I recorded the cpmgpr1d/zgpr water suppressed proton spectra for plant samples.
1.) what is the protocol/step to identify/confirmation of the exact metabolite in proton nmr spectra ?
2.) There will be always an ambiguity, when the two or more peaks of some metabolites overlapped or found in the same region ?
3.) If we find or confirm it , then how can proceed for concentration calculation of each metabolite ?
4.) Should I do manually calculation for each metabolite or there are some automated software available for this ?
5.) What should be the unit for concentration of each metabolite ?
6.) Is it always true that only TMSP play the main role for relative concentration calculation ?
1.) what is the protocol/step to identify/confirmation of the exact metabolite in proton nmr spectra ?
Typically this is a comparison with the spectra of the individual metabolites from a database such as HMDB or BMRB and others.
2.) There will be always an ambiguity, when the two or more peaks of some metabolites overlapped or found in the same region ?
Correct, good quantification of individual metabolites is only possible if at least one signal is isolated.
3.) If we find or confirm it , then how can proceed for concentration calculation of each metabolite ?
Integration against an internal or external standard. TMSP can be used as an internal standard.
4.) Should I do manually calculation for each metabolite or there are some automated software available for this ?
Some metabolomics packages will automatically report a concentrations.
5.) What should be the unit for concentration of each metabolite ?
mol/l
6.) Is it always true that only TMSP play the main role for relative concentration calculation ?
TMSP is used as its signal is not interfering with most metabolites. Other internal reference compunds such as dimethylsulfon, trimethoxy benzene or maleic acid and others can be used.
I agree with those reported by Dr Anklin.
I would only add some details.
The the parameters setting (i.e. d1 and recycle delay) for 1H-NMR experiment should permit the complete relaxation of all the different kind of protons between consecutive scansions, so to have a linear correspondence between the number of protons and the respective integrals and an adequate number of transients should be recorded so to have the lowest possible value of S/N.
To have an unequivocal assignment of resonances of a specific compound the data obtained by mono- and bidimensional experiments (homo- and heteronuclear) should be both considered and cross-checked. As example you may take a look at the method reported in the following paper:
Frezza, C., Venditti, A., Sciubba, F., Tomai, P., Antonetti, M., Franceschin, M., ... & Bianco, A. (2018). Phytochemical profile of Euphorbia peplus L. collected in Central Italy and NMR semi-quantitative analysis of the diterpenoid fraction. Journal of pharmaceutical and biomedical analysis. 160: 152-159. doi: 10.1016/j.jpba.2018.07.059
Metabolites of flavonols from plants can be quite complicated. Chromatographic techniques can isolate and separate all the [major] components and one can then use spectroscopic NMR techniques to identify each of the separated components. Surprising however in a mixture of flavonol structural analogs of components (present in unequal concentrations)how successful multi-component analysis can be without actually doing the chromatographic separation first. Having the parent compound and the mixture run under the identical NMR conditions, one can then subtract out NMR spectrum from the parent compound from the mixture. One is left with another set of peaks and if the next set of peaks is also a flavonol, if one has a standard of that compound, dissolve that same standard in the same solvent as the others and again subtract that set of NMR peaks as well. All negative peaks can be considered not real.
Chemometrics can be used in such calculations as well. Result will not be invalid if using literature reference spectra; they will just be less exact and more uncertain. When metabolites are present for which there are no standard, one gets to do structural elucidation on the unknown. One may find more data than one thought one had had.
Sumit Mishra This is a good guide for calculating concentrations via NMR. http://www.umich.edu/~chemnmr/docs/Quantitative_NMR.pdf
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