Chromatography Forum

Hello

I am using Agilent 1260 HPLC(with degasser), Mobile phase is gradient using MeOH and Ammonium formate buffer (pH=9,5), Column is waters xbridge C8 150 mm 5um and usind detection with DAD. As can be seen from the picture the baseline is not stable and I suspected that it might be an airbubble in the system somewhere. I flushed the system from every channel (2 buffers, MeCN and MeOH) for at least 20 minutes, but nothing solved the problem. I also did seal wash with no effect. Can this be a degasser problem or something else and does anyone have an idea how to fix it?


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What wavelength are you monitoring?

220, 320 and 4 or 5 others. I don´t have access to the system at the moment so I can´t tell exactly. but the problem can be seen in each and every one of them.

For me it also looks like an air bubble. If it's trapped in the flow-cell, it can be quite reluctant to go away. What you might try is, while the pump is on, close the outlet capillary of the detector with your finger for some seconds. This will create some backpressure that might compress the bubble so it can be pushed out by the eluent.
Be careful, though: Close the outlet only for some seconds or the backpressure might get so high that the flow-cell cracks .

Another possibility is insufficient degassing. Small air bubbles can hang up where the flow changes direction inside the flow cell and gradually accumulate until they start to occlude the optical path (hence the baseline moves up). When the bubble gets large enough, it breaks loose and the baseline drops back down in that characteristic "sawtooth" pattern.

2 questions: How steady is your pressure? What happens to the frequency when you change the flow rate?

2 questions: How steady is your pressure? What happens to the frequency when you change the flow rate?

Roughly +-1.5 bar. The frequency does not change when I change the flow rate.

Appling pressure to the flow-cell did the trick. Thanks for help.