Summary of the experience in the conventional operation of TLC

Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) have the same separation principle, which is based on the different adsorption and desorption rates of different substances between mobile phase and stationary phase With the development of the times, the instrument analysis gradually replaces the traditional analysis, but in the ordinary organic synthesis and fine chemical laboratory, the traditional analysis can not be completely replaced because of its flexible, convenient application and low cost TLC method has a long history With the development of economy and the increase of foreign exchange, many laboratories have used commercial thin-layer plates (such as sepaflash thin-layer chromatography silica gel plate) Compared with the self-made silicon rubber plate, the commercialized silicon rubber plate has the characteristics of stable performance and high reproducibility, which can be used for semi quantitative analysis TLC is highly sensitive, fast, simple, cost-effective, safe and easy to operate It can be widely used in pharmaceutical, chemical, biochemical, environmental protection, public security and other scientific research and production units as well as the qualitative or quantitative detection of some substances, especially for the separation and determination of some trace or complex substances The specific uses of TLC include: 1 Quality control and impurity inspection of drugs and preparations; 2 Control of chemical reaction process, inspection of reaction by-products and analysis of intermediates; 3 Exploration of separation and purification conditions; 4 Clinical and biochemical tests; 5 Toxicant analysis; 6 Authenticity inspection of traditional Chinese medicine varieties and search for substitutes 7 Sepaflash @ thin layer chromatography silica gel plate is made of ultra pure silica gel as raw material, with unique adhesive and antifoam technology Product features: 1 Excellent manufacturing process, uniform silica gel plate, no bubbles, good adhesion, no shedding 2 Silica gel raw materials with high purity and uniform particles, less impurities such as metals, small tailing, good separation effect, high number of theoretical trays 3 Stable batch, small spot, convenient for series analysis 4 Short separation time, high sensitivity, clear spots, non-proliferation 5 Rf value is constant This paper mainly introduces the conventional operation experience of TLC, including the application of TLC, the precautions and use methods of self-made silica gel plate In Pharmacopoeia thin layer chromatography, the test solution is spotted on the thin layer plate, and developed in the developing container with the developing agent, so as to separate the components contained in the test sample The chromatogram obtained is compared with the chromatogram obtained by the same method with the reference matter, and can be scanned by thin layer scanner for identification, inspection or content determination Application of thin layer chromatography: 1 To judge whether two compounds are the same (whether they have the same moving value under the same developing condition); 2 To determine the component fraction contained in the mixture; 3 To select suitable developing agent for column chromatography and monitor the separation status and effect of column chromatography; 4 To detect the reaction process; 5 Commercial thin layer plate can be used for semi quantitative analysis; 6 Determine the appropriate reaction temperature and time to prepare CMC-Na 1 First add the CMC to 8 / 10 of the required water, let it fully dissolve and swell, then heat and boil, and then slowly add the remaining water In this way, it is not easy to form particles in the boiling process, and the boiling time is short 2 The concentration of CMC solution is 0.3-0.7%, and 0.4% - 0.5% is the most practical in practical operation When the concentration is high, if there is a heating process in the future, the board is easy to blacken if it is not careful When the concentration is low, the board is not firm If it is touched lightly, the slag will fall off It is not easy to save Moreover, the sample will be very tense and easy to get out of the hole 0.5% CMC Na is dissolved in water bath until it is fully stirred and dissolved If it is not easy to dissolve, add a few drops of ethanol before swelling, which is better to dissolve, but try not to add as much as possible, because the adhesion of CMC Na is reduced after adding ethanol 4 CMC Na solution can not be mixed with cold water after boiling, otherwise, it will turn green and mildew in a few days Note that CMC Na solution which has been placed for too long may turn yellow, and mould may appear, so it can never be used again 5 In the dissolution process of CMC Na, a magnetic stirrer which can be used for heating operation can be used, and the stirring time is about 5 hours, and the result should be satisfactory In this way, CMC Na can be dissolved and the solution is more clear If there is a suction filtration device, you can filter the CMC Na solution directly, so you don't have to wait for it to precipitate and then take the supernatant There are two other advantages: one is to save the CMC Na solution, and the other is that when you pour the filtered CMC Na solution, you don't have to worry about pouring out the insolubles in the lower layer! 6 Filter the CMC Solution, lay a thin layer of absorbent cotton on the Buchner funnel, make sure that every small hole does not leak out! Wet the absorbent cotton with distilled water, start the vacuum pump, and then pour the CMC solution safely and boldly after tightening, so as to ensure that the filtered solution is clear and transparent and does not precipitate for a long time CMC Na post-processing, many people say is filtration, or suction filtration, I think it may be very slow and easy to waste My method is centrifugation, 5000rpm centrifugation for 20min Pour out the supernatant, very clear, and eliminate the possible pollution in the filtration process Requirements for thin-layer plate 1 The carrier plate shall be smooth and clean without scratches Before use, it can be washed with detergent or soapy water, and then washed with water Criteria for judging whether the carrier plate is clean: stand up with your hands, if you find that the water is not flowing down in a stream, but in a waterfall state, then your glass has been cleaned The real washed glass will soon dry 3 After cleaning, the thin-layer plate can be cleaned with washing powder and wash repeatedly for several times If the water drops still hang, the thin-layer plate can be soaked with lotion If it can't be solved, we have to give up the glass plate Requirements