During GCMS analysis, it is showing my sample contains Squalene. But whenever I am running single standard (Squalene ≥ 98% purity) as well as internal standard compound sometimes show peak and different retention times. For more detail information see the attachment(.pdf)
Dear Kiran,
generally, retention time shifts can result from incpomplete temperature equilibration before the next injection. you might give more time for the column to reach the starting temperature, once a run is completed. in addition, problems with the split and also sometimes with a leakage in the septum can cause such effects. another explanation could also be matrix effects or interacting effects of your analytes. we sometimes have slightly different retention times between pure standards and real samples, although these are not in the range of minutes.
best,
Christoph
In addition of the nice comments above, I think by GC MS you can know whether your peak is squalene or not. Squalene (EI MS) should show these m/z: 410 (M+), 341, 273, 69 (base peak), 137, 197 and 327. If those m/z do not appear ( I cannot see the M+ peak 410), I think you can reduce the ionization. If you want to do quantification just apply SIM using m/z 410 or 341. Can you work with MS MS? if yes, I think you can select as precursor ion 410, and measured the MS MS, and doing SIR/MRM for quantification.
Thank you so much for your kind response. During running the internal standard (squalene100ppm+ test sample) it is showing two different peaks and RT with approximate 1.7 min difference (Detail information ref. the attachment ). Please give me your valuable suggestion.
A few recommendations:
1. You have retention times shifting everywhere. That is typically related to injection issues (leaking septum, poor injection technique, inlet temperature, etc). 250C is really too low of an injection temperature for this analysis; you should raise the injector up to 280C. Also, there is no reason to cool down to 50C for this analysis. You can start at a higher oven temperature, which will help you cut some of the run time out of your analysis. Make sure your syringe is good and use a fast injection.
2. Your ramp rate is too low. Try running your analysis at 20C/minute or 25C/minute. Faster ramps lead to better resolution and sharper peaks.
3. Your split ratio is too low (1:5). Raise it up to 1:20. Also, you're well out of the optimum flow rate for that column. I assume from your data that you are running constant flow. If so, drop it down to 0.75 mL/minute. Lower flow and less material injected into mass spectrometers equals better performance.
4. You CAN get matrix-induced retention shifts in GC-MS. If you are still seeing the retention shifts try diluting an additional 1:5 or 1:10 prior to injection.
Good luck!
Did you initially set out to analyze for squalene or is it just showing up as a library hit? Squalene shows up as a by product of pyrolysis in the breakdown of polymers.
Thank you @ Mark Krause and Michael Toner.
In response to Michael Toner, no sir I didn't initially set out analyze for squalene.
If you are finding squalene and there is no reason to expect it then a change to conditions such as injection temperature may help.
This can be a matter of proper optimization of parameters. Also check for any likages, especially through the septum, condition of your injector liner, whether you have used the right liner ( split and splitless liners are different) etc.
The chromatograms in the attachment show multiple peaks for samples, and single peaks for the squalene standard. The simplest explanation is that the samples contain other compounds in addition to squalene.
One of the chromatograms from a sample shows an apparent retention shift for squalene. Can you confirm by MS that the labelled peak is really squalene. Is this effect repeatable with a newly spiked sample ?
I notice definite changes in the size of the solvent peak which may indicate problems with the injector or the inlet.
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