Chromatography Forum

Hey All,

I have been spending a lot of time trying to determine formic acid concentrations from anaerobic cultures. I tried methylation and analyzed my samples by GC-MS, but realized my compound is still to small and comes out with the solvent peak. I then moved to HPLC (using prevail organic acids column with KH2PO4 buffer), I can see a nice peak for formic in my standards, but as soon as I add a culture supernatant I can not differentiate the peaks (I assume Na+ and Cl- in the media are causing problems detecting such a small compound). I have since moved to sylation and detecting on a Gas GC using an FID detector. I can see formic but it is shouldering off of the solvent peak. Any suggestions for better methods? I can provide more details about my methods if that would help?

Thank you,

Colin

Which GC columns were you using? What solvent(s)?

For FA, use thick film Rtx-1. 5 microns on 30m/0.32mm and use TCD.

if you need MS, connect a 5/0.25mm retsriction to the end so you can work with 1-2mL/min and normal pumps.

jaap de zeeuw, restek corporation

I see formic acid as a nice sharp peak midway between acetic and propanoic acids on Restek PEG columns (RtxWax). The samples are wild dog urine and faeces, and so they have a multitude of bacterial fermentation products in them.

Peter

Peter, would you be willing to share a chromatogram of this analysis?

thanks
jaap

No problem (as long as the link works !!)



Peter

Thanks Peter. This looks very good. What detector was used? It looks like MS. was this a 1 micron film on 0.32mm?

Hi Jaap

This was a 0.5 micron film, 320 micron internal diameter, starting temp 40 C, programmed at 5 C/min. MS detection - formic acid is nearly invisible by FID.

Peter

Solvent is methanol?

Best regards,

AICMM

Hi AICMM

Yes, methanol solvent. We use it because it gives us the broadest range of extracted compounds form the wild dog materials. We are seeing diols and glycols that would never extract into the more usual non-polar solvents. I doubt that we would see formic acid with a non-polar solvent.

The anticipated problem with formation of methyl esters of free acids has not arisen - we always analyse within a maxium of a few hours of preparation and store at -18C. Also we do not dry the extracts (the samples themselves, except for anal gland secretion are air-dry before extraction), and I suspect that the traces of moisture inhibit esterification.

The only disadvantage is that methanol extracts urea and then gives methyl carbamate as an artifact peak.

Peter

In case anyone would doubt Peter's results (a deed, foolish as it is unlikely), formic acid does elute where Peter says it does.

Twenty years ago I was startled to make that discovery.

Thanks for sharing your chromatogram, Peter. Don't trouble yourself in sending me any of those samples. I would hate to impose.



Rodney George