The five major systems of a liquid chromatograph are as follows:
1. Injection system
Generally, a diaphragm injection sampler or high-pressure injection room is used to complete the injection operation, and the injection volume is constant.
This is beneficial for improving the reproducibility of analytical samples.
2. Infusion system
The system consists of three parts: a high-pressure pump, a mobile phase reservoir, and a gradienter. The general pressure of a high-pressure pump is 1.47~4.4X107Pa, and the flow rate is adjustable and stable. When the high-pressure mobile phase passes through the chromatography column, it can reduce the diffusion effect of the sample in the column and accelerate its movement speed in the column. This is beneficial for improving resolution, recovering the sample, and maintaining the biological activity of the sample. Mobile phase storage errors and gradienters can change the mobile phase according to the properties of the stationary phase and sample, including changing the polarity of the eluent, ion strength, pH value, or switching to other methods
3. Separation system
The system includes chromatography columns, connecting tubes, and thermostats.
The length of a chromatographic column is generally 10-50cm (if two are needed in combination, a connecting tube can be added between the two), with an inner diameter of 2-5mm. It is made of high-quality stainless steel, thick walled glass tube, titanium alloy, and other materials. The column is equipped with a diameter of 5-10 μ A fixed phase with a particle size of m (composed of matrix and fixed liquid), in which the matrix is composed of high mechanical strength resin or silica gel, both of which have the characteristics of inertness (such as the basic removal of silicate genes on the surface of silica gel), porosity, and large specific surface area, In addition, its surface is mechanically coated (similar to the preparation of stationary phases in gas chromatography) or chemically coupled with various genes (such as phosphate, quaternary amine, hydroxymethyl, phenyl, amino or alkyl groups of various length carbon chains) or ligands.
Therefore, these types of substances with different fixed relative structures have good selectivity. For example, by coupling pea lectin (PSA) on the surface of porous silica gel, a glycoprotein can be isolated from fibroblasts. In addition, the stationary phase plasmid is small, and the column bed is easy to achieve a uniform and dense state, which can greatly reduce the eddy current diffusion effect. The matrix has small particle size and shallow micropores, and the sample has short mass transfer in the microporous area. These are beneficial for reducing the bandwidth and improving resolution. According to the column efficiency theory analysis, the smaller the matrix particle size, the larger the theoretical number N of the tray.
4. Detection system
There are three commonly used detectors for liquid chromatography: UV detector, differential refractive index detector, and fluorescence detector
UV detector
This detector is suitable for detecting samples with absorption performance for ultraviolet (or visible light) light.
Its characteristics: wide range of applications (such as proteins, nucleic acids, amino acids, nucleotides, peptides, hormones, etc. can be used); High sensitivity (detection limit of 10-10g/mL); Wide linear range; Not sensitive to temperature and flow rate changes; Can detect samples eluted by gradient solution.
5. Data processing system
This system can collect, store, display, print, and process test data, enabling the proper separation, preparation, or identification of samples.
In summary, the liquid chromatograph consists of a sample system, an infusion system, a separation system, a detection system, and a data processing system. The combination of these systems makes the experiment more reliable and stable.