高效液相色谱结合化学发光的一些应用研究
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摘要
目前，高效液相色谱(HPLC)法由于对复杂样品中的分析物具有极高的分离效率而成为最有效的分离方法。将具有高灵敏度的化学发光分析法和具有高分离效率的高效液相色谱分离法相结合已引起了国内外分析化学家的极大兴趣，成为从上个世界80年代末至今分析化学研究的热点。化学发光分析法不需要外部光源，消除了杂散光及因光源发光不稳定而导致波动的缺点，从而降低了噪音，提高了信噪比，再加上灵敏的光电检测技术，使该方法具有灵敏度高、线性范围宽、设备简单、易于实现自动化等优点。但在检测复杂对象时，方法选择性不高，限制了化学发光分析方法的应用范围和应用领域。将它和色谱等分离手段结合，就可以有效地提高化学发光法的选择性。另外，和传统液相色谱常用的紫外检测法比较，化学发光检测法通常有着更高的灵敏度，可以实现对分析对象的高灵敏度检测。自1974年Hartopf等首次报道了液相色谱化学发光分析法以来，高效液相色谱化学发光分析法已成为一种分析和测量复杂基质中痕量和超痕量组分的有力工具，被广泛研究和应用于食品、化工、医药、环境、生物医学科学、临床化学等领域。
本论文由综述和研究报告两部分组成。综述部分简单概述了高效液相色谱化学发光检测的原理、仪器结构、用于高效液相色谱柱后检测的主要化学发光体系以及该技术面临的挑战和机遇等。研究报告部分包括：高效液相色谱一化学发光法测定食品中的香兰素、流动注射化学发光法测定秋水仙碱、一种气泡微量进样装置的初步研究。
一、高效液相色谱-化学发光法测定食品中的香兰素
在碱性条件下，香兰素对luminol-K_3Fe(CN)_6体系有增敏作用。根据这一现象建立了一种经高效液相色谱(HPLC)分离柱后测定香兰素的化学发光法。本方法以C_(18)为色谱柱，用乙腈和水(50：50，V/V)为流动相，在最优条件下，方法的线性范围为7．0×10~(-7)～4．0×10~(-5)mol/L，检出限为2．0×10~(-7)mol／L(3σ)，相对标准偏差为3．9％(c=4．0×10~(-6)mol/L，n=9)。该方法用于食品中香兰素含量的测定，其分析结果与高效液相-紫外光度法(HPLC-UV)比较具有很好的一致性。
二、流动注射化学发光法测定秋水仙碱
在甲醛存在时，高锰酸钾可在酸性介质中氧化秋水仙碱产生较强的化学发光。根据这一实验现象并结合流动注射技术，建立了一种高锰酸钾-甲醛-秋水仙碱化学发光测定秋水仙碱的分析方法．在最优化条件下，化学发光强度与秋水仙碱浓度在1．0×10~(-7)～3．0×10~(-5)mol／L范围内呈线性关系。方法的检出限为3．0×10~(-8)mol／L(3σ)，相对标准偏差为1．9％(c=5．0×10~(-7)mol/L,n=11)。该方法用于药物制剂中秋水仙碱含量的测定，其分析结果与药典方法比较具有很好的一致性。
三、气泡微量进样装置的初步研究
设计并构建了气泡微量进样装置，该装置把注射器、注射泵、表面活性剂(十二烷基苯磺酸钠)等引入系统，产生连续、稳定、均匀的气泡，使试剂和样品以气泡的形式经过检测器。选择经典的化学发光体系对所设计的装置进行了评价。实验结果说明，气泡微量进样分析对化学发光信号测定重现性好，在一定范围内调节流速可达到很高的分析频率。仪器结构简单，分析操作更加简化，显示出气泡微量进样分析在化学发光分析方面较大的应用潜力。
At present, high performance liquid chromatography (HPLC) has mostly been used to enhance the selectivity of analytical methods, because of its high efficiency for separation of analytes from various interfering substances. One of the major research areas about HPLC involves the development sensitive detection methods. More sensitive detection can be realized by HPLC when the analytes are subjected to the use of chemiluminescence (CL) detection. The chemiluminescence (CL) detection system combined with HPLC separation method can offer excellent analytical selectivity and sensitivity. As an important analytical method, CL has the merits of great sensitivity, wide dynamic range, simple equipment and easy automatization. However, the disadvantage of poor selectivity limites its application in many areas. So, the combination between HPLC and CL becomes a necessary consideration. Compounds are separated on the HPLC column and next mixed with CL reaction reagents, the lights emissed from the substances involved in this reaction course are recorded and conducted. Thus, the frame of HPLC-CL comes into being. This analytical method has both excellences of CL and HPLC, at the same time, succeeds in conquering the disadvantage of CL. Compare to UVdetector used in HPLC, CL detector could achieve higher sensitivity for determination of many substances. Since the first report of HPLC-CL technique by Hartkopf etal in 1974, this technique has developed rapidly. It has become a powerful tool for analysis of trace and ultra-trace ingredients and is widely used in the fields of food, chemical industry, pharmacy, environment, life science, biology and clinical medical science.
