In separation analysis, especially protein separation analysis, chromatography is a very important and quite common technique. Its principle is relatively complex and the requirements for personnel are relatively high. Only a relatively simple introduction can be made here.
1. Adsorption chromatography
1. Adsorption column chromatography Adsorption column chromatography is a chromatographic method that uses solid adsorbent as the stationary phase and organic solvent or buffer as the mobile phase to form the column.
2. Thin layer chromatography Thin layer chromatography is a chromatographic method that uses a substrate coated on a glass plate or polyester sheet as a stationary phase and a liquid as a mobile phase. This chromatographic method is to apply a material such as an adsorbent to a carrier to form a thin layer, and then spread the layer according to the paper chromatography operation.
3. Polyamide film chromatography The adsorption of polyamide on polar substances is due to its ability to form hydrogen bonds with the separated material. The strength of this hydrogen bond determines the adsorption capacity between the separated material and the polyamide film. During chromatography, the spreading agent competes with the separated product on the surface of the polyamide membrane to form hydrogen bonds. Therefore, choosing a proper spreading agent to separate the continuous process of adsorption, desorption, resorption, and desorption on the surface of the polyamide membrane can cause the separated substances to achieve the purpose of separation.
2. Ion exchange chromatography
Ion exchange chromatography is performed in a system that uses an ion exchanger as the stationary phase and a liquid as the mobile phase. The ion exchanger is composed of a matrix, a charge group and a counter ion. The reaction between the ion exchanger and the ions or ionic compounds in the aqueous solution is mainly carried out by ion exchange, or by the adsorption of the charge groups on the ion exchanger to the ions or ionic compounds in the solution. `
3. Gel filtration
Gel filtration is also called molecular sieve chromatography. The reason is that the gel has a network structure, and small molecular substances can enter its interior, while large molecular substances are excluded from the outside. When a mixed solution passes through a gel filtration chromatography column, the substances in the solution are separated according to different molecular weight sieves.
4. Affinity chromatography The principle of affinity chromatography is similar to the well-known mechanism of specific reactions such as antigen-antibody, hormone-receptor and enzyme-substrate. Just in the reaction between enzyme and substrate, specific waste (S) can combine with certain enzyme (E) to produce complex (E-S). In affinity chromatography, specific ligands have affinity with certain living macromolecules and produce complexes. The difference between affinity chromatography and enzyme-substrate reaction is that when the former is reacted, the ligand (similar to the substrate) is present in the solid phase; when the latter is reacted, the substrate is present in the liquid phase. In essence, affinity chromatography is to solidify the ligand L (the ligand for the enzyme can be a similar substrate, inhibitor, or prosthetic group, etc.) to the matrix M containing the activated group by a covalent bond ( Such as activated agarose, etc.), made affinity adsorbent M-L, or called solid phase carrier. The cured ligand still retains the ability to bind specific substances.
Therefore, after loading the peripheral phase carrier into a small chromatography column (bed volume of a few milliliters to tens of milliliters), the sample liquid to be separated is passed through the column. At this time, the substance S with affinity for the ligand in the sample can be adsorbed on the solid support by means of electrostatic attraction, van der Waals force, and structural complementation effect, while the substance without affinity or non-specific adsorption is initially buffered The liquid washes out and forms the first chromatographic peak. Then, by appropriately changing the pH value of the starting buffer, or increasing the ionic strength, or adding inhibitors and other factors, the substance S can be dissociated from the solid phase carrier, and the Mth chromatographic peak is formed (See Figure 6-2). Obviously, through this operation procedure, the effective components and impurities can be satisfactorily separated. If there are more than two substances in the sample solution that have affinity with the solid phase carrier (the size of which is different), they can also be separated by using selective buffer for elution. The used solid support can be reused after being regenerated.
The affinity chromatography method described above is also called specific ligand affinity chromatography. In addition, there is an affinity chromatography method called universal ligand affinity chromatography. Compared with the two affinity chromatography methods, the former ligands are generally complex macromolecular substances (such as antibodies, receptors, and enzyme-like substrates), which have strong adsorption selectivity and large binding force. The latter ligands are generally simple small molecules (such as metals, dyes, and amino acids), which have low cost and high adsorption capacity. By improving the adsorption and desorption conditions, the resolution of chromatography can be improved.
5. Focused chromatography
Focused chromatography is also a type of column chromatography. Therefore, like other chromatography, it usually has a mobile phase with multiple buffers as the mobile phase and multiple buffer exchangers as the stationary phase.
The principle of focusing chromatography can be explained from three aspects: formation of pH gradient solution, protein behavior and focusing effect.
1. Formation of PH gradient solution In ion exchange chromatography, the formation of PH gradient solution is achieved by a gradient mixer. For example, when using an anionic agent for chromatography, the method of preparing a gradient solution with a linear pH change from high to low is to install a high pH solution in the mixing chamber of the gradient meter (the column), while in another The chamber is filled with a low pH limit solution, and then the lower outlet of the chromatography column is opened to allow the eluent to flow continuously through the column body. At this time, the pH value of the solution changes from high to low from the upper part of the column. In focused chromatography, when the eluent flows into the multi-buffer exchanger, the pH gradient solution can be automatically formed because the exchanger has charge groups with buffer capacity.
For example, when the anion exchanger PBE94 (as a stationary phase) is loaded into the column, the initial buffer is used to equilibrate to PHg, and then the eluent containing multiple buffer substances (as mobile phase) containing PH6 is passed through the column. The most acidic component of the Shiduo buffer has a neutralizing effect in combination with the basic anion exchange pair. As the eluent is continuously added, the PH value of each point in the residence gradually decreases from high to low. According to this treatment for J period of time, a gradient of PH 6-9 was formed from the top to the bottom of the chromatography column. The pH gradient solution in the focused chromatography column is automatically formed during the washing process, but as the washing process progresses, the pH gradient will gradually migrate downward, and the pH of the effluent from the bottom will gradually decrease from 9 to 6, Finally, it is constant at this value, and the pH gradient of the chromatography column disappears.
2. The behavior of proteins The charge of a protein depends on its isoelectric point (PI) and the pH value in the chromatography column. When the pH in the column is lower than the PI of the protein, the protein is positively charged and does not bind to the anion exchanger. As the eluent moves forward, the PH value in the stationary phase changes as the eluent time increases. When the protein moves to an environment where the pH is higher than its PI, the protein changes from positively charged to negatively charged and binds to the anion exchanger. Due to the passage of the eluent, when the pH around the protein is lower than PI again, it is positively charged and desorbed from the exchanger. As the eluent migrates to the bottom of the column, the above process will be repeated, so that various proteins are washed down at their respective isoelectric points, thus achieving the purpose of separation.
Different proteins have different isoelectric points, and they move different distances before being bound by the ion exchanger, and the elution order is arranged according to the isoelectric points.
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