技术领域technical field
本发明涉及一种双酚A和壬基酚双模板分子印迹聚合物微球，直接应用于水产品、环境样品中痕量双酚A和壬基酚的富集和分析。The invention relates to a bisphenol A and nonylphenol double-template molecularly imprinted polymer microsphere, which is directly applied to the enrichment and analysis of trace amounts of bisphenol A and nonylphenol in aquatic products and environmental samples.
背景技术Background technique
双酚A（BisphenolA，简称BPA）和烷基酚（Alkylphenols）为两类常见环境激素类物质，由于结构的相似性常被归为一类研究对象，称为酚类环境雌激素。大量实验室研究和环境调查结果表明，壬基酚（NP）和双酚A（BPA）等物质对男性精子数量的减少、女性乳腺癌、子宫癌发病率的增加、雄性动物的雌性化以及鸟类、鱼类和哺乳动物生育率的下降、部分生态系统中动物雌雄比例失调等具有不容忽视的作用，给生态环境和人体健康带来严重威胁，因而成为一类新的研究和监测的重要物质。Bisphenol A (BPA for short) and alkylphenols (Alkylphenols) are two types of common environmental hormones. Because of their structural similarities, they are often classified as a class of research objects, called phenolic environmental estrogens. A large number of laboratory studies and environmental investigations have shown that substances such as nonylphenol (NP) and bisphenol A (BPA) can reduce the number of sperm in men, increase the incidence of breast cancer and uterine cancer in women, feminize male animals and bird The decline of the fertility rate of species, fish and mammals, and the imbalance of the sex ratio of animals in some ecosystems have played a role that cannot be ignored, posing a serious threat to the ecological environment and human health, so it has become a new class of important substances for research and monitoring. .
由于BPA、NP是重要的精细化工原料和中间体，广泛存在于环境土壤或江河湖泊的水体和生物体。环境样品或生物体中BPA、NP的含量极低，加上样品基体复杂，难以直接测定，常常需要经样品前处理后才能进行分析。因此，发展高效高选择性的样品前处理新方法有重要意义。Since BPA and NP are important fine chemical raw materials and intermediates, they widely exist in environmental soils or water bodies and organisms in rivers and lakes. The content of BPA and NP in environmental samples or organisms is extremely low, coupled with the complexity of the sample matrix, it is difficult to directly measure, and often requires sample pretreatment before analysis. Therefore, it is of great significance to develop new methods for sample pretreatment with high efficiency and high selectivity.
分子印迹技术（Molecular Imprinting Technique，MIT）是为获得在空间结构和结合位点上与某一分子（模板分子）完全匹配的聚合物的制备技术。分子印迹聚合物（Molecularly Imprinted Polymer，MIP）因对模板分子具有特异性选择识别，特别是用作固相萃取的填料，以实现对目标物质的浓缩、富集，消除复杂的生物样品或环境样品中基体的干扰，是当前样品前处理的一项重要研究课题。Molecular Imprinting Technique (MIT) is a preparation technology for obtaining a polymer that completely matches a certain molecule (template molecule) in terms of spatial structure and binding site. Molecularly imprinted polymers (MolecularlyImprinted Polymer (MIP) has specific selection and recognition of template molecules, especially as a filler for solid phase extraction, to achieve concentration and enrichment of target substances, and to eliminate matrix interference in complex biological samples or environmental samples. An important research topic in current sample preparation.
徐靖等人（聚氯乙烯，2010, 38(7):35-38）以壬基酚为模板分子，原位聚合法制备分子印迹聚合物用于基质固相分散萃取PVC玩具和污泥中的壬基酚，对壬基酚的萃取回收率97.8%；李永敏等人（科学技术与工程，2011, 11(25):6145-6147）利用分子印迹与溶胶凝胶技术，以氨基丙基三乙氧基硅烷为功能单体，四乙氧基硅烷为交联剂，以壬基酚为模板分子制备了壬基酚分子印迹材料，聚合物对壬基酚的最大吸附容量为3.85 mg/g；Guerreiro等人（Anal. Chim.Acta, 2008, 612(1): 99-104）以计算机模拟，壬基酚为模板分子沉淀聚合法制备分子印迹聚合物用于固相萃取水样中壬基酚，聚合物对壬基酚的最大吸附容量为231 mg/g；Núñez等人（J Separation Science, 2008, 31(13): 2492- 2499）采用本位聚合法，以甲苯为制孔剂制备分子印迹聚合物用于固相萃取复杂环境固体样品中壬基酚及其类似物，回收率60%～100%。Surong Mei等人（Microchemical Journal, 2011, 98(1):150-155）以BPA分子印迹固相萃取结合毛细管电泳测定河水、井水中痕量BPA，固相萃取回收率因基体不同在71.20%～86.23%。林福华等人（分析化学，2012, 40(2): 243-248）以BPA为模板分子，利用整体材料原位聚合技术制备分子印迹聚合物为基质的萃取饼，应用于水样BPA的萃取，回收率为86.2%～112%。Canale等人（J Separation Science, 2010, 33(11): 1644-1651）以氟双酚A衍生物4,4'-六氟苯酚作为虚拟模板制备的分子印迹聚合物应用于水样中BPA的固相萃取，回收率大于90%。江明等人（分析化学，2008, 36(8):1089-1092）采用块状聚合法，以甲苯与液体石蜡混合溶剂为致孔剂，制备一种双酚A 分子印迹微球，粒径3 μm。许志锋等人（中山大学学报（自然科学版），2005, 44(3):53-57）采用原位聚合法，以甲苯做致孔剂，以双酚A 为模板分子制备双酚A 的分子烙印聚合物，结合位点对模板分子的最大表观结合量为148.1 μmol/g （MBPA=228.28 g/mol）。杨本晓等人（南京大学学报（自然科学），2007, 43(4):351-357）采用本体聚合法，以双酚A 为模板分子、2-乙烯吡啶为功能单体、聚三甲基丙烯酸丙三醇酯为交联剂制备双酚A分子印迹聚合物，表观最大吸附量为7. 23 μmol / g。苏博（东华大学，2010）用沉淀聚合法，以甲基丙烯酸（MAA）为功能单体，二乙烯苯（DVB）为交联剂制备双酚A分子印迹聚合物，最大吸附量为606.9 μmol/g。赵美萍等人（高等学校化学学报, 2003, 24(7):1204-1206）采用原位聚合法，以双酚A为模板分子、甲基丙烯酸（MAA）为功能单体、乙腈为致孔剂，二甲基丙烯酸乙二醇酯为交联剂制备双酚A分子印迹聚合物，对双酚A的萃取回收率为101.7%。张进等人（贵州教育学院学报（自然科学），2008, 19(9): 17-19）采用表面分子印迹技术，在硅胶表面合成了基于双酚A的分子印迹聚合物，最大吸附量为1.4 μmol/g。翟美娟（南京医科大学，2011）以双酚A为模板分子，正硅酸四乙酯为交联剂，氨丙基三乙氧基硅烷为功能单体，通过溶胶凝胶聚合反应在二氧化硅小球表面反应上一层分子印迹聚合物涂层的方法，制备粒径均匀的纳米分子印迹聚合物，对双酚A的萃取回收率为97%。