Chromatography Forum

We are using a a relatively new column and injecting a pure standard dissolved in ethanol and 2ml of this solution is diluted to 10ml with mobile phase. After 5 injections with the same solution the baseline begins to slope upwards, the standard peak remains sharp and has roughly the same area as the previous peaks. However the solvent front peak (ethanol) increases in size several fold. We have tried cleaning the column and the chromatograms return to normal, but repeat the aforementioned problem after the 5th injection from the same solution. The method is a BP method with a recommended column. Any ideas?

Hello

1.Mobile phase?
2.Isocratic or gradient?
3.Detector setting? (ref wavelength?)

Tomasz Kubowicz

1) The detector wavelength is 240nm
2) Isocratic
3) mobile phase is a mixture of 6 volumes of 5%w/v of sodium dodecylsulphate in 3%v/v glacial acetic acid, 10 volumes of triethylamine, 20 volumes of butan-2-ol, 310 volumes of acetonitrile and 690 volumes of water. The mixture is then adjusted to pH 3.0 with orthophosphoric acid.
4) The column is a nucleosil C18, 5 micron.

Hello

I would try to inject 5x mobile phase and see if problem still exists. I suspect there is something coming out from column after long time (perhaps something is moving through column and it reaches detector after couple of injections)

Regards

Tomasz Kubowicz

Thanks Tomasz,

The person doing the analysis is putting the column on wash overnight and we will try tomorrow.