Column overpressure

[Ockert]

Hi

I've been working on an experiment in which my mobile phase is a gradient method consisting of acetonitrile and buffer (ammonium acetate in water, pH 4.6). I am using an Inertsil ph-3 10 cm column, and it runs fine, but when I want to run it again after a completed sampleset, the system goes over pressure. This happened with two columns now. At the end of the first succesfully completed sampleset i washed the column with Acetonitrile:water (1:1) for 60 minutes, and even now I have been washing it with methanol, water mixtures, acetonitrile mixtures and even reversed the column, yet nothing seems to help. Any help would be great!

[danko]

Let me guess, you've injected a lot of protein during your experiments!?

best Regards

[Ockert]

nope, no protein whatsoever...

[danko]

Then check the solubility of the ammonium acetate during the gradient – might be too much organic for the ammonium acetate to be in solution. In such a case you'll be seeing a lot of baseline noise and pressure increase.

Best Regards