I'm running a phosphorylation reaction to produce an organic phosphate salt as my product. Structure RPO(OH)(ONBu4). After isolating the pure product, I want to fully deprotonate the phosphate and exchange the NBu4 cation to NH4 -> structure RPO(ONH4)2.
I've seen in the literature the use of Dowex resin to perform the cation exchange; however, I need to figure out how much resin I need to convert the NBu4 cation to NH4 fully. Moreover, some procedures stir the resin with the phosphate for half an hour and filter the resin off. In other cases, they run a column loaded with the resin to perform the ion exchange.
Have you done one of these procedures? Which one works better?
Dear Matheus,
Eventually, if you try first the complete acidification of your initial phosphate (e.g. dissolving it in DCM or CHCl3, adding a 6 M HCL solution, and stirring this biphasic mixture vigorously for 0.5 - 1 hour, followed by extraction and removal of the organic phase), and then bubbling ammonia in a suitable solvent (or adding a sufficiently basic ammonium salt to neutralize the phosphoric acid), could afford you the desired ammonium phosphate.
It would help if you would specify which dowex resin you have and are using. I have a very old book (1959) on Dowex ion exchange resins. When talking about Dowex 2 (anion exchange) resin they have an interesting statement discussing selectivity. "Roughly with any such series, The affinity decreases as the size of the hydrated ion increases". I suspect that the same is true for cation exchange.
The book has an extensive information on their resins and how they work, and how to use them. You might try to locate a copy (maybe a later version?). It is a very valuable discussion. Obviously put out by Dow.
Thiago Sabino da Silva thanks for the tips. However, my substrate decomposes in the presence of acids.
John Duchek, I'll try to find a copy of the book you mentioned. I'm using Dowex 50W X8 (200-400 mesh).
I first tried the IE with a model substrate, eluting with 0.025M NH4HCO3, and I removed the TBA ion from the model product. However, my substrate is not soluble in this buffer solution. When I try to run the IE, I see a reduced amount of TBA, but I still have a good amount of it.
I also run the IE column with 3:1 buffer:iPrOH since my compound is soluble in this eluent, but by doing that, I see no ion exchange for my product.
I have not heard of anyone running an ion exchange with a buffer solution. That would introduce extra ions that would take up sites on the resin and reduce its capacity. If you wish to modify the pH of the solution a weak acid or base resin might be a solution. If you are seeing reduced amounts of TBA it sounds like a longer column with more capacity would finish the job. From the book I mentioned, one of the properties of 50w x8 is separating neutral organic molecules by size exclusion. They specifically mention glucose (p26) eluting before water (glucose doesn't penetrate the resin; water does so the glucose has a shorter path to travel through the column and elutes first. It also specifically mentions methanol as separating somewhat so you may be playing solubility games with IPA in the solution. That would complicate things pretty much.
John Duchek, thank you for the nice observation.
I was basically reproducing a published procedure, which is:
1. You first flush the resin with NH4OH:water 3:1, to add the NH4 ions to the resin.
2. Adjust the pH to 8 by washing it with this buffer solution.
3. load the sample and flush it with the buffer.
By doing this, they ended up with the mono deprotonated fosfate, with NH4+ cation in the product. As I have never worked with IE columns, I did not realized about both the size exclusion factor plus the buffer messing up with the resin capacity. Maybe I should indeed run a bigger column with only water as eluent and hopefully get the TBA ion fully removed.
Step 1 is fine. Step two I don't quite understand. If you just washed it with deionized water You would wind up with the ammonium salt of the resin wetted with water. I am not sure why you would want to put bicarb into the system. Step 3 makes less sense to me. By adding ammonium ion as a buffer while trying to elute ammonium ion off the column would seem to shift the equilibrium in the wrong direction. That is adding more ammonium ion to the solution would tend to push ammonium ion onto the column. It might not be important if the ammonium carbonate is very dilute, but it should have an effect.
By the way, the book I have been referring to and a later edition that is a different color cover is available on ebay. I saw at least 3 copies this morning. It was running $30-$40 per copy.
You should be able to roughly predict how much resin you need. If in your experiment you used 10g of resin and 50% of the ions exchanged, then 20 grams should take you to 75%, and 40 grams to about 90%. That is rough, but it gives you an idea of how to proceed.
Thanks for the nice discussion, John Duchek. I doubled the amount of resin and use only water to elute the compound, as you suggested. By doing this, I was able to fully remove the TBA salt!
I will take a look on the book you mentioned. Thanks again!
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