Chromatography Forum

Dear all, I think I have a very dirty flow cell here, Agilent G4212-60008. I'm not sure what contaminated it, I tried to flush it with pure Methanol, Acetonitrile, Isopropyl Alcohol, 10% Phosphoric acid, 10% Nitric acid, 20% Nitric acid, repeated 100uL injections of THF, but doesn't seems to work because I'm still seeing a cyclic baseline pattern.

Any idea what else to try? Thanks in advance.

Inlet and inline filters, check valves (pumps), pre-column filters, deuterium lamps aging...

New lamp, all valves and filters working as expected.

Have you done an intensity test on the lamp? Difference with the cell in and cell out can tell you if the cell is dirty.
Why would a dirty cell give a cyclic baseline?

You are mixing 50% A and 50% B. What is A and B? Is there TFA involved? Most likely you have a mixing issue. Try to add some tubing with large ID to improve mixing and check if this improves baseline stability.

That is typically for problems at your pumphead. Please check the seals and check for airbubbels. That is not a problem of your flow cell.

I was running 50% MeOH/Water.

The pump head is in good condition because I performed a test on the same HPLC with another flowcell and it works fine.

Anyway, I think I might I have identified the problem. I saw the glass solvent filter of my Methanol channel is discoloured, so there might be some unwanted stuff from there contaminating my methanol channel. I regenerated the filter and will flush the channel overnight hopefully will restore everything to working condition.

Not a flow cell problem at all. The time cycle is wrong and that is not characteristic of a dirty cell. In the future, to troubleshoot a basic instability problem, start with this article; "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)"; https://hplctips.blogspot.com/2014/01/d ... -hplc.html

Definitely a dirty flow cell. I moved the flow cell yo another instrument which is working properly, boom, same pattern.

Any good cleaning method to try? I tried 20% nitric acid but the result is too disappointing.

Not a dirty flow cell. Cyclic patterns are not from dirty cells. Far more likely to be pump or flow related (chk valve, degassing etc) as noted before. Please get some outside help to troubleshoot. BTW: Having a brand new, spare flow cell of the exact same model and type is the best way to rule out a flow cell.

I dont know how the flow cell it looke like, but if it as lachrome elite, and the ferrule is touched the glass directly.
Im afraid that you tight the the ferrule to much then you broke it.
What kind of maintenance you've done for the flow cell

This is an Agilent G4212-60008 type flow cell. Unless i force the flow cell in at a weird angle, chances of me destroying it by overtightening any ferrule is low.

Anyway, i fixed the issue by pulling about 50mL of HPLC grade acetone through it, then flush extensively with 50% water and methanol. It is weird that Nitric acid weren't able to clean things that acetone can, but whatever, saved our arse of not needing to buy any flowcell .

If its true and you fixed the issue with acetone, you very likely still have an issue with mixing. You should not have gotten this kind of pattern from a dirty flowcell alone.
What you would expect is a slowly drifting signal or a strong peak during cleaning, nothing else.

If you had a small crystal of some type that was soluble in acetone but not nitric acid, and it was floating around in the cell it could cause the problem as it circulated from top to bottom. But that would mean there is a turbulent flow within the cell which it shouldn't be.

If it happens again check to see if there is any residue on the outside of the cell. If you have a small leak, and it takes time to build up a drop, then it breaks loose and flows down the cell, that would cause the problem you were seeing, as the slow formation of the drop and the sudden flow down the outside of the cell would cause a rhythmic change in signal over time.