How To Regenerate a C18 HPLC Column

Typical Regeneration Procedure for Reversed Phase Columns:

Cleaning your column periodically can extend its lifetime, which helps to reduce the frequency of purchasing new HPLC Columns.

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

1. Disconnect column from detector and run wash solvents into a beaker.

2. Start with your mobile phase without buffer salts (water/organic). Flush 10 to 20 column volumes through the column.

3. Next, use 100% organic (methanol or acetonitrile).

4. Check pressure to see if it has returned to normal; if not, then:

5. Discard column or consider stronger conditions (for example, 75% acetonitrile/25% isopropanol)

6. Check pressure to see if it has returned to normal; if not, discard column or consider stronger conditions: Increase to 100% isopropanol, 100% methylene chloride, or 100% hexane (if you use methylene chloride or hexane, you will need to flush the column with isopropanol prior to use and 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.