Chemical Forums
Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: Babcock_Hall on December 29, 2022, 01:32:38 PM
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We have two impure samples of an aromatic glycoside. In both cases we used eight equivalents of tetrabutylammonium fluoride (TBAF) to remove tert-butyldimethylsilyl (TBDMS) groups from the four oxygen atoms of the glycosidic portion of the molecule. Our first two attempts at using Dowex-50/calcium carbonate to remove the TBA cations and to protonate the oxygen atoms went well, with over 99% being removed. This protocol was found in a 2007 J. Org. Chem. paper (9(4):723-726) written by Kaburagi and Kishi (10.1021/ol063113h). Our second two attempts removed roughly 90% of the TBA, which still leaves roughly one equivalent of TBA present. The TBA is easily observed in the H-1 NMR spectrum. We do not know why there was this decline in efficiency. We are not sure of the identity of the anion paired with TBA (it might be fluoride or bicarbonate, I suppose). Right now my thinking is to repeat the Kaburagi-Kishi step on one sample, perhaps doubling the amounts of both the Dowex-50 resin and calcium carbonate.
If this attempt fails, we can try reverse phase flash chromatography on a Teledyne ISCO system on one or both samples. Chapter 4 of their book has an example (Figure 60 on page 69 of the fifth edition of Effective Organic Compound Purification, which is Teledyne's book) in which two aromatic glycosides are purified nearly to baseline using water and acetonitrile as the mobile phase, but few details are given. We presently have a 26-gram C18 reverse phase column, and the samples are very roughly in the range of 100 milligrams, going on memory. We have no experience in preparative RP-chromatography, and I am not sure how to detect TBA during a chromatographic run (which are reasons to favor trying the Kaburagi-Kishi method first). We did obtain some information from Teledyne on changing from normal to reverse phase and back again.
Is there any point in doing preliminary TLC on RP plates to get a sense of what solvent ratio will move our compound? I never had much luck with them. Can anyone inform us about possible pitfalls that we might encounter in preparative RP chromatography? Thanks for any help or suggestions.
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You may recall from my response to one of your earlier posts, that I am a strong proponent of the use of TLC with silica gel/microscope slides to monitor carbohydrate reactions.
I would start with EtOAc/Hexane (2:1, v/v) and modify the mobile phase from there.
I used to aim for an Rf value of about 0.2-0.3 (IIRC) from a single run, and then perform three runs (letting the slide dry each time) before spraying with 5% conc H2SO4 / EtOH and heating to char the organics. This will give you a picture of how the reaction mixture will elute from a Silica column.
Use that mobile phase for dry-column chromatography.
Good Luck!
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I no longer see the TBDMS portions of the molecule (the protecting groups) by NMR, and there are four hydroxyl groups in the product. I strongly doubt that this product will move on silica or be soluble in ethyl acetate/hexane mixtures. Thank you for reminding me about sulfuric acid as a detection method.
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You are correct Re solubility and lack of movement on silica gel; I had misunderstood the status of your product.
Suitable developing phases could be,
1) EtOAc/EtOH/H2O (10:3:2 v/v/v)
or
2) 45:5:3
Regards.
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Water on acidic silica gel seems bad for TBDMS. Definitely do 2D TLC first.
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Water on acidic silica gel seems bad for TBDMS. Definitely do 2D TLC first.
Certainly, but the original objective by the poster was to remove those groups!
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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.
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Can I suggest that you run TBAF with either one of the 2 suggested aqueous phases just to determine how it behaves?
Regards
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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.
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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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Hi RedViper9,
First, thank you very much for your suggestions concerning the Teledyne CombiFlash; we can also ask for some help at our institution. Second, we are working on two glycosides which are made using very different synthetic strategies. One has a phosphonate diester as an intermediate, but this portion of the molecule will be discarded in a Horner Wadsworth Emmons reaction. We still have a few more steps to go, but I could see reverse phase used after the final deprotection step.
