Experiment 7: Isolation and Characterization of Complex Lipids from Egg Yolk

Abstract:

Lipids are a class of compounds that are soluble in organic solvents like alcohol and chloroform but are
insoluble in water and are essential components of every living cell. In this experiment several types of
lipids are isolated from the egg yolk through the Folch method The isolated lipids are then separated into
two classes: phosphorylated and non-phosphorylated through acetone precipitation. The components of
both the lipid samples and some standards like cholesterol, lecithin and galactocerebroside were
characterized with several color reaction test. These tests include the Liebermann-Burchard test and the
Salkowski test for cholesterol, Test for phosphate for phospholipids, Kraut’s test for choline in lecithin,
Ninhydrin test for primary amino group in phospatidylethanolamine and Molisch test for glycolipids. The
isolation and separation of lipids were depended on its general properties and the unique properties of
each of its components.

Introduction

Lipid ,from the Greek word “lipos” which means fat, are low molecular weight biomolecules and
nonpolar chemical substances that can be extracted from plant, microbial, and animal tissues by organic
solvent. Predominantly it is made up of carbon and hydrogen atoms rendering it nonpolar and generally
insoluble in polar solvents (i.e. water). Lipids are essential components of every living cell because it has
several biological functions like being a membrane structural material, energy storage and messenger
molecules. Based on their structural characteristics and chemical reactivities it is categorized into main
classes—simple and complex. Simple lipids are esters of fatty acids with various alcohols or simple
chains of fatty acid moieties linked together while complex lipids are characterized by the presence of
additional groups (aside from alcohol and fatty acid group) to the fatty acid ester. These groups can be
phosphate group, N-bases, carbohydrates, amines, proteins or sulfur groups (Domodoran, 2011).
Common types of complex lipids include phosphoglyceride, glycolipids and sphingolipids. Lipids are
ubiquitous in nature due to its very important roles. It can be found in every living organism, from simple
bacterial cell to complex animal cells and tissues such as the egg yolk.

In this experiment the source of lipids from the yolk of a chicken egg which is composed of 30-
36% of lipids. Egg yolk lipids contain about 65% triglycerides (Neutral Lipids), 30% phospholipids and
5% cholesterol (Mine, 2008). Egg lipids can be divided into two general classes: non-phosphorylated and
phosphorylated or phospholipids. Phospholipids contain a nitrogenous base and phosphoric acid. It is also
called phosphatids or phosphorized fats. It is an important component of membrane lipids. Furthermore,
glycolipids and hydroxyfatty acids are also found in the egg yolk. The chemical nature of the components
of the egg yolk is the basis for the technique used in isolating the lipids.

Numerous methods of separation operate in the principle that lipids are generally soluble in
nonpolar solvents and insoluble in polar ones. On this experiment the Folch method which uses a two-
component solvent system containing both polar and nonpolar groups. On the other hand, separation of
the component lipid into several lipid classes (i.e. simple, complex) can be done by taking into
consideration the reactivities of the various groups present in the lipid. Some of these techniques include
solvent precipitation and chromatography. Lastly, lipids can be detected by the traditional color reaction
tests which operates in the fact that specific groups react with chemical reagents to produce colored end
product. In this experiment, lipid components of egg yolk will be isolated and separation methods will be
used to obtain phosphorylated component and nonphosphorylated ones. Furthermore, the isolated lipids
will be characterized using various color reaction tests.

Methodology

The experiment was divided into two parts. The first part was involved in the isolation of lipids from the
egg yolk and the subsequent separation of those lipids into its phosphorylated and nonphosphorylated
components. The second part was the characterization of the complex lipids using various chemical tests.
A large-sized regular white egg was used in the experiment.

