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Extraction of Total Lipids from Chicken Egg Yolk and Analysis of Lipids by Thin Layer

Chromatography, Column Chromatography and Qualitative Tests


Cu, E., David P.P., De Leon Y., De Leon P., Del Rosario K.V., Delmendo, C.A.
ABSTRACT
The objectives of this experiment is to extract total lipids from chicken egg yolk and
analyze it using two-dimensional thin-layer chromatography which includes ninhydrin and
phosphorus test with the positive results of blue-violet coloration and blue spots on white
background respectively. Column chromatography is also performed to (SEARCHHHHH
WHYYYYY), and identify the lipids present in each of the fractions using the qualitative tests
namely: Test for ester, Test for Glycerol (Acrolein Test), and Test for Cholesterol (LiebermannBurchard Test). Positive results
. Collected eluates were subjected for the qualitative tests for lipids. 10 drops of eluates
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 phosphate, test for cholesterol also
known as Liebermann-Burchard test, test for Test for - amino acids (Ninhydrin Test) and test
for lipid unsaturation with bromine. Test for esters yielded a yellow solution for the first and
second eluate and a burgundy solution for the third eluate. Test for glycerol produced no odor for
the three eluates. Test for phosphate produced a turbid solution for the first eluate, formed white/
slightly yellowish crystals for the second eluate and turbid yellow with crystals for the third
eluate. Test for cholesterol or Liebermann-Burchard test did not show any color change for the
first and third eluate while it produced a green color on the second eluate.
INTRODUCTION
Lipids are a large and diverse group
of naturally occurring organic compounds,
found in living organisms, and are insoluble
in water but soluble in non-polar solvents
and solvents of low polarity (e.g. ether,
chloroform,
acetone
&
benzene).
(https://www2.chemistry.msu.edu/faculty/re
usch/virttxtjml/lipids.htm) This lack of
solubility in water is an important property
because our body chemistry is so firmly
based on water. Most body constituents
including carbohydrates are soluble in water
but the body also needs insoluble
compounds for many purposes, including
the separation of compartments containing

aqueous solutions from each other, thats


where lipids come in.
The water-insolubility of lipids is
due to the fact that the polar groups they
contain are much smaller than their alkanelike (nonpolar) portions. These nonpolar
portions provide the water-repellent, or
hydrophobic, property.
An important use for lipids,
especially in animals, is storage of energy.
Plant store energy in form of starch. Animals
including humans find it more economical to
use lipids (fats) instead. Although our bodies
do store some carbohydrates in the form of
glycogen for quick energy when we need it,

energy stored in the form of fats is much


more important. The reason is simply that
the burning of fats produces more than twice
as much energy as burning of an equal
weight of carbohydrates.
Composition of Chicken Egg Yolk
The yolk makes up about 33% of the
liquid weight of the egg; it contains
approximately 60 calories, three times the
caloric content of the egg white. One large
egg (50 grams in weight, 17 gram yolk)
contains approximately: 2.7g protein, 210
mg cholesterol, 0.61g carbohydrates and
4.51g total fat (USDA National Nutrient
Database). All of the fat soluble vitamins,
(A, D, E and K) are found in the egg yolk.
Egg yolks are one of the few foods naturally
containing vitamin D.
Egg yolk is also a source of lecithin,
an emulsifier and surfactant. The yellow
color is caused by lutein and zeaxanthin,
which are yellow or orange carotenoids
known as xanthophylls.

Column chromatography is another solidliquid technique in which the two phases are
a solid (stationary phase) and a liquid
(moving phase). The theory of column
chromatography is analogous to that of thinlayer chromatography. The most common
adsorbents - silica gel and alumina - are the
same ones used in TLC. The sample is
dissolved in a small quantity of solvent (the
eluent) and applied to the top of the column.
The eluent, instead of rising by capillary
action up a thin layer, flows down through
the column filled with the adsorbent. Just as
in TLC, there is an equilibrium established
between the solute adsorbed on the silica gel
or alumina and the eluting solvent flowing
down through the column.
Stationary phase (adsorbent)
The stationary phase or adsorbent in
column chromatography is a solid. The most
common stationary phase for column
chromatography is silica gel, followed by
alumina. Cellulose powder has often been
used in the past. Also possible are ion
exchange chromatography,reversed- phase
chromatography
(RP),
affinity
chromatography or expanded bed adsorption
(EBA). The stationary phases are usually
finely ground powders or gels and/or are
microporous for an increased surface,
though in EBA a fluidized bed is used.

