Rejuvinating the HPLC Column
Dirty columns are caused by the comulative effects of matrix contaminants from repeated injections.
Symptoms of Dirty Columns
1. High Back Pressure.
2. Changing Retention Times.
3. Broad Peaks and Tailing.
4. Loss of Column Resolution.
In such cases, chromatographic performance will deteriorate as more and more non-eluting
compounds build up on the column stationary phase.
HPLC Column Regeneration
First try purging the column with pure acetonitrile. If this does not work, follow the
Regeneration Procedure for Reversed-Phase Columns
1. Disconnect the column and reconnect it to the chromatograph with the flow through
the column in the reversed direction.
2. Flush out any salts/buffers with HPLC grade water. Pump 25 mL of water through the
column at 1 mL/min.
3. Flush column with 25 mL of isopropanol.
4. Flush column with 25 mL of methylene chloride.
5. Flush column with 25 mL of hexane.
6. Flush column again with 25 mL of methylene chloride.
7. Flush column again with 25 mL of isopropanol.
8. Reconnect the column to the chromatograph with the now in the proper direction.
Flush the column with the mobile phase without the buffer, then re-introduce the
9. Equilibrate the column with 25 to 50 mL of mobile phase.
10. Inject a standard or a sample to see if performance is restored.
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
1. Connect the column to the chromatograph with the flow in the reversed
2. Flush column with 50 mL of 50:50 methanol:chloroform.
3. Flush column with 50 mL of ethyl acecate.
4. Reconnect the column in the proper flow direction.
5. Equilibrate the column with 50 mL of mobile phase.
6. Inject a standard or a sample and evaluate performance.