Injector Linearity and Accuracy Test (SQT + Supplemantary Test)


I have a strange observation and I don't know the right answer why and how could it happen. Maybe somebody can help me with the right answer.

My instrument is an Acquity UPLC with classic SM-FL autosampler. The nominal loop volume is 10uL and the calibrated is 10.1uL. The sample injection mode is Partial Loop Needle Overfill (PLNO). We know, that this mode provides optimum performance when
injection volumes are maintained within a range of 10% to 75% of the actual
loop volume. That's OK.

  1. When the field service engineer runs the Injector Linearity and Sensitivity Test from SQT with 2.0, 3.0, 5.0, 6.0 and 7.0uL injecteted volume, the test is passed, and the R2 is >= 0.999. Great.
  2. BUT, I need to test the instrument with a supplementary Injector Linearity and Accuracy Test too, to check the instrument, is it able to work within the whole linearity range of 10% to 75% or not? Primarily I need to check the 75% upper limit of this range, so I inject 2.0, 3.0, 5.0 and 7.5uL sample. This supplementary test often fails, if the calibrated loop volume is less or near to 10uL (in my case it's 10.1 uL and it fails). No air bubbles in sample syringe, no dead volumes by the loop (injection valve port 1 and 4), and the system is well primed. I noticed that the test fails because of the last injection (7.5 uL) and the R2 is less than 0.998 with all four injected volumes (2.0, 3.0, 5.0 and 7.5), but R2 is greater than 0.99999 with only the first three injected volumes (2.0, 3.0, 5.0).
    When I change the loop to another one, that has a calibrated volume more then 10.5 uL, this supplementary test is passes too. And this trick always works.
Why can this happen? Could someone give me an explanation?

Thank you for your kind help.


  • MarcNoble
    edited September 2023
    Hi Balu1978,

    Have you observed this phenomenon on other loops?  The reason that I ask is that although your volume observation is interesting, it might just be coincidence.  A more likely reason for the very small difference in response could be just an anomaly in the smaller loop.  Because PLUNO requires sample to be pulled from the vial, then pushed into the loop (movement in 2 directions) it is quite sensitive to any small difference in surface tension on the walls of the loop.

    There are some very good details on PLUNO in THIS Knowledge Base article, but the slides in the attachment might also be useful.

    Good luck!

  • Hi MarcNoble,

    I observed this 2 times and only with 10uL nominal loop volume. The injector linearity with 5 and 20uL loop also passed, no problem with them. But maybe it's just coincidence or as you wrote the surface tension on the wall. Thank you for your help and the slides too. Great help.

  • Hi balu1978, have you recently re-calibrated the Air Sensor? I've had that value change a few 10ths of a uL and it makes a precision difference at those lower injection volumes. This calibration measures the path of that VDD and those few 10ths of a uL could be making the difference that you are seeing in the 10.1uL Loop characterization vs the slightly larger loop at 10.5uL.