Table of Contents

1.

Abstract ........................................................................................................................................... 2

2.

Introduction ..................................................................................................................................... 2

3.

Objective ......................................................................................................................................... 3

4.

Theory ............................................................................................................................................. 4

5.

Apparatus and Materials ................................................................................................................. 6

6.

Procedure ........................................................................................................................................ 6

7.

Result .............................................................................................................................................. 9

8.

Calculation ...................................................................................................................................... 9

9.

Discussion ..................................................................................................................................... 10

10. Conclusion..................................................................................................................................... 11
11. Recommendation........................................................................................................................... 12
12. Reference....................................................................................................................................... 12
13. Appendices .................................................................................................................................... 13

LAB 3:PURIFICATION OF LIPIDS FROM EGG YOLK

1. Abstract
The experiment was about extraction of total lipids from chicken egg yolk,
column chromatography of lipids and qualitative tests for lipids. A column
chromatographic are used to separate lipid components of serum and lipoproteins into
individual fractions containing hydrocarbons, cholesterol esters, triglycerides,
cholesterol, free fatty acids and phospholipids. This experiment determined the
components of each eluents. Lipids were based upon their polarity using column
chromatography. The extracted lipids from chicken egg yolk were used in the column
chromatography. The results obtained were analyzed and it showed that the lipids are
eluted by increasingly polar solvents. The lipids present in the crude extract were
triglyceride, cholesterol and phospholipid. In qualitative assay, there are two assay
conducted which are ester assay to detect the presence of ester in lipid and the other
one is cholesterol assay to detect the presence of cholesterol in lipid. In ester assay,
hydrochloric acid as the reagent while in cholesterol assay, acetic anhydride is used as
the reagent. The aim of this experiment is to understand and to determine the amounts
of lipid components in chicken egg yolk. In the end of this experiment we had
founded which lipid component in chicken egg yolk is more polar among all using
column chromatography.

2. Introduction
In the early sixties, the existence of lipid had been discovered by Biophysicist
Alec Bangham of the Animal Physiology Institute in Cambridge England. He made a
discovery about lipids that they they can put themselves together. When he placed
lipids from egg yolks in water they arranged themselves into double layered circles
the size of a cell, these lipid bubbles are known as liposome. After that, this discovery
been continued by Robert Boyle, Poulletier de la Salle, Antoine Franois de Fourcroy
and others in study of modern lipid chemistry began in the 17th and 18th centuries.
Then, the 19th century chemist, Chevreul, identified several fatty acids, suggested the
name cholesterine for the fatty substance in gallstones, coined the word glycerine,
and showed that fats were comprised of glycerol and fatty acids.

The 20th century

brought many advances in the understanding of lipoprotein structure and function, and
explored relationships between lipoproteins and disease states. (Barreto, M. C. ,2005).

Lipid is one of the major families of biochemical compound that are needed in
human body other than carbohydrate, protein and amino acid. Unlike other
biochemical families, lipids are characterized instead on the basis of a single physical
property and their solubility. Lipids are hydrophobic and tend to be insoluble in water,
but soluble in certain organic solvents such as benzene, chloroform and ether.
Commonly, they are divided 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).

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.

3. Objective

1. To extract total lipids from egg yolk using methanol and chloroform, then separate the
lipid fractions, triglyceride, cholesterol, and phospholipids by using column
chromatography.
2. To identify lipids present in each of the fractions using qualitative tests and
quantitative test.
3. To identified investigate the properties of lipids present in chicken egg yolks
4. To investigate the lipid components present in the crude extract using column
chromatography of the extracted lipids from chicken egg yolk.

