微萃取与在柱富集毛细管电泳联用技术及其在元素形态分析中的应用
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摘要
元素的毒性或有益作用及其在环境中的迁移途径和生物体的代谢行为不仅与其总含量有关,而且与其存在形式(形态)密切相关。因此,对某一元素进行形态分析比对其进行总量分析具有更加重要的意义。毛细管电泳(CE)是元素形态分析的一种重要分析手段。CE用于元素形态分析具有低消耗、低成本、高分离度、分离体系灵活、无需衍生等优点。但是,由于CE进样量小(纳升级),导致其浓度检出限低；此外,CE对样品基质的耐受能力弱,大量基质引入分离毛细管会导致目标分析物的迁移时间变化,从而导致分析结果不准确。因此,在CE分析前往往需要辅以合适的样品前处理技术和CE在柱富集技术。在诸多样品前处理技术中,微萃取技术如液相微萃取(LPME)、固相微萃取(SPME)和搅拌棒吸附萃取(SBSE)等因其具有微型化、环境友好、高灵敏、抗干扰能力强等优点而备受关注,将这些微萃取技术与CE联用可提高方法的检测灵敏度和净化样品基质。在柱富集技术具有模式众多、富集效率高、操作简便以及不影响CE分离度等优点,是改善CE检测灵敏度的另一种重要手段。因此,将微萃取技术与在柱富集技术分别或者二者相结合与CE联用用于实际样品中元素形态的分析具有很大的应用潜力,可拓展CE在元素形态分析领域的应用范围。
本论文的研究目的是,探索和研究适用于元素形态分析的在柱富集体系；开发和研究可直接萃取(非衍生)极性元素形态的固相微萃取和液相微萃取新体系和新方法；建立微萃取和/或在柱富集-CE/UV联用用于元素形态分析的新方法,并将其应用于环境和生物样品的分析。本论文的主要研究内容包括：
(1)建立了聚合物涂层中空纤维膜微萃取(PC-HFME)结合动态pH界面富集毛细管电泳紫外(CE/UV)检测分析环境水样中硒代氨基酸形态的新方法。对两种模式下动态pH界面在柱富集目标分析物的条件进行了优化,最后确定背景电解质(BGE)为极端酸性的磷酸缓冲溶液(pH 2.0),样品溶液基质为极端碱性的缓冲溶液(pH 12.4),样品进样量为分离毛细管总容量的90%。在此条件下,动态pH界面在柱富集目标分析物的富集倍数为430-590倍。将部分磺酸化的聚苯乙烯聚合物(PSP)涂覆在中空纤维膜上制备了PSP涂覆中空纤维膜,并将其应用于硒代氨基酸的萃取。这种PC-HFME-动态pH界面富集-CE/UV方法对硒代氨基酸的富集倍数为3171-18525倍,检出限在0.32-4.10μg/L之间。将该方法应用于自来水和东湖水样的分析,加标回收率在82.4%-106.5%的范围内。
(2)以PDMS粘附法制备了纳米Zr02涂层搅拌棒,并利用Zr02与含磷化合物之间的选择性作用,将制备的纳米Zr02涂层搅拌棒用于强极性化学战剂降解产物甲基磷酸(MPA)、甲基膦酸乙酯(EMPA和甲基膦酸片呐酯(PMPA)的直接搅拌棒吸附萃取(SBSE)。将ZrO2-SBSE与大体积样品堆积(LVSS)相结合,建立了ZrO2-SBSE-LVSS-毛细管电泳间接紫外(CE/indirect UV)分析实际环境水样中EMPA、PMPA和MPA的新方法。对影响ZrO2-SBSE萃取、解吸的因素以及影响LVSS堆积的条件分别进行了优化。在最优的萃取条件和堆积条件下,该方法对三种目标分析物的检出限在1.2-3.1μg/L之间,富集倍数为742-1583倍。该方法已成功应用于环境水样中目标分析物的分析,加标回收率在93.8%-105.3%之间。
(3)以磷酸三丁酯作为萃取溶剂,采用U型中空纤维膜-液液液微萃取(HF-LLLME)模式,实现了HF-LLLME对硫酸介质中五种极性苯砷酸类化合物的选择性萃取：提出一种阴离子耗尽进样(ASEI)堆积的新策略,即在样品电动进样前,先引入几乎整管的水柱(长度占91%的分离毛细管),从而在不改变分离度的情况下增加了样品溶液的最大进样量,提高了ASEI在柱富集苯砷酸类化合物的富集倍数。将HF-LLLME强的基质净化能力与ASEI高富集倍数的优点有机结合,建立了简便、快速、高灵敏的HF-LLLME-ASEI-CE/UV分析复杂环境样品中苯砷酸类化合物的新方法。该方法对目标分析物的检出限在0.68-6.9μg/L之间,富集倍数为155-1780倍,相对标准偏差在5.6-11.8%范围内。将所建立的方法用于当地某养猪场使用的浓缩猪饲料以及该养猪场附近环境中样品包括堆放的猪粪、耕地中的土壤、东湖水的分析并进行了加标回收实验。结果发现,该方法应用于浓缩猪饲料和东湖水中苯砷酸化合物分析的回收率在85-110%之间；堆放的猪粪样品的回收率在66.7-96.2%之间；土壤样品中各物质的回收率均低于50%。
(4)以SDS为离子对试剂,采用离子对-中空纤维膜-液液液微萃取(IP-HF-LLLME)成功实现了极性相差较大的四种甲状腺素化合物包括四碘甲状腺原氨酸(T4)、三碘甲状腺原氨酸(T3)、3,5-二碘-甲状腺素(T2)、3,3',5-三碘代-甲腺原氨酸(reverse T3, rT3)以及两种合成甲状腺素的原料3,5-二碘酪氨酸(DIT)和单碘酪氨酸(MIT)的同时萃取。据此,建立了简便、快速、高灵敏、高选择性的同时分析药物中六种目标分析物的IP-HF-LLLME-CE/UV新方法。对影响IP-HF-LLLME萃取效率的主要因素,如样品溶液pH值、有机溶剂、SDS浓度、萃取时间、搅拌速率以及盐效应等进行了系统考察。在最优的实验条件下,该方法分析六种目标分析物的检出限在0.54-1.43μg/L之间,富集倍数为183-366倍,相对标准偏差在3.19-8.98%范围内。将所建立的IP-HF-LLLME-CE/UV方法应用于两种药物中甲状腺素含量的测定,获得了满意的结果。
(5)以L-半胱氨酸作为反萃取试剂,采用U型中空纤维膜液液液微萃取(HF-LLLME)模式,实现了三种毒性较大的有机汞形态的萃取。将HF-LLLME与大体积样品堆积(LVSS)结合,建立了HF-LLLME-LVSS-CE/UV分析人发和海鱼样品中三种有机汞形态的新方法。对影响HF-LLLME的萃取条件和LVSS的电泳参数进行了优化,该方法分析甲基汞、乙基汞和苯基汞的检出限分别为0.03、0.07和0.14μg/L,富集倍数为2400-4300倍,线性范围达到两个数量级,其相关系数在0.9992-0.9999范围内。将该方法应用于标准参考物质DORM-2、人发样品和海鱼肌肉组织的分析,获得了满意的结果。该方法灵敏度高、选择性强、操作简单、分析仪器廉价,采用CE/UV可获得比CE-等离子体质谱(ICP/MS)更低的检出限水平,可应用于复杂基质样品中低含量的有机汞形态的分析。
