RT shift only for dopamin (HPLC)

[sebisbeak]

I have been trying to get my HPLC system running for a few months now but I still have a problem. I have read through the forum and haven’t seem to found a solution. Any help will be appreciated.

System: GP50 gradient pump (Dionex, Sunnyvale, USA) equipped with an autosampler FAMOS (LC packings); DC amperometry detector (Dionex, Sunnyvale, USA) with Glassy Carbon Working Electrode; C18 analytical Kinetex column.

Targeted hormones: dopamine, DOPAC, serotonin, 5HIAA (from Sigma); 1 µM

Mobile phase :75 mM NaH2PO4, 7 mM octane sulfonic acid (OSA) and 13 % MeOH, pH 2,79. Degassed with helium. Between injections, the column is reconditioned by washing with 90 % MeOH during 5 min and then re-equilibrated with buffer during 20 min.

Temperature and pressure are constant and flow rate is fine.
All my results are consistent. However, there is sometimes a shift of dopamine retention time (always a 2.5 min shift) without any impact on DOPAC, serotonin and 5HIAA RT. The peak is just moving while the mobile phase is the same (in a 2L glass), the sample is the same (same tube, maintained at 10°C) and the system is not turned off between injections.

I really appreciate if somebody could help me with suggestions…

Thank you !!

[tkubowicz]

Hello

If you have Retention Time drift just for one compound (in one direction - to longer/shorter RT) perhaps it is related to mobile phase pH. It is possible that after some time pH is changing (even 0.1 unit) retention for compound.
Perhaps your method is very pH sensitive (k vs. pH)

Regards

Tomasz Kubowicz

[Hollow]

Have a look at the thread of mine. Sounds kinda familiar...

Is the OSA you're using specified for ion-pairing chromatography?
Try different batches of that to see if it improves.

viewtopic.php?f=1&t=10064

[sebisbeak]

Thank you for your replies. The peak has been stabilized after using higher priced OSA.

However I have another issue with this assay. There is a big drop in the basal line (see picture) and this drop moves (sometimes during peaks!). I have already tried several things including :
-decrease of MeOH percentage for column washing (90% to 27%)
-increase the time of re-equilibration
-an automatic valve has been installed between column and EC detector to avoid contact of ECD with high concentrated MeOH solution (27%)
-the ECD (glassy carbon) has just been cleaned with alumina powder and washed with distilled water
-all the mobile phases have been degassed with helium for more than 24 hours
-the drop is still occuring when injecting mobile phase
-the current reference electrode was bought 2 weeks ago

Do you have any suggestion? I am running out of ideas...

Thank you in advance!!


picture : during the first 30 minutes, it is an isocratic elution

[Hollow]

Nice to read it helped you too. Would be interessting to in depth compare the differents OSA grades, to get an idea what is causing this continous drift.

But back to your new problem...
I'm not familiar with the ECD but worked a bit on Voltammetry during my study. Also haven't used ion-pairing chromatography a lot.

But first I would definitely omit a column wash between injections!
with the flush, you're changing the equilibrium of the OSA in the column. Re-equilibration then may take a lot longer than your actual ca. 10-13 column volums of mobile phase.
Does it also happens if you run your references without the wash step?

If you have late eluters in your samples, maybe a "filtration" over a C18-SPE cartridge (e.g Chromafix by Macherey-Nagel or 96-plates) may be the better option. At low pH, I expect the catecholamines showing now adsorption on the SPE cartridge.
This would also speed up your runtime by almost 2x.
If necessary, flush the column at the end of the sequence, before storage.

If the drop is related to the electrode, maybe it's possible to change the polarization to something that will electrochemically 'clean' it by oxidation or reduction reactions (-> cleaning sweeps in voltammetry). Don't know if possible or helpfull, just an idea.

[Perreman]

Do you see this baseline drop even when you are equilibrating the column without an injection made? Would be good if you could monitor the baseline after an injection-run to see if it is some equilibration issue.

[BostonFSE]

Do you see this baseline drop even when you are equilibrating the column without an injection made? Would be good if you could monitor the baseline after an injection-run to see if it is some equilibration issue.

I agree, try making a 0ul injection or just a blank vial 5 times, perhaps you need to extend your run time