My experiment includes synthesis of polymeric conjugate using hyaluronic acid and a flavonoid molecule in DMSO. Followed by purification of the conjugate by using dialysis membrane (MWCO- 12KDa) in 75% ethanol for 24hr. But as soon as I begin with the dialysis process, my conjugate gets separated into initial constituents with few hours. I have tried this process multiple times with other dialysis solutions (75% methanol, pure water) as well, still the same result. Can anyone suggest as to why this could be happening and a possible solution perhaps? Is there another way to purify my conjugate without dialysis?
Thank You
It sounds like your conjugate is unstable. You will probably have to find a more stable method of attaching the two molecules together.
Adam B Shapiro Thanks a lot. I think cross-linking of hyaluronic acid (carboxylic groups) with itself could be the reason. Is there a way to prevent this cross-linking? I'm using EDC.HCl and DMAP for the reaction.
I'm not very knowledgable on chemical synthesis. But I don't see how you could prevent crosslinking of hyaluronic acid to itself (assuming the chemical reaction favors that) except to have a very large molar excess of the flavonoid molecule over reactive groups on the hyaluronic acid.
As for removing the excess flavonoid, if you don't want to use dialysis, you could use size exclusion chromatography (also called gel filtration chromatography) instead. It's much faster. Since hyaluronic acid is a large, polymeric molecule and the flavonoid is presumably a small molecule, it should be an easy separation.
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