Experiment 3: Test of Lipids
Objective:
To explain the principle behind the specific test;
To identify specific fatty acid in a given solution;
To distinguish between identification and conformation tests; and
To enlist various tests used for identification of lipids.
Introduction:
Lipids are a heterogeneous group of compounds, including fats, oils, steroids, waxes, and
related compounds, which are related more by their physical than by their chemical properties.
Lipids classified as simple, conjugated and derived lipids. Apart from acting as energy source, lipids have
diverse functions ranging from plasma membrane formation, micelle formation, hormonal action and
thermal insulator etc. Lipids are a class of compounds distinguished by their insolubility in water
and solubility in non-polar solvents. Lipids are important in biological systems because they
form the cell membrane, a mechanical barrier that divides a cell from the external
environment. Non polar lipids, such as triglycerides, are used for energy storage and fuel. Polar
lipids, which can form a barrier with an external water environment, are used in membranes.
Polar lipids include glycerophospholipids and sphingolipids. Fatty acids are important
components of all of these lipids. Lipids have the common property of being relatively insoluble
in water and soluble in nonpolar solvents such as ether and chloroform, benzene, and hexane.
They are important dietary constituents not only because of their high energy value but also
because of the fat-soluble vitamins and the essential fatty acids contained in the fat of natural
foods.
However the availability of free floating lipids in blood and their accumulation in various vital parts of
our body is one of the major causes for life style disorders like obesity and cardiovascular diseases. In
this experiment we will be studying about the different tests performed to identify the chemical nature
of lipids i.e., saturated or unsaturated along with presence or absence of sterols.
The tests conducted to identify lipids are specific to the chemical nature and functional groups present
on them. Qualitative analysis of lipids has a significant contribution in identifying the adulteration of
edible oils and hence has vital role in industry, as well as in health sector
Materials:
Glassware: 10 mL boiling test tubes, pipettes (1-10 mL), dropper, glass rod and watch glass.
Equipment and accessories: Water bath, test tube holder, spatula and blotting paper.
Test sample: The commercially available oil samples like Coconut oil, Ghee, Palm oil, Butter and
cholesterol can be used.
Reagents and Chemicals:
Alcoholic-KOH (2% w/v KOH in ethyl alcohol): Dissolve 2 g of potassium hydroxide (KOH) in 100
mL of ethyl alcohol. Dilute potassium permanganate solution (0.05% w/v): Dissolve 50 mg of
potassium permanganate in 100 mL of distilled water.
Hubl’s Iodine Solution: To prepare this (a) dissolve 2.6 g of iodine in 40 mL of ethanol (95% v/v),
( b) dissolve 6.0 g of mercuric chloride in 40 mL of ethanol (95% v/v). Transfer both solutions ‘a’
and ‘b’ into a beaker and make up the volume to 100 mL with same ethanol.
Potassium bisulphate (solid) Bromine water acetic anhydride and Conc. sulphuric acid.
Solvents: Chloroform, ether, benzene, carbon tetrachloride, hexane.
Solubility Test:
This is a primary step to know the chemical nature of the given test sample
Principle: Due to hydrophobic of nature of lipids they are insoluble in water and are soluble in organic
solvents.
Saponification Test:
Principle: Lipids upon alkaline hydrolysis release glycerol and fatty acids. Later sodium (Na+) or
potassium (K+) ions combines with fatty acids to form “soap” (foam). Hence, this is known as
saponification reaction.
Acrolein Test:
Principle: Formation of acrolein or acrylic aldehyde that has characteristic pungent odor is the key
principle. In general lipids upon heating with potassium bisulphate produce acrolein.
Hubl’s Iodine Test (Tests for unsaturation):
This test is mainly helpful to assess the unsaturation in a given oil sample. The two principles that will
explain the reaction mechanism are given below.
Principle:
i) Bromine or iodine upon reacting with unsaturated fatty acids produce di-halo adducts.
Consumption of more bromine indicates the higher percentage of unsaturation.
ii) Decolorization of alkaline potassium permanganate is also an indirect measure of
unsaturation in fatty acids, where unsaturated fatty acids undergo incomplete oxidation.
Salkowski Test and Liebermann-Burchard Test (Test for Cholesterol):
Principle:
Cholesterol in the presence of concentrated sulphuric acid and acetic anhydride undergo dehydration
producing colored product which is blue-green in color.
Procedure for qualitative analysis of lipids
Tests for Lipids
Experiment No.
Name: ID No. Date:
Sample-
Test Observation Inference
Appearance
Solubility
Test
Saponification
Test
Acrolein Test
Tests for
Unsaturation
1. Bromine
water Test
2. Hubl’s iodine
Test
Test for
Cholesterol
1. Salkowski
Test
2. Liebermann-
Burchard test
Sample:
