CHAPTER IX: ANALYSIS OF FATS AND OILS

Fats and oils are a heterogeneous group of predominantly hydrophobic compounds. The
distinction between fats and oils does not have a chemical basis. Those fats/oils that remain liquid
at normal (ambient) temperature are generally taken as oil and those that remain solid, fats.
Analysis of fats and oils is carried out for various reasons, viz.:

1. Shelf life study (how long the item will remain without deterioration in quality under a
given set of conditions)
2. Functional quality (e.g., suitability for use in biscuits, bakery, hydrogenation, etc.)
3. Sensory quality (e.g., rancidity)
4. Nutritional quality (e.g., melting point, polyunsaturated fatty acids)
5. As an aid in controlling production operation (e.g., control of hydrogenation, recovery of
oil in mills)
6. Conformance to regulatory standards (e.g., with respect to free fatty acids, saponification
value, peroxide value, moisture)
7. Detection of adulteration (e.g., contamination with mineral oil and argemone oil,
adulteration of dairy ghee with vegetable ghee)
8. Advanced research (e.g., determination of fatty acid profile)

Some of the routine tests carried out on fats and oils are as follows:

1. Acid value/Free fatty acid (FFA)

2. Saponification value, SV (also termed Saponification number)
3. Iodine value, IV (Also termed Iodine number)
4. Unsaponifiable matter
5. Refractive index
6. Melting point (for solid and semisolid items)
7. Moisture content
8. General tests for adulteration, such as Hexabromide test for the presence of linseed oil,
Halphen test for the presence of cottonseed oil, Baudouin test for the presence of vegetable
ghee in dairy ghee, Bellier turbidity test for the presence of ground nut oil, etc.

Some of the special tests used for particular fats and oils are:

1. Crismer test for rapeseed and mustard oil

2. Reichert-Meissl, Polenske and Kirschner values for dairy ghee
3. Polybromide test for linolenic oils such as linseed oil

Some of the important physicochemical characteristics of common fats and oils are as follows:
 FOOD ANALYSIS
Fat/oil Refractive Saponification value, Iodine value, Unsaponifiable
index at 40°C mg KOH/g oil Wij’s matter, %
Soybean oil 1.466-1.470 189-195 120-143 1.5
Mustard seed oil 1.461-1.469 170-184 92-125 1.5
Maize oil 1.465-1.468 187-195 103-128 2.8
Sunflower seed oil 1.467-1.469 188-194 110-143 1.5

9.1. DETERMINATION OF FREE FATTY ACIDS (FFA) AND ACID VALUE

BACKGROUND

For the most part, natural fats and oils are in the triglyceride form when freshly extracted from
the source. With prolonged storage, however, the triglycerides begin to break down giving rise to
free fatty acids (FFA). This hydrolysis is brought about by a variety of agents: presence of
moisture in the oil, elevated temperature and, most important of all, lipases (enzyme) coming
from the source or contaminating microorganisms. Consequently, the neutral oil becomes a
mixture of triglycerides, diglycerides, monoglycerides, free fatty acids and glycerol. Some
fats/oils are relatively stable but others, such as crude rice-bran oil, are notoriously susceptible to
hydrolysis. Whichever the oil, presence of excess free fatty acids is a sure indicator to unnatural
state of oil. The presence free fatty acid in large excess, though not a health hazard is undesirable
for several reasons; some of them are:

1. The oil is no longer the same as the virgin oil

2. The oil tends to smoke during deep-frying
3. The oil is susceptible to rancidity
4. The product prepared from such oil turns rancid very soon

Rancid oils markedly lower the esthetic value of oil. Such oils also bring about health problems.
In connection with the afore-mentioned points, regulating bodies have set mandatory standards
for edible oils, for example, in Nepal:

Mandatory standards of selected fats/oils

Fat/oil FFA (as %oleic acid)

Vanaspati ≤ 0.50
Refined oil ≤ 0.25
Mustard/Rapeseed oil ≤ 3.00

The operational definitions of Acid Value and FFA are:

Acid Value: Number of milligram of KOH needed to neutralize the FFA present in 1 g of oil
FFA : Percentage of free fatty acids present in oil. It is calculated using an assumed
average molecular weight of fatty acids, usually 282 (oleic acid)

PRINCIPLE

Free fatty acids are readily soluble in rectified spirit or absolute alcohol. A suitable amount of oil
is therefore mixed with neutralized rectified spirit to extract free fatty acids and the amount of the
105
 FOOD ANALYSIS
latter calculated by titrating with standard NaOH or KOH using phenolphthalein indicator. To
facilitate extraction, the mixture may be warmed to about 70°C and swirled vigorously.
Calculation for both acid value and FFA can be carried as follows:

REQUIREMENTS

• Neutral alcohol (95%, v/v)64 • Titration arrangement

• Phenolphthalein indicator (1%, alcoholic) • Weighing arrangement
• 0.1N NaOH • Hot plate

PROCEDURE

1. Weigh out 10 g of fat/oil in a 250-ml conical flask (by difference)

2. Add 50 ml of neutral alcohol
3. Add a drop or two of phenolphthalein indicator
4. Swirl the contents and place flask on the hot plate
5. Warm the mixture to about 70°C. Swirl well
6. Titrate warm with 0.1N NaOH to persistent pink color
7. In case of doubt, tilt the mixture to allow separation of alcohol and fat fractions. Observe
the color of the alcohol fraction for persistent pink color
8. Carry out titration in triplicate

