UNIVERSITI TEKNOLOGI MARA JENGKA, PAHANG
FACULTY OF APPLIED SCIENCES
Bachelor of Science (Hons.) Biology
BIOCHEMISTRY
BIO462
Practical 7 : Qualitative Test of Lipids and Preparation of
Carboxylic Acid Salt from Lipids
No. Name Student ID
1. NURUL SYAFIKAH ATIRAH BINTI RAHIMI 2023644426
2. TUAN NORUL NAJWA BINTI TUAN 2023415044
ZAMZURI
3. SITI NORSYAHFIQAH BINTI LANA BUSU 2023479278
4. NOR AMIERA NABILA BINTI SUHAIMI 2023424256
5. SITI FATIMAH BINTI SALAHUDDIN 2023622968
Lecturer’s name: Muhammad Afif bin Yusof
Date of submission: 5/6/2024
INTRODUCTION
Lipids are chemical molecules made up of components that form chemical bonds, to carry out
certain tasks. These are compounds produced by our body that are waxy or fatty and do not
dissolve in water. Lipids are a diverse category of compounds that include waxes, fats, oils,
steroids, and other related substances that are more related to one another physically than
chemically. The solubility of lipids—a class of compounds—in non-polar solvents and their
insolubility in water. Because they contribute to the formation of the cell membrane, which is
a physical barrier that divides a cell from its surroundings, lipids are essential to biological
processes. A hydrophilic phosphate group, two hydrophobic fatty acid tails, and a glycerol
backbone usually make up the structure. Lipids found in our cell membranes regulate what
enters and exits your cells.They facilitate the synthesis of hormones, the absorption of
vitamins, and the movement and storage of energy.
Nonpolar lipids such as triglycerides are utilised as fuel and as a means of storing energy.
Polar lipids, which membranes use, have the ability to form a barrier with an external aqueous
environment. Glycerophospholipids and sphingolipids are two types of polar lipids. Fatty
acids are present in significant amounts in all of these lipids. A common property of lipids is
that they are usually insoluble in water but soluble in nonpolar solvents such as ether and
chloroform. Because they are high in calories and contain essential fatty acids and fat-soluble
vitamins found in natural food fat, they are important nutritional components. In this
experiment, the solubility, unsaturation, presence of fats and oils, and presence of cholesterol
in different lipid solutions will be examined. So, we will carry out this experiment using
various solvents and solutes and watch how the solutions react.
Triglyceride, cholesterol, phospholipid, unknown 1 and unknown 2 samples will be used to
prepare the ester assay and cholesterol assay. An organic molecule can be identified if it
contains a carboxylic acid group by using the ester assay test. An ester is produced when an
alcohol and a carboxylic acid interact in the presence of an acid. Esters usually smell sweet,
so you can determine whether the reaction occurred by sniffing the solution after it did. In
terms of the cholesterol assay, it will identify any cholesterol molecules that are present. A
chemical molecule that is part of organic matter is cholesterol. In its pure state, this white,
crystalline substance has no taste or odour. The lipids used in this experiment will also be
used to prepare the carboxylic acid salt. We refer to this procedure as saponification.
OBJECTIVE
1. To determine the presence of a functional group or molecule in triglyceride,
cholesterol, phospholipids, unknown 1 and unknown 2 by using two different assays.
2. To determine the two unknown samples.
3. To produce carboxylic acid salt and glycerol via a saponification process which makes
use of an alkali (base).
MATERIALS
A. Ester Assay
Triglyceride sample, Cholesterol sample, Phospholipids sample, unknown sample, ethanol,2M
hydroxylamine hydrochloride, 3.5M NaOH, 5M HCl, 5% ferric chloride in 0.1M HCl,dropper,
test tube and water bath.
B. Cholesterol Assay
Triglyceride sample, Cholesterol sample, Phospholipids sample, unknown sample, methylene
chloride, acetic anhydride, concentrated sulfuric acid, dropper and test tube
C. Preparation of Carboxylic Acid Salt from Lipids
Margarine (fat), NaOH, colouring agent, fragrance, microwave, beaker, pipette, glass rod and
petri dish.
PROCEDURE
A. Ester Assay
1. 2 drops of sample were put into a test tube.
2. The solvent was drying off with a gentle stream of air.
3. The sample was dissolved with 0.5 mL of ethanol.
4. Added two drops each of 2 M hydroxylamine hydrochloride and 3.5 M of NaOH.
5. The test tubes were mixed well and heated in a boiling water bath for 30 seconds.
6. 2 drops of 5 M HCI and one drop of 5% ferric chloride in 0.1 M HCI were added.
7. The sample has been mixed well and a magenta-colored complex that indicates a
positive result have been observed.
B. Cholesterol Assay
1. 2 drops of sample were put into a test tube.
2. The solvent was drying off with a gentle stream of air.
3. The sample was dissolved with 10 drops of methylene chloride.
4. 6 drops of acetic anhydride and 2 drops of concentrated sulfuric acid were added.
5. The sample has been mixed well and a greenish color after a few minutes which
shows a positive result has been observed.
C. Preparation of Carboxylic Acid Salt from Lipids
1. 20 g of fat was warmed in a clean beaker for 10 minutes.
2. 10 mL of NaOH was added into the beaker very slowly.
