Negative dip in front of impurity / main peak with CSH C18 column and 0.06% HFBA in mobile phase

<p> Hi,</p><p></p><p>I have a negative dip in the baseline in front of the main peak with a CSH C18 column and 0.06% Heptafluorobutyric acid in the mobile phase (ion-paaring reagent!!!). Please look at the sample chromatogram and the method description first. You will find both attached. The sample is a peptide with approx. 4 kDa. It is a hydrophilic peptide with an isoelectric point of 13.4 (if this is important).</p><p></p><p>During the method development I found out that this peptide is very sensitive to HFBA. The previous method was with 0.035% Trifluoracetic acid in the mobile phase but the retention on the stationary phase (CSH C18) was to low. Then I moved over to 0.06% HFBA which is nearly the same molar mass. I found out that this peptide is very sensitive to HFBA. 0.03% HFBA led to peak fronting and 0.09% HFBA led to strong peak tailing. With 0.06% HFBA the peak shape and resolution is nearly perfect. The blank solution shows a normal gradient profile but when injecting a sample solution the baseline makes a dip in front of the main peak. This makes integration not easy and small impurities are overestimated therefore the overall purity decreases. This effect was already visible during the method development but it was not very strong or annoying but during the last analyses before the method validation this effect was strong.</p><p></p><p>This effect is seen on 4 different CSH C18 column batches and with two different preparations of mobile phase. I would like to know, how this can be avoided?</p><p></p><p>We used to have a similar effect with CSH C18 columns and TFA containing mobile phases. Such an effect was visible at low TFA concentrations like 0.02%. We solved the problem by increasing the TFA content up to 0.05%. I do not know, if this is the same with HFBA and CSH C18.</p><p></p><p>I am looking forward to your feedback.</p><p></p><p>Thanks + regards</p><p>Ronald</p>

Answers

  • Hi folks,

    I've perfomed some experiments in the mean time:

    1. evaluation of 4 different column batches
    2. increase of HFBA content in the sample solvent (0.12 instead of 0.06% HFBA)
    3. increase of HFBA content in the mobile phase (0.09 instead of 0.06% HFBA)
    4. evaluation of a brand new column

    The results were:

    1. I saw this negative dip only with two column batches. One column showed a very bad resolution and the second batch showed a sufficient resolution, but was already used within different projects. The peak symmetry was completly different amoung 4 batches:
      - 0.76 (very bad resolution und deep negative dip)
      - 0.70 (good resolution und deep negative dip)
      - 0.98 (good resolution and no deep "visible" negative dip)
      - 1.31 (good resolution und no negative dip visible);
    2. The increase of HFBA content in the sample solvent did not have an influence on the negative dip in the baseline
    3. Then I increase the HFBA content up to 0.09% in the mobile phase. The peak symmetry was completly different amoung these batches.
      - I did not perform this experiment with the first "bad" column on position one
      - 1.82 (good resolution und deep negative dip)
      - 2.20 (good resolution und no deep "visible" negative dip)
      - 2.44 (good resolution and no deep "visible" negative dip)
    4. -The peak symmetry with a brand new column out of the box was
      - 2.17 with 0.06% HFBA in the mobile phase
      - 3.25 with 0.09% HFBA in the mobile phase
      The resolution was sufficient and no negative dip was visible

    Now I think, this effect has "only" something to do with the column age and column condition and not with the composition of HFBA. I think that I have to choose relatively new column and include an SST criteria after method validation.

    Thanks for your feedback

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