毛细管电泳—化学发光法用于生物与环境样品分析
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摘要
化学发光分析是近年来迅速发展的一种高灵敏的微量和痕量分析技术,以其灵敏度高、线性范围宽、仪器设备简单和容易实现自动化等优点,而成为分析化学中一个十分活跃的研究热点。毛细管电泳技术是一种高效液相分离技术,具有高效、快速、试剂和样品消耗量少等特点。毛细管电泳与化学发光的结合,兼备了毛细管电泳分离效率高和化学发光高灵敏度的优点,可直接用于复杂样品中微量组分的的分离与测定。近年来,这一联用技术已成为当前分析化学领域的研究热点之一。本文对毛细管电泳-化学发光联用技术应用于生物和环境样品中的分离分析进行了探讨,主要内容如下:
1.根据氨基酸和蛋白质在碱性溶液中具有能显著增强鲁米诺-铁氰化钾体系化学发光信号的特性,以L-精氨酸和胃蛋白酶为分析对象,对鲁米诺-铁氰化钾体系用于毛细管电泳-化学发光法直接分离测定生物分子的可行性进行了研究,并优化了化学发光和电泳条件。在优化条件下,L-精氨酸和胃蛋白酶可直接实现分离,其检测限分别为1.3×10~(-4) mol/L和7.1×10~(-7) mol/L。该方法无需复杂的衍生步骤,操作简便,应用于实际样品的分析测定,结果令人满意。
2.根据某些酚类化合物在碱性条件下具有能淬灭鲁米诺-铁氰化钾体系化学发光信号的特性,建立了毛细管电泳-间接化学发光法分离检测苯二酚异构体和苯酚的新方法。在优化的化学发光和电泳条件下,对苯二酚、间苯二酚、邻苯二酚和苯酚在10 min内可直接实现分离,其检测限(S/N=3)分别为2.9×10~(-8) mol/L,3.7×10~(-7) mol/L,8.4×10~(-8) mol/L和4.4×10~(-6) mol/L,相对标准偏差(RSD)为2.5-4.8%(n=5)。通过对染发剂实际样品和焦化废水进行分离分析,证明该方法可以用于实际样品的分析测定,结果令人满意。
Chemiluminescence has become one of the focus in the area of analytical chemistry due to its advantages such as higher sensitivity, wider linear range, simpler instrumentation, versatility of use and the easy automation. Capillary electrophoresis (CE) has gained significant acceptance in the analytical laboratory. The effciencies afforded by CE lead to high resolution separations with small sample volumes and short analysis times. Here we combined the detection method with capillary electrophoresis (CE) technique, and discussed its application for environmental and biological samples analysis. The main contents are shown as follows:
1. A novel method for the analysis of biomolecules was developed by capillary zone electrophoresis-chemiluminescence (CZE-CL) method based on the fact that some amino acids and proteins could catalyze the chemiluminescence reaction of luminol-K_3Fe(CN)_6 system. In addition, 2.5 mmol/L sodium tetraborate- 2×10~(-3)mol/L luminol and 2×10~(-4) mol/L potassium ferricyanide (pH=13.3) were chosen as optimum separation and detection condition for the analysis of L-arginine and pepsin. The detection limits (S/N=3) for L-arginine and pepsin were 1.3×10~(-4) mol/L and 7.1×10~(-7) mol/L, respectively. Finally, the presented method was used for the analysis of three real L-arginine samples successfully.
2. A capillary electrophoresis(CE) with on-line inhibited chemiluminescence (CL) detection was firstly used for the simultaneous analysis of benzenediol isomers and phenol. It was based on the quenching effect of benzenediol isomers and phenol on the chemiluminescence reaction of luminol with potassium ferricyanide in sodium hydroxide medium. Under the optimum conditions, the four phenols were baseline separated and detected in less than 10 min. The detection limits (S/N=3) for hydroquinone, resorcinol, catechol and phenol were 2.9×10~(-8) mol/L, 3.7×10~(-7) mol/L, 8.4×10~(-8) mol/L and 4.4×10~(-6) mol/L, respectively. The RSDs were between 2.5～4.8% (n=5). Finally, the presented method has been successfully applied to hair dye samples and coke plant wastewater sample.
