8. PROCEDURE
8.1 Place 2 g dried sample (W1) in a 250 mL Erlenmeyer flask or extraction
tube, add 2 mL alcohol. Stir to moisten all particles (moistening of
sample with alcohol prevents lumping on addition of acid).
8.2 Add 10 mL of the diluted 4N HCl and mix well. Set the flask on the
heater and reflux for 30 min. If the tube is used, place the tube in water
bath held at 70–80C and stir at frequent intervals until sample is
completely hydrolysed (usually 30–40 min).
8.3 Add 10 mL alcohol and cool.
8.4 If the hydrolysis has taken place in a flask, transfer the digested mixture
to extraction glassware. Rinse the flask and pour into the extraction
tube with 25 mL diethyl ether in three portions.
8.5 Close the tube with cork and shake vigorously for 1 min. Add 25 mL
petroleum ether and again shake vigorously for 1 min.
8.6 Let stand until upper liquid is practically clear.
8.7 Transfer as much as possible of the ether-fat solution into a preweighed
125 mL flask by filtering it through a funnel containing a plug of
cotton packed firmly in the stem part. Allow free passage of ether into
the flask.
8.8 Before weighing the flask, dry it in drying oven at 1005C and then let
cool in a desiccator and weigh (W2).
8.9 Repeat extraction of the liquid sample remaining in tube twice using the
same solvent. Each time, transfer the clear ether solutions through the
same funnel into the same flask. When finished, rinse inside and
outside of the funnel into the same flask.
8.10 Evaporate solvents completely on a water bath at 70-80C
8.11 Dry fat in an oven at 100+5C until constant weight is obtained.
8.12 Allow the flask to cool in a desiccator and weigh (W3).