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Extraction of Total Lipids from Chicken Egg Yolk using Ethanol, Hexane,

and Acetone and the Qualitative Analysis for the presence of Ester,
Glycerol, and Cholesterol as well as the determination of the Degree of
Saturation of the Extracts Obtained
Alabastro, Reena Avemiel; Albeza, Keeshan Gytha; Arcinas, Ann Iree;
Baello, Alexa J eatrize*; Carlos, Chamzelle; Chia, Anna Patricia
Group 1- 2GPH 2013
Faculty of Pharmacy
University of Sto. Tomas

ABSTRACT: Lipid molecules include fats, waxes, and fat-soluble vitamins such as A, D, E and K. The experiment
was about extraction of total lipids from chicken egg yolk and column chromatography of. Total lipids were extracted
from the chicken egg yolk using ethanol, hexane, and acetone. Each extract was subjected to different qualitative
tests for lipid. 10 drops of extract were subjected for each qualitative test. The qualitative tests performed were as
follows: test for ester, test for glycerol also known as acrolein test, test for cholesterol also known as Liebermann-
Burchard test, and test for lipid unsaturation with bromine. Test for esters yielded a yellow solution for the acetone
extract and a burgundy solution for the ethanol and hexane extracts. Test for glycerol produced an alcohol like odor
for the ethanol, a gas like odor for hexane extract and produced a glycogen like odor in acetone. Test for cholesterol
or Liebermann-Burchard test yielded a positive emerald green solution for acetone and hexane and a colorless
solution for ethanol. In the test for lipid unsaturation with bromine, 30 drops in the ethanol extract, 30 drops in the
Hexane extract, 32 drops in acetone extract and 33 drops in coconut oil.

The lipids are one of four major families of
biochemical compounds, the other three
being carbohydrates, proteins, and nucleic acids.
Biochemical compounds are organic compounds that
occur in living organisms. The lipids are unique among
organic and biochemical families because of the way in
which they are classified. In all other families, members
are categorized because of similar chemical structure
and similar chemical properties. Lipids are characterized
instead on the basis of a single physical property, their
solubility. Lipids are hydrophobic and tend to be
insoluble in water, but soluble in certain organic solvents
such as benzene, chloroform and ether. They are
commonly classified into three groups: simple lipids
(neutral fats, triacylglycerol or triglyceride, and waxes),
compound lipids (phospholipids such as lecithin,
glycolipids, and lipoproteins), and derived lipids (fatty
acids such as oleic acid and stearic acid, steroids such
as cholesterol and oestrogen, and hydrocarbons). The
lipid family contains a rather wide range of compounds
that are structurally quite different from each other [1].
Lipids, fats and oils, have borne the brunt of the
blame for the degenerative diseases, the heart disease

and cancer, are the major causes of death in the
developed world. The negative view of lipids has
obscured their essentiality for human health [2].
Sources of lipids found in food are mostly in
animals. Egg from a chicken could be. Egg yolk is a
source of lecithin, an emulsifier and surfactant. Lot of
lipids could be found in egg yolk although it has more
water [3].
In the experiment, the lipids present in chicken
egg yolk were isolated. The egg yolk makes up about
33% of the liquid weight of the egg and it contains
approximately 60 calories, three times the caloric
content of the egg white. All of the fat-soluble vitamins
(A, D, E, and K) are found in the egg yolk. Egg yolk is
one of the few foods naturally containing vitamin D [4].
The extract obtained from the egg yolk with the
use each of these reagents namely, acetone, ethanol,
and hexane were then subjected to different qualitative
tests. The test for ester whereby the ester is converted
to a hydroxamic acid should yield a positive result of a
burgundy coloration [5]. The next test performed was a
test for glycerol known as the acrolein test which will
give off a smell of burning fat [6]. The test for cholesterol
also known as the Libermann-Burchard test which would
yield an emerald green solution in the presence of
cholesterol [7]. The last test was the test for lipid
unsaturation with bromine which tested for the degree of
saturation of fatty acids [8]. The objectives of the
experiment were to extract total lipids from chicken egg
yolk, to analyze the lipids present in the crude extract
using column chromatography, to identify lipids present
in each of the fractions using qualitative tests and, and to
determine the degree of unsaturation of lipids by
bromine test [9].
A. Compounds Tested
The chicken egg yolk for the extraction of total
lipids and the acetone, hexane and ethanol extracts
from the Chicken egg yolk which were used in the
qualitative test.
B. Materials needed for the experiment
The materials used in the experiment are: Beaker,
Test Tubes, Pipettes, TLC Plate, and Capillary Tubes.
C. Procedures
1. Extraction of Total Lipids from Chicken
Egg Yolk. Total lipids from chicken egg yolk
were first extracted. Then an equal amount of
ethanol was added to the egg yolk to increase
the polarity of the organic solvent, and then
mixed to dehydrate and partially extract the
polar lipids. Hexane was then added and the
solution was mixed and was left to stand for 5
minutes, until two layers had formed the
fractions of polar and neutral lipids. The upper
polar fraction was then removed and an equal
amount of acetone was added to further
precipitate the polar lipids from residual
neutral ones, especially the cholesterol. The
upper layer was then collected, and was
transferred into one clean test tube. The
extractions obtained were then used for the
qualitative tests for lipids.
2. Qualitative Tests for Lipids. Test for Ester:
EtOH: 1-BuOH (3:1) with a volume of 0.5 ml
was introduced into the 10 drops of extract. 2
drops each of 2M hydroxylamine
Hydrochloride and 3M NaOH was sequentially
added and was mixed well. The samples were
allowed to stand for 5 minutes. 2 drops of 6M
HCl was added with 1 drop of 5% FeCl3. 6
H2O in 0.1M HCl and was then mixed well.
Test for Glycerol (Acrolein Test): A
pinch amount of KHSO
was added to 10
drops of the extract in the test tube. Test tube
was heated in a boiling water bath and odor
produced was noted. Burned fat odor was
observed for positive test results.
Test for Cholesterol (Liebermann-
Burchard Test): Ten drops of extract was
placed in a test tube and 0.25mL of
dichloromethane was added. Six drops of
acetic anhydride and 2 drops of concentrated
was added and mixed with the sample.
A greenish color produced after a few minutes
indicated the presence of cholesterol.
Test for Lipid Unsaturation with Br
: Ten
drops of each extract was placed in different
test tubes. Aside from the three extracts, 10
drops of Coconut oil was also used. To each
test tube 3 mL of dichloromethane was added
and mixed well. Then Br
was added drop wise
in every test tube until a desired color was
achieved. The numbers of drops dropped
were then recorded.
Extraction of Total Lipids from Chicken Egg
Yolk. Lipids are soluble in organic solvents, but sparingly
soluble or insoluble in water. The existing procedures for
the extraction of lipids from source material usually
involve selective solvent extraction and the starting
material may be subjected to drying prior to extraction.
Solubility of lipids is an important criterion for their
extraction from source material and depends heavily on
the type of lipid present, and the proportion of nonpolar
(principally triacylglycerols) and polar lipids (mainly
phospholipids and glycolipids) in the sample; therefore,
several solvent systems might be considered, depending
on the type of sample and its component. The solvents
of choice are usually hexane, chloroform, methanol or
chloroform, methanol or water. The simple lipids include
waxes, fats, and oils. These compounds are structurally
similar to each other because they consist of alcohols
combined with long organic acids known as fatty acids.
Waxes are constructed of a single molecule each of
alcohol and acid while fats and oils contain three fatty
acid molecules for each alcohol molecule. Fats are
distinguished from oils in that the former are solids and
the latter, liquids.
Table 1: Actual Results of Qualitative Tests for
Chemical Ester Glycerol
Burchard Test
Hexane Burgundy Gas-like Emerald green
solution odor solution
like odor
Emerald green

Table 1 shows the actual results for each
qualitative performed in lipids. The principles or
mechanisms behind each qualitative test are as follows:
TEST FOR ESTER: One test for esters is the
ferric hydroxamate test whereby the ester is converted to
a hydroxamic acid (HOHN-C=O) which will give a
positive ferric chloride test. The color is due to a complex
between the hydroxamic acid and the ferric ion. A deep
burgundy color is positive. Esters are common in organic
chemistry and biological materials, and often have a
characteristic pleasant, fruity odor. This leads to their
extensive use in the fragrance and flavor industry. Ester
bonds are also found in many polymers. Based on the
results obtained the only extract that was positive for the
presence for ester was ethanol. Theoretically Both
Ethanol and Hexane should have produced a positive
Acrolein (systematic name: propenal) is the simplest
unsaturated aldehyde. It is produced widely but is most
often immediately reacted with other products due to its
instability and toxicity. It has a piercing, disagreeable,
acrid smell, smell of burning fat, which is caused by
glycerine in fat decomposing.
Acrolein is a compound formed by dehydration
of glycerol, so its presence indicates the presence of a
glyceride ester (usually a triglyceride) Example: a fat or
oil. The smell is a bit like a barbecue. Acrolein test is a
test for the presence of glycerin or fats. A sample is
heated with potassium bisulfate, and acrolein is released
if the test is positive. When a fat is heated strongly in the
presence of a dehydrating agent such as KHSO
, the
glycerol portion of the molecule is dehydrated to form the
unsaturated aldehyde, acrolein (CH
=CH-CHO), which
has the peculiar odor of burnt grease. The principle
behind the acrolein test is a specific chemical reaction.
This reaction is utilized to determine the presence of
glycerin in a fat. By heating the fat sample in the
presence of potassium bisulfate (KHSO
), which acts as
a dehydrating agent, acrolein (C
O, or CH
is formed and can easily be detected by its odor.
Whenever fat is heated in the presence of a dehydrating
agent, the fat molecule will shed its glycerol in the form
of the unsaturated aldehyde acrolein. Based on the
results none of the extracts tested were positive for
glycerol. Theoretically Both Ethanol and Hexane should
have produced a burnt-fat like odor.
BURCHARD TEST): The Lieberman-Burchard or acetic
anhydride test is used for the detection of cholesterol.
The formation of a green or green blue color after a few
minutes is positive. Lieberman-Burchard is a reagent
used in a colorimetric test to detect cholesterol, which
gives a deep green color. This color begins as a
purplish, pink color and progresses through to a light
green then very dark green color. The color is due to the
hydroxyl group (-OH) of cholesterol reacting with the
reagents and increasing the conjugation of the un-
saturation in the adjacent fused ring. Based on the
results Acetone and Hexane were both positive for
Table 2: Results of Test for Unsaturation
with Br

Ethanol Hexane Acetone
of I

30 30 32 33

Table 2 shows the actual results for lipid
unsaturation with Bromine. The test for unsaturation with
bromine identifies the level of saturation and the number
of bonds an oil, fat or lipid has. The more unsaturated,
multi-bonded, the lipid is, the more it absorbs bromine.
Based on the results Acetone was the most unsaturated
not considering the coconut oil, although theoretically
Ethanol should be the most unsaturated followed by
hexane then acetone would be the most saturated.
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Biochemistry. New York: W.H. Freeman.
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quantification: A simple protocol. Journal of Chemical
Education 82(1), 103-104
[4] Chemistry of Lipids
[5] Journal of Chemical Education
[6] http://lipidlibrary.aocs.org/Lipids/whatlip/index.htm#def
[7] Gale Encyclopedia of Food & Culture: Lipids
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