Studies On The Preparation And Application Of Novel Functionalized Polymeric Monoliths And Its Recognition Mechanism

Monolithic column, which can be described as the integrated continuous porous separation media without the interparticle voids, is directly cast in tubes by free radical polymerization. Monoliths can be prepared in-situ by the copolymerization of functional monomers, cross-linkers and initiators in the presence of porogens. Compared with traditional packed columns, the major merits of monoliths are their easy preparation, low backpressure, high permeability, good mass transfer, high separation efficiency, and also that they are amenable to surface functionalization by suitable selection of monomers. In this thesis, poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) and novel poly(isocyanatoethyl methacrylate-co-methyl methacrylate-co-ethylene glycol dimethacrylate) (poly(IEM-co-MMA-co-EGDMA)) monolithic matrix were synthesized. The poly(GMA-co-EGDMA) monolithic matrix can be modified by the ring-opening reaction of epoxy groups originated from GMA. The isocyanate groups on the surface of poly(IEM-co-MMA-co-EGDMA) monolithic matrix are highly reactive and versatile, which are amenable to functionalization by hydroxyl, amine and thiol groups with high efficiency. By chemical modification with a variety of modifiers, C8-C18 functionalized hydrophobic monoliths, polyethyleneimine (PEI) functionalized anion-exchange monoliths, andβ-cyclodextrin functionalized monoliths were prepared. Radix puerariae (R.P.) crude extract, ibuprofen optical isomer and YlLIP2 crude lipase were separated and purified. The preparative conditions for monoliths were first optimized, the separation and purification methods were further, established, and the molecular recognition mechanism was finally investigated. Theβ-cyclodextrin supramolecular recognition mechanism was studied by computational modeling and nuclear magnetic resonance (NMR).Thanks to the high specific selective recognition of molecular imprinting technology (MIT) and the highly efficient preconcentration ability of solid phase extraction (SPE), molecular imprinting solid phase extraction (MISPE) was used for the selective recognition and large enrichment of dimethoate from tea leaves. The preparative conditions of molecularly imprinted polymers (MIPs) were first optimized, the thermal adsorption isotherm was further established and the selective recognition properties of MIPs were finally investigated. The systematic studies were listed below:1. The C8 group functionalized poly(GMA-co-EGDMA) hydrophobic monolith was prepared for one-step rapid separation and purification of puerarin from Radix puerariae (a crude extract of the root of Pueraria lobata) by isocratic elution. The chromatographic recognition mechanism of puerarin was also investigated. The poly(GMA-co-EGDMA) monolithic matrix was prepared in-situ by free radical co-polymerisation of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with AIBN as initiator and cyclohexanol:dodecanol=87:13 (v/v) as porogen. The monolithic matrix was then functionalized by 50%(v/v) n-octylamine ethanol solution. The monolith shows a specific surface area of 11.5 m2·g-1, average pore size of 1.6μm, and a total porosity of 57.6%. A ligand density of 2.3 mmol·g-1 was obtained for the C8 functionalized monolith, which had the binding capacity of 15 mg·g-1 for puerarin. With a sample load of 11.6 mg crude R.P per gram dry polymer and with 1% acetic acid water solution as the optimum mobile phase, a puerarin purity of around 95%(m%) with a recovery of approximate 69%(m%) was achieved by proper peak cutting. The puerarin fraction was then characterized by FTIR, LC-MS and NMR. The column can be regenerated by cleaning with 100% methanol at the flow-rate of 0.5 mL·min-1 for one hour.The puerarin, daidzin and daidzein recognition mechanisms on C8 functionalized monolith were studied by computational modeling and chromatographic evaluation using a variety of solvent systems, and were shown to be based on a mixed-mode of hydrogen bonding and hydrophobic interaction. The hydrogen bonding arises from amino groups, carboxyl groups and hydroxyl groups. The functional alkane groups (C8) produces strong hydrophobic interaction with the phenolic groups of isoflavones. By analyzing the interaction energy between polymer and analytes, the retention factor of analytes on the monolith can be predicted.2. The polyethyleneimine (PEI) modified poly(GMA-co-EGDMA) monolith was prepared by functionalization with 10%(wt%) PEI 30 kDa aqueous solution at 55℃for 12 h, and then was applied for one-step purification of YlLIP2 isoforms from Candida sp.99-125. The monolith has a specific surface area of 5.8 m2·g-1, average pore size of 1.8μm, and a total porosity of 65.6%. A ligand density of 316.8μmol·g-1 was obtained for the PEI functionalized monolith, which had the binding capacity of 45.2 mg·g-1 for bovine serum albumin (BSA). Crude lipase was separated to four isoforms with the total enzyme activity recovery of 52.2%, the maximum purification fold of 4.5, and the specific enzyme activity of 3860 U·mg-1. As analyzed on non-denaturing PAGE, the four isoforms are homogenous and have the similar molecular mass of approximate 38 kDa. The isoforms were subjected to CD and their far UV spectra 190-240 nm was obtained. The distribution of secondary structure of isoforms are different from each other, the isoforms show characteristicα/βhydrolase fold with the content of random coils of 30%. The differences of the four isoforms were further confirmed by MALDI-TOF mass analysis, which indicated molecular masses of 36 648±36,37 839±33,38 236±31 and 38 