Treating column with EDTA prior to use on LC/QTOF

We have confirmed that some our proteins are binding to active sites on our SEC column used on our MS. We need this specific column material and pore size so we cannot switch to bioinert. 

The column vendor recommended running 40 mM EDTA disodium hydrate through the column prior to use. I've used EDTA for scrubbing metals from an LC but never with a QTOF attached. I would obviously bypass the MS until the EDTA had flushed out completely but I'm concerned that it might contaminate the system with sodium and lead to increased Na+ adducts in our spectra. 

I've seen some non disodium based solutions but it's hard to tell whether there is Na/K present and non are MS-grade.

Any suggestions?

Thanks for your help!

  • So haven't ever really run into specific issues with native MS with SEC methods, though I will assume that the ammonium acetate that you are using is low metal content, so even ACS reagent grade may not be sufficient to ensure no adduct formation. Otherwise, if you have run into this issue and still see trace sodium and potassium, I would suggest flushing the LC front end with 0.1% formic acid and some ACN (5-10%); this should displace most trace metals in the flowpath. Otherwise, avoiding glassware, using high quality vials and such, this is critical as well. 

    You can use EDTA in the mobile phase eve, though you don't need a lot, 1-5 µM or so might suffice if it's still an issue. 

    Also, note the aforementioned recommendations are typically what I recommend for oligos (ion pair reversed phase). Different application I know, but a lot of the same challenges. That is, the real issue with non-RPLC methods and LC-MS is that, in most RPLC methods, you're basically running formic acid so you are displacing trace metals while running your method. However, with Native MS (or whatever you are using for your SEC-MS method), you are running likely at pH neutral conditions, so effectively you are not"passively passivating" using formate to complex heavy metals, so it's going to cause problems when working with analytes that are prone to adduct formation. 

    Hope this is useful!

  • Hi MS2N,

    Brian's given a good answer below.  There are a few other things that occur to me from my own (oligo) experiences.

    You might have some success by passivating the system, and as long as it's compatible with the column then you could apply this to the column too:

    If you google "agilent passivation for ILC" the top result is a good protocol to follow.

    I certainly would be happy to flush a column with EDTA, although I wouldn't put those concentrations into my MS!  For oligos we use 5uM EDTA in our eluents and flow that into our MS with no issues.  We use the free acid, definitely not the sodium salt (solubility is a pain, but sonicate it for most of the day and you can get it into 100% aq solution at about 20mM), you could use the conjugate base from your eluents to improve solubility if you wanted.  We also purchase trace metal grade EDTA.

    We've also seen some issues with area repoducibility in both UV and MS due to active site adsorption - we deal with this by running a few sacrificial conditioning injections of the analyte to saturate the active sites.


  • UPDATE: 

    Thank you for the input! I figured I'd share what we did in case someone else has this issue. 

    Not wanting to use the EDTA disodium salt and potentially contaminate the system with sodium ions, I went with the trace metals 99.995% pure EDTA from sigma (431788-25G) . Due to the poor solubility of the free acid I titrated to pH 8 with MS-grade ammonium hydroxide to generate the diammonium EDTA at ~40 mM. (Definitely worth doing in a hood haha) 

    I first ran the Agilent phosphoric acid passivation procedure recommended for using the InfinityLab deactivator without the column installed. Then I flushed overnight with the diammonium EDTA with the column attached. (ALL OF THIS WAS TO WASTE. NOT THE MS) 

    This did help slightly compared with earlier tests but not nearly as well as just shooting large amounts of our protein... 

    I will add that it had the side effect of making our peptide mapping look excellent (as to be expected) 

    I'll be trying the InfinityLab deactivator (medronic acid) next.

  • Appreciate the update! Proteins binding to active sites is of course common for SEC- past experience makes me blame the column frit, but proteins can indeed bind to silica-based SEC columns.

    Sufficient cosolvent/salt (typically potassium or sodium phosphate in LC-UV) suffices to minimize adsorption due to negatively charged silanols. Particularly hydrophobic proteins may then stick to the stationary phase (commonly a propyl-diol or similar), though this is commonly more an issue with high concentrations of salt in the mobile phase. 

    For Native MS applications, more often than the mobile phase is 20-50 mM ammonium acetate unadjusted; definitely not sufficient salt for particularly basic proteins. Peak tailing and/or "non-ideal" SEC behavior (elution times not corresponding to hydrodynamic radius) would indicate that was the case. In these instances, 100 mM might be required; this could potentially kill signal but there are workarounds...

    Either way, if priming helps (I'd say upwards of 20-50 µg on a 2.1x150 mm ID), then this is a good workaround. However, bear in mind that storing the column in a sufficient organic to act as a bacteriostat (e.g. 10-20% methanol) will likely strip the active sites and require priming again. 

Was this helpful?