CALCULATION

ml of alkali × N of alkali × 28.2

% FFA = (as oleic acid)
Wt. of sample (g)

ml of alkali × N of alkali × 56.1

Acid value =
Wt. of sample (g)

9.2. DETERMINATION OF SAPONIFICATION VALUE OF FAT/OIL

BACKGROUND

Saponification value of fat/oil is a very valuable test for the determination of adulteration. The
test is a rough measure of the average molecular weight of fatty acids in the oil and related thus:

561.0937 (100 − P )
M= − 12.683
Saponification value

Where M =Average molecular weight, P = percentage of unsaponifiable matter

Since the oil from a given source has a remarkably constant saponification value any deviation
found in the test is an indication to adulteration. Some of the common examples of edible oils and
their saponification values are:

64
Neutralize the acidity in the with 0.1N NaOH using phenolphthalein indicator
106
 FOOD ANALYSIS
Soybean oil: 189 – 195 Butter oil (ghee): 210 – 230
Rapeseed oil: 168 – 181

The test merits considerable attention in that successful testing is more of an art. There are at least
two titrimetric methods for the determination of saponification value. A relatively easy method
utilizes double indicator, viz., phenolphthalein and bromophenol blue.

When fat is boiled with an excess of alcoholic KOH, the glycerides irreversibly hydrolyze, giving
rise to glycerol and fatty soap (Fig. IX-1). The alkali consumed for this is a measure of
saponification value, and is defined as the number of milligram of KOH needed to saponify one
gram of oil or fat.

CH2OCO-R1 CH2OH R1-COOK

R2-COOCH + 3KOH HOCH + R2-COOK
CH2OCO-R3 CH2OH R3-COOK
Mixed triglyceride Glycerol Potassium soap

Fig. IX-1: Saponification of triglyceride

The hydrolysis is limited to glycerides, waxes and phosphatides. Sterols, hydrocarbons, pigments,
etc., although lipids, do not react with KOH under the above condition and they contribute to
what is known as unsaponifiable matter. A recapitulative presentation of the above-mentioned
points is given in Fig. IX-2.

PRINCIPLE

When the oil is saponified with a slight excess of alcoholic KOH, the reaction results in
potassium soaps, glycerol and unreacted KOH. The free KOH can be determined by titrating with
0.5N HCl using phenolphthalein as an indicator. The KOH in the form of soap is determined by
further titrating with 0.5N HCl using bromophenol blue indicator solution as the indicator.
Bromophenol blue changes from blue to permanent greenish yellow upon complete breakdown of
the soap. The amount of HCl consumed is back calculated to reflect the milligrams of KOH
consumed by one gram of fat/oil during the saponification.

CRUDE FAT
Fats KOH Potassium salts + Glycerol
KOH
Waxes Potassium salts + Alcohol
KOH
Phosphatides Potassium salts + Glycerol + K3PO4 + Amine
Non-fat Sterols
KOH
Hydrocarbon No reaction
Pigments (Unsaponifiable matter)

Fig. IX-2: Saponifiable- and unsaponifiable matter in oil

REQUIREMENTS

• Oil sample • Conical flask with condenser

• KOH pellets (pure) • Heating arrangement (hot plate or water bath)
107
 FOOD ANALYSIS
• Standard HCl (0.5N) • Titration arrangement
• Rectified spirit or absolute • Indicators: bromophenol blue (1%, alcoholic)
alcohol (aldehyde-free) and phenolphthalein (1%, alcoholic)

PROCEDURE

1. Melt the sample (if not already liquid) and filter warm. Ensure that the sample is free from
moisture and impurities
2. Weigh accurately by difference about 2 g of sample in a 250-ml conical flask
3. Add about 500 mg KOH pellet, a small amount of rectified spirit (≅ 10 ml), and emulsify
by swirling briefly
4. Add about 30 ml rectified spirit and gently reflux the whole until the oil becomes
transparent (this usually takes 25 min)
5. Add some rectified spirit (if the volume decreases) and continue refluxing till completely
saponified. The oil-alcohol mixture appears transparent at this stage
6. Slightly cool the flask and add a drop or two of phenolphthalein indicator. Intense red color
indicates the presence of excess KOH. If the color does not change, repeat the whole
process using more KOH (e.g. 600-800 mg)
7. Add a drop or two of distilled water. If a milky color develops, the sample contains
significant amounts of unsaponifiable matter or is contaminated with mineral oil
8. add more water (about 50ml) and mix well
9. Neutralize the excess KOH with 0.5N HCl. The pink color should just disappear
10. Add a drop of bromophenol blue indicator and swirl. It should give a blue color
11. Note the reading on the burette (containing the standard HCl) and titrate till a permanent
greenish-yellow color appears. If, during titration, fat-like globules suddenly appear, warm
the flask a little and continue titration to the end point
12. Note the volume of 0.5N HCl consumed (the second reading, that is) and calculate the
saponification value

CALCULATION

ml of HCl × N of HCl × 56.1

Saponification value =
Wt. of sample (g)

9.3. DETERMINATION OF IODINE VALUE OF FAT/OIL BY WIJ'S METHOD

BACKGROUND

Probably no analytical test method in all of oleochemistry has had the universal widespread use
as the measurement of unsaturation in fats and oils by iodine value determination. The first to use
this concept was Von Hubl in 1884. Since 1898 great many innovations have been made,
continuing until recently.

