Because HPLC characterization of molecularly imprinted polymers for quer-cetin has proven the existence of a substantial imprinting effect, the polymer was used as sorbent material for the solid-phase extraction of quercetin from red wine (18). The molecularly imprinted polymer enabled facile sample
cleanup prior to HPLC measurements and selective enrichment of quercetin from the red wine without any further sample cleanup procedures (see Fig. 3).
1. Prior to polymerization, 4-vinylpyridine has to be freshly distilled using a microdistillation apparatus in order to remove the inhibitor. The distilled 4-vinyl-pyridine solution can then be stored in a freezer at temperatures below -10°C for several months.
2. Depending on the type of template analyte, different functional monomers (including mixtures of functional monomers), crosslinkers, and solvents can be applied (12,29,30). One main prerequisite is adequate solubility of sufficient amounts of the selected compounds in the solvent of choice. A typical functional monomer-to-solvent ratio is 3:4 (v/v). The molar ratio selected for the imprinting approach of quercetin is 1:8:40 (template:functional monomer:crosslinker). Depending on the required amount of functional monomer and selected cross-linker, ratios typically vary between 1:4:20 and 1:10:60 (30-32). Resulting from losses during the sedimentation procedure (see Note 5), a minimum of 0.18 mmol of template in the prepolymerization mixture should be used. A minimum of 2 g of polymer material after the final sedimentation step is necessary for a successful HPLC column packing process. The functional monomer should be selected according to the potential maximum number of interaction sites, because the for mation of stable complexes between template molecules and functional monomers in the pre-polymerization mixture and during polymerization will determine the quality of the imprint. The ratio of template to functional monomer and to crosslinker for a defined system is frequently determined by trial and error after characterization of the binding properties of the resulting imprinted polymer. Methods such as nuclear magnetic resonance (NMR) studies of the pre-polymerization mixture facilitate the selection process of suitable ratios between template and functional monomer involved in the formation of complex (33,34).
3. Polymerization must be performed in an airtight glass vial, because oxygen acts as an inhibitor during a radical polymerization.
4. Depending on the properties of the template, thermal polymerization at temperatures <60°C (radical starter 2,2'-azobis-[2,4-dimethylvaleronitrile]) and ultraviolet polymerization (at room temperature or lower) offer alternatives for starting the radical copolymerization.
5. An ultrasonication processing step is applied prior to each sedimentation to ensure complete mixing between polymer particles and solvent. The accurate removal of fine particles is a crucial step, because the application of fine particles (<10-|im diameter) during packing of HPLC stationary-phase materials can result in high back-pressures in the HPLC column. The reduction of the particle size distribution is also necessary for better HPLC column performance, because using particles with a wide size distribution will lead to interfering peak-broadening effects.
6. A clear supernatant solution obtained after 20 to 30 min indicates sufficient removal of most of the fine particles.
7. Radioligand (35,36) and competitive fluorescence (37) binding assays are additional qualified techniques to evaluate the selective recognition performance of imprinted polymers. Analytical techniques such as NMR (33,34), infrared spectroscopy, or mass-sensitive devices (38) can provide supplementary information on the recognition (binding) events. However, in our personal opinion and experience, separation techniques related to affinity chromatographic concepts are particularly suitable for analyzing recognition properties of MlPs and are among the most promising applications of MIP-based recognition matrices for biomolecules.
8. The accurate and careful packing of HPLC columns is of great importance, because enclosure of air bubbles or nonuniform packed columns will result in diminished HPLC performance.
9. The proper choice of mobile phase is crucial and strongly depends on the nature of interactions between the analyte and the stationary phase. It has been shown in the literature that the application of the same solvent previously used for polymerization (39) is beneficial for maximizing the recognition properties in chromatographic applications. Although organic solvents may enhance, e.g., hydrogen bonds and ionic interactions, hydrophobic interactions are mainly amplified when applying aqueous media.
10. Capacity factors are calculated as k = (t - i0)/i0, in which t is the retention time for the compound and t0 corresponds to the retention time for the void marker
(acetone in this experiment). Separation factors are calculated as a = k^/k^, with TM indicating the template molecule and TS the respective test substance. RI is calculated as RI = aCTL/aMIP, in which MIP and CTL indicate the MIP and control polymer, respectively. The RI value for the template analyte is 1 per definition and is taken as a reference. Smaller RI values for structurally related molecules indicate less interaction among the analyte, mobile phase, and stationary phase, yielding information on the selectivity and crossreactivity of the MIP.
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