Chromatography Forum

What I meant is that it does not show that much the wavey baseline you see with pump-mixed eluents.

True enough, but the problem here is not a wavey baseline or steep baseline slope.
The real problem is inadequate sensitivity caused by the mobile phase background light absorption.
As I implied earlier if the mobile phase was transparent for light at 195 nm you could inject say 10 times less sample solution which would result in a symmetrical peak as well as narrow one. That’s my point.
My advice: remove the TFA. If the pH needs to be low, use phosphoric acid or better yet phosphate buffer. Then inject 10 – 20 µL instead of 100.
Regarding validation I understand you rationales but unfortunately still disagree. In my world validation means documentation of a working method - i.e. producing reliable results.

Best Regards
Best Regards

Hi Jörg,

You are right for UHPLC. It is a question of the optical pathway. But in standard HPLC and SEC a micro flow cell can bring up to factor 10 higher mass sensitivity.

If I had a dime for each time people use our columns wrong initially I would probably retire . It is hard to correct mistakes after validation is done and I always encourage people to consult before even choosing the column. I am here to help and don't mind even developing methods for free (although without validation)