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Purifying an aromatic glycoside from tetrabutylammonium ion

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MOTOBALL:

--- Quote from: wildfyr on January 01, 2023, 02:38:18 PM ---Water on acidic silica gel seems bad for TBDMS. Definitely do 2D TLC first.

--- End quote ---

Certainly, but the original objective by the poster was to remove those groups!

Babcock_Hall:
The TBDMS groups are gone.  The problem is that the (Bu)4N+ cations are partially but not completely removed; we started with eight equivalents of TBAF and have removed about 90% of them, based upon H-1 integrations.  The identity of the anion is uncertain at this point.

MOTOBALL:
Can I suggest that you run TBAF with either one of the 2 suggested aqueous phases just to determine how it behaves?

Regards

RedViper9:
Re: RP Chromatography on Combiflash

Typically Teledyne ISCO Combiflash systems are equipped with UV detectors. Unless your ISCO system is equipped with a Refractometer, Evaporative Light Scattering Detector (ELSD, rare) or mass spectrometer (more rare) you should not expect to visualize the tetrabutyl ammonium component. Still, assuming your target is not itself an ammonium salt, the expectation is that there will be some significant separation of the two components on C18.

One major problem may be mixed fractions due to poor peak shapes (tailing). If you want information on the separation before hand, you might try getting some C18 TLC plates or running analytical HPLC to get an idea of the retention and peak shapes.

Be sure to collect your waste stream in a large beaker rather than dumping it straight to solvent waste. Otherwise, a bad column or accidentally using the wrong solvent as the weak phase may add months of work to your isolation...

Some general recommendations if you're going in blind and want maximum info from one run:
- Use an RP column of appropriate size to the sample per manufacturer recommendations.
- Dry load your sample onto an appropriate amount of Celite.
- Run a scouting gradient, the system will suggest a generic gradient based on the size of the column you use.
- Use the collect by volume option.
- Individually check the fractions which light up by UV for TBA contamination.
- If they're clean, great you're done.
- If there are still mixed fractions, start checking fractions ahead (or behind as appropriate) of your target to determine whether you're seeing peak tailing or you just happen to have similar RF.
- ISCO RP columns can be reusable, so if the first separation fails adjust gradients to get what you need.

Remember a Combiflash is simply an automated method of running flash columns. The instrument only gives two real benefits vs. traditional glass columns: 1) More continuous pressure 2) Smaller gradient steps. That is significant for a tight separation, but will not give you HPLC levels of resolution.

RedViper9:
I just read in one of your previous posts that you were working with compounds consisting of a glycoside tethered to a phosphate. Would these two happen to be such compounds?

If so, the fact that you're getting stuck at one equivalent of tetrabutyl ammonium suggests to me that you're isolating the TBA salt of your desired compound. In this case I'd most likely focus efforts on different cation exchange resins.

RP chromatography on it's own won't separate the two salt components. Even if the ammonium salt and phosphate salt are separate salts RP may struggle. You can try adding acids to compete the ammonium off, but I'd expect you'd need trifluoroacetic acid to get any strong effects and I'm not sure the ISCO will stand up to that. You can try Preperative HPLC if available. I've resorted to multiple injections on analytical HPLC with manual fraction collection before, but that's not a great way to spend an evening.

Ion exchange chromatography will work, but automated systems are still rare in academic settings.

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