The document provides information on various analytical methods used to analyze oils and fats. It discusses determination of saponification value, acid value, ester value, iodine value, and peroxide value. Specific methods are described for determining saponification value and acid value which involve titrating the oil sample against a standard solution. Iodine value is a measure of unsaturation and different iodine methods are outlined including Wijis method using iodine monochloride and Hanus method using iodine monobromide. Reagents and calculations are also defined for various analytical techniques.

1. Analysis of oils and fats
Prepared by
Dr. N.GOPINATHAN
ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
FACULTY OF PHARMACY
SRIHER [DU]
CHENNAI-116
TAMILNADU

2. • Saponification value
• Acid value
• Ester value
• Iodine value
• Peroxide value

3. Determination of saponification value
The saponification value is the
number of milligrams of KOH
necessary to neutralise the free
acids and to saponify the esters
present in 1 g of the substance

4. Why KOH is preferred over NaOH
Potassium hydroxide is also slightly smaller than
sodium hydroxide; therefore, it can penetrate oil
molecules faster than sodium hydroxide, thus
breaking the oil’s hold on surfaces quicker. Since
they are also more soluble, the oils can be
rinsed away easier, especially when using hotter
water or steam equipment

5. TAG + 3KOH---- 3 alkali salt + Glycerol

6. Principle- saponification value
The oil sample is saponified by
refluxing with a known excess of
alcoholic potassium hydroxide
solution. The alkali required for
soaponification is determined by
titration of excess KOH against the
standard HCl

7. Saponification value- apparatus
required
• Erlenmeyer flask
• Round bottomed flask
• Reflux condenser
• Hot water bath / hot plate /
heating mantle.

8. Alcohol distillation
• Reflux 1.2 litre of ethanol with 10 g
of KOH and 6 g of granulated
aluminium or aluminium foil for 30
minutes.
• Distil it and collect 1 litre of ethanol
after discarding first 50 ml.

9. 0.5 m ethanolic KOH
• 30g of KOH in one litre of distilled
alcohol.
• Keep the temperature below 15 C while
dissolving alkali.
• Allow to stand overnight.
• Decant the clear liquid and store it in a
air tight container.

10. Phenolphthalein indicator
Dissolve 1 g of phenolphthaein in 100
ml of rectified spirit.

11. Preparation of 0.5 M HCl
Dilute 42.5 ml of concentrated HCl with water to
produce 1000 ml.

12. Standardisation of o.5M HCl
• Weigh accurately 1.5 g of anhydrous sodium
carbonate previously heated at about 270 C
for one hour.
• Dissolve it in 100 ml of water and add 0.1 ml
of methyl red solution.
• Add 0.5M HCl from burette with constant
swirling.
• Until the solution becomes faintly pink.

13. • Heat the solution to boiling , cool and
continue the titration, Heat again to boiling
and titrate further as necessary until the faint
pink colour is no longer affected by continued
boiling.
• Each ml of 0.5M HCl is equivalent to 0.02649g
of sodium carbonate

14. IP procedure
• Weight of oil mentioned in the individual
monograph otherwise
• Weigh 2 g of sample being examined into a
200 ml flask of borosilicate glass fitted with a
reflux condenser.
• Add 25 ml of 0.5 M ethanolic KOH and a little
pumice powder and boil under reflux on a
water bath for 30 minutes.

15. IP procedure
• Add 1 ml of phenolphthalein solution titrate
immediatley with 0.5 M HCl .
• Repeat the operation omitting the substance
being examined

16. Calculation
• Saponification value = 28.05 x ( b-a )/ w
• W – weight of the sample in grams.
• B- blank
• A- titre value

17. Note
• If the oil purpose of preservation has been
saturated with carbon dioxide for the purpose
of preservation, gently reflux the solution of
the oil in ethanol 95 % and ether for 10
minutes before titration.
• The oil may be freed from the CO2 by
exposing it in a shallow dish in a vacuum
desiccator for 24 hours before weighing the
sample.

18. Significance of Saponification Value
•The magnitude of saponification value gives
an idea about the average molecular weight of
the fat or oil.
•Higher the molecular weight of the fat , the
smaller is its saponification value.
•Saponification Value also indicates the length
of carbon chain of the acid present in that
particular oil or fat.
•Higher the saponification value, greater is the
percentage of the short chain acids present in
the glycerides of the oil or fats

19. Significance
• To identify the given fatty oil.
• To distinguish between fatty oils and mineral
oils.
• It also help to calculate the amount of alkali
required to convert a definite amount of fat or
oil into soap.
• In detecting the adulteration of fat or oil.
• Detection of acids containing less than 16
carbon or more than 18 carbon.