4. Theory

Lipids are known as a gru of substances of diverse structure that share the common trait
of being soluble in solvents such as ether or benzene. The major fractions of lipid are
triglycerides (68%), phospholipids (28%), and cholesterol (37%).
A triglyceride is an ester derived from glycerol and three fatty acids. It functions as a
blood lipid, which helps enable the bidirectional transference of adipose fat and blood
glucose from the liver. Triglycerides are existed by combining glycerol with three fatty
acid molecules. Alcohols have a hydroxyl (HO-) group. Organic acids have a carboxyl (COOH) group. Alcohols and organic acids join to form esters. The glycerol molecule has
three hydroxyl (HO-) groups. Each fatty acid has a carboxyl group (-COOH). In triglycerides,
the hydroxyl groups of the glycerol join the carboxyl groups of the fatty acid to
form ester bonds:

Figure 1: Example of an unsaturated fat triglyceride. Left part: glycerol,

right part from top to bottom: palmitic acid,oleic acid, alpha-linolenic
acid. Chemical formula: C55H98O6
Meanwhile,

Phospholipid

consists

of

two

section

which

are

the

'head'

is hydrophilic (attracted to water), while the hydrophobic 'tails' are repelled by water and are
forced to aggregate. The hydrophilic head contains the negatively charged phosphate group,
and glycerol. The hydrophobic tail usually consists of 2 long fatty acid hydrocarbon chains.
Phospholipid synthesis occurs in the cytosol adjacent to endoplasmic reticulum (ER)
membrane that is studded with proteins that act in synthesis (GPAT and LPAAT acyl
transferases, phosphatase and choline phosphotransferase) and allocation (flippase and
floppase). Eventually a vesicle will bud off from the ER containing phospholipids destined
for the cytoplasmic cellular membrane on its exterior leaflet and phospholipids destined for
the exoplasmic cellular membrane on its inner leaflet.

Figure 2: The left image shows a phospholipid, and the right

image shows the chemical that make up of the phospholipid
Furthermore,

cholesterol

is

a sterol(or modified steroid),

a lipid molecule

and

is biosynthesized by many animal cells because it is an essential structural component of

animal cell membranes that is required to maintain both membrane structural integrity
and fluidity. Cholesterol enables animal cells to not need a cell wall (like plants & bacteria)
to protect membrane integrity/cell-viability and thus be able to change shape and move about
(unlike bacteria and plant cells which are restricted by their cell walls). It functions to build
and maintain membranes as it modulates membrane fluidity over the range of physiological
temperatures.

Figure 3: the structure of cholesterol and its

IUPAC name is (3)-cholest-5-en-3-ol
Chromatography of lipids using a glass column filled with a suitable material is a
common and useful method for fractionation of lipid classes either on an analytical or a semipreparative scale. The retention results in a variety of mechanisms including hydrogen
bonding, Van der Waals' forces and also ionic bonding. The solid phase is relatively polar
(normal chromatography) and the more polar the lipid, the more strongly is it adsorbed. Thus,
the lipids are eluted by increasingly polar solvents. This technique has a low resolution when
used at low pressure (Solid Phase Extraction or SPE) but has a high resolution (high
performance) when run at high pressure using a stationary phase made of fine particles
(HPLC). The former is restricted to the fractionation of complex mixtures into two or three
less complex ones, the later being adopted to analyze and quantify purified fractions. The first
to be eluate is triglyceride, the second one is phospholipids and the last is cholesterol.
5

In order to calculate the lipid fraction, the formula been used:

To know the content of lipid:

5. Apparatus and Materials

1)
2)
3)
4)
5)
6)

1M NaCl
Ethyl ether
Methanol/ chloroform (2:1 by volume)
Silica gel
Petroleum ether
Egg yolk

7) Ester assay - ethanol/1-butanol (3:1 by volume, don't use denatured ethanol

which typically contains esters), 2 M hydroxylamine hydrochloride, 3 M
NaOH, 6 M HCl, 5% ferric chloride (hexahydrate) in 0.1 M HCl
8) Cholesterol assay - acetic anhydride, concentrated sulfuric acid
9) Column
10) Centrifuge tube
11) Test tubes
6. Procedure
1. Preparation of Egg Yolk Lipid Dispersion:

An egg yolk is dilute with three volumes of 1 M NaCl, mix well, and reserve about 3
mL. The rest is extracted with an equal volume of ethyl ether. It gives an outrageous
emulsion that takes some time to separate by centrifugation, but eventually it gives
two layers. Ether is removed by rotary evaporation and lipids are dispersed in the
same volume of 1 M NaCl as was extracted and mixed with the small volume of the
original dilution. That may help stabilize the dispersion and gives them some protein
in the extractions. This dispersion may stable for at least a year in the freezer.

