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;
Group 2 2B Pharmacy Biochemistry Laboratory

Abstract
Lipid molecules include fats, waxes, and fat-soluble vitamins such as A, D, E and K. A column chromatographic
procedure utilizing silica gel is described for separating lipid components of serum and lipoproteins into individual
fractions containing hydrocarbons, cholesterol esters, triglycerides, cholesterol, free fatty acids and phospholipids.
Egg-yolk lecithin has phospholipid classes and a composition that differ from soybean lecithin and may have unique
functional properties. 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 eluents used were 9:1 mixture of petroleum ether:ethyl ether, 5% methanol in dichloromethane
and dichloromethane:methanol:water (1:3:1). The results obtained were analyzed and it showed that the the lipids
are eluted by increasingly polar solvents. The lipids present in the crude extract were triacylglycerol, cholesterol and
lecithin. 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.

Introduction structure and function, and explored relationships

The existence of lipids started in the early
sixties. Biophysicist Alec Bangham of the Animal
Physiology Institute in Cambridge, England,
made a discovery about lipids that 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. David
Deamer did an experiment were he took lipids
and some DNA (with a intense fluorescent green
dye attached) and placed them in a test tube and
added a little water and when he discovered that
the DNA ended up in the liposome. Polycyclic
aromatic hydrocarbons (PAH) might have
supplied the first cells with energy, when PAH's is
exposed to light it can give off an electron and
that could have supplied the cell with energy
(That's what chlorophyll does for plants). Deamer
also took liposome loaded with polymerase and
put them into a beaker with two other molecules
- a nucleotide and protease. They placed a dye in
also that could slip through the liposome and
would attach to RNA. They discovered that the
liposome let the nucleotide in and the polymerase
assembled it into RNA.
The study of modern lipid chemistry began in
the 17th and 18th centuries with early
observations by Robert Boyle, Poulletier de la
Salle, Antoine François de Fourcroy and others.
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
between lipoproteins and disease states.
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.
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.
Lipids are important for maintenance of
human health and well-being in a number of
ways. Probably the most important function of
lipids is provision of an efficient energy source.
Fat provides 9 calories of energy per gram or
2.25 times as much as either carbohydrate or
protein. Carbohydrate is not stored in the body
and protein stores are predominantly muscle,
whose breakdown entails serious health
consequences. Fat is stored as such and can be
easily mobilized if needed. In primitive times
survival may have been possible because of
energy provided by metabolic use of stored fat.
Lipids are a group of substances of diverse
structures that share the common trait of being
soluble in solvents such as ether or benzene. The
major lipids of the body are triglycerides, which
comprise a molecule of glycerol to which three
fatty acids are bonded. Phospholipids are
substances in which glycerol carries only two
fatty acids plus phosphoric acid and an organic
base such as serine. Cholesterol is a member of
the family of large complex molecules generically
called steroids. It has the capacity to carry one
molecule of fatty acids. Cell membranes are
predominantly composed of phospholipids
and cholesterol.
Cell membranes confer stability to cells and
control entry or release of chemicals into or from
the cell. Lipids serve as effective insulators and
help in maintaining body temperature. Important
organs such as the heart, kidneys, and
reproductive organs are cushioned by fat. Nerves
are protected by a sheath, a myelin that contains
cholesterol, phospholipids, and other lipids. The
animal organism carries a number of essential
substances that catalyze chemical reactions in
cells. These are called vitamins and are
designated by letters. The B and C vitamins are
soluble in water; the others, vitamins A, D, E,
and K, are insoluble in water but soluble in fats.
They are transported in lipids in the blood and
stored in fat in the body.
Lipids occur in tissues in a variety of physical
forms, but the complex lipids are usually
constituents of membranes, where they occur in
close association with such compounds as
proteins and polysaccharides, with which they
interact by hydrophobic and van der Waals forces
and perhaps by ionic bonds. Various solvents or
solvent combinations have been suggested as
extractants, but most lipid analysts use
chloroform-methanol.
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.
One of the most useful methods for the
separation and purification of both solids and
