1、 Preparation before regeneration
Ultra liquid chromatography columnPreparation of reagents: Prepare chromatographic grade solvents such as ultrapure water, methanol, acetonitrile, isopropanol, and suitable waste collection bottles; Ensure that there are no leaks in the pump body and injection valve of the liquid chromatograph.
Chromatographic column inspection: Record the current column efficiency and peak shape data of the chromatographic column before regeneration. If there are strong residual pollutants in the column, evaluate whether the chromatographic column has regeneration value first.
Safe operation: Wear gloves and goggles during operation to avoid contact of organic solvents with the skin; Ensure good ventilation in the laboratory.
2、 Universal regeneration steps (mainly reverse phase chromatography column)
Rinse off residual mobile phase
Remove the chromatography column and rinse with ultrapure water at a low flow rate (0.2-0.3 mL/min) for 30-60 minutes to remove water-soluble impurities such as buffer salts and prevent salt precipitation from blocking the packing material.
If the mobile phase contains ion pair reagents, rinse with a 50% methanol aqueous solution for 1-2 hours, and then switch to pure methanol.
Remove strongly hydrophobic pollutants
Rinse the chromatography column with 100% methanol and 100% acetonitrile in sequence, maintaining a flow rate of 0.3-0.5 mL/min. The volume of each solvent washed should be 20-30 times the volume of the column.
If the pollutant is stubborn, isopropanol can be used for flushing (note that isopropanol has high viscosity and the flow rate needs to be reduced to prevent excessive column pressure), and then methanol can be used for transition after flushing.
Polar pollutant cleaning
For chromatography columns containing polar impurities, rinse with a mixture of methanol and water (1:1) for 60 minutes, and then rinse with pure methanol until the baseline is stable.
Balance after regeneration
Use a mobile phase equilibrium chromatography column with a target analysis method, with an equilibrium volume of no less than 10 times the column volume, until the baseline is stable before conducting sample analysis.
3、 Precautions for regenerating special types of chromatographic columns
Normal phase chromatography column: Do not rinse with aqueous solvents. During regeneration, rinse sequentially with n-hexane, dichloromethane, and ethyl acetate, and finally store at equilibrium with n-hexane.
Ion exchange chromatography column: first rinse with a buffer solution corresponding to the pH value, then rinse with deionized water until neutral, and finally store in a buffer solution containing 5% methanol.
Chiral chromatography column: Prioritize using regenerated solvents recommended by the manufacturer, avoid using strongly polar solvents, and prevent damage to the chiral stationary phase.
4、 Verification and preservation after regeneration
Performance verification:Ultra liquid chromatography columnAfter regeneration is completed, inject standard samples to test column efficiency and peak symmetry. If the peak shape improves and the column efficiency returns to more than 80% of the original level, it indicates successful regeneration.
Long term storage: The reverse phase chromatography column is sealed with pure methanol for storage, and the normal phase chromatography column is stored with n-hexane. The plugs are tightened at both ends and placed in a room temperature, dark environment.