MEASUREMENT OF OIL AND FATS

1. SEMINAR ON
ADULTERATION IN FATS AND OILS
MEASUREMENT OF SPOILAGE OF FATS AND OIL
PRESENTED BY
Manoj kumar.M
H.T.NO.636217885003
M.Pharmacy 1st year
UNDER GUIDANCE OF
Dr. Arunabha Malik
Associate professor
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
SRIKRUPA INSTITUTE OF PHARMACEUTICAL SCIENCES
[AFFILIATED TO OSMANIA UNIVERSITY]
[Approved by PCI;AICTE]

2. INTRODUCTION
 Vegetable oils and fats (VOFs) have a
big contribution in our diet as cooking
or frying oil, salad oil or in food
products formulation.
 Also, VOFs are so important by regard
to economic point of view.
 Some VOFs have high price which is
tempting for defrauders to adulterate
them with less expensive oils and fats to
get more profit.
 Issue of vegetable oils adulteration is
not a new problem and even in one
given region or country.

3.  Usually, health problems may not be an issue in
adulteration of VOFs, if edible expensive oil such as
olive oil is admixed with less expensive edible one.
 However, it has been re-ported that adulteration of
vegetable oils caused serious health problems in
some cases like Spanish olive oil syndrome due to
selling non-edible rapeseed oil as an edible rapeseed
oil and even as olive oil.
 Another example is adulteration of mustard oil with
poisonous argemone oil.

4. ADULTERATION IN FATS AND OILS
However detection of adulterant on basis of development of
the color, turbidity or precipitate is possible.
1.Detection of sesame oil as adulterant:
 To 2 ml oil, add 1 ml hydrochloric acid containing 1 per
cent sucrose and keep aside for 5 minutes.
 The presence of pink colour in the acid layer indicates
presence of sesame oil.
2.Detection of cotton seed oil as adulterant by Halphen's test:
 To 2 ml oil, add 2.5ml alcohol and 2.5 ml solution of
sulphur in carbon disulphide (1 per cent).
 Heat the mixture Formation of pink or red colour indicates
presence of cotton seed oil.

5. 3.Detection of karanja oil as adulterant:
 Take 5 ml of sample oil in a test tube; add 10 ml ethyl
alcohol to it followed by addition of 5 drops of ferric
chloride solution.
 Shake vigorusly two minutes and allow to stand for two
hours.
 The development of light blue to dark colour in the
alcoholic layer indicates the presence of karanja oil or
any other oil containing phenolic group.
4.Detection of castor oil as adulterant:
 Take 1 ml of the sample to be tested in a clean test-tube
and to it add acidified petroleum ether (60- 80°).
 Shake it thoroughly.

6.  Then to it add a drop ammonium molybdate solution.
 Development of white turbidity indicates the presence
of castor oil.
5. Detection of argemone oil as adulterant:
 It is an adulterant to mustard oil.
 Ferric chloride reacts with argemone oil in
hydrochloric acid medium to produce small needle
shaped reddish brown crystals.
 However, if content of argemone oil is less than 1 per
cent, needle-shaped cysaican be observed under
microscope.

7. 6. Detection of linseed oil as adulterant:
 About 1 ml sample is dissolved in 5 ml chloroform in a
dry test-tube and one ml liquid bromine is added.
 The tube is stoppered and cooled in ice bat hand to it
added 1.5 ml rectified spirit and 10 ml ether.
 It is then shaken thoroughly again for 30minutes.
 The appearance of precipitate (due to formation of
hexabromide) indicates presence of linseed oil.

8. 7. Detection of mineral oil as adulterant:
 To 1 ml oil or fat under test, add 25 ml alcoholic
caustic potash.
 Boil in water bath, till it becomes quite clear.
 Transfer the contents of the flask to a wide mouth
test tube and to it, add 25 ml distilled water along
the side of the test tube, keeping the tube shaken
during addition of water.
 The presence of turbidity indicates presence of
mineral oil.

9. ANALYTICAL PARAMETERS FOR OILS AND FATS
 The properties of oils and fats vary along with the
degree of un saturation, average molecular weight
and also acidity from hydrolysis.
 A number of parameters are used for their analysis
which are included under physical constants and
chemical constants.
 Physical constants include viscosity specific gravity,
refractive index, solidification point etc.
 Following is a brief idea about some of the analytical
parameters grouped under chemical constants.

10. 1. Iodine value,
2. Saponification value,
3. Hydroxyl value,
4. Acetyl value,
5. Unsaponifiable matter,
6. Acid value,
7. Peroxide value,
8. Kreis test (rancidity index),
9. Ester value,
10. Reichert Messle Value &,
11. Polenski value.

11. 1.LODINE VALUE:
 It is defined as the weight of iodine absorbed by 100
parts by weight of the sample of fat or oil.
 lodine value is a measure of the extent of unsaturation.
 Susceptibility to rancidity increases for the oil or fat
having higher iodine values.
Significance:
 Iodine number directly proportional to content of
unsaturated fatty acids.
 Lower Iodine number ,less is the degree of saturation.
 Iodine number is useful to analyze the degree of
adulteration.

