General Column Regeneration Procedure

This Information Applies To: Agilent LC Systems

Issue

After analyzing dirty or concentrated samples, some sample compounds may retain in the column and other parts of the HPLC system. If the column re-equilibration after each run is not enough to elute the retained compounds, you will notice several performance problems. 

Tailing or wider peaks, higher background signal, carry over, or ghost peaks can be a sign of a contaminated column. The use of low-quality solvents can also be the cause of those symptoms. 

As you use your column and expose it to various mobile phases, analytes, and sample matrices, these interactions will subtly affect it. Over time, there may be changes in its resolution. It’s important to keep a test chromatogram, from when your column was new, to compare and understand these changes in performance over time. If resolution degrades beyond an acceptable value for good quantitation, the column should be discarded and replaced with a new one.

Sometimes you can alleviate a high pressure or peak tailing issue by cleaning your column while back flushing it. Before cleaning your column, we recommend that you disconnect it from your detector and run your wash solvents into a beaker or other container. 

Check with your column manufacturer or review the instructions that came with your column before applying the following procedure. Start with the recommended cleaning or regeneration procedure from your column manufacturer. If it is not successful, you can use the procedure in this article as an alternative.

Steps to follow

Caution: Always use chromatographic grade solvents and wear the proper Personal Protection Equipment.

Caution: Methylene chloride, tetrahydrofuran, or dimethyl sulfoxide can swell PEEK components of the LC flow path. Confirm the material of the capillaries, fittings, or rotor seals before performing the following procedures. If any of the flow path components is made of PEEK, replace it or bypass it.

Caution: Check with your column supplier that one of the following regeneration procedures is suitable for your column and will not cause column damage before performing the following steps.

Regeneration Procedure for Reversed-Phase Silica Columns

1. Disconnect the column and reconnect it to the system, with the flow through the column in the reversed direction. Do not connect the column to the detector to avoid contamination or clogging.
2. Flush out any salts/buffers with HPLC grade water. Pump 25 mL of water through the column at 1 mL/min. For the following flushing solvents, use 1ml/min also.
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 system, with the flow in the usual direction. Flush the column with the mobile phase without the buffer, then re-introduce the buffer.
9. Equilibrate the column with 25 to 50 mL of mobile phase.
10. To see if performance is restored, inject a standard or a sample.

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

Regeneration Procedure for Normal-Phase Silica Column

1. Disconnect the column and reconnect it to the system, with the flow through the column in the reversed direction. Do not connect the column to the detector to avoid contamination or clogging.
2. Set the flow at 1 mL/min. Flush column with 50 mL of 50:50 methanol:chloroform.
3. Flush column with 50 mL of ethyl acetate.
4. Reconnect the column in the proper flow direction.
5. Equilibrate the column with 50 mL of mobile phase.
6. To see if performance is restored, inject a standard or a sample.

Tip: Follow the evolution of the signal and the background noise during the flush-out, especially before and after the regeneration procedure.

Learn how to effectively troubleshoot the Agilent LC Column: HPLC-0GEN-2201e - Chromatographic Troubleshooting for HPLC e-learning course available from Agilent Education