Xu Jing et al. (PVC, 2010,38(7):35-38) Using nonylphenol as a template molecule, molecularly imprinted polymers were prepared by in situ polymerization for solid phase dispersion extraction of nonylphenol and p-nonylphenol in PVC toys and sludge The recovery rate was 97.8%; Li Yongmin et al. (Science Technology and Engineering, 2011, 11(25):6145-6147) used molecular imprinting and sol-gel technology, using aminopropyltriethoxysilane as a functional monomer, four Ethoxysilane was used as a cross-linking agent, and nonylphenol molecularly imprinted materials were prepared with nonylphenol as a template molecule. The maximum adsorption capacity of the polymer for nonylphenol was 3.85 mg/g; Guerreiro et al. (Anal. Chim.Acta , 2008, 612(1): 99-104) Using computer simulation, nonylphenol as template molecular precipitation polymerization method to prepare molecularly imprinted polymers for solid phase extraction of nonylphenol in water samples, the polymer has the largest nonylphenol The adsorption capacity is 231mg/g; Núñez et al. (J Separation Science, 2008, 31(13): 2492- 2499) prepared molecularly imprinted polymers using toluene as a pore-forming agent by the in situ polymerization method for solid-phase extraction of nononium in complex environmental solid samples Base phenol and its analogues, the recovery rate is 60% to 100%. Surong Mei et al. (MicrochemicalJournal, 2011, 98(1):150-155) Determination of trace BPA in river water and well water by BPA molecularly imprinted solid phase extraction combined with capillary electrophoresis, the recovery rate of solid phase extraction varies from 71.20% to 86.23% depending on the matrix. Lin Fuhua et al. (Analytical Chemistry, 2012, 40(2):243-248) Using BPA as a template molecule, the extraction cake of molecularly imprinted polymer matrix was prepared by using the monolithic material in situ polymerization technology, which was applied to the extraction of BPA in water samples, and the recovery rate was 86.2%-112%. Canale et al. (J SeparationScience, 2010, 33(11): 1644-1651) Molecularly imprinted polymer prepared by using fluorobisphenol A derivative 4,4'-hexafluorophenol as a virtual template was applied to the solid phase extraction of BPA in water samples, and the recovery rate Greater than 90%. Jiang Ming et al. (Analytical Chemistry, 2008, 36(8):1089-1092) prepared a kind of bisphenol A molecularly imprinted microspheres with a particle size of 3 μm. Xu Zhifeng et al. (Journal of Sun Yat-Sen University (Natural Science Edition), 2005, 44(3):53-57) used in-situ polymerization method, using toluene as a porogen, and bisphenol A as a template molecule to prepare bisphenol A molecules imprinted polymer, the maximum apparent binding of the binding site to the template molecule is 148.1μmol/g (MBPA=228.28g/mol). Yang Benxiao et al. (Journal of Nanjing University (Natural Science), 2007,43(4):351-357) prepared molecular imprinting of bisphenol A by bulk polymerization method, using bisphenol A as template molecule, 2-vinylpyridine as functional monomer and polyglycerol trimethacrylate as crosslinking agent polymer, the apparent maximum adsorption capacity is 7. 23 μmol /g. Su Bo (Donghua University, 2010) used precipitation polymerization method to prepare bisphenol A molecularly imprinted polymer with methacrylic acid (MAA) as functional monomer and divinylbenzene (DVB) as cross-linking agent. The maximum adsorption capacity was 606.9 μmol/g. Zhao Meiping et al. (Chemical Journal of Chinese Universities, 2003, 24(7):1204-1206) used the in-situ polymerization method, using bisphenol A as a template molecule, methacrylic acid (MAA) as a functional monomer, and acetonitrile as a porogen , ethylene glycol dimethacrylate was used as cross-linking agent to prepare bisphenol A molecularly imprinted polymer, and the extraction recovery rate of bisphenol A was 101.7%. Zhang Jin et al (Journal of Guizhou Institute of Education (Natural Science), 2008,19(9): 17-19) Using surface molecular imprinting technology, a molecularly imprinted polymer based on bisphenol A was synthesized on the surface of silica gel, with a maximum adsorption capacity of 1.4 μmol/g. Zhai Meijuan (Nanjing Medical University, 2011) used bisphenol A as a template molecule, tetraethylorthosilicate as a crosslinking agent, and aminopropyltriethoxysilane as a functional monomer, and synthesized silica through sol-gel polymerization. A method of coating a layer of molecularly imprinted polymer on the surface of the bead is used to prepare a nanometer molecularly imprinted polymer with uniform particle size, and the extraction recovery rate of bisphenol A is 97%.
