2.2.27. Thin-layer chromatography(中英).doc

2.2.27. Thin-layer chromatography 薄层色谱法Thin-layer chromatography is a separation technique in which a stationary phase consisting of an appropriate material is spread in a uniform thin layer on a support (plate) of glass, metal or plastic. Solutions of analytes are deposited on the plate prior to development. The separation is based on adsorption, partition, ion-exchange or on combinations of these mechanisms and is carried out by migration (development) of solutes (solutions of analytes) in a solvent or a suitable mixture of solvents (mobile phase) through the thin-layer (stationary phase). 薄层色谱法是一种分离技术，这种技术采用由适当物质作为固定相均匀涂布在玻璃、金属或塑料的支持物上。在展开前，供试品液点样在薄层板上。分离原理是供试品溶解在溶剂中或合适的混合溶剂中（流动相）在薄层上（固定相）通过吸附、分配、离子交换或以上机制共同作用移动（展开）来实现的。APPARATUS 仪器Plates. The chromatography is carried out using pre-coated plates as described under Reagents (Error! No bookmark name given.). 薄层板 色谱法是按照4.1.1试剂项下描述的涂层板来进行的。Pre-treatment of the plates. It may be necessary to wash the plates prior to separation. This can be done by migration of an appropriate solvent. The plates may also be impregnated by procedures such as development, immersion or spraying. At the time of use, the plates may be activated, if necessary, by heating in an oven at 120 C for 20 min. 薄板预处理：在分离前用适当的溶剂来清洗薄板。可用适当的溶剂洗脱。在进行展开、浸渍或喷雾显色过程中要将薄板浸透。使用时，如果需要，要在120干燥箱中加热20min活化薄板。Chromatographic tank with a flat bottom or twin trough, of inert, transparent material, of a size suitable for the plates used and provided with a tightly fitting lid. For horizontal development the tank is provided with a trough for the mobile phase and it additionally contains a device for directing the mobile phase to the stationary phase. 层析缸：使用适合薄层板大小的无活性、透明物质制成的层析缸，并有严密的盖子，底部平整，或有双槽。水平展开的层析缸有一个装展开剂的槽，并且额外有一个装置用来引导展开剂到固定相。Micropipettes, microsyringes, calibrated disposable capillaries or other application devices suitable for the proper application of the solutions. 微量加液器、微量注射器、有刻度的一次性毛细管：或其它适合于应用溶液的装置。Fluorescence detection device to measure direct fluorescence or the inhibition of fluorescence. 荧光检测装置：用来测量直接荧光或荧光抑制Visualisation devices and reagents. Suitable devices are used for derivatisation to transfer to the plate reagents by spraying, immersion or exposure to vapour and, where applicable, to facilitate heating for visualisation of separated components. 可视化设备和试剂：合适的装置通过喷雾、置蒸汽中或浸渍来检测分离斑点，如果可以的话，加热会促进分离斑点的可视性。Documentation. A device may be used to provide documentation of the visualised chromatogram, for example a photograph or a computer file. 文件：应用能够提供可视化色谱图（例如照片或电子文件）文件的装置。METHOD 方法:Sample application. Apply the prescribed volume of the solutions at a suitable distance from the lower edge and from the sides of the plate and on a line parallel to the lower edge; allow an interval of at least 10 mm (5 mm on high-performance plates) between the centers of circular spots and 5 mm (2 mm on high-performance plates) between the edges of bands. Apply the solutions in sufficiently small portions to obtain circular spots 2-5 mm in diameter (1-2 mm on high-performance plates) or bands 10-20 mm (5-10 mm on high-performance plates) by 1-2 mm. 点样：取规定体积的溶液点样，要距离底边和侧边合适的位置上，并且点样基线与底边平行；点样点一般为圆点，各圆点中心间的距离至少为10mm（高效薄层板间距为5mm），条带间边距5mm（高效薄层板间距为2mm）。点样点应尽量小，直径一般2-5mm（高效薄层板上直径为1-2mm）或10-20mm长的条带（高校薄层板为5-10mm）。In a monograph, where both normal and high-performance plates may be used, the working conditions for high-performance plates are given in the brackets after those for normal plates. 正文中，正常板和高效薄层板都使用时，高效薄层板的展开条件应在正常板的展开条件后用 注明。