12. 2. SAPONIFICATION VALUE:
 It is defined as the number of milligrams of potassium
hydroxide required to neutralize the fatty acids resulting
from complete hydrolysis of 1 g of the sample of oil or
fatty acid present in the oil.
 This value is normally applied for butterfat, coconut oil in
which lower fatty fat.
 Saponification value occurs in an inverse proportion to
the average molecular weights of acids glycerides occur in
high content.
Significance:
 It is measure of the average molecular size of constituent
fatty acids of given fat/oil.
 Higher saponification number for fats containing short
chain fatty acids.

13. 3. HYDROXYL VALUE:
 It is defined as number of milligrams of potassium
hydroxide required to neutralize the acetic acid capable
of combining by acetylation with 1 g sample of fat or oil.
4. ACETYL VALUE:
 It is the number of milligrams of potassium hydroxide
required to neutralize acetic acid obtained when 1g
value 150), most of the oils and fats have low acetyl value
(3 -15).
Significance
 Acetyl number is a measure of number of hydroxyl
groups present.
 It is used for studying the natural properties of the fat
and to detect adulteration and rancidity.

14. 5. UNSAPONIFIABLE MATTER:
 It is the matter present in fats and oil, which after
saponification by caustic alkali and subsequent
extraction with an organic solvent, remains non-volatile
on drying at 8o°C.
 It includes sterols (phytosterol and cholesterol), oil
soluble vitamins, hydrocarbons and higher alcohols.
 Paraffin hydrocarbons can be detected by this method as
adulterants.

15. 6.ACID VALUE:
 It is defined as the number of milligrams of
potassium hydroxide required to neutralize the free
acids present in 1 g sample of fat or oil.
 Generally, rancidity causes free fatty acids
liberation, hence acid value is used as an indication
of rancid state.
Significance:
 Higher acid number mean it stored for longer
duration.

16. 7. PEROXIDE VALUE:
 It is a measure of peroxides present in oil.
 A peroxide value is generally less than 10 mEqkg in
fresh samples of oil.
 Due to temperature or storage, rancidity occurs
causing increase in peroxide values.

17. 8. KREIS TEST (RANCIDITY INDEX):
 Due to rancidity, epihydrin aldehyde or
malonaldehyde are increased which are detected by
Kreis test using phloroglucinol which produces red
colour with the oxidized fat.
 It is defined as number of milligrams of potassium
hydroxide required to combine with fatty acids
which are present in glyceride form in 1 g sample of
oil or fat.
9.ESTER VALUE:
 Difference between saponification value and acid
value is ester value.

18. 10. REICHERT MESSLE VALUE:
 This value is a measure of volatile water soluble acid
contents the fat.
 It is defined as number of milli litres N/10 potassium
hydroxide solution required to neutralize the volatile
water soluble fatty acids obtained by 5 g fat.
Significance:
 Higher content of volatile fatty acids of butter
responsible for its higher reichert-meissl number.
 It is useful in testing purity/adulteration of butter.

19. 11.POLENSKI VALUE:
 It is defined as the number of millitres of N/10
potassium hydroxide solution required to neutralize
water-insoluble, steam - distillable acids liberated by
hydrolysis of 5 gm of fat.
Significance:
 The Polenski value is an indicator of how much
volatile fatty acid can be extracted from fat through
safonification.

20. OTHER ANALYTICAL TECHNIQUE (NMR)
 Nuclear magnetic resonance (NMR) techniques to detect
olive oil adulteration with hazelnut oil.
 The detection of olive oil adulteration by NMR is based on the
qualitative and quantitative chemical information obtained
from resonance data.
 1H NMR spectra provide information on major compounds
such as fatty acids and also on minor compounds such as
aldehydes, terpenes and sterols.
 NMR is a technique that is capable of characterizing
vegetable oils according to the acyl positional distribution in
the glycerol moiety.
 An artificial neural network based on 1H- and 13C-NMR
data could be used to detect olive oil adulteration with
hazelnut oil at a level of 8%, with some limitations.

