What is the composition of liquid chromatography system

 

Liquid chromatography is composed of high-pressure infusion pump, injection system, temperature control system, etc. it is an instrument that uses the difference of mixture distribution ratio between liquid-solid or two immiscible liquids to separate the mixture first and then analyze and identify it. In daily work, the maintenance of the liquid chromatograph is very important. Pay attention not to let air enter the infusion system and the high-pressure pump. If the solution in the reservoir is not used for a long time, clean the reservoir and replace the solution. After each use of the chromatograph, the buffer should be washed with pure water to prevent the inorganic salt from escaping or depositing.

 

The system consists of a liquid reservoir, a pump, an injector, a chromatographic column, a detector and a recorder. The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector, which is loaded into the chromatographic column (stationary phase). Because the components in the sample solution have different distribution coefficients in the two phases, when the liquid chromatography moves relatively in the two phases, after repeated adsorption desorption distribution process, each component has a higher moving speed Big difference, separated into a single component, which flows out from the column in turn. When passing through the detector, the sample concentration is converted into an electrical signal and transmitted to the recorder. The data is printed out in the form of a graph. The high performance liquid chromatograph mainly includes the sample injection system, the infusion system, the separation system, the detection system and the data processing system. The following will describe their respective composition and characteristics.

 

Injection system

 

In general, diaphragm injection injector or high-pressure injection room is used to complete the injection operation, and the injection volume is constant. It is beneficial to improve the repeatability of the analytical sample.

 

Infusion system

 

Liquid chromatography includes three parts: high pressure pump, mobile phase reservoir and gradiometer. The general pressure of the high-pressure pump is 1.47-4.4x10pa, and the flow rate is adjustable and stable. When the high-pressure mobile phase passes through the chromatographic column, the diffusion effect of the sample in the column can be reduced, and the moving speed in the column can be accelerated, which is beneficial to improve the resolution, recover the sample, and maintain the biological activity of the sample. The mobile phase reservoir and gradiometer can make the mobile phase change with the properties of the stationary phase and the sample, including changing the polarity, ionic strength, pH value of the eluate, or using competitive inhibitors or denaturants. This enables the effective separation of various substances (even if there is only one group difference or isomer).

 

Separation system

 

The system includes column, connecting tube and thermostat. Generally, the length of chromatographic column is 10-50cm (if two columns are needed to be connected, a connecting tube can be added between them), the inner diameter is 2-5mm, which is made of "high-quality stainless steel or thick wall glass tube or titanium alloy and other materials, and there is a fixed phase with a diameter of 5-10 μ m (composed of matrix and solid solution). The matrix in the fixed phase is composed of resin or silica gel with high mechanical strength, both of which are composed of It has the characteristics of inert (for example, the silicic acid gene on the surface of silica gel has been basically removed), porous (pore size up to 1000?) and large specific surface area. In addition, the surface of silica gel is mechanically coated (as the preparation of stationary phase in gas chromatography), liquid chromatography or chemical coupling of various genes (such as phosphatic acid group, quaternary amine group, hydroxymethyl group, phenyl group, amino group or alkyl group with various length of carbon chain, etc ）Or ligands. Therefore, this kind of materials with different fixed relative structures have good selectivity. For example, after coupling pea agglutinin (PSA) on porous silica gel, a glycoprotein in fibroblasts can be isolated.

 

In addition, the fixed phase plasmids are small, the column bed is easy to achieve uniform and dense state, and it is easy to reduce the eddy current diffusion effect. The particle size of matrix is small, the micropore is shallow, and the mass transfer of sample in micropore is short. These are beneficial to reduce band width and improve resolution. According to the theoretical analysis of column efficiency, the smaller the matrix size is, the larger the theoretical number n of tray is. It is further proved that the resolution will be improved if the matrix particle size is small.

 

Moreover, the thermostat of liquid chromatography can adjust the temperature from room temperature to 60C. By improving the mass transfer speed and shortening the analysis time, the efficiency of the chromatographic column can be increased.