【作者】 陈军；

【导师】 柏连阳； 张裕平；

【作者基本信息】 湖南农业大学 ， 农药学， 2013， 博士

【摘要】 将分子印迹技术与常规萃取技术,色谱分离、分析技术及传感器等相结合,用于复杂样品体系中痕量目标物的富集分离分析,可克服生物及环境样品体系复杂、预处理繁琐等不利因素,为试样采集、分离纯化和分析提供极大方便。本论文是在前人学者研究基础上,对分子印迹固相(微)萃取材料,磁性复合微球、印迹敏感膜电化学传感器、色谱柱印迹填料和分子印迹搅拌吸附棒等的制备方式和应用等进行了系统的改进和评价研究,并将其用于实际环境样品中三嗪类除草剂的痕量/微量样品前处理富集与分离以及农残快速检测领域。具体研究内容如下：(1)采用远红外热引发和紫外光引发方式制备分子印迹聚合物。通过对制备聚合物的吸附性能、选择性,印迹效率、孔隙率等指标进行比较分析,探讨引发方式对聚合物性能的影响。利用平衡吸附理论分析聚合物内部形貌对其结合能、结合位点类型及选择性的影响。将聚合物研磨、过筛、洗脱模板后装填固相萃取小柱与商品化C18固相萃取柱比较分离实际环境样品。结果表明2种引发方式制备的分子印迹固相萃取柱均可用于富集水样中莠去津待测物,其回收率可达到90.1%～101.9%。与市售C18柱相比,净化更彻底,且减少了杂质峰对分析的影响,提高灵敏度。(2)以1.2μm聚苯乙烯微球为种球、莠去津为模板分子、甲基丙烯酸为功能单体、二甲基丙烯酸乙二醇酯为交联剂,采用二步种子溶胀法制备球形规整且具多孔结构的大比表面单分散分子印迹聚合物微球。通过对比制备的聚合物微球形貌考察了制备过程中溶胀比,搅拌速度、水油比、交联剂用量、乳化分散剂用量等对分子印迹聚合物微球粒径、表观形貌及孔径分布的影响。将聚合物微球作为色谱填料装填于不锈钢管柱(6.4mm I.D.×10cm)制备成液相色谱柱应用于土样中三嗪类除草剂的含量分析测定。结果表明,三嗪类除草剂在0.1～10mg/L范围内呈线性关系良好。加样回收率为92.1%～102.0%, RSD<5%(n=6)。本方法的定量限为4.13μg/L,能够满足实际样品中三嗪类除草剂限量测定的要求。(3)采用化学共沉淀法制备Fe304亚微米磁性粒子,应用溶胶-凝胶技术和表面键合修饰得到核壳结构的Fe3O4@SiO2@γ-MAPs复合微球,再以悬浮聚合法合成粒径为1.2μm的单分散莠去津分子印迹磁性微球。采用扫描电子显微镜、红外吸收光谱、磁学性质测量和能量弥散X射线分析了印迹磁性微球的表观形貌和结构特征。利用吸附等温线及Scatchard图分析表明莠去津分子印迹磁性微球存在两类不同的结合位点。建立了莠去津分子印迹磁性微球富集-高效液相色谱分析测定三嗪类除草剂莠去津、特丁津、莠灭净的分离分析方法,并将其应用于土壤样品的分离检测,检出限为在2.6μg/L～5.2μg/L,回收率为80.7%～116.6%、相对标准偏差为2.11%～6.92%。实现了简便快速富集分离三嗪类除草剂。(4)报道一种对三嗪类除草剂有识别特性分子印迹膜的制备方法,即通过循环伏安技术在金电极表面以3-硫噻吩丙二酸为功能单体,制备特丁津的分子印迹聚3-硫噻吩丙二酸敏感膜电化学传感器。考察了支持电解质、聚合圈数、扫描电位范围、酸度等对传感器的响应情况及对三嗪类结构类似化合物的选择性。建立实际样品的测定方法以及样品的重现性。实验表明,该分子印迹膜对三嗪类除草剂具有良好的选择性和较高的灵敏度。浓度在0.02mmol/L～0.12mmol/L范围内具有良好的线性关系(线性相关系数R=0.99167),检出限可达0.0025mmol/L。将此传感器用于实际西红柿和土样中三嗪类除草剂的测定,回收率在88.50%～92.0%之间。同时传感器的制备过程简便,重现性和稳定性令人满意,也满足传感器的快速响应要求和灵敏度。(5)在石英毛细管内采用微波聚合的方式制备得到了莠去津分子印迹毛细管整体柱,将其作为固相微萃取头,结合液--液萃取和中空纤维膜萃取技术,与高效液相色谱联用,优化了影响萃取效率的参数：萃取和解析溶剂、盐浓度、pH值、萃取和解析时间及搅拌速度等。建立了2种萃取结合模式直接在环境水样中萃取三嗪类除草剂并偶联高效液相色谱法的分析方法,对四种三嗪类除草剂(莠去津、特丁津、莠灭净、均三嗪)被测物的加标回收率在68.3%～113.2%之间。具备简单、快速、灵敏度高等优点,适合于实际环境水样的痕量分析。(6)以新型超顺材料钕铁硼(Nd2Fe14B)为基质,采用溶胶-凝胶技术在磁性粒子表面进行SiO2包覆,再在其表面进行分子印迹,一步热聚合物法制备了一种整体式分子印迹搅拌吸附萃取棒。将其应用于环境样品黄瓜和土壤中三嗪类除草剂的吸附萃取,通过优化萃取条件,建立了分子印迹搅拌吸附棒--液液萃取和直接萃取实际样品中三嗪类除草剂的分析方法。该方法在对水样和黄瓜样品的相对回收率达到73.6%-95.5%,成功实现了搅拌棒用于极性溶剂和非极性溶剂中的萃取分析。更多还原

【Abstract】 Molecularly imprinted Polymers (MIPs) are a new kind of macromolecular materials with molecular recognition capability. Because MIPs have the advantages of predetermination, specificity and practicability, they have been extensively used in chromatographic separation, antibody mimic, solid-phase extraction, biosensors, catalyzing reaction and other relevant fields. The combination of conventional extraction technology, chromatographic separation and sensor technology with molecular imprinting technique, have been applied for the enrichment and separation of trace analytes in the complex environmental samples. This could solve some problems such as the complexity of environment samples and cumbersome process of the pretreatment and provide great convenience for the collection, enrichment and analysis. In this work we made a systematic study and carried out some improvement and innovation focusing on the solid phase (micro) extraction material, magnetic composite microspheres and electrochemical sensor by molecular imprinting technique, chromatographic column packing and stir bar sorptive extraction combined with the molecular imprinting technique. Furthermore, these developed materials were applied for the enrichment and separation of trace and trace sample processing using triazine herbicides as representative pesticide residues. The following studies are done in this paper.1. Atrazine imprinted polymers were comparatively synthesized using identical polymer formulation by far-infrared (FIR) thermal and ultraviolet (UV) light-induced polymerization, respectively. By ultraviolet spectrophotometry, equilibrium binding experiments were carried out at room temperature with the prepared MIPs. The results showed that MIPuv resulted in three-fold reduction in polymerization time and higher specific binding to atrazine compared with their MIPFIR counterparts. Scatchard plot’s of both MIPs indicated that the affinities of the binding sites in MIPs are heterogeneous and can be approximated by two dissociation-constants