BIOMOLECULES LABORATORY

ACTIVITY 2

QUALITATIVE ANALYSIS OF LIPIDS

Introduction
Lipids are biomolecules that are structurally poorer in oxygen but made up of carbon,
hydrogen and oxygen like carbohydrates. These are groups of structurally different molecules that
are amphipathic in nature of which the majority is hydrophobic, however each molecule contains a
polar region. The hydrophobic nature of the molecule strongly determines the structures these
molecules assume in nature. Being structurally diverse, lipids have a variety of biochemical
functions, including structure, energy, energy storage, and signaling (steroids and eicosanoids). The
bulk of the mass of lipid molecules in a mammalian system are those found structurally in cellular
membranes and those involved in storage as triacylglycerides and cholesterol esters. Phospholipids
are a major component of cellular membranes and can be divided into classes based on their
headgroup and further into species based on the acyl group composition.
Based on the structure of lipid’s backbone, they are divided into two groups; complex lipids
and simple lipids. The complex lipids are esters of fatty acids. In various forms of complex lipids
like acylglycerides, phospholipids, sphingolipids and waxes, fatty acids are covalently joined via an
ester linkage to trihydroxy alcohol, glycerol, or its derivative. For example, a triglyceride or
triacylglycerol can be formed from a glycerol molecule and three fatty acids.
The property of specific solubility is an important consideration for extracting lipids from
tissues, free from any water-soluble matter, such as using organic solvents that are usually employed
to separate lipids from proteins, carbohydrates and water-soluble metabolites. However, the
subsequent analytical methods are largely individualistic.
The quality of lipids incorporated in our diet may influence our health. The triglyceride or
triacylglycerols are the major component of most foods, typically making up more than 95 to 99% of
the total lipids present. Saturated lipids are mostly solid at room temperature and could raise the
level of bad cholesterol (Low density lipoprotein or LDL) which may pose potential health
consequences. The unsaturated lipids, however, are either monounsaturated or polyunsaturated
which are both liquid at room temperature, but only the polyunsaturated lipids remain liquid even
when chilled. The monounsaturated fats are considered most desirable as they help increase the good
cholesterol (High density lipoproteins or HDL) in our body. The polyunsaturated fats on the other
hand, could lower the overall cholesterol level, but they also reduce the good cholesterol (HDL).

Learning Objectives
At the end of the activity, the students are expected to:
1. Determine the nature and structure of different lipids; and
2. Identify the activities/reaction of lipids.
3. Discuss the advantage and disadvantage of certain fats we incorporate in our diet.

Samples:
Olive oil, vegetable oil, canola oil, butter, milk, starch solution, one unknown sample
Materials/Chemicals and Reagents:
Water bath
Test Tubes
Transfer pipettes
Dilute acetic acid
Dilute potassium hydroxide
Ethanol, benzene, ether, acetone, chloroform, carbon tetrachloride
250 ml conical flasks
Bile salt
Whatman No. 1 filter paper
Bromine water, Acetic acid, H2SO4,
Acetic anhydride
Sudan IV

Pre-lab Preparations

10%Ethanol solution of KOH. Weigh 10 g of KOH. Dissolve in 50 ml of ethanol and make the
volume to 100 ml.
0.4% of Bromine water. Measure 0.4 ml of bromine and add 99.6 ml of water.
Sudan IV (0.1%). Weigh 100 mg of Sudan IV and dissolve in 100 ml of water.

Procedure
A. Paper spot test
1. Rub a bit of sample on a piece of paper.
2. If the spot on the paper assumes a translucent appearance, that means the sample under
test is a fat or oil, i.e., a lipid.

B. Solubility Test
1. Take 5 ml of water, dilute acetic acid, dilute KOH, ethanol, benzene, ether, acetone,
chloroform and carbon tetrachloride in nine test tubes separately.
2. Add a few drops of the sample in each test tube.
3. Shake well, allow the tube to stand and observe if the mixture becomes homogeneous.

C. Emulsification Test
1. Take 5 ml of distilled water, add a few drops of emulsifier (bile salt) and the same
amount of sample in a test tube.
2. Shake well to mix the contents.
3. The lipid is dispersed forming an emulsion in the form of micelles, i.e., fats, which are
broken up into minute droplets in water. (This is brought about as a result of the lowering
of the surface tension between fat and water. The bile salts or other substances that bring
about emulsification are referred to as emulsifiers.)

D. Sudan IV test
1. Take a few ml of distilled water in a test tube and add a drop or two of the sample to it.
2. Shake the tube so that the mixture is converted into an emulsion.
 3. Place a small drop of this emulsion on a micro slide and add to it a little bit Sudan IV
stain, mix and put a cover glass.
4. Examine under a microscope and note that only oily droplets are stained, this happens,
because the stain is very specific for lipids.

E. Bromine Test for unsaturation / saturation

1. Take a few ml of ethanol in a test tube and add a few drops of sample to it.
2. To this add a few drops of 0.4% bromine water drop by drop. (An orange colour is
developed.)
The intensity of the colour developed corresponds to the degree of unsaturation. If the
same test is performed with acetic acid, no colour will be developed, as it is a saturated
acid.

F. Liebermann-Buchard test for cholesterol.

1. Take 2 ml of chloroform in a test tube and dissolve a pinch of the sample in it.
2. To this add at first 8-10 drops of acetic anhydride and then a drop or two of concentrated
H2SO4 from the sides of the test tube. (A greenish or greenish blue colour will be
developed indicating the presence of cholesterol.)

Note:
Protective gloves and goggles must be worn all the time inside the laboratory and especially when
handling the following chemicals:
 acetic acid, dilute KOH, ethanol, benzene, ether, acetone, chloroform and carbon
tetrachloride
The glassware must be cleaned with distilled water, dried in hot air oven before use.
Prepare the reagents with glass distilled water.

References:

Swamy PM. 2009. Laboratory manual on biotechnology. Meerut, India: Rastogi Publications
Type of Fats. Availabe from: https://www.fediol.eu/web/type%20of%20fats/1011306087/list1187
970104/f1.html
Olive Oil Source. Chemical Characteristics. Available from: https://www.oliveoilsource.com/page/
chemical-characteristics
 BIOMOLECULES LABORATORY

ACTIVITY 2

QUALITATIVE ANALYSIS OF LIPIDS

NAME: _______________________________________ PROGRAM CODE: ____________

GROUP NO. ___________________________________ DATE: _______________________

Table 1. Positive Reactions for Lipids

Tests
Substance Unsaturated/
Paper spot Emulsification Sudan IV Cholesterol
saturated
Olive oil
Vegetable
oil
Canola oil
Butter
Milk
Starch
solution
Unknown

Legend: Presence (+); Absence (-)

Table 2. Solubility of Lipids

Samples
Solvents Unknown
Water
Acetic acid
KOH
Ethanol
Benzene
Ether
Acetone
Chloroform
Carbon
tetrachloride
Guide Questions:

1. Which test/s are confirmatory for lipids? Explain.

2. Account for the solubility of confirmed lipids in the given solvents.

3. Based on the quality of lipid samples, give an assessment as to the advantages or

disadvantages they can give to our health.

4. Give an assessment to the quality of lipid in the given unknown sample.

*Use a separate sheet of paper for the answers to the guide questions and conclusion.

Conclusion:
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

References:
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________