Brief introduction of thin layer chromatography

Directory 1 Pinyin 2 English Reference 3 Overview 4 Definition of Thin Layer Chromatography 5 Instruments and Materials of Thin Layer Chromatography 5. 1 Thin Layer Plate 5.2 Sampling Device 5.3 Spreading Container 5.4 Coloring Agent 5.5 Coloring Device 5.6 Observing Device 6 Operation Method of Thin Layer Chromatography 6. 1 Preparation of Thin Layer Plate 6.2 Sampling 6.4 Coloring and Inspection 7 System Applicability Test 7./ Kloc-0/ detection sensitivity 7.2 ratio shift 1 identification 8.2 impurity inspection 9 thin-layer scanning method 10 reference 1 pinyin báo céng sè pǔ fǎ m:

2 English Reference Thin Layer Chromatography (TLC)[ Landau Chinese-English Dictionary]

Thin layer chromatography [2 1 century bilingual dictionary of science and technology]

Landau Chinese-English dictionary

Plate chromatography y[ Xiangya medical dictionary]

Thin-layer chromogen [Xiangya Medical Dictionary]

Overview of thin layer chromatography, also known as thin layer chromatography. It is a method that the adsorbent or carrier is spread evenly on a glass plate, a plastic plate or an aluminum plate to form a uniform thin layer, and after sampling and spreading, it is compared with the chromatogram obtained by the same method with an appropriate reference substance on the same plate, and the drug identification, impurity inspection or content determination can be carried out by scanning with a thin layer scanner. According to the separation mechanism, it can be divided into adsorption, distribution, ion exchange, gel filtration and other methods. It is suitable for the separation and identification of trace samples and has the characteristics of fast separation speed and high separation efficiency.

4 Definition of thin-layer chromatography Thin-layer chromatography is a method of drug identification or impurity inspection [1]. The chromatogram obtained after the test solution is placed on a thin-layer plate and the inspection is compared with the chromatogram obtained by the same method with an appropriate reference substance.

5. Instruments and materials for TLC 5. 1 TLC plate? Unless otherwise specified, the glass plate should be smooth and flat, without water droplets after washing, and dry. The most commonly used stationary phases are silica gel G, silica gel GF254, silica gel H and silica gel HF254, followed by diatomite, diatomite G, alumina, alumina G, microcrystalline cellulose and microcrystalline cellulose F254. Its particle size is generally required to be 5 ~ 40μ m. ..

Thin-layer coating can be generally divided into two types: non-adhesive and adhesive. In the former, the stationary phase is directly coated on the glass plate, while in the latter, a certain amount of adhesive is added to the stationary phase. Generally, 10% ~ 15% calcined gypsum is commonly used (CaSO4 2H2O is heated at 140℃ for 4 hours). After mixing, add a proper amount of water for later use, or use sodium carboxymethyl cellulose aqueous solution (0.2% ~ 0.5%). The stationary phase should be coated on the glass plate in a uniform thin layer meeting the thickness requirements by using a coater.

Commercial laminate? It can be divided into ordinary thin-layer board and high-efficiency thin-layer board, such as silica gel thin-layer board, silica gel GF254 thin-layer board, polyamide film, aluminum-based thin-layer board and so on. The particle size of high-efficiency thin-layer board is generally 5 ~ 7 μ m.

5.2 Micro syringe or quantitative capillary with bracket is commonly used in sampling device, so that the sampling position should be correct and centralized.

5.3 The unfolding container shall be made of glass TLC unfolding cylinder suitable for the size of the thin-layer plate, tightly covered, with smooth bottom or double grooves.

5.4 See the regulations under each variety of chromogenic agent. It can detect spots by spraying, soaking or fumigation in suitable reagent steam.

5.5 The color developing device requires the color developing agent and compressed gas to be uniform.

Spray out in the form of mist; Soaking and color development can be replaced by special glassware or suitable glass jar; The color development of steam fumigation can be replaced by a double-tank glass cylinder or a dryer of appropriate size.

5.6 Observation device The observation device is a black box equipped with visible light, short-wave ultraviolet light (254nm), long-wave ultraviolet light (365nm) and corresponding filters, and camera equipment can be attached for shooting chromatography. The light source in the black box should have enough illumination.

6 operation method of thin layer chromatography 6. 1 preparation of thin layer plate made by ourselves? Unless otherwise specified, grind 1 part stationary phase and 3 parts water in the same direction in a mortar, remove surface bubbles, pour them into a coating machine, and the coating machine moves smoothly on a glass plate (thickness 0.2-0.3 mm) for coating, take out the glass plate coated with a thin layer, then dry it on a horizontal platform at room temperature, and then dry it at110. Check its uniformity before use (it can be observed by transmitted light and reflected light).

Commercial laminate? Generally, it should be activated at 1 10℃ for 30 minutes before use. Polyamide film does not need to be activated. The thin aluminum plate can be cut as required, but it should be noted that the silica gel layer at the bottom of the cut thin aluminum plate must not be damaged. If it is polluted by impurities in the air during storage, it can be pre-washed in a spreading container with a suitable solvent before use, activated at 1 10℃ and put into a dryer for later use.

