Periodic ripple in UV - related to pressure?

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30 posts Page 1 of 2
Dear all

I have an annoying issue on my Ultimate 3000 binary pump system: There is periodic ripple in the UV chromatogram, but I'm not sure if it is related to pressure or not. The pressure also has spikes, but these are rather small and look OK to me.

Image

Method details:
A = H2O + 0.1 % TFA
B = ACN + 0.085 % TFA

Gradient from 10 -> 25 % B over 10 min, wash step to 90 % and followed by re-equilibration.
I have read in another thread that lamp could be a problem with TFA at 220 nm. The lamp is close to 2000 h old, but passed the UV linearity and nois/drift test in the instrument PQ last week.

Thanks for any ideas,
Jörg
Good Morning Jörg,

That looks like air in your pump head, a typical picture.

Kindest regards
Gerhard
Gerhard Kratz, Kratz_Gerhard@web.de
Hello Gerhard

Thank you for the pointer. The Dionex purge valve is not really good, so I know purging is critical. I have purged the pump heads well (I think so, at least). But I will give it one more try....

Greetings
Jörg

Edit: Shouldn't the suspected bubble dissolve in the mobile phase over time? This picture remains stable over 30 h long sequences...
bunnahabhain wrote:
There is periodic ripple in the UV chromatogram, but I'm not sure if it is related to pressure or not. The pressure also has spikes, but these are rather small and look OK to me. Jörg


Turn off the pump or lower to 0.01ml/min, reduce the pressure shut off, and make a run; see if the baseline ripple in the UV chromatogram disappears or changes. Such change would tend to indicate a pumping issue.
Hi bunnahabhain,

What length and diameter of waste tubing do you have at your flow cell outlet?
Try adding ~60 cm of black PEEK tubing (0.025 cm id). You should also consider changing your lamp if it has 2000 hrs on it, especially if that includes several hundred ignitions.
Best regards,

John Guajardo
Product Manager, IC Columns
Thermo Fisher Scientific
If you haven't performed any maintenance on the pumps lately, I might also suggest changing the pump head seals. It could be air in the pump but this pattern also makes me think it could be poor seals at some point in the system - the pump head seals would be my first bet.

Also, make sure your lines aren't pulling up air bubbles as well. Sometimes the stones need to be changed - the ones I have for my PerkinElmer HPLC's seem to like to pull up air bubbles after so long, and I need to replace them.
I'm thinking air in flow cell
Thanks,
DR
Image
just do remember that in your chromatogram, the "wavelength" of the pressure pulsation is different to that of the UV signal. The pressure, which is only fluctuating very slightly, is doing so faster than the UV pulsation. Quite what that means is anyone's guess.
Thanks for all suggestions.

Here comes a short summary of what I tried so far:
- Pump seals were replaced => no change in the resulting chromatograms.
- Seal in the purge valve knob was replaced => no change.
- The flow cell was purged with isopropanol using a syringe => no change.
- Longer capillary at the flow cell output (added 40 bar pressure) => no change.
- Purged complete system with isopropanol, tested with isopropanol in both channels A and B => still periodic pressure ripples, but UV is OK
- Switched back to H2O + TFA (A) /ACN + TFA (B) => back to the initial picture

Next step is to replace the lamp (is ordered and should arrive during this week). I will post an update then.

Kind regards
Jörg
What kind of inline mixer are you using?
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
Andy Alpert wrote:
What kind of inline mixer are you using?


The Thermo standard setup: 50 µL capillary (linear mixer) plus 350µL static mixer. Should be sufficient for the flow rate 0.55 mL/min.
(Sorry, I forgot to mention the flow rate earlier...)
Purge the entire HPLC (without a column) with 100% Methanol at 5 mL/minute for 10 minutes. I suspect a bubble somewhere and IPA has 'too high' a viscosity for this purge.
HPLC chemist wrote:
Purge the entire HPLC (without a column) with 100% Methanol at 5 mL/minute for 10 minutes. I suspect a bubble somewhere and IPA has 'too high' a viscosity for this purge.


No improvement :(
Good afternoon,

Perhaps the next thing to try, if Thermo has a recommendation for a procedure, is to clean out the UV cell to ensure that "dirt" trapped within the cell is removed such that dissolved air cannot be trapped.
MattM
The new lamp also does not change the situation.

@John Guajardo: Is there a recommendation available from Thermo how to clean a flow cell? Please let me know.

Regagrds
Jörg
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