1220 infinity II decreasing absorption over course of analysis run


The pressure and the absorption at 230nm seen in the Online Signals view when I am operating the HPLC both decline steadily over the course of an injection runtime. The set up of the analysis is as follows in this literature. https://www.agilent.com/cs/library/applications/application-dedicated-cannabinoid-potency-testing-5991-9285-en-us-agilent.pdf 

Both Methanol Blanks and Standards in methanol end up declining below 0 for absorption at around the midpoint of the runtime, then bottoms out and comes back up to 0 at the end of the runtime. This dip in absorbance coincides with a dip in pressure over the course of a run. The peaks are still identified, however they are now below 0. 

We switched to a different Signal Wavelength and there was no dip in the absorbance. Wondering why we might see a dip in absorbance at one wavelength but not another when running blanks and standards? 

The Image is the first run since turning on the device. However, we ran it 20-30 times and each 9.50 min runtime looks the same. The same MeOH blank at 254 nm has a flat line absorbance for the duration of the run with the same variation in pressure.


  • The pressure and mobile phase composition seems to need more time to equilibrate before starting the run. For the first run, you should equilibrate the system for at least 30 minutes prior to analysis. Also in the method, it looks like you are missing the step of "post run" which in the technical note was set to 1.5 minutes for the method. You add this to the pump method UI which is next to where you put 9.5 minutes as the stoptime. This will allow the pressure and detector signal to stabilize prior to the next analysis in the sequence.

  • Hi Jacob

    The drop in pressure is normal in this method.  It occurs because the viscosity of the mobile phase decreases with the increase in proportion of methanol.  The dip in the VWD absorbance also follows the same path as the absorbance of the aqueous and organic mobile phase components are not matched perfectly.  Are you using exactly the same methanol, water and formic acid described in the application note?  

    The results will not be affected if the baseline dips below zero.


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