Chromatography Forum

I'm running some protein samples for FAME analysis by GCFID. My methylation step for the extracted protein samples involves heating at 95°C for 2 hours with 1mL of 14% BF3 in MeOH, then extracting in 0.5mL hexane for GC analysis.

My protein samples are fine, but I also run a "system suitability" sample that consists of just 3 individual fatty acids, dried down and subjected to the same methylation step. With these samples I see a large hump at the beginning of the chromatogram (see below). I reran this sample with MS detection to identify, and it appears to be boric acid methyl ester. The only source of boron in my method is the boron trifluoride so it must be coming from there. So why does this happen? Has anyone seen this before?

We used BF3-CH3OH for making FAME the first 25 years I worked here, didn't see that. However, we only heated for 5 minutes not two hours. Now we use H2SO4-CH3OH.

I'm working from a published paper which uses the 2h treatment step to "attack sphingomyelins".

Since the system suit's contain no protein maybe I'll reduce the heating step for those and see if that solves it.

I was just wondering whether it was excessive heating or something else in the prep that I was doing wrong.

Well I had a brainwave and realised my system suitability samples didn't have an anti-oxidant added, whereas samples do, so I prepared new ones with anti-oxidant, and the big hump is still there.

I also wondered if the BF3-MeOH was bad, so I cracked open a new bottle and tried that, and the big hump is still there.

I'll have to go back to the drawing board, but I never noticed this during method development! Start with blanks and add analytes I guess. I might also try reducing the methylation time as suggested.

If anyone else has any suggestions as to how trimethyl borate forms from BF3 in Methanol, I'm all ears.....?

contaminant from borosilicate glass?

OK I may have cracked this:

There is a tiny little step missing in the preparation of the final samples compared to the R&D prep. For the R&D methylation boron trifluoride was added and heated, then 1mL saline added and vortexed, then 0.5mL hexane added, vortexed and centrifuged and the hexane injected. For the final work the underlined part is missing.

Would this really make a difference? Why does missing the saline step suddenly result in boric acid methyl ester in the hexane?