现有技术都是以壬基酚或双酚A作为单模板分子制备壬基酚分子印迹聚合物或双酚A分子印迹聚合物，采用多为本体聚合法和原位聚合法，所得的聚合物的形貌多为不规则块状，对模板分子虽表现出较高的选择性识别能力，但吸附容量低，柱效低。当样品基体复杂时，常导致对模板分子及其结构类似物萃取回收率偏低，缺乏应用前景。The prior art uses nonylphenol or bisphenol A as a single template molecule to prepare nonylphenol molecularly imprinted polymers or bisphenol A molecularly imprinted polymers, mostly by bulk polymerization and in-situ polymerization, and the obtained polymers The morphology of most of them is irregular and blocky, although it shows high selective recognition ability for template molecules, but its adsorption capacity is low and column efficiency is low. When the sample matrix is complex, it often leads to low extraction recovery of template molecules and their structural analogues, which lacks application prospects.
发明内容Contents of the invention
为了解决现有技术的不足，本发明的目的在于提供一种双酚A和壬基酚双模板分子印迹聚合物微球，是一种选择性和吸附容量高、粒径较均一的球形分子印迹聚合物，可广泛地应用于水产品、水环境样品中双酚A和壬基酚的富集、分析，萃取回收率达99%以上。In order to solve the deficiencies of the prior art, the object of the present invention is to provide a bisphenol A and nonylphenol dual-template molecularly imprinted polymer microsphere, which is a spherical molecularly imprinted polymer with high selectivity and adsorption capacity and relatively uniform particle size. The polymer can be widely used in the enrichment and analysis of bisphenol A and nonylphenol in aquatic products and water environmental samples, with an extraction recovery rate of over 99%.
本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球，是通过以下步骤来制备的：The bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres described in the present invention are prepared through the following steps:
A）将0.2~2.0 mmol壬基酚、0.2~2.0 mmol双酚A溶于氯仿中，再加入0.8~8.0 mmol 甲基丙烯酸，超声波处理30分钟，于0-4℃温度下自组装8-12小时；A) Dissolve 0.2-2.0 mmol nonylphenol and 0.2-2.0 mmol bisphenol A in chloroform, then add 0.8-8.0 mmol methacrylic acid, ultrasonicate for 30 minutes, self-assemble at 0-4℃ for 8-12 Hour;
B）加入0.1~1.0 mmol 疏水剂、0.1~1.0 mmol的表面活性剂、20~80 mL超纯水、10.0~50.0 mmol交联剂和0.0100~0.1000 g引发剂，混合；B) Add 0.1~1.0 mmol hydrophobic agent, 0.1~1.0 mmol surfactant, 20~80Mix mL ultrapure water, 10.0~50.0 mmol crosslinking agent and 0.0100~0.1000 g initiator;
C）超声波处理10~15 分钟，通氮气除氧气8~15分钟，再超声波处理10~15分钟，利用超声波对溶液中的物质具有高分散性的特点，形成稳定乳液，于50~100℃水浴中机械搅拌，反应5~24 小时得到白色聚合物微球；C) Ultrasonic treatment for 10-15 minutes, blowing nitrogen to remove oxygen for 8-15 minutes, and then ultrasonic treatment for 10-15 minutes, using ultrasonic waves to have high dispersion of substances in the solution, forming a stable emulsion, in a water bath at 50-100 °C Medium mechanical stirring, react for 5-24 hours to obtain white polymer microspheres;
D）将得到的聚合物微球研碎，用体积比为9:1的甲醇-乙酸溶液索氏萃取至紫外检测不到壬基酚和双酚A，再用甲醇洗至中性，研磨过筛，乙腈沉降，真空干燥至恒重，得到双酚A和壬基酚双模板分子印迹聚合物。D) Grind the obtained polymer microspheres, Soxhlet extract with methanol-acetic acid solution with a volume ratio of 9:1 until nonylphenol and bisphenol A cannot be detected by ultraviolet light, wash with methanol until neutral, and grind sieve, settle in acetonitrile, and vacuum-dry to constant weight to obtain bisphenol A and nonylphenol dual-template molecularly imprinted polymer.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤A中，双酚A与壬基酚的摩尔比为1:1。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step A, the molar ratio of bisphenol A to nonylphenol is 1:1.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤A中，双酚A与甲基丙烯酸的摩尔比为1:4。According to the further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step A, the molar ratio of bisphenol A to methacrylic acid is 1:4.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤A中，壬基酚与甲基丙烯酸的摩尔比为1:4。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step A, the molar ratio of nonylphenol to methacrylic acid is 1:4.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤B中，疏水剂与表面活性剂的摩尔比为4:1。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step B, the molar ratio of the hydrophobic agent to the surfactant is 4:1.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤B中，交联剂与引发剂的比例为400:1（mmol:g）。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step B, the ratio of the crosslinking agent to the initiator is 400:1 (mmol:g).
