Chromatography Forum

I agree that it looks like an air bubble stuck in the detector.

I also have the "pleasure" of working with Beckman equipment. I have asked them numerous times about degassing and their only reply is: they have a special pump design so you don't need to degass your buffers! (amazing how a they can declare the laws of physics Null and Void)

What I do frequently, whenever I suspect bubbles in the detector, is place a 1 or 2 ml syringe on the outlet of the detector. Run the system and monitor the baseline. Allow the syringe to fill with mobile phase, then loosen the inlet to the detector (at the column) and force the flow backwards with the syringe. (I collect the few drops in a paper towel) Then tighten the connection again and observe what happens to the baseline.

I always vacuum filter my buffers, then sonicate with vacuum for ~ 10 minutes. I find that I can only run the system for about 2 days before I start to get funny baseline or pressure pulsing.

This is only a small piece of the "pleasure" I have with my current HPLC system - I can not express my frustration in language suitable for a public forum!

I feel it necessary to thank you all, from time to time for the advices... When the system will be "back to business" I will try to post "a solution" that made it work again.

Hey there HPLC-slave,

Don't know if you've figured it out yet, but I had exactly the same problem with my Dionex Bio-LC 600 HPLC-PAD using NaOH at 18mM. I had 24 minute oscillations that I could change to 12 via adjusting the waste line or putting on a restriction tubing.

It was a degassing problem. If you have an inline degasser, make sure that it is working properly. Mine was programmed to run every other minute for 20 seconds with a 2 minute startup duration. However, it was only running every 7 minutes for 15-20 seconds. If you do have a degasser, increase the frequency. If you can't manually do it, you may be having communication problems. I called Dionex and turns out there is a firmware update that I uploaded, that completely got my degasser back on track.....yeah, after 4 weeks it seems like a software/communication problem. However, there is also an electronics board on top of the pump, at least on my system, that is also replaceable.

In addition, I'd make sure that your milli-q water is the same temperature at the start and throughout degassing. Oscillations will affect gas exchange.

If you haven't done the following, also double check

1. Leaky pump heads - open them up, see if there's a leak. If not, then that eliminates the pump heads
2. Make sure the vacuum degasser is working at the specific set parameters
3. If you put restriction on the end of the wasteline (i.e. small bore PEEK tubing using a coupler to the original wasteline) and you see a change in the frequency of the oscillation, it's a degasser/air bubble problem.
4. Make sure the waste line free drips into the waste container
5. Call Beckman, ask if there are software updates
6. Make sure reference/working electrodes are in working order, if using them

Good luck!

Thanks for all the degassing advices given! Now I've pushed things a bit further, as it seems that the air-in-the-system problem is gone and I tried running a gradient on my hplc...from 100%methanol to 100 % water ph3 made with sulfuric acid.
The resulting chromatogram is shown below:

http://www.funpic.de/fotoalbum/foto,157245,0.htm

I've previously posted another strange chromatogram. Does anyone have now a clearer view of what could happen? These phenomena appear on a UV photodiode array for all wavelenghts. For smaller wavelenghts the baseline as well as the signal have a severe fluctuation!

Thanks for any suggestion!

Dear HPLCSlave:
From the latest pic, you have excessive noise. Did you check all the fittings for leak?
Also, other ideas:
1. Check and clean the check valves, and replace in-line filter.
2. Use high-purity solvent.
3. Degass MeOH (unlike IPA, MeOH has lots of bubbles!)
4. Can you switch to this gradient: A -> B, in that B = MeOH, and A =PW pH3?

Alfred.

As well as Alfred's excellent advice, I'd suggest checking your solvent reservoir filters and the gradient mixing valve performance as described by the manufacturer. I assume that you have a reasonable column in the flowline to generate normal backpressure.

Does the frequency change when you increase or decrease the flow rate for a run?. Also, try gradient runs with the same mobile phase in each reservoir, once with water, once with methanol. You should have very flat baselines with minimal noise, similar to an isocratic run. That should be the benchmark for your trials.

Please keep having fun,

Bruce Hamilton

What frequency?
HPLCslave, this latest chromatogram looks like you are trying to do gas chromatography with HPLC equipment that has a severe case of asthma or even terminal multiple sclerosis. Or, are you creating some stuff to quiz us?

*lol* @ "chromatogram"

Are you sure that it's not a check valve issue?
Last week I refused to run MeOH/H2O 60/40 mixed on pump, cause after some time of working the CV get stuck again. This even with a InLine degasser working.
After some time wasted, I switched to MeOH 60% premixed and additionally had it ultrasonic trreated. No problems afterwards. (ok, no solution for gradient runs...)

Did you observed, or even better, logged the system pressure? Does it stay constant or does it fluctuate? If second, are the frequencies correlating with the ones from the detector?

What about the flow? Maybe you can measure the flow after the detector (or everywhere) with a graduated cylinder or a volumetric flask. Is it correct?
(in the above mentioned case, I once observed +/- exactly the doubled retention times, so one pump head was not feeded cause the inlet CV was stucked)

Does the phenomen occures always at the same mixing ratio (+/-)?

For checking the gradient mixing valve I would add about 0.1% acetone to the methanol and run the gradient. Do you get what you expect?

No, unfortunately this is no quiz, even though my HPLC shows multiple severe human ill-mood-like symptoms... I've tried what Bruce Hamilton advised me to do, to run a gradient but with only one eluent. The result is better but still doubtful:



the above chromatogram is a baseline for a gradient run with only one eluent.

The main problem that persists is that when changing the composition of the eluent by varying the flow rate at the pumps, the signal is unstable for a very long time, like in the following example where after running a mixture of 50-50 methanol-water pH3, the eluent was changed to water pH3. This chromatogram is as well only a baseline:



I can mention that the measured flow has an error of max 4%.
I will try to log the pressure fluctuations which exist but I can't correlate them with the horrible fluctuation frequencies.
I'm by far not an expert but up to my knowledge this HPLC doesn't have a gradient valve but a mixing chamber, is it possible?

Should it be gas the problem, obviously?

Thank you for your hitherto patience!

I've no idea of the information in the first image - looks more like an artist's impression of a solar flare to me.

Is the data in the second image true to label?. Is it Detector 168 at 199 nm?. That region is going to be very low energy, and very susceptible to stray light etc.The drift could be standard for a low energy region. What about drift and noise at 220 and 220nm?
.
Your system could just have a mixing chamber, and what happens if you halve and/or double the flow rate for a repeat run?.

Please keep having fun,

Bruce Hamilton

This is the last time I disturb you with this nonsense...
So... There is a very sensitive region where the signal is full of perturbations.. The problem is that this region is a wide band between aprox. 190 and 230 nm...
The images below are baselines (eluent water pH3) for the following flows: 0.4 , 0.8 and 1.6 respectively at 199 nm.






Thanks once again for the kind help!

Dear HPLCSlave:
You got quite significant noise and drift. Pls perform the periodic PQ, to verify the performance of the system before further testing.
Here are some ideas (some may have been suggested already):
1. Check for leaks (by pumping at higher flow!). Replace worn seals/pistons/o-rings
2. Remove all columns, and clean system with HNO3 ~10%
3. Purge system with IPA to remove air bubbles.
Good luck
Alfred

Thank you Alfred!