Chromatography Forum

We use acetyl chloride in methnol to esterify/transesterify samples. The acetyl chloride/methanol solution is prepared freshly although it can be stable for 1 week or so.

BF3/methanol can not esterify free fatty acid.

Water content will deter reaction completeness in BF3/MEOH, while up to 10% water does not affect FAME formation in HCL/methanol. Actually, we do 1-step reaction without fatty acid extraction for most samples. For example, use 0.1ml milk in a 1ml reaction, then directly inject the upper layer into GC.

We use disposable screw glass culture tubes with Teflon lined caps, placed in a large beaker filled with water, heated on a hot plate. Either at 100C for 1 h or at 80C for 2 h.

There is this paper, http://www.jlr.org/content/51/3/635.full, talking about using concentrated HCl in methanol as a catalyst.

Please explain. Is this a typo?

"BF3/methanol can not esterify free fatty acid. "

thanks,

Rod

Sorry, I meant to say "base catalyst cannot esterify free acid acid, BF3/methanol can have incomplete reaction when water is present"

So we choose HCl+MeoH...

I agree.

HCl and Methanol has a 98+% esterification rate.

BF3 in Methanol has a 99.9+% esterification rate in 8 min reaction at 110C oven.

One has to avoid water with either or you will not get 100% conversion. One should also remove any oxygen from the reaction headspace or there will be a loss of PUFAs or if you heat longer than 10-12 minutes.

BF3 will transesterify from triglycerides in methanol readily, although you have to be careful about trans fatty acids or you can form the cis-isomer.

Rod

BF3 will transesterify from triglycerides in methanol readily

The issue there is getting the triglycerides into solution, into good contact with the BF3 and methanol. Often one sees little droplets of melted triglyceride at the bottom.

Your comment is correct.

I would add DCM or toluene to the reaction mixture.

That solved the problem

Rod

good method for trans-fats is FTIR spectroscopy, it is also approved by AOCS
no derivatisation, no 1 hr to wait

I just read an interesting paper suggesting I do as I was originally considering making a methanolic HCl solution from aqueous concentrated HCl.
http://www.jlr.org/content/51/3/635.ful ... 71c04a053f

Meanwhile I read a diatribe from AOCS against using BF3 methanol.
http://lipidlibrary.aocs.org/topics/archive/bf3.htm

The method in the first link seems really convenient though it is interesting a procedure that has been arround for so long still has so many conflicting reports.

BTW I assume the extracting solvent is not critical just some hydrocarbon solvent. I've seen hexane, pentane, toluene, benzene etc all suggested.

May I add, when transesterifying triglycerides, the use of a base catalyst is much preferred, as Dr. Christie notes the problem with isomerization of the cis/trans bonds.

I should also note, my generally satisfactory use of BF3 was due to the fast use of the reagent and the care of storage I took with the bulk, only removing a small portion of the reagent to ambient temperatures for a few hours at a time.

So there are many factors involved in choosing the better reagent for the task at hand. Choose wisely.

Rod

I just read an interesting paper suggesting I do as I was originally considering making a methanolic HCl solution from aqueous concentrated HCl.
http://www.jlr.org/content/51/3/635.ful ... 71c04a053f

Meanwhile I read a diatribe from AOCS against using BF3 methanol.
http://lipidlibrary.aocs.org/topics/archive/bf3.htm

The method in the first link seems really convenient though it is interesting a procedure that has been arround for so long still has so many conflicting reports.

BTW I assume the extracting solvent is not critical just some hydrocarbon solvent. I've seen hexane, pentane, toluene, benzene etc all suggested.

Useful links, thanks for posting.

Peter

BTW I assume the extracting solvent is not critical just some hydrocarbon solvent. I've seen hexane, pentane, toluene, benzene etc all suggested.

Probably not, I asked a similar question before on this forum. "Consumer Products Guy" said hexanes, petroleum ether could be used for extraction.

I was set to use hexanes but had solvent quality issues. Hexanes from a major brand showed many peaks after concentration, evenlthough the bottle says "also meets ACS specifications, for HPLC, pesticide residue analysis, GC and spectrophotometry". I even tested a brand new bottle by just leaving the solvent in open air and dry(to rule out N2 quality, tubing), it showed the same peaks.

For typical usage, probably it is not an issue. However, I was doing extraction and purification of FAME using silica gel column. So after all the procedure, I was basically drying ~15ml hexanes down to 0.2ml. This causes some problems in chromograms.

Because of this issue, I am using N-heptane HPLC grade for my extraction and silica gel separation. The only problem is it takes longer to dry. We don't care much about the short chain fatty acid that much, so using a difficult to evaporate solvent is not a big deal.

I can see toluene will take even longer to evaporate. Benzene is highly toxic, and I would avoid it. Pentane is probably a bit expensive. Personally I am thinking of diethyl ester although it is more flammable.

If you really want to dry it down fast use isopentane.

"Consumer Products Guy" said hexanes, petroleum ether could be used for extraction.

I was set to use hexanes but had solvent quality issues. Hexanes from a major brand showed many peaks after concentration, even though the bottle says "also meets ACS specifications, for HPLC, pesticide residue analysis, GC and spectrophotometry".

We don't concentrate, the samples we assay (soaps, fatty acids, triglycerides) are pretty high levels. So we just extract directly into the hydrocarbon solvent after esterification, and inject that directly.

We don't concentrate, the samples we assay (soaps, fatty acids, triglycerides) are pretty high levels. So we just extract directly into the hydrocarbon solvent after esterification, and inject that directly.

My lipid sources are mostly animal based. Cholesterol caused problems so I had to dry down the extracted FAME, then pass through a home-made silica gel column to remove cholesterol. Only then I found the problem. The extra peaks only happened to hexanes and a bottle of ACS grade isoproponal among all the solvents I used.

I was a little surprised that the hexanes have so many peaks as the label says " For pesticide residue analysis", which I believe including concentration steps.

Anyway, changing to heptane seems worked well for my situation.

Pesticide residue generally does NOT use a FID and the hexanes are almost invisible when doing that sort of analysis, like looking for water on a FID.

best wishes,

Rod