薄层色谱洗脱方法的研究及其在药物分析中的应用
摘要
经典薄层色谱法是一种以毛细作用力为主要推动力的色谱法。这种输液方式使它很难像柱色谱那样采用在线检测的方式。本工作最初针对这一弱点，以实现薄层色谱在线检测为目标，展开了全面研究。
首先从下行展开着手，希望在重力的作用下实现在线检测的目标。设计并实现了一种全新的下行展开输液方式。这种输液方式依靠盖板式输液分配器使流动相直接与硅胶层接触，改变了传统的依靠滤纸导引流动相的方式。工作中设计出了多种毛细引流装置以图实现在线检测，皆不理想。最终研究结果表明，在下行展开过程中流动相的主要输送力仍是毛细作用力，重力作用不占主导，因而在不依靠其他外力情况下，薄层色谱很难实现在线检测。然而在探索中，在线分离洗脱的方法被成功的发展出来。该法借助一种简单而有效的收集器在负压作用下可以进行在线分离洗脱。在此基础上，又实现了水平在线分离洗脱和原位洗脱两种方式。
这几种洗脱方式的效率很高，改变了传统的将载有样品的硅胶层刮下再洗脱的复杂操作，并被首先应用于中药纯品制备中，实现了对已分离薄层板的及时处理。实验中我们从大黄药材中分离提取出了大黄酚，大黄素甲醚和大黄素。对比了此法制备的产品与用硅胶柱层析法制备的产品的纯度。检测结果表明：原位洗脱法所得的重结晶产品纯度较高。
在线分离洗脱及原位洗脱方法较高的洗脱效率，使它成为薄层定量的一种可靠方法。本工作用这种方法对中药决明子中三种主要成分的含
沈阳药科大学硕土学位论文 摘要
量进行了测定，为薄层色谱分析处理中药提供了又一选择。
磷脂的分析是一个难点，该物质对高效液相色谱柱损害极大，而薄
层扫描法的结果又不稳定。本工作使用原位洗脱和钥蓝法对蛋黄磷脂中
磷脂酚胆碱的含量进行了测定，结果满意。
通过对薄层色谱展开方法及洗脱方式的系统研究，澄清了一些误解
和幻想，对薄层色谱的展开机理有了更深的认识。由此发展出来的在线
分离洗脱方式和原位洗脱方式为色谱工作提供了新的选择。
Capillary force is the mainly driving force of the thin-layer chromatography (TLC). For this reason, the on-line detection that used in the column chromatography is difficultly realized in the classical TLC. In view of this weakness, we started our work to solve this problem.
The descending development was studied firstly. We hoped to reach the aim with the help of the gravity. A new solvent-delivering model of descending development was developed. That changed the conventional fashion delivering the solvent by using filter paper. Many capillary-drafting instrument were designed in order to realize on-line detection, but they all cannot meet the need. The results of the experiments showed that the capillary force but not the gravity mainly delivers the solvent. So, without other outer force, it is difficult to reach the aim of on-line detection. However, with the help of a simple and effective collector and a negative pressure system, the method of separation coupling elution was developed successfully. Based on this principle, separation coupling elution in horizontal development and in-situ elution was developed.
ABSTRACT
The efficient of these elution models is high. That reduces the complex operation procedure of scraping the spots off the silica layer. This method was applied in the preparation of pure substances of traditional Chinese medicine. Three kind of anthraquinone were separated and got from rhubarb. The purities of these three substances were better than those that were got by column chromatography.
The high elution efficient of separation coupling elution and in-situ elution makes them reliable operation. The content of three anthraquinone in cassia were measured by these methods. That provides chromatographer with a new selection to analysis and deal with traditional Chinese medicine.
The analysis of phosphalipids is a difficult problem, for it is harmful to the column of high performance liquid chromatography (HPLC), and the result that got by the TLC scanner were always instable. The in-situ elution and molybdenum blue reaction was used to measure the content of the phosphacholine (PC) in the egg yolk phosphalipids. The result is satisfied.
Studied the methods of separation and elution systematically, a few of misunderstandings and fantasy were cleared up. The theory of the separation of the TLC was deeply recognized. The method that on-line elution in descending and horizontal development and the in-situ elution provide many new operations for the chromatographer.
