peak disappear after the third injection in HILIC

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

13 posts Page 1 of 1
Dear All,

I have been experienced a strange behavior in my recent work with polar pesticides (glyphosate without derivatization). The peak intensity decrease after each injection and disappears after the third injection. I have attempte various chromatographic conditions (HILIC, amide, DEA, IEX) but unsuccefully. Currently, I am following an application note using a DEA (waters) column in acid eluent. But still no more peak after 2-3 injections.

Have any of you experienced similar issues? I would greatly appreciate any feedback or suggestions on how to address this problem.

Thank you in advance.

Any feedback will be greatly appreciated!

Thank you very much
Glyphosphate and other phosphonates/carboxylates can interact with metals, although I'm not sure this would explain what you have seen. You could try a metal free column and replace some tubing with PEEK. There are also some options to passivate/deactivate the exposed metal surfaces.

What kind of detector are you using?
Glyphosphate and other phosphonates/carboxylates can interact with metals, although I'm not sure this would explain what you have seen. You could try a metal free column and replace some tubing with PEEK. There are also some options to passivate/deactivate the exposed metal surfaces.

What kind of detector are you using?

Thank you for your answer, I am using an LC-MS/MS, and I already passivate the system using EDTA and also medronic acid. Whatever the condition I use, the peak disappears after the third injection...

Thanks
HiggsBoson wrote:
Dear All,

I have been experienced a strange behavior in my recent work with polar pesticides (glyphosate without derivatization). The peak intensity decrease after each injection and disappears after the third injection. I have attempte various chromatographic conditions (HILIC, amide, DEA, IEX) but unsuccefully. Currently, I am following an application note using a DEA (waters) column in acid eluent. But still no more peak after 2-3 injections.

Have any of you experienced similar issues? I would greatly appreciate any feedback or suggestions on how to address this problem.

Thank you in advance.

Any feedback will be greatly appreciated!

Thank you very much

Can you get the peak back after flushing the system? Or do you never see the same glyphosate peak again unless you change the column?

What is the make-up of your mobile phase and which acid additive are you using?
Have you tried to replace the column with a length of tubing and see if you get your peak back?

What is your sample matrix or is the issue also with standards in mobile phase? How are you equilibrating the column after each run?

If you could post the method details that would help (column, mobile phase, gradient, etc).
wss wrote:
HiggsBoson wrote:
Dear All,

I have been experienced a strange behavior in my recent work with polar pesticides (glyphosate without derivatization). The peak intensity decrease after each injection and disappears after the third injection. I have attempte various chromatographic conditions (HILIC, amide, DEA, IEX) but unsuccefully. Currently, I am following an application note using a DEA (waters) column in acid eluent. But still no more peak after 2-3 injections.

Have any of you experienced similar issues? I would greatly appreciate any feedback or suggestions on how to address this problem.

Thank you in advance.

Any feedback will be greatly appreciated!

Thank you very much

Can you get the peak back after flushing the system? Or do you never see the same glyphosate peak again unless you change the column?

What is the make-up of your mobile phase and which acid additive are you using?



Thank you very much for your answer. Indeed, after stopping the instrument (overnight), the same phenomenon starts again in the morning (i.e. I only see the first three injections with a decreasing peak intensity at each injection to no signal at the fourth). I am using the same vial with the same method ...

I tried numerous methods, actually, I striterly follow an water application note (720006689)

Mobil phase A : H2O + 0.9 % formic acid and B : MeCN + 0.9 % formic acid,

Time %A %B Curve
0 10 90
4.5 60 40 2
8.5 60 40 6
20 10 90 1

Thanks !
lylegordon wrote:
Have you tried to replace the column with a length of tubing and see if you get your peak back?

What is your sample matrix or is the issue also with standards in mobile phase? How are you equilibrating the column after each run?

If you could post the method details that would help (column, mobile phase, gradient, etc).