This thesis includes two sections: review and research report. In the review section, informations about high performance liquid chromatography chemiluminescence detection (HPLC-CL) such as principles, HPLC-CL equipment and the main chemiluminescence reaction systems used in HPLC-CL are summarized. In the next research report section, applied reports about HPLC-CL for determination of vanillin, CL for determination of colchicine, a primary study of bubble microinjection system are reported, respectively.
1. Determination of Vanillin by High Performance Liquid Chromatography with Chemiluminescence Detection
An analytical method consisting of HPLC separation and chemiluminescence detection for determination of Vanillin is described. It is based on the chemiluminescence reaction of luminol-K_3Fe(CN)_6-vanillin. The chemiluminescence intensity is correlated linearly with concentration of vanillin in the range of 6.0×10~(-7)-4.0×10~(-5)mol/L, and the determination limit is 2.0×10~(-7)mol/L (3σ), Its relative standard deviation is 3.9% for 9 measurements (c=4.0×10~(-6)mol/L,n=9). This method has been applied to the determination of vanillin in food samples, the analysis results agrees well with those from HPLC-UVmethod. The average recoveries of vanillin (spiked at the levels of 3.0×10~(-6)-2.0×10~(-5)mol L~(-1)) in food samples ranges from 95 to 104%, and the RSDsof the quantitative results are from 0.9 to 4.5%.
2. A Chemiluminescence Flow Injection Method for the Determination of Colchicine
A chemiluminescence method for colchicine determination with flow injection is described. It is based on the chemiluminescence emitted during the oxidation of colchicine by potassium permanganate in acidic medium in the presence of formaldehyde. The linear range of determinationfor colchicine was 1.0×10~(-7) mol/L-6.0×10~(-5)mol/L with the relative standard deviation of 1.9%(c=1.0×10~(-6)mol/L, n=11). The detection limit was 3.0×10~(-8)mol/L (3σ). The recommended method has been used for determination of colchicine in pharmaceutical formulation and the analysis results agreed well with those from official method.
3. A Primary Study of Bubble Microinjection System
We design a bubble microinjection system . The system is composed of syringes, injection pump, surfactant component (SDS). It produces continuous, stable, homogeneous bubbles. Reagents and samples pass through the detector in the form of air bubbles. The characteristics of above microjectionin system have been evaluated in the presence of reactions and have shown that the proposed approach can be employed to chemilunimescence analysis for determine CL from excited state reaction product. The use of microinjection system can significantly decrease the sample and reagent consumption, promise high throughput, approach simple automation scheme.
本论文由综述和研究报告两部分组成。综述部分简单概述了高效液相色谱化学发光检测的原理、仪器结构、用于高效液相色谱柱后检测的主要化学发光体系以及该技术面临的挑战和机遇等。研究报告部分包括：高效液相色谱一化学发光法测定食品中的香兰素、流动注射化学发光法测定秋水仙碱、一种气泡微量进样装置的初步研究。
一、高效液相色谱-化学发光法测定食品中的香兰素
在碱性条件下，香兰素对luminol-K_3Fe(CN)_6体系有增敏作用。根据这一现象建立了一种经高效液相色谱(HPLC)分离柱后测定香兰素的化学发光法。本方法以C_(18)为色谱柱，用乙腈和水(50：50，V/V)为流动相，在最优条件下，方法的线性范围为7．0×10~(-7)～4．0×10~(-5)mol/L，检出限为2．0×10~(-7)mol／L(3σ)，相对标准偏差为3．9％(c=4．0×10~(-6)mol/L，n=9)。该方法用于食品中香兰素含量的测定，其分析结果与高效液相-紫外光度法(HPLC-UV)比较具有很好的一致性。
二、流动注射化学发光法测定秋水仙碱
在甲醛存在时，高锰酸钾可在酸性介质中氧化秋水仙碱产生较强的化学发光。根据这一实验现象并结合流动注射技术，建立了一种高锰酸钾-甲醛-秋水仙碱化学发光测定秋水仙碱的分析方法．在最优化条件下，化学发光强度与秋水仙碱浓度在1．0×10~(-7)～3．0×10~(-5)mol／L范围内呈线性关系。方法的检出限为3．0×10~(-8)mol／L(3σ)，相对标准偏差为1．9％(c=5．0×10~(-7)mol/L,n=11)。该方法用于药物制剂中秋水仙碱含量的测定，其分析结果与药典方法比较具有很好的一致性。
三、气泡微量进样装置的初步研究
设计并构建了气泡微量进样装置，该装置把注射器、注射泵、表面活性剂(十二烷基苯磺酸钠)等引入系统，产生连续、稳定、均匀的气泡，使试剂和样品以气泡的形式经过检测器。选择经典的化学发光体系对所设计的装置进行了评价。实验结果说明，气泡微量进样分析对化学发光信号测定重现性好，在一定范围内调节流速可达到很高的分析频率。仪器结构简单，分析操作更加简化，显示出气泡微量进样分析在化学发光分析方面较大的应用潜力。
