等离子体光谱联用技术在元素形态分析中的研究与应用
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摘要
电感耦合等离子体光谱(ICP-AES/MS)以其检测限低、精密度高、选择性好、基体效应小、线性范围宽和多元素同时测定等优点得到了广泛应用。但是随着科学的发展仅靠ICP-AES/MS常规检测已不能满足研究的要求,将ICP-AES/MS与其它分析手段联用起来可以在一定程度上将其优点与其它分析手段结合起来,甚至可以克服它们的缺点进而提高分析性能,尽量满足分析需求。
氢化物发生与ICP-AES联用技术扩展了ICP-AES对氢化物形成元素的检测能力,可使氢化物形成元素的检出限改善1～2个数量级,并且几乎消除了基体干扰,从而开辟了解决复杂基体样品分析新途径。
高效毛细管电泳具有分离效率高、分析速度快、分析模式多、试剂用量少、应用范围广、易于自动化等优点,在生命科学、生物技术、临床医学、药物分析和环境科学等领域显示了极其重要的应用前景。毛细管电泳与电感耦合等离子体光谱联用应用于元素形态分析是一种很有发展前途的分离检测方法。它可以广泛应用于环保、食品、生物、临床和中医中药等领域,也是目前研究热点——金属组学的主要方法之一。我们在测定微量元素总量的同时,应该注意到一种元素的生理、毒理影响以及生物可给性、环境行为和迁移性在很大程度上取决于它的化学形态。形态分析已成为当前环境科学、生物化学和生命科学领域中颇为活跃的前沿性课题。把氢化物发生(HG)技术引入毛细管电泳-电感耦合等离子体光谱联用中,可使分析手段的检出限得到进一步的改善。
本论文就电感耦合等离子体原子发射光谱与氢化物发生和毛细管电泳联用方法的建立及其在元素检测和形态分析中的应用进行了研究,论文的主要内容及研究结果如下:
1、最先采用超声波对大鼠红细胞进行破壁、毛细管电泳电感耦合等离子体原子发射光谱联用技术分析大鼠红细胞中钙的形态。在红细胞中加入游离钙离子以及单独对游离钙离子进行电泳分离和ICP-AES检测,对大鼠红细胞中游离钙离子进行了表征和定量。比较了SDS和超声波两种破溶红细胞方法对钙形态分析影响,结果显示选择超声波破壁更好。考察了电泳缓冲溶液浓度、pH、电泳电压及进样时间对实验的影响。红细胞样品中Ca的形态主要有七种,用峰高来定量红细胞中游离钙的含量为0.25mmol/L,其相对标准偏差为2.34%,采用以上联用技术对红细胞样品中Ca进行了加标回收测定,其加标回收率为83.2%～89.6%。
2、首次用毛细管电泳和电感耦合等离子体原子发射光谱联用技术(CE-ICP-AES)分析核酸中DNA的和RNA。考察了ICP-AES检测P分析线的选择,探讨了运行缓冲溶液、分离电压及进样时间等对毛细管电泳-ICP-AES检测的影响,得到了较为优化的测定条件。在最佳优化条件下,RNA和DNA的峰高相对标准偏差(RSD)分别为2.03%和2.21%,迁移时间的RSD分别为1.61%和1.94%,RNA和DNA的检出限分别为53.68mg/L和79.80mg/L。
3、自行设计了CE-在线氧化还原-HG-ICP-AES接口,并将该接口应用于大鼠血浆中硒代蛋氨酸的测定。探讨了电泳缓冲液的选择、酸度和硼氢化钠对Se(IV)产生氢化物的影响,考察了载气流速对硒氢化物产生的影响,得到了实验的优化条件。在实验优化条件下,相对标准偏差(RSD)为5.3%,硒代蛋氨酸检出限为6.71μg/L。
4、建立了微波消解连续氢化物发生-电感耦合等离子体原子发射光谱法同时测定牛奶中微量元素砷、硒的新方法。对等离子体工作参数和氢化物发生条件等进行了优化选择,研究了酸介质种类及浓度,硼氢化钠浓度,载气流速和试样提升量等因素对测定结果的影响。在最佳的实验条件下,砷、硒的检出限分别为0.61μg·L-1、0.50μg·L-1;相对标准偏差1.35%、1.33%;加标回收率分别为98%和101%。
Inductively coupled plasma Spectrometry (ICP-AES/MS) is a advanced multi-elements trace analysis technology.It offers extending good advantages in terms of low detection limit, high precision, good selectivity, little matrix effect, wide linear range and simultaneous multi-elements determination, so it is widely used. But the routine determination of ICP-AES/MS couldn’t meet the research demand with scientific development. The hyphenating of ICP-AES/MS with analytical instruments else would combine strongpoints of them to a certain extent and so much as improves analytical capability for conquering their shortcomings so that it meets analytical demand to the best.
