Hi everyone,
In the chemical synthesis of Guanosine diphosphate, I am using anion exchange (DEAE resins) to separate GDP from GMP and other by-products.
Sample pH is adjusted between 7.5 - 8
Column equilibrium: 50 mM TEAB pH=8
Separation: Gradient between 50-500 mM TEAB pH=8
unfortunately, all GMP and GDP come out in flow-through and there is no separation.
Is there any problem in my method? Is there any method to test the resin performance with a standard and calculate the resin capacity?
Thank you in advance
P.S. I have very trace amount of tri-butylamine, tri-butylphosphate, ortho-phosphoric acid and DMF.
If TEAB stands for triethylammonium bicarbonate you have bicarbonate ions that will compete in the anion exchange process.
You may not have the best resin here, but the most likely explanation is that the concentration of your equilibration buffer is too high.
I'd try loading a limiting amount of clean GMP standard onto a small portion of resin in 5mM Teab. Check by absorbance whether he GMP has bound. You can do the same with GDP. If one or both bind, then you can try a 5-50mM gradient, with a step (or steep gradient) up to 500mM to clean the column.
You also need to be sure the crude sample itself has an appropriate ionic strength i.e. not too high.
If it still doesn't work, I'd switch to a strong exchanger but keep the same buffer system. It's often used for this type of work.
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