At present, high performance liquid chromatography (HPLC) has mostly been used to enhance the selectivity of analytical methods, because of its high efficiency for separation of analytes from various interfering substances. One of the major research areas about HPLC involves the development sensitive detection methods. More sensitive detection can be realized by HPLC when the analytes are subjected to the use of chemiluminescence (CL) detection. The chemiluminescence (CL) detection system combined with HPLC separation method can offer excellent analytical selectivity and sensitivity. As an important analytical method, CL has the merits of great sensitivity, wide dynamic range, simple equipment and easy automatization. However, the disadvantage of poor selectivity limites its application in many areas. So, the combination between HPLC and CL becomes a necessary consideration. Compounds are separated on the HPLC column and next mixed with CL reaction reagents, the lights emissed from the substances involved in this reaction course are recorded and conducted. Thus, the frame of HPLC-CL comes into being. This analytical method has both excellences of CL and HPLC, at the same time, succeeds in conquering the disadvantage of CL. Compare to UVdetector used in HPLC, CL detector could achieve higher sensitivity for determination of many substances. Since the first report of HPLC-CL technique by Hartkopf etal in 1974, this technique has developed rapidly. It has become a powerful tool for analysis of trace and ultra-trace ingredients and is widely used in the fields of food, chemical industry, pharmacy, environment, life science, biology and clinical medical science.
This thesis includes two sections: review and research report. In the review section, informations about high performance liquid chromatography chemiluminescence detection (HPLC-CL) such as principles, HPLC-CL equipment and the main chemiluminescence reaction systems used in HPLC-CL are summarized. In the next research report section, applied reports about HPLC-CL for determination of vanillin, CL for determination of colchicine, a primary study of bubble microinjection system are reported, respectively.
1. Determination of Vanillin by High Performance Liquid Chromatography with Chemiluminescence Detection
An analytical method consisting of HPLC separation and chemiluminescence detection for determination of Vanillin is described. It is based on the chemiluminescence reaction of luminol-K_3Fe(CN)_6-vanillin. The chemiluminescence intensity is correlated linearly with concentration of vanillin in the range of 6.0×10~(-7)-4.0×10~(-5)mol/L, and the determination limit is 2.0×10~(-7)mol/L (3σ), Its relative standard deviation is 3.9% for 9 measurements (c=4.0×10~(-6)mol/L,n=9). This method has been applied to the determination of vanillin in food samples, the analysis results agrees well with those from HPLC-UVmethod. The average recoveries of vanillin (spiked at the levels of 3.0×10~(-6)-2.0×10~(-5)mol L~(-1)) in food samples ranges from 95 to 104%, and the RSDsof the quantitative results are from 0.9 to 4.5%.
2. A Chemiluminescence Flow Injection Method for the Determination of Colchicine
A chemiluminescence method for colchicine determination with flow injection is described. It is based on the chemiluminescence emitted during the oxidation of colchicine by potassium permanganate in acidic medium in the presence of formaldehyde. The linear range of determinationfor colchicine was 1.0×10~(-7) mol/L-6.0×10~(-5)mol/L with the relative standard deviation of 1.9%(c=1.0×10~(-6)mol/L, n=11). The detection limit was 3.0×10~(-8)mol/L (3σ). The recommended method has been used for determination of colchicine in pharmaceutical formulation and the analysis results agreed well with those from official method.
3. A Primary Study of Bubble Microinjection System
We design a bubble microinjection system . The system is composed of syringes, injection pump, surfactant component (SDS). It produces continuous, stable, homogeneous bubbles. Reagents and samples pass through the detector in the form of air bubbles. The characteristics of above microjectionin system have been evaluated in the presence of reactions and have shown that the proposed approach can be employed to chemilunimescence analysis for determine CL from excited state reaction product. The use of microinjection system can significantly decrease the sample and reagent consumption, promise high throughput, approach simple automation scheme.
引文
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