We have back-flushed our HPLC using methanol and then we purged it with 25mM and 5mM sulfuric acid. We have been purging it for 4 days already using 5mM sulfuric acid but still the baseline is not stable. It keeps on fluctuating. We have suspicion that it could be the RID that has a problem, one possibility. How can we troubleshoot HPLC if the baseline is not stable after purging it for a lengthy time already?
Hi.
Three major problems occur if your approaching such problem: 1) dirty system, solution loss (check all tube connections) and 3) "injured" detector. Try changing wavelength, using ACN or EtOH or both for wash the system (first without column connected). Hoping you are going to fix the problem!
RID is extremely sensitive to temperature fluctuations. Ensure that the temperature is stable. What is your stationary phase which can handle 25 mM sulfuric acid?
Change column and try it again. Use always degassed clean mobile phase. if problem still exists, this might be due to the detector lamb
Dear Rhudith:
Sulfuric Acid is not recommended for analytical columns. In case your column is dirty with salts, diluted acetic acid is the best option for cleaning up the column and also the PDA cells. Other acids can damage the column, if packing is degradeted, your background will be strong.
In case of working with organic compounds, solvents such as hexane, isopropanol, methanol and acetonitrile are highly recommeded. In fact, manufacturers recommend passing all these solvents one by one for an effective cleaning of the column when we work with organic analytes.
Dichloromethane is also recommended but this solvent is too toxic and I avoid its use in the lab.
From my opinion flushing-back the analytical column gets the resolution worse.
Best,
Revere the column and try to wash with IPA for overnight.
If doesn't work, try to connect different column and see the baseline.
Best,
Thank you everyone for your fast response.
We will try your suggestions.
Check your mobile phase temperature, it should be stable throughout the run or its temperature should not vary drastically with time during analysis.
You may have some residual salt build-up in your detector. Since you are using RID and sulfuric acid in your mobile phase, I assume you are doing some type of ion-exchange LC. Flush just the detector with dilute sulfuric and see if the baseline is stable..
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