HPLC Baseline Drifting

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Hello,

It has been in a few runs that the baseline keeps drifting down. We tried running methanol and water to wash it out. But when we run the equilibrate with PBS/Azide it just won't stop drifting. Any ideas on how to improve it?

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Hi Wevian,

I'm going to ask a few questions regarding your specific analysis:
What type of column are you using?
What mobile phase are you running along side your buffer? (if any)
When equilibrating the column, what is that peak in your chromatogram? Are you shooting a blank injection?
What is the source of your buffers, azide, and mobile phase liquids? Is there any chance that they're unclean/used in other areas of the lab with less-strict handling?
How does the impurity's absorbance look across your entire visible spectra? It seems like from your link it absorbs well at 280, but about half as much at 320nm.
Make sure that everything is filtered, HPLC grade, and degas your mobile phase.
Get back about some of those questions and I'm sure that more people will be able to help.
Thank you!

The drift happened even without the column and equilibrating with water. The photo posted we were just running PBS with 0.1% azide.
The spike just happened at the beginning of the equilibrating, maybe it's because I just put a column on. No injections just equilibrating.
All the liquids used in the system are 0.2 filtered.

TylerSmith123 wrote:
Hi Wevian,

I'm going to ask a few questions regarding your specific analysis:
What type of column are you using?
What mobile phase are you running along side your buffer? (if any)
When equilibrating the column, what is that peak in your chromatogram? Are you shooting a blank injection?
What is the source of your buffers, azide, and mobile phase liquids? Is there any chance that they're unclean/used in other areas of the lab with less-strict handling?
How does the impurity's absorbance look across your entire visible spectra? It seems like from your link it absorbs well at 280, but about half as much at 320nm.
Make sure that everything is filtered, HPLC grade, and degas your mobile phase.
Get back about some of those questions and I'm sure that more people will be able to help.
Hi Wevian,

I'm sorry if I'm interpreting this incorrectly, but that large peak is the result of an equilibration without a column? Sorry, I'm a little confused with your wording. You couldn't have placed a column on in the middle of an equilibration, correct?
By any chance, does this peak increase with longer equilibration times? I would test and see if that is the case, if so then there is definitely some contamination occurring somewhere in your LC. It seems like it is not the result of the column (if I'm interpreting what you said correctly), which could have down-stream effects like the drift you're seeing. Mayhaps your strictly-water MP bottle has some contamination in it that builds up, and then when you put your actual mobile-phase on (azide,PBS), the contamination mostly elutes (the large peak), followed by some of it slowly eluting from the source/farther up from the primary spot of contamination (ie, tubes right at the MP reservoir following your wash with water)? The peak comes out pretty late, so there seems to be a decent amount of retention (if there is in-fact a column in-line for the picture you posted). Keep the column on, try equilibrating for a longer period, and then update us following your next wash with your proper mobile phase (azide,PBS). If there is an increase in area/size, then it's safe to assume you have some contamination up-stream from the column that may be introduced through the water your are eq-ing in.
Have you attempted to switch mobile phases and bottles to different sources? I'd also try this to see if the drifting shortens or disappears. Depending on the location of this contamination however, it could be in one of your sinker frits and potentially infect other bottles, but we should know if that's the case following those few tests.

Best of luck,
Tyler Smith.
Thank you for the response! I really appreciate it.
Here's the update. We run fresh HPLC grade water only for 2 injections (650min each) overnight without the column. No azide or other chemicals and the containers are brand new. I'm not sure if you can see the photos but the first injection drifts up to about 0.016 AU in 40 min then drift down all the way to -0.014 AU. The second injection just keeps drifting from 0 to negative -0.006 AU. The events recorded around 300 min are caused by the glitch from the software, which I have no idea why the software glitch or freeze can cause AU different.
FYI, this is Water's empower.
Thank you again!

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