23/05/2019 HPLC Column Cleaning and Regeneration

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HPLC Column Cleaning and Regeneration

Column contamination from adsorption of analyte or matrix impurities at the head of the HPLC column can have deleterious chromatographic effects, such as, split peaks,
changes in selectivity, and changes in quantitation. However, before you send your column to the scrap heap it can be worth trying to clean and regenerate it.

Manufacturers will often provide methods for cleaning a specific column and these should always be consulted first, however, if this information is not available the following
protocols can be followed to regenerate a variety of column types.

Prior to washing disconnect the column and reverse the column flow direction by connecting the outlet end of the column to the pump. It is not recommended that UHPLC (1.9
µm) columns are reversed, instead they should be washed with the flow in the direction indicated by the column.

Reversed Phase Columns

Reversed phase columns include C30, C18, C8, C4, C1, CN and phenyl stationary phases. To clean and regenerate a reversed phase column flush it with the following volumes
of solvent.

20 column volumes of water/acetonitrile (95:5 v/v)

20 column volumes of acetonitrile
5 column volumes isopropanol
20 column volumes hexane
5 column volumes isopropanol
20 column volumes acetonitrile
20 column volumes of water/acetonitrile (95:5 v/v)
Re-equilibrate with the required mobile phase

Normal Phase Columns

Reversed phase columns include silica, diol, nitro, and amino stationary phases. To clean and regenerate a normal phase column flush it with the following volumes of solvent.

20 column volumes of heptane

5 column volumes of isopropanol
20 column volumes acetonitrile
20 column volumes water
20 column volumes acetonitrile
5 column volumes isopropanol
20 column volumes heptane
Re-equilibrate the column with the required mobile phase

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