Iodine Value is the number of grams of iodine absorbed per 100 g of oil or fat, when determined
using Wij'’ solution. The test is a measure of unsaturation of a given fat or oil. Since the degree of
unsaturation is more or less characteristic to oil source, the test is routinely used for the
determination of adulteration by other types of oils. Iodine values of some common edible oils
are:

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 FOOD ANALYSIS
Soybean oil: 120-143
Rapeseed oil: 94-120
Butter oil: 26-38

The test is of tremendous value in vanaspati (hydrogenated oil) plants. It is routinely used for
monitoring the degree of hydrogenation. Iodine value is also used to calculate the amount of
hydrogen used or wasted in vanaspati plants. In general, a drop in I unit of iodine value means to
the vanaspati manufacturer that 0.075 kg of hydrogen has been added to every 1000 kg oil. There
are several methods for measuring the iodine value of fats and oils. Some of the variations and /or
equivalent methods are: Hanus method, Bromine Value method, Rosenmund-Kuhnhenn method,
etc. There are some difference vis-à-vis reagent preparation in Wij’s method also.

PRINCIPLE

Halogens add across the double bonds of unsaturated fatty acids to form addition compounds.
Iodine monochloride (ICl) is allowed to react with the fat in the dark. The amount of iodine
consumed is then determined by titrating the iodine released (after adding KI) with standard
thiosulfate and comparing with blank in which the fat is omitted. The reaction occurring in the
test can be shown in Fig. IX-3.

CH CH + ICl CH CH
Unsaturated Iodine I Cl
portion of fat monochloride Addition
compound

ICl + KI KCl + I2
Residual Added after Molecular
titration iodine

Na2S2O3 + I2 2NaI + 2Na2S4O6

Na-thiosulfate Na-tetrathionate

Fig. IX-3: Reaction scheme during iodine value determination

The reaction mixture is kept in dark and the titration carried out as quickly as possible since
halogens are oxidized in the light. The amount of Wij’s reagent used in this test should be more
(usually by 150%) than shown by the stoichiometry.

REQUIREMENTS

• Carbon tetrachloride • Soluble starch (1%)65

• Potassium iodide (10%, aqueous) • Iodine value flasks
• Standard Na-thiosulfate (0.1N)66 • Pipette: 25- and 5 ml (graduated)

65
Dissolve 1g of reagent grade starch in hot water. Transfer the clear fraction into another container. Use only
fresh solution, as it is subject to microbial degradation
66
Dissolve 25g AR grade Na2S2O3.7H2O in distilled water to make 1000ml. Mix the solution thoroughly, allow to
stand for a few days, and then siphon off the clear liquid. Standardize the solution with AR grade potassium
dichromate (K2Cr2O7). Weigh 0.20 to 0.23g of K2Cr2O7 (dried for 2 hrs at 105°C). Transfer to a 250-ml beaker
using 150ml of water. Add 2g of KI and mix. Add 20ml of 1N HCl, swirl, and allow to stand for 10 min. start
109
 FOOD ANALYSIS
• Wij’s solution 67
• Measuring cylinder, 25 ml
• Electronic balance (± 1mg sensitivity) • Burette: 50 ml, 2 sets
• Oil or fat sample

PROCEDURE

1. Weigh accurately by difference suitable quantity of oil using the formula: (20.3 ÷ expected
iodine value) grams, in to a clean, dry 250-ml IV flask (see Fig. IX-4)
2. Add 10 ml of CCl4 and allow oil to dissolve
3. Add accurately 20 ml of Wij’s solution. Swirl once and close the flask with the stopper.
The stopper may be moistened with minimum of 10% KI solution
4. Stand the flask at 15-20°C for 30 min in dark
5. Add 15 ml of 10% KI solution, followed by 100 ml distilled water
6. Titrate with 0.1N Na2S2O3 using starch indicator towards the end of the titration (The
mixture turns straw color near the end point. Add two drops of starch solution. The mixture
immediately turns dark blue. Continue the titration until the blue color just disappears)
7. Carry out a blank test upon the same quantities of reagents, omitting the oil, at the same
time and under the same conditions. The excess of reagent remaining for titration in the test
must be 150% of the reagent absorbed

CALCULATION

( Blank titer − Sample titer ) ml × N of Na 2S2 O3

Iodine value = × 12.69
Wt. of sample (g)

Stopper

Fig. IX-4: Wij’s IV flask

PRECAUTIONS

1. The Wij’s solution should be ≅ 0.2 N

2. The reagent should be used in excess (150% of the amount absorbed by fat/oil)
3. Use only freshly prepared starch solution

titrating with Na2S2O3 solution from the burette, adding about 80% of the required amount. Add 1ml of starch
indicator and complete the titration to a point where the solution changes from blue-green to light green.
Calculate the strength of Na-thiosulfate as follows:
Wt. of pot - dichromate × 1000
N of sod-thiosulfate =
ml of sod - thiosulfate × 49.037
Add a pinch of Na2CO3 and 1ml of chloroform to preserve it from microbial degradation
67
Dissolve 8g iodine trichloride in 150ml glacial acetic acid and mix with 9g iodine dissolved in 350ml glacial
acetic acid. The strength of Wij’s solution, as determined by titrating with Na-thiosulfate, should not be less than
0.2N. Store the reagent in a colored bottle in dark. The solution is stable for about 30 day.
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 FOOD ANALYSIS
Note: Wij’s solution can be prepared by other methods also, viz., (i) using iodine monochloride,
and (ii) using chlorine gas and resublimed iodine. The latter method is described here.

