Incorrect calibrant masses on Waters Micromass LCT Premier

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

7 posts Page 1 of 1
Hey all,

Without going into too much detail (it’s a VERY long story), we’re over 4 months into the purchase and set-up of a “refurbished” Waters LCT Micromass Premier XE and we’re still in the set-up phase of this process. As you can guess, my boss is NOT happy….

I *finally* got signal (peaks! big beautiful peaks!) over the weekend but I couldn’t help but notice the masses were not what they’re supposed to be. I will be doing all of my analyses in negative mode (oligonucleotide analysis), so I’ve focused primarily on the negative calibrants (sodium formate, sodium cesium iodide). I’ve also used Waters’ very expensive Leucine Enkephalin standard. When I inject the Sodium Formate calibrant via syringe (flow rate = 10-20uL/min), I see peaks at 110.6, 177.1, 243.7, 310.2, 376.7….on up to 1708.2. I believe our 376.7 peak must be the formate cluster that should be present at m/z = 384.91—a full 8.1amu away!

Unfortunately, things only get weirder. The amount of error we see is proportional to the mass: for every 100m/z, our error increases by about 2.1 amu. the 112.98 peak appears at 110.6 (2.38amu lower), our 248.94 peak appears at 243.7 (5.24amu lower), and our 384.91 peak appears at 376.8 (8.1amu lower).

I have a bit of experience running oligo samples on a very well behaved, brand new Agilent 6530 but I never really had to do much optimization of the various voltages: the Agilent was installed and the ionization parameters worked great for over two years. Now, at this new job with this new (old) Waters instrument, I’m having to learn as much as I can as quickly as possible, though the manuals are only so helpful. They don’t explain what the various voltages physically do, so I can’t predict how altering a parameter will affect the spectrum. The used equipment company isn’t any help and I found out that Waters considers us a competitor (I have absolutely no idea why), so we can’t get a technician to help. If anyone can provide any hints as to what parameters I should focus on, I would be incredibly appreciative!
Sorry I am not familiar with TOF instruments, but our old Micromass Quattro Ultima has the option to roughly tune the mass positions with trim potentiometers. Maybe this can also be done for the TOFs. You have to open the side panel and look for "LM pos" and "HM pos" (or similar).
Then infuse your calibrant solution, open a tune file (be sure to select "uncal.cal" as calibration, this is very important!) and try to manually tune the peaks as close to the expected mass as possible. Not that HM position will affect LM position, while LM position has little effect on the high mass, so start with HM.
Kind regards
Jörg
I have no idea with that instrument, but with some older ToFs the calibration is up to you - you can set it up miles off. I encountered a Bruker micro-ToF that was ten-fold out one time. Yes, ten-fold. The results were about 1/10th of the expected masses, and the isotope peaks were at intervals of m/z 0.1, and the resolution was about ten times better than it should have been. The calibration, at least on that instrument, is exactly what it says on the tin: a calibration curve between time-of-flight and m/z. Time of flight is a hard physical fact, but the reported m/z means nothing to the instrument at all. The internal calibration curve could be changing time-of-flight into bananas per fortnight so far as the instrument is concerned.
So is there a possibility of doing a rough, manual calibration on a couple of peaks of which you're fairly confident, so as to make sure that the calibration curve is approximately right? Automated calibration on any instrument will generally fail if the existing calibration curve is too far wrong - because the software won't be able to find the peaks it expects, in order to calibrate itself, or will find the wrong peaks. For example, if you're only about 1 amu out, it might find the isotope peaks rather than the base peak, and calibrate itself wrongly on the isotope peak.
Leff and Veff will have significant influence on mass assignment. As said above, load uncal for whatever tune being used and start with whatever default tune file is available. Note that the triggers start and stop on the TDC settings may also effect mass assignment.
Thanks, Jörg. I looked through the manual for anything related to what you described and couldn’t find anything. I also opened up the instrument and couldn’t find anything resembling "LM pos" or "HM pos”, so it looks like I won’t be able to take that route.

lmh, yeah, I like that idea but I think I would first have to adjust one (or more) of the voltages to get rid of the variable error. I think if I were to just attempt to jump right into the calibration correction, it would correct one of the masses but the others would be adjusted by the same amount—unless it’s doing more than just offsetting the scale. Actually, now that I think about it, I don’t know what sort of algorithm they use, so perhaps it would adjust the 110.6 peak by 2.4 and it would adjust the 243.7 peak by 5.2? Any idea if their algorithm is that sophisticated or is it essentially just offsetting the scale? And any idea if there’s a voltage/parameter that essentially ‘stretches’ the spectrum so my variable error will be resolved?

Thanks for your input—it’s greatly appreciated!
Oh, it looks like I haven't hit refresh in a while—I just saw your post, mhr311. I'll check those out right now and let you know how it turns out. And thanks a lot for taking the time to reply!
mhr311, you are a GENIUS! I played around with the Flight Tube voltage and it effectively 'stretched' out the spectrum and now all of the masses line up! Beautiful! Thank-you so much!
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