Introduction
Lipids are essential fats in the body. They are a class of
organic chemical compounds that are either fatty, waxy,
or oil and are soluble in organic solvents but insoluble in
polar solvents such as water (Thompson, 2018). They
are also either hydrophobic or amphipathic and tend to
form a surface monolayer, bilayer, micelles, or vesicle
when in contact with water. Lipids include triglycerides,
phospholipids, waxes, and steroids (Ahmed et al., 2018).
They are part of the body's cell membrane that helps
regulate what goes in and out of the cells. They are
molecules mainly composed of fat and oil, yielding high,
and have a chemical composition of carbon, hydrogen,
and oxygen. Lipids serve several functions, mainly
providing structure to cell membranes, storing energy in
the body, serving as insulation, and serving as signaling
molecules.
There are three main types of lipids. These include
triacylglycerols or triglycerides, phospholipids, and
sterols. The most abundant lipid in the body is the
triacylglycerols or triglycerides, making up 95% of lipids.
This type of lipid is most commonly found in fried foods,
vegetable oil, butter, whole milk, cheese, cream cheese,
and some meats, while naturally occurring lipids can be
found in foods such as avocados, olives, corn, and nuts.
Like most fats in the body, triacylglycerols do not
dissolve in water. Another type of lipid is phospholipids
which comprise 2% of the lipids in the body. Unlike
triacylglycerols, this type of lipid is water-soluble and
can be found in animals and plants. Phospholipids are
vital in forming a protective barrier around the body
cells and are synthesized to form cell and organelle
membranes. Lastly, the most uncommon lipids are
sterols, with cholesterol as an example. Cholesterol is
essential in the body as it is a prerequisite for sex
hormones, vitamin D, and bile salt synthesis. (Libretexts,
2022).
In this experiment, lipid isolation is performed to
identify the kinds of lipids and characterize them.
Methodology
A. Materials
Isolation of Lipid extract
In the isolation of the lipid extract, the group was
assigned with isolating the lipid from the chicken (Gallus
gallus domestica) egg yolk. The egg yolk from the
organic egg, hexane:ethanol mixture with a ratio of
(2;1), anhydrous Na2SO4 (to remove any residual water),
beaker, three test tubes, graduated cylinder, pasteur
pipette, and aspirator were the materials used in this
experiment.
2D Thin Layer Chromatography (TLC) Analysis
In the 2D Thin Layer Chromatography Analysis, two
different solvent systems were used. The first solvent
was a mixture of petroleum ether, methanol, and water
in the ratio (65:25:4) and another containing petroleum
ether, methanol, and ammonium hydroxide in the same
ratio as the previous solvent. This portion also made use
of a TLC plate, a developing chamber, a spray bottle
containing a Ninhydrin spray reagent, iodine crystals,
and capillary tubes. The lipid extract was used as a
sample in this separation technique.
Column Chromatography
In the column chromatography of lipids, the lipid
extract from the chicken egg yolk was used together
with three different eluents which are the petroleum
ether:ethyl ether mixture (9:1) as Eluent A, 5%
methanol in DCM as Eluent B, and DCM:CH3OH:H2O
(1:3:1) as Eluent C. This experiment also utilized silica
gel, cotton, 5 mL syringe, three test tubes, and an iron
stand.
Qualitative Tests
The three eluates from the column chromatography
were then subjected to qualitative tests to characterize
Page 1 of 10
the lipids. Pasteur pipettes were used to add the
reagents in the microcentrifuge tubes each containing
10 drops of eluates and the cholesterol.
The hydroxamic test or test for esters made use of
ethanol:1-butanol in a 3:1 ratio, 2M NH2OH·HCl, 3M
NaOH and 5% FeCl3 ·6H2O.
In the Acrolein Test or test for glycerol, a pinch amount
of KHSO4 reagent was used, added with a small spatula.
A 100°C water bath was also utilized.
For the Kraut’s test, which is a modified Dragendoff’s
test, Bi(NO3)3 , HNO3, and KI were used as reagents.
In the Ascorbic Acid Method or the test for phosphate,
6M of HNO3, 6M NaOH, molybdate reagent, and
freshly-prepared ascorbic acid solution were needed for
the experiment
In the Liebermann-Burchard test, or test for cholesterol,
acetic anhydride, together with concentrated H2SO4,
which acted as a catalyst and dehydrating agent, was
added.
As for the test for Lipid Unsaturation, Br2 in DCM was
used. Bromine acted as the primary reagent. It was
dissolved in DCM to prevent it from fuming as it is
unstable and forms fumes when exposed to the
environment.
Quantitative Tests
The group made use of the Liebermann-Burchard
Colorimetric Essay for the quantitative analysis. Acetic
anhydride was used as a solvent and dehydrating agent.
Similarly, sulfuric acid was also used as a dehydrating
agent and an oxidizing agent.
B. Procedure
Isolation of Lipid Extract
To isolate the lipids, the egg yolk was first separated
from the egg whites. Then, two volumes of the
hexane-ethanol mixture were mixed with the yolk and
allowed to stand for five minutes until it was jelly-like. It
was then transferred to a beaker and added with
anhydrous (Na2SO4). Finally, the extract was decanted
and transferred to a clean test tube. Around 10 mL of
clear, yellow lipid extracts were isolated.
2D Thin Layer Chromatography (TLC) Analysis
The isolates were subjected to 2-D Thin Layer
Chromatography (TLC) for better separation of
compounds as compared to single chromatography. The
TLC plate is prepared by marking a 0.5cm line from the
edge of each side of the TLC plate using a pencil. The
lipid extract was spotted 10 times from the right-bottom
edge of the TLC plate. After each spot it needs to be dry
before putting another spot. Then developed in the TLC
chamber with a 65:25:4 (v/v/v) petroleum ether:
methanol: water solvent. Wait until the solvent system
rises to the top then remove the TLC plate in the
chamber. Dry after removing TLC plate then rotate it
