【作者】 张蓉；

【导师】 岳永德；

【作者基本信息】 安徽农业大学 ， 茶学， 2013， 博士

【摘要】 大量施用的有机磷农药残留于茶叶及生长环境介质中,建立满足茶叶及土壤、水环境介质中有机磷农药残留日常例行工作要求的检测方法是农药残留控制技术研究的重要内容。高效薄层技术具有多个样品同时检测、前处理要求低、快速廉价等优点,是目前液相色谱技术中可以实现极性和非极性化合物组成流动相、并可以利用不同化合物组合和配比进行多次洗脱的一项技术。研究利用高效薄层技术探求13种典型农药在茶叶中的分离方法,建立6种有机磷农药在茶叶、茶园土壤中检测方法,以及4种有机磷农药在水中的检测方法。主要研究结果如下：1.对茶叶中13种典型农药进行薄层分离,利用特殊底剂——特丁基甲基醚较好的分离了结构相似、易生成物质对的有机磷农药；两种分离方式,即全自动多项展开(AMD)和传统双槽展缸展开,均可以实现流动相成分及配比与前一次洗脱剂不同；分离8种有机磷农药,AMD可以实现23次洗脱一次完成,重复性比可以3次洗脱的传统双槽展缸展开方法优越；1次洗脱的传统双槽展缸展开方法的灵敏度比AMD多次洗脱的方法高。2.建立茶叶中6种有机磷农药残留高效薄层检测方法,并应用于样品实测,方法简单、快速。茶叶样品经过液-液萃取,串联固相柱净化；16个样品同时在1块预制高效薄板上点样,AMD实现1次分离过程中7次洗脱；洗脱溶剂化合物极性差为5.1。灵敏度在10-30ng之间；0.2-2.0mg·hg-1样品添加水平,除毒死蜱外回收率均满足日常例行的检测需求。3.建立茶园土壤中6种有机磷农药残留检测方法,应用于部分茶园土壤的实测。样品经液-液萃取,直接检测；16个样品同时在1块预制高效薄板上检测,AMD或传统双槽展缸1次分离过程中2次洗脱；灵敏度在10-30ng之间,0.1-1.0mg·kg-1样品添加水平,毒死蜱回收率较低,其余均满足日常例行的检测需求。4.建立水中4种有机磷农药检测方法,样品经液-液萃取,直接检测；4种农药5、1、0.5×10-8g·ml-1添加水平,回收率在86.05%-109.80%之间,变异系数在1.28-5.28%,满足农药残留分析要求。光解是食品和环境介质中有机磷农药重要降解途径,受到溶解性有机质(DOM)酚类衍生物的影响。茶多酚是典型DOM,但茶多酚类物质对污染物光解的影响并不明确。研究选取茶多酚主要活性成分EGCG及氧化物TF、有机磷农药模型物毒死蜱,以“高压汞灯-茶多酚-毒死蜱-水”为研究系统,建立水中毒死蜱HPLC、GC和UPLC检测方法,控制不同系统条件,调查茶多酚对水中毒死蜱光解影响；对EGCG和毒死蜱共存体系进行紫外和荧光光谱检测,并利用捕捉剂对系统中活性中间体HO·和激发三线态中间体进行捕捉,探讨茶多酚对水中毒死蜱光解影响的作用机理。结果如下：1.建立不同水溶液系统中毒死蜱的GC、HPLC和UPLC-MS-MS检测方法,比较方法特点,指出应用范围,利用建立的方法对水中毒死蜱在茶多酚类物质EGCG和TF的作用下光解进行测定。2.蒸馏水中EGCG和TF对毒死蜱光解过程的影响,在光照起始阶段使毒死蜱浓度快速下降,然后上升,之后多数情况呈现较为平稳的下降趋势；比较毒死蜱单独存在的水溶液,EGCG和TF对毒死蜱光解先敏化再抑制的作用,与浓度有关；由于光解中毒死蜱浓度的变化趋势不确定,毒死蜱光解半衰期不能用简单的模型确定。pH稳定的缓冲液中,茶多酚对毒死蜱光解的影响与pH没有递增的正相关性；EGCG比TF对毒死蜱的光解影响更为复杂与强烈；缓冲液较非缓冲溶液毒死蜱光解受EGCG和TFD影响强度小,符合一级动力学反应模型。3.紫外和荧光检测显示,水溶液中与毒死蜱共存的EGCG增加了系统对光的吸收,并展现更强的光活性,使难以接受光子即光量子产率极低的毒死蜱通过EGCG的能量传递而受到激发,对毒死蜱光解起到敏化作用；HO·捕捉实验证明,水溶液中与毒死蜱共存的EGCG可以促进系统HO·的产生,形成氧化环境,有助于毒死蜱氧化;光解中检测到毒死蜱的氧化产物——氧化毒死蜱,证明光氧化途径仍然是与EGCG共存的水溶液中毒死蜱光解的途径之一。4.利用山梨酸成功捕捉光照过程中毒死蜱激发三线态,证明毒死蜱光解途径之一是通过激发三线态转化,同时证明EGCG可以抑制这一过程；EGCG对典型激发三线态模型物光解的影响结果,佐证了EGCG对毒死蜱激发三线态的抑制作用。荧光检测显示光照时毒死蜱可能与EGCG产生光不稳定络合物,再次光照后可能释放毒死蜱;EGCG的抗氧化还原作用可能使毒死蜱母体浓度升高。总结毒死蜱光照过程中浓度上升的主要原因是EGCG对毒死蜱激发三线态的还原和抑制,络合物的生成以及EGCG的抗氧化还原作用。5.水溶液体系中溶解氧、有机溶剂、表面活性剂、腐殖酸、三价铁离子(Fe3+)、亚铁离子(Fe2+)、碳酸根离子(C032-)、碳酸氢根离子(HCO3-)等物质,可以对系统HO·产生影响,并与浓度和作用时间相关。6.EGCG影响水溶液中光解机理研究显示EGCG对毒死蜱光解具有敏化和抑制的双重作用,两种作用的竞争结果形成水溶液中毒死蜱光解浓度先下降后上升的现象更多还原

【Abstract】 Organophosphorus pesticides contaminate not only the foods such as tea, but also the other environmental media. Method for detection to satisfy the daily inspection is a focus on the pesticides resides analysis in food and environmental media. High Performance Thin-layer Chromatography (HPTLC) with multiple samples detecting at same time and a simple pre-process of samples is fast and cheap for application. This work seeks to separation of13pesticides using HPTLC, establish method for detecting6organophosphorous pesticides residues in tea and soil in tea garden, and method for4organophosphorous pesticides residues in water. The results shown as follows:1. The butyl-methyl-ether is used as special bottom agent to separate the pesticides with similar structures