A large summary of the practical techniques of thin-layer chromatography.

related topicsthin layer analysis (TLC) technologyobjective:1. Pharmacopoeia: thin layer
chromat spectrum
method will be the test solution on the thin sheet, in the expander container with expander, so that the ingredients contained in the test is separated, the resulting chromatography and the control of the matter according to the chromatography obtained by the same method, and can be scanned with a thin layer scanner for identification, inspection or content determination..2. If you are doing identification, the system applicability of thin layers is mainly to do testing limits and separation;.3. If you are doing content determination, for example, with thin layer scanning method, thin layer system applicability should do linear range, plate precision, plate precision, recovery rate;.4. Hand-paved plates are suitable only for qualitative analysis and not for separation of quantitative;.5. Chemical drugs are generally made of related substances, the need for a certain amount of drug load, so to increase the thickness appropriately;.6. Chinese medicine is generally more difficult to separate and requires sheeting to increase the degree of separation;.7. Hand-paved plates are commonly used as silicone G-plates and silicone CMC-Na plates. The former is calcined gypsum (the gypsum is baked at 140 degrees C for 3-4 hours) and silicone according to 1-1.3:10 mixed evenly. Each serving of silicone G plus water 2-3 parts into a paste, you can use. The operation of the latter has been discussed by the prawns..8. If you lay the board for analytical purposes, you must be very careful, if only natural pharmacy kind of rough examination of the fractional purity obtained by the column, it is not necessary to be so complex, that is, the speed can be fast, the requirements of the board does not need to be so high;.9. Simple hand-paved,
skills
very demanding, if there is a paver (also completely manual kind), the paved board can basically guarantee the same.10. To spray ethanol sulfate and quantify the best paving plate;11. Ethanol sulfate color for quantitative analysis of the varieties, but where the addition of CMC-Na plate is easy to bake, especially when the temperature is higher than 100 degrees, after the use of water plate without CMC auxiliary to do, there will be no baking phenomenon, so it can also be inferred that CMC is easy to paste with sulfuric acid reaction. Feel the key to the auxiliary water board is silicone G and water ratio to reach about 1:3.5, and after grinding to be coated as soon as possible, can not be easy to solidify and difficult to coat.Unfold: 12. Pharmacopeia: Unfold containers Should be expanded using a glass thin-layer chromatography suitable for the size of a thin sheet, with a tight lid, a smooth bottom, or a double groove. Up-line unfolding can generally be expanded using a dedicated flat-bottomed or double-slot expand cylinder suitable for the size of a thin sheet, which must be closed when expanded. Expand horizontally with a dedicated horizontal expand cylinder.13. Pharmacopoeia: the sample of the thin sheet into the expander of the expander, immersion in the depth of the expander 5mm from the origin is appropriate (do not invade the sample point in the expander), seal the top cover, to be expanded to a specified distance, in addition to other provisions, generally 8 to 15 cm, solvent front to reach the prescribed spread, remove the thin plate, dry, according to the provisions of the original detection.14. Choice of expander (separation monosome): (1) coarse, i.e. when your sample polarity range is relatively large, you can directly use a less polar flow phase. The silicone from the front, origin, and between the front and the origin is then scraped and extracted. In this way, the sample is divided into several parts, and the polarity difference between the parts is relatively small. These sections are then subdivided
TLC
.(2) segment. After roughing, we are generally able to count the polarity of each part. For example, samples washed to the forefront require a flow phase with a smaller polar strength, while a dead adsorption phase requires a higher intensity flow phase.we have initially determined the polar strength of the flow phase, we can design a few solvent systems of our own. When selecting the composition of the flow phase, you can refer to snyder's solvent classification, from the proton subject, proton to the body and even-polar action of the solvent to choose one, and then select a strength regulator.
, of course, can also refer to the mobile phase used in the reference. By selecting the flow phase to be used, we can obtain a ratio of the flow phase (if it is a binary flow phase) based on the polarity intensity we have initially determined. In the case of a three-way and above mobile phase, the ratio of the flow phase can be selected using the seven solvent system schemes of Glajch and Youngstrom. Then select the combination from which the separation works better.can first be groped using a micro-circular method (with the sample point in the middle and then diffusing the flow phase with capillary tubes from the origin to the circumferentry) and a small plate experiment, which can then be applied to large preparation
TLC
.I also used the ring method directly, also known as the ring unfold, to prepare. There should have been a dedicated U-shaped unfolding chamber to expand, but because we don't have this equipment here, I generally use a simple method: the sample point in the center, and then with the tip of the dropper constantly to the center of the drop to the flow phase. Samples are separated by different con center circles.
three benefits:
(1) As long as the operation is careful, the con center circles are really very round, i.e. there will be no edge effect in linear analysis, etc.
(2) separation effect is better, the trailing phenomenon is less than linear analysis.