◦
90 clockwise. After the TLC plate has dried up, it was
developed in the second TLC chamber with a 65:25:4
(v/v/v) petroleum ether: methanol: ammonium
hydroxide. Once the solvent system has reached the
top, the TLC plate was removed from the chamber and
dried again. The iodine crystals are placed inside the
beaker and the vapor inside is saturated for five
minutes. Then the TLC plate is placed inside the beaker
with iodine crystals, wait until the spots appear. Once
the spots appear, remove it from the beaker then mark
brown or yellow-colored spots with a pencil. For the
determination of amino acid in the TLC plate, ninhydrin
is sprayed on the TLC plate then dried using a hair
blower.
Column Chromatography
In the column chromatography of lipids, we removed
plunger and the needle first, then covered the base with
a cotton plug to avoid the flowing of the samples and
solvent. Afterwhich, we added the silica gel with a 1 cm
allowance on top of the barrel. A 10 mL of petroleum
ether was poured to wet the silica gel then 1 ml of the
extracted lipid was added. For the first solvent, the
petroleum ether:ethyl ether (9:1), a 5 ml of it was
poured into the barrel. We let it run through the barrel
of the syringe and waited for the eluate to be collected.
Next, the second solvent which is the 5% methanol in
DCM was also added to the barrel and collected the
second eluate afterwards. Lastly, a 5 ml of
DCM:methanol:water (1:3:1) which is the third solvent,
was added to the barrel and collected the third eluate,
Qualitative Tests
In the hydroxamic test or test for esters, 5 mL of a 3:1
ethanol:1-butanol solution was mixed with 10 drops of
each of the eluates. Then, 2M NH2OH·HCl, 3M NaOH,
and 5% FeCl3·6H2O were mixed manually in the
Page 2 of 10
microcentrifuge tubes. This was done to detect the
presence of triacylglycerides (TAGs), cholesteryl esters,
and glycerophospholipids.
In the Acrolein Test or the test for glycerol, a pinch
amount of KHSO4 was added to the 10 drops of the
different eluates that were placed on three separate
microcentrifuges. The three microcentrifuges were then
heated gently over the alcohol lamp while continuously
shaking it from side to side. This test was to determine
the presence of triacylglycerides (TAGs) and
glycerophospholipids.
The Kraut’s test or test for cholines also made us
In the Liebermann-Burchard Test or the test for
Cholesterol. A 0.25 mL of DCM was added to the 10
drops of 10 drops of the different eluates that were
placed on three separate microcentrifuges. Six drops of
acetic anhydride and two drops of concentrated sulfuric
acid were added to the mixture. This test was
accomplished to detect the presence of cholesterol and
cholesteryl esters.
Precautions
Before conducting the experiment, each group member
put on appropriate laboratory attire. These articles of
clothing include lab coats, safety goggles, head caps,
latex gloves, and masks.
Upon acquiring the materials and equipment for the
experiment, the group made sure to wash all the
glassware and disinfect the work surface with 70% ethyl
alcohol.
C. Observations
Isolation of Lipid Extract
The lipid extract appeared to be a clear and yellow
solution. The group managed to acquire approximately
15 mL of lipids from the egg yolk.
2D Thin Layer Chromatography Analysis
2D Thin Layer Chromatography was used to separate
and determine the different polarities of the ten
components of lipid extract by developing it into the
two solvent systems, petroleum ether: methanol: water
(65:25:4) and petroleum ether: methanol: NH4OH
(65:25:4), both of which have a higher Rf, indicating that
they are more polar. Visualizing agents, such as iodine
vapor, are also employed to identify unsaturated lipid
components, resulting in yellow-brown spots. Ninhydrin
was also used to determine if the lipid extract was free
of amino groups; if it showed purple spots, an amino
group was present.
After performing all of the procedures, only two
unsaturated lipid components are present, each with a
yellow brown spot, and the sample is free of amino
groups, which did not show any purple spots. Each spot
is then measured on how far the distance traveled and
the total distance traveled by the solvent systems. Rf
value is calculated using the distance of spot traveled
divided by the total distance traveled by the solvent
system. Therefore, the Rf value of the 1st spot was 0.65,
while the 2nd spot rf value was 0.80.
Column Chromatography
Column chromatography is a method of purifying
compounds based on their polarity or hydrophobicity.
(University of Toronto Scarborough, n.d.). Eluate A, the
mobile phase used was petroleum ether: ethyl ether
(9:1) that comprises TAGs and cholesterol which is the
least polar and characterized as a clear solution. For
Eluent B, the mobile phase used was 5% methanol in
DCM wherein only cholesterol is present which makes it
more polar than Eluent A, also it is more saturated than
eluate B having a blurry solution. In Eluent C, the mobile
phase used was DCM: methanol: water (1:3:1) that
comprises of glycerophospholipids and sphingolipids
wherein it was poured last because it is the most polar
than among the eluates and having a yellow color
solution. After collecting all the eluates, then this will be
further utilized in the qualitative test.
Qualitative Tests
A series of qualitative tests were conducted to
characterize the samples. A summary of all the color
reactions is shown in Table 1.
Table 1. Qualitative Tests for Lipids
QUALITATIVE TESTS FOR LIPIDS
ESTER GLYCE KRAUT’S LIEBERMAN
ROL N
BURCHARD
1st (+) (+) (-) dark (-) clear
Eluate burgu burnt orange
ndy plastic solution
w/o ppt
Page 3 of 10
 2nd (+) (+) (-) dark (-) clear Concent T1 T2 T3 AVERAG
Eluate burgu burnt orange rations E
ndy plastic solution
w/o ppt 160 1.676 2.506 1.811 1.9977
μg/mL
3rd (+) (+) (-) dark (-) clear
Eluate burgu burnt orange 80 1.639 1.755 1.824 1.7393
ndy plastic solution μg/mL
w/o ppt
40 0.529 0.623 0.417 0.523
μg/mL