首先从下行展开着手，希望在重力的作用下实现在线检测的目标。设计并实现了一种全新的下行展开输液方式。这种输液方式依靠盖板式输液分配器使流动相直接与硅胶层接触，改变了传统的依靠滤纸导引流动相的方式。工作中设计出了多种毛细引流装置以图实现在线检测，皆不理想。最终研究结果表明，在下行展开过程中流动相的主要输送力仍是毛细作用力，重力作用不占主导，因而在不依靠其他外力情况下，薄层色谱很难实现在线检测。然而在探索中，在线分离洗脱的方法被成功的发展出来。该法借助一种简单而有效的收集器在负压作用下可以进行在线分离洗脱。在此基础上，又实现了水平在线分离洗脱和原位洗脱两种方式。
这几种洗脱方式的效率很高，改变了传统的将载有样品的硅胶层刮下再洗脱的复杂操作，并被首先应用于中药纯品制备中，实现了对已分离薄层板的及时处理。实验中我们从大黄药材中分离提取出了大黄酚，大黄素甲醚和大黄素。对比了此法制备的产品与用硅胶柱层析法制备的产品的纯度。检测结果表明：原位洗脱法所得的重结晶产品纯度较高。
在线分离洗脱及原位洗脱方法较高的洗脱效率，使它成为薄层定量的一种可靠方法。本工作用这种方法对中药决明子中三种主要成分的含
沈阳药科大学硕土学位论文 摘要
量进行了测定，为薄层色谱分析处理中药提供了又一选择。
磷脂的分析是一个难点，该物质对高效液相色谱柱损害极大，而薄
层扫描法的结果又不稳定。本工作使用原位洗脱和钥蓝法对蛋黄磷脂中
磷脂酚胆碱的含量进行了测定，结果满意。
通过对薄层色谱展开方法及洗脱方式的系统研究，澄清了一些误解
和幻想，对薄层色谱的展开机理有了更深的认识。由此发展出来的在线
分离洗脱方式和原位洗脱方式为色谱工作提供了新的选择。
Capillary force is the mainly driving force of the thin-layer chromatography (TLC). For this reason, the on-line detection that used in the column chromatography is difficultly realized in the classical TLC. In view of this weakness, we started our work to solve this problem.
The descending development was studied firstly. We hoped to reach the aim with the help of the gravity. A new solvent-delivering model of descending development was developed. That changed the conventional fashion delivering the solvent by using filter paper. Many capillary-drafting instrument were designed in order to realize on-line detection, but they all cannot meet the need. The results of the experiments showed that the capillary force but not the gravity mainly delivers the solvent. So, without other outer force, it is difficult to reach the aim of on-line detection. However, with the help of a simple and effective collector and a negative pressure system, the method of separation coupling elution was developed successfully. Based on this principle, separation coupling elution in horizontal development and in-situ elution was developed.
ABSTRACT
The efficient of these elution models is high. That reduces the complex operation procedure of scraping the spots off the silica layer. This method was applied in the preparation of pure substances of traditional Chinese medicine. Three kind of anthraquinone were separated and got from rhubarb. The purities of these three substances were better than those that were got by column chromatography.
The high elution efficient of separation coupling elution and in-situ elution makes them reliable operation. The content of three anthraquinone in cassia were measured by these methods. That provides chromatographer with a new selection to analysis and deal with traditional Chinese medicine.
The analysis of phosphalipids is a difficult problem, for it is harmful to the column of high performance liquid chromatography (HPLC), and the result that got by the TLC scanner were always instable. The in-situ elution and molybdenum blue reaction was used to measure the content of the phosphacholine (PC) in the egg yolk phosphalipids. The result is satisfied.
Studied the methods of separation and elution systematically, a few of misunderstandings and fantasy were cleared up. The theory of the separation of the TLC was deeply recognized. The method that on-line elution in descending and horizontal development and the in-situ elution provide many new operations for the chromatographer.
引文
[1] Christie W.W. Has thin-layer chromatography had its day? Lipid Technology, 1990 2, 22～23.
