Chromatography Forum

Hi all,

I was told that an in-line degasser is not essential in semi-prep HPLC. Is it because semi-prep is often used in isocratic mode and with organic solvents?

If I plan to do semi-prep reverse-phase gradients, is an in-line degasser as important as for analytical HPLC?

Thanks for your answers,

Best regards

That depends on what kind of pump you have.

The "degassing" problem stems from the fact that the solubility of air in mixtures of polar organic solvents + water is less that the average of the solubilities in the starting solvents. Therefore, when you mix methanol (for example) and water, the mixture is supersaturated with air, and the excess air "precipitates" as bubbles. If this happens upstream of the pump (as it would in a "low-pressure-mixing" system), then the bubbles can interfere with pump operation. If it happens downstream of the pump (as it would in a "high-pressure-mixing" system), then the bubbles can show up as spikes on the baseline.

So, if you are using a high-pressure mixing system, and you don't mind the spikes, then you can get by without degassing.

Thanks Tom, I am planning to buy a high pressure mixing binary pump. Indeed I could do without degassing since in a first step I will not have a detector other than bioactivity

but does it affect separation for high pressure mixing?

That depends on what kind of pump you have.

The "degassing" problem stems from the fact that the solubility of air in mixtures of polar organic solvents + water is less that the average of the solubilities in the starting solvents. Therefore, when you mix methanol (for example) and water, the mixture is supersaturated with air, and the excess air "precipitates" as bubbles. If this happens upstream of the pump (as it would in a "low-pressure-mixing" system), then the bubbles can interfere with pump operation. If it happens downstream of the pump (as it would in a "high-pressure-mixing" system), then the bubbles can show up as spikes on the baseline.

So, if you are using a high-pressure mixing system, and you don't mind the spikes, then you can get by without degassing.

but does it affect separation for high pressure mixing?

Probably not. The bubbles generally will only form once the mobile phase approaches atmospheric pressure (i.e., at the outlet of the column).

I can imagine an exception if you have analytes that are easily oxidized, in which case the dissolved oxygen might have an effect.

thanks.
[
quote="tom jupille"]

but does it affect separation for high pressure mixing?

Probably not. The bubbles generally will only form once the mobile phase approaches atmospheric pressure (i.e., at the outlet of the column).

I can imagine an exception if you have analytes that are easily oxidized, in which case the dissolved oxygen might have an effect.[/quote]