Dear Experts,
I'm trying to separate a Fluorescein-like Xanthene dye (only phenolic and carboxylic charged groups) from byproducts with amino groups.
I assumed that a strong acidic cation exchanger (-SO3H, polystyrene-divinylbenzene polymer resin like Amberlyst) would do the trick. I expected the dye in the void volume and the impurity to be bound to the resin.
But nope. My dye gets stuck on the resin and only elutes with strong acid (basically the conditions used for regenerating the resin).
Experimental details: The substance (free acid, not as salt, so no cations except H+) was dissolved in an mixture of 95% EtOH and glacial HOAc (4+1 v/v), the resin was equilibrated with the same solvent and the sample applied, With this mixture, only some impurities eluted.
Next, I eluted with water, getting off some more material. The bulk however eluted with 95% EtOH / 3.6M HCl (4+1 v/v).
So i concluded that this setup won't work for my problem, but I'd like to understand why.
I'm suspecting hydrophobic interaction between the Xanthene system and the aromatic rings of the resin. Could that be the cause and is there any workaround?
I'm observing similar behaviour on Sephadex, so there might not be that much choice for alternative resins.
What would you suggest?
Thank you for your comments!
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