20 0.173 0.302 -0.054 0.1403

μg/mL
A burgundy color is indicative of a positive result In the
hydroxamic test. This confirms the presence of amides 10 -0.09 -0.154 -0.181 -0.1417
and esters. All eluates gave off positive results in this μg/mL
test.
5 μg/mL -0.245 -0.184 -0.265 -0.2313
The Acrolein test is a test for glycerol containing
LIPID EXTRACT
compounds. A positive result in this test is characterized
by an acrid or burnt fat odor. This was observed in all Absorbance (680 nm)
eluates as well.
T1 T2 T3 AVERAGE
The presence of cholein is detected through the Kraut’s
test. An orange precipitate indicates a positive result. 0.97 0.232 0.206 0.4693
None of the eluates tested positive since all results were
only dark orange solutions with no precipitate. Amount of Cholesterol
Condensation, dehydration, and epimerization are the
stated principles for this test. Additionally, this test is a 𝑦 = 0. 4693
modified version of the alkaloids test or the 𝑏 = − 0. 349
Dragendorff's test. 𝑚 = 0. 0251782

In the Liebermann-Burchard test, an emerald green or 𝑦 = 𝑚𝑥 + 𝑏

𝑦−𝑏 0.4693−(−0.349)
blue green color is expected to be the result of 𝑥= 𝑚 = 0.0251782
= 32. 5003
cholesterol and cholesteryl ester containing compounds.
Similar to the Kraut’s test, no eluates gave positive 32. 5003 𝑚𝑐𝑔 : 1000𝑚𝐿 :: 𝑥 𝑚𝑐𝑔 : 500𝑚𝐿
results in this test. 𝑥=
32.5003(500)
= 16. 25015 mcg
1000

