Determination of
Saponification Number
Saponification
On refluxing with alkali, triacylglycerols (fatty acid esters) are
hydrolyzed to give glycerol and potassium salts of fatty acids (soap).
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Saponification number
Saponification number is defined as the is the number of milligrams of KOH
required to neutralize the fatty acids resulting from the complete hydrolysis of
1g of fat.
It is an indication of the molecular weight of the fat, and is inversely
proportional to it. Human fat has a saponification number of 194-198, butter
has 210-230 and coconut oil has 253-262.
■ The saponification value gives an
indication of the nature of the fatty acids
constituent of fat and thus, depends on
the average molecular weight of the fatty
acids constituent of fat.
• The greater the molecular weight (the
longer the carbon chain), the smaller the
number of fatty acids is liberated per
gram of fat hydrolyzed and therefore, the
smaller the saponification number and
vice versa.
Materials
1- Fats and oils (olive oil, coconut oil, sesame oil, and butter)
2- Fat solvent (equal volumes of 95% ethanol and ether)
3-Alcholic KOH (0.5 mol/liter)
4-Reflux condenser.
5-Boiling water bath.
6-Phenolphethalein.
7-Hydrochloric acid (0.5 mol/liter)
8-Burettes (10 ml and 25 ml)
9-Conical flasks (250ml)
Determination of Saponification Number
0.5mol/L HCL Phenolphthalein ([Link])
•.
25 ml alcoholic KOH
3 ml fat solvent
1 ml fat
Heat flask on a boiling water bath for 30 min.
Leave to cool to room temperature
Calculations
• The difference between the blank and the test reading gives the
number of milliliters of KOH required to saponify 1g fat.
• You can use this formula to calculate the saponification value:
1ml (0.5 N HCl ) = 28.05 mg KOH
( B-T ) = S
■ Saponification value (S) =
( B-T ) x 28.05 = mg KOH/1g
Wt. of fat (1g)
Iodine Number
Iodine number of a fat is defined as the number of grams of iodine taken
up by 100 grams of fat. It is an index of the degree of unsaturation and
is directly proportional to the content of unsaturated fatty acids. Higher
the iodine number, higher is the degree of unsaturation, e.g. iodine
number of butter is 28, and that of sunflower oil is 130.
Rancidity
Rancidity of Fat Fats and oils have a tendency to become rancid. The
term rancidity refers to the appearance of an unpleasant smell and taste
for fats and oils.
✓ Hydrolytic rancidity is due to partial hydrolysis of the triacyl glycerol
molecules due to traces of hydrolytic enzymes present in naturally
occurring fats and oils.
✓Oxidative rancidity is the result of partial oxidation of unsaturated
fatty acids with resultant formation of epoxides and peroxides of small
molecular weight fatty acids by peroxides and free radicals. The same
process, if it occurs in vivo will affect the integrity of biomembranes,
leading to cell death
Many natural fats and oils may contain antioxidants (e.g. vitamin E),
which prevent the occurrence of oxidative rancidity. PUFA are more
easily oxidized; so vegetable oils with a high content of PUFA are
usually preserved with addition of antioxidants. Repeated heating of oils
would lead to the formation and polymerisation of cyclic hydrocarbons.
These will impart an unpleasant taste and color to the oil. Coconut oil
having medium chain saturated fatty acids will withstand such
polymerisation
Amphipathic Nature
✓Phospholipids in general are amphipathic, particularly Lecithin. They
have both hydrophobic and hydrophilic portion in their molecule.
✓The glycerol along with the phosphoric acid and choline constitute the
polar ‘head' of a phospholipid molecule, whereas the hydrocarbon
chains of the fatty acids represent the nonpolar ‘tail'
PL form the bilayer
Micellar Formation
When phospholipids are distributed in water, their hydrophobic parts
keep away from water, forming molecular aggregates called micelle.
These are involved in solubilization of lipids in aqueous media and help
in digestion and absorption of lipids.
Liposomes
A lipid bilayer will close on itself under appropriate conditions to form
liposomes. Unilamellar or multilamellar liposomes may be formed.
They may be prepared by sonication of mixtures of phospholipids and
cholesterol.
Liposomes are microscopic spherical vesicles. When mixed in water
under special conditions, the phospholipids arrange themselves to form
a bilayer membrane which encloses some of the water in a phospholipid
sphere. Drugs, proteins, enzymes, genes, etc. may be encapsulated by
the liposomes which could act as carriers for these substances to target
organs. Liposome-entrapped drugs exhibit superior pharmacological
properties than those observed with conventional formulations.
Liposomes have important applications in cancer chemotherapy,
antimicrobial therapy, gene therapy, vaccines and diagnostic imaging
PL form micelles and liposomes
Biomembranes
The molecules align themselves to form monolayers with the polar
heads pointing in one direction and the nonpolar tails in the opposite
direction. Only fatty acids with more than 6 carbon atoms form
monolayers. This explains their role as components of biomembranes
The self-assembly of phospholipids into bilayers is driven by
hydrophobic interaction. They also act as detergents and emulsifying
agents. In vivo, they act as pulmonary surfactants.
Action of Phospholipases
Phospholipases are enzymes that hydrolyse phospholipids. Different
phospholipases are involved in the hydrolysis of specific bonds in lecithin.
Phospholipase A2 acts on an intact lecithin molecule hydrolysing the fatty
acid esterified to the beta (second) carbon atom. The products are
Lysolecithin and fatty acid. Lysolecithin is a detergent and hemolytic agent.
The enzyme is present in the venom of viper snakes. The hemolysis and
consequent renal failure seen in viper poisoning could be thus explained. The
products formed in each case may be summarized as follows
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