corresponding to the high-and low-affinity binding sites, respectively. Moreover, several common pesticides including atrazine, cyromazine, metamitron, simazine, ametryn, terbutryn were tested to determine their specificity, and similar imprinting factor and different selectivity index were obtained for both resulted MIPs. Physical characterization of the polymers revealed that the different polymerization methods led to the slight differences in both polymer structures and performance by scanning electron microscope (SEM), Fourier transform infrared absorption (FTIR), and mercury analyzer (MA). Finally, both MIPs were used as selective sorbents for solid phase extraction (SPE) of atrazine from lake water, followed by the determination using high performance liquid chromatography (HPLC). Compared with the commercial C18SPE sorbent (86.4%-91.5%), higher recoveries of atrazine in spiked lake water were obtained in the range of93.1%-93.8%and94.3%-94.5%, for both MIPs, respectively.2. Atrazine molecularly imprinted polymer microspheres (MIPMs) were fabricated by two step seed swelling polymerization method, using atrazine as a template molecule, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EDMA) as a crosslinking agent and toluene/dodecanol as a porogenic agent, and the factors influencing on the shape, the particle size and particle size distribution of the MIPMs were detailedly studied such as swelling ratio, stirring rate, water-oil ratio, and dispersant dosage, etc. The results showed that when the swelling ratio was40, the stirring rate was200rpm, the water-oil ratio was3:1(v/v), the functional monomer MAA was6mmol, the EDMA dosage was30mmol and the PVA dosage was0.6g, a series of MIPMs with even particle size of3-5μm and good dispersivity could be obtained. We adopted an equilibrium binding experiment to study the binding capacity of the polymer with template molecule, and found through Scatchard model analysis that, for the imprinted polymers, the dissociation constant and apparent maximum binding capacity of low affinity binding site were Kd1=8.21x10-3mol/L and Qmax1=334.23μmol/g, respectively, and those of high affinity binding site were Kd2=4.36x10-4mol/L and Qmax2=38.07μmol/g, respectively. We packed the polymers into empty chromatographic steel tube columns (2.1mm I.D.x10cm) to prepare liquid chromatographic columns used for the evaluation of specificity with similar structure compounds and the determination of atrazine content in water samples. The proposed method has been successfully applied to the determination of limited atrazine in water samples. The results showed that a good linear relationship of atrazine was maintained within0.01-10mg/L (r=0.9992), the sample recovery was in a range of92.1%-102.0%, with a RSD lower than5%(n=6) and a detection limit of4.13μg/L.3. Magnetic iron oxide submicron particles were prepared by chemical co-precipitation, and then magnetic Fe3O4@SiO2@γ-MAPs composite microspheres were achieved by sol-gel technology. The magnetic molecularly imprinted beads were prepared utilizing atrazine as the template molecule by suspension polymerization. The structural characteristics and extraction performance of the atrazine magnetic molecularly imprinted beads were investigated. The imprinted beads were characterized by scanning electron microscopy, fourier transform infrared spectroscopy, vibrating sample magnetometer(VSM) and energy-dispersive X-ray spectroscopy (EDX). The results showed that the imprinted beads were in spherical shape with diameter1.2μm and the surface Was porous and rough. We adopted an equilibrium binding experiment to study the binding capacity of the MMIPs with template molecule, and found through Scatchard model analysis that, for the MMIPs, the dissociation constant and apparent maximum binding capacity of low affinity binding site were Kd1=6.306×10-4mol/L and Qmax1=22.12μmol/g, respectively, and those of high affinity binding site were Kd2=2.887×10-3mol/L and Qmax2=65.94μmol/g, respectively. The atrazine magnetic molecularly imprinted beads were coupled with HPLC for the