Why HPLC column regeneration required

This article describes the HPLC Column Regeneration procedure.

What is mean by column Regeneration for HPLC column?

column regeneration refers to the process of restoring or renewing the performance and efficiency of the chromatographic column used in the HPLC system. By regenerating the HPLC column, its separation efficiency, resolution, and sensitivity can be improved or restored, leading to more accurate and reliable analytical results. Regular column regeneration is crucial for maintaining the quality and performance of the HPLC system, ensuring accurate and reproducible analyses over time.

Over time, the HPLC column can become fouled or contaminated by sample residues, impurities, or irreversible adsorption of analytes, leading to a decline in separation efficiency, loss of resolution, and decreased sensitivity. Column regeneration is carried out to remove these accumulated substances and restore the column’s performance to its optimal state.

Why Column Regeneration Required in HPLC ?

After analyzing dirty or concentrated samples, some sample compounds can be retain in 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 may notice several different performance problems.

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

HPLC column regeneration shall be done only when the system suitability criteria not passes after several washings of HPLC Column and if new column is not available in stock or the procurement of HPLC column will take longer time than scheduled time of analysis

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.

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.

before applying regeneration procedure on column it is advisable to check with your column manufacturer or review the instructions that came with your column  or Literature of HPLC Column shall be referred.

Procedure for Column Regeneration : 

Precautions :

• If after the cleaning procedure some problems still occurred, it is then necessary to use more aggressive and lengthy washing methods.
• Since most of the impurities are retained at the head of the column, reversing the column direction (known as backflushing) will reduce
  the distance that impurities have to travel to exit the column.
• During the regeneration procedure, the column must be disconnected from the detector to avoid the contamination of the detector cell.
• The flow rate also has to be reduced to about 20 – 50 % of its typical value because some solvents or combination of solvents have higher viscosities than the normal mobile phase.
• If a buffer solution is normally used, it must be replaced with water during the procedure. Washing directly with 100 % of an organic solvent may cause buffer precipitation, thereby creating even bigger problems.

Note : Verify with your column vendor that one of the following regeneration procedures is suitable or appropriate for your column and will not cause damage to the column before executing the following steps.

Regeneration Procedure for Reversed-Phase Silica Columns :

(C18, C18-300, C8, C8-300, C4,C4-300, Phenyl, PFP, Amine, Cyano)

1. Disconnect the column and reconnect it to the system, 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. For the following flushing solvents, use 1ml/min too.
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.

Regeneration Procedure for Normal-Phase Silica Column :

(Amine, Cyano, Diol, Silica)

1. Disconnect the column and reconnect it to the system, with the flow through the column in the reversed direction.
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 25 to 50 mL of mobile phase.
6. To see if performance is restored, inject a standard or a sample.

Note : Never use water in normal phase column regeneration

Regeneration Procedure for Ion Exchange Columns :
(SCX, SAX)

1. Disconnect the column and reconnect it to the system, with the flow through the column in the reversed direction.
2. Set the flow at 1 mL/min. Flush column with Mobile phase with buffer concentration doubled (be careful for salt precipitation)
3. Flush the column with Water: methanol (90:10)
4. Flush the column with Methanol, isopropanol, methanol and water : methanol (90:10)
5. Equilibrate the column with 25 to 50 mL of mobile phase
6. To see if performance is restored, inject a standard or a sample.