| Molecular imprinting technique (MIT) is a new technique that could synthesis a kind of tailor-made polymers according to chemical and physical properties and three dimensional space structure of the target molecule. The kind of tailor-made polymers, which is so-called molecularly imprinted polymers (MIPs), can selectively recognise target analytes. The main advantage of molecularly imprinted solid phase extraction (MISPE) is that it could enrich target analytes from the complex system selectively. Moreover, MISPE overcome a shortcoming that conventional SPE sorbents usually co-extraction of interfering compounds, which improve the efficiency of enrichment and purification for the target analytes.In the present research,17β-estradiol MIPs and lysozyme MIPs were prepared with two kinds of differently functionalized monodi-sperse poly(glycidylme-thacrylate-co-ethylene dimethacrylate)(PGMA/EDMA) beads as a carrier material, and two methods had been set up coupled with HPLC-UV which were used to determine trace17β-estradiol in MCF-7cell culture solution and extract lysozyme from hen egg white.This thesis is composed of four parts:(1) Review:In this chapter, the origin, development, principle, preparation methods and applications of MIT were briefly elaborated, respectively. Then, the applications of MISPE were introduced.(2)17β-estradiol MIPs were prepared by surface imprinting technique using PGMA/EDMA beads bounded with vinyl group as carrier material, methacrylic acid and ethylene glycol dimethacrylate as functional monomer and the cross-linker, respectively. The result of scanning electron microscopy experiment showed that the particle size is uniformity and the surface of microspheres has large pore structure. Mercury intrusion method determined the average pore size of MIPs which is mainly in73.3nm. Chromatographic experiment demonstrated that17β-estradiol MIPs had higher recognizability and selectivity for target molecule than the blank imprinted polymer with good permeability.(3) The MISPE combination with HPLC method had been set up which was used to determine trace17β-estradiol from MCF-7cell culture solution. Loading solvent, washing solvent and elution solvent which affecting the extraction capacity of the MIPs have been optimized, respectively. When using acetonitrile (containing90%water, V/V) as loading solvent, methanol (containing10%water, V/V) as washing solvent, and acetonitrile (containing2%trifluoroacetic acid, V/V) as elution solvent, the extraction recoveries of17β-estradiol were reached73.0%-97.5%with RSD not more than7.5%. The calibration curves were established with R above0.9988. The linear is in range of1.00-100.00ng/mL. Limit detection (LOD) was calculated as the concentration corresponding to a signal3times the standard deviation of the baseline noise, respectively. The LOD for the17β-estradiol is0.14ng/mL. The established method was used to assay17β-estradiol from the MCF-7cell culture solution with the concentration of0.86ng/mL. Comparing with traditional C18solid extraction agent, MIPs showed higher affinity for17β-estradiol in the SPE process.(4) Lysozyme MIPs were prepared by surface imprinting technique using PGMA/EDMA beads bonding with β-cyclodextrin and vinyl group as carrier material, lysozyme as the template molecule, acrylamide combine with β-cyclodextrin as the binary functional monomers, N,N’-methylene bisacrylamide as the crosslinker, respectively. Then, the MIPs were used as a new HPLC packing material to study its recognition performance. Moreover, the effects of sodium chloride concentration in mobile phase on lysozyme retention behavior in MIPs were studied. The on-line extraction lysozme method from hen egg white was established using lysozme MIPs as the sorbing phase. The degree of purity of obtained lysozme was mensurated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The method of online extraction lysozme from hen egg white was established using lysozme MIPs as the sorbing phase. |
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