I have isolated fractions from plant extract using column chromatography. I would like to identify the plant compounds using LC-ESI-MS. But I don't know the method for development of LC-ESI-MS.
Dear Daniel, I wonder why did you chose detection wavelength 220 nm? usually it is used to detect proteins, and from my experience, phenolic compound detection can be missed easily at this wavelength..
HPLC/LC-MS Method development: The method is the collection of conditions in which the HPLC and MS operates for a given analysis Or Method development is the process of determining what conditions are adequate and/or ideal for the analysis required.
LC-ESI-MS analysis: Liquid chromatography-mass spectrometry (LC-MS) as a widely used technique for identification and quantification of molecules/compounds/analytes separated by liquid chromatography.
It provides separation of compounds and detection by MS (provide molecular weight of compounds).
LC-ESI-MS/MS analysis: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is technique in which fragmentation of molecules/compounds/analytes use to identify/ confirm the chemical structure.
Some considerations for your LC-ESI-MS analyses might include:
1. Chromatography. Depending on the purity of your samples, you may require additional separation from the LC piece? While acetic acid may give better signal in the MS, trifluoroacetic acid will give better chromatography. You may want to run both LC methods.
2. Chromatography. Because plant extracts can be very complex, you may have to work out a few LC methods in order to get separation before the MS ionization. Some compounds will suppress the ionization of other compounds.
2a. Chromatography. The cleaner your samples going in, the better the data coming out.
3. MS. The acidic modifiers in the LC methods will protonate the amines/basic compounds in your samples and give nice ions (unless suppressed by TFA), but for carboxylic acids and other aciding molecules without basic moieties, you will probably want to run some negative ion MS-methods.
4. MS. Clean your MS source periodically. You will get better ionization, among other things.
5. Structure elucidation. Remember there are limits to the confidence level from analytical methods. Even using 2-D and through-space NMR methods, people have been fooled about structures - so just do not get too arrogant with a new structure. The ultimate proof is synthesis plus analytical chem.
5a. Structure elucidation. I hope that you have access to high field NMR.
6. Structure elucidation. Make serious use of on-line databases to try to determine if you have a new structure or an old, well documented structure. It will save you a lot of time, though doing the structure elucidation on a tough molecule will teach you a lot even if it turns out to be a known compound.
Good luck and have fun.
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