It is often said that the column should be called column chromatography separation, also known as column chromatography. We usually use silica gel or alumina as the adsorption column. Because there are too many empirical components in the column, I have collected some information about the use of chromatographic column, hoping to help you.
01. The column can be divided into: pressure, atmospheric pressure and decompression
Pressure can increase the flow rate of eluent and reduce the time of product collection, but it will reduce the number of column plates. So when other conditions are the same, the atmospheric column is the most efficient, but the time is also the longest. For example, the separation of natural compounds, a column for several months.
Decompression column can reduce the use of silica gel, which can save half or even more. However, due to a large amount of air passing through silica gel, the solvent will volatilize (sometimes there is condensation of water vapor outside the column), and some things that are easy to decompose may not be available, and water pump must be used to extract air at the same time (great noise, and long time). There have been a large number of decompression columns in the past, and they have deep feelings for it, but since they tried to pressurize, they almost never thought of decompression.
The pressurized column is a better method, similar to the atmospheric column, except that the external pressure makes the drenching agent go faster. The pressure can be supplied by compressed air, double connected ball or small air pump (just supply air to the fish tank). Especially suitable for the separation of easily decomposed samples. The pressure should not be too high, or the solvent will go too fast to reduce the separation effect. I think the pressurized column is suitable for the separation of common organic compounds.
02. The size of the column should be the best one that is thick and long
When the columns are long, the corresponding number of plates will be high. When the column is thick, the origin of the sample is small (reflected in the thin sample layer on the column), which reduces the difficulty of separation. If the column is 10 cm, and the sample is 2 cm, then the difficulty of separation can be imagined, I'm afraid it will be slowly washed with a very low polarity solvent. If the sample layer is only 0.5cm, the components can be separated easily. Of course, more silica gel and solvents will be sacrificed for the use of large columns, but these costs may not be much compared with the products (some are not environmentally friendly, but the solvent recovery and re evaporation will reduce some waste).
The column diameter to height ratio seen now is generally 1:5-10. The silica gel content in the book is 30-40 times of the sample quantity. The specific selection should be analyzed in detail. If the required components and impurities are relatively open (that is, the RF of the required components is 0.2-0.4, and the difference between impurities is more than 0.1), silica gel can be used less and small columns can be used (for example, a sample of 200mg, a column of 50px × 500px); if the difference is less than 0.1, the column should be increased, I think the diameter of the column can be increased, for example, 75px It can also reduce the polarity of the eluent and so on. It is suitable for oxygen sensitive, water sensitive and easy to decompose products.
It can be wet or dry. However, the column should be saturated with solvent at least once before the sample, because the heat released when the solvent and silica gel are saturated may be product decomposition, after all, the sensitive substance should be separated, so be careful. It is also because the separated things are sensitive, so the receiving bottle must be sealable and follow Schlenk operation.
03. Wet method and dry method
It is easy to use wet method. Generally, the sample can be dissolved with eluent, dichloromethane, ethyl acetate, etc. But the less the solvent, the better, or the solvent will become the eluent. A lot of samples are sticky before the upper column, generally it doesn't matter. However, some samples will precipitate on silica gel after loading, which is generally due to the relatively large number of samples, because silica gel is saturated with the sample, and the sample itself is a relatively good solid, which should be recrystallized first, get most of the products, and then the column. If it can't be recrystallized, it doesn't matter. Just go through it directly. The sample flows with the eluent It will dissolve.
Some samples have poor solubility, and the solvent that can be dissolved can't be put on the column (for example, DMF, DMSO, etc., will go with the solvent, and the color development is a long tail removal). At this time, the dry method must be used to put on the column. There is a saying that the amount of sample and silica gel is 1:1. I think the less it is, the better. But it should be ensured that no obvious solid particles can be seen after spin drying (that means some samples are not adsorbed on silica gel).