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤B中，所述疏水剂选自：石蜡，正十二烷，正十四烷，正十六烷，正十八烷。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step B, the hydrophobic agent is selected from: paraffin, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤B中，所述交联剂选自：乙二醇二甲基丙烯酸酯，聚乙二醇二丙烯酸酯、三甲醇基丙烷三甲基丙烯酸酯（TRIM）、甲季戊四醇三丙烯酸酯（PETAR）、季戊四醇四丙烯酸酯（PETER）。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step B, the crosslinking agent is selected from: ethylene glycol dimethacrylate, polyethylene Glycol Diacrylate, Trimethylolpropane Trimethacrylate (TRIM), Pentaerythritol Triacrylate (PETAR), Pentaerythritol Tetraacrylate (PETER).
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤B中，所述引发剂选自：偶氮二异丁腈（AIBN），过硫酸钾，过硫酸铵，过硫酸盐-四甲基亚乙二胺。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step B, the initiator is selected from the group consisting of: azobisisobutyronitrile (AIBN), persulfuric acid Potassium, Ammonium Persulfate, Persulfate-Tetramethylethylenediamine.
根据本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的进一步特征，所述步骤B中，所述表面活性剂是由斯盘80与十六烷基三甲基溴化铵按1:9~5:5的比例混合而成的混合表面活性剂。According to a further feature of the bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention, in the step B, the surfactant is composed of Span 80 and hexadecyltrimethyl bromide Ammonium chloride mixed surfactant in the ratio of 1:9~5:5.
本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球，具有以下特点和优点：以双模板分子代替单模板分子，将功能单体、双模板分子（壬基酚和双酚A）在疏水溶剂中自组装；将疏水剂（正十四烷）、表面活性剂（司盘80和十六烷基三甲基溴化铵）溶于超纯水中，制成微乳液；将上述两种溶液混合，加入交联剂（EDMA）和引发剂（AIBN），在磁力搅拌下于水浴中热引发，通过静电作用、氢键作用制备得到双模板分子印迹聚合物微球。The bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres of the present invention have the following characteristics and advantages: the single-template molecule is replaced by the double-template molecule, and the functional monomer and the double-template molecule (nonylphenol and bis Phenol A) Self-assembly in a hydrophobic solvent; a microemulsion was prepared by dissolving a hydrophobic agent (n-tetradecane), a surfactant (Span 80 and cetyltrimethylammonium bromide) in ultrapure water Mix the above two solutions, add cross-linking agent (EDMA) and initiator (AIBN), thermally initiate in a water bath under magnetic stirring, and prepare double-template molecularly imprinted polymer microspheres through electrostatic interaction and hydrogen bond interaction.
本发明所制备的印迹聚合物具有球形形貌（如图2所示），粒径较均一（粒径约为0.1~0.7 μm）、选择性高、吸附容量高（经静态吸附实验测定，对双酚A和壬基酚的最大吸附量分别为42.07 mg/g 、23.65 mg/g），萃取回收率高（以制备的印迹聚合物微球为固相萃取填料，在甲醇加载、2 mL水淋洗、6 mL甲醇-乙酸（9:1,V/V）洗脱条件下，经固相萃取实验测定，对双酚A和壬基酚的萃取回收率分别为99.9%和98.1%）、制备方法简单等显著优点，非常适用于水产品和水环境样品中痕量双酚A和壬基酚的富集和分析。The imprinted polymer prepared by the present invention has spherical morphology (as shown in Figure 2), relatively uniform particle size (particle size is about 0.1-0.7 μm), high selectivity, and high adsorption capacity (measured by static adsorption experiments, for The maximum adsorption capacity of bisphenol A and nonylphenol was 42.07 mg/g and 23.65 mg/g, respectively), and the extraction recovery rate was high (the prepared imprinted polymer microspheres were used as solid-phase extraction filler, loaded in methanol, 2 mL of water Under the conditions of elution and 6 mL methanol-acetic acid (9:1, V/V), the recovery rates of bisphenol A and nonylphenol were 99.9% and 98.1%, respectively, as determined by solid phase extraction experiments), The method has obvious advantages such as simple preparation method, and is very suitable for the enrichment and analysis of trace amounts of bisphenol A and nonylphenol in aquatic products and water environment samples.
附图说明Description of drawings
图1是本发明制备双模板分子印迹聚合物的作用原理图。Fig. 1 is a schematic diagram of the present invention for preparing a double-template molecularly imprinted polymer.
图2 是本发明制备印迹聚合物（MIP）的扫描电镜图。Fig. 2 is a scanning electron micrograph of the imprinted polymer (MIP) prepared by the present invention.
图3是本发明测定肇庆市西江水的HPLC色谱图；图中，(a) 未经处理的西江水样；（b）西江水样经MIP-SPE萃取；（c）添加22.03µg/L NP、22.83µg/L BPA的西江水样经MIP-SPE萃取。Figure 3 is the HPLC chromatogram of Xijiang water in Zhaoqing City determined by the present invention; in the figure, (a) untreated Xijiang water sample; (b) Xijiang water sample extracted by MIP-SPE; (c) added 22.03μg/L NP , 22.83µg/L BPA of Xijiang water samples were extracted by MIP-SPE.
