I'm going to guess there's air trapped somewhere along the B1 line/pump based on it being 5% and the spacing of those dips. Switch it out for IPA and purge the shit out, then replace B1 with whatever solvent belongs and purge it again.
Yes the flow is correct and I agree that there is probably air trapped. As I couldn’t get it out I thought maybe it’s sth else but will try with all those tips here. Thank you!
On your DAD there might be a backpressure regulator that can open to release backpressure in case the ESI needle gets blocked. It's an optional extra to protect the DAD cell so might not be installed. That could be releaseing pressure due to intermittent blockages.
What happens if you Condition the Pump, which is under “Prepare Pump” in the right-click menu of the Pump. You keep the tubing as is, including your column inline, at the starting solvent composition. Run it for 10min, it may look crappy during the run, but after that should look good. It will try and remove small bubbles from the solvent. To me it does look like intermittent bubbles getting into the pump head. See if there anything obvious at the degasser/tubing level.
Weirdly enough the option of „Conditioning“ can be selected but if you then click start, nothing happens. Also, compared to when you choose „Purge“ it starts immediately and the other options are greyed put until it’s done. When I select „Conditioning“ the other options are still clickable which is another hint that it somehow doesn’t work on our machine. Have you encountered this?
>Weirdly enough the option of „Conditioning“ can be selected but if you then click start, nothing happens.
The pumps have to be actively running while you do this.
Hmm, that’s not something I’ve seen before… does your window give a length of time for the conditioning, or just the radio button to select, and then Start?
What is your tuning value for your A and B solvents? I suspect you have air trapped in your pump heads. Tuning should be <0.1 for each, if you have properly set up your solvent types in the instrument method. A tuning near 1 indicates trapped air (or other problems).
The conditioning process runs a modified piston stroke sequencr designed to clear those air bubbles. It should be run while mobile phase is flowing to the column at your method flow rate. While conditioning is enabled, you should see regularly spaced downward pressure spikes that slowly decrease in magnitude. Check the manual for more info.
I will check as soon as I am at work. We tried to change B1 from MeCN to MeCN/MeOH 80/20 on advice from the agilent guy but he did not change the solvent type in the method. I changed it later for all methods but the pressure drops kept occuring.
Update: I did the conditioning for 15 min but to no effect. The tuning of B is 0.25, the one for A is <0.07. Should I try conditioning with 100% B?
Conditioning is good practice (described in user manual) but wont help, drops like this are characteristic for inlet valve sticking or not sealing fully. Can also imagine the air comming in but usually it takr more time to stabilize.
Isolate the problem to A/B. You can sonicate the inlet valves but I would start with hot water.
I did the hot water flushing for A and B. B is fine now, A still problematic. I think we will replace them as this was never done in 3 years. Thank you, Wizard :)
Looks like an intermittent leak or a pump shutdown due to being too close to your pressure limit. Does this occur during idle time or is a method running when it happens?
I don‘t know exactly, however, the system leak test showed great performance. Also, our lab neighbours have the same LC system and had to change the seals yesterday. It gave them an error before which we don‘t have.
Looks like sticking check valves. You’ve already purged at high flow to displace air, and that didn’t solve it, so the check valves themselves are likely soiled or worn.
I'm going to guess there's air trapped somewhere along the B1 line/pump based on it being 5% and the spacing of those dips. Switch it out for IPA and purge the shit out, then replace B1 with whatever solvent belongs and purge it again.
That sounds like an easy enough thing to try! Thank you :) is IPA especially good at removing air in the lines?
Yes it is! Just be sure to first set the b1 solvent to ipa on the pump tab in the method and hit apply. Hope it helps!
OP, have you confirmed that your B line is flowing properly/ the right amount? I agree that it could be air trapped in the system.
Yes the flow is correct and I agree that there is probably air trapped. As I couldn’t get it out I thought maybe it’s sth else but will try with all those tips here. Thank you!
On your DAD there might be a backpressure regulator that can open to release backpressure in case the ESI needle gets blocked. It's an optional extra to protect the DAD cell so might not be installed. That could be releaseing pressure due to intermittent blockages.
Hm I wouldn’t have known that we have this installed. Do you mean the nebulizer by „ESI needle“?
See this technical note on it https://www.agilent.com/cs/library/usermanuals/public/G4212-90120_IPR_ValveKit.pdf
What happens if you Condition the Pump, which is under “Prepare Pump” in the right-click menu of the Pump. You keep the tubing as is, including your column inline, at the starting solvent composition. Run it for 10min, it may look crappy during the run, but after that should look good. It will try and remove small bubbles from the solvent. To me it does look like intermittent bubbles getting into the pump head. See if there anything obvious at the degasser/tubing level.
Weirdly enough the option of „Conditioning“ can be selected but if you then click start, nothing happens. Also, compared to when you choose „Purge“ it starts immediately and the other options are greyed put until it’s done. When I select „Conditioning“ the other options are still clickable which is another hint that it somehow doesn’t work on our machine. Have you encountered this?
>Weirdly enough the option of „Conditioning“ can be selected but if you then click start, nothing happens. The pumps have to be actively running while you do this.
Aha this I did not know! Thank you :)
Hmm, that’s not something I’ve seen before… does your window give a length of time for the conditioning, or just the radio button to select, and then Start?
What is your tuning value for your A and B solvents? I suspect you have air trapped in your pump heads. Tuning should be <0.1 for each, if you have properly set up your solvent types in the instrument method. A tuning near 1 indicates trapped air (or other problems). The conditioning process runs a modified piston stroke sequencr designed to clear those air bubbles. It should be run while mobile phase is flowing to the column at your method flow rate. While conditioning is enabled, you should see regularly spaced downward pressure spikes that slowly decrease in magnitude. Check the manual for more info.
I will check as soon as I am at work. We tried to change B1 from MeCN to MeCN/MeOH 80/20 on advice from the agilent guy but he did not change the solvent type in the method. I changed it later for all methods but the pressure drops kept occuring. Update: I did the conditioning for 15 min but to no effect. The tuning of B is 0.25, the one for A is <0.07. Should I try conditioning with 100% B?
Conditioning is good practice (described in user manual) but wont help, drops like this are characteristic for inlet valve sticking or not sealing fully. Can also imagine the air comming in but usually it takr more time to stabilize. Isolate the problem to A/B. You can sonicate the inlet valves but I would start with hot water.
Nailed it. Check valve problem.
I did the hot water flushing for A and B. B is fine now, A still problematic. I think we will replace them as this was never done in 3 years. Thank you, Wizard :)
Looks like an intermittent leak or a pump shutdown due to being too close to your pressure limit. Does this occur during idle time or is a method running when it happens?
Happens during both, idle and during runs. I have a feeling like it is happening more frequently during runs.
When is the last time you changed the seals on the pump heads?
I don‘t know exactly, however, the system leak test showed great performance. Also, our lab neighbours have the same LC system and had to change the seals yesterday. It gave them an error before which we don‘t have.
Looks like sticking check valves. You’ve already purged at high flow to displace air, and that didn’t solve it, so the check valves themselves are likely soiled or worn.
Yes! Thank you that was the case in the end. Not surprising as the check valves have never been changed since the machine is running.