How to regenerate the C18/C8 HPLC columns?
The main purpose of the regeneration of the C18/C8 HPLC column is to remove the impurities out of the HPLC column.
When do we need the regeneration of the HPLC column?
After prolonged use, the column pressure rises, or peak trailing occurs.
Main steps for the regeneration of C18/C8 column
Before cleaning the column, disconnect the column from the detector and allow the cleaning solvent to flow into the recovery vessel.
Sometimes the column can be backflushed for cleaning to allow contaminants to flow out of the column. Cleaning the column requires the use of a stronger solvent than the mobile phase. The minimum volume for the solvent used to clean the analytical column is 10 column volume.
To clean the column in the opposite direction, proceed as follows.
- Disconnect the column from the detector. Then leaving the tubing at the end of the column and place it in a beaker that receives the liquid.
- Rinse with a mobile phase without buffer salts (water/organic phase) firstly.
- Then rinse with 100% organic phase (methanol and acetonitrile).
- Check that the pressure returns to normal, and if not, proceed to the next step.
- Discard the column or consider washing with stronger conditions. E.g. 75% Acetonitrile/25% Isopropanol, 100% Isopropanol, 100% Dichloromethane, 100% Hexane
Estimated column volume for different columns:
Column Size (mm x mm)
|Column Volume (mL)||Column Size (mm x mm)|| |
Column Volume (mL)
250 x 4.6
|≈2.5||150 x 2.1|| |
150 x 4.6
|≈1.5||100 x 2.1|| |
150 x 4.0
|≈1.13||50 x 4.6|| |
|150 x 3.0||≈0.64||30 x 4.6||≈0.30|
- Whether hexane or methylene chloride is used, it must be rinsed with isopropanol before use or before resuming the reversed-phase mobile phase.
- Buffer salts may precipitate and cause backpressure within the column. Pass the column slowly with hot water to remove the precipitation.
- A rinse solution containing propanol will generate higher operating pressures due to increased viscosity. The flow rate needs to be reduced during the rinse to maintain a safe operating pressure.
- To avoid precipitation, if the system does not contain a non-volatile buffer, the system is first flushed with an aqueous buffer-free mobile phase before the pure organic solvent enters the system.
Regeneration can't recover the HPLC column with 100% efficiency
High column pressure problems may not be caused by a blocked column but by a damaged gasket in the system and particles deposited on the inline filter head, which should be replaced before cleaning the column.
Do not replace the column sieve plate at random. It may cause irreversible damage to the column.
If the deterioration in quantitative analysis is due to a decrease in separation, this column should be discarded and replaced.
The regeneration method is just for reference. For detailed information, please contact the column supplier.
Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.