Vertical development. Line the walls of the chromatographic tank with filter paper. Pour into the chromatographic tank a sufficient quantity of the mobile phase for the size of the tank to give after impregnation of the filter paper a layer of appropriate depth related to the dimension of the plate to be used. For saturation of the chromatographic tank, replace the lid and allow to stand at 20-25 C for 1 h. Unless otherwise indicated in the monograph, the chromatographic separation is performed in a saturated tank. Apply the prescribed volume of solutions as described above. When the solvent has evaporated from the applied solutions, place the plate in the chromatographic tank, ensuring that the plate is as vertical as possible and that the spots or bands are above the surface of the mobile phase. Close the chromatographic tank, maintain it at 20-25 C and protect from sunlight. Remove the plate when the mobile phase has moved over the prescribed distance, measured between the points of application and the solvent front. Dry the plate and visualise the chromatograms as prescribed. 垂直展开： 沿着层析缸的壁贴滤纸。向层析缸中加入适合层析缸大小的足够量的流动相,根据使用的薄层板的大小浸没一定深度的滤纸。为了饱和层析缸，密封顶盖，在20-25放置1h。 如果无其它说明，在饱和层析缸中进行色谱分离。应用以上提到的规定体积的溶剂。当溶剂已从点样溶液中蒸发，则将薄层板垂直置于层析缸中，确保斑点或条带要在流动相液面以上。密封层析缸，避光放置在20-25下。待展开至规定距离取出薄层板，测定点样点与溶剂前沿的距离。晾干，按规定检视色谱图。For two-dimensional chromatography, dry the plates after the first development and carry out a second development in a direction perpendicular to that of the first development. 对于双向色谱法，第一次展开后干燥薄层板，将薄层板转动90，按第一次展开方法进行第二次展开。Horizontal development. Apply the prescribed volume of the solutions as described above. When the solvent has evaporated from the applied solutions, introduce a sufficient quantity of the mobile phase into the trough of the chamber using a syringe or pipette, place the plate in the chamber after verifying that the latter is horizontal and connect the mobile phase direction device according to the manufacturers instructions. If prescribed, develop the plate starting simultaneously at both ends. Close the chamber and maintain it at 20-25 C. Remove the plate when the mobile phase has moved over the distance prescribed in the monograph. Dry the plate and visualise the chromatograms as prescribed. For two-dimensional chromatography, dry the plates after the first development and carry out a second development in a direction perpendicular to that of the first development. 水平展开 应用以上规定体积的溶剂。当溶剂已从点样溶液中蒸发，利用注射器或移液管将足量的流动相引入槽中，将薄层板水平放入分离室中，根据产品说明书连接流动相引导装置。如果有规定，则同时两侧展开薄层板。密封层析缸，避光放置在20-25下。待展开至规定距离取出薄层板，晾干，按规定检视色谱图。对于双向色谱法，第一次展开后干燥薄层板，将薄层板转动90，按第一次展开方法进行第二次展开。VISUAL EVALUATION 目测Identification. The principal spot in the chromatogram obtained with the test solution is visually compared to the corresponding spot in the chromatogram obtained with the reference solution by comparing the colour, the size and the retardation factor (RF) of both spots. 鉴别供试品溶液色谱图上的主斑点与对照品溶液色谱图上对应的斑点比较颜色、大小和比移值（Rf）。The retardation factor (RF) is defined as the ratio of the distance from the point of application to the centre of the spot and the distance travelled by the solvent front from the point of application. 比移值（Rf）的定义为是基线到展开斑点中心的距离与点样点至展开剂前沿的距离的比值。Verification of the separating power for identification. Normally the performance given by the suitability test described in Reagents (Error! No bookmark name given.) is sufficient. Only in special cases an additional performance criterion is prescribed in the monograph. 鉴别的分离能力的验证 一般4.1.1 试剂部分描述的适应性检查提供的性能就足够了。只有在特殊情况下，专论中会有其它的性能标准。