I am currently working on purifying synthesized triphosphate nucleotides using anion exchange chromatography. I have successfully found a method to separate the triphosphate product from the mono- and di-phosphate products, using 1 M ammonium bicarbonate in water as the elution buffer. Checking the right fraction on LC-MS shows a nice purification. Though after lyophilization of the chosen fractions and dissolvement in nuclease-free water, the LC-MS shows mono- and di-phosphate products present again.
Can anyone explain why?
Does it have to do with the ammonium bicarbonate buffer doing lyophilization?
Dissolvement in water after lyophilization?
Or something else?
The breakdown of triphosphate nucleotides after lyophilization may be due to hydrolysis promoted by pH changes during the process. Decomposition of ammonium bicarbonate and subsequent re-dissolution in water can contribute to this instability, leading to the formation of mono- and diphosphates.
You might want to run a desalting before evaporation, salt decomposition is frequent culprit in problems like that. Also check if your water is as nuclease-free as you think: if your sterilisation procedure isn't working properly, it will keep spoiling your product.
Thanks for the advice - I will try the desalting and checking the water
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