ANALYSIS OF FATS AND OILS

Definitions &Classification

1
 Fats and oils are a class of materials called lipids
 Lipids are natural biologically active compounds insoluble in water, used
as human food, of animal or plant origin.

Classification of Lipids:
(A) Simple Lipids:
 Esters of fatty acids with alcohols
 Fats: are esters of high molecular weight fatty acids (long-chain carboxylic
acids containing from 4 to 24 carbon atoms, mostly of saturated chains)
with gylcerol ( a triglyceride, propane1,2,3-triol)
 They are solid in nature.
 Oils: are esters of high M.W.F.A. (mostly of unsaturated chains) with
glycerol
 They are liquid in nature
 Waxes: are esters of High M.W.F.A. with alcohols other than glycerol
(Mostly monohydroxylated alcohols)

2
 H H
H OH HOOC.R1 H O.CO.R1
-3H2O
H OH + HOOC.R2 H O.COR2
H OH HOOCR3 H O.CO.R3
H H
Glycerol Fatty acids Triglyceride
Of different chains Simple or Mixed type

)
H H
H O.Palmityl H O.Oleiyl
H O.Palmityl H O.Stearyl
H O.Palmityl H O.Palmityl
H H
Simple triglyceride Mixed triglyceride

Waxes: CH3(CH2)n.CO.OCH2(CH2)m.CH3
1- n= 14, m = 14 (a waxe from spermaceti)
2- n = 24 , m = 28 or 30 (a type from beeswax)
3
(B) Compound Lipids:
 Are esters of high M.W.F.A. with glycerol also but conjugated with other
groups
Examples:
1- Phospholipids (Phosphatides):
 Are glycerol esters of fatty acids & phosphoric acid, linked to some
nitrogenous bases.
2- Glycolipids (Cerebrosides):
 Are cpds of fatty acids with carbohydrates, containing nitrogen, but no
phosphoric acid moieties.

(C) Lipid Derivatives:

 Are cpds derived from the above groups either by hydrolysis or
decomposition.
 E.g. Free fatty acids of various chains, steroids, high M.W. alcohols……etc.

4
 PHOSPHOLIPIDS
H
Lecithin Phosphatidyl Choline

)
H O.CO.R1
R1 = Staturated chain & R2= Unsturated chain
H O.COR2
H O P O CH2CH2 N CH3 3

)
H O O
H
Cephaline Phosphatidyl Serine

)
H O.CO.R1
R1 = Staturated chain & R2= Unsturated chain
H O.COR2
H O P O CH2CH2 NH3
H O O
GLYCOLIPIDS
H
CH3 CH2 CH CH OH
(

12
H CH.NH.CO. CH2 22.CH3

)
Sphingolipid O
H O H CH2OH
H H H OH
D-galactose
5 OH H
OH sugar
 Composition of fats & oils

 Natural fats & oils contain a wide variety of chemical cpds listed as:
1- Triglycerides
Defined as Saponifiable part
2- Free fatty acids of the fat or oil
 Represent about 99% of the fat or oil weight

3- Phospholipids
4- Glycolipids
Defined as
5- High M.W. alcohols & their esters & ethers
Unsaponifiable
6- Hydrocarbones part of fat or oil
7- Vitamins
8- Miscellaneous coloring matter & flavours
 Represent about 1% of the fat or oil weight

6
 (1) Triglycerides
 Result from the reaction of one molecule of glycerol & three
molecules of fatty acids
 When the fatty acid parts are identical the product is a simple
triglyceride
 A mixed triglyceride is that one containing different fatty
acid parts
 A mixed triglyceride containing three different F.A. parts has
three isomeric forms:
H H H
 H O.Pal  H O.Ole  H O.Pla
 H O.Ole  H O.Pal  H O.Stea
' H O.Stea ' H OStea ' H O.Ole
H H H
-Oleopalmitostearin -Palmitooleostearin -Stearopalmitoolein
7
 (2) Fatty Acids
 They are carboxylic acids, that can obtained by hydrolysis of
naturally occurring fats & oils
 Usually they have unbranched chains with an even number of
carbon atoms.
 They start by C4 (Butyric acid) and ended by C24 (Nervonic
acid).
 They are either saturated fatty acids or unsaturated ones
 In most cases the double bonds have cis and not trans
configuration
 Mostly occur between:
 - C9 & C10 for single double bound
 - C9-C10 & C12-C13 for 2 double bounds
 - C9-C10, C12-C13 & C15-C16 for 3 double bonds

8
 (2) Fatty Acids ,cont.
 Saturated fatty acids have the formula CnH2n+1COOH (n is
odd number)
 Unsaturated fatty acids
 With one double bond have the formula CnH2n-1COOH
 With 2 double bonds CnH2n-3COOH
 With 3 double bonds CnH2n-5COOH

 (N.B.) Carbon atoms are numbered with the carboxyl

carbon as number 1 & the double bond location is
designated by the number of the carbon atom from the
carboxyl side