3. Then, stir the mixture using a popsicle stick gently for at least 15 minutes.
4. Added 10 drops of coloring agent and 5 drops of fragrance (if available). Then, pour
the mixture into a petri dish and leave it in the lab for several days.
RESULTS
Results (positive or negative)
Sample Ester Assay Cholesterol Assay
Triglyceride Positive Negative
Cholesterol Negative Positive
Phospholipids Negative Negative
Unknown 1 Positive Negative
Unknown 2 Positive Negative
1. Why is soap referred to as a 'salt' ?
Because it is produced through a chemical reaction called saponification, which
involves the reaction of a fatty acid with an alkali, such as sodium hydroxide (NaOH)
or potassium hydroxide (KOH). This reaction forms salts, which are the basic
components of soap. So, in essence, soap is a type of salt formed from the reaction
between fatty acids and alkalis.
2. What kind of molecular properties enable substances to dissolve
in water ?
Substances that dissolve in water typically have molecular properties that allow them
to interact with water molecules through hydrogen bonding or other polar interactions.
These properties include having polar or ionic bonds within the molecule, which
allows them to form favourable interactions with water molecules, leading to
dissolution. Additionally, molecules with smaller sizes and lower molecular weights
tend to dissolve more readily in water due to their ability to fit between water
molecules and disrupt hydrogen bonding.
3. Show the structures of reaction for saponification of ethyl acetate.
CH3COOC2H5(aq) + NaOH(aq) → CH3COONa(aq) + CH3CH2OH(aq)
DISCUSSION
The experiment was done to determine qualitative tests for as well as prepare carboxylic acid
salts from the lipids. The two tests, the ester assay and the cholesterol assay, were used to
determine the presence of a functional group or molecule in triglyceride, cholesterol,
phospholipid and two unknown samples.
According to the results, triglyceride, unknown 1 and unknown 2 showed a positive result
while cholesterol and phospholipid showed a negative result with the ester assay. The
hydroxyl group and carboxyl group will react to each other and form an ester bond that results
in a colour change. The ester then reacts with hydroxylamine to form hydroxamic acid. A
positive result is indicated by a maroon or burgundy complex between the hydroxamic acid
and the ferric ion (Chickos et al., 2020). Both the unknowns contain esters as they showed a
positive result.
Next, the cholesterol assay only showed a positive reaction with cholesterol while
triglyceride, phospholipid, unknown 1 and unknown 2 are negative. Acetic anhydride is used
as solvent and dehydrating agents, and the sulfuric acid is used as a dehydrating and oxidising
agent. The positive solution changes from colourless to greenish after a few minutes. This
colour is due to the –OH group of cholesterol and the unsaturation found in the adjacent fused
ring (Moorpark College, 2017). Since unknown 1 and 2 showed a negative result, we can
safely assume they do not contain any cholesterol.
Based on the two tests done on the unknowns, we can assume unknown 1 is corn oil as it is a
lipid that contains triglyceride but not cholesterol (National Center for Biotechnology
Information, 2024) while unknown 2 is mustard oil because it contains cholesterol-reducing
elements.
Producing carboxylic acid from lipids is a process called saponification. Saponification is the
hydrolysis of an ester to form an alcohol and the salt of a carboxylic acid in acidic or essential
conditions. Due to the nature of lipids, they are insoluble in water and thus the use of NaOH
in the experiment deprotonated the fatty acids to carboxylate salts that are more soluble in
water. Ester reacts with an inorganic base during saponification to create alcohol and soap. It
normally happens as potassium or sodium hydroxide (lye) reacts to triglycerides to create
glycerol and fatty acid salt, called ‘soap’ (BYJU's, 2017).
CONCLUSION
In conclusion, the ester assay test and the cholesterol assay test can be to determine the
presence of the functional group or molecule in triglyceride, cholesterol, phospholipids and in
two unknown samples. Based on the ester assay test, the results show a positive results in
triglyceride, unknown 1 and unknown 2 meanwhile the cholesterol show negative result in the
ester assay test. In the cholesterol test, positive results only shown in cholesterol and the
negative results shown in triglyceride, unknown 1 and unknown 2. The positivity of the result
can be measured by observing the change of color and precipitation of the solution. In
conclusion all of the objective for the experiment were achieved.
REFERENCES
1. Chickos, J., Garin, D., & D'Souza, V. (2020, June 29). 8: Identification of Unknowns
(Experiment). Chemistry LibreTexts. Retrieved June 1, 2024, from
https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/Wet_Lab_Ex
periments/Organic_Chemistry_Labs/Experiments/8%3A_Identification_of_Unknowns
_(Experiment)
2. Moorpark College. (2017, November 9). Experiment 16 - Analysis of Lipids.
Chemistry M11 Laboratory Manual. Retrieved June 1, 2024, from
https://www.moorparkcollege.edu/sites/moorparkcollege/files/media/pdf_document/20
20/chem_11_expt16.pdf
3. National Center for Biotechnology Information (2024). PubChem Compound
Summary for Corn Oil. Retrieved May 31, 2024 from
https://pubchem.ncbi.nlm.nih.gov/compound/Corn-Oil.
4. BYJU's. (2017, November 9). Saponification. Retrieved June 1, 2024, from
https://byjus.com/chemistry/saponification/