for grinding and plate laying 1 Take the ratio of the weight of silica gel in thin layer chromatography to the volume of CMC Na, which is 1:4 (the ratio can make the effect of laying out silica gel plate better, not to drop slag and not to make the plate too hard), mix and grind fully until the mixture is picked up and the hammer can see that there is certain adhesion between the mixture and the hammer That's good 2 The grinding of silica gel must be in one direction A certain amount of anhydrous ethanol or acetone can be added to defoaming, or it can be properly stirred and placed in a clean container for ultrasound The effect is good 3 According to the needs of the thin-layer plate, pour a proper amount of the developed adsorbent onto the thin-layer plate, shake the adsorbent evenly with a small hammer, tilt the chromatography plate to make the adsorbent flow to one side of the chromatography plate, and then tilt the chromatography plate to make the adsorbent flow back after the adsorbent accumulates a certain amount; then repeat the above operations in the other two directions, and slightly shake the thin-layer plate 4 When laying the board, it can be poured along the middle of the board, or along a certain edge, or it can be spread on the glass board with the solution led by a glass rod, and it should be noted not to introduce small bubbles during pouring If necessary, you can hold the glass plate with 10 fingers of both hands, which makes the paste silica gel evenly distributed Especially the four corners of the carrier plate are easy to be higher than other parts of the glass plate, so special attention should be paid The surface of the bumped board should be smooth and flat without pores After the thin-layer board is laid, it must be placed on the flat table, otherwise it is difficult to ensure the uniform thickness of silica gel on the board 5 After the thin layer board is dried slightly, no obvious watermark can be seen Put it into the oven and dry it for 30 minutes with the temperature below 50 ° C and open the air, and then heat it up and dry it to dryness Pay attention to the phenomenon that the temperature rises too fast in the process of use, which is not conducive to separation The above method has better practical effect and takes less time 6 Add 3 drops of 95% ethanol to 10g adsorbent to drive away bubbles As for the developer 1 The acid of the separated sample is relatively large Generally, add acid in the developer 2 Add formic acid because the sample is acidic The amount of acid added is directly proportional to the acid of the substance Add water maybe because your sample is glycoside and buffer it with acid water The purpose is to make the spots smooth, without tail off and good spread 3 Saturation is also very important If the edge effect is very serious, you can stick the filter paper immersed in the developing agent at the bottom to the inner wall of the developing cylinder, so the saturation effect will be better 4.1) apply appropriate amount of Vaseline on the opening of chromatography cylinder to increase the sealing property; 2) determine the length of the equilibrium time of the developer by the size of the edge effect of the developer, which is generally 30 minutes 5 The polarity of organic solvent, methanol > chloroform, so in chloroform: methanol: ammonia water (10:1:0.6), if the polarity is slightly larger, the proportion of methanol can be appropriately reduced; if the polarity is too small, the proportion of methanol can be appropriately increased In addition, there is another problem In this developing agent, the amount of methanol is small, and methanol is volatile, which is easy to produce edge effect Pay special attention to the balance of developing agent and the sealing of chromatography cylinder 6 Different development systems mean that at least one of them should have different solvents (preferably solvents of different classification groups), rather than different proportions Or use different stationary phases In the work, we have studied the varieties of sulfuric acid ethanol for quantitative analysis, but all the plates with CMC are easy to be pasted, especially when the temperature is higher than 100 ℃, then we use the water plate without CMC to do it, there will be no paste phenomenon, so we can also infer that CMC is easy to react with sulfuric acid 2 However, all the plates with CMC are easy to be pasted, especially when the temperature is higher than 100 ℃, if the water plate without CMC is used instead, there will be no pasted phenomenon, but the plates without CMC are too soft, so it is easy to spot holes when sampling A good way is to adjust the concentration of CMC to 0.1%, so it is not easy to be blackened 3 It is easy to solve the problem of heating and blackening the thin-layer plate: after spraying the developer, it is not necessary to dry it in the oven, but it can be developed by blowing it on the back of the plate with a hair dryer This method is generally used in our laboratory It is simple and quick to clean If you do not want to use the oven to dry, you must use the one with a glass window When you see the color development, take it out Otherwise, it is difficult to control the color development time If the time is too long, CMC is easy to carbonize and turn black 4 The blackening of the thin layer plate is related to the excessive concentration of CMC Na If you pay attention to it, you will find that when there is concentrated sulfuric acid in the developer, it will turn black after a little longer heating time (other developers are OK) My teacher said that this is because concentrated sulfuric acid carbonizes CMC Na In this case, you only need to reduce the concentration of CMC Na properly Of course, if CMC Na is not added It's easy to break the board 5 Generally, I use a hair dryer after color rendering I need to blow it evenly The hair dryer should be far away from the board to prevent part of it from being black In addition, pay attention to the color developing agent If the color developing agent contains sulfuric acid, the heating time must be well controlled, not too long About point sample 1 I don't have any skills in sampling My tutor taught me a good way to use it Write it out for everyone to share That is, when sampling, put the index finger on the upper end of the sampling tube When the lower end of the sampling tube contacts with the silica gel plate, slightly loosen the index finger on the upper end The solution will come out of the sampling tube naturally and quickly lift the sampling tube In this way, the spots produced by repeated operation are small and even However, the sample solution should not be too concentrated If the concentration is too high, please