9
 Nomenclature of fatty acids
 Most of them have common names usually derived from the
plant or animal origin separated from it.
 The official systematic names consists of:
 The number of carbon atoms followed by
 ……noic acid.
 If the fatty acid containing double bonds, the name started by
configuration of double bonds, followed by the position of
them & then the number of carbon atoms present followed by
number of double bonds & then ..enoic acid

 N.B. Other substituents must be indicated

10
 1) The Saturated fatty acids:
No Common Systematic Numerical Formula
name name symbol
1 Butyric acid Butanoic acid 4:0 C3H7COOH

2 Caproic acid Hexanoic acid 6:0 C5H11COOH

3 Caprylic acid Octanoic acid 8:0 C7H15COOH

4 Capric acid Decanoic acid 10:0 C9H19COOH

5 Lauric acid Dodecanoic 12:0 C11H23COOH

6 Myristic acid Butadecanoic 14:0 C13H27COOH

7 Palmitic acid Hexadecanoic 16:0 C15H31COOH

8 Stearic acid Octadecanoic 18:0 C17H35COOH

11
9 Arachidic acid Icosanoic acid 20:0 C19H39COOH
 2) Unsaturated & substituted fatty acids:

No Common Systematic name Numerical Formula

name symbol
1 Oleic acid Cis-9-octadecaenoic acid 18:1(9) C17H33COOH

2 Linoleic Cis,cis-9,12- 18:2(9,12) C17H31COOH

acid octadecadienoic acid
3 Linolenic Cis,cis,cis-9,12,15- 18:3(9,12,15) C17H29COOH
acid octadecatrienoic acid
4 Arachidonic Cis,cis,cis,cis-5,8,11,14- 20:4(5,8,11,1 C19H31COOH
acid Icosatetraenoic acid 4)
5 Palmitoleic Cis-9-hexadecaenoic 16:1(9) C15H29COOH
acid acid
6 Ricinoleic 12-hydroxy-cis-9- 18:1(9),(12- C17H32(OH)C
acid octadecaenoic acid OH) OOH
7 Erucic acid Cis-13-doicosaenoic acid 22:1(13) C21H41COOH

12
 (3) Phospholipids (Phosphatides)
 Consists of a polyhydric alcohol (Usually but not always glycerol) esterified with
fatty acids & also phosphoric acid
 Phosphoric acid usually combined with a basic nitrogenous base (e.g. Choline &
serene bases)
 They are usually surface active compounds of amphoteric nature (having both
acidic and basic functions)
 They play a vital rule in many biochemical processes e.g. Fatty acids transfer, protein
transfer as well as in ion transfer.
 The most common phosphatides are Lecithins (choline base derivatives) and
Cephalins (Serene derivatives)
 Cephalins have antioxidant activities.

CH2 O.CO.R1 CH2 O.CO.R1

CH O.CO.R2 CH O.CO.R2
O CH3 O
CH2 O P O CH2CH2 N CH3 CH2 O P O CH2CH2 NH3
O CH3 O
13 Lecithines Cephalins
 (4) Glycolipids (Cerebrosides)
 Mostly are derivatives of sphingosine e.g. sphingomyelin and cerebroside
 Sphingolipids do not yield glycerol when they are hydrolyzed, they give
sphingosine, phosphoric acid, a 24-carbon fatty acid called lignoceric acid and
choline base or a carbohydrate moiety.
 Sphingolipids together with proteins and some polysaccharides, make up
(Myeline) which is the protective coating that encloses nerve fibers.

CH3. CH2 C H
(

)
12
H C CH OH

Sphingosine CH NH CO. CH2 22CH3 CH2OH

)
O OH
In case of
CH2 OH H H
OH Cerebroside
H
OH H
O CH3
In case of
O P O CH2CH2 N CH3
14 Sphingomyelin
O CH3
 (5) Terpenes and Terpenoids (Hydrocarbons)
 One class of the important constituents of essential oils are a type of hydrocarbons
known as terpenes and the oxygen containing derivatives of it which known as
terpenoids.
 Most terpenes present have skeletons of 10, 15, 20 or 30 carbon atoms as the
following table:
Number of carbons Class
10 Monoterpenes
15 Sesquiterpenes
20 Diterpenes
30 Triterpenes
 Terpenes is built from a 5-carbon units known as isoprene units (2-methyl-1,3-
butadiene)
Head CH
Head 3
CH2 C
Tail CH CH2 Tail

15 Isoprene unit
 Examples of the most important terpenes are:
1- Squalene (C30H50), which is a triterpene obtained from shark
liver oil
2- ,,g-Carotenes which are naturally occurring tetraterpenes.
 They are present in almost all green plants & serve as precursors
for vitamin A.
T
T H H
T H T
H T
H
SQUALENE
H T

H T
H T H T H T
T H T H T H
T H
16
-CAROTENE
 (6) Sterols

 Crystalline
 Naturally occuring unsaponifiable alcohols of high melting
points.
 Comprise the bulk of the unsaponifiable matter in many fats &
oils.
 Relatively inert, although they have some importance
 Because they constitute the starting materials for synthesis of sex-
hormones & vitamin D2.
 The most important sterol in animal fats is cholesterol
(C27H46O).
 Sterols of plant origin are mixtures of sterols known
collectively as phytosterols
 E.g. Ergosterol (which known as plant cholesterol).

