Anybody have some good advice for mobile phase for isolation of nucleoside triphosphate (like UTP and ATP)using DNAPac PA100 ion exchange column? thanks!

The September issue of the LC-GC applications notebook shows a separtation of these components on the DNAPac PA100. The application resolves all of the dNTPs and NTPs from one another in ~ 9 minutes.
A ten minute gradient of of ~4-18 mM NaClO4 in 20 mM NaOH will separate ATP and UTP with ~ 3 minutes of baseline between the peaks.

thanks, Jim.
I tried to use 0-800mM NH4Cl in 25mM Tris base to seperate UTP and UDP but they came out together. The retention time is 1.2 min. So I guess the triphosphates were not binded to the column I tried the pure water as the eluent. The triphosphates came out at the same time again and the retention time still 1.2min. What do you think? Can you give me some advice? Thanks!
linna

Dear Linna,

I suspect that there are residual salts in the eluent, you are operating at a pH value that is too low, or that the column you are using has lost capacity. The DNAPac column comes with a test chromatogram that you can use to evaluate your pump, sampler and detector, and to test (periodically) how much capactiy is left in the column. If you are certain the NTPs and NDPs elute in the column void after washing out system salts with several column volumes of water, I would evaluate the column for isocratic ion-exchange capacity using the test method shipped with the column. If that isocratic chromatogram (7 anion standard eluted with NaHCO3 and Na2CO3) verifies that the column has capacity, then you have a residual salt (system) problem, or are operating at a pH that minimizes the charge difference between UDP and UTP. These compounds should be well resolved at pH ~9, but are better resolved at pH 10-12.