(6)设计了简单的自动化动态中空纤维膜-液液液微萃取(AD-HF-LLLME)装置,该装置采用流动注射仪辅助,通过程序控制可实现上样、清洗、动态萃取步骤的自动化。采用18-冠-6作为给予相络合试剂,L-半胱氨酸作为接受相反萃取试剂实现了无机汞和有机汞的同时萃取。其中,实验发现在萃取过程中存在乙基汞形态转化为无机汞的现象。据此,将AD-HF-LLLME与大体积样品堆积(LVSS)技术相结合,建立了AD-HF-LLLME-LVSS-CE/UV分析人发样品和环境水样中甲基汞、苯基汞和无机汞形态的新方法。该方法具有装置简单、自动化程度高、重现性好和样品基质净化能力强等优点；与静态萃取模式相比,AD-HF-LLLME的萃取动力学更快,所需萃取平衡时间更短,并且可获得较低的检出限和较高的富集倍数。
(7)提出了一种基于相转移原理的液相微萃取新技术-相转移液液液微萃取(PT-LLLME)。采用乙腈作为分散剂,DDA作为络合试剂,该技术成功实现了无机汞和有机汞的同时萃取,具有操作简单、萃取动力学快、萃取平衡时间短等优点,为拓展LPME在元素及其形态分析领域中的应用提供了一条新的思路。为实现PT-LLLME,设计了新的膜支撑-液液液微萃取(MS-LLLME)装置,该装置可允许大体积的接受相液滴,既可满足商用毛细管电泳仪自动进样的要求,又可以与HPLC等其他仪器大体积进样相匹配。据此,将PT/MS-LLLME与大体积样品堆积(LVSS)毛细管电泳紫外(CE/UV)检测联用,建立了PT/MS-LLLME-LVSS-CE/UV同时分析无机汞和有机汞形态的新方法,并将其成功的应用于复杂基质样品包括环境和生物样品的分析。
It has been recognized that the toxicity, bioavailability, transportation, metabolism, and other specific biological functions of a given element are highly dependent on its physicochemical forms in environmental and biological samples. Therefore, identification and quantification of different elemental species, termed as elemental speciation, are more important than the determination of its total content of an element. Capillary electrophoresis (CE) has been demonstrated to be one of the effective analytical techniques for elemental speciation due to the merits of low cost, low-sample consumption, high resolution, rapid separation and no need for derivatization. However, insufficient detection sensitivity and low tolerance of sample matrix have limited the application of CE in real sample analysis. To overcome such limitations, effective sample pretreatment techniques and on-column preconcentration methods are highly demanded. An appropriate sample pretreatment technique could isolate the target analytes from the sample matrix and concentrate the analytes simultaneously. In recent years, a variety of novel sample pretreatment techniques such as liquid phase microextraction (LPME), solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE) have been developing rapidly and gained great interest in analytical community. On the other hand, on-column preconcentration techniques such as staking, sweeping, dynamic pH junction and t-isotachophoresis are also excellent alternative methods to improve the detection sensitivity of CE, and they deserve special attention for their simple operation and high enrichment factor. However, to the best of our knowledge, the research on the combination of effective sample pretreatment techniques and/or on-column preconcentration methods with CE is very scarce until to present for elemental speciation.
