毛细管电泳—电化学检测联用技术及其应用研究
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摘要
毛细管电泳(Capillary Electrophoresis,CE)是近二十年来发展最快的分离分析技术之一,是分析科学中继高效液相色谱之后的又一重大进展,进样量从微升水平进入纳升水平。作为一种经典电泳技术与现代微柱分离有机结合的新兴分离技术,其研究和应用涉及环境分析、药物分离、生化分析等几乎所有的分析领域。极细的毛细管内径带来了很高的分离效能,但同时也给样品组分的检测带来困难,因此对检测技术相应提出了较高的要求,发展了许多类型的检测器,有光学、电化学、质谱学检测器及化学发光等。电化学检测中的安培检测技术,具有比紫外检测更高的灵敏度,且仪器简单、价格成本低、线性范围宽、操作简便,因而其与毛细管电泳联用后在分析化学领域得到了广泛的研究和应用,研究对象大到生命大分子,小到单细胞分析和无机离子分析。
近年来,随着人们对生活质量的关注,环境分析和药物分析也成为分析化学研究的重点和热点。本论文探讨了毛细管电泳—安培检测联用技术在环境分析、药物分析等领域中的一些应用,主要内容分为以下六章:
第一章绪论
简单地概述了毛细管电泳(CE)的特点、分离模式、理论基础、联用的检测器、发展趋势以及毛细管电泳安培检测的理论研究和应用现状等,并简单介绍了本论文的目的和意义。
第二章毛细管电泳安培检测分离测定硝基苯胺位置异构体的研究
采用简单的CZE分离模式,不加任何添加剂,在极端pH值条件下,成功地分离了环境中硝基苯胺类化合物。研究了背景缓冲溶液、pH值、分离电压及进样时间等因素对分离的影响,在选定的实验条件下18 min内实现了3种苯胺类化合物的分离,得到分析物的标准曲线、线性范围及加样回收率,同时测定了2种染料废水样品中被测物的含量。结果表明该方法快速,准确,重现性好。
第三章毛细管电泳安培检测联用检测印染废水中的硝基苯酚异构体
本文利用毛细管电泳—安培检测联用技术,通过在缓冲液中添加有机溶剂,成功地实现了硝基酚异构体的分离。研究了不同有机溶剂对分离效果的影响,发现10%的甲醇作添加剂能实现很好的电泳分离。探讨了影响分离的一些实验条件,得出了三种样品的标准曲线、线性范围及加样回收率。该方法简便、快速、重现性好,并直接用于染料废水的测定,取得了满意的结果。
第四章毛细管区带电泳-安培检测联用检测地锦草中的活性成分
对中药地锦草中的五种活性成分芦丁、木樨草素、山奈酚、芹菜素和没食子酸进行研究。首次采用毛细管电泳-安培检测法成功地分离了地锦草中的芦丁、木樨草素、山奈酚、芹菜素和没食子酸,并优化了分离和检测条件。在本实验最佳优化条件下,分析物质在pH 8.8,20 mmol/L的硼砂缓冲溶液中20 min内实现了完全快速地分离;采用碳圆盘电极,所有待测物在工作电极电位为+0.95 V(vs.SCE)下有良好的响应;线性范围在信噪比为3下跨越三个数量级。本方法简单快速、灵敏度高,可用于地锦草的定性和定量的研究,分析结果可靠,令人满意。
第五章毛细管电泳安培检测联用测定中草药杠板归中的活性成分
采用毛细管电泳-电化学检测法(CE-ED)同时分离测定了中药杠板归中的阿魏酸、香草酸、槲皮素、咖啡酸、原儿茶酸等主要生物活性成分的含量。考察了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间等实验参数对实验结果的影响。在优化的实验条件下,以直径300μm的碳圆盘电极为工作电极,检测电位为+0.95V(vs.SCE),在10mmol/L磷酸盐(pH 9.2)的运行缓冲溶液中,五个分析物能够在17min内实现很好的基线分离,被测物浓度与峰电流在3个数量级呈良好的线性,检测限(S/N=3)范围从7.1×10~(-8)到9.3×10~(-8)g/mL。该方法已应用于实际样品的分析,样品处理简单,获得了令人满意的结果。
第六章毛细管电泳-电化学检测联用测定动物组织中的β-激动剂
本文采用毛细管电泳—电化学检测技术,通过在缓冲液中添加有机溶剂,成功地分离了四种β—激动剂:盐酸克伦特罗、硫酸特布他林、盐酸莱克多巴胺和硫酸沙丁胺醇,并考察了缓冲液酸度和浓度、分离电压、氧化电位和进样时间等对分离检测的影响。得出最佳实验条件如下:工作电极为直径300μm的碳圆盘电极,检测电位为+0.95V(vs.SCE),缓冲液为60 mmol/L硼砂溶液(pH=8.7),分离电压17 kV,该方法已成功地应用于实际样品中β—激动剂残留状况的分析,结果令人满意。
Capillary Electrophoresis(CE) is one of the most important and rapidly growing separation techniques in recent twenty years,It absorbed the virtues of both the conventional electrophoresis and modem High Performance Liquid Chromatography (HPLC).However,the theoretical plate number is great developed to tens of thousands and even millions and the volume Of sample injection reaches nano milliliter level in CE analysi.In recent years,capillary electrophoresis(CE) has been extensively studied and applied as a highly effective analytical method in modern analysis.And excellent prospects of CE have been shown in the fields of pharmaceutical analysis,biochemical analysis,food analysis,and environmental analysis.It has many advantages such as much lower sampling volume,cheaper instrumentation and higher separation efficiency.However,the small sample injection volume and very thin capillary bring about difficulties to detections.The common used detection methods are Ultra-violet(UV),Laser Induced Fluorescence(LIF), Luminescence Detection,Mass Spectra(MS).Because amperometric detector(AD) is more sensitive than UV-visible detector and much cheaper than LIF detector,it is preferred to couple with CE in many analysis areas.