(3) because the center-expanded Rfc is the square root of the linearly expanded RF, which is greater than the linearly expanded rf, the division is more open. The downside is that you need to be more careful when scraping individual components off the board so that they don't mix. 15. Solvent demission occurs when the polarity of the expander varies widely, especially when the proportion of the polar components is very small. In this case, there is no significant difference between the saturated and unsaturated unsaturated unsaturated unscathed, and similar layering tends to occur on thin plates, so the system can only be adjusted by changing the unroving system. 16. Methanol consumption is small, and methanol is volatile, easy to produce edge effects, pay special attention to the balance of the expander and the sealing of the analyzer. 17. If there is still enough expander left in the unfolder after one unride, can the second plate be expanded? think it's best not to. Generally speaking, in order to get a better separation effect, should first saturate, the second use if also saturated will cause the other side of the residual expander attached to the board, resulting in an impact. Moreover
organic
solvents are generally more volatile. 18. How can the thin sheet of their own hand shop not buy the efficient plate to use, after use can also use a more polar expander will expand the point, the second spread run, so that it can be reused. Coloring: 19. Pharmacopoeia: Coloring device Spray coloring requires the use of compressed gas to make the color agent evenly finely mist sprayed out, impregnated color can be replaced by special glassware or with a suitable glass cylinder, steam fumigation color can be replaced by a two-slot glass cylinder or a suitable size
drying
. 20. The problem of thin sheet
heating
turning black is actually easy to solve: when the color-showing agent is sprayed, you don't have to bake in the oven, you can use an electric hair dryer to blow on the back of the plate to show color. We generally use this method in our laboratory, simple and clean. If you do not want to use the oven oven, be sure to use a glass window, when you see the color on the take out, otherwise it is not good to control the coloring time, too long, CMC easy to carbonize black. 21. In my experience, the thin-layer version blackening is related to the concentration of CMC-Na is too large, if you pay attention, you will find that when the color agent has a strong sulfuric acid, the heating time will be slightly black (other colorants are all right), my teacher said that this is because the thick sulfuric acid carbonized CMC-Na, in fact, you only need to reduce the concentration of CMC-Na, of course, if you do not add CMC-Na easy to break the plate. 22. Colorant R6: 100mgDC Red 19 (dye index number No 45170) dissolved in 150 ml of diethylene ether, 70 ml of 95% ethanol and 15 ml of water. This solution can be stored for one week. Immediately after the thin sheet spray colorant R6, observe and record at 366nm. problems and applications: 23. The plate will crack, one may be because the proportion of silicone is too large, and the other may be that the plate will be dried at room temperature, and then resuspended in the oven. If the paving is soon at a higher temperature, the chance of a crack is higher. 24. After the plate is laid, the natural cool drying is best, must be from the back of the glass plate is also dry, pay attention to be leveled, the best control of air flow; 25. "How did the drying plate break up in the oven and there's any way to avoid it?" Your plate is not completely dry, the surface is dry, but the middle is not dry, so you put directly into the oven at 105 degrees baking, of course, will crack, so you should first use low temperature about 40 degrees about 30 minutes, and then with 105 degrees of live, you can solve this problem. 26. Plate cracking may also be caused by floc-like precipitation in CMC-Na. 27. But any plate with CMC is easy to bake, especially when the temperature is above 100 degrees, if you use water plate without CMC auxiliary to do, there will be no baking phenomenon, but not CMC auxiliary plate is too soft, point-and-point easy to point holes, there is a good way to adjust the concentration of CMC to 0.1%, it is not easy to blacken. 28. System applicability: If you are doing identification, the system applicability of thin layers is mainly to do detection limits and separation. if you are doing content measurement, for example, with thin-layer scanning method, you should do linear range, plate precision, plate precision, recovery rate. . CMC-Na solution preparation: 29. Pharmacopeia stipulates that THEC-Na concentration is 0.2% to 0.5%, and 0.4% to 0.5% is most practical in practice; The solvent of the CMC-Na solution is applied to distilled water to minimize contamination. 30. The preparation of CMC-Na solution, according to practical experience, the solution concentration is generally rationed to 0.3%, the need to lay a thick plate can be rationed to about 0.5%, the sheet can be rationed to 0.2%. CMC-Na dissolution is best naturally dissolved (concentration is not too high), can also be heated in the water bath solubility (when used or
filtration
a good, to avoid the spread of the plate has a hemp pit). 31. When the CMC-Na solution is made, the distilled water can be taken first, and then the weighed CMC-Na is evenly sprinkled in the water, stirred with a glass rod, if the operation is good, can be dissolved without heating very well; the first key point, your CMC-Na solution must be well-made and well placed, and can only be cleaned after complete layering. On the liquid to clarify transparent, too long CMC-Na may yellow, if mold appears, absolutely can not be used; 33. If you have a filtration device you can filter the CMC-Na solution directly, you don't have to wait for it to precipitate and then take the liquid (hey hey, I'm an acute child), and there are two benefits: one is to save the CMC-Na solution, and the other is to reverse the filtered CMC-Na solution without having to worry about pouring out the insolubles in the lower layer. 34. During the dissolution of the CMC-Na, a magnetic agitator that can be used for heating operation can be used, stirring for approximately 5 hours, with satisfactory results. And this can make CMC-Na dissolve, and the solution is more clarified; CMC-Na reprocessing, many people say filtering, or filtration, I think it may be slow, and easy to waste. My approach is centrifugation, 5000rpm centrifugation 20min. Pour out the liquid (very clear, but also eliminate possible contamination during filtration). Even more valuable is that I can collect CMC-Na below that is not fully dissolved. Continue to add to the water, you can also make. 35. Preparation of 4% to 5% of methyl cellulose (CMC): called CMC, dissolved in cold water, stirred while heated, until it becomes a clear, transparent solution. 36. There's a way to filter the CMC-Na solution, tile a thin layer of skimmed cotton on the Breitling
flepper
, and make sure that every hole doesn't miss out! Wet the degreased cotton with distilled water, start the
spoulation pump
, after pumping can rest assured that the bold inverted CMC-Na solution, to ensure that the filtered solution clear and transparent, and long-term placement does not precipitate. < br.