The system consists of a reservoir, a pump, an injector, a column, a detector, a recorder, and the like. The mobile phase in the reservoir is driven into the system by a high pressure pump. The sample solution enters the mobile phase through the injector and is loaded into the column (stationary phase) by the mobile phase, since each component in the sample solution has two phases. Different distribution coefficients, in the relative motion of the two phases, after repeated adsorption-desorption distribution processes, the components have a large difference in the moving speed, and are separated into individual components and sequentially flow out from the column. When passing through the detector, the sample concentration is converted into an electrical signal and sent to the recorder. The data is printed in the form of a map. The high performance liquid chromatograph mainly has a sample introduction system, an infusion system, a separation system, a detection system, and a data processing system. Describe their respective compositions and characteristics.
1. Separation system The system includes a column, a connecting tube, a thermostat, and the like. The column is generally 10 to 50 cm in length (when two are used together, a connecting tube can be added between them), and the inner diameter is 2 to 5 mm. It is made of "high-quality stainless steel or thick-walled glass tube or titanium alloy. The housing contains a stationary phase with a particle size of 5-10 μm (consisting of matrix and fixative). The matrix in the stationary phase consists of a resin with high mechanical strength or silica gel, which are inert (such as the basic silicic acid gene on the surface of silica gel). Has been removed), porosity (pore size up to 1000?) and large specific surface area, plus the surface is mechanically coated (as in the preparation of stationary phase in gas chromatography), or chemically coupled to various genes (such as An organic compound of a phosphate group, a quaternary amino group, a methylol group, a phenyl group, an amino group or an alkyl group of various lengths of a carbon chain, or the like. Therefore, such a substance having a fixed relative structure is excellent in selectivity. After pea agglutinin (PSA) is coupled to the surface of the porous silica gel, a glycoprotein in the fibroblast can be isolated. In addition, the stationary phase-based plasmid is small, and the column bed can easily reach a uniform and dense state. It is easy to reduce the eddy diffusion effect. The matrix particle size is small, the micropores are shallow, and the sample has short mass transfer in the microporous region. These are beneficial for reducing the band width and improving the resolution. According to the column effect theory analysis, the matrix particle size is small, the tray The larger the theoretical number N, this further proves that the small particle size of the matrix will improve the resolution. Furthermore, the thermostat of the high performance liquid chromatography can adjust the temperature from room temperature to 60C, and improve the mass transfer rate and shorten the analysis time. , can increase the efficiency of the column.
2. The introduction system liquid chromatograph generally uses a diaphragm injection injector or a high pressure injection chamber to complete the injection operation, and the injection volume is constant. This is beneficial for improving the repeatability of the analytical sample. 3. Infusion system The system consists of a high pressure pump, a mobile phase reservoir and a gradiometer. The general pressure of the high pressure pump is l. 47～4.4X10Pa, the flow rate is adjustable and stable. When the high-pressure mobile phase passes through the column, the diffusion effect of the sample in the column can be reduced, and the moving speed in the column can be accelerated, which improves the resolution and recovers the sample. It is advantageous to maintain the biological activity of the sample and the like. Mobile phase reservoirs and gradiometers allow the mobile phase to vary with the nature of the stationary phase and sample, including changing the polarity of the eluent, ionic strength, pH, or switching to competitive inhibitors or denaturants. This allows efficient separation of various substances (even if there is only one group difference or isomer).
1. Separation system The system includes a column, a connecting tube, a thermostat, and the like. The column is generally 10 to 50 cm in length (when two are used together, a connecting tube can be added between them), and the inner diameter is 2 to 5 mm. It is made of "high-quality stainless steel or thick-walled glass tube or titanium alloy. The housing contains a stationary phase with a particle size of 5-10 μm (consisting of matrix and fixative). The matrix in the stationary phase consists of a resin with high mechanical strength or silica gel, which are inert (such as the basic silicic acid gene on the surface of silica gel). Has been removed), porosity (pore size up to 1000?) and large specific surface area, plus the surface is mechanically coated (as in the preparation of stationary phase in gas chromatography), or chemically coupled to various genes (such as An organic compound of a phosphate group, a quaternary amino group, a methylol group, a phenyl group, an amino group or an alkyl group of various lengths of a carbon chain, or the like. Therefore, such a substance having a fixed relative structure is excellent in selectivity. After pea agglutinin (PSA) is coupled to the surface of the porous silica gel, a glycoprotein in the fibroblast can be isolated. In addition, the stationary phase-based plasmid is small, and the column bed can easily reach a uniform and dense state. It is easy to reduce the eddy diffusion effect. The matrix particle size is small, the micropores are shallow, and the sample has short mass transfer in the microporous region. These are beneficial for reducing the band width and improving the resolution. According to the column effect theory analysis, the matrix particle size is small, the tray The larger the theoretical number N, this further proves that the small particle size of the matrix will improve the resolution. Furthermore, the thermostat of the high performance liquid chromatography can adjust the temperature from room temperature to 60C, and improve the mass transfer rate and shorten the analysis time. , can increase the efficiency of the column.
2. The introduction system liquid chromatograph generally uses a diaphragm injection injector or a high pressure injection chamber to complete the injection operation, and the injection volume is constant. This is beneficial for improving the repeatability of the analytical sample.
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