[2] Christie W.W. and Dobson, G. Thin-layer chromatography-revisited. Lipid Technology, 1999 11, 64～66
[3] Su P., Wang D.Y., Lan M.N., Half-way development—the technique and the device, J. Planar Chromatogra., 2001, (14),3,203～207
[4] 苏萍，薄层色谱创新技术及其在药物分析中的应用，沈阳药科大学硕士学位论文，沈阳药科大学，2000
[5] Lan M. N., Wang D. Y., Han J.; Improvement of two dimensional development of TLC; J. Planar Chromatogra., 2002, 15(2), 144～146
[6] 兰梦宁，薄层色谱新技术及其在中药分析中的应用，沈阳药科大学硕士学位论文，沈阳药科大学，2002
[7] 孙毓庆，现代色谱法及在医药中的应用，人民卫生出版社，1998，300～309
[8] 何丽一，平面色谱的方法及应用，化学工业出版社，2000，162～165
[9] Chang T. T., Lay J. O., Franced R.T., Jr., etc. Direct analysis of thin-layer chromatography spots by FAB Mass spectrometry, Anal. Chem., 1984,56,109～111
[10] Crecelius A, Clench M.R., Richards D.S., etc. Analysis of UK-224,671 and Related Substances by Thin Layer Chromatography/Matrix Assisted Laser Desorption Ionisation/Time of Flight Mass Spectrometry, J. Planar Chromatogra., 2000, 13(1), 76～81
[11] 汪瑗，国占生，王英锋等，小檗碱在TLC原位的FT—SERS研究，光谱学与光谱分析，2002，22(5)，745～748
[12] 傅强，梁明金，常春，薄层色谱-化学衍生荧光分光光度法测定槐米中芦丁的含量，中国现代应用药学，2002，19(2)，142～144
[13] 杨春欣，孙丽霞，许根英等，薄层色谱—分光光度法测定黄芪粉针剂中黄芪甲苷的含量，中草药，2001，32(4)，312～314
[14] Petrovic S. C., Dewald H. D., On-plate square-wave stripping valtammety of Cd(Ⅱ) Cu (Ⅱ) and Pb(Ⅱ) at the mercury-film ultmmicroelectrode after separation by TLC, J. Planar chromatogra., 1996, 9(6), 269～272
[15] Banks C. T., Development and application of coupled HPLC-TLC for pharmaceutical analysis, J. Pharm. Biomed. Anal., 1993, 8(11), 705～710
[16] Tyrpien K., Bodzek D., Janoszks B., Application of TLC and GC-MS to the identification of Azaavenes in sewage scudges, J. Planar chromatogram., 1995, 8(1), 75～77
[17] Skett P., The design and operation of a novel device for collecting fractions from TLC plates for biological and spectroscopic analysis, J. Planar Chromatogra., 1997, (10), 4,236～266
[18] Roubal W. T., Semiautomated indexing device for removal of adsorbent from thin-layer chromatography plate, Anal. Chem., 1984, 56,595～597
[19] Kirchner J. G, The Thin-layer Chromatography, 2nd, John Wiley & Sons. Inc 1978 New York
[20] Seikel M. K., Semi-micro preparative thin-layer chromatography, J. Chromatogra., 1964, 15, (1), 115～118
[21] Pe W., Use of descending TLC for identification of cannabinoids, Bull Narc, 1985, 37, 83～86 (Anal. Abstract 1986 10e23)
[22] Pelick N., Descending thin layer chromatography apparatus, U.S.A., USP, 3449083, 1969
[23] 齐登福，荧光光度法测定中药芦荟大黄素，山东工程学院学报，2002，16(2)5～8
[24] 杨成勇，刘丽娜，分光光度法测定决明子中大黄素的含量，重庆中草药研究，2001，4(3)，45～48
[25] 缪月英，戚晓菲，双波长薄层扫描法测定决明子中蒽醌含量，佳木斯医学院学报，1996，19(1)，30～31
[26] 曹爱民，沙明，孟淑智，高效液相色谱法测定决明子中大黄酚的含量，中国中药杂志，1997，22(2)，107～108
[27] 于超，兰红梅，王宇，HPLC法测定不同产地决明子中五种葸醌类化合物，基层中药杂志，2002，16(2)，8～10