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 H3C H3C
CH3 CH3
CH3 H CH3 H

CH3 H CH3 CH3 H CH3 CH3

H H H H
HO HO
H H
Cholesterol Ergosterol

H3C
CH3
CH3 H
CH2 CH3 CH3

H
HO
Vitamin D2
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 (7) Vitamins
 Fat-soluble vitamins include: A, D & E

(a) Vitamin A:
 Fish liver oils are very rich in this vitamin
 Plant oils containing b-carotene which considered pro-
vitamin A,
 That produces a considerable amount of Vitamin A by cleavage of the
molecule (Each molecule give 2 Vitamin A molecules)

(b) Vitamin D (Calciferol):

 Mainly derived from the natural sterols
 E.g. Cholesterol & ergosterol

OH

19
Vitamin A
(c) Vitamin E (Tocoferols):
• Composed of at least 7 types of tochopherol derivatives
• Tochopherols designated -, -, & g- tochopherols
• They act as antioxidants & prevent rancidity of the oils
• Easily oxidized to tochoquinone
CH3
HO -Tochopherol
CH3
H3C O
CH3 CH3 CH3
CH3

Oxidation
CH3
O
HO CH3
H3C O
CH3 CH3 CH3
CH3
20
-Tochoquinone
 (8) Components affecting the appearance (Color) of fats & oils
 One of the well known classes of oily pigments is the carotenoids, which give
fats & oils their characteristic yellow colors.
 About 75 carotenoids are now known.
 The most important derivatives of them are -, -, and g- carotenes, lycopene &
lutein.
 The chromogenic properties of the carotenoids can be seriously affected by
oxidation, heating & hydrogenation.

(9) Mineral contents of fats & oils

Fats & oils may also contain:

 Traces of phosphorous from the residuals of phospholipids
 Traces of sodium left after alkaline refining &
 Traces of As, Cu, Mn, Zn & Fe cpds in some marine & vegetable oils.

21
 Steps for industrial preparation of an oil or fat
1- Collection of the suitable materials
 E.g. Fruits, nuts, roots, animal tissues, fish parts …. etc.
2- Removal of foreign matter
 E.g. hairs from seeds & shell from nuts …. etc.
3- Crushing, grounding or powdering to obtain a product suitable for oil
recovery.
4- Recovery of the oil by hydraulic pressing, extraction by organic solvents
or by continuous milling & pressing.
5- Purification & refinement of crude oil:
 Done by addition of sodium hydroxide which dissolves proteins &
saponify the free fatty acids.
 Filter to separate suspended matter & wash with water to free the oil
from alkalis.
 Now the oil becomes clearer & much fainter in color

22
 Steps for industrial preparation of an oil or fat (cont.)
6- For bleaching of the oil color, the oil is treated with aluminium silicate,
charcoal or similar substances on which the pigments are adsorbed and
clear oil is obtained by filtration or decantation.
7- The oil then decolorized by passing superheated steam in the oil under
readuced pressure to prevent hydrolysis of oil
8- Wintering of the oils:
 Some oils (e.g. Olive oil and cotton seed oil) contain soild esters of
stearic acid, high paraffin hydrocarbons, & high alcohols (stearoptenes)
 These oils during winter (cold weather) become turbid due to
precipitation of these compounds, so these solids should be removed
(By cooling the oil & filtration) before the oil is delivered or used.

23
 Collection of Materials Removal of foreign matter

Recovery of the oil Crushing & grounding

Industry
Purification & refinement Technical oils

Alkali treatment (Proteins &

Degumming (Hydration)
free fatty acids splitting)

Bleaching & Filtration Industry

Soap industry or
Technical F.A. for
De-Odorisation candles & paint
industries

Oil recovery (food oils)

Mixing with
other Hydrogenated oils
ingredients, (Food products)
salts & vitamins
Medicinal oils
24 Emulsification
Margarine (Food Product)
 Oil Hydrogenation
 Complete hydrogenation of unsaturated fatty acids is recommended due
to the elimination of the so formed undesired fatty acids (e.g. iso-oleic
acid)
 But produce a hard fats with higher melting points

Catalyst Ni0

)
R CH CH R' + H2 R CH2 CH2 R'

Oil Liquid Gas Solid or Semi-solid

(

)
C8H17 C C C7H14 COOH Oleic acid

)
H H

Full Partial hydrogenation

Hydrogenation Isomerization occur

)
H H H
C8H17 C C C7H14 COOH C8H17 C C C7H14 COOH
H H H
Stearic acid Iso-oleic acid
25
 Oil Hydrogenation (cont.)
Linoleic acid Oleic acid

 Linolenic acid Stearic acid

Iso-linoleic acid Iso-oleic acid

(3 D.B.) (2 D.B.) (1 D.B.) (0 D.B.)
Patrial Complete
Hydrogenation Hydrogenation
 The rate of hydrogenation depends on the temperature, pressure, catalyst
type, mixing process, purity of the oil & hydrogen gas.

