Lipids and Preparation of Derivatives

TLC has found its principal application in the fractionation of complex lipids into compoxmd classes (see p. 374). Further subdivision of these lipid classes is not as a rule carried out, although good methods are available (see p. 394) on the contrary, an aliquot is hydrolysed and the composition of the fatty acids and other components determined gas chromatographically. Even the identification of a pure compound, e. g., a triglyceride, requires qualitative and quantitative analysis of both the lipophilic and hydrophilic hydrolysis products (fatty acids and 

Alkaline hydrolysis ( saponification ) of fatty materials yields the water-soluble alkali salts of the fatty acids in these lipids, plus non-saponifiable neutral compounds such as hydrocarbons and alcohols. The non-saponifiable matter is extracted from the alkaline aqueous-alcoholic phase with non-polar solvents. The fatty acids are then liberated by acidification and likewise extracted with organic solvents. 

Procedure 1 part potassium hydroxide is dissolved in 1 part by weight of distilled water and, after cooling, this concentrated solution is diluted with 3 to 4 parts of methanol. The lipid sample is mixed with about 10 times its amoimt of this potassium hydroxide solution and left standing overnight at room temperature. 

For monitoring the hydrolysis, 1— 5 jd of the reaction mixture is applied to silica gel G layers on narrow plates and developed with petrol ether (60—70° C)-diethyl ether-acetic acid (70 + 30 + 2). The lipid fractions are visualised by spraying the chromatogram with the chromic acid-sulphuric acid reagent (No. 46) and heating. At the beginning of saponification, spots of substances of low polarity are visible ahead of the fatty acid spot. After complete hydrolysis, only the spots of the acids, of the non-polar compounds (hydrocarbons) ahead of them and of the strongly polar compoimds (mono- and polyalcohols) behind them are visible. 

The aqueous solution of the potassium salts of the higher fatty acids which has been freed from neutral compounds, is acidified with 5N sulphuric acid under a layer of diethyl ether. The solution of fatty acids thus liberated is washed with distilled, air-free water and then dried over anhydrous sodium sulphate. The ether is evaporated off and the fatty acids sealed into a vessel in an atmosphere of nitrogen or dissolved in petroleum ether and stored in a refrigerator. 

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