具体实施方式Detailed ways
实施例一：本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的制备Example 1: Preparation of bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres according to the present invention
本实施例所述的双酚A和壬基酚双模板分子印迹聚合物微球的制备的原理图如图1所示，制备方法包括以下步骤：The schematic diagram of the preparation of bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres described in this example is shown in Figure 1, and the preparation method includes the following steps:
A）将1.0 mmol壬基酚、1.0 mmol双酚A溶于氯仿中，4.0 mmol MAA，超声30分钟，于0-4℃温度下自组装8-12小时； A) Dissolve 1.0 mmol nonylphenol, 1.0 mmol bisphenol A in chloroform, 4.0 mmol MAA, sonicate for 30 minutes, self-assemble at 0-4°C for 8-12 hours;
B）加入0.8 mmol 正十四烷，0.2 mmol混合表面活性剂、50 mL超纯水、20.0 mmol乙二醇二甲基丙烯酸酯（EDMA）和0.0500 g偶氮二异丁腈（AIBN）混合； B) Add 0.8 mmol n-tetradecane, 0.2 mmol mixed surfactant, 50 mL ultrapure water, 20.0 mmol ethylene glycol dimethacrylate (EDMA) and 0.0500 g azobisisobutyronitrile (AIBN);
C）超声10 min，通氮气除气泡5 min，再超声10 min确保形成乳液后于80℃水浴中机械搅拌，反应14 h得到聚合物，聚合物的形貌如图2所示； C) Sonicate for 10 minutes, pass nitrogen gas to remove bubbles for 5 minutes, and then sonicate for 10 minutes to ensure that the emulsion is formed, then mechanically stir in a water bath at 80°C, and react for 14 hours to obtain a polymer. The morphology of the polymer is shown in Figure 2;
D）将聚合物研碎，用体积比为9:1的甲醇-乙酸溶液索氏萃取至紫外检测不到壬基酚和双酚A，再用甲醇洗至中性，研磨过筛，乙腈沉降，真空干燥至恒重，得到双酚A和壬基酚双模板分子印迹聚合物。D) Grind the polymer, Soxhlet extract with methanol-acetic acid solution with a volume ratio of 9:1 until nonylphenol and bisphenol A cannot be detected by UV, then wash with methanol until neutral, grind and sieve, and settle with acetonitrile , and vacuum-dried to constant weight to obtain bisphenol A and nonylphenol dual-template molecularly imprinted polymer.
上述制备工艺中，所采用的表面活性剂优选是由斯盘80与十六烷基三甲基溴化铵（CTAB）按3:7的比例混合而成的混合表面活性剂。In the above preparation process, the surfactant used is preferably a mixed surfactant obtained by mixing Span 80 and cetyltrimethylammonium bromide (CTAB) at a ratio of 3:7.
实施例二：本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的制备Example 2: Preparation of bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres according to the present invention
本实施例所述的双酚A和壬基酚双模板分子印迹聚合物微球的制备的原理图如图1所示，制备方法包括以下步骤：The schematic diagram of the preparation of bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres described in this example is shown in Figure 1, and the preparation method includes the following steps:
A）将0.2 mmol壬基酚、0.2 mmol双酚A溶于氯仿中，0.8 mmol甲基丙烯酸（MAA），超声30分钟，于0-4℃温度下自组装8-12小时； A) Dissolve 0.2 mmol nonylphenol, 0.2 mmol bisphenol A in chloroform, 0.8 mmol methacrylic acid (MAA), sonicate for 30 minutes, self-assemble at 0-4°C for 8-12 hours;
B）加入0.1 mmol石蜡，0.1 mmol混合表面活性剂、20 mL超纯水、10.0 mmol聚乙二醇二丙烯酸酯和0.0100 g过硫酸盐-四甲基亚乙二胺混合； B) Add 0.1 mmol paraffin, 0.1 mmol mixed surfactant, 20 mL ultrapure water, 10.0 mmol polyethylene glycol diacrylate and 0.0100 g persulfate-tetramethylethylenediamine to mix;
C）超声12min，通氮气除气泡8 min，再超声12 min确保形成乳液后于50℃水浴中机械搅拌，反应5 h得到聚合物，聚合物的形貌如图2所示； C) Sonicate for 12 minutes, pass nitrogen gas to remove bubbles for 8 minutes, and then sonicate for 12 minutes to ensure that the emulsion is formed, then mechanically stir in a water bath at 50°C, and react for 5 hours to obtain a polymer. The morphology of the polymer is shown in Figure 2;
D）将聚合物研碎，用体积比为9:1的甲醇-乙酸溶液索氏萃取至紫外检测不到壬基酚和双酚A，再用甲醇洗至中性，研磨过筛，乙腈沉降，真空干燥至恒重，得到双酚A和壬基酚双模板分子印迹聚合物。D) Grind the polymer, Soxhlet extract with methanol-acetic acid solution with a volume ratio of 9:1 until nonylphenol and bisphenol A cannot be detected by UV, then wash with methanol until neutral, grind and sieve, and settle with acetonitrile , and vacuum-dried to constant weight to obtain bisphenol A and nonylphenol dual-template molecularly imprinted polymer.