Figure 3: the chicken egg yolk

2. Extraction of Lipids:

Mix 1.6 mL of the dispersion with 6 mL of methanol/chloroform (2:1 by volume) in a

test tube (16 x 125 mm) with a Teflon-lined screw-cap. After shaking the tube, add 2
mL each of 1 M NaCl and chloroform. Again the tube is capped and shaken, then
centrifuged to separate the phases. After removing and discarding the upper phase,
remove the lower phase to a clean preweighed tube. Solvent is removed from the tube
by drying under a stream of nitrogen or air in a warm water bath. When the residue is
dry the tube is weighed. The residue is the total lipid pool from the yolk and can be
stored cold if necessary.

3. Column Chromatography:
To separate the lipid fractions, prepare a small column by pouring a slurry of 0.5 g of
silica gel in 4 mL of petroleum ether into a glass column with a tapered end plugged
with glass wool. Dissolve the lipid residue in 2 mL of petroleum ether and pour it into
the column, saving the run-through, which contains some triglyceride, in a preweighed
tube. To complete elution of triglyceride, wash the column with 9 mL of a 9:1 mixture
of petroleum ether and ethyl ether, collecting the eluate in the same tube as the runthrough. Cholesterol is then eluted into a clean preweighed tube using 9 mL of 5%
methanol in chloroform. Finally, to completely remove phospholipids, elute the column
into a screw-capped tube with 9 mL of chloroform/methanol/water (1:3:1 by volume).
Then add 2 mL of chloroform and 2 mL of 1 M NaCl to break phases and get rid of the
water; the lower phase is removed to a clean pre-weighed tube. Solvent is removed
from the tubes again as above and the tubes are weighed. Fractions are saved for
qualitative analysis

Figure 4: the column chromatography

4. Qualitative Tests for Lipids:

a) Ester assay:
Place two drops of sample in a test tube and add 0.5 mL of ethanol/1-butanol (3:1
by volume). Add sequentially two drops each of 2 M hydroxylamine hydrochloride
and of 3 M NaOH and mix well. After allowing the samples to stand for 5 minutes,
add two drops of 6 M HCl and one drop of 5% ferric chloride (hexahydrate) in 0.1
M HCl and mix well. Carboxylate esters are converted to hydroxamic acids which
form a magenta-coloured complex with ferric ion.

b) Cholesterol assay:
Place two drops of sample in a test tube and add 0.25 mL of chloroform. Then add
six drops of acetic anhydride and two drops of concentrated sulfuric acid and mix
well. A greenish color produced after a few minutes is indicative of cholesterol.
Results are quite sensitive to the presence of water, so keep everything dry.

7. Result
i.

ii.

Quantitative result:
Samples

Weight (g)

Lipid fraction (%)

Triglyceride (bottle A)

1.5

7.66

Phospholipids (bottle B)

5.345

65.06

Cholesterol (bottle C)

12.475

27.28

Qualitative result:
Assays

Observations

Ester

Before
White

After
Light brown

Cholesterol

White

No change (white)

8. Calculation

9. Discussion

Chicken egg yolk composed of 33% of the liquid weight of the egg; 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. The composition (by
weight) of the most prevalent fatty acids in egg yolk is typically as follows:
Unsaturated fatty acids (Oleic acid 47 %, Linoleic acid 16 %, Palmitoleic acid 5 %,
Linolenic acid 2 %), Saturated fatty acids (Palmitic acid 23 %, Stearic acid 4 %,
Myristic acid 1 %). Naturally, egg yolks are contains vitamin D. The yellow color is
caused by lutein and zeaxanthin, which are yellow or orange carotenoids known as
xanthophylls.

The first important step in this experiment is the extraction of egg yolk. One of
the important factor that must be considered in extraction is the solubility of lipids,
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. Thus, several solvent systems might be well thought-out, depending on
the type of sample and its component. The solvents of choice are usually hexane,
chloroform, methanol or chloroform, methanol or water.

