Chromatography Forum

Hello everybody,

I want to analyse the supernatant after quenching my bacteria with 20-30% cold glycerol and subsequent centrifugation. So I have a high glycerol concentration and low metabolite concentrations at the same time. Does it make any sense to inject the sample (2,5 uL) directly - with such a high glycerol concentration? In our group we argue whether this investigation is sensible at all. Did anyone of you try such a method?

David

We have a DIONEX ICS3000 System with a CarboPac PA1 and an IonPac AS11.

I guess the most severe problem would be the viscosity of the sample? That will cause problem both in the injector and on the column.

I agree with Mattias. You could try to dilute your sample solution with solvent or mix of solvents weaker than your mobile phase (to avoid peak broadening).
Because your metabolites are present in low concentration, you should find the appropriate ratio between the sample solution and diluent. You could use, also, a narrow-bore column (e.g. 2.1mm i.d) to enhance the sensitivity of your analyses.

Regards

What are quenched bacteria?

Hi, thank you for your answers!
I will try with a 3% solution of glycerol and tell you about possible problems.

"Quenching bacteria" is not precise - you quench the metabolism of the bacteria with e.g. cold glycerol - you stop the metabolism. Glycerol is favorable because the intracellular metabolites stay "inside", the cells do not burst - "cold shock phenomenon".
The "only" problem is the high glycerol content in the supernatant for analysis of the extracellular metabolites.

Chromadave,

I am not an LC guy here, but what about a GPC cleanup and collect the bacterial fraction? Glycerol should come off pretty late, bacteria pretty early, it is often low pressure chromatography so the viscosity should be less of an issue, and you can build one pretty cheap (having done it once...)

Just a thought.

Best regards.

I have injected up to 5% glycerol containing samples in C18 columns so 3 % should work...

Seems to me that one can withdraw extracellular (extrabacterial) fluid faster than stopping the metabolism? So, maybe there is no need for glycerol?