conversion of gradient

[tilly]

i'm trying to shortern the run time of a gradient method by using a shorter column. Are there any easy calculators/converters that can be used to work out the new gradient. The existing column is 250mm x 4.6mm and the new is 50mm x 4.6mm.

[elsico]

It is not simple because you use gradient.
But probably depending on compounds it would be for example not 5 minutes but 3... There is no any rules and calculations, need to try and find it yourself

[AA]

A simple but complex question. If every thing else is still the same, column chemistry, solvents, flow rates, column ID, particle size, temperature, and all you are changing is the column length then: the same number of column volumes of mobile phase will elute your compound, in your case all the gradient times reduce by 5 times (ie if your old gradient was 10-90% over 60 minutes the new would be 10-90% over 12 minutes, simple. Problem though, peak resolution now also reduces by the same amount, so unless you have tons of resolution to spare, you will likely fail without changing other parameters (particle size is the one that could help you here). All of the major vendors have literature discribing how to shorten your runs using modern small particle chemistry in its various forms (RRLC, UPLC, UFLC and others).

[tilly]

thank you for your replies - it seems to make sense. i'll give it a go and see.

[lmh]

... and addition to AA's answer. If you are shortening your column length so much, you will also reduce your backpressure enormously, so you can, if you wish, increase your flow rate enormously, which speeds things up still more. You need to check that your detector is still collecting data fast enough.

Another option to benefit from shorter columns is to get narrower bore columns at the same time. You may not want to operate at 4mL per minute! The narrower bore means you retain the slower flow but use a lot less solvent, and decrease the amount of sample you need to get the same signal (remember, many common detectors, including UV, detect concentration, not amount; reducing the flow rate by a factor of 5 increases the concentration, and therefore sensitivity, by the same factor. Manufacturers would give their eye-teeth to get an increase of 5-fold over the competition).

If you don't trust separation on your existing 4.6mm by 50mm column at phenomenal flow-rates, you can still ramp up the flow rate during column reequilibration (if you're running a gradient).

In scaling, I usually increase my flow rate proportional to the length of the column. In theory I follow AA's advice of same-volume-gradients, but in practice I find it doesn't quite work, and to get best resolution, I usually have to make my gradient slope a little more shallow than I would expect on shortening a column. Practically, I'd recommend running a couple of extra gradients steeper and flatter than your guess, and see which works best.

To calculate expected elution times, you will also have to allow for the dead volume of the system, which is basically a constant time-per-flow-rate.

[zokitano]

You can also try the on-line Agilent Method translator:

http://www.chem.agilent.com/en-us/produ ... 60931.aspx

Regards