[28] 王威，张彤，周立春，高效液相色谱一蒸发光散射检测器法对大豆磷脂原料药中各种成分的含量测定，首都医药．2002，9(8)，49～50
[29] 王立新，吴启南，吴德康等，钼蓝比色法测定不同产地合子草中总磷脂的含量，中国野生植物资源，2001，20(3)，50～51
[30] 雷厉，宋志宏，屠鹏飞等，反相高效液相色谱法制备松果菊苷标准晶，色谱，2001，
19(3)，200～202
[31] 陈培榕，廖志勤，薄层层析-干柱层析技术在分离制备石油生物标志物-卟啉中的应用，分析试验室，1997，16(2)，39～41
[32] 任海彧，查丽杭，秦川，大孔树脂分离提取麻黄碱的研究离子交换与吸附，2002，18(2)，97～104
[33] 李良学，刘继德，尹业高等，雷公藤内酯甲的制备和晶体结构，高等学校化学学报，2001，22(4)，573～576
[34] 赖宇红，陈浩桉，固相萃取法在中药分析中的应用前景及其建立优化新方法的策略与步骤 广东药学，2002，12(2)，12～16
[35] Hah J., Wang D.Y., Wang D.et.al., Coupling development and elution, a new thin-layer chromatography technique, J. Chromatogr. A (im press)
[36] 王东援，金海英，薄层色谱展开机理的探讨，色谱，1995，13(1)，74～74
[37] Maxwell R.J., Yeisley S.W., Unruh J., The importance of effective predevelopment plate washing techniques ing thin-layer chromatography, J. liquid Chromatography, 1990, 13, (10), 2001～2011
[38] 徐友锋，李灵芝，陈虹，大黄中提取游离葸醌的工艺改进，武警医学院学报，2002，11(2)，76～77
[39] 陈琼华，戴汉松，苏学良，中药大黄的综合研究 ⅩⅩⅪ．大黄葸醌衍生物系统分离的改进方法，天然产物研究与开发，2001，13(3)，58～60
[40] Sadtler Standard Infrared Grating Spectra, 42597P
[41] Fairbaim J. W., El-Muhtadi F.J., Chemotaxonomy of anthraquinones in Rumex. Phytochemistry, 1972, 11(1), 263～268
[42] Sadtler Standard Infrared Grating Spectra, 42616P
[43] Choline glycerophospholipids, http//www. Cyberlipid. org
[44] Separation ofphospholipids by TLC, http//www. Cyberlipid. org
[2] Christie W.W. and Dobson, G. Thin-layer chromatography-revisited. Lipid Technology, 1999 11, 64～66
[3] Su P., Wang D.Y., Lan M.N., Half-way development—the technique and the device, J. Planar Chromatogra., 2001, (14),3,203～207
[4] 苏萍，薄层色谱创新技术及其在药物分析中的应用，沈阳药科大学硕士学位论文，沈阳药科大学，2000
[5] Lan M. N., Wang D. Y., Han J.; Improvement of two dimensional development of TLC; J. Planar Chromatogra., 2002, 15(2), 144～146
[6] 兰梦宁，薄层色谱新技术及其在中药分析中的应用，沈阳药科大学硕士学位论文，沈阳药科大学，2002
[7] 孙毓庆，现代色谱法及在医药中的应用，人民卫生出版社，1998，300～309
[8] 何丽一，平面色谱的方法及应用，化学工业出版社，2000，162～165
[9] Chang T. T., Lay J. O., Franced R.T., Jr., etc. Direct analysis of thin-layer chromatography spots by FAB Mass spectrometry, Anal. Chem., 1984,56,109～111
[10] Crecelius A, Clench M.R., Richards D.S., etc. Analysis of UK-224,671 and Related Substances by Thin Layer Chromatography/Matrix Assisted Laser Desorption Ionisation/Time of Flight Mass Spectrometry, J. Planar Chromatogra., 2000, 13(1), 76～81
[11] 汪瑗，国占生，王英锋等，小檗碱在TLC原位的FT—SERS研究，光谱学与光谱分析，2002，22(5)，745～748
[12] 傅强，梁明金，常春，薄层色谱-化学衍生荧光分光光度法测定槐米中芦丁的含量，中国现代应用药学，2002，19(2)，142～144
[13] 杨春欣，孙丽霞，许根英等，薄层色谱—分光光度法测定黄芪粉针剂中黄芪甲苷的含量，中草药，2001，32(4)，312～314
[14] Petrovic S. C., Dewald H. D., On-plate square-wave stripping valtammety of Cd(Ⅱ) Cu (Ⅱ) and Pb(Ⅱ) at the mercury-film ultmmicroelectrode after separation by TLC, J. Planar chromatogra., 1996, 9(6), 269～272