 Advantages of hydrogenation:
1- Improves keeping qualities, odor, taste & color
2- Destroys the objectionable odor & taste of marine oils &
3- Produces different stages of hardened oils which suitable for margarine,
soap & candle manufacturing

26
 Oil Hydrogenation (cont.)
 The hydrogenated oils usually:
 Colorless
 Odorless
 Tasteless
 Solid or semi-solid
 Lower iodine value
 Lower refractive index &
 Higher melting point than original oil

27
 Detection of hydrogenated oils:
1- Test for Iso-oleic acid:
 Depends on the following facts:
 Lead salts of unsaturated F.As are soluble in ether
 Lead salts of saturated F.As & also iso-oleic acid are ether insoluble.
 Iodine value of saturated F.As approximately = zero
 Iodine value for iso-oleic acid about 90
 M.P. for iso-oleic acid about 340C
2- Test for Nickel:
 By extraction and testing with dimethylglyoxime give rose red
color.
 N.B. Negative results do not mean the absence of hydrogenated
oils (Catalyst other than nickel may be used)

28
 Oil Rancidity
 When oils & fats exposed to the atmospheric conditions (moisture, light,
oxygen & high temperature) for long time,
 They acquire unpleasant odor & taste ---- said to be RANCID
 This is due to:
1- Hydrolysis produced by micro-organisms & moulds present in the air
2- Hydrolysis produced by the hydrolytic splitting factors such as enzymes
(lipases), commonly present in the original plants or animals.
3- Auto-oxidation of the double bonds of unsaturated fatty acids.
 Moisture, air, light & temperature are accelerating factors for rancidity.

 Rancidity has two types:

(a) Hydrolytic rancidity
(b) Oxidative rancidity

29
 (a) Hydrolytic Rancidity:

 It is usually associated with a high acid value (free fatty acid content)
 Change in the refractive index & specific gravity

R.CH2-CH2.R’.COO-glycerol

hydrolysis followed by
oxidation

R.COOH + CH3.R’.COO-glycerol
free fatty acid Combined-shorter chain F.A.

30
(b) Oxidative Rancidity:
 More common than the hydrolytic rancidity
 Result from auto-oxidation of double bonds of unsat. F.A.
 All fats & oils containing unsat. F.A. are subjected to the oxidative rancidity
 Oxidation process proceed via free radicals chain reaction involving one or
more carbon atoms adjacent to D. bond.
 Original free radicals are formed by the catalytic influence of Pro-oxidants,
usually metal ions that present in trace amounts in oils & fats.
 Peroxides, hydroperoxides, carbonyl compounds & fatty acids of reduced
chains are main compounds produced in oxidative rancidity.
 In early stages of rancidity, the peroxides & hydro-peroxides are formed.
 In advanced stages, peroxides & hydro-peroxides undergo some
transformations & isomerization forming new unsaturated compounds which
add O2 forming a series of oxy-compounds which finally polymerized to give
solid polymers.

31
 R- CH2 – CH = CH . CH2 . R’
Unsaturated Fatty Acid
(1) Pro-oxidants initiation step
Free radical formation
R– *CH–CH = CH.CH2. R’ &/Or R.CH2–CH = CH. *CH.R’
Carbon free radicals
+ O2 Propagation step
(2) (Two stages)
R–CH – CH = CH – CH2 . R’ Or R – CH2 – CH = CH – CH.R’
O – O* Peroxy radicals *O - O
R-CH2-CH = CH.CH2.R’ +O2

R-CH-CH = CH.CH2.R’ + R.*CH-CH = CH.CH2.R’

O – OH Carbon free radicals
Hydro-peroxides (3) Decomposition of hydro-
peroxides
R.CH2.CHO + R’.CH2.COOH +
R.CH = CH.CHO + R’.CH2.OH
Aldehydes , Acids , Alcohols
32
 In some types of rancidity the epihydrin aldehyde is formed (such type of
rancidity is called “ Aldehydic rancidity.
R.CH2.CH = CH.CH2.R’
[O]
R. CH2.CH – CH.CH2.R’

[O]
R.CH2.CH – CH.CHO Epihydrin aldehyde

 Ketonic Rancidity:
 Ketones usually result from b-oxidation of the saturated fatty acids by the
activity of the enzyme peroxidase which present in certain moulds.

