Generic Reversed Phase Column Clean

Generic Reversed Phase Column Clean

An LC column is one of the most critical parameters in reversed phase chromatographic separation. The buffered mobile phases, corrosive additives and any strongly bound compounds need to be removed to prevent precipitation and degradation of the silica bed. As such, efforts should be made to ensure the longevity of the stationary phase, by performing simple column cleaning gradients and appropriate column flushes at the end of every batch and for long term storage. The following is a generic guide to flushing and storing reversed phase columns, therefore please refer to your laboratory SOP and column manufacturer information for more details.

Column Clean:

1. Add HPLC grade water and acetonitrile to Lines A and B, respectively. Use LCMS grade solvents if a MS is in series.
2. Purge the mobile phase lines to remove any air bubbles
3. Ensure the column is connected securely to minimise leaks.
4. Set up the LC method with the following conditions.

Method Conditions

Mobile Phase A:   Water

Mobile Phase B:   Acetonitrile

Gradient:               Time       %B  .

                                0.0          5

                                5.0          5

                                15           95

                                20           95

                                20.1        5

                                25           5

Flow Rate:             1 mL/min (adjust for column ID)

5. Stop the pump and removed the column from the flow path.

6. Replace the column end stops to prevent evaporation of solvent which can cause the stationary phase to dry out.

7. If the column is going into long term storage, perform the column storage conditions or adjust the gradient to end on 50% acetonitrile.