Extraction of Lipids from Chicken Egg Yolk, Column Chromatography, and

Qualitative Test for Lipids

Kristel Van Cuarentas, Jesspeed Marvel Delos Santos, Kathrina Antheia Dimaano, Gia Directo,
Clare Ducut, Toni Encinares
Group 3, 2 G Pharmacy, Pharmaceutical Biochemistry Laboratory
Abstract
The experiment involves the extraction of chicken egg yolk, column chromatography and the
qualitative test for lipids. Lipids were extracted from the egg yolk using ethanol, hexane and
acetone. The extracted lipids then undergone column chromatography to separate the different
lipids contained in the egg yolk. Three eluates were separated and were tested for the presence
of ester, glycerol and cholesterol. The eluates were also tested for the lipid unsaturation with
bromine. Eluates 1 and 3 were positive from the test for the presence of ester and glycerol, and
were negative in the test for presence of cholesterol. Eluate 2 was negative from the test for the
presence of ester and glycerol while it showed positive result in the test for the presence of
cholesterol. All the eluates were positive from the test for lipid unsaturation with bromine. Based
on the qualitative test performed, it can be concluded that eluate 1 is triglyceride/triacylglycerol,
eluate 2 is cholesterol and eluate 3 is lecithin.

Introduction
Lipids are naturally occurring
organic molecules that have limited
solubility in water and can be isolated
from organisms by extraction with
nonpolar organic solvents.
Classified according to their
chemical nature, lipids fall into two main
groups. One group consists of openchain compounds with polar head
groups and long nonpolar tails includes
fatty acids, triacylglycerol, sphingolipids,
phosphoacylglycerols, and glycolipids.
The second major group consists of
fused-ring compounds, the steroids; an
important representative of this group is
cholesterol.

The egg yolk is a fat-in-water

emulsion with about 50% dry weight. It
consists of 65% lipids, 31% proteins and
4%
carbohydrates,
vitamins
and
minerals. Table 1 shows the lipid
composition of an egg yolk.
Table 1: Egg Yolk Lipid

Column Chromatography is used for the

separation and purification of solids and

liquids when carrying out microscale

experiments

chromatography, (3) identify the lipids

present in each of the fraction\s using
qualitative tests and (4) to determine the
degree of unsaturation of lipids through
bromine test.
Methodology
Extraction of Total Lipids from Chicken
Egg Yolk

Figure 1: Column Chromatography Set-up

Column Chromatography involves

two phases: the stationary phase and
the mobile phase. The mobile phase
flows over the stationary materials and
carries the sample to be separated on it.

Chicken egg yolk was separated

from the chicken egg and its volume
was determined.
An equal amount of ethanol was
added and mixed to dehydrate and
partially extract the polar lipids. Hexane
was added, mixed and let stand for 5
minutes until two layers form. This
resulted in fractions of polar and neutral
lipids. The upper polar fraction was
removed and an equal amount of
acetone was added to further precipitate
the polar lipids from residual neutral
ones, specially cholesterol.
The upper layer was collected
and transferred into a clean test tube.
Column Chromatography of Lipids
A small column was prepared by
pouring a slurry of 0.5 g silica gel in 4
mL of petroleum into a glass column
(Pasteur pipette). The glass column had
a tapered end plugged with glass wool.

Figure 2: The Principles of Column

Chromatography

The experiment aims to: (1)

extract total lipids from chicken egg yolk,
(2) analyze the lipids present in the
crude
extract
using
column

One (1) mL lipid extract (collected

from the extraction of total lipids from
chicken egg yolk) was poured into the
column; the run-through was saved in a
clean test tube. The column with 5 mL
9:1 mixture of petroleum ether: ethyl
ether was washed, the eluate in the
same tube as run-through was

collected. The column with the second

eluent (5 mL 5% methanol in
dichloromethane) was washed, the
eluate was collected in another clean
test tube. Finally, the column with the
last
eluent,
5
mL
dichloromethane:methanol:water (1:3:1)
was washed, and the eluate was
collected in another test tube. The
different eluates were saved for
qualitative analysis.
Qualitative Test
Test for Ester
Ten (10) drops of the eluates
were placed in separate test tubes. 0.5
mL ethanol: 1-butanol (3:1) was added.
Then 2 drops each 2 M NH2OH HCl and
3 M NaOH were sequentially added.
The samples were allowed to stand for 5
minutes. 2 drops 6 M HCl and 1 drop
5% FeCl36H2O in 0.1 M HCl, were
added, and mixed well. The samples
with esters produced a burgundy color.
Test For Glycerol (Acrolein Test)
A pinch amount of KHSO was
added to 10 drops of the eluate in a test
tube. The test tube was heated in a
boiling water bath and the odor
produced was noted. The burnt fat odor
indicated the presence of glycerol.
Test For Cholesterol
Burchard Test)

