high background noise with methanol at 450nm

<p>Hi,</p><p></p><p>Using UPLC-DAD (elambda), I have observed a strong increase of background noise in the high region of UV spectrum (about 400-600nm) when mobile phase composition gets close to 100% MeOH (approximately 1.3x10-1 AU at 450 nm). With acetonitrile, this doesn't occur. Methanol used is of ULC-MS grade from Biosolve. Have anyone experienced it?</p><p></p><p>Thanks</p>


  • I have experienced things similar to this but it has always been reproducible with Acetonitrile.  It seems like the most probable cause is your methanol is somehow contaminated.  The most similar instance I've experienced was a contaminant in our formic acid (Part of phase A) that would elute when methanol concentration was raised.  What is your other phase?    

  • Thanks, that was my thought too. Mobile phase is composed of pure water and MeOH. I have now tested 2 brands of MeOH and results were similar.

    However when I bypassed the column (set a flow restrictor instead) and performed a water:MeOH linear gradient, the noise increase was not linear and appeared again late during the gradient, which makes unlikely that contaminants were trapped on the column and then eluted at high % MeOH. I also thought of an issue with the degasser and generation of bubbles at 100% MeOH. I have attached a figure showing the trace at 450nm without and with column, and a DAD spectrum.

    I will have a try on another UHPLC system, hopefully things will get clearer.