Recently,with more attention paid to the quality of life,the analysis in environment and medicine field has become a hotspot of the analytical chemistry research.The goal of this dissertation is to explore some applications in environmental analysis,pharmaceutical analysis and food analysis etc.by CE-ED. The contents of this dissertation include six chapters:
Chapter 1.Preface
The characteristics of CE,the separation models,the basic theories of CE,the detectors,the studies and applications of CE-AD are introduced briefly.The goal and significance of this dissertation are also introduced.
Chapter 2.Separation and determination of nitroaniline isomers by capillary zone electrophoresis with amperometric detection
Capillary zone electrophoresis with amperometric detection(CZE-AD) was firstly applied to the simultaneous separation and determination of nitroaniline positional isomers.The three analytes could be perfectly analyzed by using the buffer of extreme pH.The effects of several important factors were investigated to find optimum conditions.A carbon-disk electrode was used as working electrode.The optimal conditions were 40 mmol/L tartaric acid-sodium tartrate(pH 1.2) as running buffer, 17 kV as separation voltage and 1.10 V(versus saturated calomel reference electrode, SCE) as detection potential.Under the optimum conditions,o-,m- and p-nitroaniline were separated successfully and good linearity,reproducibility and recovery results were obtained.The detection limit for m-nitroaniline was as low as at 9.06×10~(-9) mol/L. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 1.8%for migration time and 1.1%for peak areas.The utility of this method was demonstrated by monitoring dyestuff wastewater and the assay results were satisfactory.
Chapter 3.Separation and determination of nitrophenol isomers by capillary electrophoresis with amperometric detection
In this paper,capillary zone electrophoresis with amperometric detection(CZE-AD) was applied to determine the nitrophenol isomers in dyestuff wastewater.The analytes could be perfectly separated by only adjusting the run buffer with little organic modifier.The effect of certain percentage of organic solvents present in the electrophoretic buffer on the separation of three phenols was investigated.10%(V/V) methanol and the same level of 1:1(V/V) methanol-acetonitrile mixture were found equally effective for sensitivity enhancement in detection and efficiency,but 10%methanol solvent may offer better baseline and peak shapes. The detection limit was as low as 7.50×10~(-8) mol/L,which was much lower than that in CZE-UV method.Analyzing wastewater by this method has some merits of speed,simple instrumentation and operation,high sensitivity and high reproducibility.The experimental results showed that this CZE-AD method was very practical to determine analogous substances in Environmental Monitoring.
