Operation method of thin layer chromatography
Thin layer chromatography is the application of a suitable stationary phase to a glass plate, plastic or aluminum substrate to form a uniform thin layer. After being sampled and expanded, it is compared with a suitable control according to the chromatogram obtained by the same method for the identification of the drug, the impurity inspection or the content determination method.
(1) Preparation of thin layer plate Unless otherwise specified, 1 part of stationary phase and 3 parts of water are ground and mixed in one direction in a mortar to remove bubbles on the surface, and then poured into a coater to be smooth on the glass plate. Ground the applicator for coating (thickness 0.2-0.3mm), remove the thin-coated glass plate, dry it on the water platform at room temperature, and then dry at 110 ° C for 30 minutes, that is, desiccant In the dry box for spare. Check the uniformity before use (viewable by transmitted light and reflected light).
(2) Spotting Unless otherwise specified, spotting on a thin layer plate with a spotter is generally a dot. The base of the sample is 2.0 cm from the bottom edge. The diameter of the sample and the distance between the points are the same as paper chromatography. The distance between the visible spots and the spread of the spots is not affected by the detection. Care must be taken not to damage the surface of the thin layer when spotting.
(3) If the unfolding cylinder is to be saturated with the developing agent in advance, a sufficient amount of developing agent can be added to the cylinder, and two filter strips of the same height and width as the cylinder are attached to the wall, one end is immersed in the developing agent, and the sealing cylinder is sealed. The top cover allows the system to be balanced or operated as specified by the text. Place the thin layer plate of the sample into the developing agent of the developing cylinder, and immerse the developing agent at a depth of 0.5 to 1.0 cm from the bottom edge of the thin plate (do not immerse the sample in the developing agent), seal the cylinder head, and wait Expand to the specified distance (usually 10 to 15 cm), take out the thin layer plate, dry it, and test according to the regulations under each item.
(4) Thin layer scanning can be used if a chromatographic spot is scanned by a thin layer scanner or the chromatographic spot is directly quantified on a thin layer. The thin layer scanning method can be based on the structural characteristics and usage instructions of various thin layer scanners unless otherwise specified.
In combination with the specific case, the absorption method or the fluorescence method is selected to scan with a dual wavelength or a single wavelength. Since there are many factors affecting the results of thin layer scanning, the test sample and the reference substance should be scanned on the same thin layer after the spot of the test sample is linear within a certain concentration range, and then compared. Calculate the quantification to reduce the error. For all kinds of test samples, only the thin layer chromatography with good resolution and reproducibility can obtain satisfactory results.
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