R.CH2.CH2.COOH R.CH(OH).CH2COOH

R.CO.CH3 + CO2 R.CO.CH2.COOH

33
 Tests for Rancidity
1- Peroxide test:
 Give quantitative measure of the amount of peroxide present in the fat or oil
 Mainly used for early stages of rancidity
 Peroxide Value: It is the number of milliliters of 0.002N Na2S2O3 required to
titrate I2 liberated from one gram of the fat or oil.
CHCl3/KI
R.CH.CH = CH.CH2.R’ R.CH.CH = CH.CH2.R’
O – OH CH3COOH OH + H2O + I2

 Peroxide Value = ml’s of 0.002 N Na2S2O3 X 100

Wt. of the oil (gm)

34
 Tests for Rancidity (cont.)
2- Thiobarbituric acid test:

 Mainly used for detection of advanced rancidity

 The Test:
 The oil dissolved in chloroform or benzene is mixed with thiobarbituric acid
in aqueous acetic acid and shaken well for some time and then separated.
 The aqueous layer is heated for 30 minutes at boiling water bath, where a red
color is produced
 The intensity of the red color can be used as a quantitative measure of
rancidity.
 The red colour produced due to the reaction of thiobarbutric acid with the
aldehydic compounds resulted in the advanced stage of rancidity.

3- Kreiss test (Kreiss-Epihydrin aldehyde test):

 Oil (in ether) + Phloroglycinol (in ether) + Conc. HCl
 Give a pink or red color due to the interaction between epihydrin and
phloroglycinol.
35
 Antioxidants
 Are substances which protect oils & fats against oxidation & tend to reduce
or inhibit rancidity.
They have two types:
(A) Naturally Occurring Antioxidants:
 Vitamin E, Vitamin C, Vitamin A, Carotenes, Lecithins, Hydroquinones,
Sesamol (alcoholic compound obtained from sesam oil) are some of the
powerful natural antioxidants present in many natural sources.
 Phosphoric acid & other acidic substances stimulate the action of phenolic
antioxidants.
 Chelating agents e.g. EDTA also enhances the performance of antioxidants.
(B) Synthetic Antioxidants:
 Propylgallate, p-diphenyl-p-phenelene diamine and nor-dihydroguaiaretic
acid and many other phenolic compounds are used as synthetic
antioxidants.

36
 Analysis of Fats & Oils
(a) Physical examination:
 E.g. Specific gravity, moisture content, melting behavior & refractive
index.
(b) Chemical examination:
1- Acid, saponification & ester values.
2- Fatty acids analysis, including:
 Determination of unsaturation content.
 Determination of hydroxy-fatty acids content.
 Determination of volatile fatty acids content.
 Separation of saturated fatty acids from unsaturated fatty acids by lead salt-ether
method.
3- Determination of unsaponifiable matter content.
(c) Selective color tests for individual fats & oils
(d) Detection of adulteration in fats & oils.

37
 (A) Physical Examination
1- Specific gravity:
 The ratio between the weight of a given volume of an oil measured at a given
temperature and that of the same volume of water measured at 15.5 0C
 Approximately all essential oils have specific gravities less than unity (1.00)
 Determination of the specific gravity for an oil is done by using Westphal
Balance

Graduated beam

Weights

Screw Knife edge

Brass
Weight Sinker

oil
38
 (A) Physical Examination (cont.)
2- Moisture content:
 Determined by Karl-Fischer method
 Karl-Fischer reagent composed of 2-Methoxy ethanol & pyridine (4+1)
 Solvent system; Chloroform and methanol (1+1)
 Method:
 Titrate a specific weight of the oil with Karl-Fischer reagent to electrometric
end point.
 Carry out blank determination (Solvent system only without oil sample)

(ml’s of reagent for sample-blank)

% water =
Weight of oil sample (gm)

39
 (A) Physical Examination (cont.)
3- Melting Behavior:
 Melting range of a fat : Is the difference between the temperature at the point
of incipient fusion and complete fusion of the fat.
 The capillary tube method is usually used for determination of the melting
range of a fat.

4- Refractive Index:
 R.I. of oils & fats can be measured directly by using Abbe Refractometer.
 Where a thin film of oil or molten fat is placed between two prisms of glass
and the degree of light refraction is then measured.
 (N.B.) The more the unsaturated fatty acid content present the higher the R.I.
of an oil or fat.

40
 (B) Chemical Examination
1- Acid Value:
 It is the number of milligrams of KOH required to neutralize the free fatty
acids in one gram of the fat or oil dissolved in 95% ethanol.

 ACID VALUE = V * N * 56.1

Weight of fat or oil
 V, N – volume (ml) & normality of KOH

 Acidity sometimes required in terms of certain fatty acids

 E.g. Oleic acid (M.W. = 282), or Lauric acid (M.W. = 200)
 In such cases:
 % Oleic acid = % acidity = V * N * 28.2
Weight of oil sample
 It is a measure of free fatty acids in a given oil or fat.

