Doc.

No : AIL/COR/QC / OIL-01
Rev. No : 00
SOP for Unsaponificable value
Date : 23.12.2020
Page |1

Principle:

Unsaponifiable matter is defined as the substances soluble in the oil, which after saponification
are insoluble in water but soluble in the solvent used for the determination. It includes lipids of natural
origin such as sterols, higher aliphatic alcohols, pigments, vitamins, and hydrocarbons as well as any
foreign organic matter non-volatile at 100°C e.g. (mineral oil). Light Petroleum or diethyl ether is used as
a solvent but in most cases results will differ according to the solvent selected and generally the use of
diethyl ether will give a higher result.

Apparatus:

a) Flat bottom flask or conical flask with a ground glass joint, 250 mL capacity

b) Air condenser 1 meter long to fit the flask

c) Separating funnel, 500 mL capacity

d) Weighing balance

The weighing balance should be accurately calibrated to measure 10 mg of sample on a tare

weigh of 100 gm.

Reagents:

a. Alcoholic potassium hydroxide solution: Dissolve 7 to 8 gm of potassium hydroxide in an equal

quantity of distilled water and add sufficient aldehyde free ethyl alcohol and make up to 100 mL.

b. Ethyl alcohol: Ninety-five percent

c. Phenolphthalein indicator solution: Dissolve one gram of phenolphthalein in 100 mL of ethyl

alcohol. d. Petroleum ether (40 - 60°C): Analytical reagent grade

e. Aqueous alcohol: 10% of ethyl alcohol in water

f. Standard sodium hydroxide solution: Approximately 0.02N g. Acetone: Analytical reagent

grade

h. Anhydrous sodium sulphate

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 Doc. No : AIL/COR/QC / OIL-01
Rev. No : 00
SOP for Unsaponificable value
Date : 23.12.2020
Page |2

Procedure:

 Weigh accurately 5 gm of well mixed oil/fat sample into a 250 mL conical flask

 Add 50 mL of alcoholic potassium hydroxide solution. Boil the content under reflux air
condenser for one hour or until the saponification is complete (complete saponification
gives a homogeneous and transparent medium).

 Take care to avoid loss of ethyl alcohol during the saponification. Wash the condenser
with about 10 mL of ethyl alcohol.

 Transfer the saponified mixture while still warm to a separating funnel, wash the
saponification flask first with some ethyl alcohol and then with cold water, using a total
of 50 mL of water to rinse the flask. Cool to 20 to 25°C

 Add to the flask 50 mL of petroleum ether, shake vigorously, and allow the layers to
separate.

 Transfers the lower soap layer into another separating funnel and repeat the ether
extraction 3 times, using 50 mL portions of petroleum ether.

 Some oils high in unsaponifiable matter, e.g., marine oils, may require more than three
extractions to completely remove Unsaponifiable matter

 Wash the combined ether extract three times with 25 mL portions of aqueous alcohol
followed by washing with 25 mL portions of distilled water to ensure ether extract is
free of alkali (washing are no longer alkaline to phenolphthalein).

 Transfer washed ether extract to 250 mL beaker, rinse separator with ether, and add
rinsing to main solution. Evaporate to about 5mL and transfer quantitatively using
several portions of ether to a previously dried and weighed 50 mL Erlenmeyer flask.
Evaporate ether.

 When all ether has been removed add 2-3 mL acetone and while heating on steam or
water bath completely remove solvent under a gentle air. To remove last traces of
ether, dry at 100°C for 30 minutes till constant weight.

 Dissolve residue in 50 mL of warm ethanol, which has been neutralised to a

phenolphthalein end point. Titrate with 0.02N Sodium hydroxide.

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 Doc. No : AIL/COR/QC / OIL-01
Rev. No : 00
SOP for Unsaponificable value
Date : 23.12.2020
Page |3

Calculation:

Weight in g of the free fatty acids in the extract as oleic acid = 0.282 V N

Where,

V = Volume in mL of standard sodium hydroxide solution

N = Normality of standard sodium hydroxide solution

Unsaponifiable matter = 100 (A-B) /W

Where,

A = Weight of the residue in gm

B = Weight of free fatty acids in the extract in gm

W = Weight of the sample in gm

(Ref: - ISI Handbook of Food Analysis (Part XIII)-1984, page 67 /AOAC 17th edn, 2000, Official method
933.08, Residue (unsaponifiable) of oils and fats)

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