A. Isolation of Complex Lipids

An egg was cracked and the egg yolk was separated from the egg white. The egg yolk was
placed in a clean 250-mL beaker. The yolk was stirred with 100mL of a solvent mixture (a
2:1 by volume mixture of chloroform [CHCl3] and methanol [CH3OH]). The mixture was
allowed to stand for 10 minutes. The mixture was then filtrated through a filter paper to a
separatory funnel and was extracted with proximately 100 ml of 1% NaCl solution. The
separatory funnel was swirled and the pressure build up inside it was subsequently released
by opening the vent. The organic layer (bottom layer) is separated from the aqueous layer.
The aqueous layer was discarded. The organic layer is placed in a graduated cylinder and its
volume measured. The bottom organic layer was collected in 250 mL Erlenmeyer flask and
dried with anhydrous Na2SO4. After which, the solution was filtered and the filtrate was
collected in a 50 mL clean Erlenmeyer flask. A pinch of hydroquinone was added to the
solution and was mixed thoroughly. The solution was then transferred to an evaporating dish
which was placed on a steam bath inside the hood. Evaporation was allowed to occur until the
solution was transformed in a sticky yellow residue. It was then cooled in an ice bath for 15
minutes. The Acetone solution is carefully decanted through a filter paper and the filtrate was
collected in a clean Erlenmeyer flask. The precipitate or residue was washed with 5mL of
cold acetone. The solution was decanted and filtered. The residue is kept for used later on.
The acetone solution was evaporated to dryness in a water bath in the hood. The residue is
dissolved in 3mL of solvent mixture and added with a pinch of hydroquinone. The resulting
solution was transferred to a clean large test tube, was sealed with cork, was labeled as PL
(phosphorylated lipid) and was refrigerated. All of the acetone solution collected in the
Erlenmeyer flask was evaporated to dryness in the steam bath inside the hood. After which,
the residue on the evaporating dish was allowed to cool and was dissolved in 3 mL of the
solvent mixture (described earlier). A pinch of hydroquinone was added. The solution was
then transferred to a clean Erlenmeyer flask and was mixed, was sealed with cork, was
labeled as NPL (nonphosphorylated lipid) and was refrigerated.

B. Characterization of Complex Lipids

Six tests were performed for both the lipid samples (PL and NPL) and the s
tandards (cholesterol, lecithin and galactocerebroside).
B.1 Liebermann-Burchard Test

An amount of 0.5mL of each of the isolated lipid and standards were placed in a separate
test tube. Ten drops of acetic anhydride was added to each test tube and was gently
swirled. Four drops of concentrated sulfuric acid (H2SO4) was carefully added to each
test tube and was mixed well. The color produced was noted.

B.2 Salkowski Test

Ten drops of the lipid samples were placed in small test tubes. Then, twenty drops of
concentrated sulfuric acid (H2SO4) were carefully added down the sides of each tube.
Two layers were observed and were not disturbed. The color of the interphase (the area
between the upper layer and the lower layer) were noted.

B.3 Test for Phosphate

In a crucible, 0.5mL of the phosphorylated lipids was mixed with fusion mixture (5 times
its bulk). The mixture is ignited over a free flame until all the organic matter is burned
away and the mixture turned into a grayish or colorless liquid or a white or gray ash is
obtained. The mixture was allowed to cool and dissolved in 3mL of warm water. The
solution is transferred to a test tube and acidified with 3M nitric acid. The solution was
heated to 65˚C. An amount of 3mL of 2.5% ammonium molybdate was added and the
solution was warmed. The color of the solution and precipitate was noted. The same
procedures were done for non-phosphorylated lipid solution and the standards solutions.

B.4 Kraut’s Test

Ten drops of the lipid samples were placed in large test tubes. Then, the tubes were
placed in a boiling water bath inside the fume hood so as to evaporate the solvent. The
resulting dried lipid was suspended in ten drops of distilled water. Afterwards, fifteen
drops of Kraut’s reagent (diluted bismuth subnitrate in 3M nitric acid [HNO3] with
potassium iodide [KI]) were added. Finally, the tubes were warmed for 1-2 minutes and
the color of the solution and precipitate were noted.

B.5 Ninhydrin test

Ten drops of the lipids solution and standards were placed into separate small test tubes.
Five drops of ninhydrin in ethanol was added to each of the test tube. The solutions were
warmed for 1 to 2 minutes and the color of the solutions was notes.

B.6 Molisch Test

Ten drops of the lipid samples were placed in large test tubes. Then, the tubes were
placed in a boiling water bath inside the fume hood so as to evaporate the solvent. The
dried lipid is suspended in twenty drops of distilled water. Twenty drops of concentrated
H2SO4 was slowly added to the side of the tube. The color of the interphase was noted.
Results And Dicussion

Table 1. Components of the phosphorylated (PL) and the nonphosphorylated lipids (NPL)

Phosphorylated (PL) Nonphosphorylated lipids (NPL)

Phosphatidylcholine (lecithin) Triglycerides
Phospahtidylethanolamine Cholesterol
Glycolipids

Table 2. The results in the various chemical tests performed in the standards and samples.

