Affinity chromatography, also known as affinity chromatography, is a chromatographic method that utilizes the binding properties of a stationary phase to separate molecules. Affinity chromatography is to connect molecules with certain binding capacity to the substances to be separated on the gel filtration chromatography column. Their binding is reversible. When the mobile phase conditions change, they can separate from each other. Affinity chromatography can be used to purify or concentrate molecules from a mixture, or to remove or reduce the content of molecules in the mixture.

General process

The substances separated by affinity chromatography are usually mixed in solution, such as cell contents, culture medium, or plasma. When the molecules to be separated pass through the chromatographic column, they are captured by functional groups on the stationary phase or medium, while other substances in the solution can smoothly pass through the chromatographic column. Then remove the solid matrix for elution, and the target molecule is immediately eluted. If the purpose of separation is to remove some molecules from the solution, then as long as the molecules can bind to the medium, there is no need for elution. During regeneration, the mobile phase is adjusted to elute impurities.

Special applications

Affinity chromatography has a wide range of applications, including the separation and purification of nucleic acids and proteins from cell extracts, as well as the separation of antibodies from plasma. Affinity chromatography is commonly used for separating recombinant proteins. By genetic modification, artificial features are added to proteins to selectively bind with ligands, thereby achieving the goal of separation. Another important use of affinity chromatography is to separate antibodies from plasma.

The affinity chromatography medium should meet the following four conditions: ① porous network structure ② low non-specific adsorption, inert substrate chemical properties, and low surface charge; (3) The physical and chemical properties are stable and will not change due to changes in covalent coupling reaction conditions and adsorption conditions; ④ The substrate must be activated or functional.