Quantitative Tests
The Liebermann-Burchard colorimetric assay was The amount of cholesterol was computed using the
utilized as a quantitative test since it is a chemical values garnered from absorbance at 680 nm after the
estimation of cholesterol.
assay was subjected to the spectrophotometer.
Table 2. Quantitative Analysis of Lipids
As seen in Table 2, the amount of cholesterol was
QUANTITATIVE ANALYSIS denoted as x or concentration. It was determined from
CHOLESTEROL STANDARD CURVE the using the values of the linear regression equation
with y as the value of absorbance. The lipid extract
Absorbance (680 nm) amounted to 16.025 mcg of cholesterol.

Page 4 of 10
IX. Conclusion
Lipids are nutritional and dietary necessities found
abundantly in animal sources. Egg yolks in particular
contain cholesterol, cholesterol esters,
phosphatidylcholine, phosphatidylethanolamine,
sphingomyelin, lysophosphatidylcholine,
lysophosphatidylethanolamine, phosphatidylinositol,
phosphatidylglycerol, and phosphatidylserine (Du et al.,
2020).
Isolating the lipids from egg yolk did not yield large
amounts despite the varied lipids it contains since they
are susceptible to oxidation and peroxidation.
The 2D TLC Chromatography analysis only gave two dots
despite egg yolks having eight to ten types of lipids.
Some may not be present for they are only available in
trace amounts. The second spot had a higher Rf value as
compared to the first spot which indicated that the
former is more nonpolar. The second spot could either
be phosphatidylethanolamine, phosphatidylcholine, and
sphingomyelin, while the first could be either
cholesteryl esters, TAGs, free fatty acids, or cholesterol.
In column chromatography, TAGs and cholesterol were
present in Eluent A which makes the least polar, hence
appearing as a clear solution. The more polar the
eluent, the more blurry the solution. Thus, Eluent B,
which contains solely cholesterol, was more turbid as
compared to Eluent A. Eluent C, which was the most
turbid, is the most polar among the three due to the
solvents that comprises it. This eluate contains
glycerophospholipids.
In the Hydroxamic test, esters are detected from the
samples. All eluates gave positive results in this test due
to the complexation of the ferric hydroxamate reagent.
For the Acrolein test, all eluates gave a positive result.
However, Eluate B should have tested negative. The
false positive result may be due to the residues of Eluate
A that remained in the syringe since only Eluate A and
Eluate B contained glycerol. The principle behind this
test is dehydration due to the acidic reagents.
The presence of choline is detected in the Kraut’s test
through condensation, dehydration, and epimerization
reactions. None of the eluates gave positive results in
this test.
In the Liebermann-Burchard test, the presence of
cholesterol, particularly the
cyclopentanoperhydrophenanthrene (CPPP) nucleus,
and cholesteryl esters were tested. The complexation
reaction due to the shifting of the positions of the
double bonds within the CPPP nucleus. None of the
eluates gave off positive results in this test.
In the Liebermann-Burchard colorimetric assay, the
chemical estimation of cholesterol occurred due to the
esterification of the third lipid carbon attached to a
hydroxyl group and esterification of the double bond at
the fifth carbon position. The darker the emerald green
color indicated higher intensity and concentration of
cholesterol.
References:
Ahmed, S., Ahmed, O., & Shah, P. (2018, October 27).
Biochemistry, Lipids. Nih.gov; StatPearls Publishing.
https://www.ncbi.nlm.nih.gov/books
/NBK525952/
Du, H., Xiao, N., Zhao, Y., Yao, Y., Wu, N., Xu, M., & Tu,
Y. (2020). Biological activities of egg yolk lipids:
A review. Journal of agricultural and food
chemistry, 68(7), 1948-1957.
Libretexts. (2022, May 23). 6.2: What Are Lipids?
Medicine LibreTexts.
https://med.libretexts.org/Courses/American_P
ublic_University/APU:_Basic_Foundation_of_Nu
trition_for_Sports_Performance_(Byerley)/06:_
Lipids_Basics_-_Another_Energy_Source_for_th
e_Athlete/6.02:_What_Are_Lipids
Thompson, T. E. (2022, December 2). Lipid | Definition,
Structure, Examples, Functions, Types, & Facts.
Encyclopedia Britannica.
https://www.britannica.com/science/lipid
University of Toronto Scarborough (n.d.). Column
Chromatography Theory.
https://www.utsc.utoronto.ca/webapps
/chemistryonline/production/column.php
Appendices:
Appendix A. Schematic Diagram
Page 5 of 10
 Figure 4. Schematic diagram of the Qualitative Test of
Figure 1. Schematic diagram of the isolation of Lipids the Test for Esters

