环境激素辛基酚的电化学检测.doc
环境激素辛基酚的电化学检测目 录摘要11 前言31.1 环境内分泌干扰物31.2 碳糊电极的特点及应用41.3 循环伏安法的特点及应用41.4 生物样品分析52 实验部分52.1 实验仪器及试剂52.2 碳糊电极的制备52.3 实验方法63 结果与讨论63.1 底液的选择63.2 底液pH的影响73.3 富集时间的影响83.4 扫描速度的影响83.5 线性范围、校正曲线93.6 电极的重现性、稳定性103.7 干扰实验103.8 回收率及样品的测定114 结论11参考文献12致谢13附录13环境激素辛基酚的电化学检测 摘要:本文研究了辛基酚在碳糊电极上的电化学性质,在-0.21.2V的电位范围内进行了循环伏安扫描,辛基酚在0.7V处有一明显的氧化峰,根据该氧化峰的电流与浓度的关系建立了循环伏安法测定辛基酚的电化学分析方法。对辛基酚检测方法的条件进行了优化。结果表明:测定的最佳底液是pH=5的磷酸盐缓冲溶液PBS,扫描速率为100mV/s。辛基酚浓度与氧化峰电流在7.0×10-73.1×10-5mol/L范围内呈良好的线性关系,线性方程为:ipa (A)= 2.0×10-6+0.4598 c mol/L,线性相关系数r= 0.9966,该方法的检测限为4.0×10-7 mol/L。用标准加入法对辛基酚样品的回收率进行测定,回收率范围在96.5%105.4% 之间,平均回收率为99.7%。将该方法用于鲫鱼体内辛基酚含量的测定取得较好的结果。该方法具有电极制作简单,操作简便,测定结果准确等优点。关键词:碳糊电极;辛基酚;循环伏安法;鲫鱼Electrochemical Detection of Environmental Hormone Octyl Phenol Abstract: This paper had investigated the electrochemical properties of the octyl phenol in carbon paste electrodes, cyclic voltammetric scanning was proceeded in -0.21.2V potential range, meanwhile, octyl phenol was an obvious oxidation peak at 0.7V place, according to the relationship between the oxidation peak current and concentration, a method of electrochemical analysis called cyclic voltammetry was established to detect octyl phenol. The conditions of detection method for octyl phenol were optimized. The results showed that the optimal determination of the bottom fluid was the phosphate buffer solution (pH=5.0) and scanning rate was 100mV/s. In the range of 7.0×10-73.1×10-5mol/L, concentration and oxidation peak currents of ocytl phenol presented a good linear relationship, the linear equation was: ipa (A)= 2.0×10-6+0.4598c (mol/L), the linear correlation coefficient was r=0.9966 and this detection limit of this method was 4.0×10-7mol/L. Standard addition method was used to the detection of recovery of octyl phenol samples, the recovery range was 96.5% 105.4%, and the average recovery was 99.7%. The method applied to detect the content of octyl phenol in carp body had taken good results. The advantages of this method were convenient, preparation for electrode was simple and determination result was accurate, etc. Keywords: Carbon paste electrodes; Octyl phenol; Cyclic voltammetry;Carp 1 前言1.1 环境内分泌干扰物烷基酚化合物污染主要来源于炼油、炼焦、造纸、制药和化工等工业排放的污水。低碳数的烷基酚是通过石油开采及处理过程进入水环境中,其中含碳数少的烷基酚的浓度高于含碳数高的烷基酚。随着烷基酚碳数的增加,水中的溶解度和降解速度减小,而生物体内蓄积性增强。烷基酚污染的另一个主要来源是被广泛用于涂料、清洁剂、杀虫剂生产的非离子表面活性剂烷基酚聚氧乙烯醚的降解产物壬基酚(NP)、辛基酚(OP),研究表明NP、OP都具有较强的环境雌激素活性,是一种内分泌干扰物质。环境内分泌干扰物(EEDs)是指一类干扰体内与生物体的正常行为及生殖、发育相关的正常激素的合成、贮存、分泌、体内运输、结合及清除等过程的外来物质。目前,环境内分泌干扰物被列为继臭氧层空洞和地球变暖之后的又一新的环境污染问题,成为当前国际上研究的热点。在众多的环境内分泌干扰物中,烷基酚等酚类物质由于其存在的广泛性和雌激素效应逐渐引起世界各国环境科学工作者的关注。许多研究表明, EEDs可以导致生物体生殖细胞的畸变和繁殖率的下降,并能引起雄性生物的雌性化1。已知和疑似的环境内分泌干扰物主要是指人工合成的化学物质,1997 年世界野生动物基金会(WWF)列出68种,据化学结构分为多卤联苯类、邻苯二甲酸酯类、酚类、重金属类、有机锡类化合物等。