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