The aim of this dissertation is to explore efficient on-column methods for elemental speciation and investigate the concentration mechanism; to develop new microextraction methods for the direct extraction of polar elemental species; and to establish new methods by combining microextraction techniques and/or on-column preconcentration methods with CE/UV detection for elemental speciation in environmental and biological samples. The major contents of this dissertation are described as follows:
(1) A novel method of polymer-coated hollow fiber microextraction (PC-HFME) combined with dynamic pH junction-CE/UV detection was developed for the speciation of four selenoamino acids. The enrichment resulted from dynamic pH junction is attributed to the migration velocity decreases of target analytes ions when migrating from a high-pH sample zone (pH 12.4) to a low-pH (pH 2.0) back ground electrolyte (BGE) buffer. In comparison to standard injection mode, the dynamic pH junction alone provided 430- to 590-fold improvement in terms of sensitivity. In PC-HFME, partially sulfonated polystyrene (PSP) coated hollow fibers were prepared for the simultaneous extraction of four selenoamino acids in acid media based on electrostatic interaction. With two-step enrichment procedure of dynamic pH junction and PC-HFME, enhancement factors (EFs) of 3171 to 19388 folds were obtained, and the limits of detections (LODs) (S/N=3) of 0.32-4.10μg/L were achieved for CE/UV determination of target selenoamino acids. The application potential of this method was successfully demonstrated by the analysis of selenoamino acids in environmental water samples.
(2) Zirconia (ZrO2) coated stir bar sorptive extraction (SBSE) coupled with on-column large volume sample injection (LVSS)-CE with indirect UV detection was developed for the direct analysis of strong polar chemical warfare agent degradation products of methylphosphonic acid (MPA), ethyl methylphosphonic acid (EMPA) and pinacolyl methylphosphonate (PMPA). Nanometer-sized ZrO2 coated stir bars were prepared by a PDMS adhesion method, and successfully applied in the extraction of the target strongly polar analytes due to the high affinity of ZrO2 to electronegative phosphonate group containing compounds. By combination of ZrO2-SBSE with LVSS-CE/ indirect UV, the LODs were found to be 1.4,1.2 and 3.1μg/L for PMPA, EMPA, and MPA, respectively. The reproducibility (RSD) was in the range of 9.0-11.8%. The EFs were up to 1583-fold. The proposed ZrO2-SBSE-LVSS-CE/indirect UV method has been applied to the analysis of three target analytes in different environmental water samples with recoveries ranging from 93.8% to 105.3%.
