Lets assume that i used the SIM to quantify a mixture having 2 compounds, A and B. Based only on peak's area, can I say that compound A has higher concentration than compound B? Or should I do the calibration first to draw such a conclusion.
I'm sure that with classic chromatographic detectors, such as FID for CG or UV in HPLC, we can’t say this since the peak area depends on compound’s quantity in the mixture and its sensitivity toward the detector. So I wonder if it is the same in case of GC/MS or HPLC/MS.
Thank for your timegg
Short answer: You cannot make a determination that A has a higher concentration than B unless each compund is very similar in structure. If you think about the "original" mass spectrum of A, the peak you chose for your SIM quantification calculation peak might be the only significant peak - Like the parent 78 peak in benzene is roughly 1.5x the intensities of all of the other spectral peaks combined - Whereas for hexane the parent 57 peak is only about a quarter of the intensity of all of the other peaks combined. This is ignoring other variables like the relative efficiency of ionization. You must do a calibration unless the spectra are almost identical, as they would be for, say, different dichlorobenzene isomers. See: hexane and benzene spectra links.
http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=JP009416&site=10
http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=JP002347&site=10
Short answer: You cannot make a determination that A has a higher concentration than B unless each compund is very similar in structure. If you think about the "original" mass spectrum of A, the peak you chose for your SIM quantification calculation peak might be the only significant peak - Like the parent 78 peak in benzene is roughly 1.5x the intensities of all of the other spectral peaks combined - Whereas for hexane the parent 57 peak is only about a quarter of the intensity of all of the other peaks combined. This is ignoring other variables like the relative efficiency of ionization. You must do a calibration unless the spectra are almost identical, as they would be for, say, different dichlorobenzene isomers. See: hexane and benzene spectra links.
http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=JP009416&site=10
http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=JP002347&site=10
NO! NO! NO!. Even molecules that have a common ion fragment (SIM) can have a different response to the detector.
Imagine an MS like a 'hammer'. A drinking glass will shatter when hit by the hammer but the hammer will bounce back when a 'bowling ball' is hit. But both are made of 'glass (almost).
In SIM you absolutely must calibrate, as Tim explained so well. In full scan mode you can treat an MS similarly to a FID. You can semi quantify for similar compounds, such as alkanes or aromatics. The differences in response factors for most hydrocarbons in full scan mode in MS are relatively similar, so your quantification will be pretty close. Where you do see larger differences is in highly electronegative heteroatoms (halogens, for example). Some of the highly chlorinated or fluorinated compounds will give you response factors 10-50 times lower than hydrocarbons, which is also true for a FID.
Thank you for the more clarification Mark. How about polyphenols (Ferulic Acid, Palmatic Acid, Oleic Acid, Linolic Acid, Cafeic Acid etc.......), can i consider them similar and use gallic Acid as internal standard to quantify them?
Why not just get a fatty acid mixture (or FAME mixture) from Restek (or equivalent)? They are NIST traceable. Why reinvent the wheel?
No Bruce, my question is can we assume that polyphenols belong to the same family? I guess they are having differents chemical structures although the may exibit in some cases same physical proprities! So my guess, they will not have the same response using an MS detector!!
No. Apart from the different response factors of the MS (especially in SIM), you also have to consider differences in recovery during sample prep, and at trace levels differences in losses during analysis.
GC-FID is one of the rare detectors in chromatography where the response is related to the concentration (i.e. the carbon content) of the compound, therefore this is a helpful to semiquantitatively determine or compare the concentration of 2 unknown compounds A and B. Therefore i cannot agree with the last sentences of Moncefs question.
You should be also aware of the fact that different response factors are not as problematic in Electron impact ionization (EI) on GC-MS as they are in LC-MS. If You perform an unknown screening based on a wide range scan it is much easier to perform a semiquantitative approach based on EI due to the fact that this is a more universal approach for ionization. LC-MS response factors may vary in some orders of magnitudes.
- Badges
- Science method
- Similar topics
- Chemistry
- General Biochemistry
- Biochemical Techniques
- Analytical Chemistry