FAT ANALYSIS - SOXHLET METHOD

PRINCIPLE

Fat is extracted, semicontinuously, with an organic solvent. Solvent is heated and volatilized, then is
condensed above the sample. Solvent drips onto the sample and soaks it to extract the fat. At 15–20
min intervals, the solvent is siphoned to the heating flask, to start the process again. Fat content is
measured by weight loss of sample or weight of fat removed.

Chemicals
Petroleum ether

Hazards and Precautions

Petroleum ether and ethyl ether are fire hazards; avoid open flames, breathing vapors, and contact with
skin. Ether is extremely flammable, is hygroscopic, and may form explosive peroxides. Otherwise, adhere
to normal laboratory safety procedures. Wear gloves and safety glasses at all times.

Supplies
3 Aluminum weighing pans; Beaker, 250 ml; Cellulose extraction thimbles; Glass boiling beads; Filter
paper; Graduated cylinder, 500 ml; Mortar and pestle; Plastic gloves; Snack foods (need to be fairly dry
and able to be ground with a mortar and pestle); Tape (to label beaker); Tongs.

Equipment
Analytical balance; Oven; Soxhlet extractor, with glassware

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 PROCEDURE
1. Record the fat content of your snack food
product as reported on the package label. Also
record serving size so you can calculate g fat/100
g product.
2. Slightly grind ~30 g sample with mortar and
pestle (excessive grinding will lead to greater loss
of fat in mortar).
Soxhlet extraction unit. Sample in thimble will be
placed in the beaker after extraction and before
drying.
5. Remove thimble from the Soxhlet extractor
using tongs, dry in an oven until gain constant
weight. Cool dried sample then reweigh.
6. Correct for moisture content of product as
follows:

3. Weigh on an analytical balance ~2–3 g of
sample (to the nearest 0.001 g) in a filter paper.
Pack and place in a thimble. Reweigh.
4. Place the sample in a Soxhlet extractor. Put
~350 ml petroleum ether in the flask, add several
glass boiling beads, and extract for 6 h or longer.
Place a 250-ml beaker below your sample on the
(a) Using the remainder of the ground sample and
three dried, labeled, and weighed aluminum
sample pans, prepare triplicate 2–3 g samples for
moisture analysis.
(b) Dry sample at 70°C.
(c) Reweigh after drying, and calculate moisture
content of the sample.

Observation table
Using the weights recorded in the tables below, calculate the percent fat (wt/wt) on a wet weight basis
as determined by the Soxhlet extraction method. If the fat content of the food you analyzed was given
on the label, report this theoretical value.
Name of Snack Food:
Label g fat/serving:
Label serving size (g):
Label g fat/100 g product:

Data from Soxhlet extraction

Wet sample + Dry sample +
Thimble + filter Thimble + filter % (Fat +
Rep Wet sample (g) paper (g) paper (g) Moisture)

Data from moisture analysis

Pan + Wet Pan + Dried
Rep Pan (g) sample (g) sample (g) % Moisture

Question
If the fat content measured here differed from that reported on the nutrition label, how might this be
explained?
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 FAT ANALYSIS - PEROXIDE VALUE METHOD

AIM: Determine the peroxide value of fats and oils, as an indicator of oxidative rancidity.

PRINCIPLE

Peroxide value is defined as the milliequivalents of peroxide per kilogram of fat, as determined in a
titration procedure to measure the amount of peroxide or hydroperoxide groups. To a known amount
of fat or oil, excess potassium iodide is added, which reacts with the peroxides in the sample. The
iodine liberated is titrated with standardized sodium thiosulfate using a starch indicator. The
calculated amount of potassium iodide required to react with the peroxide present is used to
determine the peroxide value.

Reagents
Acetic acid-chloroform solution. Mix three volumes of concentrated acetic acid with two volumes of
chloroform.
Potassium iodide solution, saturated
Sodium thiosulfate, 0.1 N, standard solution
Starch indicator solution, 1%

Hazards and Precautions

Use hydrochloric acid in a fume hood. Otherwise, adhere to normal laboratory safety procedures.
Wear gloves and safety glasses at all times.

Supplies
Beaker, 250 ml (to melt fat); Buret, 25 ml or 50 ml; 4 Erlenmeyer flasks, 250 ml; glass stoppered; 2
Graduated cylinders, 50 ml; Pipette; Fat and/or oil samples

Equipment
Analytical balance; Hot plate

Procedure 5. Slowly titrate samples with 0.1 N sodium

1. Melt any samples that are solid at room
temperature by heating to a maximum of 15°C
above the melting point.
2. Accurately weigh ca. 5 g fat or oil (to the
nearest 0.01 g) into each of two 250-ml glass-
stoppered Erlenmeyer flasks.
3. Add 30 ml acetic acid-chloroform solution
and swirl to dissolve.
4. Add 0.5 ml saturated KI solution. Let stand
with occasional shaking for 1 min. Add 30 ml dd
water.
thiosulfate solution, with vigorous shaking until
yellow color is almost gone.
6. Add ca. 0.5 ml 1% starch solution, and
continue titration, shaking vigorously to release
all iodine from chloroform layer, until blue color
just disappears. Record the volume of titrant
used.
7. Prepare (omitting only the oil) and titrate a
blank sample. Record the volume of titrant used.

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Data and Calculations

Rep Weight (g) Titrant volume (ml) Peroxide value

Calculate the peroxide value of each sample as follows:

(S − B) x N
Peroxide value = x1000
W

where:
Peroxide value = mEq peroxide per kg of sample
S = volume of titrant (ml) for sample
B = volume of titrant (ml) for blank
N = normality of Na2S2O3 solution (mEq/ml)
1000 = conversion of units (g/kg)
W = sample mass (g)

Question
1. The peroxide value method was developed for fat or oil samples. What must be done to a food
sample before measuring its peroxide value using this method?

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Protein Analysis: Folin – Lowry Method

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Protein Analysis: Bradford Method

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 REDUCING SUGAR ANALYSIS – DNSA METHOD

SAMPLE PREPARATION
− Thoroughly remove all foreign mater and rapidly grind or chop material into fine
pieces.
− Place 5 g sample (to the nearest 0.01 g) in 100 mL beaker. Add 50 ml 80% alcohol by
volume, mix thoroughly. Heat nearly to boiling point on water bath 30 minutes,
stirring frequently.
− Cool and filter extraction (through filter paper to remove impurities) into a
Erlenmeyer flask. Rinse beaker throughly with water.
− Boil the flask in water bath until there is no alcohol residue.
− Transfer to 50 ml volumetric flask and rinse Erlenmeyer thoroughly with water,
adding rinsing to flask. This is your unknown sample.

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