Developing a gradient method II

Basic questions from students; resources for projects and reports.

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Hi all,

I already posted on this topic, and it was really useful for me. Now, I have a second question on this.

I did a gradient starting with 95:5 (A:B) for 30 min, and with this, I have a peak around 1.4min and the other at 5.3 min.
Next step is to reduce the total run time proportionally, but I also would like to reduce the time between them.

Any well-structured workflow about how to do it?

Hope you can help me!!
If there is nothing eluting after that 5.3 minute peak, you can simply stop the gradient after that. You will need to know:
- flow rate
- the gradient steepness (you didn't say what your final composition was) -- %B/minute
- the column volume (which you can estimate from 0.5 * L * dc^2, where L is the column length, and dc is the column internal diameter)
- the dwell volume (gradient delay volume)
Use that information to estimate what the %B is at 5.3 minutes, add 5% or so to be safe, and end the gradient at that point.

If there are additional peaks eluting after 5.3 minutes, but you don't care about them, then ramp up to the % indicated above, and then simply put a "step" up to wherever you are ending now and allow a few minutes to wash that junk off the column.
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374

Thank you very much for your answer.
It's been great!!

But now I have realised that 2 of my compounds are eluting at the same retention time :(

With this initial run, I used 100% Methanol. Do you think trying with 50:50 Methanol:ACN would be a good idea? And thus trying different % of Methanol:ACN?

Thank you again!!!
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