We are having a recurring problem with performing gradient analysis on a Waters 2690. When we get to the washing stage of the method (85% Methanol/ACN :15% 15 mM KH2PO4) we lose prime on the pump. This has occurred on three separate systems.

Water's suggested we allow the mobile phase to sit in the system at 0 mL/min with the degasser on continious to ensure proper degassing. We have performed this, on top of helium sparge, to ensure proper degassing but have continued to see the problem.

So far, to get the pump pumping again we have to remove the check valves and open the pump head. We have tried replacing seals and check valves but the problem persists. But this does not ensure that it will continue to operate for any length of time. Any suggestions or are we the only ones seeing this?

Thanks.

At some solvent composition and pH you will run into problems with the solubility of the KH2PO4. This is especially suspicious, since you ran into the same problem with 3 systems. What happens if you reduce the KH2PO4 concetration by a factor of 2? BTW, what is the pH, and why do you need it?

My guess is that you are precipitating your buffer at the high organic concentration. Since this happens on the low pressure side of the pump, the blockage then causes the pump to be starved for solvent causing the lost prime error. I do not belive that this has anything to do with degassing.

Your problem is precipitation in the checkvalves.
Take 2 graduated cylinders and measure out the proportions of issue. Upon mixing, you'll observe the problem. Either lower the molarity of the buffer or reduce the % of organic.

Thanks for the information. However, I am confident that it is not a buffer precipitation. I had suspected this at first. I have performed mixing of my buffer and organic in a beaker to ensure that there was not a precipitation problem. One thing I failed to note is that when this analysis is performed on a HP 1050, we have never seen any loss of prime issues. Although this may solve the problem, we have only one 1050 and 12 Waters 2690 instruments.

As far as my buffer, I am using 15 mM KH2PO4 at pH=2.5. pH is necessary due to the pKa of the molecule. The organic portion is 50/50 methanol:ACN.

Thanks

don't be to sure it is not precepitation of the buffer. i had the same effect with a water 600. when i mixed in a beaker there was no problem but running the gradient there whas the problem. lowering the concentration of phosfaat solved the problem. try it!