Best way to equilibrate HSS T3?

<p>Just curious about opinions regarding best way to equilibrate an HSS T3 column.  I know it's 100% aqueous compatible, but is it better to have a touch of organic in there?  Which one (ACN, MeOH...)?  I run at 40oC.  Reason I ask is because I feel like I may have "lost" an analyte, in other words I had 20 good runs in a row where I saw two analytes, now I am only seeing one.  Did my column give up the ghost or does it just need a stern talking to, i.e. perhaps a good long cleaning (in what?) or just a good long equilibration (in what?).  Any other wisdom to help me find my lost analyte?  Thanks!</p>


  • Beowulf

    Without knowing your analytes or molbile phase, your question is a bit hard to answer.  In general, the care and use guide for your column should be sufficient; mobile phase equilibration with 10-20 column volumes should be sufficient and cleaning with 100% of whatever organic you use in your mobile phase. A more thorough description is in the HSS care and use guide.  If you are missing and analyte after about 20 injections, that is another whole issue.  It could be sticking on the column, not being retained at all, or co-eluting with the peak you do see.  Consider:

    Sample solvent organic higher concetration that mobile phase?

    Dirty samples?

    Unfilterd mobile phase?  HPLC-UV grade is filthy.

    Are you using guard column?

    Have you monitored the column pressure? Does pressure increase during use?


  • Cleaning with a mix of  1% Formic Acid, 24% Water, 25%Methanol, 25% Acetonitrile, 25% Isopropanol for 10-20 CV works wonders (most of the time).

    After this, wash with 20 CV organic phase and re-equilibrate with your aqueous phase.

    Good Luck!

  • symes7

    Thanks much to all for your suggestions. Your time is appreciated! I will give these ideas a try.

  • Lusby

    10 - 20 column volumes of what you are equilibrating to should do the trick. I have seen this exact column destroyed by running under high aqueous conditions for long periods in an attempt to equilibrate.Examination of the column in these cases reveals silica dissolution in the head of the column every time.

    This column is not as robust to acidic aqueous conditions as the marketing suggests.

  • dougm

    A void at the head of the column is due to either a mechanical void of the packed bed or dissolved particles. Acidic conditions do not dissolve silica particles nor do such conditions cause a column void. Unbonded silica can be used in highly acidic conditions for extended periods of time without issue.

    Highly acidic mobile phases cleave the siloxane bond that connects the ligand (e.g., C18, CN, PFP, etc) to the particle. We empirically set the lower pH limit of a given stationary phase based upon accelerated acid stability testing. The type and density of bonding as well as endcapping determine how well/poorly the column lasts under acidic conditions. Here is a graph of such accelerated acid stability testing (smaller bar is better).

    Details describing this test are found in this reference: Trammell, B. C.; Boissel, C. A.; Carignan, C.; O’Shea, D. J.; Hudalla, C. J.; Neue, U. D.; Iraneta, P. C. J. Chromatogr. A, 2004, 1060, 153. You can also see a description of this test in the attached CSH Technology Whitepaper.

    Acid Stability.jpg

    We are measuring the loss in retention for the neutral probe methyl paraben under extreme, accelerated 'column killing' conditions. As you lose bonded phase (e.g., C18, PFP, CN, etc) and endcapping you lose retention for neutral compounds. Retention loss directly correlates to ligand loss. We are not dissolving particles (and creating a void).

    So, if you are experiencing voided columns, the reason is not due to acid stability. Something else is going on.

  • Lusby

    Okay, thanks for the paper.

    Yep silica is acid resistant, thats why we have sandy beaches. But if you do want to discuss this further....

    Equlibrating under pH 4.5 conditions produces a void in the column head every time. To be honest, I dont care about the mechanism, but what i do care about is the fact that this was promoted as a column that is stable under acidic aqueous conditions, but it isnt, and has had serious consequnces for a new method, where after validating with the T3 we had to re-validate with a YMC column because the T3 was only lasting 200 - 300 injections (delaying a new API project!!!!)

    The buffer is quite concentrated, but is only sodium acetate at pH 4.5 with acetonitrile as the modifier. Any info on why this failure is occuring would be appreciated. We had contacted waters previously regarding this, but the feedback was that the acetonitrile possibly contains an impurity that destroyed the column. A scenario that seemed very unlikely, and was not helped by trying different brands.