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
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 ColumnsConnect 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.