由于极少量的环境内分泌干扰物即可以对生物体造成危害,因此需要采用高灵敏性和特异性的分析方法来检测环境介质中内分泌干扰物的含量,才能准确把握内分泌干扰物质对生物体的危害。内分泌干扰物质的分析检测一般包括样品预处理和样品检测两个过程。样品预处理有提取、净化、浓缩和衍生化等步骤,可以起到富集痕量组分、消除基体干扰、提高方法灵敏度的作用2。从复杂基体中分析痕量环境内分泌干扰物质需要高灵敏度的分析仪器,随着现代科学技术的进步,各种新的分析仪器和方法在适用性和灵敏度方面都有很大的提高,特别是带有质谱检测器的液相色谱和气相色谱3的普及大大推动了环境内分泌干扰物的准确分析。如今,测定烷基酚等类环境内分泌干扰物的方法有气相色谱法4、气相色谱-质谱分析法5、液相色谱法6-7、液相色谱-质谱分析法,其中气相色谱法的灵敏度要高于液相色谱法。辛基酚(OP)是非离子表面活性剂辛基酚聚氧乙烯醚的生物代谢产物,在环境中很难进一步降解,属环境激素类物质,同时也是一种环境内分泌干扰物质8。环境内分泌干扰物(EEDs)是迫切需要治理的新一代环境污染物。辛基酚是已经得到确证的EEDs之一9,值得关注和研究。辛基酚在环境中性质稳定,不易降解。多个实验表明,辛基酚具有类雌激素样作用,并能对雄性动物生殖系统产生影响。虽然国内辛基酚对人类影响的报道不多,但从有关于世界目前的污染状况的报道来看10-11,辛基酚对人的健康影响值得注意,而对其含量监测也应引起重视。1.2 碳糊电极的特点及应用电化学分析具有快速、简便、灵敏的特点,其中固体电极特别是碳糊电极的优点尤其突出,主要表现在:无毒、电位窗口宽(依实验条件,电位范围为-1.4+1.3V,最高至+1.7 V)、残余电流小、制作简单、表面易更新、价格便宜,备受电化学分析工作者的青睐12。因而碳糊电极广泛用于测定无机离子、有机物。本文主要通过碳糊电极研究了辛基酚的电化学行为及其应用。1.3 循环伏安法的特点及应用循环伏安法(CV)是一种常用的电化学研究方法。该法控制电极电势以不同的速率,随时间以三角波形一次或多次反复扫描,电势范围是使电极上能交替发生不同的还原和氧化反应,并记录电流-电势曲线。根据曲线形状可以判断电极反应的可逆程度,中间体、相界吸附或相形成的可能性,以及偶联化学反应的性质等。常用来测量电极反应参数,判断其控制步骤和反应机理,并观察整个电势扫描范围内发生哪些反应,及其性质如何。对于一个新的电化学体系,首选的研究方法往往就是循环伏安法,可称之为“电化学的谱图”。此方法可用于电极反应的性质、机理和电极过程动力学参数的研究等。1.4 生物样品分析生物活性分子电化学行为的研究仍是电化学和电分析化学中一个十分重要的领域,愈来愈受到人们的广泛重视。辛基酚是环境基体中存在的含量较低的有机物之一,目前具有的检测方法有:气相色谱法、高效液相色谱法、气相色谱-质谱法、胶束电动色谱法13 等。辛基酚(OP)等环境酚类内分泌干扰物由于它们具有明显的雌激素效应、生产量巨大、应用范围广和环境中无处不在的特点引起了广泛的关注。对于治理环境内分泌干扰物的污染显得尤为迫切,因此本文采用了电位分析法对辛基酚的含量进行了测定。在对鲫鱼样品进行测定时,先要对样品进行预处理,主要包括提取、净化和浓缩步骤,可以起到富集痕量组分、消除基体干扰、提高方法灵敏度的作用。2 实验部分2.1 实验仪器及试剂实验仪器:电化学工作站CHI615c( 上海辰华仪器公司 ,上海);PHS-25 型酸度计(上海雷磁仪器厂);三电极系统:自制的碳糊电极(CPE)为工作电极,饱和甘汞电极为参比电极,铂丝为对电极;测量均在室温下进行。实验试剂:辛基酚(进口,Alfa Aesar ),乙醇(分析纯,国药集团),磷酸氢二钠(分析纯,国药集团),磷酸二氢钠(分析纯,国药集团),石墨粉(光谱纯),石蜡油(化学纯,上海化学试剂公司),二次蒸馏水。配制pH=3.0至 pH=8.0的磷酸盐缓冲溶液(PBS)按文献14 配制。配制1.0×10-2 mol/L的辛基酚溶液:准确称取辛基酚0.0516g于小烧杯中,用乙醇溶解,转移到25mL容量瓶中,用乙醇定容。2.2 碳糊电极的制备将石蜡油:石墨粉=3:7(质量比)混合,并在玛瑙研钵中研磨5min,使其充分混合,将此碳糊均匀地涂在直径为6mm的石墨电极表面上。 2.3 实验方法用循环伏安法分别对含有辛基酚的PBS溶液和PBS空白溶液进行扫描。图1为在含有1.0×10-5mol/L的辛基酚的PBS缓冲溶液(pH =5.0)中的循环伏安图。从图中可以看出,辛基酚在碳糊电极上发生了氧化还原反应,在0.7V处有一明显的氧化峰,在0.1V处有一还原峰,根据0.7V的氧化峰对辛基酚进行电化学检测。图1 辛基酚在PBS缓冲溶液(pH =5.0)中的循环伏安图1空白PBS缓冲溶液(pH =5.0)2含有1.0×10-5mol/L的辛基酚的PBS缓冲溶液(pH =5.0)3 结果与讨论3.1 底液的选择在选择底液时遵循的原则是:1. 辛基酚在溶液中应该溶解;2. 在该液中易发生氧化反应;3. 发生氧化反应的电位应尽量小;4. 尽可能的有较少的干扰。在查找的文献中,大多数对酚类等有机物进行检测时用的底液是磷酸盐的缓冲性溶液。本实验中用循环伏安法分别在2mol/L的盐酸溶液、1mol/L的硫酸溶液、0.2mol/L的氢氧化钠、3mol/L 的醋酸溶液、pH=2的HAc-NaAc溶液以及pH=5的PBS缓冲溶液进行空白扫描和含辛基酚溶液的扫描对比,发现辛基酚在弱酸性溶液和弱碱性溶液中出峰,其中在pH=5PBS的缓冲溶液中辛基酚氧化峰较好,本实验选择用pH=5PBS的缓冲溶液作底液。并进一步研究了不同pH对辛基酚氧化峰电流及峰电位的影响。3.2 底液pH的影响 辛基酚浓度为1.0×10-5mol/L,在pH=38的PBS缓冲溶液中进行循环伏安扫描,得到循环伏安图(图2),并以峰电流对溶液的pH作图(图3),从图3中可以看出:当底液pH<5.0时,随着溶液pH的升高,峰电流逐渐增大,pH>5.0时,随着溶液pH的升高,峰电流逐渐减小。故本实验选用pH =5.0 PBS的溶液。图2 辛基酚在不同pH的PBS缓冲溶液中的循环伏安图图3底液pH对峰电流的影响3.3 富集时间的影响在PBS缓冲溶液中,加入一定量的辛基酚,搅拌一定时间后进行测定,研究了搅拌时间辛基酚对峰电流的影响,结果表明:搅拌60s后,峰电流即达到最大值。