Chromatography Forum

Dear all,
I am running a size exclusion chromatography in my HPLC. I am using chlorofrom as eluent at 1mL/min. The oven temperature is at 31C and the detector temperature is at 35C. The problem is that the baseline present at random times sudden drops (using RI detector). It is not a peak, but a drop in the baseline.
This is a normal chromatogram:
These are some wrong chromatograms:




I have an online degasser connected (membrane degasser), and I have also tried degassing the eluent with sonication or helium (still connected to the degaser), and have not seen any improve.
The pressure in the system does not seem to fluctuate (only from 963-967 psi), and also the temperature in the cell of the RI and the temperature in the oven are constant.
Any ideas?

Thanks in advance,
Anna

Hello.

Please fix images attached. Try to connect pump directly to detector (check for maximal pressure!) and see if there are still baseline problems.

Do you dehydrate mobile phase somehow?

Did you flush the reference cell with fresh mobile phase ?

Thank you both for your replies.

Answering to Uzman:
- yes, I flush with fresh mobile phase every time that I change eluent, for more than one retention time of the column. Then I balance the cells. Actuallly when I have the flow on the reference cell on, the signal is much more stable than when I turn it off, what makes sense, but reaffirms that the problem is not in the detector but somehow in the eluent flow.
- When I disconnect the pump the signal of the detector is very stable. So it is not a problem of the detector I would guess.

Asnwering to dap:
- What is not clear in the images attached? I can see them well in my screen.
- I will try disconnecting the column and seeing the signals in the detector. Thanks for your advise.
- I am not overpassing the maximal pressure of the column if that is what you meant with "check maximal pressure".

Many thanks and regards,
Anna

Dear both,
I have tried removing the column from the system and the unstabilities are still present. The baseline is very stable for a period, but then the unstabilities appear randomly.

Also in response to your previous comment, no I am not dehydrating the eluent, and I was not aware that i had to do it or how to do it. I am using HPLC grade chloroform.

Best regards,
Anna

Hello!
Try to bypass degasser and check if there are any air/temperature fluctuations f.e. conditioner flow cycling.

Other suggestions:

Bypass the column with a capillary tubing , and wash the whole system with IPA.
This may remove any contaminants and also the water , if present.

Change the piston seals ,even if the pressure is stable, chloroform is a very aggressive solvent , and it may dissolve the junk at the backside of the piston seals.

Honestly, when I first saw this post I assumed someone was playing a joke on the members. The "chromatogram" shown above appears to show the two RID detector leads wired in reverse (wrong polarity). The negative peaks you refer to look just like peaks from a normal isocratic run, BUT upside down. It looks like you have sample peaks eluting normally. The peaks seen may be sourced or caused by: cross contamination from a dirty or poorly maintained injector seal or rotary seal (inspect the needle seat & valve and service) or the In-line DEGASSER.

You did not tell us what brand and model of in-line degasser you are using, but several of them are not compatible with chloroform (plus warm chloroform can dissolve all kinds of material into your HPLC system and contaminate everything. If the degasser is the source, then the problem may not go away after you remove it (use Helium sparging instead). The inside of the vacuum degasser MAY be damaged (the chloroform vapors go right through the lines into the vacuum system contaminating the vacuum pump and other parts. That contamination is then passed back into your mobile phase).