Chromatography Forum

I am using RP-HPLC with DAD and FLD detection for the analysis of olive oil polar phenols obtained with liquid-liquid extraction (60% aqueous methanol).
I have run the anlysis for many samples without problem and suddenly a small broad peak (at a ~18% of organic solvent in the gradient) which is also fluorescent (280/320 nm) started to appear and does not permit an accurate intergration of one of the peaks of intterest. Despite the fact that I have washed the column with 50% organic solvent (1:1 Methanol/acetonitrile) overnight it is still there. I replaced the column with an older one used for the same type of analysis and the peak also appeared. I removed the column and i put a joint and run the gradient but nothing appeared so it does not seem to be something in the solvent.
Any idea? Thanks in advance

Is the size of the mystery peak proportional to the amount of time the system is held at initial conditions? If so, search this forum for "Empore Extraction" for more on the cause & fix.

What kind of injection system has your HPLC system, injection valve or syringe system? Maybe you have to check your autosampler and/or injection system.

I have washed the column with 50% organic solvent (1:1 Methanol/acetonitrile) overnight it is still there. Any idea?

Sorry: I can't understand if you mean 50% water by "50% organic solvent" or 50% methanol/50% acetonitrile.

If the former, wash out with 90% acetonitrile afterwards, maybe program in a high-organic cleanout at the end of each run.

Hello.
Please also try blank run (without injection) and extraction liquid injection run. If peak reproduces you should try to flush the system from pump to the injector with hot water and than with organic solvent and finally try to remake mobile phase.