另外,富集时,加一定的电位,更有利于富集的进行,实验结果表明,在-0.1V搅拌富集60s,峰电流即达最大值,故本实验选用-0.1V搅拌富集时间60s。3.4 扫描速度的影响 在1.1×10-6 mol/L的辛基酚溶液中,研究扫描速度对辛基酚峰电流的影响,结果表明,在80400mV/s的扫描速率范围内,辛基酚的氧化峰电流与扫描速率呈线性关系(图4B),说明辛基酚在碳糊电极上是吸附控制15。其中扫描速度为100mV/s时峰形最好,故本实验选用扫描速度为100mV/s。图4 (A) 辛基酚在不同扫描速率下的循环伏安图(a-i: 80, 100, 150, 180, 270, 360 mV/s)(B) 峰电流与扫描速率的关系曲线3.5 线性范围、校正曲线在pH=5.0的PBS介质中,辛基酚在碳糊电极上的氧化电流与其浓度在7×10-73.11×10-5mol/L范围内呈良好线性关系(图5B),其线性回归方程:ipa (A)= 2.0×10-6+0.4598 c(mol/L),线性相关系数r= 0.9966。检测下限为4.0×10-7 mol/L。图 5(A) 碳糊电极在不同浓度辛基酚溶液中的循环伏安图(a:7.00×10-7,b: 3.60×10-6, c: 8.60×10-6, d: 5.95×10-5, e: 1.66×10-5, f: 3.11×10-5mol/L )( B )辛基酚峰电流与浓度的关系曲线3.6 电极的重现性、稳定性用碳糊电极对6.6×10-6mol/L辛基酚溶液连续测定8次,测得结果的平均值为5.125×10-6A,RSD=2.4%。表该电极对辛基酚的测定有较好的稳定性。碳糊电极直接用二次蒸馏水洗净进行重复测定,重复8次,对辛基酚的响应电流基本不变,说明该电极具有较好的重现性。3.7 干扰实验对浓度为5.0×10-6mol/L的辛基酚溶液进行测定,允许测定误差控制在±5%以内,实验表明5.0×10-4mol/L以上的K+、Na+、Hg2+、Pb2+ 、Ca2+、柠檬酸钠、甘氨酸、L-半胱氨酸、葡萄糖等对辛基酚溶液的测定均无干扰。3.8 回收率及样品的测定取鲫鱼样品6.2g,先加入10mL的乙醇,再加入100L5.0×10-3mol/L的辛基酚于样品中,混合溶液搅拌均匀,浸泡2小时后抽滤,再将滤液水浴蒸发至2mL。分别对浓度在7×10-73×10-5mol/L范围内的8份含辛基酚的鱼样溶液以及辛基酚的标样用标准加入法测定,回收率在96.5%105.4% 之间,平均回收率为99.7%,相对标准偏差RSD=2.60%。结果见表1。表1 辛基酚回收率的测定鱼样测定量标样测定量回收率鱼样测定量标样测定量回收率7.00×10-79.00×10-71.10×10-63.60×10-67.38×10-79.21×10-71.07×10-63.56×10-5105.4%102.3%97.3%98.9%8.60×10-61.36×10-51.86×10-52.11×10-58.30×10-61.38×10-61.83×10-52.05×10-596.5%101.5%98.4%97.2%4 结论本实验研究了辛基酚在碳糊电极上的电化学性质,建立了循环伏安法测定辛基酚的电化学分析方法。并探究了辛基酚检测的最佳条件,研究了测定的线性范围,测定方法的检测限等。将该方法用于鲫鱼体内辛基酚含量的测定取得较好的结果。该检测方法具有电极制作简单,操作简便,测定结果准确等优点。参考文献 1Andersen H R. 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Rev., 1997, 97: 32483272.12董绍俊, 车广礼, 谢远武. 化学修饰电极M. 北京: 科学出版社, 2003, 2.13蔡亚岐, 江桂斌, 周庆祥. 胶束电动色谱法分离和测定双酚A、辛基酚和壬基酚J. 分析化学, 2004, 32(9): 11791181.14中南矿冶学院分析化学教研室等, 化学分析手册M. 北京: 科学出版社, 1997, 381.15Fred A.黄慰曾译. 电化学与电分析化学M. 北京: 北京大学出版社,1983,86.致 谢这篇论文是在导师胡效亚教授的悉心指导下完成的。导师在科研选题、研究方法和治学态度等方面给予作者谆谆教导。导师扎实的理论基础,严谨的治学态度和精益求精的科研风格使作者受益非浅。在本论文选题、具体实验和写作过程中,胡老师付出了辛勤劳动和无私帮助。在此,仅向导师致以衷心的感谢和深深的敬意!同时还要特别感谢金根娣老师以及实验室的师兄和师姐们给予我的极大的支持与帮助,在此向他们表示衷心的感谢!回顾四年学习期间的一千多个日日夜夜,自己为有机会摆脱各种烦恼与浮躁,静心钻研,潜心研究,并取得初步研究成果而感到欣慰。欣慰之余,我要向关心和支持我学习的所有朋友们表示真挚的谢意!感谢他们对我的关心、关注和支持! 在即将毕业离校之际,我要感谢老师及同学们在生活上给予我的关心和帮助以及学业上的切磋和指点。同窗之谊和手足之情,我将终生难忘! 雄关漫道真如铁,而今迈步从头越。我愿在未来的学习和工作过程中,以更加丰厚的成果来答谢曾经关心、帮助和支持过我的老师、同学和朋友。附 录Determination of some endocrine disrupter chemicals in urban wastewater samples using liquid chromatographymass spectrometryAbstract:An analytical method for the simultaneous determination of trace amounts of fourteen endocrine disrupter chemicals (EDCs) in urban wastewater samples has been developed. The studied compounds were: bisphenol A and its chlorinated derivatives (monochloro, dichloro, trichloro and tretrachlorobisphenol A), three alkylphenols (4-n-nonyl, 4-n-octyl