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How to Regenerate Heavily Fouled Columns

Regeneration Procedure for Reversed-Phase Columns

  • Disconnect the column and reconnect it to the chromatograph with the flow through the column in the reversed direction.
  • Flush out any salts/buffers with HPLC grade water. Pump 25 mL of water through the column at 1 mL/min.
  • Flush column with 25 mL of isopropanol.
  • Flush column with 25 mL of methylene chloride.
  • Flush column with 25 mL of hexane.
  • Flush column again with 25 mL of methylene chloride.
  • Flush column again with 25 mL of isopropanol.
  • Reconnect the column to the chromatograph with the flow in the proper direction. Flush the column with the mobile phase without the buffer, the re-introduce the buffer.
  • Equilibrate the column with 25 to 50 mL of mobile phase.
  • Inject a standard or a sample to see if performance is restored.

Note: For some retained compounds that have fouled the column, dimethyl-formamide may be a better "cleaning" solvent than methylene chloride and hexane.

Regeneration Procedure for Normal-Phase Columns

  • Connect the column to the chromatograph with the flow in the reversed direction.
  • Flush column with 50 mL of 50:50 methanol:chloroform.
  • Flush column with 50 mL of ethyl acetate.
  • Reconnect the column in the proper flow direction.
  • Equilibrate the column with 50 mL of mobile phase.
  • Inject a standard or a sample and evaluate performance.

Caution: In general, it is worth trying to clean and back flush an HPLC column with appropriate solvents before trying to replace the frit. There is always a danger that small amounts of packing material may be lost when a frit is replaced and column efficiency will decrease. In addition, the use of a 0.5Ám in-line filter to capture particulate material and routine column washes with an appropriate strong solvent are highly recommended.