Regenerating or Cleaning Your HPLC Columns

THESE PROCEDURES DO NOT APPLY TO CHIRAL COLUMNS.  Refer to the manufacturer's recommended steps.

Reversed-Phase Columns

• Connect the column in the reversed direction and bypass the detector.
• Flush out any salts/buffers with 20 column volumes of HPLC grade water at 1mL/min.
• Flush column with 20 column volumes of isopropanol.
• Flush column with 20 column volumes of methylene chloride.
• Flush column with 20 column volumes of hexane.
• Flush column again with 20 column volumes of methylene chloride or dimethyl-formamide.
• Flush column again with 20 column volumes of isopropanol.
• Connect the column in the correct direction of flow.Flush the column with the 10 columns volumes of mobile phase without any buffer.
• Equilibrate the column with 15 - 20 column volumes of mobile phase.
• Inject a standard or a sample to see if performance is restored.

Normal-Phase Columns

• Connect the column in the reversed direction and bypass the detector.
• Flush column with 20 column volumes of 50:50 methanol:chloroform.
• Flush column with 20 column volumes of ethyl acetate.
• Reconnect the column in the proper flow direction.
• Equilibrate the column with 15 - 20 column volumes of mobile phase.
• Inject a standard or a sample and evaluate performance.

Reverse-Phase Protein/Peptide Columns

• Connect the column in the reversed direction and bypass the detector.
• Rinse with 20 column volumes of mobile phase without buffer.
• Flush column with 20 column volumes of 0.1% TFA in water.
• Flush column with 20 column volumes of 0.1% TFA in Acetonitrile:Isopropanol (1:2).
• Reconnect the column in the proper flow direction.
• Equilibrate the column with 15 - 20 column volumes of mobile phase.
• Inject a standard or a sample and evaluate performance.

Ion-Exchange Columns

• Connect the column in the reversed direction and bypass the detector.
• Rinse with 10 column volumes of each of the following:

1. 500mM Phosphate Buffer, pH 7
2. 10% Aq Acetic Acid
3. Water
4. Methanol (if for protein removal, use 5M Urea instead)
5. Water

• Reconnect the column in the proper flow direction.
• Equilibrate the column with 15 - 20 column volumes of mobile phase.
• Inject a standard or a sample and evaluate performance.

Note:  We do not recommend attempting to replace the frits or repack the head of today's analytical columns.  Always consult each manufactures washing instructions prior to choice of conditions.  4CHROM's procedures are for guidance only; no 4CHROM responsibility will be accepted for use of these suggestions, as usage of these or other procedures is the sole responsibility of the user.