﻿ Developing a gradient method II - Chromatography Forum

## Developing a gradient method II

Basic questions from students; resources for projects and reports.

### Developing a gradient method II

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!!
Thanks!!

### Re: Developing a gradient method II

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
tjupille@lcresources.com
+ 1 (925) 297-5374

### Re: Developing a gradient method II

Hi,

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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