Which Column to Choose?

<p>Hello ,</p><p></p><p>I have a new UPLC method for analysing a non-polar compund using a BEH C8 ,1.7µm ,2.1*100 mm column and</p><p></p><p>using Eluent containing :Water:ACN:Perchloric acid (700:300:1 v/v/v) .The problem with is that it takes a very long conditioning time for equilibrating</p><p></p><p>the column in order to achieve system suitability .If the column is conditioned for relatively short time the peak is not eluted .</p><p></p><p>only after injecting for several times the peak starts to appear (sometimes with high tailing) .</p><p></p><p>Am I using the wrong column type for non-polar compound or should I change Eluent ?</p><p></p><p>Regards ,</p><p></p><p>Maher  </p>


  • Hello,

    To be honest, I am not that familiar with the routine use of perchloric acid (PCA) as a mobile phase additive. It it acts like other acidic modifiers such as formic acid or phosphoric acid, there may be an equilibration/re-equilibration issue when used at concentrations such as those that you use (0.1%) when retaining and separating basic compounds.

    I have some questions.

    Is your analyte of interest a base?

    How flexible are you in choosing a mobile phase? One suggestion that could improve equilibration time is using a salt such as (e.g., sodium phosphate, ammonium formate, etc.) instead of the PCA additive. That would likely decrease your equilibration time, assuming the reason for this issue is the low ionic strength, acidic modifier that you are using. I know that sodium perchlorate is commercially available in HPLC grade, but I am unfamiliar with its routine use in LC.

    How flexible are you in choosing a column? Another option would be to use an ACQUITY UPLC CSH column (C18, Phenyl-Hexyl or Fluoro-Phenyl). CSH columns are designed to equilibrate rapildly in mobile phases such as yours (see HERE for more info).

    Again, my suggestions are based on two things: 1) your analyte is basic in nature; 2) your equilibration issues are due to the acidic, low ionic strength mobile phase that you are using.

    I hope this information helps,


  • Perchloric acid is an ion-pairing agent. That might explain the strange chromatography (including the long equilibration times)



  • hi,

    maybe u can simply take more ACN  (4:6 insteat of 3:7 and change from percloric to phosphoric acid). dont know how it works, or maybe u tried this, but i would change this parameter first.

  • We have the same issues with TFA in mobile phases (0.02 - 0.1%). The solution was, that we store all UPLC columns in the mobile phase, or ACN/H2O 7:3 (V/V) + 0.05% TFA. You should try this. Take your column, flush it with your mobile phase containing Perchloric acid store it overnight and then run the method again. After that, store it in your mobile phase and your column will be well equilibrated for the next time.

  • If you want to protect your column life time you should never ever store it in a solvent ocntaining an ion pairing agent or acids/bases/buffer salts.

    Most of these UPLC columns should be stored in 100% ACN.

  • This is your opinion. We are following this approach since a long time and we do not see any disadvantages concerning column life time. Maybe columns stored in 100% ACN have a longer column lifetime, but we see more advantages in storing the columns in 0.05% TFA. We do peptide analyses. TFA as ion paring reagent is used in 95% in our methods. If we take a column, that was only stored in ACN/H2O, it sometimes takes up to 1 week for equilibration. Therefore we store the columns in the mobile phase and we always have well equilibrated columns that show their ideal performance at once and not a week later! We had a long discusion with Dr. Uwe Neue from Waters and he recommended this approach.

    Please follow this discussion here

    http://forums.waters.com/message/3855#3855 and here


  • I would like to emphasis, that my approach is recommended for methods using ion paring reagents in the mobile phase and here is a useful publication . . .

  • According to BEH column care and use manual it takes 100-200 column volumes to fully equilibrate if low-concentration mobile phase additives are used.

    Ronald may be right that it would be more effective to store column in mobile phase (or storage buffer with additive present) than bother with long equilibration times.

  • Yes, you are correct that this is indeed my opinion.

