Quantify large and small peaks - overloading problem

Dear forum,

I am attempting to perform untargeted GCMS on plant extracts from ~20 different resurrection plants. My issue is that one peak, namely sucrose, is many orders of magnitude larger than the others in almost all species which is typical for such plants under stress. I extracted from 15mg dry plant tissue with 1 ml 80% MeOH and dried down 300 ul of this extract which produced an overloaded sucrose peak in most species with split 9:1 and gain factor 2. When I dry down 100 ul and increase split ratio to 25:1 it is still overloaded in some species. I also tried lowering gain factor to 1 only over the time interval of the sucrose peak but its still overloaded so I need to load a lot less. However, I am worried that loading even less will result in loosing too many other small peaks of interest as in some of the species the first dilution already lost some interesting low abundant analytes.

My questions concern using the gain factor can be used to deal with this issue.

Firstly, will lowering the gain factor at the sucrose peak even further help prevent overloading? My understanding is that if the detector or even column etc is overloaded then changing the gain factor should not make a difference as it would just lower the peak signal but change the flat top of the peak? However, I have seen it mentioned that it does affect overloading in some references.

Alternatively, I am thinking to load even less such that sucrose is no longer overloaded but then substantially increase the gain factor for the retention times around the sucrose peak to boost the signal of the other peaks and so potentially avoid loosing as many. Not sure how well this would work though.

Any advice on how to maximize peak detection, would be appreciated. 

P.S. other people have overcome this problem by running each sample twice at a higher and lower dilution but this is not in budget for me as there are 500 samples. 

Thanks in advance,

Michael

Instrument: GC Agilent 7890A

  • Overloading and too high a signal are slightly different. Inlet, column, ion source, and electron multiplier overload can happen even if the maximum signal output is less than the 2x10^6 recommended highest ion signal.  Peaks that are too big cause issues with additional necessary inlet, liner, gold seal, column, ion source, and vacuum manifold maintenance.  Sucrose is sticky and degrades, too, so you want to minimize the mess it will cause if injected.  Most papers online show that sucrose is derivatized before being run on a GCMS system.

    9:1 split ratio is too low for the inlet to have good pressure/flow control and reasonable split ratio and retention time reproducibility. The total flow should never be below 20 ml/min for the inlet to function properly.  

    How many counts tall are your smallest peaks of interest? They only need to be 3x to 5x the RMS average baseline noise, see this blog article: (+) Agilent Community

    You may be able to see them just fine with a significantly higher split ratio to reduce the sucrose peak size.

    Gain controls the electron multiplier voltage setting as run in acquisition.  Running higher gain increases the voltage which increases the size of the signal response but also increases the background signal response, so there is a trade-off and requires some experimentation.  Higher EM voltage also reduces EM lifetime...eventually.

    If the sucrose peak is so large that it is flat-topped, you may be causing damage to the filament.  Make sure you are using a Solvent Delay so that the detector is off when that large peak elutes to reduce filament damage.  You can also use a timed event to turn off the detector when the peak will elute and turn it back on after it does.

  • Thanks for the useful info! Yes I am derivatizing with MOX and MSTFA.

    Overloading solved by drying down only 50 ul of a 1ml extract and increasing split ratio to 24:1. However, relative to the more contrated runs, some of the smallest peaks of interest are now barely visible and so hard to annotate as their m/z's are maybe only double that of baseline. However, I will run some pooled samples at a higher concentration (with detector off at sucrose as suggested) to use for peak picking and annotation and then extract these signals from more dilute runs so hopefully I won't loose much info.

  • Which liner is being used?  Check out: GC Liner Selector | Agilent   You should think about trying one with a frit instead of glass wool, like this one: 5190-5105 | Agilent    Or a single tapered, ultra inert liner  5190-2295 | Agilent .

    Splt/Splitless Inlet or MMI?  For the SSI, make sure that the column is installed about 8mm past the end of the ferrule. For the MMI, make sure that the column is installed about 14 mm past the end of the ferrule.  See: (+) Column Installation - Split/Splitless and Multimode inlets =and= SQ and TQ MS transferlines - Files - GC/MS - Agilent Community  

    Maybe you want to try to dry down more and use a higher split ratio. The higher total flow with higher split ratios increases the speed of the vaporized sample through the liner and will change the way the splitting happens. There's a lot going on in the inlet during this very short time and for some applications you're playing with temperature/flow/volume/expansion/speed/timing/distance...a dance with many partners again.

  • Sorry for the slow response, I only managed to check out the liner with our technician today.

    We are in fact using the single tapered, ultra inert liner  5190-2295 | Agilent and it still looks very clean. We will consider trying a different one for future experiments though, since this is a recurring problem.

    In terms of splitting, I tried 60:1 with the 200 ul extract but that was still overloaded. Probably could get a nice peak with 100ul but ended up sticking with the 25:1 using 50ul.

    Out of curiosity, would it generally be better for instrument performance to run with a higher split ratio?

    Thanks again for the tips!

  • Contamination on the liner is already a problem before it becomes visible.  I would probably try higher than 100:1 with the 200µL extract or something like that.   25:1 with 50µL should equal 50:1 with 100µL, 100:1 with 200µL, 1000:1 with 2000µL....

    Split works best between, oh, 60 ml/min and 400 ml/min or so.  The split liners are about 800µL volume. Sweeping the liner volume quickly past the end of the column where the split happens works better.  Sure it will still split down to 20 ml/min of total flow yet will also work up to over 1000 ml/min, but both ends are not as optimal.

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