(3) Off-line hollow fiber liquid liquid liquid microextraction (HF-LLLME) combined with on-column anion selective exhaustive injection (ASEI)-CE/UV detection was proposed for the speciation of phenylarsenic compounds. By the use of tributyl phosphate (TBP) as the organic phase and 0.8 mmol/L Tris solution as acceptor phase in HF-LLLME, a simultaneous preconcentration of target analytes in pH 2.15 H2SO4 medium as the donor phase was realized. In ASEI, a large plug of water (91% length of total capillary) was introduced into the separation capillary before sample injection in order to prolong the sample injection time and thus enhance the stacking efficiency of ASEI. Under the optimized conditions, up to 236-fold of EF was obtained for the ASEI-CE/UV determination of target phenylarsenic compounds. Compared with reported LVSS technique, ASEI is more effective. By combining HF-LLLME with ASEI-CE/UV, the obtained LODs were in the range of 0.68-6.90μg/L for five phenylarsenic compounds with RSDs (n=5) of 5.6-11.8%. The proposed HF-LLLME-ASEI-CE/UV method was applied for the determination of target phenylarsenic compounds in pig feed, stored pig litter, soil and water samples obtained in a local pig farm.
(4) Taking four thyroid hormones including thyroxine (T4),3,5,3-triiodo-L-thyronine (T3), reverse 3,3,5-triiodo-L-thyronine (rT3) and 3,5-diiodo-L-thyronine dihydrate (T2), and two other related compounds of 3,5-diiodo-L-tyrosine (DIT) and 3-iodo-L-tyrosine (MIT) as the target analytes, a new method of ion pair based hollow fiber liquid-liquid-liquid microextraction (IP-HF-LLLME) combined with CE-UV detection was developed for the simultaneous determination of six target analytes in pharmaceutical formulations. The extraction was facilitated by adding sodium dodecyl sulfate (SDS) in the donor phase to form ion pairs with target analytes. The factors affecting the extraction efficiency of IP-HF-LLLME were optimized. Under the optimum conditions, the LODs (S/N=3) for six target analytes were in the range of 0.54-1.43μg/L with RSDs (n=7) ranging from 3.19 to 8.98%. The enrichment factors obtained by this method ranged from 183 to 366 folds. The proposed method is simple, inexpensive, high selective and sensitive for the simultaneous analysis of thyroid hormones and related compounds.
(5) U-shaped hollow fiber-based liquid-liquid-liquid microextraction (HF-LLLME) combined with large-volume sample stacking (LVSS)-CE/UV detection has been proposed for the speciation of organomercury in biological samples. In LVSS, a polarity switch mode was applied. In HF-LLLME, the analytes were extracted from 12 mL sample solution (pH 3.0) into an acceptor solution of L-cysteine (15μL,0.02% w/v) inside the hollow fiber through bromobenzene impregnated in the pores of the hollow fiber. Under the optimized conditions, EFs of 2610-4580 were achieved with the LODs in the range of 0.03-0.14μg/L. The developed method has been validated using a certified reference material (DORM-2, dogfish muscle), and the determined values coincided very well with the certified values. The developed method was also applied to the speciation of organomercury in real fish samples and human hair samples.
(6) A simple automatic dynamic hollow fiber based liquid liquid liquid (AD-HF-LLLME) device was designed for the mercury speciation by using a programmable flow injection analyser. With 18-crown-6 as the complexing reagent, mercury species including methyl-, ethyl-, phenyl- and inorganic mercury were first extracted into the organic phase (chlorobenzene), and then transferred into 0.1%(m/v) 3-mercapto-l-propanesulfonic acid (MPS) aqueous solution in the hollow fiber lumen as acceptor phase. However, ethylmercury was found to be partially decomposed during the AD-HF-LLLME process, and was not included in the developed method. Based on it, a new method of AD-HF-LLLME combined with LVSS-CE/UV detection was established for the simultaneous analysis of methyl-, phenyl- and inorganic mercury species in water and human hair samples.