Chapter 4.Simultaneous determination of active ingredients in Herba Euphorbiae Humifusae by capillary zone eleetrophoresis with amperometric detection
In this chapter,the determination of active ingredients(rutin,quercatin,gallic acid and chlorogenic acid) of Euphorbia Humifusa Willd by capillary electrophoresis with amperometric detection were studied.CE-AD was successfully used to separate and detect the five active ingrediences for the first time,and the experiment conditions were optimized.Under the optimum conditions,the analytes could be separated in a 20 mmol/L borate buffer(pH=8.8) within 20 min.A 300μm diameter carbon disk electrode has a good response at +0.95 V(vs.SCE) for all analytes.The response was linear over three orders of magnitude with detection limits(S/N=3).The method could be used in the rapid determination of practical samples.
Chapter 5.Determination of bioactive components in Polygonum perfoliatum L.by Capillary Electrophoresis with Electrochemical Detection
The major bioactive components in a Chinese herb named Polygonum perfoliatum L.including ferulic acid,vanillic acid,quercetin,caffeic acid and protocatechuic acid were simultaneously determined by capillary electrophoresis with electrochemical detection(CE-ED) in this paper.The effects of working electrode potential,pH and concentration of running buffer,separation voltage,and injection time on CE-ED were investigated.Under the optimum conditions,the five analytes could be separated within 17 min at a separation voltage of 18 kV in 10 mmol/L phosphate buffer(pH 9.2).A 300μm diameter carbon disk electrode generated good responses at +0.95V (vs.SCE) for the five analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) ranging from 7.1×10~(-8) to 9.3×10~(-8)g/mL for the analytes.This proposed method could be successfully applied to the analysis of the real samples with relatively simple extraction procedures,and the assay results were satisfactory.
Chapter 6.Separation and Determination ofβ-Agonists in Animal Tissue by Capillary Electrophoresis with Electrochemical Detection.
A high-performance capillary electrophoresis with electrochemical detection (CE-ED) method had been applied for the determination ofβ-agonists,Clenbuterol hydrochloride,Terbutaline sulfate,Ractopamine hydrochloride and Salbutamol sulfate,in meat produces.The effects of several factors such as the acidity and concentration of running buffer,the separation voltage,the applied potential and the injection time on CE-ED were investigated.Under the optimum conditions,these four components could be separated successfully at the separation voltage of 17 kV in a 60 mmol/L borax running buffer(pH 8.7).A 300μm diameter carbon disk electrode was used as the working electrode positioned carefully opposite the outlet of the capillary in a wall-jet configuration at potential of +0.95V(vs.SCE).The method had been successfully applied to analyzeβ-agonists in real-life samples,and the assay results were satisfactory.
近年来,随着人们对生活质量的关注,环境分析和药物分析也成为分析化学研究的重点和热点。本论文探讨了毛细管电泳—安培检测联用技术在环境分析、药物分析等领域中的一些应用,主要内容分为以下六章:
第一章绪论
简单地概述了毛细管电泳(CE)的特点、分离模式、理论基础、联用的检测器、发展趋势以及毛细管电泳安培检测的理论研究和应用现状等,并简单介绍了本论文的目的和意义。