and 4-(tert-octyl) phenol) and six well known phthalate esters (dimethyl, diethyl, di-n-butyl, butylbenzyl, bis(2-ethylhexyl) and di-n-octyl phthalate). The method involves extraction from the samples and preconcentration of the analytes using a solid-phase extraction (SPE) procedure and subsequent liquid chromatographic separation with mass spectrometric detection (LC-MS). Bisphenol F was used as a surrogate. Quantification limits found ranged between 12ngL-1 for diethylphthalate and 69ngL-1for 4-(tert-octyl) phenol. The method was satisfactorily used for determination of these chemicals in urban wastewater samples of Granada City (Spain) and validated using a recovery assay with spiked samples.Keywords: Alkylphenols; Bisphenol A; Bisphenol A chlorinated derivatives; Phthalate esters; Liquid chromatographymass spectrometry (LCMS); Wastewater analysis1. IntroductionIn recent years, a specific category of human made chemicals, called commonly endocrine disrupter chemicals (EDCs), has attracted a great deal of public and scientific attention because of their suspected carcinogenic and estrogenic properties. In 1996, the European Union (EU) defined this class of compounds as “exogenous substances that cause adverse health effects in an intact organism, or its progeny, consequent to changes in endocrine function” 1. Emerging evidence from wildlife and laboratory studies indicates that some synthetic chemicals may interfere with the endocrine system in a broad variety of ways, including the initiation of hormones, blocking their action or accelerating their breakdown 2.Aware of the problem, both, the EU and the US Environmental Protection Agency (EPA) have authored a“priority” list of substances for further evaluation of their role in endocrine disruption 3,4 indicating the need to assess the levels and effects of EDCs. A report from the EU 5 proposed a candidate list of 533 substances for further evaluation of their role in endocrine disruption. Moreover, incremental growth in dysfunctions related to male reproductive health has been demonstrated 6, 7. Both, human and wildlife are exposed to complex mixtures of these compounds. Effects are cumulative and it could be that they will only appear in subsequent generations. Meantime the resulting effects may be irreversible, threatening sustainable human development.Most EDCs are introduced into the environment by way of anthropogenic inputs and are therefore ubiquitous in aquatic environments receiving wastewater effluents. Although it is possible that a large amount of EDCs remain yet unidentified, it is clear that there are some identified families of compounds showing potential estrogenic activity. Plastic