(7) A novel extraction technique termed phase transfer based liquid-liquid-liquid microextraction (PT-LLLME) was proposed for the simultaneous extraction of inorganic mercury and three organic mercury species. To ensure a maximum contact between the target mercury species and the complexing reagent of dodecylamine (DDA) in donor phase, an intermediate solvent, which is miscible with water, was added into the sample solution. Furthermore, a membrane supported liquid-liquid-liquid phase microextraction (MS-LLLME) unit was designed. By using nylon membrane as supporting carrier, larger than 50μL of acceptor solution could be hung up in MS-LLLME unit. Parameters affecting the extraction efficiency of PT/MS-LLLME were investigated in details. Under the optimized conditions, EFs ranging from 160- to 478-fold were obtained for the mercury species by PT/MS-LLLME. The acceptor phase was directly injected into CE for LVSS-CE/UV analysis. By combination of PT/MS-LLLME with LVSS-CE/UV, the limits of detection (LODs) at lowμg/L level were achieved with the EFs up to 12138- fold. This newly established approach of PT/MS-LVSS-LLLME-CE/UV was successfully applied to simultaneous determination of inorganic and organic mercury species in biological samples and environmental water samples.
本论文的研究目的是,探索和研究适用于元素形态分析的在柱富集体系；开发和研究可直接萃取(非衍生)极性元素形态的固相微萃取和液相微萃取新体系和新方法；建立微萃取和/或在柱富集-CE/UV联用用于元素形态分析的新方法,并将其应用于环境和生物样品的分析。本论文的主要研究内容包括：
(1)建立了聚合物涂层中空纤维膜微萃取(PC-HFME)结合动态pH界面富集毛细管电泳紫外(CE/UV)检测分析环境水样中硒代氨基酸形态的新方法。对两种模式下动态pH界面在柱富集目标分析物的条件进行了优化,最后确定背景电解质(BGE)为极端酸性的磷酸缓冲溶液(pH 2.0),样品溶液基质为极端碱性的缓冲溶液(pH 12.4),样品进样量为分离毛细管总容量的90%。在此条件下,动态pH界面在柱富集目标分析物的富集倍数为430-590倍。将部分磺酸化的聚苯乙烯聚合物(PSP)涂覆在中空纤维膜上制备了PSP涂覆中空纤维膜,并将其应用于硒代氨基酸的萃取。这种PC-HFME-动态pH界面富集-CE/UV方法对硒代氨基酸的富集倍数为3171-18525倍,检出限在0.32-4.10μg/L之间。将该方法应用于自来水和东湖水样的分析,加标回收率在82.4%-106.5%的范围内。
(2)以PDMS粘附法制备了纳米Zr02涂层搅拌棒,并利用Zr02与含磷化合物之间的选择性作用,将制备的纳米Zr02涂层搅拌棒用于强极性化学战剂降解产物甲基磷酸(MPA)、甲基膦酸乙酯(EMPA和甲基膦酸片呐酯(PMPA)的直接搅拌棒吸附萃取(SBSE)。将ZrO2-SBSE与大体积样品堆积(LVSS)相结合,建立了ZrO2-SBSE-LVSS-毛细管电泳间接紫外(CE/indirect UV)分析实际环境水样中EMPA、PMPA和MPA的新方法。对影响ZrO2-SBSE萃取、解吸的因素以及影响LVSS堆积的条件分别进行了优化。在最优的萃取条件和堆积条件下,该方法对三种目标分析物的检出限在1.2-3.1μg/L之间,富集倍数为742-1583倍。该方法已成功应用于环境水样中目标分析物的分析,加标回收率在93.8%-105.3%之间。
(3)以磷酸三丁酯作为萃取溶剂,采用U型中空纤维膜-液液液微萃取(HF-LLLME)模式,实现了HF-LLLME对硫酸介质中五种极性苯砷酸类化合物的选择性萃取：提出一种阴离子耗尽进样(ASEI)堆积的新策略,即在样品电动进样前,先引入几乎整管的水柱(长度占91%的分离毛细管),从而在不改变分离度的情况下增加了样品溶液的最大进样量,提高了ASEI在柱富集苯砷酸类化合物的富集倍数。将HF-LLLME强的基质净化能力与ASEI高富集倍数的优点有机结合,建立了简便、快速、高灵敏的HF-LLLME-ASEI-CE/UV分析复杂环境样品中苯砷酸类化合物的新方法。该方法对目标分析物的检出限在0.68-6.9μg/L之间,富集倍数为155-1780倍,相对标准偏差在5.6-11.8%范围内。将所建立的方法用于当地某养猪场使用的浓缩猪饲料以及该养猪场附近环境中样品包括堆放的猪粪、耕地中的土壤、东湖水的分析并进行了加标回收实验。结果发现,该方法应用于浓缩猪饲料和东湖水中苯砷酸化合物分析的回收率在85-110%之间；堆放的猪粪样品的回收率在66.7-96.2%之间；土壤样品中各物质的回收率均低于50%。
(4)以SDS为离子对试剂,采用离子对-中空纤维膜-液液液微萃取(IP-HF-LLLME)成功实现了极性相差较大的四种甲状腺素化合物包括四碘甲状腺原氨酸(T4)、三碘甲状腺原氨酸(T3)、3,5-二碘-甲状腺素(T2)、3,3',5-三碘代-甲腺原氨酸(reverse T3, rT3)以及两种合成甲状腺素的原料3,5-二碘酪氨酸(DIT)和单碘酪氨酸(MIT)的同时萃取。据此,建立了简便、快速、高灵敏、高选择性的同时分析药物中六种目标分析物的IP-HF-LLLME-CE/UV新方法。对影响IP-HF-LLLME萃取效率的主要因素,如样品溶液pH值、有机溶剂、SDS浓度、萃取时间、搅拌速率以及盐效应等进行了系统考察。在最优的实验条件下,该方法分析六种目标分析物的检出限在0.54-1.43μg/L之间,富集倍数为183-366倍,相对标准偏差在3.19-8.98%范围内。将所建立的IP-HF-LLLME-CE/UV方法应用于两种药物中甲状腺素含量的测定,获得了满意的结果。