OBSERVATION

STANDARDS SAMPLES
TESTS
Non-
Cholesterol Lecithin Galactocerebroside Phosphorylated
phosphorylated

Dark
Light-Brown Emerald green Red- Brown
green Clear Solution
Liebermann- soulution solution
solution
Burchard Solution (-)
(-) (+) (-)
(-)

Red-violet Red-violet Red-violet Red-violet Clear turbid

Salkowski interphase interphase interphase interphase solution

(+) (+) (+) (+) (-)

Red orange Brick-Red Red orange Red orange

Red orange solution
solution solution solution solution
w/black
w/black and w/brown w/brown
Kraut’s
ppt.
ppt. ppt. ppt. ppt.
(-)
(-) (+) (-) (-)

Phosphate N/A N/A N/A N/A N/A

Clear Maroon
Blue-violet solution Brown solution Violet solution
Ninhydrin solution solution
w/white ppt (-) (-) (+)
(-)
 (-).

Clear turbid Turbid

solution w/ solution Clear turbid solution Yellow turbid Yellow turbid
brown w/violet w/ violet interphase solution solution
Molisch
bottom Ring (+) (-) (-)
(-) (+)

The Folch method was used to isolate the lipids in this experiment. In this method, a 2:1 by volume ratio

of chloroform to methanol was used as a solvent to extract the lipids found in the egg yolk. This solvent

mixture is improvised by Folch’s group. The lipids are hard to isolate since they do not occur as free

molecules and are covalently bonded to proteins or carbohydrates. Lipids are generally less polar than

other cell constituents. This is why organic solvent can be used to extract lipids. The properties of each

solvent in the system can be accounted for its ability to separate the lipids from the nonlipid contaminants.

Chloroform (CHCl3), which possess only a weak dipole moment, are only weakly attracted to water and

nonlipid contaminants hence will mostly be in the phase where there is less water and consequently more

lipid. On the other hand, the more polar methanol (CH3OH) can attract more water and nonlipid

contaminants than the chloroform and hence can be associated in a single phase. Basically, the solvent

mixture causes the non-lipid components to transfer to the solvent system partly by ionic interactions.

Two phases were exhibited in this experiment: the methanol phase which contains mostly the nonlipid

substances and a small amount of lipids, and the chloroform phase which consists of pure lipids from the

sample. Since the main goal of this step is to isolate, at most, the lipids present in the sample at high

efficiency, then the remaining amount of lipids in the methanol phase should also be isolated. This is

accomplished by extracting the sample mixture with dilute salt solution (i.e. NaCl). Sodium chloride is an

inorganic salt that can extract the lipids in aqueous solution that are non-covalently attached to proteins or

carbohydrate. In the methanol phase, these salts exist as dissociated form while it exists undissociated in

the chloroform phase. These forms are in equilibrium with each other in the extraction system. Hence, the

addition of salts (i.e. NaCl) containing cations would decrease the rate of dissociation of lipids due to the
shift in the position of the equilibrium from right to left applying Le Chatelier’s principle. This would in

turn shift the lipids from the methanol phase to the chloroform phase efficiently. The mineral salts

together with the nonlipid contaminants will remain in the aqueous layer and can be discarded. The final

organic layer is composed of lipids, portion of the solvents and small amounts of water. Theoretically,

Multiple extractions should be done for more efficient extraction of lipids but in this experiment the

extraction was done only once which means that some of the lipids may remain in the aqueous layer. The

water can be removed by adding anhydrous sodium sulfate (Na2SO4). The anhydrous sodium sulfate traps

and removes water molecules present in the organic layer. The solution is filtered off to remove the

hydrated sodium sulfate. Antioxidants (i.e. hydroquinone) are used before further processing of the

organic layer. There is a need for an addition of antioxidant because of the reason that lipids can auto-

oxidize upon exposure to air or sunlight. Since the hydroquinone is added it is oxidized first before the

lipids. . The solution is then evaporated dryness in a evaporating dish in a warm water bath in the hood.

This is done to remove or evaporate the solvents.

On the other hand, acetone precipitation was used to separate phosphorylated (PL) and

nonphosphorylated lipids (NPL) present in the organic layer. Cold acetone provides a mild but rapid

method of dehydrating tissues and as the water content decreases the acetone extracts fats, sterols and

other simple lipids. It is effective due to the fact that surface active lecithins and disaturated phosphatidyl

complexes are less soluble to cold acetone as compared to other lipid classes. