Chromatography Forum

Hi,

We are experiencing an issue where our known check standard results will fluctuate. Tried setting up a run of 20 check standards at 16 ug/ml. So far we've had 10 good ones right at/around 16. The 11th came out at 9 ug/ml.

Pump pressure fluctuates from sample to sample and sometimes during a sample run. Pressure goes anywhere from 135 - 160 bar. Causing our check standards to be > 20% off target. We have replaced nearly all items in auto-sampler (not the needle) and replaced the active inlet valve on the pump. This gave us better results for a while, but now the results are starting to fluctuate much more.

Any feedback or ideas would be appreciated.

Did you check pistons and seals ? Any leakage from the pump heads ?

We did not check or replace the pistons or seals yet. There is no noticeable leakage from the pump heads.

Now, it doesn't seem as if the pressure fluctuation is affecting the result. The last 5 check standards ran, the pressure remained right around 140 bar, and my results were 16, 9, 16, 5, 8 ug/ml (target 16 ug/ml)

Go back and overlay the chromatograms. Are you seeing fluctuations in retention time (which might suggest a proportioning problem) or does the different quantitation come largely from peak width (which would point at erratic flow). Also look at t0 (if that's fluctuating, you definitely have a flow problem).

We did not check or replace the pistons or seals yet. There is no noticeable leakage from the pump heads.

Now, it doesn't seem as if the pressure fluctuation is affecting the result. The last 5 check standards ran, the pressure remained right around 140 bar, and my results were 16, 9, 16, 5, 8 ug/ml (target 16 ug/ml)

Not necessarily all problems related to piston seals result in visible leaks. If you have a seal wash, you won't notice at all because all leaking solvent will end up in the was line. As the piston seals are relatively cheap, this is really worth a try. What I also do from time to time is to flush the whole system (bypass the column, of course) using water at 60 °C, followed by 5 % nitric acid, then water again and back to solvent. This will clean out most salt buildupt anywhere in the system.

Good luck
Jörg

Go back and overlay the chromatograms. Are you seeing fluctuations in retention time (which might suggest a proportioning problem) or does the different quantitation come largely from peak width (which would point at erratic flow). Also look at t0 (if that's fluctuating, you definitely have a flow problem).

Our retention times are holding steady, which is what was puzzling to me. Basically, our area counts are differentiating a lot when running the check standards back to back.

We went ahead and replaced the piston seals and cleaned up any residue (not much.) Going to see if that helps.

Thanks.

We did not check or replace the pistons or seals yet. There is no noticeable leakage from the pump heads.

Now, it doesn't seem as if the pressure fluctuation is affecting the result. The last 5 check standards ran, the pressure remained right around 140 bar, and my results were 16, 9, 16, 5, 8 ug/ml (target 16 ug/ml)

Not necessarily all problems related to piston seals result in visible leaks. If you have a seal wash, you won't notice at all because all leaking solvent will end up in the was line. As the piston seals are relatively cheap, this is really worth a try. What I also do from time to time is to flush the whole system (bypass the column, of course) using water at 60 °C, followed by 5 % nitric acid, then water again and back to solvent. This will clean out most salt buildupt anywhere in the system.

Good luck
Jörg

Thanks, Jörg.

We replaced the piston seals and cleaned up the little residue that was present on the pump head. Going to see if this works.

Pump pressure fluctuates from sample to sample and sometimes during a sample run. Pressure goes anywhere from 135 - 160 bar.

This is really bad. Pressure shouldn't fluctuate more than +/- like 3 bar at most. Is your degasser operating OK? Are your reservoir inlet frits all clean and free-flowing. And - important - did you purge out ALL four channels even though you might only be using two? If only using one channel (pre-mixed mobile phase) - did you try bypassing the multi-channel mixing valve with the Agilent adapter?

Pump pressure fluctuates from sample to sample and sometimes during a sample run. Pressure goes anywhere from 135 - 160 bar.

This is really bad. Pressure shouldn't fluctuate more than +/- like 3 bar at most. Is your degasser operating OK? Are your reservoir inlet frits all clean and free-flowing. And - important - did you purge out ALL four channels even though you might only be using two? If only using one channel (pre-mixed mobile phase) - did you try bypassing the multi-channel mixing valve with the Agilent adapter?

As far as we can tell, the inlet frits are all clean. Normal run conditions use 40% from A channel and 60% from B channel. We are going to try and swap to C and D channels.

Replacing the piston seals did not solve the issue. Our first check standard failed at around 50% recovery, retention time seemed to hold steady though.

In the first injection there is a rise >10 bars in pressure after sample valve switch from bypass o mainpass. As concentration is lower then it should be my guess is It could be some blockage in valve, metering device, loop or needle

Yeah, once you get about a minute into the run, it looks like the pressure holds pretty steady until the next ALS cycle. So like some others, I would suspect first the ALS automated valve/rotor seal.