Preparation of Wij’s solution by chlorination

Before anything else, prepare standard sod-thiosulfate, conc sulfuric acid, 10% KI solution and
starch indicator. Assemble pipettes, burettes and other glassware needed for iodometric titration.

• Take 13g resublimed iodine in a 1-liter beaker

• Add 200ml glacial acetic acid and dissolve by gentle heating (along with stirring). Iodine
dissolves very slowly and the complete dissolution can be carried out in stages by using
small portions of glacial acetic acid
• Transfer the dissolved portion to 1-liter volumetric flask
• Add more glacial acetic acid (~ 200ml) to the undissolved iodine in the beaker and heat
gently (as previously done) to affect dissolution
• Transfer the dissolved portion to the volumetric flask (to pool the solution) again
• Carry out this operation until iodine is completely dissolved. However, do not exceed the
total volume of 1000ml. If some space is available, make up the volume to 1000ml by
glacial acetic acid. Mix the solution well. Take out about 25ml solution and set aside (as a
reserve) in a separate flask (you will need this later)
• Transfer the bulk iodine solution in a Woulfe bottle and assemble the parts as in Fig. IX-5
• Generate chlorine68 and pass through the iodine solution to form iodine monochloride
• Continue passing the chlorine until the characteristic color of free iodine is discharged
(solution suddenly lightens because of free chlorine)
• Stop passing chlorine and test the Wij’s solution for dismantle the assembly for
chlorination
• Add small amounts of iodine solution (reserved earlier) until the free chlorine has been
destroyed (the color again darkens). A slight excess of iodine does little or no harm but
excess chlorine must be avoided. Typically, the iodine/chlorine ratio should be 1.1± 0.1
and this can be ascertained by determining iodine content and total halogen content as
follows:

Iodine content:

o Take 150ml of Chlorine-saturated water in a 500-ml conical flask and add some
glass beads
o Add 5ml of Wij’s solution
o Mix, and heat to boiling for 10 min
o Cool and add 30ml of 2% H2SO4
o Add 15ml of 15% fresh KI solution
o Titrate with 0.1N sod-thiosulfate to starch end point
o Note the titer (say A)

68
Chlorine is generated in the laboratory by reacting KMnO4 and con. HCl:
2KMnO4 + 16HCl → 2KCl + 2MnCl2 + 8H2O + 5Cl2
The amounts of HCl and KMnO4 needed for chlorination are not very large. However some amounts of chlorine go
waste during chlorination. Besides, sufficient amounts of chlorine must be produced to force itself through the
solution. To take this into account, use about 5-10g of KMnO4 and 50-100ml of conc HCl.
111
 FOOD ANALYSIS
Total halogen content:

o Take 150ml of recently boiled, cooled water in a 500-ml conical flask

o Add 15ml of 15% KI solution
o Pipette 20ml Wij’s solution
o Titrate immediately with 0.1N sod-thiosulfate to starch end point
o Note the titer (say B)
o Iodine/Chlorine ratio as follows:

2A
I/Cl = , the Wij’s solution thus prepared should consume approximately
( 3B − 2 A )
double the amount of 0.1N sod-thiosulfate

Delivery tube Conc. HCl

Small vent to release

excess pressure

KMnO4
Iodine solution

Chlorine bubbles

Fig. IX-5: Preparation of Wij’s solution

9.4. DETERMINATION OF PEROXIDE VALUE

BACKGROUND

Peroxide value (PV) is a very sensitive indicator of the early stages of oxidative deterioration of
fats and oils. PV therefore provides a means of predicting the risk of the development of flavor
rancidity. There are numerous analytical procedures for the measurement of peroxide value. In all
cases the results and accuracy of the test depend on the experimental conditions, as the method is
highly empirical. The most common methods are those based on the iodometric titration
originally reported by Lea and Wheeler, which measure the iodine produced from potassium
iodide by the peroxides present in the oil. It has been contended that the two principal sources of
error in these methods are the absorption of iodine at unsaturated bonds in the fatty acids on the
one hand, and on the other, the liberation of iodine from potassium iodide by oxygen present in
the solution to be titrated. Other types of error which can arise include variation in weight of the
sample, the type and grade of solvent used, variation in the reaction conditions such as time and
temperature, and the constitution and reactivity of the peroxides present in the oil.

Other methods have been recommended for peroxide value determination and these include a
colorimetric method based on the oxidation of ferrous to ferric ion and the determination of the
latter as ferric thiocyanate; a variation in the iodometric method reported by Swoboda and Lea, in
which the liberated iodine is converted into a blue starch iodine complex; and the Sully method,
in which the mixture is boiled.

112
 FOOD ANALYSIS
Peroxide value of an oil or fat is the amount of peroxides present and expressed as milli-
equivalents of peroxide per 1,000g of sample.

