Chromatography Forum

Hi all,
I am very new to HPLC and have only run a couple gel-filtration so far.
We have AKTA systems. Now I would like to determine the amounts of ATP, ADP, and AMP on my reaction mixture (protein crystallization which started with only ATP, and I hope the ATPs are stable over weeks). I found literatures which indicate the columns that the authors used (e.g., Vydac 302IC (Ion Chromatography, 300A pore size), Targa C18 5um 250mm*4.6, and etc). No one in the lab has done ATP assay using HPLC, and I simply don’t know which column I should purchase. So I am desperately seeking your suggestions. Protocols? I would really be happy.
The reaction volumes are very small and the MW of the protein is about 200kDa.
Thank you all, and I really appreciate your time and help.

cuny.

We have a complete protocol for nucleotides by ion exchange: http://www1.dionex.com/en-us/webdocs/7441_AN162_V22.pdf

This method runs at pH 13, so make sure your pump and injector are rated for that; if not, there are substitute components to make it so.

No matter whose method you use, you will want to remove the protein before analyzing for ATP.

I have used a specialty C-18 column from Supelco with good results. You can contact them for their nucleotide application. Other vendors may have such applications also. Check those you are familiar with. If you do choose a C-18 column, temperature control of the column and mobile phase compostion are critical. Good luck!

You can also analyze nucleotides on our Unison UK C18 column:

http://www.imtakt.com/TecInfo/TI133E.pdf

Thanks a lot to all of you.
I haven't decided which one to purchase. I will give a list of columns and my adviser will decide which one to buy.

Once again, I appreciate your help.

cuny

Cuny,

We also have an application note for the separation of AMP, ADP and ATP on an anion exchange column. It is not on our web site but I can e-mail you a PDF with the conditions and the chromatogram.

rhaefele(at)hamiltoncompany(dot)com

best regards