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Do reverse phase HPLC columns really need conditioning?

13 February 2014
  • Nancy Schwartz
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Well, it’s partly a matter of semantics sometimes, but there is usually nothing drastic that you need to do to get your HPLC column going if it is new.  There is no baking at high temperatures involved like you might do for GC or that sort of thing. You do, however, need to equilibrate in the mobile phase that you will be using. If you will be doing isocratic runs and your mobile phase is similar to the solvent used for storage and shipping (usually 50/50 methanol and water), you should only need to pump 7 -10 column volumes of mobile phase through the column.  That would be the first thing to try. If you need help determining column volume, click on the link below to go through the calculations:

How do I determine total column volume or void volume for LC?

If you are using a gradient, using a mobile phase that is higher in organic content than your storage solvent, or if you are using a mobile phase that has a different organic solvent than what it was stored in (for example, acetonitrile instead of methanol), you should pump for a while with a solution that contains the organic solvent you will be using in a slightly higher (10-20% higher) content than what your method calls for. This is a precaution in case there is anything present that has a higher solubility in the new solvent versus the previous one(s). After you do this, you can gradually begin pumping your mobile phase to equilibrate.

Pumping a slightly higher organic content as described above is also a good practice to use for columns that have sat idle for a while, especially those that have had dirty sample matrices loaded. It is also a good practice to use if you are noticing an elevated baseline by UV that you suspect might be column bleed. This usually minimizes those effects if they occur.

However, a word of caution: If you are using buffer salts, make sure to flush them out with at least 50% but no higher than 90% aqueous solution before gradually increasing the organic content. (If using an Aqueous C18 phase, you may use up to 100% aqueous if needed). Failure to remove the buffer salt will cause it to precipitate out of solution at the next step, which will result in a pressure increase and clogging of column frits.

Even after proper equilibration, many analysts still notice more interference or inaccuracy with the first injection after a system has been sitting idle. There are many debates and theories about why this happens, but it may involve elimination of contents sitting in valves (or portions of such valves) and tubing that doesn’t get flushed well until a full cycle of injection and analysis occurs. Most of us regard this first injection as somewhat of a “primer” and this is not reported. Many of us have gone so far as to label such data as “equilibration” and I venture to say that quite a few SOPs have been written globally to address such things.

I hope these suggestions will help you get started if you’re feeling anxious about it.  Let us know if you have any issues or questions along these lines. Thanks for reading.


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Fri, Jul 24, 2015

Thank you Mr. Bob Weidemer for sending me the link.