PRINCIPLE

When a rancid fat or oil sample is treated with potassium iodide after dissolving in an appropriate
solvent, peroxides present in the fat liberate iodine. The test is a volumetric one where I2, formed
from KI in the presence of peroxide is determined by titrating with sodium thiosulfate and
conducting a blank determination.

Now, milliequivalent peroxide = milliequivalent thiosulfate at the equivalence point

Again, milliequivalent = (strength × volume), when volume is in milliliter

Therefore, PV = milliequivalent thiosulfate / kg sample

REQUIREMENTS

• Oil or fat sample • Iodine flasks: 250ml cap

• Acetic acid-chloroform solvent69 • Burette: 25-50ml cap
• Saturated potassium iodide70 • Pipette: 25ml cap
• 0.01N and 0.1N sod-thiosulfate (see • 0.5% starch indicator (see determination
determination of Iodine Value) of Iodine Value)
• Measuring cylinder: 25ml cap • Weighing arrangement

PROCEDURE

1. Weigh accurately (by difference) 5g of fat or oil sample in the Iodine flask
2. Add 25ml of solvent and displace the air with CO2
3. Add 1ml of KI solution, stopper the flask, and allow it to stand for 1min (with gentle
shaking)
4. Add 35ml of distilled water and a few drops of starch indicator. Appearance of blue color
on addition of starch indicates presence of free iodine
5. Titrate the liberated iodine with 0.01N or 0.1N sod-thiosulfate until the blue color just
vanishes
6. Carry out a blank determination simultaneously (omitting oil)
7. Calculate Peroxide value using following equation:

N × (VS - VB ) × 1000
PV (meq/kg) =
Wt. of sample (g)

Where,

N = normality of sod-thiosulfate, VS = sod-thiosulfate consumed by sample (ml), and VB =

sod-thiosulfate consumed by blank (ml).

69
2 volumes of acetic acid and 1 volume of chloroform
70
4 parts of pure KI in 3 parts of distilled water. Keep in brown bottle
113
 FOOD ANALYSIS
9.5. MELTING POINT OF FAT BY OPEN-TUBE CAPILLARY METHOD

BACKGROUND

Fats do not melt sharply because they contain different types of fatty acids with different melting
points. Melting point of fat increases with an increase in the degree of saturation and chain length
of fatty acid. Unsaturated bonds produce kinks in the fatty acid chain and therefore allow very
loose molecular packing. This facilitates faster slipping away of molecules, thereby resulting in
low melting point. Melting point also depends on isomeric forms and polymorphism in fatty
acids. Trans isomers of fatty acids (that usually form during hydrogenation process) have higher
melting point because the chains are less kinked.

Polymorphism in fats and oils refers to existence of more than one crystalline form of fatty acid
or glyceride. Three such polymorphic forms, viz., alpha (α), beta prime (β’) and beta (β) have
been identified. Polymorphism results from different patterns of molecular packing in fat crystals.
A comparison of the three polymorphs is given below:

Polymorph Crystal size Melting point Stability

Alpha (α) 5µm Lowest Least stable
Beta (β) 20-50µm Highest Most stable
Beta prime (β’) 1-2µm Intermediate Intermediate

The stability of fat is related to the polymorphic form and the associated melting point. The
melting points of α-, β’-, and β forms of tristearin are 55°C, 64°C, and 73°C, respectively.
Polymorphic transformations occur from α to β’ to β and are irreversible. When fat is cooled
rapidly the α polymorph is produced, which is usually quickly converted to the β’ form. These
polymorphic forms also affect the appearance and texture of fat. β’ form gives a smooth texture
whereas β form results in a very coarse granules. It is therefore very important to control the
balance of polymorphic forms in the production of fat and fatty foods like margarine (needs β’
form), ghee (needs β form), etc.

Because of the reasons described above, melting point as such is not very reliable for establishing
identity of the fat and oil. However, it is extensively used in controlling process operation (e.g.,
hydrogenation), quality control, and determining suitability of fat for a particular purpose. The
methods used for the determination of melting point vary considerably. A typical method used in
vanaspati manufacture is the open-tube capillary method. The melting point is therefore defined
by the specific conditions of the method by which it is determined.

PRINCIPLE

The temperature at which the oil or fat softens or becomes sufficiently fluid to slip or run as
determined by the open-tube capillary-slip method.

REQUIREMENTS

• Capillary tubes71 • Beaker or Thiele tube

71
Thin-walled with uniform bore capillary glass tubes opn at both ends with following dimensions:
Length = 50-60mm
114
 FOOD ANALYSIS
• Thermometer (0.2°C sensitivity) • Heat source (gas burner or spirit lamp)

PROCEDURE

1. Melt the sample and filter it through a filter paper to remove any impurities and last traces
of moisture
2. Make sure that the sample is absolutely dry
3. Mix the sample thoroughly
4. Introduce a capillary tube into the molten sample, so that a column of the sample, about
10mm long, is sucked into the tube
5. Chill the tube containing the sample immediately by touching the tube, against a piece of
ice until the fat solidifies
6. Place the tube in a small beaker and hold it for one hour either in a refrigerator or in water
maintained at 4-10°C

