The problem of peak draft for help

<p>The problem of peak draft</p><p></p><p>Instrument: ACQUITY Xevo TQ MS</p><p></p><p>LC condition</p><p>Channel A1: H2O + 0.1%FA</p><p>Channel B1: Methanol + 0.1%FA</p><p>Gradient: </p><table border="1"><tbody><tr><td><p>Time (min)</p></td><td><p>Flow (mL/min)</p></td><td><p>%A</p></td><td><p>%B</p></td><td><p>Curve</p></td></tr><tr><td></td><td><p>0.60</p></td><td><p>70.0</p></td><td><p>30.0</p></td><td></td></tr><tr><td><p>0.20</p></td><td><p>0.60</p></td><td><p>70.0</p></td><td><p>30.0</p></td><td><p>6</p></td></tr><tr><td><p>1.50</p></td><td><p>0.60</p></td><td><p>10.0</p></td><td><p>90.0</p></td><td><p>6</p></td></tr><tr><td><p>2.00</p></td><td><p>0.60</p></td><td><p>10.0</p></td><td><p>90.0</p></td><td><p>6</p></td></tr><tr><td><p>3.00</p></td><td><p>0.60</p></td><td><p>70.0</p></td><td><p>30.0</p></td><td><p>6</p></td></tr><tr><td><p>4.00</p></td><td><p>0.60</p></td><td><p>70.0</p></td><td><p>30.0</p></td><td><p>6</p></td></tr></tbody></table><p></p><p>MS condition: MRM 4mins</p><p></p><p>Problem: </p><p>The retention time of injections number from 001 to 037 was draft from 1.53min to 1.60min.</p><p>And the other RT of injections number from 038 to 180 was draft from 1.60min to 1.72min.</p><p></p><p>Would you please give me some advice for this problem and tell me the reason why the peak was draft and how to avoid.</p><p></p><p>thank you </p>

Answers

  • Did I get you right when I understand your problem as drift of retention time?

    If yes then would you like to post your column and tell us something about your compounds and sample matrix?

  • Dear Newman768,

    Thanks for your reply.

    You get right understand. additional my retention time back to 1.65min when injections went on 200 number. so the retention time from 1.53min moved to 1.73 min and then moved back to 1.65min. i have no solution for this problem till now.

    column infor: ACQUITY UPLC BEH C18 1.7um 2.1×50mm

    compound: reserpine

    matrix: no

    thanks again

  • I have an explanation for this. Maybe this explains your problem:

    I like the BEH material, but it has some "strange behavors" like rt shifts when the surface of the column was "modified" by lots of injections and/or mobile phase. (This is not proofed but I never ever learned of a better explanation for the symptom).

    You are using acetic or formic acid, so purge the column with a mobile phase of approx 20 mmol of ammoniumbicarbonate, adjusted to pH 9 with ammonium hydroxyde and add 30% of acetonitril. Run this for 30 min or so over your column and then reequilibrate system and column back to your mobile phase and check rt.

    If the rt stays the same for the next dozend of analysis then it it very likely you run into the effect I mentioned above.

  • thank you so much.

    i will try to do it as your explanation.

    best regards

  • Hello:

    I also want to point out before looking further, I would chceck into the column re-equilibration.

    Your column is 2.1 x 50 mm and has a volume of 174 uL and your flow rate is 600 uL a minute. Your final step seems a bit short to me. You need to explore what your final hold volume needs to be before doing anything else.

    The traditional 5 column volumes and 3 system volumes says you need 300 uL for the system and 870 uL for the column. That is 1170 uL so your final hold needs to be at least 2 mins. You need to play conditions so you can tell what your final hold should be.

    Try this:

    Go to your initial conditions for at least 3 or 4 mins, make an injection and run the gradient change the final step and hold the final reequilibration step for 5 or 6 mins, (ie 4 minutes becomes 6 mins), repeat, does the drift stabilize?

    If it does, reduce the final step by a minute per injection until you see the drift return and this will tell you what you can get away with. You could of course try a higher flow rate and increase the number of column volumes at each step in this manner.

    I think this is a simple experiment just to check that equilibration is really occurring.

    Liz

  • This might make it easier. This is your gradient. I think both your hold and re-equilibration may be problematic

    StepTime (min)Flow (mL/min)%A%BCurve
    1 0.67030
    20.20.670306
    31.50.610906
    42 (a) may be too short0.610906
    530.670306
    64 (b) may be too short0.670306

    Here is the first experiment - make re-equilibration longer

    StepTime (min)Flow (mL/min)%A%BCurve
    1 0.67030
    20.20.670306
    31.50.610906
    420.610906
    530.670306
    660.670306

    Here is the second experiment - make final hold longer

    StepTime (min)Flow (mL/min)%A%BCurve
    1 0.67030
    20.20.670306
    31.50.610906
    440.610906
    550.670306
    660.670306

    Hope that helps

    Liz

  • I agree with Eizabeth, it seens the equilibration time is not enough to re-equilibration column.

  • Imagine my surprise and delight when i come back and see these replies.

    I will remember this method for next project.

    Thanks for your help.

  • Are you controlling the temperature of the column ?

    Uni-directional (as opposed to random) retention time changes are often the result of temperature changes.

    For example, on a system that is not controlling the temperature of the column, it is not uncommon for retention times to drift "shorter" during the day as activity in the lab and rising outside temperatures raise the temperature in the lab.

    Conversely, at night when the lab cools off retention times will drift longer.

    Retention Time consistency requires that column temperature be held constant.

  • Shanghai,

    you have received many good suggestions for your drift problem...

    Since we do not know the purpose or final goal of your analysis ( you have said no matrix) is it possible that you could run an isocratic method?

    Years ago i ran reserpine with good retention and peak shape under isocratic conditions 50/50 water/ACN buffered to pH 3 (4.6x150 mm, RT=4.8min, 40C, 1ml/min). Reserpine has pKa ~6.5 so you can scale these conditions to your column using FA and see how your peak looks. If you also want to keep MeOH you could start at 40/60 water/MeOH.

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