20. Acid value

21. Introduction
Free fatty acids are a source of flavors and aromas.
short chain of free fatty acids tend to be water soluble and
volatile with characteristic smell.
long chain saturated and unsaturated fatty acids are more
prone to oxidation in their free form and their breakdown
products (aldehydes, ketones, alcohols, and organic acids)
provide characteristic flavors and aromas
Different fat samples may contain varying amount of fatty
acids. In addition, the fats often become rancid during
storage and this rancidity is caused by chemical and
enzymatic hydrolysis of fats into free acids and glycerol

22. Acid value
• It is the number which expresses in milligrams
the amount of KOH necessary to neutralise
the free acid present in 1 g of the substance.

23. Principle
RCOOH + KOH → ROO- K+ + H2O
AV is also known as
neutralization number or acid number or
acidity
The acid value is determined by directly
titrating the oil/fat in an alcoholic medium
against standard potassium
hydroxide/sodium hydroxide solution.

24. Ip procedure
• Dissolve about 10 g of substance [ weight
mentioned in the individual monograph ] being
examined accurately weighed, in a 50 ml of
mixture of equal volumes of ethanol 95 % and
ether, previously neutralised with 0.1 m KOH to
phenolphthalein solution.
• If the sample does not dissolve in the cold
solvent, connect the flask with a reflux condenser
and warm slowly with a frequent shaking until
the sample dissolves.

25. • Add 1 ml of phenolphthalein solution titrate
against 0.1 M KOH until the solution remains
faintly pink after shaking for 30 seconds.

26. Ethanol-ether solution
Prepare a mixture of ethanol and
diethyl ether (1:1, v/v). Neutralize with
sodium hydroxide titrant and add 1.0
mL of phenolphthalein indicator until
pink colouration is observed. Freshly
prepare the solution.

27. Phenolphthalein indicator
Dissolve 1 g of phenolphthaein in 100
ml of rectified spirit.

28. 0.1M KOH
• Dissolve about 6 g of KOH in distilled water to
produce 1000 ml

29. 95% ethanol
• 95ml of ethanol to 100 ml with water.

30. calculation
• Acid value = 5.61 n/w
• N= the number of ml of 0.1 M KOH required.
• W= the weight in g of the substance.

31. Note
• If the oil purpose of preservation has been
saturated with carbon dioxide for the purpose
of preservation, gently reflux the solution of
the oil in ethanol 95 % and ether for 10
minutes before titration.
• The oil may be freed from the CO2 by
exposing it in a shallow dish in a vacuum
desiccator for 24 hours before weighing the
sample.

32. Significance and application of Acid
value
• The acid value (AV) is a common parameter in
the specification of fats and oils.
• it signifies due to attack of atmospheric
oxygen, hot moist air or microorganisms.
• It gives how much generation of free fatty acid
has taken place which leads to rancidity.
• It gives an indication about the age and extent
of its deterioration.

33. Significance and application of Acid
value
• Find rancid state, triglycerides are converted into fatty
acids and glycerol, causing an increase in acid number.
• It is a measure of the free fatty acids (FFA) in a sample
of oil or fat indicates hydrolysis of triglycerides. Such
reaction occurs by the action of lipase enzyme
• it is an indicator of inadequate processing and storage
conditions (i.e. high temperature, and relative
humidity, tissue damage)
• The acid number is a measure of the amount of
carboxylic acid groups in a chemical compound, such as
a fatty acid, or in a mixture of compounds

34. Drawbacks of non-aqueous solvents in
determination of acid value
• The majority of national and international
standards for AV determination in fat and oils are
based on the acid-base titration techniques in
non-aqueous solvents. However, these
techniques have a number of drawbacks :
• Currently used non-aqueous solvents are toxic,
such as ethanol or isopropanol heated up to 60ºC
or higher or diethyl ether-ethanol solvent.
• Incomplete solubility of test oil portion in alcohol
(even under heating) caused by the formation of
a dispersed system.