Hydride Generation-Inductively Coupled Plasma Atomic Emission Spectrometry (HG-ICP-AES) enhance detection power of ICP-AES to hydride-forming elements. The detection limits of hydride-forming elements can be improved by one or two orders of magnitude over conventional nebulizer, and nearly eliminated matrix spectrum interference. Therefore, it provides a new way to solve complex matrix interference and speciation analysis .
Capillary electrophoresis (CE) has many advantages: high efficiency , quick analysis speed , many analysis modes, little reagent consumption, wide application, easy automation, etc. Thus it shows extremely important application to life sciences, biotechnology, clinical medicine, medicine analysis and environmental science and so on. The hyphenating of capillary electrophoresis and inductively coupling plasma spectrometry applying to element speciation analysis is a kind of rising separation and detection method. It can be widely applied to environmental protection, food, biology, clinic and Chinese Traditional Medicine (CTM), etc. We should be conscious of that the physiology, toxicity effect, biogeochemical behavior, environmental behavior and transformation of a element are highly depended on their chemical speciation to some great extent when determining the total amount of trace elements. Speciation analysis has already become an active frontier in environmental sciences, biochemistry and life sciences at present. When HG coupled with CE-ICP, the detection limits of analytical method can be improved.
In this paper, the research has been carried on foundation of hyphenating of inductively coupling plasma atomic emission spectrometry with hydride generation and capillary electrophoresis. The application of coupling method to determination and speciation analysis of elements has been studied, too. The dominating contents and researching results of the paper are as follows:
1:Using ultrasonic wave to bread red blood cell, the speciation of calcium in red blood cell was studied by capillary electrophoresis-inductively coupled plasma atomic emission spectrometry. The speciation of free calcium in red blood cell was identified using free standard Ca2+.Comparing the breading effect of SDS with ultrasonic wave, the ultrasonic method is more efficient than that of SDS. The effects of running buffer concentration and pH was researched, separation voltage and injection time on the CE-ICP-AES determination were optimized. The forms of calcium in rat red blood cells have seven different species, and quantification of free calcium concentration by peak height which was 0.25 mmol/L. The precisions (relative standard deviation, RSD, n=11) was 2.34%. The recoveries of the free calcium species in red blood cells were 83.2% to 89.6%.
2:The separation of DNA and RNA using on-line coupling capillary electrophoresis (CE) with inductively coupled plasma atomic emission spectrometry(ICP-AES) was reported in the paper for the first time. The wavelength of P detected using ICP-AES was researched. The effects of running buffer, separation voltage and injection time on the CE-ICP-AES determination were investigated. The optimal condition was received. The relative standard deviation (RSD) of peak height of RNA and DNA was 2.03% and 2.21%, RSD of migration time for 1.61% and 1.94%, Detection limit RNA and DNA for 53.68mg/L and 79.80mg/L, respectively.
3:An interface for CE- on-line redox-HG-ICP-AES has been designed. It was applied to determination of selenomethionine in rat plasma. The effects of separation buffer pH, the acidity of HCl, NaBH4 concentration and nebulizer flow rate on Se hydride signal intensity were discussed. The optimal condition has been gained. The relative standard deviation (RSD) was 5.3%, Detection limit of selenomethionine was 6.71μg/L.
4:A new method was proposed for simultaneous determination of trace arsenic and selenium in milk by microwave-assisted digestion-HG-ICP-AES. The operation condition of hydride generation and inductively coupled plasma atomic emission spectrometry was investigated and optimized. The effects of acid concentration, NaBH4 concentration, nebulizer flow rate and sample volume was discussed. Under the optimal condition, the detection limits of arsenic and selenium are 0.61μg·L-1 and 0.50μg·L-1,respectively. The relative standard deviations are 1.35% and 1.33%, respectively. The recovery are 98% and 101%, respectively.
