I have tested different organic fractions of crude methanol extract of Parthenium hysterophorus leaves. Ethyl acetate fraction showed highest toxic activity. Now I would like to find out the most toxic compound form this fraction. I have LC-MS, FT-IR and TLC in access. What sequence should I adopt to identify and quantify the responsible compound?
You need to first get pure or semi-pure components of tha active fraction/extract by column or counter current chromatography. Test all to identify the active component or fraction. If no single one is active then do addition/substraction bioassay to identify the components which are critical for the biological activity. You then need to undertake chemical characterization of the identified bioactive compounds: mp/bp, UV, CD, Optical Rotation, IR, 1 & 2D NMR, MS, X-Ray Crystallography. Finally, you have to undertake in vitro and in vivo toxicology tests.
Run TLC to determine a solvent system that appears to give the best separation of your compounds; I realize that what you see on the plate may not correspond to your active compound. Then use column chromatography to resolve the compounds, using a gradient from weak to strong solvents.
One can run "wide-polarity range" chromatography to purify the compounds as well. I do have a poster on column screening I can share if you wish using this method, but here is an earlier poster: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf
You will need to see which fractions exhibit your desired biology. The fractions may be pure enough for LC-MS, or you may need to purify them some more.
The brief steps are:
Conduct preliminary analysis using laboratory chemical test methods. Then run TLC to know the type of fractions. After that Column Chromatography with HPLC, after isolated pure fraction run FT-IR, LC-MS, HNMR, CNMR for structure elucidation. You can read my papers too.
Run TLC to determine a solvent system that appears to give the best separation of your compounds. Then try to separate all the compound in preparative chromatography. Then collect each band and tried it for your activity. You may get some conclusion on toxic compound.
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