35. • Conditions for accurate acid-base titration in hot
amphoteric solvents might deteriorate due to the
increase of the solvolysis constant for anion
titrable weak acids with an increase in
temperature. This conclusion follows from the
fact that the solvent auto-protolysis constant
increases, and the acid dissociation constant
decreases with increased temperature.
• Need to previously neutralize the solvents

36. Ester value
• Ester value is the number of milligrams of KOH
required to saponify the esters present in 1g
of the substance
• Ester value = saponification value – Acid value

37. Iodine value

38. Iodine value
The iodine value is the number which expresses in grams
the quantity of halogen, calculated as iodine which is
absorbed by 100 g of substance under the described
condition. It may be determined by any of the following
methods.
It also known as iodine adsorption value or iodine
number or iodine index
Saturated fatty acids will not give the halogenation
reaction
Oil /fat sample+ ICl or I2 in ethanol and in presence of
mercuric chloride

39. Significance
The iodine value is a measure of the degree of
unsaturation in an oil. It is constant for a particular
oil or fat.
Iodine value is a useful parameter in studying
oxidative rancidity of oils since higher the
unsaturation the greater the possibility of the oils
to go rancid.
Iodine numbers are often used to determine the
amount of unsaturation in fatty acids. This
unsaturation is in the form of double bonds, which
react with iodine compounds.

40. Significance
The higher the iodine number, the more C=C
bonds are present in the fat
If the iodine number is between 0-70, it will
be a fat and if the value exceeds 70 it is an oil.
Starch is used as the indicator for this reaction
so that the liberated iodine will react with starch
to give purple coloured product and thus the
endpoint can be observed.

41. Method
1. Iodine monochloride method or Wijis
method
2. Iodine monobromide method or Hanus
method
3. Pyridine bromide method

42. Method A- iodine mono chloride
method
Principle
• The oil/fat sample taken in carbon tetrachloride is
reacted with an excess of iodine monochloride solution
(Wjis solution). Unsaturated fatty acids undergo
halogenation reaction resulting in the addition of an
iodine atom to one carbon of the double bond.
• On completion of the reaction, the remaining iodine
monochloride reacts with potassium iodide leading to
the formation of molecular iodine. The liberated iodine
is then evaluated by titration with a standard solution
of sodium thiosulphate using starch as indicator.
Mercuric ions are added to fasten the reaction

43. Method A- iodine mono chloride
method

44. Wijis reagent
• Iodine monochloride is produced simply by
combining the halogens in a 1:1 molar ratio,
according to the equation
I2 + Cl2 → 2 ICl
• When chlorine gas is passed through iodine
crystals, one observes the brown vapour of iodine
mono chloride. Dark brown iodine mono chloride
liquid is collected.
• Excess chlorine converts iodine monochloride
into iodine trichloride in a reversible reaction:
ICl + Cl2 ⇌ ICl3

45. IP procedure
• Place an accurately weighed quantity of the substance
(100 g)under examination in a dry 500ml iodine flask,
add 10ml of carbontetrachloride and dissolve.
• Add 20ml of iodine monochloride solution , insert the
stopper and allow to stand in the dark at a temperature
between 15°C and 25°C for 30 minutes.
• Place 15ml of potassium iodide solution in the cup top
, carefully remove the stopper , rinse the stopper and
the sides of the flask with 100ml of water, shake and
titrate with 0.1M sodium thiosulphate using starch
solution , added towards the end of the titration , as
indicator .

46. IP procedure
• Note the number of ml of 0.1M sodium
thiosulphate (a).
• Repeat the operation without the substance
under examination and note the number of ml
required(b). (BLANK)
• SPECIAL SAFETY NOTE Carbon tetrachloride is a
carcinogen in laboratory animals. Avoid breathing
vapors or skin contact. Work in a well ventilated
hood but avoid dispersing to atmosphere

47. Calculation
• Calculate the iodine value from the expression
Iodine value = 1.269 (b-a) / w
• Where w= weight in grams od the substance
• a = the number of ml of 0.1M sodium
thiosulphate
• b = the number of ml of 0.1M sodium
thiosulphate with out substance
• w =weight of the substance in gram
• 1.269 is atomic mass of iodine

48. Iodine monobromide method or
Hanus method
• Some of the IBr reacts with the double bonds in the
unsaturated lipids, while the rest remains:
R-CH=CH-R + IBr(excess)→ R-CHI-CHBr-R + IBr(remaining )
• The amount of IBr remaining is determined by
adding excess potassium iodide to the solution to
liberate iodine, and then titrating with a sodium
thiosulfate (Na2S2O3) solution in the presence of
starch to determine the concentration of iodine
released:

49. IBr(remaining) + 2KI → KBr + KI + I
I2 + starch + 2Na2S2O3 (blue)
2NaI + starch + Na2S4O6

50. Haus solution
Hanus solution (for iodine number).
• Dissolve 13.2 g of resublimed iodine in 1 L of glacial acetic
acid which will pass the dichromate test for reducible
matter. Add sufficient bromine to double the halogen
content, determined by titration (3 mL is about the proper
amount). The iodine may be dissolved by the aid of heat,
but the solution should be cold when the bromine is added.
• Store the hanus solution in a amber bottle and away from
light. The Hanus solution can be used for a year.
• Extremely hazardous in case of eye contact (corrosive) and
Causes severe eye burns. Extremely hazardous in case of
skin contact (corrosive).