(5)以L-半胱氨酸作为反萃取试剂,采用U型中空纤维膜液液液微萃取(HF-LLLME)模式,实现了三种毒性较大的有机汞形态的萃取。将HF-LLLME与大体积样品堆积(LVSS)结合,建立了HF-LLLME-LVSS-CE/UV分析人发和海鱼样品中三种有机汞形态的新方法。对影响HF-LLLME的萃取条件和LVSS的电泳参数进行了优化,该方法分析甲基汞、乙基汞和苯基汞的检出限分别为0.03、0.07和0.14μg/L,富集倍数为2400-4300倍,线性范围达到两个数量级,其相关系数在0.9992-0.9999范围内。将该方法应用于标准参考物质DORM-2、人发样品和海鱼肌肉组织的分析,获得了满意的结果。该方法灵敏度高、选择性强、操作简单、分析仪器廉价,采用CE/UV可获得比CE-等离子体质谱(ICP/MS)更低的检出限水平,可应用于复杂基质样品中低含量的有机汞形态的分析。
(6)设计了简单的自动化动态中空纤维膜-液液液微萃取(AD-HF-LLLME)装置,该装置采用流动注射仪辅助,通过程序控制可实现上样、清洗、动态萃取步骤的自动化。采用18-冠-6作为给予相络合试剂,L-半胱氨酸作为接受相反萃取试剂实现了无机汞和有机汞的同时萃取。其中,实验发现在萃取过程中存在乙基汞形态转化为无机汞的现象。据此,将AD-HF-LLLME与大体积样品堆积(LVSS)技术相结合,建立了AD-HF-LLLME-LVSS-CE/UV分析人发样品和环境水样中甲基汞、苯基汞和无机汞形态的新方法。该方法具有装置简单、自动化程度高、重现性好和样品基质净化能力强等优点；与静态萃取模式相比,AD-HF-LLLME的萃取动力学更快,所需萃取平衡时间更短,并且可获得较低的检出限和较高的富集倍数。
(7)提出了一种基于相转移原理的液相微萃取新技术-相转移液液液微萃取(PT-LLLME)。采用乙腈作为分散剂,DDA作为络合试剂,该技术成功实现了无机汞和有机汞的同时萃取,具有操作简单、萃取动力学快、萃取平衡时间短等优点,为拓展LPME在元素及其形态分析领域中的应用提供了一条新的思路。为实现PT-LLLME,设计了新的膜支撑-液液液微萃取(MS-LLLME)装置,该装置可允许大体积的接受相液滴,既可满足商用毛细管电泳仪自动进样的要求,又可以与HPLC等其他仪器大体积进样相匹配。据此,将PT/MS-LLLME与大体积样品堆积(LVSS)毛细管电泳紫外(CE/UV)检测联用,建立了PT/MS-LLLME-LVSS-CE/UV同时分析无机汞和有机汞形态的新方法,并将其成功的应用于复杂基质样品包括环境和生物样品的分析。
It has been recognized that the toxicity, bioavailability, transportation, metabolism, and other specific biological functions of a given element are highly dependent on its physicochemical forms in environmental and biological samples. Therefore, identification and quantification of different elemental species, termed as elemental speciation, are more important than the determination of its total content of an element. Capillary electrophoresis (CE) has been demonstrated to be one of the effective analytical techniques for elemental speciation due to the merits of low cost, low-sample consumption, high resolution, rapid separation and no need for derivatization. However, insufficient detection sensitivity and low tolerance of sample matrix have limited the application of CE in real sample analysis. To overcome such limitations, effective sample pretreatment techniques and on-column preconcentration methods are highly demanded. An appropriate sample pretreatment technique could isolate the target analytes from the sample matrix and concentrate the analytes simultaneously. In recent years, a variety of novel sample pretreatment techniques such as liquid phase microextraction (LPME), solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE) have been developing rapidly and gained great interest in analytical community. On the other hand, on-column preconcentration techniques such as staking, sweeping, dynamic pH junction and t-isotachophoresis are also excellent alternative methods to improve the detection sensitivity of CE, and they deserve special attention for their simple operation and high enrichment factor. However, to the best of our knowledge, the research on the combination of effective sample pretreatment techniques and/or on-column preconcentration methods with CE is very scarce until to present for elemental speciation.