Figure 1. Chicken egg yolk


Column Chromatography
Column chromatography is one of
the most useful methods for the separation
and purification of both solids and liquids
when carrying out small-scale experiments.

Figure 2. Column Chromatography


Mobile phase (eluent)

The mobile phase or eluent is either a pure


solvent or a mixture of different solvents. It
is chosen so that the retention factor value of
the compound of interest is roughly around
0.2 - 0.3 in order to minimize the time and
the amount of eluent to run the
chromatography. The eluent has also been
chosen so that the different compounds can
be separated effectively. The eluent is
optimized in small scale pretests, often using
thin layer chromatography (TLC) with the
same stationary phase.
A faster flow rate of the eluent minimizes
the time required to run a column and
thereby minimizes diffusion, resulting in a
better separation, see Van Deemter's
equation. A simple laboratory column runs
by gravity flow. The flow rate of such a
column can be increased by extending the
fresh eluent filled column above the top of
the stationary phase or decreased by the tap
controls. Better flow rates can be achieved
by using a pump or by using compressed gas
(e.g. air, nitrogen, or argon) to push the
solvent through the column (flash column
chromatography).
The particle size of the stationary phase is
generally
finer
in
flash
column
chromatography than in gravity column
chromatography. For example, one of the
most widely used silica gel grades in the
former technique is mesh 230 400 (40 63
m), while the latter technique typically
requires mesh 70 230 (63 200 m) silica
gel.
A spreadsheet that assists in the successful
development of flash columns has been
developed. The spreadsheet estimates the
retention volume and band volume of

analytes, the fraction numbers expected to


contain each analyte, and the resolution
between adjacent peaks. This information
allows users to select optimal parameters for
preparative-scale separations before the
flash column itself is attempted.
The objectives of the experiment are as
follows: (1) to extract total lipids from
chicken egg yolk, (2) to analyze the lipids
present in the crude extract using column
chromatography (3) to identify lipids present
in each of the fractions using qualitative
tests and, (4) to determine the degree of
unsaturation of lipids by bromine test.
METHODOLOGY
Materials and Compounds Used
Samples to be tested:
The following samples were
subjected
to
test
using
column
chromatography and qualitative test for
lipids: Chicken egg yolk, Coconut oil,
Canola oil, Corn oil, Olive Oil, Vegetable
oil, Cholesterol and Lecithin.
Extraction of total lipids from chicken egg
yolk
Chicken egg yolk, equal amount of
ethanol, hexane and acetone were used for
the Extraction of total lipids from chicken
egg yolk
Thin Layer Chromatography Analysis from
Egg Yolk
Two separate beakers with TLC
solvent mixtures namely: a. 65:25:4 (v/v/v)
petroleum ether:methanol:water and b.
65:25:4
(v/v/v)
petroleum

ether:methanol:NH4OH were prepared along


with two clean TLC plates, iodine (I2)
crystals and vapour, ninhydrin and
phosphorus.
Column Chromatography of Lipids
The following were used for the
column chromatography of lipids: extracted
total lipids from chicken egg yolk, 0.5 g
silica gel, 4ml of petroleum ether, 5ml of 9:1
mixture of petroleum ether and ethyl ether, 5
ml 5% methanol in dichloromethane, and
5ml CH2Cl2: CH3OH: H2O (1:3:1).
Test for Ester

It was then diluted with an equal amount of


ethanol and was mixed to dehydrate and
partially extract the polar lipids. After
dilution, hexane was added and stands for 5
minutes until two layers formed. This results
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 ones, especially cholesterol. The
upper layer was collected and transferred
into a clean test tube. The thin-layer
chromatography (TLC) and column
chromatography (CC) were performed using
this.

