Short chain fatty acid analysis using Stabilwax-DA

[Nut1]

Dear All

I am presently setting up a GC method to analyse SCFA levels (acetic, propionic and butyric acid) in plasma/serum samples (which will be deproteinised prior to injection). I started off following a procedure that was previously set up in other labs, however I have already made a few changes to the method and still have a number of issues with it.

We are using a Stabilwax-DA column (30m x 0.53mm id, 0.5um df) from Restek, which is similar to the Agilent HP-FFAP columns, and does not require you to derivatise the acids. I have been running standards at the levels you would expect in plasma (1, 2, 3, 5, 8, 10, 20, 50uM propionic and butyric acids; 5, 10, 15, 20, 30, 50, 100, 200 uM acetic acid) made up with DD water. The standards include ~1% w/v formic acid (which was advised in the procedure we are using). Our internal standard is isovaleric acid.

While results for butyrate and acetate have been good, my problem is that the formic acid peak is running into the propionic acid peak, making accurate quantification difficult - a problem has not been solved by changing the oven T ramping.


So... my questions are:

1. Is the formic acid necessary, or could I use an alternate acid to acidify my samples? I had considered HCl, but having read other posts on this forum I'm not so sure. Any suggestions would be most welcome.

2. Would the Stabilwax-DA able to tolerate samples that had not been acidified (apart from the SCFAs present)? In other words could it be problematic/damage the column by not adding formic acid or any other acid at all?

Any other suggestions/comments would be most welcome, as I am very new to GC analysis!

Thank you
Julia

[chromatographer1]

I would not change the chemistry of your analysis without a lot of research confirming the equivalency of any changes.

The problem is with your chromatography. I would deal with it instead.

Try increasing your film thickness or try a different vendor and see if things improve. You could also just lengthen the column type you are presently using.

First step would be to try a 60m column (you can always cut it in half and have two usable columns, nothing wasted).

Then I would try a 1 micron film column of the same length.

Then I would try another vendor. (I work for one of the alternatives).

Good luck,

Rod

[Nut1]

Dear Rod

Thank you for your prompt reply. I do agree that it is better not to change the chemistry of the existing technique in an ideal world. However unfortunately as I am a PhD student with a very limited budget (especially as we have just paid out ~£500 for the current column!), and this method development is only a 'small' part of my PhD (most of my time/budget is spent carrying out human nutrition studies which are also expensive...) it is really not an option for me to buy another column until I have tried out the cheaper options of optimising the preparation method and using the column I have already got.

So with that in mind I was wondering if you or anyone else knows if it is necessary for formic acid to be run with SCFA-containing samples when using a Stabilwax-DA column, and if there are any thoughts / ideas on alternate acids to acidify my samples? It seems from the literature that some groups add the formic acid with the thought that it improves resolution and reduces the appearance of ghost peaks, and the method I was referring to initially used it for that reason.

I may even consider acidifying my samples (e.g. with HCl) and then extracting into a solvent (e.g. ether) after the deproteinisation step or have even considered looking into using SPME (something I know absolutely nothing about....

Thanks again,
Julia

[chromatographer1]

The reason for the addition of formic acid is to keep the other organic acids in an uniform molecular state (no anionic state) so a quanitative elution can be performed. The formic acid also occupies any active sites in the injection port or column.

If you can isolate the SCFA species in another solvent, a aprotonic solvent would be preferred. Also, try to eliminate the presence of any water.

You can of course try to derivatize the acids for quantification.

My preference is to do ion chromatography instead of GC.

I wish you well in your research.

Rod