which is difficult to be separated on account of forming substance pair. Two separation methods, i.e. automatic multiple development (AMD) and the traditional development with two-chamber, can realize the mobile phase composition and proportion is different with the previous eluent; AMD can achieve a development with23-step elution and a good repeatability; Traditional development with two-chamber can achieve3-step elution but the repeatable operation is poor compared with AMD. AMD with multiple developments is not better at sensitivity than Traditional development with one-step elution. A simple and rapid detection method of6organophosphorous pesticides residues in tea is established and applied to the sample test. After liquid-liquid extraction, the tea samples is clean-up with a tandem SPE column;16samples can be applied on one preproduction stationary, separated by AMD with7-step elutions; The polarity of compounds composing the elution solvent is difference of5.1. The sensitivity is between10-30ng; The recoveries of fortifications with0.2～2.0mg· kg-1satisfied for the testing of daily routine, except for Chorpyrifos.2. It is also developed for a method to detecting6organophosphorous pesticides residues in soil of tea garden. After liquid-liquid extraction, the samples is determined directly without clean-up and separated by AMD or traditional development with2-step elutions with16samples processed on one stationary plate. The sensitivity is between10～30ng; The recoveries of fortifications with0.1～1.0mg· kg-1satisfied for the testing of daily routine, except for Chorpyrifos.3. A method for detecting4organophosphorous pesticides residues in water is achieved. After liquid-liquid extraction, the samples is determined directly without clean-up The recoveries of fortifications with0.1～1.0mg· kg-1is from86.05%to109.80%with a coefficient of variation of1.28～5.28%and satisfied for the testing of water.Photolysis is an important pathway of organophosphorus pesticides degradation in food and environmental media and influenced by phenolic derivatives, a dissolved organic matter (DOM). The tea phenols (TP) are typical DOM. However, the effect of TP on pollutions photolysis is unclear. To investigate the interaction of TP and organophosphorus pesticides in light, the main active ingredient in TP, EGCG, and its oxidant, Theafalvin, are chosen to compose the controlling photo-system with Chlopyrifos in water using high press mercury lamp. After establishing the detection method of Chlorpyrifos, the effect of tea polyphenols on chlorpyrifos photolysis in water is investigated by controlling the system conditions. The mechanism is discussed by analysis the interactions between the intermediates, i.e. HO·and triplet material, and the tea phenols while the spectrum of the system was scanned by fluorescence and ultraviolet spectrometer. The results indicate as follows:1. Methods, including