Ion exchange chromatography, gel filtration chromatography and affinity chromatography are the three classical chromatographic techniques for separating proteins, depending on the packing matrix and the sample distribution exchange principle.
2. Techniques and methods
1. Selection of column chromatography operation method
At present, the operation mode of column chromatography mainly includes three modes: normal pressure separation, reduced pressure separation and pressure separation. Atmospheric separation is the simplest separation mode. It is convenient and simple, but the elution time is long.
Although the decompression separation can save the amount of packing, the solvent will volatilize due to a large amount of air passing through the packing, and sometimes there will be condensation outside the column, and some easily decomposed compounds are also difficult to obtain, and it is necessary to use a water pump or a water pump at the same time. Vacuum pump.
Pressure separation can speed up the flow rate of the eluent and shorten the elution time of the sample. The supply of pressure can be compressed air, double ball or small air pump.
2. Selection of column specifications
There are various specifications of column chromatography separation columns on the market. The longer the column, the higher the corresponding number of plates, and the separation is good. The diameter-to-height ratio of columns currently on the market is generally in the range of 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 required separation of components and impurities is large, the amount of packing can be reduced, and a column with a relatively small inner diameter (such as a 2 cm × 20 cm column) is used; if the difference in Rf is less than 0.1, the column should be enlarged. Increase the amount of packing, eg, use a 3 cm id column.
There are two main types of packing for column chromatography: wet method and dry method. Wet method saves trouble. Generally, eluent is used to dissolve the sample. Dichloromethane, ethyl acetate, etc. can also be used, but the less solvent, the better, otherwise The solvent becomes the eluent. The piston at the bottom of the column must not be lubricated, otherwise it will be brought into the eluent by the eluent, and a valve made of polytetrafluoroethylene can be used.
There is no substantial difference between dry and wet packing, as long as the column can be packed well. The packed column should be reasonably tight (too dense, eluent flow is too slow), and must be uniform, otherwise the sample will flow diagonally from one side.
At the same time, there can be no large bubbles in the column. In most cases, some small bubbles have little effect, because the bubbles can disappear as long as the pressure is applied. But the column is more taboo is cracking, cracking will affect the separation effect, or even scrapped.
4. Choice of solvent
Choosing an appropriate solvent system is the key to column chromatography separations. When choosing an eluent for column chromatography, three factors must first be considered: solubility (Solubil2ity), affinity (Affinity) and resolution (Resolution).
The solvent should be cheap, safe and environmentally friendly, such as petroleum ether, ethyl acetate, dichloromethane, diethyl ether, methanol and n-hexane. However, the price of n-hexane is relatively high, ether is very volatile, and the adsorption of methylene chloride and methanol on silica gel is an exothermic process, which is easy to cause bubbles in the column. Other solvents are used relatively little and should be selected according to different needs.
It is also worth mentioning that since we are conducting trace analysis, we must pay attention to the purity of the eluent. Generally, pesticide residue grade or HPLC grade is used. If it is analytically pure, it must be refined. At the same time, it is best to recycle the solvent after passing through the column. On the one hand, it is environmentally friendly, and on the other hand, it can also save some funds.
5. Loading the sample
Dissolve the sample with a small amount of solvent and add the sample. After adding, open the piston at the bottom. When the solvent layer drops to the quartz sand surface, add a small amount of low-polarity solvent, and then open the piston. Basically white.
Add the eluent, do not pressurize at first, wait until the solvent that dissolves the sample and the sample layer have a certain distance (2~4 cm), and then pressurize, so as to avoid the solvent (such as dichloromethane, etc.) entraining the sample to descend rapidly.
Many samples are viscous before being loaded on the column, and they will precipitate on the column after loading. This is generally caused by a relatively large number of samples, because the adsorption of the filler to the sample is saturated. Some samples have poor solubility, and soluble solvents (such as DMF, DMSO, etc.) cannot be loaded on the column, so the column must be loaded by dry method.
6. Collection and concentration of eluent
The principle of using silica gel as the stationary phase to pass through the column is an equilibrium of adsorption and desorption. If the adsorption of the sample to the silica gel is relatively strong, it is not easy to flow out. At this time, alumina can be used as the stationary phase.
The eluent after column chromatography must be concentrated due to the use of a large amount of solvent. If the analyte has a certain volatility, it is best to use a volatile solvent at normal pressure, otherwise it will easily lead to low detection results.