liquids when carrying out small-scale
experiments is column chromatography. It is
another solid-liquid technique in which the two
phases are a solid (stationary phase) and a liquid
(moving phase).
The general approach is to extract total lipids
from egg yolk using methanol and chloroform,
then separate the lipid fractions, triglyceride,
cholesterol, and phospholipids, by
chromatography on silica gel. This experiment
will investigate the properties of lipids present in
chicken egg yolks, the lipid components present
in the crude extract using column
chromatography of the extracted lipids from
chicken egg yolk.
Materials and Procedures
The materials we have used were test tubes,
beaker, stirring rod, Pasteur pipette, hot plate,
iron stand and iron clamp. We started the
procedures by extracting total lipids from chicken
egg yolk. We added an equal amount of ethanol
to the egg yolk to increase the polarity of the
organic solvent, and mixed it to dehydrate and
partially extract the polar lipids. We added
hexane and then mixed it again and we had let it
stood for 5 minutes, until two layers were
formed, the fractions of polar and neutral lipids.
We removed the upper polar fraction and added
an equal amount of acetone to further precipitate
the polar lipids from residual neutral ones,
especially the cholesterol. We had collected the
upper layer and transferred it into a clean
beaker.
After we had extracted the lipids from chicken
egg yolk, we performed the column
chromatography on the collected upper layer
placed in the beaker (lipid extract). First, we
prepared a small column by pouring a slurry of
0.5 silica gel in 4 mL of petroleum ether into a
Pasteur pipette with the end tapered and plugged
with glass wool. Then we poured 1 mL of lipid
extract into the column, we saved the run-
through in a clean test tube. We have washed the
column with 5 mL 9:1 mixture of petroleum
ether:ethyl ether, and we collected the eluate in
the same test tube as the run-through. We have
washed again the column with the second eluate
(5 mL 5% methanol in dichloromethane) and
then we collected again the eluate in another
clean test tube. Lastly, we have washed the
column with the last eluent, 5 mL
dichloromethane:methanol:water (1:3:1) and we
collected the eluate in another test tube. We
ended up the procedures by saving the different
eluates.
Results and Discussion
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.
Compound lipids consist of a simple lipid and
some other group, such as a phosphoric acid
fragment or a nitrogen-containing alcohol.
Members of this family have many important
functions in biological systems. For example, one
group of compound lipids, the glycolipids, occur
in the membranes of brain and nerve cells.
Because the compound lipids are so complex and
because they occur in such small concentrations,
little has been known about them until recently.
One of the best known compound lipids is
lecithin.
The steroids are a fascinating group
of compounds that includes such diverse
representatives as cholic acid, a component
of bile; estrone, progesterone, testosterone, and
other sex hormones; vitamin D; cortisone; and
cholesterol.
The terpenes are another very large class of
naturally-occurring compounds, many with
characteristic and pleasing aromas. The oils of
camphor, menthol, lemon, orange, basil and
geranium are examples of terpenes, as is natural
rubber.
Lipoprotiens are organic compounds composed
of both protein and a lipid. There are at least four
groups of lipoproteins present in plasma: High-
density lipoproteins (HDL), low-density
lipoproteins (LDL), very low density lipoproteins
(VLDL), and chlyomicrons. The different densities
refer to the relative amounts of lipid and protein.
The higher the density, the higher the protein to
lipid ratio. LDLs transport cholesterol to cells and
deposit excess cholesterol in the blood vessels,
which increases the risk of arteriosclerosis. HDLs,
however, transport cholesterol from the tissues
to the liver where it is excreted, lowering the risk
of arteriosclerosis. A high HDL to total
cholesterol ratio is the best indication of
decreased risk of arteriosclerosis. HDL levels vary
from person to person and can be influenced by
such things as heredity, sex, age, and physical
activity. Smoking and obesity have been shown
to decrease plasma HDL levels.
The composition of chicken egg 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. 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 %). Egg yolks are one of the few foods
naturally containing vitamin D. The yellow color is
caused by lutein and zeaxanthin, which are
yellow or orange carotenoids known as
xanthophylls.
Column Chromatography of Lipids. The table
below (Figure1) gives us the results obtained in
this experiment. 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 triacylglyceride, second
eluate was cholesterol, and the third eluate is the
lecithin.
Eluates Components
1st eluate Triacylglycerol
2nd eluate Cholesterol
3rd eluate Lecithin
Figure1. Components of Eluates