上述制备工艺中，所采用的表面活性剂优选是由斯盘80与十六烷基三甲基溴化铵（CTAB）按1:9的比例混合而成的混合表面活性剂。In the above preparation process, the surfactant used is preferably a mixed surfactant obtained by mixing Span 80 and cetyltrimethylammonium bromide (CTAB) at a ratio of 1:9.
实施例三：本发明所述的双酚A和壬基酚双模板分子印迹聚合物微球的制备Example 3: Preparation of bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres according to the present invention
本实施例所述的双酚A和壬基酚双模板分子印迹聚合物微球的制备的原理图如图1所示，制备方法包括以下步骤：The schematic diagram of the preparation of bisphenol A and nonylphenol dual-template molecularly imprinted polymer microspheres described in this example is shown in Figure 1, and the preparation method includes the following steps:
A）将2.0 mmol壬基酚、2.0 mmol双酚A溶于氯仿中，8.0 mmol甲基丙烯酸（MAA），超声30分钟，于0-4℃温度下自组装8-12小时； A) Dissolve 2.0 mmol nonylphenol and 2.0 mmol bisphenol A in chloroform, 8.0 mmol methacrylic acid (MAA), sonicate for 30 minutes, and self-assemble at 0-4°C for 8-12 hours;
B）加入1.0 mmol正十六烷（或正十八烷），1.0 mmol混合表面活性剂、80 mL超纯水、50.0 mmol TRIM（或40.0 mmol PETAR或PETER）和0. 1000 g过硫酸钾（或过硫酸铵）混合； B) Add 1.0 mmol n-hexadecane (or n-octadecane), 1.0 mmol mixed surfactant, 80 mL ultrapure water, 50.0 mmol TRIM (or 40.0 mmol PETAR or PETER) and 0. 1000 g potassium persulfate ( or ammonium persulfate) mixed;
C）超声15 min，通氮气除气泡15 min，再超声15 min确保形成乳液后于100℃水浴中机械搅拌，反应24 h得到聚合物，聚合物的形貌如图2所示；C) Sonicate for 15 minutes, blow nitrogen to remove air bubbles for 15 minutesmin, and then ultrasonic for 15 min to ensure that the emulsion was formed, mechanically stirred in a water bath at 100°C, and reacted for 24 h to obtain a polymer. The morphology of the polymer is shown in Figure 2;
D）将聚合物研碎，用体积比为9:1的甲醇-乙酸溶液索氏萃取至紫外检测不到壬基酚和双酚A，再用甲醇洗至中性，研磨过筛，乙腈沉降，真空干燥至恒重，得到双酚A和壬基酚双模板分子印迹聚合物。D) Grind the polymer, Soxhlet extract with methanol-acetic acid solution with a volume ratio of 9:1 until nonylphenol and bisphenol A cannot be detected by UV, then wash with methanol until neutral, grind and sieve, and settle with acetonitrile , and vacuum-dried to constant weight to obtain bisphenol A and nonylphenol dual-template molecularly imprinted polymer.
上述制备工艺中，所采用的表面活性剂优选是由斯盘80与十六烷基三甲基溴化铵（CTAB）按5:5的比例混合而成的混合表面活性剂。In the above preparation process, the surfactant used is preferably a mixed surfactant obtained by mixing Span 80 and cetyltrimethylammonium bromide (CTAB) at a ratio of 5:5.
实施例四：本发明所述的双酚A和壬基酚双模板分子印迹聚合物用于肇庆市西江水样的检测Example 4: The bisphenol A and nonylphenol dual-template molecularly imprinted polymer of the present invention is used in the detection of Xijiang water samples in Zhaoqing City
西江水样（2mL，肇庆市端州区江滨三路西江边），过0.22 μm滤膜后，分别直接进行以下两组检测：（1）HPLC直接检测；（2）MIP-SPE-HPLC-FID分离检测，其中分离检测柱的固相萃取填料为实施例一所制备的双酚A和壬基酚双模板分子印迹聚合物。Xijiang water samples (2mL, along the Xijiang River, Jiangbin 3rd Road, Duanzhou District, Zhaoqing City), after passing through a 0.22 μm filter membrane, were directly subjected to the following two groups of tests: (1) HPLC direct detection; (2) MIP-SPE-HPLC -FID separation and detection, wherein the solid phase extraction filler of the separation and detection column is the bisphenol A and nonylphenol dual-template molecularly imprinted polymer prepared in Example 1.
结果见图3。由图3可以看出，西江水样直接进行HPLC测定的色谱图（图3a），在双酚A色谱峰（2.587 min）附近有基体干扰；经分子印迹固相萃取（MIP-SPE）处理后的HPLC色谱图（图3b），在双酚A（2.587 min）、壬基酚（4.395 min）色谱峰附近没有观察到明显的干扰峰；添加22.03µg/L NP、22.83µg/L BPA的西江水样经MIP-SPE处理后的HPLC色谱图（图3c）没有机体的干扰，在2.587 min（双酚A）、4.395 min（壬基酚）处观察到明显的色谱峰。本发明所述的分子印迹聚合物可用于水样品中的双酚A和壬基酚的检测分析。The results are shown in Figure 3. It can be seen from Figure 3 that the chromatogram of the Xijiang water sample directly measured by HPLC (Figure 3a) has matrix interference near the bisphenol A chromatographic peak (2.587 min); The HPLC chromatogram (Figure 3b), no obvious interference peaks were observed near the chromatographic peaks of bisphenol A (2.587 min) and nonylphenol (4.395 min); The HPLC chromatogram (Fig. 3c) of the water sample treated by MIP-SPE had no interference from the organism, and obvious chromatographic peaks were observed at 2.587 min (bisphenol A) and 4.395 min (nonylphenol). The molecularly imprinted polymer of the invention can be used for the detection and analysis of bisphenol A and nonylphenol in water samples.
