GC-FID for free Volatile Fatty Acid quantification - issues

Discussions about GC and other "gas phase" separation techniques.

25 posts Page 1 of 2
Hi everyone,

I use the GC-FID with the StabilWax DA column (http://www.restek.com/chromatogram/view/GC_CH00284) for volatile fatty acid (VFA) quantification (C2 - C7 acids). For this, I use 2% formic acid to acidify the samples and standards before injecting them. This is to acidify them below the pka of the VFAs. I use the VFA standard mix (https://www.sigmaaldrich.com/catalog/pr ... &region=US) to prepare my standards and dilute them in 2% formic acid. I also dilute my samples in 2% formic acid.

However, recently, I did a small test and noticed that the area of the standards changes drastically when I use 2%, 5% and 10% formic acid for the same concentration (E.g., areas for 5 mM VFA with 2% formic acid is drastically different from the area of 5mM VFA with 5% formic acid).

In the beginning, I assumed that the formic acid concentration doesn't matter as long as the pH of the sample (or standard) is below the pKa value of the VFAs (Typically, the pH is around 3 after acidifying). But now, it looks like the formic acid concentration is affecting the area!

This is concerning to me mainly because if I inject samples with different dilution factors (say 10 vs 50), the amount of 2% formic acid I add to make the dilutions will be different... ergo, the formic acid concentrations will be different too. So, how can I justify that the areas I observe are because of the actual VFA concentration and not because of formic acid?

This comes back to the idea about if this procedure is right and if there is something critical I am missing. I am reaching out to this community for help and advice. Thank you!
Assuming that you are always at pH3, in which direction do your results go with increasing formic acid level to be "drastically different" ?

The more formic acid the lower or higher the C2-C7 vfa areas?

Do they all change to the same degree?

Have you analysed the formic acid itself to check for other vfa impurities?

You are correct in adjusting the pH of your sample to around 3 but, out of interest, why are you using formic acid rather than an inorganic acid?

Some more details of your sample, sample prep and the background to the analysis would be very helpful
Regards

Ralph
I addition to what Ralph wrote; the difference in formic acid content over the range of sample dilutions that you do is less than the range in your tests; because the 2% formic acid is the main constituent it changes from 90% to 98% in the dilutes samples, a relative change of only 0.09. Between 2% and 5% is a relative change of 2.5.

Consequently I doubt that you have a real problem, but you do now know that the results are vulnerable to formic acid concentration, and you can take the appropriate precautions.

Peter
Peter Apps
Thanks Ralph and Peter. Great points!

Ralph:
Although not consistent across all the acid species, in general, the areas increase with increase in formic acid conc.

That's a good point about contamination... I will go back and look into that as well.

We use formic acid since we know it cannot be detected in the GC-FID (thereby, avoiding a peak) and since we know it is similar to the other VFAs, that it will not adversely affect the column. Perhaps an inorganic acid can resolve this issue with the peaks? I can explore that option too.

My samples are from an anaerobic fermentation system. The are aqueous and I usually filter them in a 20u PTFE filter. I then dilute them appropriately with 2% formic acid and inject them into the GC-FID (with the procedure I provided the link to). There is no other form of sample preparation. My objective is to quantify C2 - C6 acids.

Peter:
I understand your point about the concentration difference. At this point, I'm wondering if I should adjust the formic acid such that the FINAL concentration is 2%. That's a more tedious way to prep the samples and standards but will, at least, provide consistency across the board.

Other suggestions and advice are most welcome! Thanks so much!
Rather than fiddle with final concentrations I would validate the robustness of the method to the differences in final concentration that will occur in normal use, plus a little bit for safety. By doing that you will retrospectively validate the results you have already generated also.

An advantage of using formic acid is that it is volatile and so deactivates the system with each injection.

Peter
Peter Apps
Ah I see. By robustness, do you mean the error range?

