PHR 326_ Oil Analysis-Power Point--

Prof. MOHAMMED HAFIZ 

12/10/2012 1 

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Chemical structure of lipids 

Lipid is an ester of fatty acid and alcohol. 

If glycerol is alcohol the ester is known as 

triglyceride which almost constitutes oils and fats 

(LIPIDS). 

If alcohol is a long chain of high molecular weight 

monohydroxy alcohol the ester is known as wax 

e.g., beeswax. CH 2 - OCOR 1 

R 2OCO 

C 

* 

H 

CH 2 - OCOR 3 

oils and fats 

CH 3 - (CH 2) 12 - OCO - (CH 2) 14 - CH 3 

beewax 

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Composition of lipids 

Lipid is a solution, its solvent is the glycerides 

(saponifiable part) and its solute is non-glycerides (nonsaponifiable 

part). 

1) Non-saponifiables (or Unsaponifiables) 

It is that part of lipid which is not affected by 

alkali-hydroxides during saponificaiton of lipid and can be 

extracted by organic solvents after saponifcation (as 

vitamins, sterols and resins). They are non-volatile on 

drying at 80 o C. It rarely exceeds 2%. 

2) Saponifiable part (glyceride part) 

They are esters of fatty acids and glycerol which 

upon saponification with alkali gives glycerol and alkali salt of 

fatty acids (soap). It is the major part of lipid as it 

constitutes about 99% of lipid. Therefore the chemical and 

physical properties of lipids vary with variations of the 

glyceride part (fatty acid composition). 

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Glycerides 

Fats and oils are ESTERS of glycerol and 

long chain carboxylic acids 

Glycerides 

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Saponifiable 

Part(Glyceride) 

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Saponifiable 

Part(Glyceride) 

Triglycerides may be: 

-Simple glycerides: the same fatty acid 

radicals 

-Mixed glycerides: different fatty acid 

radicals 

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Composition of lipids 

2) Saponifiable part (glyceride part) 

CH 2 OCOR CH 2 OH 

heat 

CHOCOR + KOH CHOH + 3 RCOOK 

CH 2 OCOR CH 2 OH 

Glycerol 

Immiscible with Miscible with 

aqueous solution aqueous solution 

Soap 

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I- Physical examination 

vDetermination of water 

The moisture content of oils and fats is determined by Karl- 

Fischer reagent. 

vMelting point (M.P.) 

Melting point of lipid is directly proportional to the chain 

length of fatty acids and inversely proportional to the number 

of double bonds. 

vSpecific gravity 

weight of certain volume of oil 

Specific gravity (Sp.gr.) = 

weight of the same volume of water 

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II- II Chemical Examination 

The chemical examinations of lipids involve 

reactions with ester linkage of the glycerides or 

free carboxylic acid, hydroxyl group of hydroxyl 

acids as well as the double bonds of the 

hydrocarbon chain of fatty acids, in order to 

differentiate between the different types of 

lipids, through determination of some chemical 

constants. 

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Acid value 

The acid value, AV, is the number of milligrams of 

KOH required to neutralize the free fatty acids (FFA) 

present in 1 g of the substance. 

Determination 

Dissolve the substance examined in ethanol/ether 1:1, 

titrate against 0.1M KOH, ph.ph. indicator 

mL KOH×M× 56 

Acid value = ______________ 

sample wt in gm 

-Edible oil contain > 1% 

-Pharmaceutical oil must not have any acidity. 

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Acid value 

Significance 

Acid value is the measure of 

hydrolytic rancidity (action of lipase on 

triglycerides to give FFA). In general, it 

gives an indication about freshness and 

edibility of the lipid 

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The saponifcation value is the number of mg of KOH 

required to neutralize the free fatty acids and to 

saponify the esters in 1 g of the substance. 

SPV = FFA+combined FA 

Saponification value 

Determination 

A certain weight of the substance is refluxed with 

known volume of 0.5M ethanolic KOH, then the excess 

unreacted 0.5M KOH is titrated against 0.5 M HCl, 

using ph.ph. indicator. 

(Total – e.p.) ×M×56 

Saponifcation value = _______________________ 

sample wt in gm 

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Saponification value 

Significance 

Saponification value is inversely proportional to the 

mean molecular weight of fatty acids (or chain length). 