41
 2- Saponification and Ester Values:

 Saponification value is the number of milligrams of KOH required to

neutralize the free fatty acids and to sponify the esters in one gram of the oil
or fat dissolved in 95% ethanol.
 Sapon. Value = (E – B) * N * 56.1
weight of oil sample
 Where B = Blank determination (Without oil sample)
 E = Experiment (With oil sample)
 N = Normality of the standard (KOH solution)
 It is a measure of both free & combined fatty acids

 To obtain the combined fatty acids only (As Esters)

 The Ester Value = Sapon. Value – Acid Value

42
3- Fatty Acids Content:
(A) Determination of Un-saturation content:
(i) Iodine Value:
 It is the number of parts of I2 absorbed by 100 parts of the fat or oil.
 It is a measure of the unsaturated fatty acids present in a fat or oil.
 The determination is done by allowing suitable weight of the oil (100 gm) to
stand in contact with a known excess of the specified reagent for a sufficient
period of time.
 The solution is then diluted with water & KI is added.
 The liberated I2 is then titrated with standard solution of Na2S2O3 using
starch as indicator.
 The greater the degree of unsaturation, the higher the iodine value.
 Iodine value is directly proportional to the degree of unsaturation of fatty
acids Examples:

43
 For zero D.B. e.g. Stearic acid I.V. = zero
 For 1 D.B. e.g. Oleic acid I.V. = 90
 For 2 D.B. e.g. Linoleic acid I.V. = 180
 For 3 D.B. e.g. Linolenic acid I.V. = 270-275

 Inner Iodine Value:

 It is the iodine value of the unsaturated fatty acids of a fat or oil determined
on the acids obtained after separation of saturated fatty acids.

 Reagents used for determination of I2 values:

Reagent Composition Reaction Time
1- Wijs ICl in gl. Acetic acid 30-60 min
2- Hanus IBr in gl. Acetic acid 60-120 min
3- Hubl I2 + HgCl2 in 95% alcohol 5-15 hours
4- Dams Pyridine SO4 + Br2 in CH3COOH 15-20 min
5- Bromine/Dioxan Br2 + Dioxan in CHCl3 1 min

44
  The last two solutions adds Bromine instead of I2 to the double bonds
 The addition of bromine is more quantitative and more rapid than
iodine.

Br Br
O O
Enhance the addition of
+ 2 Br2 Bromine to the D.B. 1 min

)
O O
Br Br
Dioxan Tetra bromo dioxan

Br Br
O
H H H H
+ R C C R' R C C R'
O
Br Br Br Br
The excess bromine is treated with KI and the liberated
iodine is titrated with sodium thiosulfate.
Br2 + 2 KI I2 + 2 KBr
45
I2 + 2 Na2S2O3 2 NaI + Na2S4O6
 (ii) Thiocyanogen Value
 It is the % of thiocyanogen (Calculated as I2) absorbed by an oil of fat
under the conditions of the test described.
 Determined by allowing the oil to react with known excess amount of
thiocyanogen in anhydrous CH3COOH,
 Then add KI and the liberated iodine is back titrated with sodium
thiosulfate using starch as indicator.

SCN H H H H
SCN + R C C R' R C C R'
SCN SCN

46
 The addition of thiocyanogen to double bonds is more
selective than iodine.
Thiocyanogen is added to 1 D.B. of Oleic acid (T.V.=90)
added to 1 D.B. of linoleic acid (T.V.=90)
added to 2 D.B. of linolenic acid (T.v.=180)
Thus by using both iodine and thiocyanogen values of a
given fat or oil , we can calculate the % of oleic, linoleic
and linolenic acids in such oil.

(iii) Bromine Value:

Calculated as the % increase in weight of oil or fat by
addition of bromine, also calculated as iodine value
numbers
It is similar to iodine value in importance.

47
(iv) Diene Value ( Maleic Anhydride value):
 It is the % of maleic anhydride (Calculated as iodine) which reacts with an oil
under the conditions described.
 It is useful for oils containing Fatty acids with conjugated double bonds.

H O H R O
H C C C H C
C H C C
H C R Reflux, 100 C in
+ O O
H C R' Benzene for 3 hours
C H C C H C C
C C
H O H R' H
O
After the reaction is completed, the solution is diluted with water
and extracted with ether and the aqueous solution is titrated with
standard alkali (for the excess maleic acid), a blank experiment
must be carried out.
Diene Value (%) = C.C. takn (B-E) X N X Equiv. X1.296
Weight of oil in grams
N=Normality of standard alkali, 1.296 = conversion factor between
maleic acid and iodine (127/98)
48
N.B. All Unsaturation values are calculated as I2 equivalents
(B) Determination of hydroxy Fatty Acids Content:
(i) Hydroxyl Value:
 It is the number of milligrams of KOH required to neutralize the acetic acid
which can combine with one gram of an oil upon acetylation process.

O
H3C C
Reflux R CH CH2 R'
R CH CH2 R' + O + CH3COOH
Pyridine O.CO.CH3
OH H3C C
O
After reflux the liberated acetic acid is titrated with standard
KOH.
A blank determination must be carried out
Hydroxyl Value = (B – E) X 56.1
Weight of the oil in grams
49
(ii) Acetyl Value:
 It is the number of milligrams of KOH required to neutralize the acetic acid
obtained when 1 gram of the acetylated oil is saponified.