1.根据氨基酸和蛋白质在碱性溶液中具有能显著增强鲁米诺-铁氰化钾体系化学发光信号的特性,以L-精氨酸和胃蛋白酶为分析对象,对鲁米诺-铁氰化钾体系用于毛细管电泳-化学发光法直接分离测定生物分子的可行性进行了研究,并优化了化学发光和电泳条件。在优化条件下,L-精氨酸和胃蛋白酶可直接实现分离,其检测限分别为1.3×10~(-4) mol/L和7.1×10~(-7) mol/L。该方法无需复杂的衍生步骤,操作简便,应用于实际样品的分析测定,结果令人满意。
2.根据某些酚类化合物在碱性条件下具有能淬灭鲁米诺-铁氰化钾体系化学发光信号的特性,建立了毛细管电泳-间接化学发光法分离检测苯二酚异构体和苯酚的新方法。在优化的化学发光和电泳条件下,对苯二酚、间苯二酚、邻苯二酚和苯酚在10 min内可直接实现分离,其检测限(S/N=3)分别为2.9×10~(-8) mol/L,3.7×10~(-7) mol/L,8.4×10~(-8) mol/L和4.4×10~(-6) mol/L,相对标准偏差(RSD)为2.5-4.8%(n=5)。通过对染发剂实际样品和焦化废水进行分离分析,证明该方法可以用于实际样品的分析测定,结果令人满意。
Chemiluminescence has become one of the focus in the area of analytical chemistry due to its advantages such as higher sensitivity, wider linear range, simpler instrumentation, versatility of use and the easy automation. Capillary electrophoresis (CE) has gained significant acceptance in the analytical laboratory. The effciencies afforded by CE lead to high resolution separations with small sample volumes and short analysis times. Here we combined the detection method with capillary electrophoresis (CE) technique, and discussed its application for environmental and biological samples analysis. The main contents are shown as follows:
1. A novel method for the analysis of biomolecules was developed by capillary zone electrophoresis-chemiluminescence (CZE-CL) method based on the fact that some amino acids and proteins could catalyze the chemiluminescence reaction of luminol-K_3Fe(CN)_6 system. In addition, 2.5 mmol/L sodium tetraborate- 2×10~(-3)mol/L luminol and 2×10~(-4) mol/L potassium ferricyanide (pH=13.3) were chosen as optimum separation and detection condition for the analysis of L-arginine and pepsin. The detection limits (S/N=3) for L-arginine and pepsin were 1.3×10~(-4) mol/L and 7.1×10~(-7) mol/L, respectively. Finally, the presented method was used for the analysis of three real L-arginine samples successfully.
2. A capillary electrophoresis(CE) with on-line inhibited chemiluminescence (CL) detection was firstly used for the simultaneous analysis of benzenediol isomers and phenol. It was based on the quenching effect of benzenediol isomers and phenol on the chemiluminescence reaction of luminol with potassium ferricyanide in sodium hydroxide medium. Under the optimum conditions, the four phenols were baseline separated and detected in less than 10 min. The detection limits (S/N=3) for hydroquinone, resorcinol, catechol and phenol were 2.9×10~(-8) mol/L, 3.7×10~(-7) mol/L, 8.4×10~(-8) mol/L and 4.4×10~(-6) mol/L, respectively. The RSDs were between 2.5～4.8% (n=5). Finally, the presented method has been successfully applied to hair dye samples and coke plant wastewater sample.
引文
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[2]李晓燕,何治珂,曾云鹗.化学发光分析法在环境分析中的应用[J].分析科学学学报, 1999, 15(3): 258-262
[3] Kricka L.J.. Clinical applications of chemiluminescence[J]. Anal.Chim.Acta, 2003, 500: 279-286
[4] Liu Y.M., Cheng J.K. Ultrasensitive chemiluminescence detection in capillary electrophoresis[J]. J. Chromatogr. A, 2002, 959:1–13
[5] Kuyper C., Milofsky R.. Recent developments in chemiluminescence and photochemical reaction detection for capillary electrophoresis[J]. Trends in Anal. Chem., 2001, 20: 232-240
[6] Famei L., Caihua Z., Xingjie G., et al. Chemiluminescence detection in HPLC and CE for pharmaceutical and biomedical analysis[J]. Biomedical Chromatography, 2003, 17: 96-105
[7]林金明.化学发光色谱柱后检测技术及其应用[J].色谱, 2003, 21(4): 324-331
[8] Huang X.J., Fang Z.L.. Chemiluminescence detection in capillary electrophoresis[J]. Anal.Chim.Acta, 2000, 414: 1-14
[9] Zhang Y., Huang B., Cheng J.K.. On-line indirect chemiluminescence detection in capillary electrophoresis [J]. Anal. Chim. Acta, 1998, 363: 157-163
[10] Safavi A., Karimi M. A.. Flow injection chemiluminescence determination of sulfide by oxidation with N-bromosuccinimide and N-chlorosuccinimide[J]. Talanta, 2002, 57: 491-500
[11] Yaqoob M., Nabi A., Worsfold P.J.. Determination of silicate in freshwaters using flow injection with luminol chemiluminescence detection[J]. Anal.Chim.Acta, 2004, 519: 137-142
[12] Yaqoob M., Nabi A., Worsfold P.J.. Determination of nanomolar concentrations of phosphate in freshwaters using flow injection with luminol chemiluminescence detection[J]. Anal.Chim.Acta, 2004, 510: 213-218
[13] Yaqoob M., Nabi A., Waseem A.. Determination of sulphite using an immobilized enzyme with flow injection chemiluminescence detection[J]. Luminescence, 2004, 19(1): 26-30
[14] Nagoshi T., Ohno O., Kotake T., et al.. Chemiluminescence of iron-chlorophyllin [J]. Luminescence, 2005, 20: 401-404
[15] Nie F., Lu J., He Y., et al. Use of molecule imprinting–chemiluminescence method for the determination of tamoxifen in breast cancer sufferers’urine[J]. Luminescence, 2005, 20: 315-320
[16] Yang M., Liu R., Hou D., et al. A novel flow-injection chemiluminescence system for the determination of guanine[J]. Luminescence, 2005, 20: 307–310
[17]武竟存,章竹君,吕九如.液相色谱化学发光检测法的新进展[J].分析化学, 1994, 22(4):396-405
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