S.V. of butter fat and vegetable fats ~ 220 – 250 

S.V. of fixed oils (vegetable oils) ~ 195 

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Ester value 

The ester value is the number of milligrams 

of KOH required to saponify the esters present in 

1 g of the substance. 

It is calculated from the saponification value and 

the acid value 

Ester value = saponification value (SPV)-Acid value (AV) 

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Acetyl value 

It is the number of mg KOH required to neutralize the 

acetic acid liberated by the hydrolysis of one g of 

acetylated oil. 

Determination 

non acetylated oil determine its SPV 

Oil sample 

+(CH 3 CO) 2 O acetylated oil +CH 3 COOH 

determine its SPV 

Acetyl value=SPV of acetylated part-SPV of non 

acetylated part 

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Significance 

Acetyl value 

Acetyl value is the measure of hydroxy acids 

in lipids. 

Acetyl value of castor oil (Recin oil) = 150, 

because it is rich in recinoleic acid. 

Acetyl value of Oils containing no hydroxyl 

fatty acids = 5-15 

Oxidative rancidity formation of hydroxy 

acids rise acetyl value 

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Iodine value I.V. 

It is the number of parts of I 2 absorbed by 100 

parts of lipid by weight. (1gm of fat absorbed 

1.5 gm I 2 , I.V.=150) 

The following reagents can be used as halogenating 

agents during the determination of I.V. 

Halogenating agent Composition Time of addition 

1) Wij's reagent I 2 /Cl 2 

2) Hanus reagent I 2 /Br 2 

in gl. acetic acid 30-60 minutes 

in gl. acetic acid 60-120 minutes 

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Determination 


Take a certain weight of substance examined, 

dissolve in organic solvent, add known volume of 

halogenating agent, keep in dark for 60 min. Add 

KI solution and titrate the libarated I 2against 

st. Na 2S 2O 3 

IBr + KI I 2 + KBr 

I 2 + 2Na 2 S 2 O 3 2I - + 2Na 2 S 4 O 6 

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vSignificance 

I.V. is the measure of the degree of 

unsaturation of fatty acids composing the 

glycerides of oils or fats. 

I.V. is directly proportional to the degree of 

unsaturation (No of d.b.) and inversely proportional 

to the melting point (m.p.) of lipid. An increase in 

I.V. indicates high susceptibility of lipid to 

oxidative rancidity due to high degree of 

unsaturation. 

1 

I.V. a No of d.b a ________ Iodine value I.V. 

a succeptibility to 

M.P. oxidative rancidity 

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According to I.V. oils can be classified into 

the following classes: 

Class I.V. The main 

1- Non-drying 

oils 

2- Semidrying 

oils 

unsaturated acid 

Examples 

80-100 Oleic acid (C 18:1) Olive oil, Castor oil, 

almond oil 

100-140 Linoleic (C 18:2) Cottton seed oil, 

sesame oil 

3- Drying oils 140-200 Linolenic (C 18:3) Linseed oil, 

Fish oil, sunflower oil 

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Rancidity is a process which is accompanied 

by the formation of unpleasant odour, 

taste, as a result of the action of 

moisture, air (O 2 ) and enzymes 

Types of rancidity 

1- Hydrolytic rancidity (lipolytic rancidity) 

2- Oxidative rancidity 

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1- hydrolytic (lipolytic) rancidity 

T.G. Moisture 

Lipase 

1,2-diglyceride 

2,3-diglyceride 

Free fatty acid 

2-monoglycerides 

Free fatty acids 

Glycerol 

free fatty acids 

To guard against this type, we have to protect 

lipid from moisture and direct light as well as 

its storage must be in a cold place to 

deactivate lipase. 

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2- Oxidative rancidity 

It is caused by the attack of oxygen on the 

unsaturation centers in oils and fats with the 

formation of peroxides (early stage or incipient 

rancidity) 

When the concentration of peroxides increases 

(later stages or progressive rancidity), some 

chemical changes occurs with the formation of 

epihydrine aldehydes, hydroxy and keto acids 

which are responsible for rancid taste and 

odor. 

The greater the degree of unsaturation, the 

greater the liability of the lipid to oxidative 

rancidity. 

