Stop Flushing Arbitrarily! These Procedures Effectively Regenerates Your HPLC Columns

In HPLC, columns are the heart to laboratory analyses. But most analysts might face such phenomena: even with no changes to the methods, over time, the retention time drifts, the peak tails, and the resolution falls. And even a new column could lose efficiency after only a few runs.

These problems are not caused by the method, but by inadequate flushing!

A single column costs hundreds to thousands of dollars. Frequent replacement not only wastes money but disrupts schedules and undermines reliability. Most column efficiency loss can be prevented through scientific, standardized flushing and maintenance instead.

Different mobile phase systems require different flushing procedures and duration. All following procedures in this article are for reversed-phase columns only. For procedures for other columns, please refer to our column care and use manuals.

• No acids/bases/salts: Flush with 80% methanol.
• Acids/bases/salts present: 10% methanol followed by 80% methanol.
• Ion-pair reagents: 10%, 50%, 80% methanol in sequence.

All flushes are under method flow rate.

Flush durations are differentiated by column length. 

• 100 mm or below: 30 minutes each. 
• Above 100 mm: 40 minutes each.

If column pressure rises, peaks shapes are abnormal, or resolution drops, don't throw it away. Flush it reversedly, first with 10% methanol to remove salts, then with methanol, acetonitrile, isopropanol, and acetonitrile in sequence, all under 1/4 of method flow rate. 

• Column length 100 mm or below: 100 minutes each. 
• Above 100 mm: 120 minutes each.

Note: after backflushing, remember to return the column to forward direction. If used reversedly, the column ends will block, preventing future flushing and use.

After flushing, remember to store the column in a high-organic solvent, tighten the plugs, and keep it in a cool, dry place. This avoids the column bed from cracking and the stationary phase from collapsing, allowing the column to "sleep" safely.

Tips:

1. Methanol can be substituted with acetonitrile.
2. Isopropanol has high viscosity; reduce flow rate to avoid overpressure.