Standard operating procedure for thin-layer chromatography.

: The Second Edition of the 2000 Edition of the Pharmacopoeia of the People's Republic of China.
content: . 1. Brief description:
thin layer
chromatography
method, is the appropriate fixed phase coated on the glass plate, into a uniform thin layer. After the sample is equally sampled and unfolded, it is compared with the chromatography obtained by the appropriate control in accordance with the same method for the identification of drugs, impurity examination or content determination methods. 2. Instruments and materials:
2.1 glass plate: unless otherwise specified, with 5cm×20cm, 10cm×20cm or 20cm×20cm specifications, requiring smooth, flat, washed without water beads, drying.
2.2 Fixed phase or carrier: the most commonly used are silicone G, silicone GF, silicone H, silicone HF254, followed by diatomite, diatom g, alumina, alumina G, microcrystalline cellulose, microcrystalline cellulose F254 and so on. Its particle size, generally requires a diameter of 10 to 40 m. Thin coating, generally can be divided into non-adhesive and adhesive-containing two, the former is fixed phase directly coated on the glass plate, the latter is tied to the fixed phase to add a certain amount of adhesive, generally commonly used 10-15% caster (CaSO4) 2H2O baked at 140 degrees C for 4 hours), mixed with water in moderation, or with methyl cellulose sodium water solution (0.5-0.7%) in moderation into a paste, evenly coated on the glass plate. There is also a thin layer containing a certain amount of unfolding fluid or buffer.
2.3 Coater: The fixed phase or carrier should be coated on the glass plate with a uniform thin layer that meets the thickness requirements.
2.4 point sampler: a microsysring device or timed capillary commonly used with
sort stand
should enable the point sample position to be properly concentrated.
2.5 Unfold Chamber: The thin-layer chromatography cylinder should be expanded using glass suitable for the size of a thin sheet plate, with a tight lid, unless otherwise specified, the bottom should be flat and smooth and easy to observe. . 3. Operation method:
3.1 thin sheet preparation: unless otherwise specified, 1 fixed phase and 3 parts of water in the research in one direction grinding mix, remove the surface of the bubble, poured into the coater, in the Move the coater smoothly on the glass plate for coating (thickness 0.2-0.3mm), remove the thin layered glass plate, dry it at room temperature on a water platform, and bake at 110 degrees C for 30 minutes. That is,
drying
drying agent in
drying
the drying box. Check its uniformity before use (which can be viewed by transmission and reflected light).
3.2 points sample: unless otherwise specified, with dot sample sample on the thin sheet, generally a dot, dot-like baseline from the bottom edge 2.0cm, dot-like diameter of 2-4mm, point-to-point distance of about 1.5-2.0cm, point-to-point distance visual speckle diffusion situation to detect without impact. Care must be taken not to damage the thin surface when ordering.
3.3 Unfolding: If the unfolder needs to be saturated with an expander in advance, a sufficient amount of expander can be added to the chamber and two
filter papers
as high and wide as the chamber can be attached to the wall, one end immersed in the expander, sealing the cover on the top of the chamber, so that the system is balanced or operated as specified in the body. Place the thin sheet of the sample in the expander of the unfolder, immerse the expander at a depth of 0.5-1.0cm from the bottom edge of the thin sheet (do not immerse the sample point in the expander), seal the chamber cover, etc. to the specified distance (generally) For 10-15cm), take out the thin sheet, dry, according to the provisions of each variety of detection, if you need to use a thin layer scanner for scanning out of chromatography spots, or directly on the thin layer of chromatography spots for scanning quantification, you can use thin layer scanning method. The method of thin layer scanning, unless otherwise specified, can be based on the structural characteristics of various thin-layer scanners and instructions for use, combined with specific circumstances, the selection of absorption or fluorescence method, with double-wavelength or single-wavelength scanning. Because there are many factors affecting the results of thin layer scanning, it is possible to expand the test subject and
control product
on the same thin layer after scanning, comparison and calculation of quantification, in order to reduce the error. A variety of test products, only to obtain a good degree of separation and reproducible thin layer chromatography, in order to obtain satisfactory results.
4. Precautions:
4.1 glass plate should be washed without hanging water beads, smooth and flat.
4.2 paving should be uniform, suitable thickness, and at room temperature after drying at 110 degrees C to be active for 30 minutes, placed in a drying box with desiccant or dryer spare.
4.3-point sample point is generally a dot and cannot be too large.
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