实施例五：本发明所述的双酚A和壬基酚双模板分子印迹聚合物用于啤酒的检测Example 5: The bisphenol A and nonylphenol dual-template molecularly imprinted polymer of the present invention is used for the detection of beer
取超市购买的啤酒样品2mL过0.22 μm滤膜后，分别直接进行以下两组检测：（1）HPLC直接检测；（2）MIP-SPE-HPLC-FID分离检测，其中采用实施例二所制备的的双酚A和壬基酚双模板分子印迹聚合物作为分子印迹固相萃取（MIP-SPE）的填料。测试结果见表1。After passing 2 mL of beer samples purchased in supermarkets through a 0.22 μm filter membrane, the following two groups of tests were directly carried out: (1) direct detection by HPLC; (2) separation and detection by MIP-SPE-HPLC-FID, in which the Bisphenol A and nonylphenol dual-template molecularly imprinted polymers were used as fillers for molecularly imprinted solid-phase extraction (MIP-SPE). The test results are shown in Table 1.
表1 啤酒样品中BPA和NP的测定结果（n=3）Table 1 Determination results of BPA and NP in beer samples (n=3)
ND：无检测。ND: Not detected.
由表1可以看出，原啤酒样品检测不到BPA或NP；在啤酒样品中加入BPA或NP后，采用MIP-SPE-HPLC检测到BPA和NP的回收率分别为99.0%和97.0%，RSD小于4.4%，说明采用本发明所述的双酚A和壬基酚双模板分子印迹聚合物作为分子印迹固相萃取（MIP-SPE）的填料，对于双酚A和壬基酚的吸附回收率极高，可以适用于对啤酒等饮料样品的双酚A和壬基酚的监测分析。It can be seen from Table 1 that no BPA or NP could be detected in the original beer sample; after adding BPA or NP to the beer sample, the recoveries of BPA and NP detected by MIP-SPE-HPLC were 99.0% and 97.0%, respectively, RSD Less than 4.4%, indicating that the bisphenol A and nonylphenol dual-template molecularly imprinted polymer used in the present invention is used as the filler for molecularly imprinted solid phase extraction (MIP-SPE), and the adsorption recovery rate for bisphenol A and nonylphenol Extremely high, it can be applied to the monitoring and analysis of bisphenol A and nonylphenol in beverage samples such as beer.
实施例六：本发明所述的双酚A和壬基酚双模板分子印迹聚合物用于鲫鱼的检测Example 6: The bisphenol A and nonylphenol dual-template molecularly imprinted polymer of the present invention is used in the detection of crucian carp
市购鲫鱼7尾，重量500±200 g。其中2尾作空白鱼，5尾暴露在添加壬基酚和双酚A的西江水样中一周，进行毒理实验探究。将鱼去鳞去内脏后，分别取鱼鳃、适量鱼肉片放在绞肉机中捣碎，称重。25 mL乙醚提取，震荡3 h，弃渣取清夜，涡旋离心去蛋白，氮气吹干后溶于5 mL甲醇，进行分子印迹固相萃取（采用实施例三所制备的双酚A和壬基酚双模板分子印迹聚合物作为分子印迹固相萃取（MIP-SPE）的填料），过0.22 μm滤膜，稀释10倍进行MIP-SPE-HPLC 检测，结果见表2。Seven crucian carp were purchased in the market, weighing 500±200 g. Among them, 2 fish were used as blank fish, and 5 fish were exposed to Xijiang water samples added with nonylphenol and bisphenol A for a week to conduct toxicological experiments. After removing the scales and viscera of the fish, take the fish gills and appropriate amount of fish slices, mash them in a meat grinder, and weigh them. Extract with 25 mL of diethyl ether, shake for 3 h, discard the residue and take the clear night, vortex centrifuge to remove protein, blow dry with nitrogen, dissolve in 5 mL of methanol, and carry out molecular imprinted solid phase extraction (using bisphenol A and nonyl The phenolic double-template molecularly imprinted polymer was used as the filler for molecularly imprinted solid phase extraction (MIP-SPE), passed through a 0.22 μm filter membrane, and diluted 10 times for MIP-SPE-HPLC detection. The results are shown in Table 2.
表2 市售的鲫鱼中BPA和NP的测定结果（n=3）Table 2 Determination results of BPA and NP in commercial crucian carp (n=3)
ND：无检测。ND: Not detected.
