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

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 there is any question about the proper way to store, please see the LC column usage and care instructions.

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. To do this, please see our  LC Cleaning Recommendations (Note: Pumping in a backward direction is not recommended for UHPLC -any columns with 2.0 µm particle size or smaller, 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.

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3 Responses to “Resurrecting an old reverse phase LC column- should I, could I, how would I???”

  1. Rusty (Robert) Bryant says:

    Hi Nancy,
    I liked your comments. I’m a “retired biochemist” from big pharma but am now voluteering as at a small college. I’m trying to restore performance on some columns which now seem to be trapping the organic samples. They are getting stuck on the ccolumn even when running methanol

  2. Robert "Rusty" Bryant says:

    Hi Nancy,
    I likes your comments above. Have you ever had success with acetone or acetone:acetic acid to clean c18 columns which have gotten clogged with organics. I’m thinking of reversing them to flush the “organic plug” back out. I’ve been running hop acids (humulus lupulus) over them and they are not coming out well.
    You guys used to be Supelco, right. I used to use your silylation reagents for our prostaglandin research many years ago.

  3. Hello Rusty, thanks for your comments and participation. We are a separate company, not affiliated with Supelco. Really the best way to clean the columns is the method approved by our applications chemist, found here on our website: Most of our C18 columns have a lower pH limit of 2.0 or 2.5, so we would have to be careful about using any acid to clean the phase. Usually that is not the desired technique. I have not used acetone, it has a polarity between isopropanol and hexane, so would be OK to use for C18 if you use it in the right sequence with the other solvents listed in the instructions I just mentioned. Please note that acetone or any solvent containing an aldehyde or ketone should never be used with amino phases, as it will permanently alter the phase. I hope that helps to answer your questions. If you need to discuss further, please feel free to contact Support ( Thank you!

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