%area of related compounds depending on the injection technique

BJ
BJ
in Instrumentation and Mass Spectrometry
<p>Hi,</p><p></p><p>I found this issue for an API by accident:</p><p>When I inject in Full Loop, 10µl, I get 0.57%area for our major degradation peak, no peaks overloaded, 0.25AU max for main peak.</p><p>When I inject Partial loop Needle Overfill 5µl, I only get 0.34%.</p><p>I injected in both methods a few times to get the results confirmed.</p><p></p><p>I also injected 3,4,5,6,7,8µl PLUNO as a series, all with the same result: 0.34%.</p><p></p><p>Any ideas about what is going on here?</p><p></p><p>Best regards</p><p></p><p>Bart</p><p></p><p></p><p></p>

Answers

  • lizh
    lizh

    Hello BJ

    It is the case that each injection mode is different in the way that it injects and therefore are recoveries, while reproducible are different.

    will differ. The following explanation is attached. This is a truth, that you must embrace. Have three different modes, is like having three different injectors. The responses cannot be compared, or indeed put on the same calibration curve. Full loop will always give the best recovery and that recovery will be dependent on the actual volume of the loop and no two loop ID's are exactly the same.

    Liz.

  • BJ
    BJ

    Liz,

    thanks for your reply but it looks like I was a bit unclear.

    I used %area as it's a normalisation procedure.

    So whether the system injects 10µl or 9.9µl, the %area should be the same.

    So I was wondering why I get such a strange results with different injection mode, as the %area should be the same

    Bart

  • lizh
    lizh

    Bart:

    Apologies, should have asked whether was accuracy or recovery. Please confirm the recovery is the issue and I do need more help in definition around your concern. Can you confirm for one compound or all peaks you see different recovery across the injection modes? Can you confirm that Full Loop is best and can you share the instrument methods, sample diluent and washes. I am guessing that the difference in how the modes operate is affecting the result, could be aspiration speed, weak wash, which is why details in the method would assist us. For full loop the loop is simply overfilled and there is little influence from the other factors.

    Best regards,

    Liz

  • BJ
    BJ

    Hi Liz,

    there seems to be a recovery problem, but with only one peak. There are a few other peaks which doesn't behave like this.

    Full loop seems to give 100% recovery.

    Partial loop with needle overfill gives only about 60% recovery for that particular peak while the other peaks still have 100%.

    The instrument method was set to the defaults. WNW: 90% water 10% MeOH, SNW 100% MeOH.

    I talked yesterday to Ilse Hoes and she came up with the idea of migration of one peak in the mobile phase/WNW (I'm not sure anymore).

    She suggested to increase the needle overfill flush to 30µl so that it will be comparable to FL.

    The results looks promising: 94% recovery (damned, still not 100).

    But I will lower the flush and see if recovery also decrease.

    I will keep you informed

    BJ

  • lizh
    lizh

    Perfect, Ilse is the best, you are in good hands!!

    Liz

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