Use of ion exchange chromatography buffer
Release time:
2021-10-29
Ion exchange chromatography is a technology that uses ion exchange cellulose or ion exchange dextran gel as a stationary phase and proteins or other samples as a mobile phase to separate and purify proteins. In addition to proteins, samples in the mobile box can also be nucleic acids, enzymes, hormones, polysaccharides, etc. Most of these substances are pH sensitive, and a suitable buffer must be prepared to maintain the pH of the system.
The relationship between protein and buffer pH
Protein purification is more typical in ion exchange chromatography, which is used as an example for illustration. Protein is formed by the dehydration and condensation of a variety of amino acids. It is amphoteric and has acidic carboxyl groups and basic amino groups. When the isoelectric point of the protein is equal to the pH of the buffer, the electrostatic charge is 0; when the pH of the buffer is greater than the isoelectric point of the protein, the carboxyl groups of the protein are free and the protein is negatively charged; when the pH of the buffer is lower than the isoelectric point of the protein At this time, the amino group ionizes and the protein is positively charged. The greater the difference between the pH value of the buffer and the isoelectric point of the protein, the more charge the protein carries, and vice versa. Serum proteins are all negatively charged, but the degree of negative charge carried by various proteins is different, among which albumin is the most, followed by globulin.
The relationship between buffer and ion exchanger
The ion exchangers in ion exchange chromatography are divided into anionic and cationic types, which can adsorb negatively charged proteins and positively charged proteins respectively. The ions in the buffer or other ions in the system are similar to the colloidal ions of proteins, and they all have the ability to exchange adsorption. In order to reduce interference, try to choose a buffer with the opposite ion type to the ion exchanger. For example, anion exchange chromatography uses cationic Tris buffer; cation exchange chromatography uses anionic HIPES and other buffers.
Effect of buffer on ion adsorption
When the pH of the buffer increases, the cationization of the protein is inhibited, and its adsorption to the cation exchanger is weakened; when the pH is lowered, the anionization of the protein is inhibited, and other things are reduced, and then the adsorption of the anion exchanger is reduced. When an anion exchanger is used, increasing salt ions lowers the pH value.
In addition to the pH of the buffer, which affects the chromatography, other ions will also be competitively exchanged and adsorbed with the protein. Increasing the ionic strength can also achieve the purpose of separating the protein. Desheng is a manufacturer of biological buffers and can provide Tris, Bicine, MOPS and other buffers to meet the needs of different ion chromatography.
News
2023/09/27
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