Thermometer

Capillary Rubber band

Heat source
Fat
Theile tube

Chilled water

Fig. IX-6: Arrangement for melting point determination of fat

7. Remove the tube and attach with a rubber band to the thermometer bulb, so that the lower
end of the capillary tube and the thermometer bulb are at the same level
8. Take water at 10°C in the Thiele tube and immerse the thermometer with the capillary tube
containing the sample of fat (see Fig. IX-6)
9. Gradually increase the temperature by heating at the side-tube of the Thiele tube at the rate
of 2°C pen min, till the temperature reaches 25°C, and thereafter at the rate of 0.5°C per
min
10. Note the temperature of the water when the sample column begins to rise in the capillary
tube
11. Report the average of two such separate determinations as the melting point, provided that
the readings do not differ by more than 0.5°C

Inside diameter = 0.8-1.1mm

Outside diameter1.2-1.5mm
115
 FOOD ANALYSIS
9.6. TESTS FOR THE ADULATERATION OF FATS AND OILS

Physicochemical properties of fats and oils are often used to identify them. Usually, more than
one property is measured so that the identification can be made with some assurance since natural
fats and oils vary somewhat in their properties. A few special tests are now available for the
unequivocal determination of adulteration in fats and oils. Some of these tests are described next.

9.6.1. DETERMINATION OF REICHERT-MEISSL, POLENSKE AND KIRSCHNER

VALUES

BACKGROUND

These tests are widely used for identification and test of adulteration of butter. The tests are based
on the quantitative measurement of low molecular weight fatty acids (C4-C14) that are
predominant in butter. Although the RM value varies for butter with season, nutrition, and time
in the lactational cycle of the cow, it is usually between 24 and 34, higher than other edible oils.

The definitions of the terms are:

Reichert-Meissl value: It is the number of milliliters of 0.1N NaOH required to neutralize the
steam-volatile, water-soluble fatty acids distilled from 5g sample of fat under precise conditions
specified in the method. This test measures the quantity of C4 and C6 fatty acids.

Polenske value: It is the number of milliliters of 0.1N NaOH required to neutralize the steam-
volatile, water-insoluble fatty acids distilled from 5g sample of fat under precise conditions
specified in the method. This test measures the quantity of C8 to C14 fatty acids.

Kirschner value: It is a measure of steam-volatile, water-soluble fatty acids forming water-

soluble silver salts (which is the property of butyric acid). In recent years, this analysis is not
usually done.

PRINCIPLE

Steam-volatile fatty acids can be collected by saponification and steam-distillation of oil. Reichert
Meissl value can be determined by titrating the steam condensate (that contains water-soluble
fatty acids) with0.1N NaOH. Polenske value can be determined by eluting the fatty acids
adhering on the condenser with neutral ethanol and titrating with 0.1N NaOH. Determination of
Kirschner value involves neutralization of the water-soluble fatty acids with barium hydroxide,
preparation of their silver salts, separation of the water-soluble butyric acid salt by filtration,
liberation of butyric acid by acidification, separation by steam distillation, and quantification by
titrating again with barium hydroxide.

REQUIREMENTS

• Fat sample • 90% neutral ethyl alcohol (v/v)

• Glycerol • 0.05 N barium hydroxide
• 50% NaOH • Finely powdered silver sulfate
• Pumice powder: 1.4-2.0mm in diameter • Titration arrangement

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 FOOD ANALYSIS
• Dilute H2SO4 (25ml H2SO4 + 1000ml H2O) • Weighing arrangement
• RM-Polenske-Kirschner apparatus72 • Phenolphthalein indicator
• 0.1N NaOH (not to be used, if Kirschner • Heating arrangement
value is to be determined)

PROCEDURE

1. Melt the fat sample if solid but do not heat above 50°C
2. Weigh 5±0.01g of fat sample into a Polenske flask
3. Add 20g of glycerol and 2ml of 50% NaOH solution from a burette which is protected
from CO2 pick up. Wet the tip of the burette before adding alkali to free it of carbonate
deposit and reject the first 0.5ml of NaOH
4. Heat the mixture over a low flame with wire gauze until the liquid becomes clear and the
fat has saponified. Do not overheat at this stage which causes discoloration
5. When all the fat has saponified, cover the flask with a watch glass, and allow to cool
6. Add 93ml of boiling distilled water which is free of CO2 and mix. The solution must be
completely clear at this stage and pale yellow in color. If the solution is not clear which
indicates incomplete saponification, or if it is darker which indicates overheating, repeat
the procedure with a fresh sample. An old sample of oil or fat may behave similarly
7. Add 0.1g of pumice powder and 50ml of dilute H2SO4
8. Connect to the distillation apparatus (Fig. IX-7)
9. Warm the mixture until any insoluble material which may be present melts
10. Increase the heat and distil 110ml of solution in 19 to 21 min. The distillation is considered
to begin when the first drop forms in the shill-head
11. Stop heating soon after 110ml has distilled over, and replace the graduated flask by a
measuring cylinder to collect drippings from the condenser
12. Close the graduated flask with the stopper. Do not mix
13. Place the flask in a water bath at 15°C for 10 min and ensure that the 110ml graduation is
below the water level
14. Mix and filter through a 9cm Whatman No. 4 paper. Reject the first 2-3ml of the filtrate
and collect the rest in a dry flask
15. Wash the condenser, still head and the 110ml graduated flask with three lots of 15ml
distilled water passing each washing through the measuring cylinder, 100ml flask and
stopper
16. Filter through the same filter paper ensuring that all insoluble matter is transferred to the
paper. Discard the filtrate. Do not mix with filtrate of the distillate got in the previous step.
The filtrate should be free from water insoluble fatty acids
17. Pipette out 100ml of the filtrate to a dry 250ml conical flask and titrate with 0.1N NaOH
using phenolphthalein as indicator
18. Calculate RM value as follows:

RM value = (Sample titer – Blank titer)ml × N of NaOH × 11

The factor 11 has been obtained as follows:

72
Apparatus consisting of flat-bottom boiling flask, still head (10.7cm wide and 18cm high), condenser (52cm long
with 30cm cooling length and 7cm entry tube) and a receiver (with graduations at 100ml and 110ml)
117
 FOOD ANALYSIS
Total volume collected (ml) 110
= = 11
Aliqout taken (ml)× Required N of NaOH (i.e.,0.1) 100× 0.1

19. Dissolve the insoluble fatty acids by three washings of the condenser, the measuring
cylinder, the 110ml flask with stopper and the filter paper containing the main bulk with 3
similar washings as before using 15ml portions of neutral ethanol
20. Collect the alcoholic washings (45ml) in a clean dry flask and titrate with 0.1N NaOH
using phenolphthalein indicator

110ml
100ml

Fig. IX-7: Reichert-Meissl ditillation apparatus (not to scale)

21. Carry our a blank determination similarly

22. Calculate Polenske value as follows:

Polenske value = (Sample titer – Blank titer)ml ×N of NaOH ×10

RM and Polenske values are affected by low barometric pressures which occur at high altitudes.
Under such conditions, correct the readings as follows:

(Observed RM -10) log 760

Corrected RM value = +10
log p

Observed value × (760 - 45)

Corrected Polenske value =
p - 45

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 FOOD ANALYSIS
Where, p = barometric pressure in mm of Hg at the place of determination

23. For Kirschner value, proceed as in RM value determination but replace 0.1N NaOH with
0.05N Ba(OH)2 for titration
24. After determination of RM value, add 0.5g of finely powdered silver sulfate to the solution
25. Keep in a dark place for 1hr with intermittent shaking
26. Filter through a dry Whatman No. 4 paper
27. Add 35ml of cold, CO2-free distilled water, 10ml of dilute H2SO4 and 0.1g of pumice
powder
28. Connect to the distillation apparatus, and distil 110ml in 19-21 min
29. Cool the distillate at 15°C for 10 min, mix and filter through 9cm Whatman No. 4 filter
paper as before
30. Titrate 100ml of the filtrate as in RM value using 0.05N barium hydroxide
31. Carry out a blank determination similarly
32. Calculate Kirschner value as follows:

⎡100 + ( Tr - Ta )121⎤⎦
Kirschner value = ( Tk - Tb ) ⎣
10,000

Where,

Tk and Tb = sample and blank titer respectively in Kirschner value determination

Tr and Tb = sample and blank titer respectively in the RM value determination

9.6.2. BOUDOUIN TEST

This test is useful in the detection of adulteration of dairy ghee with vanaspati ghee. The test is
based on the color reaction between sesamolin (a compound present in sesame oil) and furfural In
Nepal, use of sesame oil in vanaspati is mandatory. Dairy ghee containing sesamolin gives a
positive Baudouin test, thereby indicating the presence of vanaspati ghee.

PRINCIPLE

The development of pink color with furfural solution in the presence of hydrochloric acid
indicates the presence of sesame oil. The color is produced on account of reaction with sesamolin
present in sesame oil.

REQUIREMENTS

• Glass-stoppered test tube/measuring cylinder • Furfural solution73

• Con. Hydrochloric acid • Oil sample

PROCEDURE

1. Take 5ml of the oil or melted fat in a 25-ml measuring cylinder (or test tube) provided
with a glass stopper

73
2% furfural, freshly distilled in ethyl alcohol
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 FOOD ANALYSIS
2. Add 5ml of conc. hydrochloric acid
3. Add 0.4ml of furfural solution
4. Insert the glass stopper and shake vigorously for two minutes
5. Let is stand and allow the mixture to separate. The development of pink or red color in the
lower acid layer indicates presence of sesame oil
6. Confirm by adding 5ml of water and shaking again
7. If the color in acid layer persists, sesame oil is present, if the color disappears it is absent

9.6.3. HEXABROMIDE TEST

This test is of importance for detecting adulteration of edible oil with linseed oil (which is
inedible). The test is based on the formation of insoluble polybromides when unsaturated fatty
acids are brominated. Di- and tetrabromides that result from oleic and linoleic acids are soluble
and therefore do not interfere with this visual test.

PRINCIPLE

The formation of a precipitate of hexabromide when the oil in chloroform is treated with bromine
and then with alcohol and ether in cold condition indicates the presence of linseed oil.

REQUIREMENTS

• Boiling tubes • Ice water bath

• Chloroform • Ethyl alcohol
• Liquid bromine • Diethyl ether

PROCEDURE

1. Pipette 1ml of oil into a boiling tube (wide-mouthed, 100ml cap)

2. All 5ml chloroform and about 1ml of bromine drop-wise till the mixture becomes deep red
in color
3. Cool the test tube in an ice water-bath
4. Add about 1.5ml of rectified spirit drop-wise while shaking the mixture until the
precipitate which was first formed just dissolves
5. Add 10ml diethyl ether
6. Mix the contents and place the tube within the ice water-bath for 20 min
7. Appearance of precipitates indicates the presence of linseed oil

The sensitivity of this test is about 1% if linseed oil in other other oils. The test has some
limitations. It is not suitable for test in mahua oils. Besides, marine oils, which contain
polyunsaturated fatty acids, also give insoluble polybromide precipitate.

