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Resurrecting an old reverse phase LC column - should I, could I, how would I???

8 Apr 2014

Well, if we had an Easter egg hunt and you found an egg that was from last year or the year before, you could probably tell the difference and you would know pretty quickly if it had gone bad.  Let’s just say that is not so easy with HPLC columns. If you’ve dealt with this much, you’re aware that it is difficult to fully know the history of column usage for one that you find in a drawer somewhere. That is the biggest problem.  Maybe it was in good shape when placed in storage or maybe not. Maybe it was stored properly or maybe it wasn’t. 

If it is a rugged column phase such as a C18 and it truly has not been used, its condition will depend on how well it was sealed on the ends, the climate/temperature and what solvent it contained. Theoretically it should be good as new if sealed well, stored properly and no extreme high temperatures are encountered. If it has been used, then its future is not so full of promise.

The worst case scenario would be if it was stored in mobile phase containing buffer salts and then it dried out. A dead giveaway for something like this would be extreme pressure issues if you were to start pumping solvent through it. Things almost never end well when this happens. Other minor issues could occur relating to leakage of solvent over time and drying of the packing material. The result of this could be pressure issues and/or some things that look like phase collapse.


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If you are determined save a column that you’ve just pulled out of storage, try the following:

  1. At a reduced flow rate, pump several column volumes of 40:60 ACN/water (in case any buffer salts are inside). Observe the pressure rating throughout this process.
  2. If you do experience any difficulties with high backpressure at this point, stop the flow and let the pressure dissipate.  Try it again at a lower flow rate and give it some more time.  If the pressure is still excessively high, skip to #5.
  3. Once your pressure is in normal range, increase the flow rate gradually to your normal operating flow rate or close to it.
  4. Pump several column volumes of ACN (For rewetting in case the phase has started to collapse or the packing is dried out). Continue to observe pressure. You should see it decrease as more of the water is replaced by organic solvent.
  5. If you continue to have trouble with pressure, for some columns, you can try flushing in a backward direction. (Note: Pumping in a backward direction is not recommended for UHPLC -1.9 µm Pinnacle DB and Raptor Columns, although you can still pump through a series of solvents as described in a forward direction).  For these purposes, you can use ACN and MeOH interchangeably.
  6. If you do not (or no longer) experience high pressures, proceed to pump the mobile phase that you plan to use. Equilibrate with at least 7 column volumes before attempting any injections.
  7. If it does not seem that you are making any progress, contact us before proceeding. It may or may not be worth continuing.

If you have tried these suggestions and still have issues with backpressure or poor resolution, then the column has exceeded its lifetime and not likely to be revived. It’s time to replace the column and go home.