Equilibration/resolution problems with new Acquity columns (using TFA-containing eluents)

<p><span style="font-size: 10pt; font-family: Arial;">Hi,</span></p><p></p><p>I want to introduce myself shortly. My name is Ronald, I am Project Chemist and develop UPLC methods for purity and assay for peptides with approx. 1000 - 6000 dalton. One basic requirement for our methods is that the mobile phases must be suitable for MS. This means we are using mainly TFA containing eluents with 0.02 or 0.05% TFA in water and acetonitril (alternatively also formic acid, ammonium acetate or ammonium formate buffers). Since 1 year we are confronted with a problem when using new Acquity columns for our analyses. <strong>After an equillibration procedure</strong> (see attachment) <strong>we do not obtain sufficient resolution with new columns</strong>. We see these problems with BEH PST columns, as well as with BEH Shield or BEH phenyl columns. Further <strong>we loose resolution, when we flush the columns afterwards with ACN/H2O 7:3 before storage (see attachment)</strong>. This leeds to a enormous efforts for several days to gain again resolution of the column.</p><p></p><p>We do not fully understand the problem. This problem is also present in other departments and at our customers, when the methods are transferred to them. One approach is, to flush the column with ACN/H2O 7:3 + 0.5% TFA at higher temperatures (60 - 80°C) for one day or over weekend. And we start to store our columns in ACN/H2O 7:3 + 0.05% TFA to "save" resolution (not to loose resolution again). We think that the problem might have something to do with ionparing/TFA/Column/Ligand/Peptide.</p><p></p><p>Maybe someone of you has similar problems? I would like to know:</p><ul><li><span style="font-size: 10pt; font-family: Arial;">Which equilibration procedure is adequate to gain resolution with new UPLC columns (using TFA-containing eluents afterwards)</span>
</li><li><span style="font-size: 10pt; font-family: Arial;">What happens exactly on the column with TFA?</span>
</li><li><span style="font-size: 10pt; font-family: Arial;">What is the secret behind i</span><span style="font-size: 10pt; font-family: Arial;">onparing/TFA/column/ligand/peptide?</span>
</li><li><span style="font-size: 10pt; font-family: Arial;">Why do we loose resolution after flushing the columns with ACN/H2O 7:3 before storage?</span>
</li></ul><p></p><p>I am looking forward to your feedback.</p><p></p><p>Kind regards</p><p>Ronald</p>


  • Ronald:

    TFA is as an ion-pairing reagent that enhances binding of peptides to the column in two ways.  As an acid, it will neutraize the positive charges on the peptides.  The TFA will also increase the hydrophobicity of the peptide-TFA pair, increasing the retention time and increasing resolution by improving peak shape.  Ion-pairing reagents also interact with the column packing.  It requires a long time for the TFA to establish an equilibrium between the column and the mobile phase because it must penetrate the pores of the packing material.  By washing the column in acetonitrile-water before storage, you remove the TFA from the column.

    If you are using a mass spectrometer, I would recommend you try 0.1% formic acid in the mobile phases instead of TFA to increase sensitivity by reducing ion suppression.  Formic acid will reequilibrate quickly.  ACQUITY columns, unlike may older silica based columns, usually does not require ion-pairing agents for good peak shape.  If you need greater retention of the hydrophilic peptides, you could try ACQUITY HSS column.

  • Hello everybody,

    I was terribly busy during the last couple of weeks. Therefore I could not make any further experiments. But now I have time today and received the required test columns from Waters (Acquity PST 130 BEH C18; 150x2.1mm; 1.7µm; batch 193). I set up a design of experiments, that I discussed with Uwe Neue (from Waters) and I want to start today:

    At first I want to analyzed my special peptide method, that is sensitive for this equilibration problem. This method will be run with all columns to obtain chromatograms for T0 (not well equilibrated columns).

    Method parameters: 0.02% TFA in water/acetonitrile mixture; temp: 60°C, flow rate 0.25 mL/min

    Starting position:

    30min equilibration at starting conditions

    22min gradient run without injection (inject immediate samples)

    22min blank run

    22min sample run

    This will be performed for 3 new columns (0 injections) and one well equilibrated column of the same batch (145 injections).

    After that, the 3 new columns will be stored different:

    1: flush column with water/acetonitrile 3/7 + 0.02% TFA for 30min, stop flow, storage for 1 week at 25°C

    2: flush column with water/acetonitrile 3/7 + 0.05% TFA for 30min, stop flow, storage for 1 week at 25°C

    3: flush column with water/acetonitrile 3/7 + 0.05% TFA for 30min, stop flow, storage for 1 week at 40°C (stability chamber)

    After the first week the sequence for the starting position will be repeated again to see, which of the different slow equilibration procedures was successful.

    I keep you updated.


  • Ronald:

    I will be very interested in the results of your experiment on the column storage.


  • Agreed

  • As do I.

    Remember that TFA is an ion-pairing agent. When you strip an ion-pairing agent from any LC column (such as using/storing it without TFA), it will require a period of re-equilibration. The same can be said for any classic ion pair such as octane sulfonic acid (OSA) or tetrabutyl ammonium chloride. When I was in the lab, I used ion-pairing agents all the time. When installing a new column (or a previously used column) that I wished to use with an ion-pairing agent, I assumed that I needed to equilibrate overnight. I never performed a formal study to see if this period of time was overkill, but I knew that when I came in the next day that the RTs for my compounds would be rock solid. It just made method development a bit easier as that was one element that I did not have to worry about. Also, upon storage, the column was labeled with the ion pair I used so that nobody would use the column unless they wished to use a forever-changed LC column. As we used to say: once ion paired, always ion paired.

    Not to sound too commercial, but our newest family of HPLC and UPLC columns (XSelect and ACQUITY UPLC CSH) offer much of the performance that you are looking for with TFA (improved shape peaks for bases, etc.) with formic acid without the associated TFA drawbacks such as column equilibration/re-equilibration and MS ion suppression. If you are at all interested, please go here: http://www.waters.com/csh.

    Please post your findings and good luck!!


  • TX Doug!


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  • Hello everybody,

    It was a while ago since I've started the experiments but I was busy and I was not able to take care for this issue. The outcome of my experiments were:

    One week equilibration with water/acetonitrile 3/7 + 0.02% TFA at room temperature was not enough to reach the required resolution of the column.

    One week equilibration with water/acetonitrile 3/7 + 0.05% TFA at room temperature was sufficient to reach the required resolution of the column.

    One week equilibration with water/acetonitrile 3/7 + 0.05% TFA at 40 °C in a stability chamber was as good as equilibration with water/acetonitrile 3/7 + 0.05% TFA at room temperature to reach the required resolution of the column.

    My conclusions are:

    Temperature is not necessary for the equilibration of the columns. 0.05% TFA is better than 0.02% TFA for the equilibration of UPLC columns to use them for ion-paaring chromatography with TFA. To retain the resolution with an well equilibrated column, we never flush the column without TFA. We do also store it in water/acetonitrile 3/7 + 0.05% TFA. Otherwise, we have to start again the long equilibration procedure. The equilibration is time depending and not depending on how much solvent is used for equilibration. It is similar with good Whiskey, it takes 10Y and not ony 3Y to obtain good Whiskey.

    Kind regards