Chromatography Forum

Hi,

I would need some advice on how to construct a gradient, please.

I am working with and UPLC-UV.
I have started with a gradient (A:B) of (95:5) to (0:100) for 20min at a flow of 0.3 mL/min. My peaks of interests are at 3.4 and 5 min.

How should I proced, to reduce the time run?
I mean, maybe one option is (A:B) of (95:5) to (70:30) for 10min at a flow of 0.3 mL/min?

Otherwise, I think I should increase the flow, at least at 0.5 mL/min. Do you agree?

Please, all help will be welcome!!!
Thanks a lot!!

both could be possible even together.

if there aren't any peaks after 5 min then, you can just trim your gradient after the peak came out, but keep the slope of your gradient (%/min) the same. Otherwise you may change the separation

So in your case you could trim your gradient from 5% to 33.5%B in 6 min
If there are some unwanted compounds after 5 min, keep your gradient slope the same until all peaks of interest are out then fast increase the %B to 100% to flush everything out. eg. 0/5%, 6/33.5%, 6.2/100%

Second you can increase the flow rate as much as your system and column can handle, but then further reduce the gradient step to keep the gradient volume (time gradient x flow) the same.

So with 6 min x 0.3 ml/min = 1.8 ml
-> with 0.5 ml/min, your new gradient would be 5% to 33.5%B in 3.6 min

This should keep your separation constant if there aren't any other effects from dwell volume or pressure issues

Hi!!

Thank you very much for this great explanation!!
It has worked!!

Thanks again,

Welcome

Thanks for feedback