By using method of Column Chromatography of Lipids, with the use of the

collected lipid extract from chicken egg yolk, the lipids present in the crude extract
was analyzed and the first eluate was triglyceride, second eluate was cholesterol, and
the third eluate is the phospholipids. Chromatography of lipids using a glass column
filled with silica gel mix with petroleum ether, is a common and useful method for
fractionation of lipid classes either on an analytical or a semi-preparative scale. The
retention results in a variety of mechanisms including hydrogen bonding, Van der
Waals' forces and also ionic bonding. The solid phase is relatively polar (normal
chromatography) and the more polar the lipid, the more strongly is it adsorbed. Thus,
10

the lipids are eluted by increasingly polar solvents. From the obtained data
phospholipids is the most polar among the three eluates, next is cholesterol and last is
triglyceride. The lipid fractions obtained from the experiment are 7.66% for
triglyceride, 65.06% for phospholipids and 27.28% for cholesterol.
For qualitative method, the dry been residue is divided into two and each of it
undergoes ester assay and cholesterol assay. In the cholesterol assay, 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. Acetic anhydride 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. Unfortunately, in this experiment, there is no change in colour due to error
during the experiment undergoes. Meanwhile, ester assay involves acid hydrolysis as
it uses hydrochloric 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 colour but in this experiment it more to
light brown colour.
Possible sources of errors for the experiment were the use of incorrect or wrong
reagents and the lack of precision and accuracy in measuring samples or reagents. In
order to minimize the errors, the reagent should be labelled correctly and do not mix
the spatula or the dropper of the reagent with other reagent. This will increase the
accuracy of the result obtained.

10. Conclusion
As the conclusion, the lipids extracted from the chicken egg yolk are separated
based on its differences in solubility. Lipid solubility is depending on chemical nature
of the molecule, atomic or molecular formula weight, valence or charge or sphere of
hydration, charge density and sphere of hydration. Furthermore, in analyzing the
lipids present in the crude extract using column chromatography, it is necessary to
first isolate them quantitatively from nonlipid components. Extraction of lipids from
11

source materials, such as food, animal and plant tissues,or microorganism, should be
carried out in a manner that avoids changes in the lipids or leads to the formation of
artifacts it has eluates and each was differentiated by its components.
Based on the experiment, it can be concluded that phospholipid has the highest
percentage of lipid fraction in the egg yolk followed by cholesterol and triglyceride.
In addition, the presence of ester in the lipid is proved by test the lipid by ester assay
using hydrochloric acid. After undergoes the assay, the white residue (lipid) should
turn to dark green, however in this experiment, there is no change due to errors.
Meanwhile in order to detect the presence of cholesterol, cholesterol assay using
acetic anhydride. The white residue changes to light brown and this proved that the
presence of cholesterol in lipid.

11. Recommendation
1)

Keep the column in a clean and dust free place.

2)

Do not disturb the column till the separation is complete.

3)

Avoid gaps within the stationary phase packing.

4)

Results are quite sensitive to the presence of water, so keep everything dry.

12. Reference
1. Extraction Of Total Lipid From Chicken Egg Yolk, Column Chromatography And
Qualitative Test For Lipids, Maria Feliza C. Abesamis, Marie Em Clarisse P.
Acosta and Marilu Jane H. Bagiscan.
2. Extraction of Total Lipids from Chicken Egg Yolk and Column Chromatography
of Lipids, Bialen, Mary Camille Joyce; Biscarra, Prince-Jerome; Calubad,
Lareina; Cansino, Anjeanette; Chua, Norlene.
3. Triglyceride, retrieved from http://en.wikipedia.org/wiki/Triglyceride
4. Cholesterol, retrieved from http://en.wikipedia.org/wiki/Cholesterol
5. Phospholipids, retrieved from http://en.wikipedia.org/wiki/Phospholipid
6. Purification

of

lipids

from

egg

yolk,

http://faculty.mansfield.edu/bganong/biochemistry/pcpurif.htm

12

retrieved

from

13. Appendices

Figure 1 : the column chromatography been used to eluent the lipid

Figure 2 : the result obtain from ester assay.

Figure 3: the result obtain from cholesterol assay.

13