minutes which indicated the presence of

cholesterol.
Test For Lipid Unsaturation With Br2
Ten (10) drops eluate were
placed in a test tube.
3 mL
dichloromethane were added and mixed
well. Under a fume hood, added was
5% Br2 in dichloromethane dropwise into
the test tube, shaken after each
additional until a reddish brown color
persisted. The added number of drops
of 5% Br2 in dichloromethane was
recorded. The procedure was repeated
and the results were compared with the
following: 8 drops each of coconut,
canola, corn, and olive oil.
Results and Discussion
Lipids are separated based on
differences in solubility. Neutral lipids
are relatively soluble in non-polar
solvents.
Polar lipids are further
separated by increasing the polarity of
the organic solvent.
Lipid was extracted from the
chicken egg yolk and had undergone
the column chromatography.

(Liebermann-

Ten (10) drops of eluate were

placed in a test tube.
0.25 mL
dichloromethane was added. 6 drops
acetic anhydride and 2 drops conc.
H2SO4 were added and mixed well. A
greenish color was produced after a few

Figure 3: Column Chromatography of

Egg Yolk

Three eluates were collected

from the chromatography and had
undergone the qualitative test for lipids.
Table 2 shows the result for the
test for ester.
Table 2: Test for Ester
Eluate
1
2
3

Test for Ester

Burgundy Solution
Yellow Solution
Burgundy Solution

The presence of ester is

determined by the classic ferric
hydroxamate test. Positive result for the
test for ester yields a burgundy color.
The burgundy color is due to the
formation of ferric hydroxamate complex
Based on table 1, eluate 1 and eluate 3
gave a positive result while eluate 2
gave a negative result.
Table 3 shows the result for the
Acrolein test or test for glycerol.
Table 3: Acrolein Test
Eluate
1
2
3

Acrolein Test
Burnt fat odor
odorless
burn fat odor

Acrolein test is a test for the

presence of glycerol or fats. Glycerol,
either in free form or as an ester of fatty
acids, is heated with potassium
hydrogen sulfate till it gets dehydrated to
an unsaturated aldehyde called acrolein.
Acrolein can be identified by its

characteristic burn fat odor. Based on

the result, eluates 1 and 3 produced a
positive result while eluate 2 produced
negative result.
Table 4 shows the result for the
Liebermann-Burchard Test or the test for
cholesterol.
Table 4: Liebermann-Burchard Test
Eluat
e
1
2
3

Leibermann-Burchard Test
no color change
greenish color produced
no color change

Liebermann-Burchard Test is
used to test the presence of cholesterol
in the eluate. This test involves the
reaction of the cholesterol with
concentrated sulfuric acid in the
presence of acetic anhydride to give a
blue color, which immediately gets
converted into green color. These color
changes are attributed to dehydration,
condensation,
and
isomerization
reactions.
Table 5 shows the result for the
test for lipid unsaturation with bromine
Table 5: Test for Lipid Unsaturation
Eluate
1
2
3

Test for Lipid Unsaturation

reddish brown color (8 drops)
reddish brown color(15 drops)
reddish brown color(12 drops)

The fatty acid residues may also

differ according to the extent of
saturation (number of unsaturated or

double
bonds)
present
in
the
hydrocarbon chain. The extent of
unsaturated bonds can be demonstrated
by the degree of decolorization of a
halogen solution (iodine, bromine), this
is usually measured by the number of
bromine drops. The more double bonds
a fat contains, the more bromine
required for the addition reaction; thus, a
high bromine number means a high
degree of unsaturation. Thus, eluate 2 is
the most unsaturated followed by eluate
3 then eluate 1.
Conclusion
The eluates were identified based
on their qualitative color reaction. The
eluates were:
1st eluate : triglyceride/triacylglyceride
2nd eluate: cholesterol
3rd eluate: lecithin
The lack of accuracy in
measuring samples or reagent and
contaminated reagents contributes to
possible error in the experiment.
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