Introduction to column chromatography

Introduction to column chromatography
First, the introduction

Ion exchange chromatography, gel filtration chromatography and affinity chromatography are three classical chromatographic techniques for separating proteins, depending on the principle of packing matrix and sample exchange.



Second, skills and methods

1. Selection of column chromatography operation methods

At present, the operation mode of column chromatography separation mainly includes three modes of atmospheric pressure separation, vacuum separation and pressure separation. Atmospheric separation is the simplest separation mode that is convenient and simple, but has a long elution time.

Although the separation under reduced pressure can save the amount of filler used, the solvent evaporates due to the large amount of air passing through the packing, and sometimes there is condensation on the outside of the column, and some easily decomposable compounds are difficult to obtain, and it is also necessary to use a water pump or Vacuum pumping.

Pressurization separation can speed up the flow rate of the eluent and shorten the elution time of the sample. It is a better method, similar to the atmospheric column, except that the pressure is applied to make the eluent elute faster. The pressure can be supplied as compressed air, double-ball or small air pump.



2. Selection of column specifications

There are various types of column chromatography separation columns available on the market. When the column is long, the corresponding number of plates is high, and the separation is good. At present, the diameter of the column on the market generally ranges from 1:5 to 10. In actual use, the amount of filler is generally 30 to 40 times that of the sample. The specific selection should be analyzed according to the nature and content of the sample.

If the separation of the required components and impurities is large, the amount of filler can be reduced, using a column with a relatively small inner diameter (for example, a column of 2 cm × 20 cm); if the difference of Rf is less than 0.1, the column is enlarged. Increase the amount of filler, such as a 3 cm inner diameter column.



3. Packing column

There are two kinds of column chromatography column, wet method and dry method. The wet method is convenient. Generally, the sample is dissolved by eluent. Methylene chloride, ethyl acetate, etc. can also be used, but the less the solvent, the better. The solvent becomes the eluent. The piston at the bottom of the column must not be coated with lubricant, otherwise it will be carried into the eluent by the eluent. A valve made of Teflon can be used.

There is no substantial difference between dry and wet packing, as long as the column can be installed. The packed column should be moderately tight (too dense to the eluent flow rate) and must be uniform, otherwise the sample will flow obliquely from one side.

At the same time, there should be no large bubbles in the column. In most cases, some small bubbles do not have much influence, because as long as the pressurized bubbles disappear. But the more taboo is the cracking, which will affect the separation effect and even scrap.



4. Solvent selection

Choosing a suitable solvent system is the key to column chromatography separation. There are three factors to consider when choosing a column chromatography eluent: Solubil2ity, Affinity, and Resolution.

Solvents should be chosen at low cost, safe and environmentally friendly. Petroleum ether, ethyl acetate, dichloromethane, diethyl ether, methanol and n-hexane can be considered. However, the price of n-hexane is relatively high, and ether is very volatile. The adsorption of methylene chloride and methanol with silica gel is an exothermic process, which tends to cause bubbles in the column. Other solvents are used in relatively small amounts and should be selected according to different needs.

It is also worth mentioning that since we carry out trace analysis, the purity of the eluent must be paid attention to. Generally, the pesticide residue grade or HPLC grade is used. If it is analytically pure, it must be refined. At the same time, the solvent is best recycled after passing through the column. On the one hand, it is environmentally friendly, on the other hand, it can save some money.



5. Loading

Dissolve the sample with a small amount of solvent, and open the bottom piston after the addition. When the solvent layer drops to the quartz sand surface, add a small amount of low-polar solvent, then open the piston again, so two or three times, the general quartz sand Basically it is white.

Add the eluent, do not pressurize at the beginning, wait for the solvent and sample layer of the dissolved sample to have a distance (2~4 cm), and then pressurize, so as to avoid the rapid downward flow of the entrained sample (such as dichloromethane).

Many samples are more viscous before the upper column, and will precipitate on the column after loading. This is generally a relatively large number of samples, which is caused by the saturation of the adsorption of the sample. Some samples have poor solubility, and soluble solvents (such as DMF, DMSO, etc.) can not be applied to the column, so the dry column must be used.



6. Eluent collection and concentration

The principle of using silica gel as a stationary phase through a column is a balance of adsorption and desorption. If the adsorption of the sample and the silica gel is relatively strong, it is not easy to flow out, and alumina can be used as the stationary phase.

After the column chromatography, the eluent must be concentrated due to the use of more solvents. If the analyte has a certain volatility, it is best to use a normal pressure volatile solvent, otherwise the detection result may be low.




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