Dear colleagues,
I am fractionating hexane-extracts of some biological sample, and identifying resulting compounds. Curiously at the end of my extractions, I obtain an oily emulsion which is no longer soluble in hexane and in fact now water-soluble!
I am eager to ID this compound or mixture but the extract shows no peak at the GC-MS, meaning it is either of a very high boiling point or just being burnt upon injection. Given it is water-soluble, I was thinking of injecting this into LC-MS instead. However I have no experience with such system.
Would anyone have ideas of which kind of compound these could be? I believe it strongly interacts with apolar substances thus why it was joint-extracted by hexane, but once separated it got no longer soluble in apolar environment. It is yellowish, oily, abundant, and Draggendorf-negative.
Thanks
Oh, just some fundamental details. The emulsion comes out from silica columns separation with increasing acetone-TEA concentrations. It will also be obtained with methanol.
Hello Michael, I work with ants. We are extracting whole-body in hexane. I know it is fairly unexplored grounds thus this is why I am looking into more general chromatography guesses. We did TLC before and after the column samples. The TLC is dyed with Draggendorf's reagent, which is aimed at alkaloids with some demi-false-positives (e.g. will stain phtalates on a different hue). The mix will not dilute in hexane, but will carreate on acetone and poorly on methanol, but it is hard to guess much for I cannot see it properly. As mentioned, the "goo" is negative at Draggendorf there, thus I believe it has no nitrogenated ring. I am very naive on this topic -- could you please add detail on the references on chemotaxonomy, e.g. Hegnauer? I do not know of other useful and easy-to-prepare specific reagents. Please provide me with a few. Do you think LC-MS is an option? We have one available, it is just that I would not know which protocol to apply. Thanks!
Dear Michael I see no reason to apologise, your feedback is positive. I am afraid NMR depends on a pure fraction, which I am afraid I do not have nor currently know on how to check. I will look into the book and reagents you mention. I've evaporated the solvent with N2, please mind the resulting mix is completely water soluble. I know it's odd but it would explain a lot. I now need to know the composition
Dear Edouardo and Michael,
I really wonder if you were not facing another case of NaDES (Natural Deep Eutectic Solvent) extraction. Have you ever heard of these "natural solvents"? They were described for the first time by Pr Robert Verpoorte (Pharmacognosy, Leiden) in a seminal paper in Plant Physiology, August 2011, Vol. 156, pp. 1701–1705, which was entitled "Are Natural Deep Eutectic Solvents the Missing Link in Understanding Cellular Metabolism and Physiology?".
To my opinion, if you read carefully the related story in this paper, it is the occasion to totally "rethink" our extractive processes: for instance, in my own case, I was very much surprised, after having evaporated the mixture of ethanol/water used as solvent to extract quite easily anthocyanins from grape, to get to an "oily" material which I could never dry thoroughly. I always thought that it was the water molecules which were "sequestrated" (not removable) by the anthocyanins! Rather than to run NMR analysis on these crude extracts, I always tried to purify them by column chromatography: unfortunately, I then was unable to dissolve back in any solvent, the solid material obtained upon evaporation of the solvent of some fractions.
The best explanation came from the "giant" researcher, Rob, who pushed me to go back to the bench ... and to confirm that a precise molar ratio mixture of "natural" abundant metabolites (with melting point above 150°C) were able to form deep eutectic liquids until -80°C, which were much better solvents than either the lipidic or the aqueous phase... As soon as you purify the crude extracts, whatever they are, you destroy the eutectic by changing the molar ratio conditions and thus the solvent properties of them.
For sure one have to transpose to your special case, when extracting lipophilic materials from the ants, but, this is totally conceivable since many many kinds of eutectic can be acheived from the lipidic phase of the tissues (phospholipids, cardiolipins, cholesteryl stuff,...). Maybe, the chitin material could play also a role in this phenomenum.
Anyway, the absence of signal in GC/MS analysis supports this hypothesis since your extract would contain too high molecular weight molecules to pass through the column. To investigate further the content, proton NMR analysis, better than any other technique, would give you an informative insight on it: whatever their nature, it would tell you that there are organic molecules. If you don't have such facilities, just send me a few mgs of the crude material and I will run a 500 MHz spectrum for you.
Hope that this will be helpful...
Sincerely,
Joseph Vercauteren
Dear Joseph,
I should add that in fact the sample I am dealing with is described to work as the first naturally-occurring reported ionic liquid under special conditions. I am not sure prior to reading further but I suspect an ionic liquid is akin to what you describe. This was one the first things I suspected of when I saw the unexpected behaviour. Please allow me to emphasise that it is not like there is no CG-MS signal whatsoever, but it returns peaks of some of the compounds which should be in there (Draggendorf-positive alkaloids) but they are too small for the concentration of injected extract. Therefore my suspicion there is something else in there that doesn't show.
However Michael has a point here that maybe the mixture may behave differently in the machine; I am not sure.
At this point I am tying to reproduce the phenomenon with a related sample. If it proves true and reproducible I am planning on applying to a special protocol I am designing, which is dependent on water-soluble compounds.
Thanks for all this interesting account -- these things are the colours of lab research.
The fact that your compound is water soluble and has no peak in the GC suggests that it might be ionic, i.e. you have an ionic liquid. I believe. It is known that some ants produce ionic liquids, http://onlinelibrary.wiley.com/doi/10.1002/anie.201404402/full, i wonder if that is your case as well. Anyway, an NMR and ESI-MS (both positive and negative) might help you figure out what it is.
Dear friend,
In hexane fraction you will only get non-polar compounds or fats and oils. so your sample is oily viscous. so, if you want to identify their constituents,you need to esterify first then run it by GC-FID or GC-MS , you will get the composition. Otherwise you need to run it on the LC-MS,. Fats and Oils composition is different from other organic constituents, so it may be triglycerides, sterols, Fatty acids, longer chain compounds.These compounds definitely have higher boiling points.
Hi
Of course, you can use NMR: 1H NMR and 13C NMR. As the following paper
https://doi.org/10.1007/s12161-021-02203-0
I hope this helps!
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