第二章毛细管电泳安培检测分离测定硝基苯胺位置异构体的研究
采用简单的CZE分离模式,不加任何添加剂,在极端pH值条件下,成功地分离了环境中硝基苯胺类化合物。研究了背景缓冲溶液、pH值、分离电压及进样时间等因素对分离的影响,在选定的实验条件下18 min内实现了3种苯胺类化合物的分离,得到分析物的标准曲线、线性范围及加样回收率,同时测定了2种染料废水样品中被测物的含量。结果表明该方法快速,准确,重现性好。
第三章毛细管电泳安培检测联用检测印染废水中的硝基苯酚异构体
本文利用毛细管电泳—安培检测联用技术,通过在缓冲液中添加有机溶剂,成功地实现了硝基酚异构体的分离。研究了不同有机溶剂对分离效果的影响,发现10%的甲醇作添加剂能实现很好的电泳分离。探讨了影响分离的一些实验条件,得出了三种样品的标准曲线、线性范围及加样回收率。该方法简便、快速、重现性好,并直接用于染料废水的测定,取得了满意的结果。
第四章毛细管区带电泳-安培检测联用检测地锦草中的活性成分
对中药地锦草中的五种活性成分芦丁、木樨草素、山奈酚、芹菜素和没食子酸进行研究。首次采用毛细管电泳-安培检测法成功地分离了地锦草中的芦丁、木樨草素、山奈酚、芹菜素和没食子酸,并优化了分离和检测条件。在本实验最佳优化条件下,分析物质在pH 8.8,20 mmol/L的硼砂缓冲溶液中20 min内实现了完全快速地分离;采用碳圆盘电极,所有待测物在工作电极电位为+0.95 V(vs.SCE)下有良好的响应;线性范围在信噪比为3下跨越三个数量级。本方法简单快速、灵敏度高,可用于地锦草的定性和定量的研究,分析结果可靠,令人满意。
第五章毛细管电泳安培检测联用测定中草药杠板归中的活性成分
采用毛细管电泳-电化学检测法(CE-ED)同时分离测定了中药杠板归中的阿魏酸、香草酸、槲皮素、咖啡酸、原儿茶酸等主要生物活性成分的含量。考察了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间等实验参数对实验结果的影响。在优化的实验条件下,以直径300μm的碳圆盘电极为工作电极,检测电位为+0.95V(vs.SCE),在10mmol/L磷酸盐(pH 9.2)的运行缓冲溶液中,五个分析物能够在17min内实现很好的基线分离,被测物浓度与峰电流在3个数量级呈良好的线性,检测限(S/N=3)范围从7.1×10~(-8)到9.3×10~(-8)g/mL。该方法已应用于实际样品的分析,样品处理简单,获得了令人满意的结果。
第六章毛细管电泳-电化学检测联用测定动物组织中的β-激动剂
本文采用毛细管电泳—电化学检测技术,通过在缓冲液中添加有机溶剂,成功地分离了四种β—激动剂:盐酸克伦特罗、硫酸特布他林、盐酸莱克多巴胺和硫酸沙丁胺醇,并考察了缓冲液酸度和浓度、分离电压、氧化电位和进样时间等对分离检测的影响。得出最佳实验条件如下:工作电极为直径300μm的碳圆盘电极,检测电位为+0.95V(vs.SCE),缓冲液为60 mmol/L硼砂溶液(pH=8.7),分离电压17 kV,该方法已成功地应用于实际样品中β—激动剂残留状况的分析,结果令人满意。
Capillary Electrophoresis(CE) is one of the most important and rapidly growing separation techniques in recent twenty years,It absorbed the virtues of both the conventional electrophoresis and modem High Performance Liquid Chromatography (HPLC).However,the theoretical plate number is great developed to tens of thousands and even millions and the volume Of sample injection reaches nano milliliter level in CE analysi.In recent years,capillary electrophoresis(CE) has been extensively studied and applied as a highly effective analytical method in modern analysis.And excellent prospects of CE have been shown in the fields of pharmaceutical analysis,biochemical analysis,food analysis,and environmental analysis.It has many advantages such as much lower sampling volume,cheaper instrumentation and higher separation efficiency.However,the small sample injection volume and very thin capillary bring about difficulties to detections.The common used detection methods are Ultra-violet(UV),Laser Induced Fluorescence(LIF), Luminescence Detection,Mass Spectra(MS).Because amperometric detector(AD) is more sensitive than UV-visible detector and much cheaper than LIF detector,it is preferred to couple with CE in many analysis areas.
Recently,with more attention paid to the quality of life,the analysis in environment and medicine field has become a hotspot of the analytical chemistry research.The goal of this dissertation is to explore some applications in environmental analysis,pharmaceutical analysis and food analysis etc.by CE-ED. The contents of this dissertation include six chapters:
Chapter 1.Preface
The characteristics of CE,the separation models,the basic theories of CE,the detectors,the studies and applications of CE-AD are introduced briefly.The goal and significance of this dissertation are also introduced.
