Initial fractions from Cosmosil column gave a precipitate which dissolves in MeOH or EtOH. But I cant find any spot on the TLC plate via Anisaldehyde-Sulfuric acid or Iodine vapour or Ceric sulfate-sulfuric acid spraying methods. No success in UV also. I would like to know if there is a way to detect these kind of compounds by any other detection methods. Also i also like to know whether it is worth to spend so much time after this fraction if it is really a complex one to deal with.
There are lots of stains you could try. Check this document for several examples:
http://www.chemistry.mcmaster.ca/adronov/resources/Stains_for_Developing_TLC_Plates.pdf
You can find more examples on internet. It would be very helpful to know roughly what kind of functionalities could have your sample (I guess polar things like OH or NH if it is soluble in MeOH or EtOH).
Whether it is worth or not to spend time on it... it's up to you, really.
After spraying with the sulfuric acids, did you heat the plate? Some compounds need a little heat to char.
Try a water spray, although this works better for more lipophilic compounds that you seem to describe (yours are soluble in MeOH or EtOH). It's non-destructive so it doesn't hurt to try this.
Can't say whether is it worth your time; depends on your research needs.
Also try spotting the plate and spraying without running it- this makes sure the spot didn't run with the solvent front and get diluted or otherwise lost somehow.
@Jack Silver Thank you for the response. Yes I do heat the plate after spraying. And I hope water spray may not be applicable here as my fraction is higly polar. Or any chance of lipophilic compounds still present in the polar fraction? And for your final point: When I use less polar mobile phase system the spots doesnt move and after spraying and heating I could see some color change in the sitting spot. But after developing and spraying I am not able to see anything. But tailing is seen under UV before spraying the plate.
Since that fraction is soluble in MeOH or EtOH, it can be tried in electrospray ionization mass spectrometer. If any component(s) ionize(s), then molecular mass can be determined. From molecular mass, it could be possible to know, what kind of molecule(s) is/are present in the sample. Also, NMR spectroscopy can be attempted, if mass spectrometry is not useful. Further, if this fraction is soluble in some volatile solvents such as hexane, chloroform, Gas Chromatography - Mass Spectrometry can also be tried. By the way, what (Cosmosil) column have you used: is it C18?
What type of detection was used to monitor the eluents from C18 column? Was it UV detection; if so, at what wavelength, the absorbance was monitored? What solvents did you use for C18 column; MeOH & H2O or ACN & H2O or any other solvent system? Did you get to see the precipitate (i.e. some kind of turbid solution) while elution? Or did you begin to see the precipitate after the fractions were collected? Did you see the precipitates in all the eluted fractions or only in some fractions?
I didn't know it was a C18 TLC plate; it's helpful to know such things from the beginning. Using sulfuric acid sprays won't work on C18 (or other bonded phases) because the C18 (and other bonded phases) also chars (remember doing this myself). The water spray won't work either.
You can try the reagents in Alejendro's reference. As an alternative, try running a silica plate with DCM/methanol (or ethanol) (you should be able to run up to 100% methanol), and try the ceric acid/sulfuric acid char or iodine again. You _may_ be able to run methanol with a small amount of water as well, the binder may fall apart on the TLC plate. Running silica with water is akin to the HILIC that is often run on HPLC.
What is the sample or extract? From which source, i.e., plant or animal or microbe is the sample obtained? What kind of compound are you looking for: carbohydrate or protein/peptide or lipid? How are you extracting? I guess, you are loading the extract on the Cosmosil (C18) column and those eluents of this column are subjected to TLC; am I right?
Why don't you try some other solvents other than methanol for extracting secondary metabolites? You can try hexane, chloroform, ethyl acetate, where non-polar compounds can be expected to be extracted. There are several non-polar secondary metabolites known from plant sources. Also, instead of using only methanol for extraction, you may try a combination of two or more solvents, for e.g. methanol & water in different proportions, chloroform & methanol in different proportions, etc. And then, you may verify, which extract gives better chromatogram on C18 column. Subsequently, you can test those C18 eluents on TLC.
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