[15] Banks C. T., Development and application of coupled HPLC-TLC for pharmaceutical analysis, J. Pharm. Biomed. Anal., 1993, 8(11), 705～710
[16] Tyrpien K., Bodzek D., Janoszks B., Application of TLC and GC-MS to the identification of Azaavenes in sewage scudges, J. Planar chromatogram., 1995, 8(1), 75～77
[17] Skett P., The design and operation of a novel device for collecting fractions from TLC plates for biological and spectroscopic analysis, J. Planar Chromatogra., 1997, (10), 4,236～266
[18] Roubal W. T., Semiautomated indexing device for removal of adsorbent from thin-layer chromatography plate, Anal. Chem., 1984, 56,595～597
[19] Kirchner J. G, The Thin-layer Chromatography, 2nd, John Wiley & Sons. Inc 1978 New York
[20] Seikel M. K., Semi-micro preparative thin-layer chromatography, J. Chromatogra., 1964, 15, (1), 115～118
[21] Pe W., Use of descending TLC for identification of cannabinoids, Bull Narc, 1985, 37, 83～86 (Anal. Abstract 1986 10e23)
[22] Pelick N., Descending thin layer chromatography apparatus, U.S.A., USP, 3449083, 1969
[23] 齐登福，荧光光度法测定中药芦荟大黄素，山东工程学院学报，2002，16(2)5～8
[24] 杨成勇，刘丽娜，分光光度法测定决明子中大黄素的含量，重庆中草药研究，2001，4(3)，45～48
[25] 缪月英，戚晓菲，双波长薄层扫描法测定决明子中蒽醌含量，佳木斯医学院学报，1996，19(1)，30～31
[26] 曹爱民，沙明，孟淑智，高效液相色谱法测定决明子中大黄酚的含量，中国中药杂志，1997，22(2)，107～108
[27] 于超，兰红梅，王宇，HPLC法测定不同产地决明子中五种葸醌类化合物，基层中药杂志，2002，16(2)，8～10
[28] 王威，张彤，周立春，高效液相色谱一蒸发光散射检测器法对大豆磷脂原料药中各种成分的含量测定，首都医药．2002，9(8)，49～50
[29] 王立新，吴启南，吴德康等，钼蓝比色法测定不同产地合子草中总磷脂的含量，中国野生植物资源，2001，20(3)，50～51
[30] 雷厉，宋志宏，屠鹏飞等，反相高效液相色谱法制备松果菊苷标准晶，色谱，2001，
19(3)，200～202
[31] 陈培榕，廖志勤，薄层层析-干柱层析技术在分离制备石油生物标志物-卟啉中的应用，分析试验室，1997，16(2)，39～41
[32] 任海彧，查丽杭，秦川，大孔树脂分离提取麻黄碱的研究离子交换与吸附，2002，18(2)，97～104
[33] 李良学，刘继德，尹业高等，雷公藤内酯甲的制备和晶体结构，高等学校化学学报，2001，22(4)，573～576
[34] 赖宇红，陈浩桉，固相萃取法在中药分析中的应用前景及其建立优化新方法的策略与步骤 广东药学，2002，12(2)，12～16
[35] Hah J., Wang D.Y., Wang D.et.al., Coupling development and elution, a new thin-layer chromatography technique, J. Chromatogr. A (im press)
[36] 王东援，金海英，薄层色谱展开机理的探讨，色谱，1995，13(1)，74～74
[37] Maxwell R.J., Yeisley S.W., Unruh J., The importance of effective predevelopment plate washing techniques ing thin-layer chromatography, J. liquid Chromatography, 1990, 13, (10), 2001～2011
[38] 徐友锋，李灵芝，陈虹，大黄中提取游离葸醌的工艺改进，武警医学院学报，2002，11(2)，76～77
[39] 陈琼华，戴汉松，苏学良，中药大黄的综合研究 ⅩⅩⅪ．大黄葸醌衍生物系统分离的改进方法，天然产物研究与开发，2001，13(3)，58～60
[40] Sadtler Standard Infrared Grating Spectra, 42597P
[41] Fairbaim J. W., El-Muhtadi F.J., Chemotaxonomy of anthraquinones in Rumex. Phytochemistry, 1972, 11(1), 263～268
[42] Sadtler Standard Infrared Grating Spectra, 42616P
[43] Choline glycerophospholipids, http//www. Cyberlipid. org
[44] Separation ofphospholipids by TLC, http//www. Cyberlipid. org