Purifying and Concentrating ATP

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Sat Jul 26 08:54:36 EST 2003


Bhadresh Rami wrote:

> 
> Hello Folks,
> 
>        I am trying to purify ATP (commercially supplied - Sigma/
> Calbiochem) from its hydrolysis product ADP (even 3-5% ADP contamination
> is not acceptable for my experiments).
> 
>        Running a KCl gradient on an ion-exchange column separates ADP
> from ATP. 

I have once dabbled with ion pairing chromatography of nucleotides on
C18-columns, using 0-1 M diethylamin bicarbonat as buffer. This buffer
is volatile and can be removed by lyophilisation. If the gradient slope
is adjusted properly, ATP, ADP and AMP/Pi can be base-line separated in
under 15 min.

The problem with this buffer is that it is difficult to get the pH below
7.5, which is required to keep the silica backbone in the column from
dissolving. Soda-Stream soda-water generators from your local food store
sort of work.

In theory, resin-based C18 columns would solve the problem, as they are
not pH sensitive. However, I did not have the money to try this out.

I have also tried diethylamine formiate and acetate buffers for ion
exchange and ion pairing, but found that these acids are far less
volatile than the diethylamine. During lyophilisation you end up with
your product in concentrated acetic of formic acid, with predictable
consequences.



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