UPLC Gradient

[smartin]

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!!

[Hollow]

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

[smartin]

Hi!!

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

Thanks again,

[Hollow]

Welcome

Thanks for feedback