R CH CH2 R' Known excess of R CH CH2 R'

OH + CH3COOK + H2O
O.CO.CH3 Alcoholic KOH
 The excess KOH is then titrated against standard acid solution.
 Acetyl Value = (B - E) X 56.1
Weight of acetylated oil in grams

 Castor oil is the only oil having a significant acetyl value (about 150) due to
the presence of ricinoleic acid
 [ C6H13CH(OH)CH2CH=CH(CH2)7COOH]
 Acetyl values of most of fats and oils ranged from zero up to 20 units.
 Acetyl Value = Hydroxyl Value
1 + (0.00075 X hydroxyl value)

50
(C) Determination of Volatile Fatty Acids Content:
 Fatty acids up to 10 carbon atoms are volatile in steam but they differ from
each other in their solubility in water and alcohol.

No Fatty Acid Carbon Volatility in Solubility in Solubility in

numbers steam water alcohol
1 Butyric 4 Volatile Soluble Soluble

2 Caproic 6 Volatile Slight.Sol Soluble

3 Caprylic 8 Volatile Slight.Sol Soluble

4 Capric 10 Volatile Very.S.Sol Soluble

5 Lauric 12 Slight.Vol Insoluble Soluble

6 Myristic 14 Non-Vol Insoluble Insoluble

7 Palmitic 16 Non-Vol Insoluble Insoluble

8 Stearic 18 Non-Vol Insoluble Insoluble

51
(i) Reichert Value

 It is the number of milliliters of 0.1 N aqueous standard alkali required to

neutralize the steam-volatile, water soluble fatty acids distilled from 5 grams
of the fat or oil.
 Mainly determine the Butyric acid content and slightly caproic and caprylic
acids.

 5 grams of the oil is treated with 5 ml of 50% NaOH

 Make under reflux to complete the saponification of oil
 Saponified oil is treated with sulfuric acid and heating to liberate the fatty
acids.

 Steam distill the liberated fatty acids and collect the volatile fatty acids
which is either === Water soluble
=== or water insoluble (Alcohol soluble)

52
(ii) Kirschner Value:

 It is the number of milliliters of 0.1 N aqueous standard alkali required to

neutralize the steam-volatile, water soluble fatty acids which form water
soluble silver salts distilled from 5 grams of the fat or oil.
 It is a measure for Butyric acid only, so it is very characteristic for detection
of adulteration in butter fat.
 * Caproic & Caprylic acids content = Reichert V. – Kirschner V.

(iii) Polenske Value:

 It is the number of milliliters of 0.1 N aqueous standard alkali required to

neutralize the steam-volatile, water insoluble fatty acids distilled from 5
grams of fat or oil.
 It is mainly determine the content of Caproic, Caprylic and Capric acids, and
slightly the Lauric acid content.

53
  Reichert, Kirschner and Polenske Values are very important in analysis
of butter fat and some other fats of vegetable origin (Coconut and palm
oils) which also contain the lower molecular weight fatty acids and can
be used as adulterants or substitutes to butter or butter fat.

 Fatty Acid Principal Source

 Butyric acid Milk fats
 Caproic acid Milk fats
 Caprylic acid Milk fats & nut oils
 Capric acid Milk fats, nut & palm oils
 Lauric acid Nut & palm oils

54
(D) Separation of Saturated fatty acids from Unsaturated ones by
Lead-Salt-Ether Method:

 1- 10 grams of the fat or oil is treated with alcoholic KOH and reflux to
obtain the saponified oil
 2- Neutralize the excess KOH by using acetic acid.
 3- Evaporate to dryness and then add 10% lead acetate solution to form lead
salts of fatty acids (All of them are water insoluble)
 4- Filter to separate the other components from the lead salts of fatty acids.
 5- To the residue add ether and reflux, cool, and filter.
 * Residue is the lead salts of saturated fatty acids
 * Filtrate is the lead salts of unsaturated fatty acids.
 6- Residue or Filtrate is treated with HCl to give free F.A.
 7- Extract with ether, separate the ether layer
 8- Ether layer evaporate to dryness to obtain the free Fatty acids.
 9- Weigh and calculate the % content in 10 grams for each type
gravimetrically.

55
(4) Determination of Unsaponifiable Matter in a fat or
oil:
 It is the % of materials not saponified by at least 0.5 N alcoholic KOH and is
insoluble in water soluble in ether and not volatile at 80 0C
 It includes all the fat or oil components accept triglycerides and free fatty
acids.
 They represent about 1-2% of the oil or fat content.

Separation and Determination:

 To 10 grams of fat or oil add excess of 1 N alc. KOH and reflux for 30 minutes
to saponify the oil
 In a separating funnel, shake the saponified oil with ether to separate the
unsaponifiable matter (ether soluble) from the saponifiable one (water
soluble)
 Transfer the ether layer to a weighed flask and distilled off the ether and dry
the residue at 100 0C for 30 min.
 Weigh the flask and determine the % of the unsaponifiable matter present.

