I am new to GC-MS and mass spec in general. Currently, I am attempting to quantify the uracil base content in cells by derivatizing it with 3,5-Bis(trifluoromethyl)benzyl bromide, where two of the derivatizing reagent bind to a single uracil. I will be comparing it to an internal standard uracil isotope to determine how much uracil there is. The literature uses the NCI mode for this, but our campus does not have it, so I have been attempting to use EI for it. The GC portion of the setup is starting at 45C and ramping up to 280C at 30C per minute. I then added a post run at 300C for 10 minutes to try to clean it between runs. I think the max column temp is 325C. It appears I am only getting a single fragment, which seems ideal, so I set it to SIM for that mass. However, I am getting at least two (perhaps more less abundant ones) retention times for this mass. Preferably, I would like these to be a single more abundant peak, because my uracil masses will likely be down in the 0.1-10 pg range. I am guessing that this happens because I have two forms of the derivatized uracil in my sample (maybe singly and doubly reacted), but I don't know enough about it GC-MS to be sure about that. Does anyone have suggestions on how I can have a single peak, if possible? Or can confirm my thoughts on it? Thank you in advance!
Noel W Davies Thank you for your answer. I ran my samples with low amounts of uracil in the full scan mode and could only see those ions if I zoomed in pretty far. However, using SIM on each of those ions, I would get a peak at 294, and the others were at or near background. The 294 ion had two main retention times though rather than all eluting at the same time, and I was unsure what might be the cause of that and if there's a way to make it a single elution time.
I had also used TLC in the past to verify that the derivatization reaction was being successful, and it appeared to be, even for much higher mass (mg) of uracil compared to the pg amounts I'm using now.
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