The following were used for the test


for ester: Ethanol:1-BuOH (3:1) with 10
drops of eluate, 2 drops each of 2M
hydroxylamine hydrochloride and 3M
NaOH, 2 drops of 6M HCl, 1 drop of 5%
FeCl36 H2O in 0.1M HCl.
Test for Glycerol (Acrolein Test)
For this test, 10 drops of eluate with
a pinch amount of KHSO4 were prepared.
Test for Cholesterol (Lieberman-Burchard
Test)
The following were used for the test
for cholesterol (Lieberman-Burchard Test):
10 drops of eluate, 0.25 ml dichloromethane,
6 drops of acetic anhydride and 2 drops of
concentrated H2SO4.
B. Procedures
Extraction of Total Lipids from Chicken Egg
Yolk
The egg yolk was separated from the
chicken egg and its volume was determined.

Figure 1. Egg Yolk Isolate (Upper layer) for


TLC and CC
Thin Layer Chromatography Analysis of
Lipids from Egg Yolk
Two separate beakers were prepared
and used to separate beakers were prepared
and used to equilibrate the following TLC
solvent mixture: a. 65:25:4 (v/v/v)
petroleum ether:methanol:water and b.

65:25:4
(v/v/v)
ether:methanol:NH4OH.

petroleum

ninhydrin test, the TLC plates were


completely sprayed with phosphorus and
placed on the hot plate for 30 seconds.
Phosphorus containing compounds will
appear as blue spots on white background.
Additional heating will char unsaturated
compounds.
Column Chromatography of Lipids

Figure 2. The two solvent mixtures for TLC


Two TLC plates were placed on the hot plate
with the silica side up for approximately 3
minutes to reactivate silica. Remove the
TLC plates from heat. The extract (from the
extraction of Total Lipids from Chicken Egg
Yolk) was used to spot at least 1 cm from
the edge of the TLC plates. The TLC plates
were developed in the first solvent mixture
(65:25:4
(v/v/v)
petroleum
ether:methanol:water). The TLC plates were
then removed when the solvent front was
almost inch from the top and the plates
were transferred to the second solvent
mixture
(65:25:4
(v/v/v)
petroleum
ether:methanol:NH4OH). Iodine crystals
were placed in a separate beaker and the
containers were saturated with I2 vapor for 5
minutes. TLC plates were transferred in the
beaker with iodine until spots appeared. The
plates were then placed on a hot plate to
remove excess I2 and completely sprayed
with ninhydrin. The TLC plates were placed
again on the hot plate for 1 minute. A blueviolet coloration indicated the presence of amino acids in the ninhydrin test. After the

Small column was prepared by


pouring a slurry of 0.5 g silica gel in 4ml of
petroleum ether into the glass column
(Pasteur pipette) with a tapered end plugged
with glass wool. The lipid extract from
chicken egg yolk with a volume of 1 ml was
then introduced into the column, saving the
run-through in a clean test tube. The column
was washed with 5ml 9:1 mixture of
petroleum ether and ethyl ether, collecting
the eluate in the same tube as the runthrough. The column was again washed with
the second eluent (5 ml 5% methanol in
dichloromethane) the eluate was then
collected in another clean test tube. The
column was washed with the last eluent, 5ml
dichloromethane:methanol:water (1:3:1) and
eluate was collected in another test tube. The
different eluates culled were saved for
qualitative analysis.
Qualitative Tests for lipids
Test for Ester
Ethanol:1-butanol (3:1) with a
volume of 0.5 ml was introduced into the 10
drops of eluate. 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% FeCl36 H2O in


0.1M HCl and was ensured to be wellmixed. Color was noted. Samples with
positive results gave a burgundy color.

has traditionally been the chromatography of


choice.
(http://www2.onu.edu/~kbroekemeier/Chem314/IsolationCharacteriza
tionLipids.pdf)

Test for Glycerol (Acrolein Test)

The eluates identified based on the


chemical test performed are as follows:

A pinch amount of KHSO4 was


added to 10 drops of the eluate in a test tube.
The test tube was heated in a boiling water
bath and odor produced was noted. Burnt fat
odor was observed for positive test results.
Test for Cholesterol (Lieberman-Burchard
Test)
A volume of 0.25 mdichloromethane
was introduced into the 10 drops of eluate. 6
drops of acetic anhydride was then added
with 2 drops of concentrated H2SO4 and well
mixed. Color was noted. Positive results
produced a greenish color which indicated
the presence of cholesterol.
RESULTS AND DISCUSSION
Thin Layer Chromatography Analysis of
Lipids from Egg Yolk
The lipid composition of cells and
membranes can differ significantly with
respect to lipid group, phospholipids class
and acyl group identity. Separation of polar
lipids is further characterized through
adsorption chromatography using mixtures
of organic solvents in varying proportions to
separate phospholipids based on head group
polarity and charge. Organic solvents are
employed to separate lipids from proteins,
carbohydrates
and
water
soluble
metabolites. Lipids are often subsequently
separated into groups through the use of
chromatography. Thin layer chromatography

