​Cleaning Your C18 HPLC Column: A Guide

C18 columns are widely used reversed-phase HPLC columns. However, over time sample residues and contaminants can build up, degrading performance. Appropriately cleaning your C18 column preserves integrity, extends lifespan and restores separating power. Follow these best practices for cleaning:

Solvents

• Use a mix of water, acetonitrile (ACN), isopropanol (IPA) and methanol, like 25:25:25:25. The blend helps solubilize diverse sample residuals. Also add ~1% formic acid if processing basic compounds.

Method

• Set a slow flow rate and pump the cleaning solvent mix overnight. This allows sufficient contact time for dissolving residues. Reverse flowing can also aid cleaning, if your system supports it. Follow manufacturer directions for reverse flowing suitability.

Avoid Overloading

• Always use guard columns and 0.45um pre-injection filters. These dramatically reduce particulate matter and contaminants passing through the column itself. Change filters often to prevent clogging. Guard columns extend the usable life of the analytical column.

Post-Cleaning Checks

• After cleaning, run an analytical method and blank injections with fresh solvents to check. The column is sufficiently clean when blank chromatograms show only low, flat baselines with no spurious peaks. If residual peaks remain, repeat cleaning as needed.

Proper Storage

• Once satisfactorily cleaned, flush the C18 column with a weak solvent strength mobile phase (60:40 water:ACN typical) compatible with planned applications. Cap column tightly. Store upright in this storage mobile phase for ready future use.

Following this routine maintenance, your C18 columns will deliver reliable, trouble-free service for many injections to come. Let the retention times and chromatographic separations continue!