795±96 Da for isoform A, isoform B, isoform C and isoform D, respectively.3. Theβ-cyclodextrin (β-CD) functionalized poly(GMA-co-EGDM A) monolith was prepared by thermal grafting of 20%(wt%) EDA-β-CD in 0.2 M Na2CO3 (pH=12) aqueous solution at 60℃for 12 h. The monolithic polymer exhibits a specific surface area of 5.1 m2·g-1, average pore size of 1.1μm, and a total porosity of 65.6%. The modified monolith shows aβ-CD ligand density of 680μmol·g-1. The resultant monolith was used for the chiral separation of ibuprofen optical isomer with methanol//0.5%TEAA=30/70 (v/v) (pH=4) as the mobile phase by isocratic elution. The resolution is 2.0 and the selectivity factor is 6.1.Theβ-CD/ibuprofen supramolecular enantioselective recognition mechanism was further investigated by computational modeling. The preferred penetration mode found corresponds to a geometry where the polar group (-COOH) of ibuprofen faces the narrower cavity of the (3-CD (P-N). Studies on the interaction energy, hydrogen bonding and competitive binding between S- and R-ibuprofen demonstrate that S-ibuprofen forms more stable inclusion complex withβ-CD, and consequently has a stronger retardation on the monolith. Compared with free-stateβ-CD, bondedβ-CD has better enantioselectivity for S- and R-ibuprofen.4. The copolymer poly(isocyanatoethyl methacrylate-co-methyl methacrylate-co-ethylene glycol dimethacrylate) (poly(IEM-co-MMA-c o-EGDMA)) was developed as a novel, facile, highly reactive and versatile monolithic matrix, which was amenable to surface functionalization with a variety of nucleophilic modifiers based on the reactive isocyanate groups, producing hydrophobic chromatography andβ-CD functionalized monoliths. The molar ratio of IEM, MMA and EGDMA was 12:1:1. A mixture of toluene and heptane with a volume ratio of 0.5 to 1 was used as the porogen. The monolith shows a specific surface area of 11.5 m2·g-1, average pore size of 1.6μm, and a total porosity of 57.6%. The success of the chemical modification of the monolithic matrix was confirmed by FTIR, solid state 13C NMR and XPS elemental analysis, showing the high ligand density of the modified monoliths. A ligand density of up to 2.33 mmol·mL-1 was obtained fo the 1-octanol modified monolith with an isocyanate group conversion of 96.9%, indicating the high efficiency of the modification reaction. The potential applications of the monoliths are demonstrated by the separation of a series of model compounds and Radix puerariae, and show promising results.5. Study on the recognition mechanism of puerarin, daidzin and daidzein onβ-CD functionalized monolith by chromatographic evaluation, computational modeling and NMR.β-CD exhibits the smallest interaction energy with puerarin, and the largest interaction energy with daidzein, which corresponds to the chromatographic elution result that the elution order is puerarin, daidzin and daidzein. Larger interaction energy means stronger interaction and more stable inclusion complex, and consequently longer retention time on the monolith. By simulating and analyzing the interaction energy betweenβ-CD and guest molecules, the retention property and elution order of guest molecules on theβ-CD functionalized monolith can be well predicted. Blank monolithic matrix has no selective recognition for puerarin, daidzin and daidzein. As a result, P-CD functionality plays a major role for the separation. The adsorption behaviour of puerarin onβ-CD functionalized monolith, has been investigated using a variety of solvent systems and shown to be based on a mixed-mode of hydrogen bonding and hydrophobic interaction.1H NMR and 2D ROESY NMR study shows that puerarin forms a tight 1:1 inclusion complex withβ-CD by penetrating its B, C and A rings into the hydrophobic cavity from the wider rim ofβ-CD, which is consistent with the simulation result.6. Selective recognition and large enrichment of trace dimethoate (organophosphorus pesticides, OPs) from tea leaves by molecular imprinting solid phase extraction (MISPE). The preparative conditions for dimethoate imprinting polymer were optimized by molecular dynamics modeling and chromatographic evaluation. Butyl methacrylate (BMA) was used as functional monomer, ethylene glycol dimethacrylate (EGDMA) was cross-linker, AIBN was initiator, and tetrahydrofuron (THF) was porogen. The molar ratio of template, BMA and EGDMA was 1:4:20. The polymerization was performed at 60℃for 24 h. MIP shows highly selective recognition property for the dimethoate template, and its adsorption isotherm is well presented by Langmuir model. Recoveries of dimethoate from tea samples by MIP and blank polymer are 99% and 26%, respectively, with a large enrichment factor of 100 for MIP. Two parameters, the imprinting factor indicator (IFindicator) and the competitive factor indicator (CFindicator), were defined according to the interaction energy difference to give an insight into the imprinting selectivity of MIPs to dimethoate versus other structurally related organophosphorus pesticides (OPs). A higher value of IFindicator means a better specificity and a higher value of CFindicator indicates a bigger structural difference between dimethoate and its analogues. A higher IFindicator value corresponds to a lower CFindicator value. By considering the two parameters, the selective recognition property of MIPs can be determined. The influences of the rebinding solvents on the adsorption properties of MIPs were also investigated. A good rebinding solvent should have a good solubility for the monomers and template, and less affinity with both template and polymer. A large self-association among solvent molecules could enhance the affinity between template and MIPs.