Chromatography Forum

I'm having a tough time fixing an unusual backpressure issue, hoping someone here could offer me some advice.

I have an Agilent 1100 quaternary pump (low-pressure mixing, micro-degasser) where the backpressure will stay stable for the first 8-10 minutes or so then suddenly begin fluctuating in a manner that indicates there is air in the lines. Purging the system with solvents on both A and B for an extended length of time doesn't resolve the problem -- still comes back same as before. The system passes a leak test, suggesting the check valve, AIV and seals are fine (replaced the seals earlier this year anyway). The degasser is used but was purchased recently and seems to be working fine. The multi-channel gradient valve is also fairly new and it's pretty unlikely that would be the problem anyway.

Any thoughts? What am I missing here?

I'm probably wrong, but I'll assume that this is a gradient run. If so, detail what the A and B solvents are, and also what the gradient profile is.

Also, if you think this may be a degassing issue, did you try to degas manually using membrane filtration or ultrasonic to eliminate the degasser as the source of the problem?

That's where it gets a little more bizarre...I'm just running in isocratic, water + 0.1% TFA as mobile phase A and acetonitrile + 0.1% TFA as mobile phase B. So, no gradient involved, no obvious reason why the backpressure would suddenly destabilize.

I haven't tried sparging the solvents or anything to rule out the degasser -- I will say it's not giving a warning light; I ran a couple simple diagnostic tests using the Agilent auxiliary cable and the vacuum pump on the degasser seemed based on that to be working fine.

Hello

I think that your problem is linked to:
-solvents and degassing

Quaternary pump is mixing solvents before pump head so mixing is not as good as for binary pump.

And main thing: air has different solubility in separate solvents (acetonitrile and buffer) and different in solvent mixture. So when you mix solvents air solubility is lower and that is why you see air bubbles.

I'd recommend:

1.Use degasser with bigger vacuum chamber
2.Do not use 100% buffer - instead use as mobile phase B 95:5 buffer:ACN

Regards

Tomasz Kubowicz

You may also try removing the inlet filters , temporarily ( may be clogged ) , purging all four lines ( even not in use ) , checking purge valve outlet ( if leaking ) .

Have you replaced the OBV? (Outlet ball valve) its the check valve on top of the pump. Its the first thing I would look at when you have pressure fluctuations for all HPLCs. If you dont have one, you can try sonicating in methanol.
Observe the skinny clear tube from the middle of the mcgv out (Gradient valve) going into the AIV (active inlet valve) when its having its pressure fluctuation
If you observe stubborn air bubbles that will not go into the AIV, you need to replace the AIV cartridge.

Also do a test of the mixing solenoids by placing the lines in graduated cylinders and set the mixture to 50/50% or 25/25/25/25% and run on bypass for an hour and see if the levels drop at the same rate. If not, then you have a solenoid valve that is not operating properly. If it does not open enough, then you could be getting air bubbles from cavitation. Also as mentioned above, the same problem can happen when the inlet frits have algae growth on them, especially the aqueous one, it will cause air bubbles to form because of vacuum pulled on the solvent.