氢化物发生与ICP-AES联用技术扩展了ICP-AES对氢化物形成元素的检测能力,可使氢化物形成元素的检出限改善1～2个数量级,并且几乎消除了基体干扰,从而开辟了解决复杂基体样品分析新途径。
高效毛细管电泳具有分离效率高、分析速度快、分析模式多、试剂用量少、应用范围广、易于自动化等优点,在生命科学、生物技术、临床医学、药物分析和环境科学等领域显示了极其重要的应用前景。毛细管电泳与电感耦合等离子体光谱联用应用于元素形态分析是一种很有发展前途的分离检测方法。它可以广泛应用于环保、食品、生物、临床和中医中药等领域,也是目前研究热点——金属组学的主要方法之一。我们在测定微量元素总量的同时,应该注意到一种元素的生理、毒理影响以及生物可给性、环境行为和迁移性在很大程度上取决于它的化学形态。形态分析已成为当前环境科学、生物化学和生命科学领域中颇为活跃的前沿性课题。把氢化物发生(HG)技术引入毛细管电泳-电感耦合等离子体光谱联用中,可使分析手段的检出限得到进一步的改善。
本论文就电感耦合等离子体原子发射光谱与氢化物发生和毛细管电泳联用方法的建立及其在元素检测和形态分析中的应用进行了研究,论文的主要内容及研究结果如下:
1、最先采用超声波对大鼠红细胞进行破壁、毛细管电泳电感耦合等离子体原子发射光谱联用技术分析大鼠红细胞中钙的形态。在红细胞中加入游离钙离子以及单独对游离钙离子进行电泳分离和ICP-AES检测,对大鼠红细胞中游离钙离子进行了表征和定量。比较了SDS和超声波两种破溶红细胞方法对钙形态分析影响,结果显示选择超声波破壁更好。考察了电泳缓冲溶液浓度、pH、电泳电压及进样时间对实验的影响。红细胞样品中Ca的形态主要有七种,用峰高来定量红细胞中游离钙的含量为0.25mmol/L,其相对标准偏差为2.34%,采用以上联用技术对红细胞样品中Ca进行了加标回收测定,其加标回收率为83.2%～89.6%。
2、首次用毛细管电泳和电感耦合等离子体原子发射光谱联用技术(CE-ICP-AES)分析核酸中DNA的和RNA。考察了ICP-AES检测P分析线的选择,探讨了运行缓冲溶液、分离电压及进样时间等对毛细管电泳-ICP-AES检测的影响,得到了较为优化的测定条件。在最佳优化条件下,RNA和DNA的峰高相对标准偏差(RSD)分别为2.03%和2.21%,迁移时间的RSD分别为1.61%和1.94%,RNA和DNA的检出限分别为53.68mg/L和79.80mg/L。
3、自行设计了CE-在线氧化还原-HG-ICP-AES接口,并将该接口应用于大鼠血浆中硒代蛋氨酸的测定。探讨了电泳缓冲液的选择、酸度和硼氢化钠对Se(IV)产生氢化物的影响,考察了载气流速对硒氢化物产生的影响,得到了实验的优化条件。在实验优化条件下,相对标准偏差(RSD)为5.3%,硒代蛋氨酸检出限为6.71μg/L。
4、建立了微波消解连续氢化物发生-电感耦合等离子体原子发射光谱法同时测定牛奶中微量元素砷、硒的新方法。对等离子体工作参数和氢化物发生条件等进行了优化选择,研究了酸介质种类及浓度,硼氢化钠浓度,载气流速和试样提升量等因素对测定结果的影响。在最佳的实验条件下,砷、硒的检出限分别为0.61μg·L-1、0.50μg·L-1;相对标准偏差1.35%、1.33%;加标回收率分别为98%和101%。
Inductively coupled plasma Spectrometry (ICP-AES/MS) is a advanced multi-elements trace analysis technology.It offers extending good advantages in terms of low detection limit, high precision, good selectivity, little matrix effect, wide linear range and simultaneous multi-elements determination, so it is widely used. But the routine determination of ICP-AES/MS couldn’t meet the research demand with scientific development. The hyphenating of ICP-AES/MS with analytical instruments else would combine strongpoints of them to a certain extent and so much as improves analytical capability for conquering their shortcomings so that it meets analytical demand to the best.
