2.2.3 Determination of Unsaponifiable Matter
Determination of unsaponifiable matter is a rigorous experiment that required a level of accuracy. Experimental procedure according to Lester (1928) was adopted, and this is in conjunction with the ACOS official method Ca 6a-40 and Ca 6b-53.
Dissolved PFAD weighed at 20g was emptied into a round-bottom flask of 300ml. 40ml of ethanol and 10ml of 40% aqueous sodium hydroxide were added to the content of the flask and heated under reflux condenser for one hour. The mixture was rinsed into a litre separating funnel with 150ml of water. It was allowed to cool and later shaken vigorously with 300ml of laboratory grade ether as the separator was closed with a glass stopper. The separation was completed in few minutes. Soap solution formed was extracted twice with 250ml of ether, and washed the combined extracts with 20ml of water. The ethereal solution was transferred into a distilling flask and most of the ether was removed. Several other extractions of soap solution (a sixth time) were performed with 250ml portions of ether, washed and combined with the wash water previously used. The resultant mixture was transferred to the same distilling flask as before, and distilled to small bulk, and the extract was carefully rinsed into a 250ml separating funnel with 100ml of ether. 75ml of ether was poured into another separating funnel for the purpose of re-extraction of all the wash-liquors from the separator. This helped to prevent the loss of any unsaponified matter which dissolved in the soapy washings and of any portions of the upper layer which may have been emulsified in or accidentally run off with the lower layer. The extract was washed again with 5ml of wash liquor, using first water (twice), then 2N sodium hydroxide in 10% ethanol followed by two water washes. This extract procedure was repeated twice more. Finally, the resulting mixture was washed with 0.5N hydrochloric acid to decompose any remaining soap, including traces of metal (calcium, magnesium, etc.) soap, and to remove the basic impurities often present in the PFAD. About 100ml of water was used to wash out the ethanol from the ether layer. As per precautionary method, any emulsion which appeared at the interface during washing was treated as part of the upper layer, hence it need hardly be mentioned that no lubricant of greasy nature is allowed on the separator taps. The ethereal extract was poured into a suitable weighing flask. Again, the first separator was rinsed with ether from the second. The mixture was distilled to remove water globules that were present, by evaporation of ethanol, and taken to read-off constant weight in a vacuum hot-plate desiccator. About 10ml of ethanol was added, neutralized to phenolphthalein end point, warmed to dissolve the unsaponifiable matter, and titrated with 1N sodium hydroxide, and phenolphthalein was used as indicator.
\begin{equation} Unsaponifiable\ Matter=\ \frac{R-\ \left(B+F\right)\ .\ 100}{W}\nonumber \\ \end{equation}
where R is the weight of residue (g), F is the weight of fatty acid (g), B is the weight of blank (g), and W is the weight of sample (g).