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Hints on this purification

  1. Jul 18, 2012 #1
    Basically I took Mannose Amine and acylated it with benzoic acid using EDC ( 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide). The problem is the fact that I can not, no matter how hard I try, separate out the EDC and final product doing a column running 15% DCM:MeOH. I extracted out impurties by dissolving the product in water and extracting with ethyl acetate (the compound dissolves in water). The problem is that EDC is also water soluble. Anyone have any ideas on how to get the EDC out? Would a SPE cartidge work that would bind the EDC amine and flush through my product? That's probably the last thing I could think of in order to purify my material. I'm definitely not a rookie when it comes to running chromatography, I really don't think it is going to separate out on a flash column very easily.
     
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  3. Jul 21, 2012 #2

    chemisttree

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    Is it EDC or the urea?
     
  4. Jul 21, 2012 #3
    As hinted by chemistree, you should be talking about removing the byproduct of the EDC...

    We are also assuming that you (by some selectivity) did not get a complex mixture of the various benzoic esters of the sugar OH's, and the desired benzamide, that complicates things further (follow by TLC and visualize the sugars with a sugar specific reagent spray?).

    Why not start over with DIC or DCC as coupling reagents; the byproducts should be extractable to an organic solvent, and leave your water soluble product and any starting material. A quench step during workup should ensure no starting coupling reagent is around for the extraction steps.
     
  5. Jul 22, 2012 #4

    chemisttree

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    MrSid,

    Grave is a talented synthetic chemist, IMO, so he is likely not asking a simple question here. I believe that the amide will form much faster than an ester under these conditions and I assume that he is using EDC because he knows it is much less likely to racemize than DCC. If the reaction went to completion then he is likely dealing with the urea. My question was meant to clarify that the reaction had indeed gone to completion with respect to the reactive carbodiimide. If so, then he is purifying a water-soluble urea (HCl salt?) which has a tertiary amine or ammonium functionality in the presence of a fairly greasy but still water-soluble amide/alcohol. He didn't mention what the stationary phase would be but I assume he is using something like Florisil. He could be limited to silica for some reason. Amines are notoriously difficult to chromatograph... lucky he is going for the amide! He hasn't mentioned TLC results or visualization methods but I assume that he gets some separation there which isn't translating well to the flash column. I would be a bit surprised if a simple aqueous acid workup followed immediately by filtration through a plug of silica with something like THF as the eluant wouldn't pretty effectively stick the urea and allow the desired product through but then I don't know what he's tried yet.
     
  6. Jul 22, 2012 #5
    By NMR it looks like it is both the urea and EDC impurities. There doesn't seem to be any ester formation, I'm following a procedure that has already been published for something very similar. The TLC is pretty clean for the product, but it if very difficult to visualize where the impurities are by UV. The stationary phase I"m using for columns silica, but I have been spiking my eluents with 1% TEA. I have tons of experience purifying amines and know how difficult they can be on columns. I'm wondering if there exists some sort of solid phase extraction product out there that can remove both EDC starting material and the urea while allowing me to simply flush through my product very easily.


    Also, if I simply acetylated the -OHs and then purified it on the column, could I just remove the esters with MeOH and TEA? Then just rotavap everything off?

    Thanks for the advice.
     
    Last edited: Jul 22, 2012
  7. Jul 22, 2012 #6

    chemisttree

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    First thing I would do is to develop a good TLC technique that allowed me to visualize everything. That means using glass-backed silica without fluorescent binder and either H2SO4 or chromic acid for development. Once you have a better separation of the amide vs the amines you can move to the column. If silica won't work maybe you can try basic alumina or florisil (if you can find florisil TLC plates). Redi-Sep SCX (Teledyne Isco) might be useful but I'm not sure if it would also retain an amide. Probably not and using the SCX might be a great way to perform a first step cleanup of any remaining EDC, EDC-urea or unreacted mannose amine.
     
    Last edited: Jul 22, 2012
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