How to purify ATP in a large quantity, like 1 gram? Is it possible to run a silica gel column instead of a ion-exchange column? Thanks.
I've eluted ATP from silica columns using HILIC- A solvent is acetonitrile, B = water. I don't know if it would resolve your mixture, try a small column first.
Start with 1-2% water in acetonitrile, also use dry-loading. Gradient up to 50% water should work.
Thanks, Jack. Your answer is very helpful. I am wondering which kinds of HILIC stationary you used. It seems there are five kinds:
1. simple unbonded silica silanol or diol bonded phases. 2. amino or anionic bonded phases. 3. amide bonded phases. 4.cationic bonded phases 5. zwitterionic bonded phases. Where did you buy it? Thanks.---Qing
@Qing- I just used unmodified silica, RediSep Gold silica columns.
Please note that I do work for Teledyne Isco, which makes these columns. 457-63-A is ATP, 468-09 is plain adenosine. Methanol didn't work for me, please note that one run was on a flash column, and the adenosine was run on a silica prep HPLC column.
Hi Jack, thanks again for your advice which is very helpful. I just tried to use regular TLC plate to test whether I can separate ATP from AMP using 1:1 ACN/water plus 0.1% TFA. Indeed, both of them can migrate with AMP migrate much faster. The only problem is that ATP spot show a long tail while AMP showed a nice spot. Do you have any suggestion on how to overcome this? In addition, you use 0.1% FTA in your gradient, can you tell me why? My concern is whether ATP is stable in such acidic solution. Do you think it better to use 1:1 ACN/0.1 M TEAA (or TEAB) instead? Since I need to purify ATP analogue in a large quantity, I plan to do silica gel column chromatography which I will install my own column so that it can have enough capacity. The only problem now is how to optimize the eluting solution to avoid the tailing problem, otherwise, I am afraid significant amount of ATP will be hard to be eluted.
Qing
@Qing- Sorry for the late reply!
I'm a little surprised the TLC ran with an aqueous system; the binders used to keep the silica in place often don't stand up to water, but that is plate dependent. The TFA is probably not needed for these compounds as they have phosphate groups, but I was running only the basic part of the molecule as well. You don't want to go above pH 7 or 7.5 on silica because it will dissolve in such polar solvent systems under basic pH.
You may also consider using a diol bonded phase instead of silica to possibly avoid the tailing problem. Although it is more expensive, it is reusable. Something less expensive to try is alumina, same solvent system as the silica (also run diol the same way too). These can be purchased as TLC plates as well.
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