9.5.4. TEST FOR THE PRESENCE OF ANIMAL FAT BY MICROSCOPIC EXAMINATION

Animal body fats such as beef tallow and lard have been shown to contain trisaturated glycerides.
On crystallization these glycerides exhibit a characteristic crystalline appearance when viewed
under microscope. The procedure recommended by Williams Sutton for the microscopy of fat
crystals have been suitably modified and given.

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 FOOD ANALYSIS
REQUIREMENTS

• Fat sample • Test tubes

• Ice water-bath • Filtration unit
• Ethyl alcohol • Glycerin
• Microscope

PROCEDURE

1. Take about 2 g of melted fat samples in test tubes

2. Add 10 ml of diethyl ether and mix
3. Plug the tubes with cotton and allow to stand for 30 min in ice water or 24 hrs at 20ºC
(slow crystallization gives bigger crystals). In certain cases it is preferable to first
crystallize with a stronger solution of fat from a mixture of ether and ethyl alcohol (1:1). In
such cases separate the crystals by filtration and recrystallized in ether
4. Place the crystals on a drop of glycerin previously taken on a microscopic slide
5. Cover the crystals immediately with cover glass
6. Examine the crystals under ×160 and finally ×400 magnifications. The typical appearance
of beef tallow crystallized into characteristic fan like tufts, the ends of which are more or
less pointed can be seen. Lard crystals are chisel-shaped. Hydrogenated fats deposit
smaller size crystals. The size and shape of the crystals depend upon the strength of
solution, amount of fat taken and the time allowed for crystallization

9.6.5. TEST FOR PRESENCE OF ARGEMONE OIL

Argemone (Argemone maxicana L.), yellow poppy, is a wild herb, which grows in mustard field
and bears capsules full of brown black seeds. Because of its resemblance with black mustard, it is
often used as an adulterant. The oil is reported to cause glaucoma, dropsy and sometimes total
blindness due to the presence of alkaloids namely, sanguinarine and dihydrosanguinarine.

PRINCIPLE

The hydrochloric acid extract of the oil sample containing argemone oil when subjected to TLC
for separation of alkaloid gives fluorescent spot under UV light.

• Standard argemone oil extract • UV chamber (long wave, 366nm)

• Pear-shaped flask • Solvent mixture (moblile phase)
• Hot water bath • Diethyl ether
• Separating funnel, 50ml cap. • Conc. HCl, sp. grav. = 1.19
• Glass beaker, 10ml cap • Chloroform:Acetic acid:Water =
• Aqueous NaOH, 20% 70:20:10 (v/v)
• TLC plates coated with silica gel G or • Chloroform:Acetic acid (90:10, v/v)
precoated ready made plates cut to mixture
suitable size

121
 FOOD ANALYSIS
PROCEDURE

1. Take 10 ml sample in a separating funnel and dissolve in 15 ml Diethyl ether

2. Add 5 ml conc HCL and shake vigorously for 2 – 3 minutes. Allow to separate. Contents
of the separatory funnel may be heated cautiously over the vent of heating water bath for
some time for quick separation
3. Transfer the acid layer to a 25 ml beaker
4. Place the beaker into a boiling water bath and evaporate till dryness
5. Dissolve the residue obtained after evaporation of hydrochloric acid in 1 ml of a mixture
of chloroform and acetic acid (9:1)
6. Spot on TLC plate with the help of spotting capillary. Spot side by side standard
Argemone oil extract (0.1 % in ether)
7. Develop the plate in (a) Butanol:Acetic acid:water; or (b) Hexane:Acetone mixture
8. Allow the solvent front to move up a distance of 10 cm
9. Allow the plate to dry
10. Place the plate under UV light in the visualization chamber
11. Bright yellow or orange yellow fluorescent spots having Rf similar to the standard
argemone oil will confirm presence of argemone oil. The spot gives blue florescence under
UV-light if plate is sprayed with 20% aqueous sodium hydroxide solution

The method is very sensitive and can detect argemone oil upto 50 ppm level.

9.6.6. KRIES TEST FOR RANCIDITY IN FATS/OILS

Kries test is a very rapid test for the assessment of rancidity in fats and oils. It can be carried out
quantitatively as well as quantitatively. The qualitative method involves vigorous mixing of 5ml
of oil with 5ml of 0.1% phloroglucinol solution (in diethyl ether), adding 5ml of conc HCl and
observing for pink color as the evidence of incipient rancidity.

PROCEDURE FOR QUANTITATIVE METHOD

1. Shake 5ml of oil and 5 ml chloroform in a stoppered test tube

2. Add 10ml of a 30% solution of trichloroacetic acid (in glacial acetic acid)
3. Add 1ml of 1% solution of phloroglucinol (in glacial acetic acid)
4. Incubate the test tube at 45ºC for 15 min
5. Add 4ml of ethanol and immediately measure the absorbance at 545 nm.

Absorbance values below 0.15 indicate no rancidity. Absorbance values greater than 0.2 denote
incipient rancidity, and absorbance values around 1.0 show that the sample is highly rancid.

122