Hydride Generation-Inductively Coupled Plasma Atomic Emission Spectrometry (HG-ICP-AES) enhance detection power of ICP-AES to hydride-forming elements. The detection limits of hydride-forming elements can be improved by one or two orders of magnitude over conventional nebulizer, and nearly eliminated matrix spectrum interference. Therefore, it provides a new way to solve complex matrix interference and speciation analysis .
Capillary electrophoresis (CE) has many advantages: high efficiency , quick analysis speed , many analysis modes, little reagent consumption, wide application, easy automation, etc. Thus it shows extremely important application to life sciences, biotechnology, clinical medicine, medicine analysis and environmental science and so on. The hyphenating of capillary electrophoresis and inductively coupling plasma spectrometry applying to element speciation analysis is a kind of rising separation and detection method. It can be widely applied to environmental protection, food, biology, clinic and Chinese Traditional Medicine (CTM), etc. We should be conscious of that the physiology, toxicity effect, biogeochemical behavior, environmental behavior and transformation of a element are highly depended on their chemical speciation to some great extent when determining the total amount of trace elements. Speciation analysis has already become an active frontier in environmental sciences, biochemistry and life sciences at present. When HG coupled with CE-ICP, the detection limits of analytical method can be improved.
In this paper, the research has been carried on foundation of hyphenating of inductively coupling plasma atomic emission spectrometry with hydride generation and capillary electrophoresis. The application of coupling method to determination and speciation analysis of elements has been studied, too. The dominating contents and researching results of the paper are as follows:
1:Using ultrasonic wave to bread red blood cell, the speciation of calcium in red blood cell was studied by capillary electrophoresis-inductively coupled plasma atomic emission spectrometry. The speciation of free calcium in red blood cell was identified using free standard Ca2+.Comparing the breading effect of SDS with ultrasonic wave, the ultrasonic method is more efficient than that of SDS. The effects of running buffer concentration and pH was researched, separation voltage and injection time on the CE-ICP-AES determination were optimized. The forms of calcium in rat red blood cells have seven different species, and quantification of free calcium concentration by peak height which was 0.25 mmol/L. The precisions (relative standard deviation, RSD, n=11) was 2.34%. The recoveries of the free calcium species in red blood cells were 83.2% to 89.6%.
2:The separation of DNA and RNA using on-line coupling capillary electrophoresis (CE) with inductively coupled plasma atomic emission spectrometry(ICP-AES) was reported in the paper for the first time. The wavelength of P detected using ICP-AES was researched. The effects of running buffer, separation voltage and injection time on the CE-ICP-AES determination were investigated. The optimal condition was received. The relative standard deviation (RSD) of peak height of RNA and DNA was 2.03% and 2.21%, RSD of migration time for 1.61% and 1.94%, Detection limit RNA and DNA for 53.68mg/L and 79.80mg/L, respectively.
3:An interface for CE- on-line redox-HG-ICP-AES has been designed. It was applied to determination of selenomethionine in rat plasma. The effects of separation buffer pH, the acidity of HCl, NaBH4 concentration and nebulizer flow rate on Se hydride signal intensity were discussed. The optimal condition has been gained. The relative standard deviation (RSD) was 5.3%, Detection limit of selenomethionine was 6.71μg/L.
4:A new method was proposed for simultaneous determination of trace arsenic and selenium in milk by microwave-assisted digestion-HG-ICP-AES. The operation condition of hydride generation and inductively coupled plasma atomic emission spectrometry was investigated and optimized. The effects of acid concentration, NaBH4 concentration, nebulizer flow rate and sample volume was discussed. Under the optimal condition, the detection limits of arsenic and selenium are 0.61μg·L-1 and 0.50μg·L-1,respectively. The relative standard deviations are 1.35% and 1.33%, respectively. The recovery are 98% and 101%, respectively.
引文
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