    I've followed Dr. Neue's work closely in both his publications and on ChromForum. I was unable to find where he recommended using this approach you have described. That must have happened in your offline discussions.

    I also just don't see the benefit of this approach (personally). A typical 2.1 x 100 mm column has an empty volume of about 350 uL. 100 times this amount is 35 mL of mobile phase that needs to be run through the column. I understand well that ion-pairing methods take significantly longer to equilibrate. I've not done any of these methods on a UPLC yet, but they HPLC methods I've developed, validated, and ran took maybe 3-4 hours max. So you start the system up and go make samples. By the time you are done your system is ready.

    If this is taking a week for equilibration then, being completely igonrant of your situiation, I would be inclined to think that something else must be at play. I would also not recommend this as a initial approach for everyone.

    Having said that, if you have actually been doing this and have not experienced any negatives to date ... obviously it is working out for you. And why try to fix what is not broken...

  • Yes you are right. We discussed a lot of things offline via email.

    1 week equilibration is only necessary when installing a brand new column. What I also found out is, that it is not necessary to flush the column 100 or 1000 times the column volume, flow does not speed it up. It is sufficient to flush the column for about 30 min and then we can stop the flow or store it on the lab bench. After 1 day (or 3 days after weekend; or up to 1 week for brand new columns) the column is equilibrated and ready to use with TFA. I made a cross comparison, equilibration with flow and without. The storage time (equilibration time) in TFA was the key factor.

  • I'm really curious here as this is quite contrary to what we have been told all along. I hope you do not mind...

    Would you happen to remember/mind sharing why Dr. Neue eventually ended up stating that a column can be stroed in TFA without in essence killing the column?

  • I'm really curious here as this is quite contrary to what we have been told all along. I hope you do not mind...

    Would you happen to remember/mind sharing why Dr. Neue eventually ended up stating that a column can be stored in TFA without in essence killing the column?


    That will not be so easy. At first, when I explained my problems and my trials, he pointed out, that I will kill the columns with 0.5% TFA. That was a procedure we tried out at the beginning. Later we decreased the TFA content down to 0.05%, like the concentration in most of our mobile phases. 0.5% TFA did not speed the equilibration process up. Therefore it was not necessary. During some discussions within my company I found out, that our lab for in process control never flush their columns with ACN/H2O. They always store them in 0.05% TFA ACN/H2O 7:3. They said, they do not see any disadvantages concerning a shortage of column life time. But Waters would never say that. They want to guarantee a maximum column life time. It is for sure, that a storage at pH2 (0.05% TFA) reduces column life time, but we do not see a rapid decrease. For us it is acceptable. We rather purchase a new column, than having issues with long equilibration times again and again and again.. As long as we store the columns in TFA, we can run the methods immediately after setting up the system, because the columns are already well equilibrated. 2 blank runs are sufficient. After that, the baseline is stable. I discussed my experiments concerning equilibration of columns with Uwe and my results indicated, that our updated procedure with flushing the column with ACN/H2O 7:3 + 0.05% TFA for 30 min and storage in the same solution, might be the best way for us, to avoid equilibration problems. BECAUSE 95% our methods are with TFA.

    BUT this procedure is only appropriate for methods with TFA containing eluents. If you have other mobile phases with other ion paring reagents, store the column in these solvents. If the pH value of the mobile phase is to agressive, decrease the amount of acid in the wash/storage solution. If we have a method with e.g. ammonium format buffer, for do not follow this approach. For these cases, we store the columns with ACN/H2O 7:3 only. Our experiences are, never use columns with ion paring reagents for methods without. It takes a very long time, to flush down the ion paring reagents down the column. The best way seems to be to reserve columns for specific methods.

  • Thank you  for your answer Ronald. That's a fairly neat trick that I might need to look into.

    I very clearly see your point and why you have chosen to proceed in this manner. Even if you are getting a slight reduction in column life, the cost savings from not having to equilibrate everytime must be incredible. Especially when the majority of your methods use TFA.