6.2 Unless otherwise specified, use a spotter to spot the sample on a thin plate, usually a point. The base line of the spot is 2.0cm from the bottom, and the spot diameter is 2 ~ 4 mm (1 ~ 2~4mm for high-efficiency thin-layer plate). The spacing between points shall not affect the detection, and is generally 1.0 ~ 2.0 cm (the high-efficiency thin-layer plate shall not be less than 5mm). Care must be taken not to damage the surface of the sheet when sampling.

6.3 If the unfolding drum needs to be filled with developer in advance, enough developer can be added to the drum. When necessary, stick two filter paper strips with the same height and width as the cylinder on the wall, one end of which is immersed in the developing agent and covered with a top cover to make the system balanced or operate according to the regulations under each variety.

Put the thin-layer plate containing the test sample into the developing agent cylinder, immerse it in the developing agent to a depth of 0.5 ~ 1.0 cm at the bottom of the thin-layer plate (do not immerse the sample in the developing agent), and seal the top cover until it is developed to a suitable development distance (for a thin-layer plate of 20cm, the development distance is generally10 ~15 cm). 10cm high-efficiency thin-layer board, the laying distance is generally about 5cm), take out the thin-layer board, dry it, and inspect it according to the regulations under each variety.

Can be expanded in one direction, that is, in one direction; It can also be unfolded in two directions, that is, unfolded and taken out in one direction. After the developer is completely volatilized, rotate the thin-layer plate by 90 degrees, and then unfold it with the original developer or another developer. It can also be expanded many times.

6.4 Fluorescence quenching method can be used for color development and inspection of fluorescent thin-layer plates; Ordinary thin-layer plates, colored substances can be directly tested, and colorless substances can be tested by physical or chemical methods. The physical method is to detect the fluorescence color and intensity of spots; Chemical method generally uses chemical reagent to develop color, and immediately covers the glass sheet with the same size for inspection.

7 System Applicability Test According to the requirements of various varieties, the system applicability test of thin-layer chromatography was carried out, which made the detection sensitivity, specific shift value (Rf) and spot separation efficiency meet the requirements.

7. 1 detection sensitivity refers to the minimum amount of substances to be detected in the test solution during impurity inspection. Generally, the solution is diluted several times with the control solution, and the test solution and the control solution are sampled and unfolded. Under the specified chromatographic conditions, they are examined on the same thin-layer plate, and the former should show clear spots.

7.2 The specific displacement value (Rf) refers to the ratio of the distance from the baseline to the center of the developing spot to the distance from the baseline to the front end of the developer. In identification, the specific displacement values of the main spots of the test solution and the reference solution can be compared, or the position of the main spots or impurity spots can be explained by the specific displacement values.

Unless otherwise specified, the specific displacement value (Rf) should be between 0.2 and 0.8.

7.3 When identifying the separation efficiency, the chromatogram of the mixed control solution made of reference substance and drug reference substance with similar structure should show two obviously separated spots. When choosing the impurity inspection method, the impurity reference substance can be dissolved in the diluted control solution of the test sample itself to make a mixed control solution, or the impurity reference substance can be dissolved in the control solution of the component to be tested to make a mixed control solution, or the solution obtained by properly degrading the test sample can be used. After the above solution is spotted and spread out, the chromatogram should show two clearly separated points.

8 determination method 8. 1 TLC identification, the sample and the control solution with the same concentration can be placed on the same thin layer plate, unfolded and checked. The color (or fluorescence) and position (Rf) of the main spots displayed by the control solution should be consistent with the main spots of the control solution, and the size and color depth of the main spots should be roughly the same. Or the test solution is mixed with the reference solution in equal volume, which should be a single and compact spot; Or compare the reference substance with similar chemical structure to the test solution with the main spots of the test solution, and the Rf of the two should be different, or mix the above two solutions in equal volume, and two obviously separated spots should be displayed.

8.2 Impurity inspection When TLC is used for impurity inspection, impurity reference method, self-dilution control method of test solution or both methods can be used. Other spots except the main spots of the test solution should be compared with the corresponding main spots of the impurity control solution or the impurity control solution with a series concentration, or with the corresponding main spots of the test solution diluted by itself or the control solution with a series concentration, and should not be deeper.

Usually, the number of spots and the amount of individual impurities should be specified, and when a series of self-dilution control solutions are used, the estimated total amount of impurities can also be specified.

Thin-layer scanning method refers to irradiating a thin-layer plate with light with a certain wavelength, scanning spots that absorb ultraviolet light or visible light or emit fluorescence after excitation, and using scanning atlas and integral data for drug identification, impurity inspection or content determination.

Unless otherwise specified, thin-layer scanning method can choose reflection method, absorption method, fluorescence method, dual-wavelength or single-wavelength scanning according to the structural characteristics and instructions of various thin-layer scanners and combined with specific conditions. The determination methods include internal standard method and external standard method. Because there are many factors affecting the results of TLC scanning, the quantitative determination of TLC scanning should be carried out on the same plate as the reference substance under the conditions of ensuring the number of spots on the calibration curve, unfolding, scanning, determination and calculation.