I want to determine the structure of extracellular metabolite of plant extract
Janitha Nipun , is your metabiolite isolated or you have a difficult mixture of many compounds?
If you detect this compound in a mixture, analysed by HPLC/UPLC-MS or GC-MS, use the corresponding Mass Spectra.
If you can isolate and purify the compound, you can combine this information with the one got from the classical spectroscopy techniques (IR, 1H and 13C NMR).
If it is a solid and you can get a single crystal, use X-ray Crystallography.
@Ekaterina E. Khramtsova
It is a mixture of compounds, I need to identify their structure
@ Abel J S C Vieira
Basically it is a plant extract and compounds are unknown. In order to find out which compounds present in extract I need to elucide the structure of metabolites present in the extract
Firstly you need to isolate each and every single compound and then go for structure elucidation
Janitha Nipun if it is a mixture, you shoul try to isolate every one and to study their NMR, IR, MS and etc. But if you do not have such opportunity, you can use HPLC or GC with MS (4 quadrupole is preffered) and a special programme and database for them.
Ekaterina E. Khramtsova If I carried out HPLC, how may I know whether my eluted fractions contain only one compound. Is it okay to use TLC to determine how many components present in the each eluted fraction.
Janitha Senevirathna HPLC will show you how many components your mixture contains.
TLC is good for this purpose, but it is less sensitive than HPLC.
Ekaterina E. Khramtsova Suppose if the chromatogram in HPLC shows three peaks. How I separately elute each of the component from HPLC?
Janitha Senevirathna Do you need to separate your components or to elucidate their structures without separation?
For separation you can you column chromatography.
Dear Janitha, this is a very interesting technical question which is certainly of broad general interest to many other RG members working with plant extracts. Since you have a mixture of components, I strongly suggest that you have a look at the following very useful article entitled:
Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample
Article Protocol: A fast, comprehensive and reproducible one-step ex...
Fortunately this paper has been posted as public full text on RG. Thus you can freely download it as pdf file. I hope it helps.
Good luck with your research and best wishes, Frank Edelmann
Ekaterina E. Khramtsova I want to separate the compounds first and then undergo bioassay to determine which of them are bioactive. Then the compounds which shows significant bioactivity, I need to elute their structure.
If the substance is pure, NMR or IR can be used to identify the structure of the substance. If it is not pure, you can use GC-MS or LC-MS first.
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