Chromatography Forum

Hi.
I have a problem when I try to separate an organic product using an iscocratic method (in this case 30% organic phase, 70% aquous phase)
Organic phase: 1:1 Acetonitrile:Methanol
Aquous phase: 65mM Amonium Acetate (9:1 Amonium Acetate:Acetonitrile)
In an analitical case (column size: 100x4,6 mm) I obtain a more or less good separation, but in preparative, the result is bad (column size: 100x30 mm).
Tech Data:
- Analytical: flow 3,014ml/min in a 7 minutes running time. Injection volume: 5ul. Injection amount: 2mg see analytical injection
- Preparative: flow 50ml/min in a 20 minutes running time. Injection volume: 5000ul. Injection amount: 40mg see preparative injection
Do you know what I can do to obtain a better separation in the preparative case?

Thanks

PS: the link for the prep one doesn't work...

What is your sample solvent?
I guess it is too strong for injecting 5 ml (=7% of the column volume!)
This would translate to about 125 µL on the analytical column...
(and the translated flow on the analytical one would be about 1.2 ml/min)

Try to dilute the sample solvent, so it is the same or even weaker than your mobile phase.
If it's not possible, reduce the injection volume.

Maybe you can make a loadability study on the analytical column to find out how much you can inject with this sample solvent, before it starts to affect the separation too much, than scale in means of the column volume.

Maybe you could even improve the separation factor "a" for your product peak first.

Or collect your preparative run in narrow time based fractions and re-chromatograph the impure ones.

in prep if you want to see the same picture as in the analytical run you need to make sure of several things

1. is it the same chemistry? some vendors do not have a prep size column for the analytical phase and you are using a different chemistry and so different results
2.the column particle size is the same, if not, if bigger then you generally compensate by injecting less in order to get the same picture
3. you need to transfer the flow rate and injection volume by checking the ration of the columns for the ID
in your case it is 900/21.16=42.53
so if the flow was 3.014 i the analytical then you need to make it 128.18ml/min to keep things equal, and it is not
for the injection volume, it should be 212.65ul

so basically you are working at a different linear velocity, overloading your column. no real reason to expect the same picture
can you give more details of the columns