The present synthesis used four TBDMS groups on the four carbohydrate oxygen atoms (at carbons 2, 3, 4, and 6). The final step was the use of 8 equivalents of TBAF to remove these protecting groups. We have performed this step several times with only one failure. Then we followed the procedure of Kaburagi and Kishi (J. Org. Chem. 2007) to remove the remaining fluoride ions and the tetrabutylammonium ions, some of which may be pairing with carbonate or bicarbonate anions. In brief calcium ions form CaF, and the Dowex-50 provides protons to oxygen and binds to the TBA ions. This step was partially successful, removing roughly 90% of the TBA ions by H-1 NMR (it has previously been more successful in our hands). Our job now is to remove the majority of the remaining TBA ions. Our product is electrically neutral, but I would describe it as amphipathic, having an aromatic portion and a glycosidic portion.
If I had a charged product, I might be tempted to try low pressure ion exchange using TEAB, which is volatile.
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Can you use a different fluoride source that doesn't leave the NBu4 to deal with? HF.Pyridine?
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clarkstill,
Yes, that is an alternative but it would require us to repeat a three-step synthesis, and I am short on person power. However, we have previously been able to get to better than 99% removal on two occasions when we synthesized a structural isomer. We presently have two samples of the desired glycoside, both of which have about 10% remaining TBAX (where X is the anion).
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Tetrabutylammonium will stick very hard to RP-gel, it will not move. Just 50%MeOH/H2O will do the trick I think. Load the column with pure MeOH, then wash a little with 50%MeOH/H2O before loading the sample.
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It should be Pack the column with pure MeOH, not Load the Column with pure MeOH,
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Hi Rolnor,
Good to hear from you again! Since I have two samples, I will try repeating the Kaburagi-Kishi method on one and using reverse phase methodology on the other. On my shelf I have an old, probably unused 20-gram sample from JT Baker, of Bonded phase-octadecyl (C18), having an average particle diameter of 40-µm and described as being for flash chromatography. I also have a product from Analtech (catalog #50010) that is RPS hydrocarbon impregnated silica gel, 35-75 micron. I found a description at Amazon. If I am reading this correctly, the hydrocarbon impregnated product is not covalently linked to the silica and would come off in a sufficiently non polar solvent (if one is not careful).
"Silica gel and bonded phases are based upon a 150A pore silica gel, 35-75um particle. SPICE Packings are available in bulk quantities of 100 grams, 500 grams, 5 kg, and 10 kg. For larger quantities call for a price quote. Analtech offers two types of reversed phase packings for column chromatography: A C18 octadecyl bonded silica gel and a hydrocarbon impregnated silica gel. The most common reversed phase adsorbent is the octadecyl (C18) bonded silica gel. The C18 hydrocarbon is chemically anchored to the silica gel support. This material is compatible with all aqueous and organic solvent combinations. The carbon content is about 14%. Pore size is 150 Å Particle size is 35-75 microns. The hydrocarbon impregnated silica gel is a column grade equivalent of Analtech's unique RPS UNIPLATE. The long chain hydrocarbon is not chemically bonded to the silican get but is adsorbed onto the surface. This material is compatible with classical reversed phase eluting solvents such as MeOH/Water and Acetonitrile/Water. Less polar organic solvents will solubilize the hydrocarbon. The Reversed Phase Separation (RPS) column material is ideal for prep scale separations established on RPS UNIPLATES. Carbon content is about 5%. Pore size is 60 Å. Particle size is 35-75 microns."
I seem to recall from a previous discussion that packing and running a column of reverse phase material is quite different from running a silica column, perhaps needing more pressure.
Is one of these two products suitable for our application? If not, what should I buy?