The aim of this dissertation is to explore efficient on-column methods for elemental speciation and investigate the concentration mechanism; to develop new microextraction methods for the direct extraction of polar elemental species; and to establish new methods by combining microextraction techniques and/or on-column preconcentration methods with CE/UV detection for elemental speciation in environmental and biological samples. The major contents of this dissertation are described as follows:
(1) A novel method of polymer-coated hollow fiber microextraction (PC-HFME) combined with dynamic pH junction-CE/UV detection was developed for the speciation of four selenoamino acids. The enrichment resulted from dynamic pH junction is attributed to the migration velocity decreases of target analytes ions when migrating from a high-pH sample zone (pH 12.4) to a low-pH (pH 2.0) back ground electrolyte (BGE) buffer. In comparison to standard injection mode, the dynamic pH junction alone provided 430- to 590-fold improvement in terms of sensitivity. In PC-HFME, partially sulfonated polystyrene (PSP) coated hollow fibers were prepared for the simultaneous extraction of four selenoamino acids in acid media based on electrostatic interaction. With two-step enrichment procedure of dynamic pH junction and PC-HFME, enhancement factors (EFs) of 3171 to 19388 folds were obtained, and the limits of detections (LODs) (S/N=3) of 0.32-4.10μg/L were achieved for CE/UV determination of target selenoamino acids. The application potential of this method was successfully demonstrated by the analysis of selenoamino acids in environmental water samples.
(2) Zirconia (ZrO2) coated stir bar sorptive extraction (SBSE) coupled with on-column large volume sample injection (LVSS)-CE with indirect UV detection was developed for the direct analysis of strong polar chemical warfare agent degradation products of methylphosphonic acid (MPA), ethyl methylphosphonic acid (EMPA) and pinacolyl methylphosphonate (PMPA). Nanometer-sized ZrO2 coated stir bars were prepared by a PDMS adhesion method, and successfully applied in the extraction of the target strongly polar analytes due to the high affinity of ZrO2 to electronegative phosphonate group containing compounds. By combination of ZrO2-SBSE with LVSS-CE/ indirect UV, the LODs were found to be 1.4,1.2 and 3.1μg/L for PMPA, EMPA, and MPA, respectively. The reproducibility (RSD) was in the range of 9.0-11.8%. The EFs were up to 1583-fold. The proposed ZrO2-SBSE-LVSS-CE/indirect UV method has been applied to the analysis of three target analytes in different environmental water samples with recoveries ranging from 93.8% to 105.3%.
(3) Off-line hollow fiber liquid liquid liquid microextraction (HF-LLLME) combined with on-column anion selective exhaustive injection (ASEI)-CE/UV detection was proposed for the speciation of phenylarsenic compounds. By the use of tributyl phosphate (TBP) as the organic phase and 0.8 mmol/L Tris solution as acceptor phase in HF-LLLME, a simultaneous preconcentration of target analytes in pH 2.15 H2SO4 medium as the donor phase was realized. In ASEI, a large plug of water (91% length of total capillary) was introduced into the separation capillary before sample injection in order to prolong the sample injection time and thus enhance the stacking efficiency of ASEI. Under the optimized conditions, up to 236-fold of EF was obtained for the ASEI-CE/UV determination of target phenylarsenic compounds. Compared with reported LVSS technique, ASEI is more effective. By combining HF-LLLME with ASEI-CE/UV, the obtained LODs were in the range of 0.68-6.90μg/L for five phenylarsenic compounds with RSDs (n=5) of 5.6-11.8%. The proposed HF-LLLME-ASEI-CE/UV method was applied for the determination of target phenylarsenic compounds in pig feed, stored pig litter, soil and water samples obtained in a local pig farm.
