GROUP 3

Illustrated Laboratory Activity 3: Qualitative Test for Lipids

Introduction:
Lipids being insoluble in water known as hydrophobic biomolecules, but they are soluble in organic solvents like
benzene, chloroform, hexane and diethyl ether. It is well known fact that lipids are high energy compounds and are
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. 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. However 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 Required:
• 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.

Procedure for Qualitative Analysis of Lipids

TEST OBSERVATION INFERENCE
Solubility test:
To small quantities of fat/fatty acid
taken in a separate test tube add the
following organic solvents separately:
1. a) Chloroform, I.) Completely soluble in both I.) The samples are miscible in the
b.) ethyl alcohol solvents chloroform and alcohol, indicating
the presence of oil or fat in the
samples.
2. Distilled water II.) Found small floating droplets on II.) The sample is miscible in distilled
water. water, indicating the presence of oil
or fat in the samples.
Saponification test: All the samples form a lower layer in
Take approximately 100 mg of oil or Appearance of alcohol, when a small quantity of
fat in a test tube. Add 3 mL of foamy solution ethyl alcohol pour into test tubes.
alcoholic-KOH and mix well. Place the And when the test tubes is heated in
tube in a boiling water bath for 15-20 a boiling water bath, the samples are
min. dissolved on heating, indicating the
presence of oi or fat in the samples.
Acrolein test: The sample indicates the presence of
Take approximately 100 mg of test Release of glycerol. When oil or fat is treated
sample in a test tube and add a pinch pungent odor strongly in the presence of
of potassium bisulphate and mix well. dehydrating agent like potassium
Heat the mixture over a Bunsen bisulphate, the glycerol portion of
burner for 1-2 minutes. the molecule is dehydrated to form
an unsaturated aldehyde acrolein
which has a pungent irritating odor.
 Tests for unsaturation:
Hubl’s iodine test:
Take 1 mL of oil or 100 mg of fat in a i) Decolorization of potassium The violet color of iodine fades away
test tube add 2 mL of nonpolar permanganate or bromine water is in test tube B but it does not fade
organic solvent (Refer solubility test) observed away in test tube A. This indicate that
and dissolve completely. Add Hubl’s linseed oil is more unsaturated than
reagent drop wise mixing the ii) No decolorization observed. cotton seed oil.
contents.

Summary or Conclusion:
• The given test or the solubility test gives us an idea about the nature of the samples. Like it is used to
determine a compounds ability to dissolve in a solvent which is usually are liquids.
• Saponification Test is used to determine the average relative molecular mass of oils and fats.
• In acrolein test if there is a pungent irritating odor then the presence of fats or oil is confirmed.
• Hubl’s iodine test confirms the presence or absence of unsaturation in the given sample.

Laboratory Guide Questions:

1. Why is there a need perform qualitative test for lipids?
➢ It is useful in determining the presence or absence of lipid depending upon the color change of the
lipids reactivity with the chemical reagents, and classifying the heterogenous lipids (like wax, steroids,
triglycerides, fats, etc.) based on the properties like solubility, emulsification, oxidation etc.

2. Enumerate some emulsifying agents and why they are so called emulsifiers?
➢ Phospholipids and bile salts are emulsifying agents because they have a hydrophilic head that is
attracted to water and a hydrophobic tail that is attracted to lipid molecules.

3. Why are fats soluble in organic solvents but not in water?

➢ Fats are wide and diverse group of biological compounds defined by their inability to dissolve in water,
or hydrophobicity. The numerous nonpolar covalent bonds in lipids give them their hydrophobic
properties. Water, on the other hand, possesses polar covalent bonds and hence only mixes well with
polar or charged substances.
4. What is the importance of iodine test?
➢ The iodine test is useful for determining how much unsaturation is present in fatty acids. Double bonds
react with iodine compounds, causing unsaturation. The more unsaturated fatty acid linkages there are
in a fat, the higher the iodine value.
5. Give the reasons why fats develop rancid odor.
➢ Fats containing unsaturated fatty acids are more susceptible to rancidity. Rancidity occurs when fats
and oils are exposed to air, moisture, light, bacteria etc. Hydrolytic rancidity occurs due to partial
hydrolysis of triacylglycerol by bacterial enzymes. Oxidative rancidity is due to oxidation of unsaturated
fatty acids. This results in the formation of unpleasant products such as dicarboxylic acids, aldehydes
and ketones.