Figure 2. Schematic diagram of the 2D TLC Analysis of Figure 5. Schematic diagrams of the Qualitative Test of
Lipids the Test for Glycerol and Test for Choline

Figure 3. Schematic diagram of the Column Figure 6. Schematic diagram of the Qualitative Test of
Chromatography of Lipid Extract the Test for Phosphate

Page 6 of 10
 Photo of extract

Figure 7. Schematic diagrams of the Qualitative Test of

the Test for Cholesterol and Test for Lipid Unsaturation
with Bromine

Characteristics Clear yellow solution

Appendix C. Column Chromatography Eluates

Figure 8. Schematic diagram of the Quantitative Test of Eluate A

the Liebermann-Burchard Assay

Eluate B

Figure 9. Schematic diagrams of the Computation for

the Quantitative Test of the Liebermann-Burchard Assay

Appendix B. Isolated Lipid Extract Sample

Table 1. Characteristics of Lipid Extract
LIPID EXTRACT FROM EGG YOLK

Page 7 of 10
 Eluate C Eluate burgundy plastic orange
solution w/o
ppt

Table 2.2 Qualitative Data

QUALITATIVE TESTS FOR LIPIDS

Appendix D. Qualitative Analysis of Lipids PHOSPHATE LIEBERMANN LIPID

BURCHARD UNSATURATION
Table 2.1 Qualitative Data
QUALITATIVE TESTS FOR LIPIDS - (-) clear -

ESTER GLYCEROL KRAUT’S

1st (+) (+) burnt (-) dark

Eluate burgundy plastic orange
solution w/o
ppt

- (-) clear -

2nd (+) (+) burnt (-) dark

Eluate burgundy plastic orange - (-) clear -
solution w/o
ppt

3rd (+) (+) burnt (-) dark

Page 8 of 10
Appendix E. 2D Thin Layer Chromatography

QUANTITATIVE ANALYSIS

CHOLESTEROL STANDARD CURVE

Absorbance (680 nm)

Concent
rations T1 T2 T3 AVERAG
E

160 1.676 2.506 1.811 1.9977

Image 1. Group 1’s 2D Thin Layer Chromatography μg/mL

80 1.639 1.755 1.824 1.7393

μg/mL
Total Distance:
1st Distance: 4.0 cm
40 0.529 0.623 0.417 0.523
2nd Distance: 5.5 cm
μg/mL
Distance spots traveled:
1st spot: 2.6 cm 20 0.173 0.302 -0.054 0.1403
2nd spot: 4.4 cm μg/mL
𝑅𝑓𝑣𝑎𝑙𝑢𝑒𝑠:
2.6 𝑐𝑚 10 -0.09 -0.154 -0.181 -0.1417
1st 𝑅𝑓𝑣𝑎𝑙𝑢𝑒𝑠 : 4.0 𝑐𝑚
= 0.65
μg/mL
4.4 𝑐𝑚
2nd 𝑅𝑓𝑣𝑎𝑙𝑢𝑒𝑠 : 5.0 𝑐𝑚 = 0.80
5 μg/mL -0.245 -0.184 -0.265 -0.2313
Number of spots: 1 spot in each mobile phase
LIPID EXTRACT
A: 1 spot
B: 1 spot Absorbance (680 nm)

Appendix F. Quantitative Analysis of Carbohydrates T1 T2 T3 AVERAGE

0.97 0.232 0.206 0.4693

Amount of Cholesterol

𝑦 = 0. 4693
𝑏 = − 0. 349
𝑚 = 0. 0251782

𝑦 = 𝑚𝑥 + 𝑏
𝑦−𝑏 0.4693−(−0.349)
𝑥= 𝑚 = 0.0251782
= 32. 5003
Image 2. 96-well Microplate of Liebermann-Burchard
Method
32. 5003 𝑚𝑐𝑔 : 1000𝑚𝐿 :: 𝑥 𝑚𝑐𝑔 : 500𝑚𝐿
32.5003(500)
𝑥= 1000
= 16. 25015

Image 3. Regression Line Chart of 96-well Microplate of

Liebermann-Burchard Method

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Grading Rubrics Formal Report
Group: 1 Section: 2BPH

Page 10 of 10