21. MEASUREMENT OF SPOILAGE OF FATS AND OIL
Rancidity is a term generally used to denote unpleasant
odors and flavors in foods resulting from deterioration
in the fat or oil portion of a food.
Three different mechanisms of rancidity may occur.
These are oxidative, hydrolytic, and ketonic. Oxidative
Rancidity.
Test for presence of Rancidity:
 In routine work apart from the free fatty acids
determination, the analysis should include the
determination of Peroxide Value, Kries Test,
Ultra-violet Absorption at 234 nm and 268 nm to
establish rancidity

22. PEROXIDE VALUE
 This is an indication of the extent of oxidation suffered by
an oil.
Reagents:
i) Acetic acid - chloroform solvent mixture (3: 2). Mix 3
volumes of glacial acetic acid with 2 volumes of
chloroform.
ii) Freshly prepared saturated potassium iodide solution.
iii) 0.I N and 0.0I N sodium thiosulphate solutions.
Weigh 25 g of sodium thiosulphate and dissolve in 1 L of
distilled water. Boil and cool, filter if necessary. Standardiz
-e against standard potassium dichromate solution.
iv) Starch solution - 1% water-soluble starch solution

23. Procedure:
 Weigh 5 g (±50 mg) sample into a 250 ml stoppered conical
flask.
 Add 30 ml acetic acid chloroform solvent mixture and swirl to
dissolve.
 Add 0.5 ml saturated potassium iodide solution with a mohr
pipette. Let stand for 1min in dark with occasional shaking,
then add about 30 ml of water.
 Slowly titrate the liberated iodine with 0.1 N sodium
thiosulphate solution, with vigorous shaking until yellow color
is almost gone.
 Using Add about 0.5 ml starch solution as indicator and
continue titration shaking vigorously to release all I 2 from
CHCl3 layer until blue color disappears. If less than 0.5 ml of
0.1 N Na2S2O3 is used repeat using 0.01 N Na2S2O3. Conduct
blank determination ( must be less than 0.1 ml 0.1 N Na2S2O3).

24. Calculation:
 Peroxide value expressed as milli equivalent of peroxide
oxygen per kg sample (meq/kg):
Where,
Titre value= ml of Sodium Thiosulphate Used
(blank corrected)
N = Normality of sodium thiosulphate solution.
 Fresh oils usually have peroxide values well below 10 meq/kg.
A rancid taste often begins to be noticeable when the peroxide
value is above 20 meq/kg. (between 20 – 40 meq / Kg).
 In interpreting such figures, however, it is necessary to take
into account the particular oil or fat.
Peroxide Value = Titre Value X N X 100/ Weight Of The Sample

25. KRIES TEST
 TWO TESTS
1.Qualitative
2.Quantitative
1.Qualitative
 Shake 5 ml of the oil vigorously with 5 ml of
0.1% phloroglucinol solution in diethyl ether and
add 5 ml of conc. hydrochloric acid.
 A pink colour indicates incipient rancidity.

26. 2.Quantitative: (method:1)
Weigh 0.8 – 1.02 gm of oil or fat into a 100 ml beaker.
Melt sample of fat and add slowly with stirring 20 ml of
phloroglucinol ( 0.1 gm in100 ml of diethyl ether,
freshly prepared) until sample dissolved.
 Transfer solution to a separating funnel, add 10 ml
conc HCl, shake well and allow to separate.
Run off acid layer into a 1inch (2.54mm) Lovibond cell
and match the colour using red , yellow and blue
glasses.
Express result as red Lovibond units.
Up to 3 red units indicates incipient rancidity, between
3and 8 units indicates the end of induction period, over
8 units indicates definite rancidity.

27. Quantitative (method:2)
Shake 5 ml of oil and 5 ml chloroform in a stoppered test
tube.
 Add 10 ml of a 30% solution of trichloroacetic acid in
glacial acetic acid and 1 ml of 1 percent solution of
phloroglucinol in glacial acetic acid.
 Incubate the test tube at 45ºC for 15 min. After
incubation, add 4 ml 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.

28. ULTRA-VIOLET ABSORPTION
 Oxidised fatty acids containing conjugated double bonds
absorb UV strongly between 230 and 375 nm, dienes
absorbing at 234 nm and trienes at 268 nm.
 Conjugated trienes may be formed by industrial processing,
E.g. decolorising with bleaching earths.
 A secondary absorption by trienes occurs at about 278 nm. In
the early stages of oxidation the UV absorption increases
somewhat proportionately to the uptake of oxygen and the
formation of peroxides.
 The UV absorption curve forms plateau just before the end of
the induction period.
 The magnitude of UV absorbance is not readily related to the
amount of oxidation; so the method is best applicable to
detecting relative changes in oxidation of an oil in comparison
experiments or stability tests.

29. Procedure :
 Weigh accurately into a 25 ml volumetric flask, an amount
of the oil sample so that the absorbance of its solution in iso-
octane in a 10 mm quartz cell lies between 0.2 and 0.8.
Trace the absorption curve against iso-octane between 220
and 320 nm and select the wavelength l max of maximum
absorption near 230, 268 and 278 nm, and the absorbance
(A) at these points.
 The specific absorbance
E1 cm1% (λ max) = A = c x d
 Where,
‘c’ is the concentration of the sample solution (g/100 ml)
‘d” is the cell length in c.

30. REFERENCES
1. A Book of Pharmacognosy by
C.K. KOKATE, A.P. PUROHIT, S.B. GOKHALE
49TH Edition. Pg no:11.2 to 11.8
2. Adulterations in Some Edible Oils and Fats and
Their Detection Methods from: jfqhc.ssu.ac.ir