Hi,

if you plan do analyze total FA composition of your sample (including free and bound FA) you can do an acidic transesterification which ends up in fatty acid methylesters of all FAs.If you want only free FAs u can either isolate them by TLC or use a derivatization which does not hydrolyze bound FAs e.g. silylation.

Saponification describes the procedure of an alkaline hydrolysis, usally of TGs which yields the sodium salt of the bound FAs -- as "soap".

Good luck

Thanks Thomas. I understand. I will do acidic transesterification.

But could you explain me why we need saponification or alkaline hydrolysis in FA analysis. Is this for any specific compound?

Hi, I am not sure which protocol u r using, we never do alkaline hydrolysis prior to analysis.

We only use the "saponification" to generate the water-soluble sodium salts of FAs e.g. for cell culture.

Fatty acids are found generally in cell as triacylglycerol and phospholipid. For fatty acid analysis, fatty acids must be free.For this purpose, we can neede saponification

That is absolutely correct. For a complete FA analysis you need hydrolysis of bound FAs. However you can do it acidic or alkaline.

Maybe me answer was unclear. We never use alkaline hydrolysis (saponification) for full FA analysis because we do acidic transmethylation. Sorry for that.

 Thanks for the replies. It means that if I do acid esterification then saponification is unnecessary.

Yes, when u do an acid-catalyzed reaction e.g. with BF3 you do not need prior saponification.

Check this link: http://www.cyberlipid.org/fattyt/fatt0001.htm#1

There you can find a comparison of commonly used methods as well as the according descriptions.

Good luck

Hello Syed,

You can still do the saponifcation in non aqueous  solvent such as methanol with sodium or potassium hydroxide. The purpose of saponification to separate fatty material from non fatty materials such as sterols, sugars , proteins  etc. The saponified  matter (soap)  can be esterified using acid catalysis such as 0.5-1% sulphuric acid in methanol or lewis acids such as BF3/methanol or BCl3/Methanol. The most commonly used are BF3/Methanol solution. The alkaline saponifcation is facile and quick compared to acid hydrolysis followed by transesterification. 

Transesertifcation is also recommended  that be can be applied directly with triglycerides followed by extraction with suitable solvent such as hexanes/ heptanes 

All the answers are right ...it depends what you want to do. (and what your analytical method is). Eichmann's procedure works... but if it was my GLC ... (and I didn't want to crap up the column) I'd follow a normal BF3/MeOH transesterification protocol, then do a cleanup by partitioning the resultant solution between 25% aq MeOH and hexane and filter the hexane solution (after concentration) before injection into GC. 

Robert is absolutely right. Our procedure also includes a final double-extraction of methylesters into hexane to get rid of impurities. 

    You can use acid-methanol or boron trifluoride-methanol successful transesterification (instead of two-stage saponification + methylation method)  operating ONLY with triglycerides, phospholipids and esters of FA and primary alcohols: these compounds are transesterified in relatively mild conditions  (75-85C, 1-2 h). But you cannot use it in order to obtain Me-esters of FA starting from cholesterol esters of FA, esters of secondary, cyclic, sterically hindered alcohols as well as phenols -  it is well documented that  transesterification of such compounds requires several hours of heating in sealed ampule (110-140C). Such procedure seems to be extremely inconvenient and displays certain  symptoms of degradation and isomerization of  several polyunsaturated acids such as 22:6 or 20:4. Nevertheless it is possible to use such mehod sometimes  to avoid the application of strongly basic  reagents  such as sodium hydroxide or methoxide . But two stage saponification-methylation method is usually  thought to be preferable in most cases  for it is extremely mild.  Complete saponification  of ALL type esters   is obtained after 3-5 minutes (64-70C) in NaOH-methanol-toluene mixture, complete conversion of resulting free FAs into their Me-esters also requires 5-10 minutes (70-75C). I prefer such procedure being analyzing serum lipids FA composition as well as composition of sertain cell membranes (significant portions of cholesterol esters are present) although high temperature transesterification gives compatible results. Remember that it is quite difficult to seal an ampule filled with methanol solution - such manipulation needs definite experience of investigator!  

Thomas and I are 'singing from the same hymn sheet' best wishes RLB

You can saponify and make methyl esters in one go, does that help?

   It would be nice to saponify lipid and make Me-esters in one step.  ALMOUST all lipid types (such as glycerides, phospholipids and cholesteryl  esters) are smoothly transesterified with the use of methanolic 1M sodium methoxide - toluene 2:1 mixture, 70C, 10-15 min. But there is an exeption:  free fatty acids are not alkylated absolutely in such conditions!  Even traces of esters corresponding to free FA (present in the sample) cannot be observed in chromatogram. But this subcluss of lipids is extremely significant  - for example, human blood serum  lipids contain 10-15% of free FA,  and concentration of free FA in some fungal lipids may be close to 40%! Certainly one step transesterification using NaOMe in MeOH may be used successfully during the analysis of food oils containing no free FA. According to my own experience the use of sodium methoxide is preferable (a portion of  non-alkylated free FA is usually produced  upon transesterification by means of sodium hydroxide  methanol solution). 

Alexander is quite right that methoxide treatment doesn't always work.  The chemistry of the problem is well known: if the fatty acid carboxylate anion is formed (i.e. a fatty acid that has been de protonated), then it can't be attacked by a nucleophile like methoxide.  methoxide is a strong base and so will abstract the proton on the fatty acid, producing the caboxylate anion.  This not only reduces the amount of fatty acid that is esterified, it also reduces the amount of methoxide to do it.  This is why approaches using acid are good, however both acid and base may be a problem around certain fatty acids, such as those comprising cyclopropyl groups (though I haven't tested this myself).