It is accelerated by exposure to heat and light 

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Detection and determination of oxidative 

rancidity 

I- Qualitative test (Kreis-Kerr test) 

Used for detection of epihydrine aldehydes 

(progressive rancidity). 

Reagent : Phloroglucinol in alcohol or ether. 

Test 

n Oil is treated with the reagent and conc. HCl. 

Shake and leave to stand for one hour. 

Appearance of pink color indicates the presence 

of aldehydes (rancidity). 

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II- Quantitative determination of oxidative 

rancidity 

Peroxide Value 

n It is a measure of peroxides which indicates 

incipient rancidity. 

n The peroxide value is the number of milliequivalents 

of active oxygen that expresses 

the quantity of peroxide contained in 1000 g 

of the substance. 

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Peroxide Value 

Determination 

Wt of the substance, add (KI/ gl.acetic) and 

leave for one hour, KI/ gl.acetic reacts with 

bond oxygen with the liberation of equivalent 

amount of I 2 which is determined by 

titration against st. Na 2S 2O 3. 

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Genuine or adulterated sample? 

qThe close similarity between the physical 

constants for various oils and fats leads to 

adulteration of more expensive oils and fats 

with cheaper ones 

qAdultration is not always detected by 

determination of chemical constants (S.V & 

I.V), some special tests (Identification tests) 

are set to detect a number of oils used for 

adultration, so we can conclude, whether the 

sample is genuine or adulterated or completely 

substituted. 

qBellier’s test (Peanut oil) 

qHalphen’s test (cotton seed oil) 

qBaudouin’s test (sesame oil) 

qHalphen’s insolube bromide test (Fish oil) 

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I-Identification 

tests 

Bellier’s test 

It is used for the detection of peanut oil 

(or arachies oil), peanut oil in cotton 

seed oil, olive oil, corn oil and soyabean 

oil. 

Test 

Saponify one g of oil by reflux with 5 ml (1.5 

N) alcoholic KOH for 3 minutes, add 50 ml 

70% alcohol and 0.8 ml HCl (sp. gr. 1.16). 

Heat to dissolve any ppt. Cool in temperature 

rate 1 o C/minute. If turbidity appears at 9 o C, 

this indicates the presence of peanut oil in 

olive oil. 

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tests 

Halphen’s test 

It is used for the detection of cotton seed oil 

Halphen reagent : It is composed of equal 

parts of amyl alcohol and carbon disulphide 

(CS 2) containing 1% S o . 

Test 

Equal parts of oil and reagent are heated 

together for 30-60 minutes. Appearance of 

red colour indicates the presence of gossypol 

(phenolic compound present in cotton seed oil) 

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tests 

Baudouin’s test 

It is used for the detection of sesame oil. 

Reagent : It is composed of 2% alcoholic 

furfural or (sucrose in conc. HCl). 

Test 

1 ml oil is treated with 1 ml conc. HCl, 

shake, add the reagent and shake for 15 

minutes. Appearance of pink color indicates 

the presence of sesame oil (due to the 

presence of the phenolic compound sesamol). 

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tests 

Halphen’s insoluble bromide test 

It depends on the fact that fish oils contain glycerides 

of highly unsaturated fatty acids (>4 db), upon 

bromination it gives insoluble polybromides which 

blacken but do not melt above 200 o C. 

Test 

Oil sample, dissolve in dry ether, add Br 2 /gl. Acetic , 

leave to stand in ice where polybromides ppted. 

N.B. Vegetable oils give soluble bromides, except 

linseed oil which gives insoluble bromides but they melt 

without blackening at 180 o C. 

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II- 

Chemical constants 

1-Iodine value (I.V.) 

I.V. may be increased or decreased, according 

to the I.V. of the adulterant: 

(a) If the adulterant is mineral oil (liquid 

paraffin) which has I.V. zero, the I.V. of 

analysed sample will be decreased according to 

the percentage of adulteration. 

(b) If drying oil is adulterated with nondrying or 

semidrying oil, its I.V. will be decreased and 

vice versa. 

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II- 

Chemical constants 

2- Saponification value (S.V.) 

