photodiode array detection

Abstract Over 90% of the lethal cases of mushroom toxin poisoning in man are caused by a species of amanita. The amatoxins (especially α- and β-amanitin) found in amanita deserve special attention, because of their high pharmacological potency, their high natural concentration and their high chemical and thermal stability. Measures can be taken to improve the survival rates (aggressive gastroenteric decontamination, liver protection therapy) if the poisoning is diagnosed correctly and as early as possible. The standard assay for α-amanitin is a radioimmunoassay (RIA). Among other reagents, this assay uses 125 I-labelled α-amanitin, which has a low shelf life. The assay is therefore not available at all hospitals and all year round. In this paper, a first attempt to employ capillary zone electrophoresis (CZE) to quantify amatoxins α- and β-amanitin in urine samples of afflicted patients and in toadstool extracts is described. Diode array detection is used for identification of the resolved substances in the electropherogram. An analysis require 20 min. The detection limits is 1 μg/ml, i.e., 5 pg absolute. Relative standard deviations are between 1 and 2% for the calibration standards (peak height and area) and ca. 7.5% for the real samples. Advantages of the CZE over the RIA include lower cost, the possibility of quantifying several toxins in one analysis, less consumption of potentially harmful reagents (no radio-labelled substances, no addition of α-amanitin as reagent) and, most importantly, all-year-round availability of the assay. The detection limit is still somewhat high and does not cover the entire clinically relevant range. Attempts to lower the detection limit by the necessary order of magnitude are currently under way in our laboratory. These include application of laser-induced fluorescence detection, liquid chromatography-CZE and CZE-mass spectrometry techniques.

Determination of polycyclic aromatic hydrocarbons in soil samples by micellar electrokinetic capillary chromatography with photodiode-array detection

The reliable quantification even of trace amounts of polycyclic aromatic hydrocarbons (PAHs) is of great concern in environmental and also in medical analysis. PAHs are typically small, uncharged, hydrophobic molecules which do not dissolve well in water. Several methods were investigated and compared for the determination of such substances by capillary electrophoresis, including systems where the analytes are provided with a charge (tetraalkylammonium ions) via solvophobic interaction and systems based on micellar electrokinetic capillary electrophoresis (MECC) using sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide as micelle-forming agents. Diode-array detection permitted the positive identification of the separated pure substances via their prerecorded UV-Vis spectra. By using an aqueous-organic electrophoresis buffer [8.5 mM borate, 85 mM SDS, 50% (v/v) acetonitrile, pH 9.9], a mixture of seven standard PAHs could be separated and quantified within 10 min. The detection limit was 10 pg. The calibration graph was linear over five orders of magnitude. Compared with the chromatographic analysis used so far, the MECC method is faster, has a higher mass sensitivity and requires a smaller sample volume. The method was used to quantify the PAH content of soil samples (heath sand) deliberately contaminated with a mixture of standard PAHs and with machine oil. Two PAHs (anthracene and chrysene) could be determined in samples collected during a biological soil decontamination process.

Selective recognition and separation of β-lactam antibiotics using molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) to penicillin V or oxacillin were prepared, and their recognition properties were investigated, both in organic and aqueous phases. The resulting MIPs proved to be efficient in distinguishing between different penicillins (penicillin V, penicillin G and oxacillin), but could also be prepared to recognise the penicillins as a group. The results indicate that MIPs can be used to prepare either selective or general recognition matrices for penicillins, which can be of potential use in separation and detection applications.