04. Solvent selection
Of course, it's the cheapest, safest and most environmentally friendly, so most of them choose petroleum ether and ethyl acetate. In the literature, it is written that n-hexane is too expensive. Unless you need it especially, the silver splashes. It flows faster than the lotion. However, because the polarity is very small, sometimes it is necessary. Ether can also be used, but it's easy to sleep. Keep awake and don't let the solvent run dry. Then the column will be uncomfortable.
Dichloromethane is also useful, but, you know, its adsorption with silica gel is an exothermic process, so in summer, bubbles are often generated in the column, and it will be better when the weather is cold. Methanol is said to be able to dissolve part of silica gel. Therefore, if you want to analyze the elements of the product, you should pay attention to it. It should go through subsequent treatment, such as recrystallization. Other solvents are relatively less used and should be selected according to individual needs.
Due to some reasons, most of the washing agents used are large packages (cheap). We use 10 or 25 liter plastic barrels here. We should pay attention to the low purity of these industrial products. Often can see the colored impurity from the bottom of the big barrel, other impurities can be imagined, so in a more strict column, we need to re steam the solvent. Of course, this step can be avoided after the raw materials. Anyway, there are purification methods below.
In addition, it is better to recycle the solvent after passing through the column. On the one hand, it is environmentally friendly, on the other hand, it can save part of the cost. The disadvantage is to consume a certain amount of labor. What should be noted here is that, generally, the column passes through at the same time through decompression and rotary evaporation. The proportion of petroleum ether and ethyl acetate will lead to the change of polarity due to the different volatility, which will generally lead to the increase of polarity. It is suitable for gradient elution, just the polarity is increasing. After passing the column, the solvent should be recovered at atmospheric pressure. Because some impurities with low boiling point will come out together when the column is rotated at reduced pressure. This phenomenon will be reduced when the column is rotated at atmospheric pressure. If the impurities react with the sample you want to pass below, it will be terrible.
05. About operation
1. loading column:
The piston under the column must not be coated with lubricant, which will be brought into the product by the eluent, and can be Teflon gate. There is no difference between the dry method and the wet method of column installation, as long as the column can be installed firmly. The packed column should be moderately compact (too dense, too slow for the eluent), and be uniform (otherwise, the sample will fall obliquely from one side). In the book, we can't see bubbles. I think in most cases, some small bubbles don't have a great influence, and they all come down as soon as they are pressurized. Of course, if the columns you install always have bubbles, you need to practice more. But the column is more taboo is cracking, regardless of vertical or horizontal, will affect the separation effect, or even void!
2. sample added:
Use a small amount of solvent to dissolve the sample and add the sample. After adding, open the piston below. When the solvent layer drops to the quartz sand surface, add a small amount of low-polar solvent, and then open the piston. So, two or three times, the quartz sand is basically white. Add the eluent, do not pressurize at first, the solvent of isosoluble sample has a distance from the sample layer (2-100px is enough), and then pressurize, so as to avoid the fast downward direction of the sample entrained by the solvent (such as dichloromethane).
3. Selection of eluent:
It's better to make the required points around rf0.2-0.3. Do not think that the pole climbing on the board is relatively open. If the Rf is 0.6, even if the difference is 0.2, it is not easy to separate on the pole, because the pole is in the state of climbing board many times.
You can compare the formula:
The resolution of 0.6/0.8 is certainly not as high as the third or fourth power of (0.2 / 0.3).
4. Collection of samples:
The principle of using silica gel as stationary phase passing through the column is a balance of adsorption and desorption, so if the adsorption of sample and silica gel is relatively strong, it is not easy to flow out. This will happen, with the back points coming out first and the front points coming out later. Alumina can be used as the stationary phase.
In addition, the size of the collected tube depends on the sample quantity, especially the small sample. If a large tube is used, three samples may be received for one tube. Woo woo, if you use small test tubes, it's too much work.
5. Final treatment:
As a result of the use of a large number of solvents, impurities in the products after column separation will also accumulate in the products. Therefore, if you want to send them for analysis, you'd better wash them with a small amount of solvent, because most impurities are dissolved in the solvent, and they will be basically gone once washed, and recrystallization will be carried out if necessary.