Experiment No.
- 05
SCT 222 – 2
Biochemistry
Properties of Lipids
UWU/SCT/20/035
Rameesh Ishak
2022.11.28
Date - 2022.11.21
Experiment No. – 05
Experiment – Properties of lipids
Theory -
Fats and lipids are an essential component of the homeostatic function of the human body. Lipids contribute
to some of the body’s most vital processes. Lipids are fatty, waxy, or oily compounds that are soluble in
organic solvents and insoluble in polar solvents such as water. Lipids include:
• Fats and oils (triglycerides)
• Phospholipids
• Waxes
• Steroids
Fats and oils are esters made up of glycerol (a 3-carbon sugar alcohol/polyol) and 3 fatty acids. Fatty acids
are hydrocarbon chains of differing lengths with various degrees of saturation that end with carboxylic acid
groups. Additionally, fatty acid double bonds can either be cis or trans, creating many different types of fatty
acids. Fatty acids in biological systems usually contain an even number of carbon atoms and are typically 14
carbons to 24 carbons long. Triglycerides store energy, provide insulation to cells, and aid in the absorption
of fat-soluble vitamins. Fats are normally solid at room temperature, while oils are generally liquid.
In this practical we extract lipids from a lipid containing food and carry out some test to check the composition
of the lipid extraction. These test are,
• Acrolein Test
• Bromine Test
• Lieberman – Burchard Test
• Solubility in water and Hexane
Materials and Chemicals –
• 6.0g of Peanuts
• 20.0 ml of Hexane
• 125 ml Erlenmeyer flask
• Mortar and pestle
• Filter funnel
• Filter paper
• Graduated cylinder
• Electrical Balance
• Albumin
• Vegetable oil
• Glycerol
• Margarine
Procedure –
1. Lipid Extraction
First, 6.0 g of peanuts were weighed out and coarsely powdered for three to four minutes using a mortar
and pestle. Then a 125 ml Erlenmeyer flask was filled with 3.0g of powder that had been powdered out. The
Erlenmeyer flask was then filled with 20.0 ml of hexane and sealed. Following that, the mixture in the
Erlenmeyer flask was periodically stirred for the following 15 minutes. The filter paper's weight was first
measured. The filter funnel was then covered with filter paper. Then the solution in the Erlenmeyer flask
was filtered with the funnel that was prepared. Then the weight residue and the filter paper were
measured and the weight of the reside was calculated. The filtrate (lipid extraction) was subjected to
further test to identify its’ composition.
2. Solubility in water
Five test tubes were prepared which each contained one of the samples of 3 drops vegetable oil, 3 drops of
margarine, 3 drops of lipid extracted from the lipid extraction, 3 drops of albumin and 3 drops of glycerol.
Then 2 ml of water was added to each test tube. Then the observations were recorded and for clear
observation 5 drops of Sudan III was added.
3. Solubility in hexane
Five test tubes were prepared which each contained one of the samples of 3 drops vegetable oil, 3 drops of
margarine, 3 drops of lipid extracted from the lipid extraction, 3 drops of albumin and 3 drops of glycerol.
Then 2 ml of hexane was added to each test tube. Then the solubility of the samples in hexane was
observed and the results were recorded. The test tubes were saved for the bromine test.
4. Bromine Test
They saved test tubes that had previously been used to measure the solubility of hexanes. The next step
involved adding 2.0 ml of hexane to a control test tube that was left empty. Five drops of bromine in
dichloromethane solution were added to each test under the fume hood, and the tubes were gently spun.
Each test's color was noted after 10 seconds, 30 seconds, and 60 seconds.
5. Acrolein Test
Four clean, dry test tubes were taken and labeled as vegetable oil, margarine, glycerol, and albumin. Two
drops of the albumin, glycerol, and vegetable oil samples, as well as a small pinch of the margarine, were
added to each test tube. 0.45g of potassium bisulfate was then added to each test tube.
The mixture was agitated while being moved in and out of the flame using a test tube holder until the smell
of the vapor was recorded. Each test tube's bottom was heated over a Bunsen burner flame at a 45 o angle.
6. Lieberman-Burchard Test
Each of the five thoroughly cleaned and dry test tubes received three drops of the albumin, glycerol,
vegetable oil, and lipid extraction samples as well as a little pinch of margarine. Each test tube was first
filled with 2.0 ml of chloroform in the fume hood. Then each test tube received 10 drops of acetic
anhydride. Then each test tube received 2 drops of concentrated sulfuric, which was thoroughly mixed. The
color and relative intensity of each test tube's color were noted after few minutes.
