How To Regenerate a C18 HPLC Column

Typical Regeneration Procedure for Reversed Phase Columns:

Regularly cleaning your column can help extend its lifespan, reducing the need for frequent replacements of HPLC Columns.

For columns that can be backflushed (typically reversed-phase particles >1.8), begin with a stronger (less polar) solvent.

1. Disconnect the column from the detector and direct the wash solvents into a beaker.
2. Start by flushing 10 to 20 column volumes of your mobile phase without buffer salts (water/organic) through the column.
3. Next, use 100% organic solvent (methanol or acetonitrile).
4. Check the pressure to see if it has returned to normal; if not, proceed to the next step.
5. If the pressure is still abnormal, consider discarding the column or using stronger conditions, such as a 75% acetonitrile/25% isopropanol mixture.
6. If the pressure has not normalized, discard the column or try even stronger conditions by increasing to 100% isopropanol, methylene chloride, or hexane. (Note: If using methylene chloride or hexane, flush the column with isopropanol before returning to your reversed-phase mobile phase.)

For columns with particles <1.8µm, do no backflush the column – replace the column.

Storage recommendations

Long-term storage of silica-based, bonded phase columns should be in a pure, organic solvent, such as acetonitrile. If the column has previously been used with a buffered mobile phase, the buffer should first be removed by purging the column with 20 to 30 column volumes of a 50:50 mixture of methanol or acetonitrile and water, followed by 20 to 30 column volumes of the pure solvent. Before storing, end‑fittings should be tightly capped with end-plugs to prevent packing from drying out.

To protect equipment, remove salts from the instrument and column by purging the column with the same mobile phase without the buffer (for example, using 60:40 ACN/H20 to remove a 60:40 ACN/0.02 M phosphate buffered mobile phase). Re-equilibration is rapid with the original mobile phase when using this approach, and any danger of corrosion from the salts is eliminated.

Consider a standalone HPLC Pump to Regenerate HPLC Columns

If you are looking to regenerate or prepare the column for storage but you don’t want to waste valuable HPLC time as you have samples to run, consider a standalone HPLC pump. Chrom Tech offers economical HPLC Pumps that can be plumbed with a few simple HPLC Accessories, including HPLC Stainless Steel Tubing.

For overnight regeneration, you can use a low-pressure solvent selection valve to automate the solvent selection. For more information, see our Regenerated Columns white paper and reach out to our technical support team. One of our chromatography specialists is here to help.