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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
(
错误！未指定书签。
).
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.
<
/p>
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.
<
/p>
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
< br>
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<
/p>
5mm
（高效薄层板间距为
2mm
）。点样点应尽量
小，直径一般
2-5mm<
/p>
（高效薄层板上直径为
1-2mm
）或<
/p>
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
℃<
/p>
-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 manufacturer’s
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.
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