the assays of HPLC, GC and UPLC, are established to meet the target substances detection in water. The method applicability is discussed and suggested for different analysis.2. The concentration of chlorpyrifos rapid decline, then up, finally for the most part shows relatively steady downward trend in the stage of initial photolysis process of chlorpyrifos in distilled water influenced by EGCG and TF. EGCG and TF were sensitized and obstacles, and associated with concentration; Due to the fluctuations of chlorpyrifos concentration, it is difficult to model the half-life of chlorpyrifos using the first-rate kinetic model. There are no increasing positive correlation between the concentration of chlorpyrifos impacted by tea polyphenols and the pH. The influence of EGCG is more complex than TF effect on photolysis of chlorpyrifos and strong. Compared with in the distilled water, the influence of EGCG and TF is slight and the photolysis of chlorpyrifos corresponde to the first-rate kinetic model.3. The results of UV and FL detection show that EGCG coexisted with chlorpyrifos increases the energy of aqueous solution system by absorpting light, and has stronger activity excited by chlorpyrifos. It helps chlorpyrifos, which with low light quantity, accepts the energe and is sensitized. EGCG promotes system to produce HO·forming a system with strong oxidizing and promote chlorpyrifos oxidation. That chlorpyrifos-oxon, a product of chlorpyrifos oxidation, was detected in the photolysis indicates that photooxidation is a pathway of chlorpyrifos degradation in water containing EGCG and TF.4. Sorbic acid isomerization proves one of pathway of chlorpyrifos photolysis is by the transformation of excited-triplet state and EGCG could inhibit this process, successfully. It is also proved by EGCG affecting the transformation of excited-triplet state of model chemical in water. Therefore, EGCG reducts and inhibits the transformation of chlorpyrifos excited-triplet state is the main reason for the concentration rise of chlorpyrifos after a sharp decreased in the beginning of photolysis. In addition chlorpyrifos may form an unstable complex compound with EGCG and is released after light irritation. It resulted in the concentration of chlorpyrifos increasing. The last, reduction of EGCG may lead to the concentration of chlorpyrifos increasing.5. Dissolved oxygen, organic solvents, surfactants, humic acid, ferric iron ion (Fe3+), ferrous ion (Fe2+), carbonate ions (CO32-), and bicarbonate ions (HCO3-) can impact on the production of HO in system. The concentration of the substances and the action time play roles in the process.6. Studies on mechanism of EGCG influencing chlorpyrifos photolysis have shown that EGCG in aqueous solution not only sensitized but also inhibit the chlorpyrifos photolysis. The competition of two roles resulted in the phenomenon of chlorpyrifos photolysis in aqueous solution.更多还原