Chromatography Forum

Hi all,

I am currently experiencing an issue with big square blocks of signal appearing in my chromatograms. They are always ~100mAU and there is no disturbance or anything out of the ordinary with the pump pressure at the same point in the run. The blocks themselves are raggedy, not clean, so I think it must be a detector issue.
Things I have tried:
I have tried multiple columns, eluents and buffers and the issue occurs sporadically with all of them.
I have replaced the UV-Vis lamps with our back-up and that has not resolved the issue.
I have purged each solvent bottle twice before running.
I have left the system equilibrating for ~2 hours, still hasn't resolved it.

I think it may be a bubble in the detector that is occasionally oscillating in front of the lamp, causing a signal disturbance for 1-3mins, then moving back out, this makes sense as the pressure does not fluctuate. If this is the problem, what is the best way to wash it out? Would disconnecting the column and washing it through continuously be the best plan of action?

Thanks a lot!

Would you please provide some chromatograms?

If I understood you correctly you are right when you suspect the detector. Usually an old or almost defect lamp will cause such issues. Also a defect of the detector is possible or an EMC problem (Electromagnetic compatibility).

Do you observe differences when only the UV or the VIS Lamp is used during a run?

If it is really an air bubble, try to repeatedly inject isopropanol (without column) and run at high flow rate, pure or high organic (no methanol/water, pum mixed!). Also gently tapping the flow cell can help to wiggle bubbles out.
Good luck
Jörg

Before chasing bubbles, run the detector for a couple of hours with the pump shut off. If the problem shows up then, it's an electronic or lamp problem.

This could be an air bubble in the detector flowcell, as you suggest. Some systems appear to be particularly prone to this problem, and in these systems it often appears when running gradients.

You can buy back pressure regulators to fit on the detector waste line which maintain a slightly elevated pressure in the flowcell and are effective at preventing this problem. Alternatively you could make your own with a length of very narrow bore HPLC tubing.

Chris