The basic working principle of liquid chromatography is based on the difference in distribution coefficients between the stationary and mobile phases of each component in the sample to achieve separation. After the mixed sample is injected into the liquid chromatography system, it will enter the chromatographic column containing the stationary phase (such as silica gel, chemically bonded phase, polymer, etc.) along with the mobile phase (such as water, methanol, acetonitrile, etc.). Due to the differences in the interaction forces between different components and the stationary phase (such as adsorption, distribution, exclusion, affinity, etc.), their residence times in the stationary phase are also different. Specifically, components with weaker interactions with the stationary phase will flow out of the chromatographic column first as they move faster with the mobile phase; The components tightly bound to the stationary phase move slower and then flow out of the chromatographic column. Through this process, each component in the mixture is separated one by one. In addition, the separation efficiency depends not only on the difference in solute distribution coefficients between the two phases (thermodynamic factors), but also on dynamic factors such as diffusion coefficient, fixed phase particle size, column packing, and mobile phase flow rate.

Precautions for using liquid chromatography:

1. Selection and treatment of mobile phase

-HPLC grade or higher solvents should be selected, and secondary distilled water or ultrapure water should be used for water, and it should be prepared and used immediately to prevent microbial growth and contamination of the system.

-After preparing the mobile phase, it should be restored to room temperature before use to avoid unstable performance caused by temperature differences.

-When using buffer salt solution, it must be filtered and the flow path must be cleaned with pure water before and after use. It is forbidden to leave the buffer salt solution in the flow path and let it stand. At the same time, the chromatographic column needs to be washed with a low proportion organic phase (organic phase content not less than 5) first, and then stored with a high proportion organic phase to avoid buffer salt precipitation. Try to avoid using high concentration phosphate buffer salts (>10mmol/L) as much as possible.

-Try to avoid using pure acetonitrile as the mobile phase to prevent liquid absorption caused by acetonitrile polymerization. When necessary, it is recommended to store in brown bottles.

2. Operating standards

-It is prohibited to disassemble the high-pressure pipeline connection under pressure.

-Be cautious when adjusting the flow rate to avoid sudden changes in pressure and temperature, and to prevent impact on the packing inside the column.

-When changing the solvent composition of the test sample solution, it should be done gradually, especially in reverse phase chromatography. Organic solvents should not be directly replaced with pure water, or vice versa.

-The UV detector light source needs to cool down for 30 minutes before shutting down.

3. Security maintenance

-Waste liquid needs to be classified and collected, and organic waste liquid cannot be directly discharged.

-Regularly maintain the instrument, such as cleaning the injection needle, replacing the sealing gasket, etc.