56
 (C) Special color Tests For Some Selected Fats &
Oils
1- Test for Cotton-Seed oil (Halphen’s Test):
 Cotton seed oil + CS2 + 1% S0 + Amyl alcohol
 Heat at 120 0C for 1-2 hours using an air condenser
 A Crimson red color of Halphen pigment is produced.

2- Halphen-Insoluble bromide test:

 It is useful test for differentiation between drying, semi-drying and non-
drying oils.
 Oil + Br2 + Glacial acetic acid + Nitrobenzene (as a solvent)
 Non-Drying oils give Dibromo derivatives which is completely soluble in
nitrobenzene (No ppt)
 Semi-Drying oils give Tertabromo derivatives which is partially soluble in the
solvent (Slight turbidity)
 Drying oils give Hexabromo derivatives which is insoluble in nitrobenzene
(Give a marked ppt)
57
 Examples:
 Drying oils Linseed oil
 Semi-Drying oils Cotton-seed, sesame and maize oils
 Non-Drying oils Olive, arachis, almond and castor oils

Oil Type Myrist Palmiti Steari Oleic Linolei Linole I2

ic % c% c% % c% nic % Value

Olive Non- 1 10 3 75 11 0 30-90

drying

Cotton- Semi- 1 21 1 27 50 0 100-

Seed drying 180

Lin- Drying 0 5 3 22 20 50 190-

Seed 250

58
3- Test for Sesame Oil (Baudouin’s Test):
 This test mainly for sesamol alcohol present in the sesame oil which result
from hydrolysis of sesamoline
 The oil is shacked with conc. HCl and few drops of 2% alcoholic furfural
solution for 30 sec.
 A deep red color is formed in the acidic layer

Furfural
OH
H OH
O
Hydrolysis O CHO Red Color
HO H
O Conc. HCl O
OH O O
O C
Sesamoline O C
Sesamol

59
4- Test for Drying Oils (Elaidin Test):
 5 ml of oil + 1.5 ml HNO3 + 3.5 ml H2O in a test tube
 Dip in a piece of copper wire so that a portion of it remains above the liquid level
 Stand for 24 hours and observe the result:
 Non drying and semi drying oils remains liquid as it is
 Drying oils start to solidify (Give solid mass attached to the copper wire)

Copper wire
Non or Semi
Drying oil Drying oil
Aqueous solution

5- Test for Liver oils (Cod-Liver oil):

• Conc. Sulfuric acid is poured down on the side of a tube containing
the oil sample dissolved in CS2 (20% v/v)
• A blue color at the junction of the oil and acid layers indicate the
60
presence of the liver oils.
 (D) Adulteration Detection
 Some Common Examples for Adulteration of high cost and
medicinal oils:

 (A) Detection of Hydrogenated oils as substitute or adulterant

to butter fat:
 Butter fat composition:

 Butyric Caproic caprylic Capric Lauric Myristic

 4% 2% 1% 3% 3% 11%
 Palmitic Stearic Palmitoleic Oleic Linoleic
 25% 10% 5% 30% 3%
 Linolenic Non-Saponifiable matter
 zero% 3% (The highest %)
61
  Due to adulteration the following are the main parameters that
can be affected:

 1- Refractive Index (Decreased) Why ??

 2- Saponification value (Decreased) Why??
 3- Iodine Value (Decreased) Why??
 4- Thiocyanogen value (Decreased) Why??
 5- Volatile fatty acids content [Reichert, Kirschner & Polenske values] (All
are decreased) Why??
 6- Iso oleic acid test positive result
 7- Test for Nickel positive result (May be not)
 8- Individual color tests ( One of them at least +ve)
 9- Non-Saponifiable matter content (Decreased)

62
(B) Cocoa Butter adulterated with paraffin oil:
 Due to adulteration the following are the main parameters that
can be affected:

 Acid, saponification & ester Values (decreased)

 Iodine and the other unsaturation values (decreased)
 Volatile fatty acids content (decreased)
 All the physical parameters (affected)

(C) Cocoa Butter adulterated with cotton-seed or soybean oils:

 Due to adulteration the following are the main parameters that
can be affected:
 Iodine and other unsaturation values (increased)
 Referactive Index (increased)
 Polenske Value (Decreased)
 Halphen’s test +ve result
 Halphen insoluble-bromide test (Slight turbidity)
63
(D) Castor oil adulterated with other semi-drying oils:
Due to adulteration the following are the main
parameters that can be affected:
 Specific Gravity (Decreased)
Ricinoleic acid content (Hydroxyl and Acetyl values
 decreased)
Color tests for vegetable oils (sesame, cotton seed or soybean
oils) one or more give +ve result

(E) Animal fats adulterated with vegetable oils:

Refractive Index (Increased)

Iodine and other un-saturation values (Increased)
Non-Saponifiable matter content (Decreased)

64