analysis of three triazines (atrazine、ametryn、 terbuthylazine). The proposed method presented good linearity and the detection limits was2.6μg/L～5.2μg/L. This method was used to the simultaneous quantification of three triazines in soil samples. The recoveries were from80.7%to116.6%for the spiked soil respectively, with the RSD of2.11%～6.92%.4. A novel sensor for the determination of terbuthylazine (TBA) based on3-thiophenemalonic acid (3-TMA) as functional monomer was fabricated by molecularly imprinted technology. The polymeric film was obtained on the gold electrode surface by electrocopolymerization of3-TMA in the presence of the template molecule of TBA, through the use of cyclic voltammetry (CV). Several important parameters controlling the performance of the molecularly imprinted polymer modified sensor were investigated in detail such as the monomer concentration, the electropolymerization cycles and pH. The obtained MIP sensor is highly specific towards newly added TBA and the recognition can be quantitatively analyzed by the differential pulse voltammetry (DPV) to verify the changes in oxidation currents of ferricyanide. In the optimal condition, the response of the imprinted sensor to TBA was linearly proportional to its concentration over the range2.5×10-7mol/L to1.2×10-4mol/L, with high selectivity, good stability and reproducibility (RSD,2.05%). The method has been successfully applied to the determination of TBA in real samples, with a recovery ranging from71.2%to84.9%and a detection limit of2.0×10-8mol/L (S/N=3).5. A novel method to fast prepare molecularly imprinted polymer monolithic fibers is developed with the help of microwave irradiation and used to overcome the well known water-compatibility problem of MIP by liquid-liquid-solid microextraction (LLSME) and the developed hollow fiber-liquid-solid microextraction (HFLSME) system. The resulted atrazine MIP fiber was obtained after silica being etched away with a controlled length of1cm, and subsequently characterized by scanning electron microscope. Main factors affecting the selective extraction including extraction time, desorption time, extraction and desorption solvents were investigated for LLSME and HFLSME procedures in detail. The selectivity was also evaluated using the atrazine template molecule and its structurally-related compounds including2-amino-4-methoxy-6-methyl-1,3,5-triazine, terbuthylazine, ametryn and metribuzin pesticides. The extraction ratio for target pesticides was increased to more than10times in atrazine-imprinted polymeric monolithic fiber compared to non-imprinted polymeric monolithic fiber. The resulted fibers were successfully applied to detect atrazine and its analogues pesticides in lake water coupled to HPLC and resulted in recoveries in the range of68.3%to113.2%, respectively. It demonstrated that the proposed technique could overcome the problem of disturbance from water when the MIP monolithic fiber was exposed directly to non-polar solvent above the aqueous solution.6. To overcome the shortcomings of commercial Stir-bar sorptive extraction, a novel stir bar for sorption extaction based on monolithic material was prepared. The new matrix high magnetic and super paramagnetic material Nd2Fe14B magnetic powder as a muddler magnetic supply, then magnetic powder by TEOS cladding y-MAPs by surface grafting were achieved by sol-gel technology. The molecularly imprinted stir bar sorptive extraction were prepared utilizing atrazine as the template molecule, MAA as the functional monomer, EDMA as the crosslinker, chloroform as the porogen, by in-situ stepwise polymerization. It was then used to extract compound triazine herbicides in the environmental soil and cucumber sample. A method was established by optimizing the experimental conditions such as adsorption curve, stir rate, extraction time, desorption mode and desorption solvent. The method had a good linear relationship in the concentration range of0.01-0.3μg/mL with a correlation coefficient of0.9993. The recovery was from73.6%to95.5%. The SBSE was successfully used to extract triazine herbicides.更多还原