Characterize Needle and Loop Volumes fails - Waters M-class UPLC

I am currently unable to get our M-class sample manager to characterize the needle and loop volume using the default (automatic) settings and would appreciate any feedback or help.

For reference, we're experiencing a reduction in signal of a standard peptide (and digests) by MS which we suspect is related to the needle or sample loop volumes.  We also noticed a slight delay in retention time which might be unrelated to the main issue (loss of signal).

Our mobile phases consist of water + 0.1% FA and acetonitrile + 0.1%. Strong and weak needle wash are acetonitrile and water, respectively. As far as we can tell, there are no leaks and everything is primed. We can calibrate the volume detection device and needle seal passes, but the needle and loop volume characterization fails even after changing the needle, sample loop and inject valve. 

With that being said, we're able to get the characterization to pass by first running a method with a pre and post-air gap of 6uL or more, and then proceeding to characterize the needle and loop volumes. However, based on some test runs after the characterization passes with a 6uL air-gap, we noticed the signal our standard peptide by MS is negatively affected by increasing the air gap volume (from 2, 4, 6 and 8uL). I'm not sure why this is the case but it appears the air gaps affect our signal and I suspect there may be an issue with our sensor or volumes that lead to loss of sample.

In the past, we characterized the needle using the automatic air-gap settings but now we have to change it to 6uL to pass. Also, in the instrument configuration for our Q exactive plus and m-class, the measured volume for the sample manger says 10uL, although I it should be 9.7uL according to the values reported when it passed with the 6uL air-gap. So I don't know if the sample manager is saving the correct volumes in its memory either.

If anyone has information or suggestions please let me know,



  • Update: 

    I can no longer get the characterization to pass by changing the air gaps from automatic to 6uL or 8uL.

    We also had a service technician look at it today but we could not identify the cause of the issue. 

    Maybe the pre or post sample air-gap is not being introduced properly since the sample syringe keeps drawing liquid during characterization until it gives an error.
  • MarcNoble
    edited April 2019
    Has any work been done on the injector pod?  A leak in the system, or added void volume (sometimes created when tubing is removed and/or replaced) can cause the characterization to fail. 

    you could also check out these Knowledge base articles -