Related substances test. The secondary spot(s) in the chromatogram obtained with the test solution is (are) visually compared to either the corresponding spot(s) in the chromatogram obtained with the reference solution containing the impurity(ies) or the spot in the chromatogram obtained with the reference solution prepared from a dilution of the test solution. 有关物质检查供试品溶液色谱中的除主斑点外的其它斑点与含杂质对照品溶液和供试品溶液自身稀释得到的对照品溶液中的相应斑点比较。Verification of the separating power. The requirements for the verification of the separating power are prescribed in the monographs concerned. 分离能力的验证 分离能力验证的要求在相关专论中有提供。Verification of the detecting power. The detecting power is satisfactory if a spot or band is clearly visible in the chromatogram obtained with the most dilute reference solution. 检测能力的验证 在大多稀释的对照品溶液的色谱图上的斑点或条带如果清晰可见，则认为检测能力良好。QUANTITATIVE MEASUREMENT 定量测定The requirements for resolution and separation are prescribed in the monographs concerned. 分辨率和分离度的要求在相关专论中有描述。Substances separated by thin-layer chromatography and responding to UV-Vis irradiation can be determined directly on the plate, using appropriate instrumentation. While moving the plate or the measuring device, examine the plate by measuring the reflectance of the incident light. Similarly, fluorescence may be measured using an appropriate optical system. Substances containing radionuclides can be quantified in 3 ways: either directly by moving the plate alongside a suitable counter or vice versa (see Error! No bookmark name given.), by cutting the plates into strips and measuring the radioactivity on each individual strip using a suitable counter or by scraping off the stationary phase, dissolving it in a suitable scintillation cocktail and measuring the radioactivity using a liquid scintillation counter. 可由薄层色谱分离并且对紫外照射有反应的物质可以通过合适的仪器来直接在薄层板上检测。移动薄层板或检测仪器，通过测量入射光的反射率或透光率来检测薄层板。同样利用合适的光学系统则可以测量荧光。含有放射性核素的物质可以通过三种方式进行定量：可直接通过移动薄层板靠在一合适计数器旁或反之（见放射性药品制备 0125），或通过将薄层板切成小条，利用一合适的计数器测量每个小条的放射性，或刮去固定相，将其溶解于合适的闪烁体中，利用液体闪烁计数器测量放射性。Apparatus. The apparatus for direct measurement on the plate consists of: 仪器 直接测量薄层板的仪器包括： a device for exact positioning and reproducible dispensing of the amount of substances onto the plate; 一个可以准确定位，并且重复分配薄层板上物质的量的仪器； a mechanical device to move the plate or the measuring device along the x-axis or the y-axis; 一个可以移动薄层板的机械装置或者沿着X-轴或Y-轴的测量设备； a recorder and a suitable integrator or a computer; 记录仪和一个合适的积分仪或计算机； for substances responding to UV-Vis irradiation: a photometer with a source of light, an optical device able to generate monochromatic light and a photo cell of adequate sensitivity are used for the measurement of reflectance or transmittance; if fluorescence is measured, a suitable filter is required to prevent light used for excitation from reaching the detector while permitting emitted light or a specific portion thereof to pass; 对紫外照射有反应的物质：有光源的光度计，一个可以产生单色光的光学设备和一个灵敏的光电元件用来测量反射率或透光率。如果测量荧光，额外还需要一个单色光滤光器，来选择一个特殊的发光光谱区。 for substances containing radionuclides: a suitable counter for radioactivity. The linearity range of the counting device is to be verified. 含放射性的物质：放射性计数器。计数设备的线性范围要进行验证。Method. Prepare the solution of the substance to be examined (test solution) as prescribed in the monograph and, if necessary, prepare the reference solutions of the substance to be determined using the same solvent as in the test solution. Apply the same volume of each solution to the plate and develop. 方法 按规定制备供试品溶液，如果必要，用同样溶剂制备对照品溶液。点样相同体积的每种溶液于薄层板上，展开。Substances responding to UV-Vis irradiation. Prepare and apply not fewer than 3 reference solutions of the substance to be examined, the concentrations of which span the expected value in the test solution