Periodic ripple in UV - related to pressure?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

30 posts Page 2 of 2
Are there any internal lamp diagnostics you can run through your software? Maybe it's an issue with power or a dirty mirror inside the detector? Is there a wavelength verification test, or holmium/erbium filter test, etc... It might help point you into a proper direction.

Could be an issue with the power to the lamp, so regardless of the lamp, there's an issue. Has there been any kind of electrical disturbance in the area? Significant power outage, construction in the area or in the building that may cause electrical issues to the instrument? Anything like this that may have occurred between before and after you've been making this observation. I've seen things like this happen in the past.

As for cleaning the flow cell, you should be able to follow the tubing into the detector to see where the flow cell is located, it's just a matter of taking it out, using a combination of Methanol or Ethanol, lint free wipes, and Nitrogen to clean, wipe and dry it respectively.
What happens if you
- change flow rate? -> change in frequency of ripple?
- change (increase / decrease) the concentration of TFA in one of the mobile phases ->change in amplitude?
- put both solvent lines in same reservoir
- test a different lot/brand of TFA and/or ACN
- remove solvent reservoir frits

- have you done a gradient accuracy/mixing test?

My thoughts to see if the ripple is correlated to one solvent channel or solvent.
...or mixing quality (remove and/or add one mixer)

And what if you additionally degas the mobile phases offline maybe with He?
Hollow wrote:
What happens if you
- change flow rate? -> change in frequency of ripple?
Assessment of frequency is a bit difficult because it increases during the chromatogram, parallel to the increase of %B: At 0.55 mL/min flow using channels A and B, I have measured the following values (each time point is a "minimum" in the chromatogram):
Time %B
4.13 16.2
5.12 17.7
6.03 19.1
6.87 20.3
7.65 21.5
8.37 22.6
9.07 23.6
9.68 24.5

I changed the flow rate to 0.4 mL/min (decrease to 75 % of the initial value), the result was roughly a decrease of the frequency by factor 1.4 (reciprocal of 73 %), so correlation to flow rate and gradient composition is for sure.
- change (increase / decrease) the concentration of TFA in one of the mobile phases -> change in amplitude?
No change in amplitude when H2O + 0.1 % TFA stays constand and TFA in ACN is increased from 0.085 to 0.1 % TFA. Only the baseline drift is much greater (as expected due to the different extinction coefficient of TFA in water and ACN).
- put both solvent lines in same reservoir
No ripple at all
- test a different lot/brand of TFA and/or ACN
pending, all bottles in house are from the same batch
- remove solvent reservoir frits
These were already replaced => No change
[edit] Tried without frits => no change[/edit]
- have you done a gradient accuracy/mixing test?
Yes, accuracy is sensationally good with calculated deviation < 0.05 % and ripple < 0.005 % (Thermo PQ procedure), reproducibility is 0.006 % RSD between three runs.
My thoughts to see if the ripple is correlated to one solvent channel or solvent.

I also tried C vs. D instead of A vs. B => Amplitude and frequency remain almost identical.
Next experiment B vs. A instead of A vs. B => Amplitude and frequency remain almost identical.
...or mixing quality (remove and/or add one mixer)
pending
And what if you additionally degas the mobile phases offline maybe with He?

Unfortunately we don't have helium.

This seems to be a difficult one :lol:
lmh wrote:
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.


I plotted the retention time of UV ripple and pressure ripple against retention time:
Image
So the frequency is identical, but the extrema occur at different retention times.

Zoraku wrote:
Are there any internal lamp diagnostics you can run through your software? Maybe it's an issue with power or a dirty mirror inside the detector? Is there a wavelength verification test, or holmium/erbium filter test, etc... It might help point you into a proper direction.

Both tests are well within the instrument specification.
Zoraku wrote:
Could be an issue with the power to the lamp, so regardless of the lamp, there's an issue. Has there been any kind of electrical disturbance in the area? Significant power outage, construction in the area or in the building that may cause electrical issues to the instrument? Anything like this that may have occurred between before and after you've been making this observation. I've seen things like this happen in the past.

The method is new to this instrument. It ran before on an Agilent 1100 quat pump without this issue, so there is no before/after I could base such investigations on.
However, with the clearly demonstrated correlation between pressure and signal ripple, I think electronics are not the cause.
I will take care of the flow cell on Monday.

Thanks for your input.
So maybe there is nothing wrong with the system. Other than insufficient mixing quality for this application.
New systems are designed and optimized for low extra column volumes which is reciprocal to mixing.
Similar to the volume overload effects that can occur when going from an Alliance to a 1260/90

Maybe it may already help to add some of the ACN to phase A (or water to B). Idea behind, altering the surface tension before the solvents meet in the mixer.