industry intermediates as bisphenol A or phthalate esters, detergent degradation compounds, as 4-n-nonylphenol, 4-n-octylphenol and 4-(tert-octyl)phenol are described in scientific literature. It is now known that phthalate esters, alkylphenols and bisphenol A (BPA) are potential endocrine disrupter chemicals 8, 9. Let us see some of their uses till now. Bisphenol A is mainly used as a monomer in the preparation of epoxide resins, polycarbonate plastic and as an antioxidant in polyvinylchloride 10. Levels of BPA have been found in different matrices such as body fluids, canned food and environmental samples as wastewaters. The use of chlorine for disinfecting wastewaters or drinking water has become widespread over the years. BPA present in waters may react with residual chlorine originally used as a disinfectant, producing chlorinated BPA derivatives 11. Polychlorinated BPA has been recently identified and biodegradation tests using activated sludge revealed that it is not easily biodegraded 12, and chlorinated BPA being more cytotoxic than BPA 13.Alkylphenols such as 4-n-nonylphenol, 4-n-octylphenol and 4-(tert-octyl) phenol are widely used as intermediates to produce surfactants (anionic and non-ion surfactants) and as stabilizers of ethyl cellulose resin, oil-soluble phenol resin and esters 14, 15.Phthalate esters are one of the most abundant man-made environmental pollutants. They are widely used as additives in the manufacture of plastics to make them flexible. Because of their properties as plasticizers in plastic production and because its use has increased significantly in recent years, they are often found in environmental matrices such as waters and soils 16. Six of the most commonly used phthalates are dimethyl, diethyl, di-n-butyl, butylbenzyl, bis(2-ethylhexyl) and di-n-octyl phthalate. Bis(2-ethylhexyl) phthalate is the most used and accounts for up to one quarter of the total production of plasticizers 17. There is a high industrial activity associated with them and relatively large amounts of these compounds are released into the environment, wastewater being the most common way to introduce these contaminants into the environment 1619.Gas chromatography (GC) and liquid chromatography (LC), after previous concentration and clean-up, have been used for determining these compounds in water samples. Commonly, their preconcentration and clean-up involves liquidliquid extraction (LLE) with cyclohexane 20 or dichloromethane 21, 22, solid-phase extraction (SPE) 1619, 2325 or solid-phase microextraction (SPME) 26. After an extensive bibliographic revision, SPE seems to be the most used procedure for the isolation, preconcentration an