The term “unsaponifiable matter” in oils or fats refers to those substances that are not saponifiable by alkali hydroxides but are soluble in the ordinary fat solvents, and to products of saponification that are soluble in such solvents.
Transfer about 5.0 g of the oil or fat, accurately weighed, to a 250-mL conical flask, add 50 mL of an alcoholic potassium hydroxide solution prepared by dissolving 12 g of potassium hydroxide in 10 mL of water and diluting this solution with alcohol to 100 mL, and heat the flask on a steam bath under a suitable condenser to maintain reflux for 1 h, swirling frequently. Cool to a temperature below 25°, and transfer the contents of the flask to a separator with a polytetrafluoroethylene stopcock, rinsing the flask with two 50-mL portions of water that are added to the separator (do not use grease on stopcock). Extract with three 100-mL portions of ether, combining the ether extracts in another separator containing 40 mL of water. Gently rotate or shake the separator for a few minutes. [Note—Violent agitation may result in the formation of a difficult-to-separate emulsion.] Allow the mixture to separate, and discard the lower aqueous phase. Wash the ether extract with two additional 40-mL portions of water, and discard the lower aqueous phase. Wash the ether extract successively with a 40-mL portion of potassium hydroxide solution (3 in 100) and a 40-mL portion of water. Repeat this potassium hydroxide solution–water wash sequence three times. Wash the ether extract with 40-mL portions of water until the last washing is not reddened by the addition of 2 drops of phenolphthalein TS. Transfer the ether extract to a tared flask, and rinse the separator with 10 mL of ether, adding the rinsings to the flask. Evaporate the ether on a steam bath, and add 6 mL of acetone to the residue. Remove the acetone in a current of air, and dry the residue at 105° until successive weighings differ by NMT 1 mg. Calculate the percentage of unsaponifiable matter in the portion of oil or fat taken:
Result = 100 × (WR/WS)
= weight of the residue (g)
= weight of the oil or fat taken for the test (g)
Dissolve the residue in 20 mL of alcohol, previously neutralized to the phenolphthalein endpoint, add phenolphthalein TS, and titrate with 0.1 N alcoholic sodium hydroxide VS to the first appearance of a faint pink color that persists for NLT 30 s. If the volume of 0.1 N alcoholic sodium hydroxide required is greater than 0.2 mL, the separation of the layers was incomplete; the residue weighed cannot be considered “unsaponifiable matter,” and the test must be repeated.