determination of fatty acids

[nina]

Hi
I want to determine the fatty acids in cod liver oil with GC/FID.
I esterifide these fatty acids with BF3/MeOH and another catalyst.
I dont have any standard except a internal standard, oleic acid, methyl oleate and menhaden fish oil.
how saponification the oil? is it essential?
is it correct that I compare these two method?
can I determine the DHA & EPA only with Is?
I know that this question is foolishly.
thanks

[chromatographer1]

It is important in the fatty acid analysis of fish oil that highly unsaturated fatty acids are not destroyed by the esterification process. This happens if an acidic procedure that requires temperature elevated reactions are used.

The Organic base transesterification of glycerides is less likely to damage the highly unsaturated components of fish oil than the BF3/Methanol transesterification.

This is done by adding TBAH (tetrabutylammoniumhydroxide) to an ether solution of the fish oil and allowing it to react at 37°C overnight in a nitrogen purged tightly sealed ampule.

Any free acids in the fish oil will not be esterified but these are not present in any large amount and can be separated before transesterification and esterified by an acidic procedure if this is deemed necessary.

[chromatographer1]

The tetra methyl ammonium hydroxide is in a methanolic solution and must be added to the ether solution of the fish oil for the transesterification reaction to take place.

Call or email Sigma Chemical Technical service for assistance if you do not have the procedure for this derivitization reaction.

[nina]

Dear chromatographer1
following the preview posts I esterified the fish oil:
100 mg of fish oil extracted with MeOH/CHCl3 ( can. j. Biochem.physiol, 37;911-917)then esterified with 1 ml BF3/MeOH at 90C for 15 min.
is it possible thatDHA or EPA arent esterified with this procedure?
I dont saponificate the oil before esterification, is it correct?
I hope state well my mind.
thanks

[chromatographer1]

I think 15 minutes will destroy a good part of the tri and tetra unsaturated fatty acids. I hope you purged your reaction vessel with nitrogen before reacting with the BF3/Methanol. I would heat no longer than 6 minutes, 8 on the outside. I routinely tested these fatty acids for a major chemical supplier for years for purity. Ijt was never required to derivitize simple fatty acids longer than 10 minutes.

[HW Mueller]

Chromatographer1,
do you have unequivocal evidence for normal (no electron withdrawing substituent on the double bond(s)) unsat. FA reacting with BF3/MeOH? When we did these we never saw anything happening. (I am talking about addition of MeOH, etc., not autoxidation, which prefers basic conditions, though)

[gcguy]

There is an interesting article in issue 18 of The Reporter(Europe) from Supelco about FAME analysis using fast GC. Not sure if the European version is online. I can scan it in and post it as a pdf if anyone is interested.

[chromatographer1]

http://www.sigmaaldrich.com/img/assets/ ... _GC_1M.pdf

[chromatographer1]

It was not my intent to imply that BF3/MeOH reacts with unsaturated Fatty acids other than to esterify them.

I do know that without doing a scrupulous purging of oxygen from reactants and the solvents involved a good deal of the fatty acids were consumed doing a typical derivitization that lasted more than 10 min.

I routinely derivitized Linolenic acid, Arachidonic acid, Docosahexaenoic acid etc and examined the TLC, HPLC, and GC analyses of these reactions and witnessed the amount of 'decomposition' products grow and the amount of esterified acid decrease.

A slightly yellowing of the solution is followed by a darkening of the solution if heating time is extended.

That is my experience over many years of lab work and I am glad to share it for what it is worth.

[HW Mueller]

OK, most likely O2.....