Chromatography Forum

For AOAC method of Fatty Acid Analysis, Dairy products such as milk and
cheese, alkaline hydrolysis (NH4OH) is adopted for extraction of Free Fatty Acid, while acid hydrolysis (HCl) is used for other sample.

Do any body know why?

Thank you

Due to my product types, I don't have to use official methods for my fatty acid analyses. That said: if I have mono-, di-, or tri-glycerides, I saponify with either NaOH in CH3OH or KOH in CH3OH, then acidify with either BF3/CH3OH or H2SO4/CH3OH to esterify. I haven't found any business need to do as a one-step approach.

I don't know why, but my guesses would be:-

Dairy products often contain significant amounts of Short Chain ( C4 ) fatty acid, which is complete esterified by both base and acid catalysed transesterification, however it is virtually impossible to quantitatively extract the free acid from water. Base catalysts will not normally esterify the dominant longer chain ( C12-C24 ) free fatty acids in most oils.

So, if you want a quick method for oils with low levels of longer chain FFA, base catalysts are very convenient. If you want a method that captures FFA as well, then acid-catalyst is preferred - except where volatile fatty acids are present.

The above is from memory, but the reasons are quite complex, and I highly recommend referring to either W.W.Christie's " Lipid Analysis ", or his very informative www site.

Bruce Hamilton

Base catalysis of lipids involved here strictly does a saponification. Once the carboxylate is formed the reaction is at an end. With acid catalysis one gets an equilibrium between esters and carboxylate, so if you have a large excess of MeOH you get a large preponderance of methyl esters of the acids.

The AOAC method involves three parts,

1. Fatty Acid extraction from sample : reflux sample /w acid/alkaline at 80 C (8.3M HCl/NH4OH + 2ml ethanol) , then ether extraction

2. Transesterification (methylation) /w BF3/ethanol

3. GC analysis

It seems that in step 1. , only breaking down of sample matrix is the main target. Does "transesterification" or "saponification" already occur in step 1. ?

Step one is saponification ( aka as hydrolysis ) in that procedure, forming free fatty acids, nonsaponifiable materials and water-soluble saponification products, such as glycerol.

The comments I made about extraction of volatile short chain fatty acids from acidic aqueous solutions apply to saponification.

Free fatty acids are esterified and O-acyl lipids present will be transesterified by heating them with a large excess of anhydrous methanol and an acidic catalyst. If water is present, it may prevent the reaction going to completion. N-Acyl lipids are not ususally transesterified with the common acidiic reagents.

O-Acyl lipids are also transesterified very rapidly in anhydrous methanol with a basic catalyst. Free fatty acids are not normally
esterified, so water should be excluded from the reaction medium to prevent FFA formation from the hydrolysis of lipids.

Most analysts would avoid saponification when short chain length are involved, because volatile free fatty acid losses are very hard to control. If you have to use saponification, the losses of volatiles have to be carefully monitored.

I prefer transesterification according to Bannon, but from memory, Cristie prefers sodium methoxide in methanol for 5 minutes at ambient temperature, followed by acetic acid - as proposed by Christopherson and Glass, and modified by Christie for his work.

Christie was also less keen on saponification, especially when short chain acids were present and, if I recall correctly, rather scornful of standard methods that use saponification.

As I noted, W.W. Christie is the recognised expert, and his book is a must have for people routinely doing lipid analysis.

Bruce Hamilton

For most of odd balls I do
I saponify dilute with water acidify and ether extract.
Ether extract is evap to dryness in dry gas stream at room temp.
In same tube an excess of methanol is added and small amount of Toluene sulphonic acid catalyst is also added. Tube is closed and placed in 60 C water bath 12 hr.
At the end of this time sample is diluted with water extracted with small amount of ether and sample of this goes to GC.

Use of appropriate acid internal standard means single extraction and not multiple extractions are required for Quantitation.

I find T sulphonic acid is a much better catalyst than sulphuric acid as it gives much cleaner esterification particularly when esterifying diols and polyols in glacial acetic acid.

Bob M

As I noted, W.W. Christie is the recognised expert, and his book is a must have for people routinely doing lipid analysis.

OK, A quick look over the weekend weekend revealed that Christie has an article summarising preparation of FAME on his WWW site..

http://www.lipidlibrary.co.uk/topics/ester_93/index.htm

I especially refer interested people to scroll down to section I ( Special Cases ) and part 1 on short-chain fatty acids.

Edit - A further trawl around his www site has found that his 191 page 1989 book "Gas Chromatography and Lipids" is freely available on the site, and also as a free pdf ( 2.04MB ) from the publisher's ( Oily Press ) web site. Christie's site points to the publisher.

http://www.lipidlibrary.co.uk/gc_lip.html

Analysts working with lipids may already have the hard copy, but if not, you should download it. It complements the later editions of Lipid Analysis.

Bruce Hamilton

Bruce, ok there is the diffrence in "schools" that have crept in here. An old physical organic chemist like me does not consider a reaction as catalyzed when the so called catalyst is consumed in the reaction (like methoxide). But in "Bio", where a Michaelis-Menton Equation is regarded highly, it does not deter anybody from calling a reagent a catalyst. So I concede that I will not convince the "Bios" to call their catalyst a reagent instead.