Is it a pure binary system or is it a low pressure mixing system? (You wrote about Channel C & D which I correlate with low pressure gradient systems)
So if you could mix 3 channels then maybe you could use a 1%TFA phase and keep it constant at 10%. Then use pure solvent in A and B.
bunnahabhain wrote:
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


Hi Jörg, section 7.4.2 of the VWD manual recommends flushing with methanol first, then 0.1 M nitric acid, followed by water.
Best regards,

John Guajardo
Senior Product Manager
Thermo Fisher Scientific
Hollow wrote:
So maybe there is nothing wrong with the system. Other than insufficient mixing quality for this application.
New systems are designed and optimized for low extra column volumes which is reciprocal to mixing.
Similar to the volume overload effects that can occur when going from an Alliance to a 1260/90

Yes, I really think this is the problem here. I added a steel capillary with large ID (don't know exactly, but about 50 cm with 500µL volume) and the ripple decreases greatly:

Image
Maybe it may already help to add some of the ACN to phase A (or water to B). Idea behind, altering the surface tension before the solvents meet in the mixer.

This might be the best way to go from a scientific and practical point of view, but this is not an option because the method is taken from an OECD guideline, and customers don't like changes in official methods.
Is it a pure binary system or is it a low pressure mixing system? (You wrote about Channel C & D which I correlate with low pressure gradient systems)
So if you could mix 3 channels then maybe you could use a 1%TFA phase and keep it constant at 10%. Then use pure solvent in A and B.

It is a binary system with solvent selection valves, so I can mix A + (B or D) or B + (A or C) etc.
So maybe I should add a static mixer to the system, any recommendations?

Kind regards
Jörg
Good afternoon,

Why not just stick with Thermo parts--check UltiMate 3000 Series Pump Series Operating Instructions (Original Instructions), Revision: 1.3, Date: November 2011 © 2011 Thermo Fisher Scientific, Doc No. 4820.3551, Section 8.1, p. 136.

Otherwise, Mott makes good mixers: (100 uL worked well for Acquity-H systems, better than the OEM device)

https://mottcorp.com/mixer
MattM
The pressure difference seen is very, very small. The signal seen is NOT due to air. It looks a lot like what happens when you use old, contaminated TFA solution. If you have not done so already, please purchase a brand new, high purity bottle of TFA. Flush the entire HPLC flow path with MeOH/Water, then pure HPLC grade MeOH (Fully DEGASSED!!!). Next, run the individual pump pressure tests. Do the A and B pump heads pass the test with ease? Make sure they do. Also, try premixing the mobile phase and running a solution on just one pump head. How do this compare to online mixing using A and B? Differences should be expected, but in this case you need to look at specific signal patterns and fluctuations. This is needed to confirm that adequate mixing is taking place with the ACN and TFA solutions between the two pump heads. Be sure to use FRESH solutions for the tests to be valid.
Multidimensional wrote:
... high purity bottle of TFA ...

We use Merck Uvasol (Cat No. 1.08262) for this application. It is about one year old bottle. Should I buy the same quality again or go for the Peptide synthesis grade (Cat No. 1.08178) or even a different manufacturer?

Kind regards
Jörg
I'm not too familiar with TFA but would go with some "HPLC" labled quality for this application. But UVasol sounds not so bad neither, because it's specified for spectroscopy, but use fresh one.

Depending on how often you need the TFA, you may think of buying TFA in 1 ml glass ampules. Or buy small bottles of ca. 25 ml so it won't get too old.
E.g Sigma-Aldrich 302031-10X1ML, or 91707-10X1ML
Re; TFA "It is about one year old bottle."

WOW, that certainly is one of the reasons for your issues.

*We recommend purchasing the highest purity, smallest volume possible (You are not saving money buying a larger bottle because it quickly goes bad after opening). Better yet, please do not use TFA. It is overused, too strong and not needed in most applications. A weaker and higher purity acid often works better and results in less contamination of the whole HPLC system (e.g. acetic, formic or other HPLC grade acids).
If 100% mp A or mp B exhibits no ripple effect, the issue is highly likely related to the mixing. This is especially true if the method performs well with a quatery pump system (using the same mobile phases). No matter what, fresh TFA should be used for mobile phase preparation.
Update:
We tried the new batch of TFA, but it does not improve the ripple effect at all. So, for me it is clear that mixing is the problem. At first, I will see if I can live with it (i.e. if integration is OK despite of the ripple) then I will decide if I want to spend the money for an additional mixer.

Kind regards
Jörg
I still do not see any troubleshooting data or results....

"Next, run the individual pump pressure tests. Do the A and B pump heads pass the test with ease? Make sure they do. Also, try premixing the mobile phase and running a solution on just one pump head. How do this compare to online mixing using A and B? Differences should be expected, but in this case you need to look at specific signal patterns and fluctuations. This is needed to confirm that adequate mixing is taking place with the ACN and TFA solutions between the two pump heads. Be sure to use FRESH solutions for the tests to be valid."
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