Rsing Baseline in UHPLC

[Wellyana]

Hi,

Recently I have attempted to transfer an existing HPLC method for a list of 11 analytes to UHPLC, in the hope of reducing the analysis time. I am currently using Thermo ODS Hypersil 250 x 4.6 mm, 5 micron column. The system that I am using is Shimadzu NEXERA X2 LC-30AD, Fluorescence detector.

I have since managed to develop the method, with one major problem, that is the rising baseline after t= 20 mins. I am using acetonitrile and water as mobile phase, gradient method (with 30% acetonitrile from the beginning to 70% acetonitrile in 35 mins, plus 3 more minutes of re-equilibration). Current flowrate is at 1ml/min and pressure is slightly over 100 bars.

So, I would like to know what could possibly be the reason for the rising baseline and how can I try to solve/reduce this?

Thank you.

PS: I have tried to use other columns such as Agilent RRHD 2.1 x 150 mm, 1.8 micron column, but it was no good as there are two particular analytes that tend to co-elute when using a shorter column.

[danko]

First, but not very importantly 250 x 4.6 mm, 5 micron is not exactly a column associated with the so called UHPLC mode. Neither the 1 mL/min flow rate.
Anyway…..if you utilize fluorescence detection I wouldn’t expect rising baseline unless something fluorescent elutes during the gradient, which is not that probable given your mobile phase composition. It could be from the column but still not very likely.
Something else: You say the re- equilibration is supposed to find place in 3 minutes at 1 mL/min. Taken the column dimensions into account I don’t think that’s possible. You’ll have to at least double the re- equilibration time. And maybe you’ll se a flat baseline.

Best Regards