600 fold variability in peak areas between injections from same vial

Hi All,

Background: Bile acid assay online SPE setup with 1290 and 1260 pumps and 1290 II multisampler on a 6470B. 3ul injection from vial inserts with multiwash using IPA for 10 seconds for seat and needle wash and then starting mobile phase for 10 seconds for seat and needle wash.

A new issue arose this week. Approximately 1 out of every 5-10 injections is "failing" and looking like it didn't inject the full amount of sample (very low peak areas for all compounds, but not as low as blanks). When rerunning the exact same line in the worklist file with a "2" added to the end of the data file name, sometimes the peak areas will return to normal. Specifically looking at a known amount of internal standards added to every sample. In all other samples, the peak areas for these compounds will be 30-35k. Then in the "failed" runs the peak areas will be anywhere from 50-2000 for these same compounds. But on reinjection from the same vial, will get 30k peak area. 

troubleshooting steps so far:

  1. there is definitely enough liquid sample in the vials - 100ul in 250ul inserts and default offset
  2. peak areas are normal on reinjection (sometimes takes 2 times reinjecting)
    1. not likely something inherently wrong with the sample
  3. pressure curves are identical between "failed" runs and good runs
    1. likely not from sample debris blocking the needle
  4. no pattern in the reinjections - i.e., for one sample, first injection had peak areas of 2000, second had peak areas of 50, third had peak areas of 30,000. 
    1. sample isnt concentrating from evaporation on the autosampler
  5. Tune is good - abundances in positive unit dropped by half from 1 month ago, but negative mode unit abundances are near same, and vast majority of compounds in this assay are in neg mode <500 m/z. Cleaned the source and examined nebulizer with magnifying tool- all good.

Any ideas on what else could be causing this? Last week, we were testing 1ul vs 3ul injections and the ratios looked spot on. So if there is suddenly a problem in the metering device, how does one test that?

Thank you for any suggestions!

Parents Reply Children
  • Ah, my favourite vial inserts!

    With highly aqueous samples in these inserts I often find that some of them trap a small air bubble in the narrow part at the bottom. Could this be the cause of your random low responses?

    I can think of a couple of solutions... make sure that air bubbles are displaced by what I can only describe as "hand-centrifuging" each sample after it is transferred to the insert.

    Could always set a higher needle height offset to keep away from the air bubble at the bottom.  How high would depend on how big the bubbles are.

    Does any of that make any sense?


  • I know exactly what you mean! We slide the whole plastic tray sideways into a wall a couple of times before moving them to the autosampler tray to dislodge any bubbles. Maybe this didn't work for a few of them? Since posting, we have run a couple hundred more samples and are steady at about 1 in 20 misfiring. We can manually check each vial for bubbles next run and see if we still get failed injections. 

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