I am conjugating a phospholipid with a free terminal amine with a carboxylic acid using PyBOP methodology (employing DIPEA) for the formation of the amide conjugate.
After the reaction, the crude was extracted with HCl aq., and Brine and the organic phase is further purified by flash chromatography using MeOH:DCM.
However, given the extreme polarity of the product, the desired phospholipid appears right next to another spot in the TLC (Rf 0.70 vs 0.65 in 10:1) which makes it very difficult to purify. After NMR (see image) I thing the impurity is a pyrrolidine derivative from the PyBOP.
I have tried further extraction of the impure fraction of the final product with K2CO3 10 % aq. and after also more HCl aq. but I was not able to make the pyrrolidine go the water phase.
Does anybody have any ideas on how to remove this impurity using a water extraction?
Thanks!
As understood from the literature data in the amidation reaction should be avoided medium with pH 8.5, to avoid by-products of the reaction
Two dimensional TLC (with 2 sets of solvents systems ) may help you to identify the exact separable conditions of solvent systems to separate two closely moving spots on TLC plate.
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