(4) Taking four thyroid hormones including thyroxine (T4),3,5,3-triiodo-L-thyronine (T3), reverse 3,3,5-triiodo-L-thyronine (rT3) and 3,5-diiodo-L-thyronine dihydrate (T2), and two other related compounds of 3,5-diiodo-L-tyrosine (DIT) and 3-iodo-L-tyrosine (MIT) as the target analytes, a new method of ion pair based hollow fiber liquid-liquid-liquid microextraction (IP-HF-LLLME) combined with CE-UV detection was developed for the simultaneous determination of six target analytes in pharmaceutical formulations. The extraction was facilitated by adding sodium dodecyl sulfate (SDS) in the donor phase to form ion pairs with target analytes. The factors affecting the extraction efficiency of IP-HF-LLLME were optimized. Under the optimum conditions, the LODs (S/N=3) for six target analytes were in the range of 0.54-1.43μg/L with RSDs (n=7) ranging from 3.19 to 8.98%. The enrichment factors obtained by this method ranged from 183 to 366 folds. The proposed method is simple, inexpensive, high selective and sensitive for the simultaneous analysis of thyroid hormones and related compounds.
(5) U-shaped hollow fiber-based liquid-liquid-liquid microextraction (HF-LLLME) combined with large-volume sample stacking (LVSS)-CE/UV detection has been proposed for the speciation of organomercury in biological samples. In LVSS, a polarity switch mode was applied. In HF-LLLME, the analytes were extracted from 12 mL sample solution (pH 3.0) into an acceptor solution of L-cysteine (15μL,0.02% w/v) inside the hollow fiber through bromobenzene impregnated in the pores of the hollow fiber. Under the optimized conditions, EFs of 2610-4580 were achieved with the LODs in the range of 0.03-0.14μg/L. The developed method has been validated using a certified reference material (DORM-2, dogfish muscle), and the determined values coincided very well with the certified values. The developed method was also applied to the speciation of organomercury in real fish samples and human hair samples.
(6) A simple automatic dynamic hollow fiber based liquid liquid liquid (AD-HF-LLLME) device was designed for the mercury speciation by using a programmable flow injection analyser. With 18-crown-6 as the complexing reagent, mercury species including methyl-, ethyl-, phenyl- and inorganic mercury were first extracted into the organic phase (chlorobenzene), and then transferred into 0.1%(m/v) 3-mercapto-l-propanesulfonic acid (MPS) aqueous solution in the hollow fiber lumen as acceptor phase. However, ethylmercury was found to be partially decomposed during the AD-HF-LLLME process, and was not included in the developed method. Based on it, a new method of AD-HF-LLLME combined with LVSS-CE/UV detection was established for the simultaneous analysis of methyl-, phenyl- and inorganic mercury species in water and human hair samples.
(7) A novel extraction technique termed phase transfer based liquid-liquid-liquid microextraction (PT-LLLME) was proposed for the simultaneous extraction of inorganic mercury and three organic mercury species. To ensure a maximum contact between the target mercury species and the complexing reagent of dodecylamine (DDA) in donor phase, an intermediate solvent, which is miscible with water, was added into the sample solution. Furthermore, a membrane supported liquid-liquid-liquid phase microextraction (MS-LLLME) unit was designed. By using nylon membrane as supporting carrier, larger than 50μL of acceptor solution could be hung up in MS-LLLME unit. Parameters affecting the extraction efficiency of PT/MS-LLLME were investigated in details. Under the optimized conditions, EFs ranging from 160- to 478-fold were obtained for the mercury species by PT/MS-LLLME. The acceptor phase was directly injected into CE for LVSS-CE/UV analysis. By combination of PT/MS-LLLME with LVSS-CE/UV, the limits of detection (LODs) at lowμg/L level were achieved with the EFs up to 12138- fold. This newly established approach of PT/MS-LVSS-LLLME-CE/UV was successfully applied to simultaneous determination of inorganic and organic mercury species in biological samples and environmental water samples.
引文
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