Chromatography Forum

I am currently trying to analyse VFA (volatile fatty acids) from manure samples (very glamourous). I have a free acid mix which I acidify to pH2 and extract with chloroform, then do a 2ul injection with a 13:1 split onto a deactivated wax column. Chromatography for standards is fine.
However when prepping samples the manure is suspended in water, acidifed and then centrifuged. The supernatant is passed through a 0.45um syringe filter into the chloroform, at which point a gel forms (possibly proteins?).
Does anyone have experience of this sort of testing?
One option is to use water injections (ie skip the chloroform extraction) but that would limit my injection volume to 0.5ul due to flashback, and I'm not wild about putting any water through the FID.

Thoughts?

Mike

Hi Mike

Ah the joys of analysing poo !

Try leaving the acidificiation until after you have added the aqueoeus phase to the chloroform. By adding acid while the lumpy bits are still mixed in you actually decrease the concentration of acids in the liquid phase.

Also, if you can, try a less toxic solvent.

Either of these might help with the gel formation as well.

Peter

Hi Peter,
Further to the water based method. By fiddling with the inlet pressure, I have been able to inject 1ul withour exceeding the liner volume. Chromatography looked good, and has better linearity than the extraction but the reproducibility was poor.

So here's a good question, which is more important, the linearity or the reproducibilty?
(I don't expect an answer by the way, I 'm just trying to think how to explain this to a non-scientist manager)

Thanks for the sugestions, I'm looking into alternative solvents now.

Michael