I guess I could try HCl but, as you said, I am not sure if it will leave residue in the column and if the column can take such a strong acid. Would you know anything about that?
Robustness is a test of how much you can vary the method and still get good results - the test you already did with the different percentages was a robustness test. Do the same again with a slightly wider range of formic concentrations than will occur in real samples, with enough replicates to get an estimate of standard deviation.

I use phosphoric as an inorganic acid because I know for absolute certain that it cannot give off acidic vapour that will eat my column. Stick with formic - don't fix what ain't broke.

Peter
Peter Apps
Since we now know that your samples are aqueous with aqueous injections then Peter has made a good point

I use phosphoric as an inorganic acid because I know for absolute certain that it cannot give off acidic vapour that will eat my column. Stick with formic - don't fix what ain't broke.


It may seem an obvious question but are you using a pH meter to adjust and control your pH to 3 rather than relying on calculations ?
Regards

Ralph
Ya, I was worried about fumes damaging the column.

I do not specifically adjust the pH to 3. I just make sure that the pH is below the pKa of all the acids. Typically, my samples have a pH between 2 - 3.
Apart from possible added vfas from your formic acid , which you still need to check, if you are not adjusting to exactly the same measured pH with a properly calibrated pH meter ( rather than using indicator strips? ) for your aqueous samples with added different levels of added formic acid then that may explain part of your problem.

pH is a logarithmic scale and not a linear one
Regards

Ralph
As Ralph reminded me that you are doing aqueous injections then formic acid is by far the best choice, all the inorganic acids will eat your column away or deposit in the inlet liner and mess everything up.

Peter
Peter Apps
Hi everyone!

I did a second test today with the following. This was more to confirm that I am preparing the standards correctly. The standards were:
2% formic acid with 0.5, 1, 2, 3 and 7 mM VFAs
5% formic acid with 0.5, 1, 2, 3 and 7 mM VFAs
10% formic acid with 0.5, 1, 2, 3 and 7 mM VFAs

The 0.5 mM was clearly outside the detection limit of the system. However, when I plotted the others, the strangest thing happened.

The 2% formic acid standards look ok (still not the best R2 value but at least they look fine). However, for the 5% and 10% formic acid standards, the calibration curve for acetic acid had a negative slope!! The other VFA calibration slopes are positive though. And, like before, the areas increase with formic acid concentration.

I have been told (although I do not know this myself), that formic acid elutes out after acetic acid on this column. I wonder if that has anything to do with this peculiar disparity. Or maybe this method is just not robust enough for acetic acid?
I have been told (although I do not know this myself), that formic acid elutes out after acetic acid on this column.


Why not try it for yourself and suggest ( yet again!) run your formic acid to find out if that is so on your wax phase and share the information. It would be faster for you to do compared to me looking up RIs for you.

See this link, page 5 , which gives you some idea of the relative response by FID of formic acid compared to higher acids

https://www.agilent.com/cs/library/appl ... 0845EN.pdf

I wonder if that has anything to do with this peculiar disparity.


I can't see how the relative retention time of formic acid would in itself have an effect providing that you get a good separation from higher vfas but others may wish to comment
Regards

Ralph
Formic elutes after acetic on a wax phase. With an MS you see the peak, but the response with an FID is very weak. You may be seeing the acetic peak being distorted by the higher concentrations of formic, making it difficult to integrate.

Peter
Peter Apps
Hello Peter and Ralph,

I ran the formic acid as you suggested and you are absolutely right. It seems that since the FID signal for formic acid is low, it is not apparent it but is distorting the acetic and propionic acid peaks at higher concentrations. So, it seems like keeping the formic acid concentration low (2%) is both helping the peak separation and shape (through reducing pH) while not heavily distorting the peaks.

The only follow up I have for this is... The Restek tech person said this column is designed for pH 5 - 9. Would it be a problem to run my samples at pH 2 -3?

It was also suggested to me that wax columns in general cannot take aqueous samples very well. I currently use a 1 uL injection with 50:1 split. But I am now testing out a 0.5 uL injection just to limit the water entering the column. Perhaps this will help with better peak separation as well. What to you think? I am testing it now and will have results tomorrow.
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