How To Regenerate a C18 HPLC Column

Typical Regeneration Procedure for Reversed-Phase HPLC Columns

Regular cleaning of your column helps extend its lifespan and reduces the need for frequent replacements of HPLC columns.

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

1. Disconnect the column from the detector and direct the wash solvents into a beaker.
2. Flush 10–20 column volumes of mobile phase without buffer salts (water/organic mixture) through the column.
3. Use 100% organic solvent, such as methanol or acetonitrile.
4. Check the column pressure. If it returns to normal, regeneration is complete. Otherwise, continue to the next step.
5. If pressure remains abnormal, try stronger conditions (e.g., 75% acetonitrile / 25% isopropanol).
6. If still abnormal, increase solvent strength to 100% isopropanol, methylene chloride, or hexane. Note: When using methylene chloride or hexane, flush with isopropanol before returning to the reversed-phase mobile phase.

For columns with particles <1.8 µm, do not backflush; replace the column instead.

Column Regeneration Quick Reference Table

Storage Recommendations for Reversed-Phase Columns

Silica-based bonded phase columns should be stored long-term in a pure organic solvent, such as acetonitrile. If the column has been used with a buffered mobile phase, remove the buffer first:

• Purge the column with 20–30 column volumes of a 50:50 mixture of methanol (or acetonitrile) and water.
• Follow with 20–30 column volumes of pure organic solvent.
• Cap the end-fittings tightly with end-plugs to prevent packing from drying out.

To protect your HPLC equipment, remove salts from the instrument and column by purging with mobile phase without buffer (e.g., 60:40 ACN/H₂O to remove 60:40 ACN/0.02 M phosphate buffer). This ensures rapid re-equilibration and prevents corrosion.

Consider a Standalone HPLC Pump for Column Regeneration

If you want to regenerate or prepare your column for storage without using valuable HPLC time for samples, a standalone HPLC pump is an efficient solution. Chrom Tech offers economical HPLC pumps and accessories, including stainless steel tubing.

For overnight regeneration, a low-pressure solvent selection valve can automate solvent switching. For detailed guidance, see our Regenerated Columns white paper or contact our technical support team—our chromatography specialists are ready to assist.

Frequently Asked Questions (FAQ)

Why Should I Regenerate My HPLC Column?

Regular regeneration removes contaminants and salts, helping maintain column performance, extend lifespan, and reduce replacement costs.

Can All HPLC Columns Be Backflushed?

No. Only reversed-phase columns with particle sizes >1.8 µm are typically safe for backflushing. Smaller particle columns should not be backflushed to avoid damage.

What Solvents Should I Use for Regeneration?

Start with the mobile phase without buffer salts. Use 100% organic solvents such as methanol or acetonitrile. For stubborn contaminants, stronger solvents like isopropanol, methylene chloride, or hexane can be used following proper precautions.

How Should I Store Columns Long-Term?

Store silica-based columns in a pure organic solvent. Ensure any buffered mobile phase is removed, and tightly cap the end-fittings to prevent the packing from drying out.

Can I Regenerate Columns Without Using My Analytical HPLC System?

Yes. A standalone HPLC pump can be used to flush columns with minimal disruption to your analytical workflow, and automation tools like low-pressure solvent valves can assist with overnight regeneration.