1st eluate: triglyceride/triacylglycerol


2nd eluate: cholesterol
3rd eluate: phospholipid (Lecithin)
Qualitative Tests for Lipids
Test for Ester
Esterification is the general name for a
chemical reaction in which two reactants
(typically an alcohol and an acid) form an
ester as the reaction product. 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.
Esterification is a reversible reaction.
Hydrolysisliterally "water splitting"
involves adding water and a catalyst
(commonly NaOH) to an ester to get the
sodium salt of the carboxylic acid and
alcohol. As a result of this reversibility,
many esterification reactions are equilibrium
reactions and therefore need to be driven to
completion according to Le Chatelier's
principle. Esterifications are among the
simplest and most often performed organic
transformations.
The most common esterification processes
involve nucleophilic acyl substitution where

the carbonyl compound is used as an


electrophile and is attacked by a
nucleophilic alcohol. However, other
processes are possible; esterification by
alkylation reverses the roles of "classic"
carbonyl chemistry: a carboxylate anion is
used as a nucleophile that displaces a halide
ion in an SN2 reaction.
Acid hydrolysis using sulphuric acid and
water (equilibrium reaction). The ester splits
into a carboxylic acid and alcohol, protons
are donated from the acid. The solution can
then be distilled and the remaining acid can
be checked using UV indicator.
Positive results for the test for ester yields a
burgundy color. Based on Table 2, the first
and second eluate yielded yellow solution
which is a negative result for ester while the
third eluate gave a burgundy solution which
is a positive result and shows the presence of
ester.
Test for Glycerol (Acrolein Test)
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 KHSO4, the glycerol portion
of the molecule is dehydrated to form the
unsaturated aldehyde, acrolein (CH2=CHCHO), which has the peculiar odor of burnt
grease.
Based on the results that were culled (Table
2), the first second and third eluate did not
produce any odor hence indicates the
absence of glycerol for each eluates.

Test for cholesterol (Lieberman-Burchard


Test)
Cholesterol is a lipid with a structure
quite different from that of phospholipids. It
is a steroid, built from four linked
hydrocarbon rings. A hydrocarbon tail is
linked to the steroid at one end, and a
hydroxyl group is attached at the other end.
In membranes, the molecule is oriented
parallel to the fatty acid chains of the
phospholipids, and the hydroxyl group
interacts with the nearby phospholipid head
groups.
Cholesterol is absent from prokaryotes but is
found to varying degrees in virtually all
animal membranes. It constitutes almost
25% of the membrane lipids in certain nerve
cells but is essentially absent from some
intracellular membranes.

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 that were culled (Table
2), the first and third eluate did not produce
any change in color. The second eluate

produced a greenish color which indicated


the presence of cholesterol.

REFERENCES:

Analysis of Lipids in Egg Yolk and Milk.


(2012, May 2). Retrieved May 11, 2016,
from
http://faculty.mansfield.edu/bganong/bioche
mistry/liptlc2.htm

BOOKS:

Column Chromatography
http://orgchem.colorado.edu/hndbksupport/c
olchrom/colchrom.html

Bettelheim,F.A.,
March,J.
(1990).
Introduction to organic and biochemistry.
Philadelphia: Saunders College.

Retrieved: March 8, 2010

Lipid Library
Heftman, E. (1967). Chromatography. New
York: Reinhold Publishing Corporation

http://lipidlibrary.aocs.org/Lipids/whatlip/in
dex.htm#def
Retrieved: March 8, 2010

Lehninger,
A.L.
(2008).
Legninger
Principles of Biochemistry. New York: W.H.
Freeman.

McKee.
(2003).
Biochemistry:
The
Molecular Basis of Life. Boston: McGrawHill.

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