How to clean and regenerate the column after contamination?

Date:2021-11-25 16:42:09 Thursday

Summary:When a chromatographic column is contaminated, its chromatographic behavior will be somewhat different from that of an uncontaminated column. Contaminated columns can cause back pressure problems. The contaminated reversed-phase column must be cleane......

When a chromatographic column is contaminated, its chromatographic behavior will be somewhat different from that of an uncontaminated column. Contaminated columns can cause back pressure problems. The contaminated reversed-phase column must be cleaned and regenerated to restore the original operating conditions.
　　The key to regenerating a contaminated HPLC column is to know the nature of the contaminant and find an appropriate solvent to remove it. If the contamination is caused by the accumulation of strongly retained substances during repeated injections, simple steps to remove these contaminants can often restore the chromatographic behavior. Sometimes, after many operations, the chromatographic column is washed with 90-100% solvent B (the stronger solvent in the two-solvent reverse phase system) for 20 volumes to eliminate contaminants. For example, non-aqueous solvents such as methanol, acetonitrile, and tetrahydrofuran can be used for the residual lipids in the column.

　　 Generally speaking, all cleaning methods have similar forms. The solvents used increase with solvent strength. Often the last solvent is very hydrophobic (such as ethyl acetate or even hydrocarbon) and can be used to dissolve non-polar substances such as lipids and oils. We must ensure that each solvent in a series of solvents can be mixed with the next solvent.
At the end of the cleaning process, it is necessary to return to the original solvent system with the aid of a medium-strength miscible solvent. For example, isopropanol is a very good solvent as an intermediate step because it is miscible with n-hexane or dichloromethane and also with water phase solvents. However, the viscosity of isopropanol is very high, and a low flow rate must be ensured to avoid excessive pump pressure. When flushing the chromatographic column, the flushing liquid is directly connected to the waste bottle from the outlet end of the chromatographic column, and cannot flow through the detection cell to avoid contamination of the detection cell.
　　Reversed-phase chromatographic column does not use buffer solution, please use the following solvent series to regenerate:
100% methanol → 100% acetonitrile → 75% acetonitrile-25% isopropanol → 100% isopropanol → 100% methylene chloride → 100% n-hexane → 100% methylene chloride → 100% isopropanol → 100% acetonitrile .
　　Reverse phase chromatography column uses buffer solution, please use the following solvent series to regenerate:
　　 water → acetonitrile → chloroform (or isopropanol) → acetonitrile → water.