Chromatography Forum

Hello everyone
I am trying to determine the fatty acids present in muscle.
I perform methylation so that FAMEs are formedby following the procedure from this paper.....Direct transesterification of all classes of lipids in
a one-step reaction by Lepage and Roy.

4:1 ratio of methanol: benzene is added to the samples along with acetyl chloride. Then the samples are incubated at 100C for 1 hr and neutralized with KOH. The benzene is separated and used for GC analysis.

My question is what is the role of acetyl chloride in this procedure?

Regards
Sankella

An acid catalyst is needed to esterify FA to Methyl esters in a timely fashion.

Some use H2SO4, others use HCl , and others use BF3, in methanol.

You are using acetyl chloride in methanol.

Methyl acetate and HCl are also formed in this reaction. These are separated by the extraction with aqueous KOH (KCl is also formed).

Rodney George

Unless I'm dealing only with soaps or fatty acids, I always saponify using either NaOH or KOH prepared in methanol. After saponification, I acidify with either BF3-methanol or H2SO4-methanol, heat 10 minutes, cool, add hexane and swirl, then add saturated sodium chloride and mix. I inject some of the hexane layer.

Thanks for the input guys

Hi Consumer Product Guys,

You mention about adding saturated NaCl at the end of the FAME preparation. What is the purpose of adding the saturated sodium chloride. Is this step very critical in the preparation?

Thank you

Hi Consumer Product Guys,

You mention about adding saturated NaCl at the end of the FAME preparation. What is the purpose of adding the saturated sodium chloride. Is this step very critical in the preparation?

Thank you

Addition of saturated NaCl makes the methanol-water lower layer very ionic, helps force smaller fatty acid esters into the hexane layer, keeps most of the methanol in the aqueous layer, and speeds the separation of the hexane from the methanol-water layer. The procedure is from AOAC and publications of L. Metcalfe.