51. Procedure
• Unless otherwise specified, weigh accurately the quantity of
substance under examination, stated in the table ,
• place it in a dry 300ml iodine flask or which has been rinsed
with glacial acetic acid unless otherwise specified in the
monograph. Add 15ml of chloroform and dissolve
Presumed iodine value Quantity of the substance
Less than 20 1.0
20 to 60 0.25 to 0.5
61 to 100 0.15 to 0.25
More than 100 0.10 to 0.15

52. Procedure
• Add slowly from a burette 25.0 ml of iodine monobromide
solution , insert the stopper allow to stand the dark for
30minutes , unless otherwise specified in the monograph,
shaking frequently.
• Add 10ml of potassium iodide solution and 100 ml of water
and titrate with 0.1M sodium thiosulphate using stach
solution , added towards the end od the titration, as
indicator,
• note the number of ml required (a).
• Repeat the operation without the substance under
examination and note the number of ml required(b).
Calculate the iodine value from the expression given under
method A

53. Calculation
• Calculate the iodine value from the expression
Iodine value = 1.269 (b-a) / w
• Where w= weight in grams od the substance
• a = the number of ml of 0.1M sodium
thiosulphate
• b = the number of ml of 0.1M sodium
thiosulphate with out substance
• w =weight of the substance in gram
• 1.269 is atomic mass of iodine

54. Pyridine bromide method
• Place an accurately weighed quantity of the
substance under examination in a dry iodine
flask, add 10ml of carbon tetrachloride and
dissolve. Add 25ml of pyridine bromide
solution, allow to stand for 10minutes in the
dark and complete the determination
described under method A beginning at the
words “place 15ml of….”

55. The approximate weight in gram of the
substance to be taken may be calculated by
dividing 12.5 by the highest expected iodine
value. If more than half the available halogen is
absorbed, the test must be repeated with a
smaller quantity of the substance.

61. Peroxide value

62. Peroxide value
It is the number of milli
equivalents of active oxygen
that expresses the amount of
peroxide contained in 1000 g of
the substance.

63. Procedure -IP
• Weight unless otherwise mentioned in the
individual monograph .
• Weigh 5 g of the substance being examined,
accurately weighed into a 250ml glass
stoppered conical flask, add 30ml of a mixture
of 3 volumes of glacial acetic acid and 2
volumes of chloroform swirl until dissolved
and add 0.5 ml of saturated potassium iodide
solution.

64. • Allow to stand for exactly one minute with
occassional shaking add 30 ml of water and
titrate gradually with continious and vigorous
shaking, with 0.01M sodium thio sulphate
until yellow colour almost disappears.
• Add 0.5 ml of starch solution and continue the
titration shaking vigorously until the blue
colour just disappear [ a ml]

65. • Repeat the operation omitting the substance
being examined [ b ml].
• The volume of 0.01M sodium thio sulphate in
the blank determination must not exceed 0.1
ml

66. Calculation
• Peroxide value = 10 ( a-b) /w
• Where w = weight in g of the substance

67. Autoxidation
• The process of oxidation induced by air at
room temperature referred to as
“autoxidation”.

68. Rancidity
• Rancidity is a term generally used to denote
unpleasant odours and flavours in foods resulting
from deterioration in the fat or oil portion of a
food.
• There are two basic types or causes of rancidity
that cause and/or contribute to the degradation
of stored edible oils: oxidative and hydrolytic
• Oxidative rancidity, known as autoxidation,
occurs when oxygen is absorbed from the
environment

70. • Hydrolytic rancidity, also called hydrolysis or enzymatic
oxidation, occurs in the absence of air, but with moisture
present.
• Enzymes found naturally in plant oils
• (i.e., lipoxygenase, cyclooxygenase)
• and animal fats (i.e., lipase)
• can catalyze reactions
• between water and oil
• Microbial rancidity, in which micro-organisms such as
bacteria, molds and yeast use their enzymes to break down
chemical structures in the oil, producing unwanted odors
and flavors. Water needs to be present for microbial growth
to occur.