Thank you very much for your answer. After stopping the instrument (overnight), the same phenomenon starts again in the morning (i.e. I only see the first three injections with a decreasing peak intensity at each injection to no signal at the fourth). I am using the same vial with the same method ...

The issue is with the standards ..

I tried numerous methods, actually, I follow waters application note (720006689)

Column TORUS DEA

Mobil phase A : H2O + 0.9 % formic acid and B : MeCN + 0.9 % formic acid,

Time %A %B Curve
0 10 90
4.5 60 40 2
8.5 60 40 6
20 10 90 1

Thanks !
We also had trouble with glyphosate. One thing which helped was not only to passivate the system with EDTA but also to add a very little bit of it in the standards and the samples. However, at acidic pH, EDTA readily precipitates, especially at high organic composition, so be careful. Another thing is that if you work with a relatively complex matrix, and if you can get a blank matrix, it may be worth to run matrix-matched standard curves. Usually the peak shape for glyphosate is better in the matrix than in neat solutions.

Do you have a flow-through needle or fixed-loop injector? In our case it worked better on a fixed-loop with a PEEK needle. Also, we have two fixed-loop systems and were surprised that one worked much better than the other for glyphosate. Probably something to do with different degrees of passivation of the systems.
Gaetan Glauser wrote:
We also had trouble with glyphosate. One thing which helped was not only to passivate the system with EDTA but also to add a very little bit of it in the standards and the samples. However, at acidic pH, EDTA readily precipitates, especially at high organic composition, so be careful. Another thing is that if you work with a relatively complex matrix, and if you can get a blank matrix, it may be worth to run matrix-matched standard curves. Usually the peak shape for glyphosate is better in the matrix than in neat solutions.

Do you have a flow-through needle or fixed-loop injector? In our case it worked better on a fixed-loop with a PEEK needle. Also, we have two fixed-loop systems and were surprised that one worked much better than the other for glyphosate. Probably something to do with different degrees of passivation of the systems.


THank you very much for your answer!! Actually, I tried to use a small amount in the vial also, but it will no more works after the fifth injection .. I have both FTN and FL. I am currently working with the FL. Will try again to passivate the system..
Do you have this issue with samples that contain matrix or also with standards in mobile phase?

If its matrix perhaps the matrix is accumulating on the column and eluting by the third injection and causing ion suppression. Maybe you need to flush/wash the column more between injections?
lylegordon wrote:
Do you have this issue with samples that contain matrix or also with standards in mobile phase?

If its matrix perhaps the matrix is accumulating on the column and eluting by the third injection and causing ion suppression. Maybe you need to flush/wash the column more between injections?


Thanks, I am using standard in mobile phase
I found a start up guide from waters that suggests a different mobile phase A from the one you are using (suggests 50mM ammonium formate pH=2.9)

https://www.waters.com/webassets/cms/su ... 6156en.pdf

The guide also contains this declaimer "NOTE: Torus DEA Columns are manufactured and QC-tested for supercritical chromatography,
not for the hydrophilic chromatographic separation of polar pesticides." but then proceeds to describe its use in hydrophilic separation so you should be OK, I would highly advise using the EDTA column wash solution from this method and the suggested mobile phase

only other thing I havn't seen mentioned is double check your column dimensions and flow rates, HILIC columns need many column volumes of re-equilibration between injections, the above application uses your same gradient timings but these will be off if you have different column dimensions.
We have been having severe issues with dropped response for some analytes with one of our LC-MS systems recently. Unfortunately it is a mystery and the instrument has been out of action for some months now.

It's probably a chromatographic issue in your case with glyphosate, but the 'resetting' of the glyphosate response the following morning followed by a rapidly diminishing response is very similar to what we're seeing with our LC-MS. Our suspicion is that something is leaching out of the LC system somewhere which causes increasing ion suppression after a few runs but disappears after leaving the system idle (not pumping) for a while. Still occurs when all solvents, etc are changed and tunes are perfectly fine. Just a left-field thought, but you might want to do an overhaul of your LC system (valve seals etc) and see if the response changes.
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