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 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, 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.
Lecithin is the most polar among the three
eluates, next is cholesterol and last is
triacylglycerol.
The first eluate was triacylglycerol or
triglyceride. The eluent was 5 mL 9:1 mixture of
petroleum ether:ethyl ether. Triacylglycerols are
the main components of animal and plant lipids.
They are an ester of three fatty acids and
glycerol. They are the most concentrated source
of energy in the human body and are stored in
subcutaneous fat deposits where they contribute
to insulation. Fat deposits contain over 70 000
kcal of stored energy, but the triacylglycerol is
not immediately accessible for muscle respiration
because it must be broken down into its basic
components for transport in the blood and then
oxidized before entry into the krebs cycle.
Triglycerides have lower densities than water
(they float on water), and at normal room
temperatures may be solid or liquid. When solid,
they are called "fats" or "butters" and when liquid
they are called "oils".
Independent studies of biosynthesis of fatty
acid and glycerol components of glycerolipids
exhibited that 3H-leucine was mainly consumed
in synthesis of glycerol moiety of phospholipids
and triacylglycerols, whereas 14C-acetate was
utilized in synthesis of fatty acids. Ethanol
activated most distinctly the synthesis of glycerol
moiety as compared with the synthesis of
triacylglycerol fatty acids. Ethanol activated more
effectively esterification of fatty acids with
formation of triacylglycerols as compared with
phospholipids. Incorporation of the label into
glycerol molecule occurred in response to
activation of glycero-glyconeogenesis by
ethanol. The image below (Figure2) gives us the
structure of triacylglyceride.
Figure2. Structure of Triacylglyceride
The second eluate was cholesterol. The eluent
was 5 mL 5% methanol in dichloromethane.
Cholesterol is a chemical compound that is
naturally produced by the body and is a
combination of lipid (fat) and steroid. Cholesterol
is a building block for cell membranes and for
hormones like estrogen and testosterone. About
80% of the body's cholesterol is produced by the
liver, while the rest comes from our diet.
Cholesterol is only one of several lipids (fats)
circulating in our blood stream. Its components,
Triglycerides are an additional form of fat (3 fatty
acids plus glycerol) circulating in the blood.
Cholesterol and Triglycerides cannot dissolve in
water due to being lipids, or fats. Because our
blood is comprised primarily of water, for
Cholesterol and Triglycerides to circulate through
your blood, the Cholesterol and Triglycerides
must be carried by protein packages called
Apoproteins.
The combination of Lipids and Apoproteins is
known as Lipoprotein. Lipoproteins in turn are
divided into two types: the first being Low
Density Lipoprotein (LDL), which is a combination
of 25% Apoproteins and 45% Cholesterol (also
commonly called “bad Cholesterol”). LDL
provides Cholesterol for necessary body
functions, but in excess promotes potentially
damaging Cholesterol accumulation in the artery
walls.
The second Lipoprotein type is High-density
Cholesterol (HDL), comprised of 50% Apoproteins
and 20% Cholesterol (generally known as “good
Cholesterol”). HDL tends to help remove excess
Cholesterol from your blood. Therefore, a
relatively low ratio of LDL to HDL is desirable for
lowering your risk for development of coronary
artery disease. To calculate your Total
Cholesterol Level, add HDL, plus LDL Cholesterol
levels, plus 20% of the Triglycerides level. The
chart in this report gives the ranges of the
various Cholesterol components with desirable to
dangerous ranges for each.

Figure3. Structure of Cholesterol
The third eluate was lecithin. The eluent was 5
mL dichloromethane:methanol:water (1:3:1).
Lecithin is a lipid that consists mostly
of choline, but also includes inositol,
phosphorus, and linoleic acid. Lecithin helps
to prevent arteriosclerosis, protects against
cardiovascular disease, improves brain
function, helps keep the liver and kidneys
healthy, aids in thiamin and vitamin A materials, such as food, animal and plant tissues,
absorption, and can even help to repair liver
damage caused by alcoholism, this nutrient
is essential to every living cell in the human
body.
One of the various functions of lecithin is to
keep cholesterol in line. Its ability to emulsify oils
and hold them in solution plays a major role in
preventing gall stone formation. Together with
bile and bile salts, it comprises the three major
constituents of bile. Bile is mostly made up of
fats, which lecithin keeps in liquid form in order
to prevent gall stones from forming. On the other
hand, cholesterol holds a delicate balance with
the bile salts. If the balance is tipped on either
side, the result could stone formation. By keeping
cholesterol in check, lecithin helps prevent stone
formation.
As a component of the enzyme lecithin
cholesterol acyl tranferase, the compound is said
to help in the metabolism of cholesterol to it’s by
products. As mentioned earlier, this substance is
also called phosphatidylcholine and is an
excellent source of choline. Much of the medical
benefits of lecithin, particularly on high
cholesterol-related conditions have been
attributed to the presence of choline. The image
below (Figure4) gives us the structure of lecithin.
Figure4. Structure of Lecithin
We have concluded that the lipids extracted
from the chicken egg yolk are separated based
on its differences in solubility. There are factors
affecting lipid solubility like chemical nature of
the molecule, atomic or molecular formula
weight, valence or charge or sphere of hydration,
charge density and sphere of hydration. Also, 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 source
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.
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Purification of Lipids from egg yolk

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