Chromatography Forum

Hi,

Using a linear gradient H2O-MeOH in reverse-phase UHPLC, I have observed a strong increase of background noise in the high region of the UV-VIS spectrum (about 400-600nm) when mobile phase composition gets close to 100% MeOH (approximately 1.3x10-1 AU at 450 nm). The column is a Waters BEH C18, temperature is set to 50°C. With acetonitrile, this doesn't occur, hence I suspect methanol to be the cause. All solvents used are of ULC-MS grade from Biosolve. Has anyone experienced it as well?

Thanks

If you haven't already done it, overlay a couple of chromatograms and see if the "noise" is not actually garbage peaks (i.e., the pattern reproduces from run to run). If it is, then the odds are it's junk in your methanol. You can confirm that by running the "three blank gradients" test and checking if the size of the peaks increases with increasing equilibration time. 450 nm is a rather long wavelength for random garbage peaks, but it's *possible*.

If it is truly noise, the most likely culprit is dissolved air coming out of solution. The first thing to check there is the degassing system (obviously). If that's not the problem, remove the column and replace it with a capillary restrictor. If the noise goes away, then there is something coming off your column. If it doesn't, then the only other thing I can think of is something stuck in your detector cell.