Observations –
Substance Solubility Sudan 3 Solubility Bromine test Observat Observatio
in water test in ion of n of the
and Hexane Acrolein Lieberman
10s 30s 60s
miscibility test -Burchard
test
Albumin Soluble Colorless Insoluble Bright Bright Yellow No Colorless
yellow yellow color observati
on
Glycerol Soluble Colorless Insoluble Bright Bright Yellow Pungent Red color
yellow yellow color odor and
black
colored
sample
Vegetable Insoluble Red color Soluble Yellow Light Colorl Pungent Colorless
oil where yellow ess odor
lipids
were
present
Margarine Insoluble Red color Soluble Bright Light Colorl Pungent Pink Color
where yellow yellow ess odor
lipids
were
present
Lipid Insoluble Red color Soluble - - - - Colorless
extraction where
lipids
were
present
Lipid Extraction –
Weight of the filter paper = 0.91g
Weight of filter paper and the residue = 3.18g
Residue weight = 2.27g
Lipid percentage = (3.00 – 2.27)g / 3.00g x 100% = 24.34%
Residue color = White yellowish color precipitate
Figure 01 Figure 02
Solubility in water Sudan III test
Figure 03 Figure 04
Lipid Extraction solubility in water Lipid Extraction Sudan III test
Figure 05 Figure 06
Solubility in Hexane Bromine Test after 60s
Figure 07 Figure 08
Acrolein Test Lieberman-Burchard Test
Discussion –
The lipids are insoluble in water due to water being polar and lipids are insoluble in polar solvents. In the
solubility in water test, it is hard to observe the insolubility of lipids. Due to this reason, we added Sudan III
to the same test tube, then we could observe red color on the wall of the test tube and above water, where
the lipids are present. Hexane is a non-polar solvent therefore lipids are soluble in it while glycerol and
albumin are insoluble in hexane.
In the Bromine test, we check for the presence of double bond, that is the saturation of the sample is
tested. If double bonds are present, they are unsaturated and if double bonds are not present, they are
saturated. In the bromine test, for unsaturated samples it gives a positive result that is, the yellow color of
bromine disappears over time.
In the acrolein test, the presence of glycerol is checked. If the sample contains glycerol, it gives a pungent
odor. The reaction occurring in this test as follows,
CH2OH KHSO4 CH2
CHOH CH + 2H2O
CH2OH CHO
Glycerol Acrolein
The smell of the acrolein gives pungent odor observed in this test.
A solution called Lieberman-Burchard, which produces a deep green color, is used in a test to identify
cholesterol. This color shifts from a pinkish-purplish hue to a light green and finally a very dark green. The
color is caused by a reaction between the chemicals and the hydroxyl group (-OH) of cholesterol, which
increases the conjugation of the un-saturation in the neighboring fused ring. Because sulfuric acid and
acetic anhydride are used in this test as reagents, care must be taken to prevent serious burns.
Pre-lab questions –
1. Presence of double bond (aldehyde functional group)
Positive result – The yellow color of bromine disappears over time.
2. Glycerol
Positive result – A pungent odor
3. Cholesterol (OH functional group)
Positive result – Green color solution
4.
Name Structure
Bromine Br2
Potassium Bisulfate KHSO4
Stearic acid
Chloroform (trichloroethane)
Hexane
Oleic acid
Sulfuric acid H2SO4
Sodium Hydroxide NaOH
Acetic Anhydride
Post-Lab questions –
1. Glycerol and albumin
2. The lipids vegetable oil, margarine has less density than water due to them being floating on water
as observed in the solubility in water test and the Sudan III test. Glycerol is denser than water due to
the ability to form H-bonds more than water. In albumin it is closer to water in density.
3. Lipid extracted from peanuts, margarine, vegetable oil is soluble in hexane solution and glycerol and
albumin are not soluble in hexane. Since glycerol has both polar and non- polar characteristics, it is
insoluble in hexane.
4. Vegetable oil, margarine and lipid extraction are insoluble in water. All these lipids are dissolved in
Hexane. That means these three lipids agree with the property of lipids which states that due to
non-polar nature of lipids they do not dissolve in polar solvents such as water but dissolve in non-
polar solvents such as hexane.
5. Albumins does not show positive result for any test except the solubility in water test. This is due to
albumin being a protein and the tests conducted are for the composition of lipids. Glycerol is a
starting material which combines with fatty acids to form a lipid molecule. Therefore, glycerol is not
a lipid as proven by the solubility in water test.
6. –
7. From the bromine test, vegetable oil and margarine give positive result. Therefore, vegetable oil and
margarine are unsaturated. Glycerol and albumin are saturated.
8. –
9. Glycerol, vegetable oil and margarine gave positive results for the acrolein test due to the presence
of glycerol. Albumin showed negative results for the acrolein test due to the absence of glycerol.
Conclusion –
The extracted lipids form 3.0 g of peanuts showed positive results for solubility in hexane, Sudan III,
bromine test. Which conclude that the extracted lipids are unsaturated and is soluble in non-polar solvents.
References –
• BYJUS. (n.d.). Tests for Unsaturation - Chemistry Practicals Class 12. [online] Available at:
[Link]
• BYJUS. (n.d.). Tests of Oils and Fats - Chemistry Practicals Class 12. [online] Available at:
[Link]
fats/#:~:text=(c)%20Acrolein%20Test%3A&text=A%20pungent%20irritating%20odour%20or.