Vegetable (or fixed) oils have nearly the 

same S.V., so adulteration of oil with 

another oil, shows no significant change in 

S.V., but the presence of mineral oil which 

has zero S.V. will markedly decrease S.V., 

also incomplete saponifcation of the 

analysed sample is observed. (If the sample 

is adultrated with paraffin oil, a long chain 

hydrocarbon, both S.V. and I.V. will 

decrease and after saponification two 

layers are obtained as part will remain 

unsaponifiable). 

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•Fats and oils found many applications in 

pharmacy 

•Several fats and oils are official in different 

pharmacopoeias 

Refined Olive Oil 

Acid value: not more than 0.5 

Peroxide value: not more than 10. 

If intended for use in the manufacture of 

parentral dosage forms, not more than 0.5. 

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Pharmacopoeial 

Monographs 


DEFINITION 

Refined olive oil is the fatty oil obtained by refining of crude olive oil, obtained 

by cold expression or other suitable mechanical means from the ripe 

drupes of Olea europaea L. A suitable antioxidant may be added. 

CHARACTERS 

A clear , colourless or greenish-yellow, transparent liquid, practically insoluble 

in alcohol, miscible with light petroleum (50 °C to 70 °C). 

When cooled, it begins to become cloudy at 10 °C and becomes a butter-like 

mass at about 0 °C. It has a relative density of about 0.913. 

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TESTS 

Pharmacopoeial 

Monographs 

Acid value (2.5.1) 

Not more than 0.5, determined on 10.0 g. 

Peroxide value (2.5.5, Method A) 

Not more than 10.0. If intended for use in the manufacture of 

parenteral dosage forms, not more than 5.0. 

Unsaponifiable matter 

Not more than 1.5 per cent. 


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Pharmacopoeial Monographs 


Composition of fatty acids (2.4.22, Method A) 

—saturated fatty acids of chain length less than C16 : not more than 

0.1 per cent, 

— palmitic acid : 7.5 per cent to 20.0 per cent, 

—palmitoleic acid (equivalent chain length on polyethyleneglycol 

adipate 16.3): not more than 3.5 per cent, 

— stearic acid : 0.5 per cent to 5.0 per cent, 

—oleic acid (equivalent chain length on polyethyleneglycol adipate 

18.3): 56.0 per cent to 85.0 per cent, 

—linoleic acid (equivalent chain length on polyethyleneglycol adipate 

18.9): 3.5 per cent to 20.0 per cent, 

—linolenic acid (equivalent chain length on polyethyleneglycol 


—arachidic acid: not more than 0.7 per cent, 

—eicosenoic acid (equivalent chain length on polyethyleneglycol 


—behenic acid: not more than 0.2 per cent, 

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Action and use 

Pharmaceutical aid. 

DEFINITION 

Virgin oil obtained by cold expression from ripe seeds of Linum 

usitatissimum L. A suitable antioxidant may be added. 

CHARACTERS 

Clear , yellow or brownish-yellow liquid, on exposure to air turning dark 

and gradually thickening. When cooled, it becomes a soft mass at 

about - 20 °C. 

Relative density 

About 0.931. 

Refractive index 

About 1.480. 

Virgin Linseed Oil 

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TESTS 

Acid value ( 2.5.1 ) 

Maximum 4.5. 

Iodine value ( 2.5.4 ) 

160 to 200. 

Peroxide value ( 2.5.5 ) 

Maximum 15.0. 


Saponification value ( 2.5.6 ) 

188 to 195. Carry out the saponification for 1 h. 

Unsaponifiable matter ( 2.5.7 ) 

Maximum 1.5 per cent, determined on 5.0 g. 

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Composition of fatty acids 


—fatty acids with a chain length less than C 16 : maximum 1.0 

per cent, 

— palmitic acid : 3.0 per cent to 8.0 per cent, 

—palmitoleic acid (equivalent chain length on 

polyethyleneglycol adipate 16.3): maximum 1.0 per cent, 

— stearic acid : 2.0 per cent to 8.0 per cent, 

—oleic acid (equivalent chain length on polyethyleneglycol 

adipate 18.3): 11.0 per cent to 35.0 per cent, 

—linoleic acid (equivalent chain length on polyethyleneglycol 


—linolenic acid (equivalent chain length on polyethyleneglycol 


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PHR 326_ Oil Analysis-Power Point--

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