Quantify large and small peaks - overloading problem

Mikki 10 days ago

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

0 paulsalverda 7 days ago

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.
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