由表2可以看出，空白组中鲫鱼鱼肉及鱼鳃中均未检测到NP，但均检测到BPA；而实验组中检测到BPA和NP的回收率均大于96.0%，RSD小于5.9%，说明采用本发明所述的双酚A和壬基酚双模板分子印迹聚合物作为分子印迹固相萃取（MIP-SPE）的填料，对于双酚A和壬基酚的吸附回收率极高，可以适用于对鱼等水产品的双酚A和壬基酚的监测分析。As can be seen from Table 2, NP was not detected in crucian carp meat and gills in the blank group, but BPA was detected; while the recoveries of BPA and NP detected in the experimental group were both greater than 96.0%, and the RSD was less than 5.9%. It shows that the bisphenol A and nonylphenol dual-template molecularly imprinted polymer used in the present invention is used as the filler of molecularly imprinted solid phase extraction (MIP-SPE), and the adsorption recovery rate for bisphenol A and nonylphenol is extremely high, which can It is suitable for the monitoring and analysis of bisphenol A and nonylphenol in fish and other aquatic products.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103736468B (en)* | 2014-01-16 | 2016-05-11 | 昆明理工大学 | A kind of preparation method of bimodulus plate molecular imprinting-absorbing extraction stirring rod |
| CN104086795A (en)* | 2014-07-10 | 2014-10-08 | 南京理工大学 | A magnetic molecularly imprinted polymer, its preparation and application |
| CN105348437B (en)* | 2015-10-27 | 2017-11-24 | 成都市食品药品检验研究院 | A kind of double-template molecularly imprinted polymer and preparation method and application |
| CN107840928A (en)* | 2017-11-21 | 2018-03-27 | 石家庄君乐宝乳业有限公司 | The preparation method of nonyl phenol and bisphenol-A double-template molecularly imprinted polymer, nonyl phenol and bisphenol A molecular engram post and its application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177500A (en)* | 2007-10-30 | 2008-05-14 | 湖北出入境检验检疫局检验检疫技术中心 | Method for preparing bisphenol A molecular engram polymer |
| CN101857664A (en)* | 2010-06-21 | 2010-10-13 | 湖北出入境检验检疫局检验检疫技术中心 | Preparation method of molecular imprinting polymer with specific recognition capability to tetracycline family |
| CN102532390A (en)* | 2011-12-05 | 2012-07-04 | 中国农业科学院农业质量标准与检测技术研究所 | Triazine weedicide, and metabolite molecular engram polymer microspheres, preparation method and application thereof |
| CN102702429A (en)* | 2012-06-19 | 2012-10-03 | 苏州市汉微环保科技有限公司 | Method for preparing bisphenol A molecularly imprinted polymer |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7830699B2 (en)* | 2006-04-12 | 2010-11-09 | Samsung Electronics Co., Ltd. | Resistance variable memory device reducing word line voltage |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177500A (en)* | 2007-10-30 | 2008-05-14 | 湖北出入境检验检疫局检验检疫技术中心 | Method for preparing bisphenol A molecular engram polymer |
| CN101857664A (en)* | 2010-06-21 | 2010-10-13 | 湖北出入境检验检疫局检验检疫技术中心 | Preparation method of molecular imprinting polymer with specific recognition capability to tetracycline family |
| CN102532390A (en)* | 2011-12-05 | 2012-07-04 | 中国农业科学院农业质量标准与检测技术研究所 | Triazine weedicide, and metabolite molecular engram polymer microspheres, preparation method and application thereof |
| CN102702429A (en)* | 2012-06-19 | 2012-10-03 | 苏州市汉微环保科技有限公司 | Method for preparing bisphenol A molecularly imprinted polymer |
| Title |
|---|
| Antonio Guerreiro等.Preliminary evaluation of new polymer matrix for solid-phase extraction of nonylphenol from water samples.《analytica chimica acta》.2008,第612卷第99-104页. |
| Direct enrichment and high performance liquid chromatography analysis of ultra-trace Bisphenol A in water samples with narrowly dispersibleBisphenol A imprinted polymeric microspheres column;Ming Jiang等;《Journal of Chromatography A》;20060203;第1110卷;第27-34页* |
| Migaku Kawaguchi等.Molecularly imprinted solid phase extraction using stable isotope labeled compounds as template and liquid chromatography–mass spectrometryfor trace analysis of bisphenol A in water sample.《Analytica Chimica Acta》.2005,第539卷第83-89页. |
| Ming Jiang等.Direct enrichment and high performance liquid chromatography analysis of ultra-trace Bisphenol A in water samples with narrowly dispersibleBisphenol A imprinted polymeric microspheres column.《Journal of Chromatography A》.2006,第1110卷第27-34页. |
| Molecularly imprinted solid phase extraction using stable isotope labeled compounds as template and liquid chromatography–mass spectrometryfor trace analysis of bisphenol A in water sample;Migaku Kawaguchi等;《Analytica Chimica Acta》;20050408;第539卷;第83-89页* |
| Preliminary evaluation of new polymer matrix for solid-phase extraction of nonylphenol from water samples;Antonio Guerreiro等;《analytica chimica acta》;20080212;第612卷;第99-104页* |
| 分子印迹基质固相分散-超高效液相色谱法测定儿童玩具中双酚A;徐靖等;《理化检验-化学分册》;20111231;第47卷;第665-667、671页* |
| 双酚A 分子印迹聚合物的制备及其在水相中的吸附性能;苏博等;《化工环保》;20111231;第31卷(第2期);第167-171页* |
| 双酚A 分子印迹聚合物的制备及其在环境监测中的应用初探;杨本晓等;《河南科学》;20071231;第25卷(第6期);第1047-1051页* |
| 徐靖等.分子印迹基质固相分散-超高效液相色谱法测定儿童玩具中双酚A.《理化检验-化学分册》.2011,第47卷第665-667、671页. |
| 杨本晓等.双酚A 分子印迹聚合物的制备及其在环境监测中的应用初探.《河南科学》.2007,第25卷(第6期),第1047-1051页. |
| 苏博等.双酚A 分子印迹聚合物的制备及其在水相中的吸附性能.《化工环保》.2011,第31卷(第2期),第167-171页. |
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