Chapter 2.Separation and determination of nitroaniline isomers by capillary zone electrophoresis with amperometric detection
Capillary zone electrophoresis with amperometric detection(CZE-AD) was firstly applied to the simultaneous separation and determination of nitroaniline positional isomers.The three analytes could be perfectly analyzed by using the buffer of extreme pH.The effects of several important factors were investigated to find optimum conditions.A carbon-disk electrode was used as working electrode.The optimal conditions were 40 mmol/L tartaric acid-sodium tartrate(pH 1.2) as running buffer, 17 kV as separation voltage and 1.10 V(versus saturated calomel reference electrode, SCE) as detection potential.Under the optimum conditions,o-,m- and p-nitroaniline were separated successfully and good linearity,reproducibility and recovery results were obtained.The detection limit for m-nitroaniline was as low as at 9.06×10~(-9) mol/L. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 1.8%for migration time and 1.1%for peak areas.The utility of this method was demonstrated by monitoring dyestuff wastewater and the assay results were satisfactory.
Chapter 3.Separation and determination of nitrophenol isomers by capillary electrophoresis with amperometric detection
In this paper,capillary zone electrophoresis with amperometric detection(CZE-AD) was applied to determine the nitrophenol isomers in dyestuff wastewater.The analytes could be perfectly separated by only adjusting the run buffer with little organic modifier.The effect of certain percentage of organic solvents present in the electrophoretic buffer on the separation of three phenols was investigated.10%(V/V) methanol and the same level of 1:1(V/V) methanol-acetonitrile mixture were found equally effective for sensitivity enhancement in detection and efficiency,but 10%methanol solvent may offer better baseline and peak shapes. The detection limit was as low as 7.50×10~(-8) mol/L,which was much lower than that in CZE-UV method.Analyzing wastewater by this method has some merits of speed,simple instrumentation and operation,high sensitivity and high reproducibility.The experimental results showed that this CZE-AD method was very practical to determine analogous substances in Environmental Monitoring.
Chapter 4.Simultaneous determination of active ingredients in Herba Euphorbiae Humifusae by capillary zone eleetrophoresis with amperometric detection
In this chapter,the determination of active ingredients(rutin,quercatin,gallic acid and chlorogenic acid) of Euphorbia Humifusa Willd by capillary electrophoresis with amperometric detection were studied.CE-AD was successfully used to separate and detect the five active ingrediences for the first time,and the experiment conditions were optimized.Under the optimum conditions,the analytes could be separated in a 20 mmol/L borate buffer(pH=8.8) within 20 min.A 300μm diameter carbon disk electrode has a good response at +0.95 V(vs.SCE) for all analytes.The response was linear over three orders of magnitude with detection limits(S/N=3).The method could be used in the rapid determination of practical samples.
Chapter 5.Determination of bioactive components in Polygonum perfoliatum L.by Capillary Electrophoresis with Electrochemical Detection
The major bioactive components in a Chinese herb named Polygonum perfoliatum L.including ferulic acid,vanillic acid,quercetin,caffeic acid and protocatechuic acid were simultaneously determined by capillary electrophoresis with electrochemical detection(CE-ED) in this paper.The effects of working electrode potential,pH and concentration of running buffer,separation voltage,and injection time on CE-ED were investigated.Under the optimum conditions,the five analytes could be separated within 17 min at a separation voltage of 18 kV in 10 mmol/L phosphate buffer(pH 9.2).A 300μm diameter carbon disk electrode generated good responses at +0.95V (vs.SCE) for the five analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) ranging from 7.1×10~(-8) to 9.3×10~(-8)g/mL for the analytes.This proposed method could be successfully applied to the analysis of the real samples with relatively simple extraction procedures,and the assay results were satisfactory.
Chapter 6.Separation and Determination ofβ-Agonists in Animal Tissue by Capillary Electrophoresis with Electrochemical Detection.
A high-performance capillary electrophoresis with electrochemical detection (CE-ED) method had been applied for the determination ofβ-agonists,Clenbuterol hydrochloride,Terbutaline sulfate,Ractopamine hydrochloride and Salbutamol sulfate,in meat produces.The effects of several factors such as the acidity and concentration of running buffer,the separation voltage,the applied potential and the injection time on CE-ED were investigated.Under the optimum conditions,these four components could be separated successfully at the separation voltage of 17 kV in a 60 mmol/L borax running buffer(pH 8.7).A 300μm diameter carbon disk electrode was used as the working electrode positioned carefully opposite the outlet of the capillary in a wall-jet configuration at potential of +0.95V(vs.SCE).The method had been successfully applied to analyzeβ-agonists in real-life samples,and the assay results were satisfactory.
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