negative peak column Amminex

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Dear Anna,
We just installed yesterday a new column HPX_87H (amminex Bio-rad) and precolumn 125019. In case it matters, we also had yesterday some service in the equipment to change some parts that had been running too long: plunger seal, low pressure valve and lamp of the PDA detector.
We have been running for years with this column and precolumn setting with the following conditions:
Flow: 0.6mL/min, temp: 63C, injection volume 40uL, eluent: H2SO4 12mM.

Our analysis runs for 43 min, and we always observed a small negative peak at around 20 min (RI detector). This was generally not an issue, but with the new column this peak has become bigger, so it covers the peak corresponding to one of our analytes.
Image
link to the image:https://ibb.co/zJyXFKY
In the picture attached you will see a chromatogram of the same sample runned last week (black- with the previous column) and today (blue- with the new column). The peaks are: lactic acid, glycerol, acetic acid, propionic acid and butyric acid. As you can see, we do not see anymore the peak of butyric acid because it is covered by the negative peak.
For further information, I have to say that this big negative peak has always appeared as well in blank samples (eluent h2SO4 12mM), but it was generally much smaller…
The sample that you see in the chromatogram is the compounds I mentioned at 0.05 g/L prepared in 12mM H2SO4. And to make it clear, it is exactly the same sample (two aliquots of the same sample saved in the freezer).

Do you have any explanation?
Might it be that the column is not properly installed?
Do you know what is this peak? Is it unretained stuff from the injection?

Any help would be very very much appreciated.

Thanks in advance,
Anna
Column problems generally manifest as poor peak shape or incorrect retention times (neither of which seem to be a problem in those chromatograms).

In my experience, negative peaks occur when you inject a sample that does *not* contain something in the mobile phase (in effect, the result of "vacancy chromatography". You are correct that the elution time of this peak corresponds to the dead time of the column (the separation mechanism involves "ion exclusion", which means that your organic acids actually elute before the dead time). One possible cause for a negative peak with RI detection is dissolved air.

If this were my problem, the first thing I would do would be to make up a fresh batch of mobile phase. If the problem persists try injecting a mobile phase blank again to see if you still get the large peak. Since the onset of the problem coincides with service work done on the system, I'd be inclined to look in that direction for the cause:
- are you degassing your mobile phase?
- is the degasser working properly?
- are you degassing both the samples and the mobile phase?
- can you borrow another instrument and see if the problem shows up there as well?
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Given that you recently had service, most likely the system had a different mobile phase flushed throughout the flow path for proper maintenance. It is possible on some models of autosamplers to have fluid that remains static in the system if not properly flushed. This could potentially be something you are injecting, and the symptom would be a change in its negative peak size from injection to injection.

I would recommend following the user manual's guide for proper flushing / priming of any wash lines or dead volume space for your particular module.

(Waters alliance - run a sample purge, Agilent 1313, inject maximum volume blank a few times, etc.
Scott Allison
HawkEye Analytical, LLC
www.hawkeye-analytical.com
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