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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
 

Ion Exchange Columns
Ion exchange phases can be either anion or cation exchangers (WCX, SCX, WAX, and SAX).  To clean and regenerate an ion exchange column flush it with the following volumes of solvent.

  • 20 column volumes of the same eluent at double the buffer concentration
  • 20 column volumes of water
  • 20 column volumes of acetonitrile
  • 5 column volumes isopropanol
  • 20 column volumes heptane
  • 5 column volumes isopropanol
  • 20 column volumes acetonitrile
  • 20 column volumes of water
  • Re-equilibrate the column with the required mobile phase

HILIC Columns

  • Rinse with 10 Column Volumes each of:
  • 95 % Water/5 % Acetonitrile (for buffer removal)
  • 95 % 100 mM Ammonium Acetate, pH 5.8/5 % Acetonitrile
  • 95 % Water/5 % Acetonitrile
  • Re-equilibrate the column with the required mobile phase
 

To calculate the column volume (Vc) use Equation 1.

Where: 
d = diameter
l = length
Keep all dimensions in mm and the result will be in μL.

 
 

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