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I attempted a second Kaburagi-Kishi cleanup on one of the two samples, in which there were more moles of tetrabutylammonium ion than product. Now there are fewer moles of tetrabutylammonium ion than product, but the impurity is still present. I don't see the point in running this method a third time. Instead, I will try one of the two reverse phase methods outlined in my previous message. If I get a satisfactory result on one sample, I will also purify the second sample. Is either of these products suitable for use in a gravity or low pressure column format? Does anyone have any sort of procedural reference? Thanks for any ideas.
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Tetrabutylammonium will stick very hard to RP-gel, it will not move. Just 50%MeOH/H2O will do the trick I think. Load the column with pure MeOH, then wash a little with 50%MeOH/H2O before loading the sample.
A few weeks ago we repeated the Kaburagi-Kishi cleanup, and we see a lower integral of tetrabutylammonium ion than we saw before. In round numbers we went from 0.6 mole fraction to 0.4 mole fraction TBA ion. Today we tried two kinds of RP C-18 TLC plates. In 50% aqueous methanol the Rf of the presumed product is close to 0.5. On my shelf I have an old, probably unused 20-gram sample from JT Baker, of Bonded phase-octadecyl (C18), having an average particle diameter of 40-µm and described as being for flash chromatography." I was planning to try a purification with this material as the stationary phase.
One, will using pressure that is typical for flash chromatography produce a sufficient flow rate?
Two, I seem to recall that the capacity is lower for C18, but I don't remember where I heard this. I was thinking about using C18 silica to product at a 100:1 ratio by mass. Does this sound about right?
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Yes, I agree, the capacity is lower, att least for low-pressure RP-chromatoghraphy.
Thanx! I have had some serious problem with blackmoldtoxinepoisoning, it got in my livingroom via a pipe down to the basement, catastrophic. I am much better now, got some "progressive brainfog".
1:100 is probably good!
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I am not sure if the flow will be good enough, I dont recall the diameter of the RP-gel I have used, sorry.
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We will try dipping our plates in PMA to see if we can find where the TBA is. Our long-term goal is an anti-fungal compound...
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Nice! Anti-fungal, is this a co-project with the industry? Is the compound meant to be oraly active? The TBA can be hard to spot, it does not react with just about anything, maybe molybdate-spray can visualize it.
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The PMA apparently stains the C-18 stationary phase, and we did not see any unambiguous spots above background. Perhaps there is another stain that would work, but the more I think about it, the less likely it seems.
The most promising version of our compound would not be orally active, but it might be suitable in the form of an injection. That is a long way off, however.
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Interim update: We tried using C-18 coated silica twice. The first time we isolated what appears to be the aromatic portion of the molecule minus the glycosidic portion. The second time we obtained two small fractions, each of which appear to include the glycosidic portion. But the two fractions are not identical by H-1 NMR. There are additional fractions that we have not analyzed yet, but by their R(f) values, these might contain the aromatic portion of the molecule. The good news is that I don't see evidence of the tetrabutylammonium ion. My tentative conclusion is that over the course of multiple purification attempts, some hydrolysis of the bond between glucose and the aromatic ring has occurred. I am looking into the possibility that this bond isomerized.
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Ninhydrine would work if you have a amide/amine I think.
Great that the TBA is gone! Maybe you need to control the pH, if you use reagents like TBDMSOTf and the use water in the workup you need NaHCO3 or TEA as acid scavenger, also if you have trace of triflate left after workup, the acid may form later and hydrolyse upon storage/evaporation.
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We checked the fractions with reverse phase TLC, and we mainly detected with UV light. PMA stains the whole plate, if I recall correctly. There is no amine, but there is a vinyl group present. There is a chance that KMnO4 would work as a stain.
I think that the acid may come from Dowex-50 in the Kaburagi-Kishi technique of removing the TBA ions. Perhaps adding a greater proportion of CaCO3 relative to Dowex-50 would buffer this.
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KMnO4 will react with the carbon in the C-18 but initially the vinyl will stain, it reacts faster.
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There is anisaldehyde/H2SO4/MeOH i think, that stains almost anything, it could work.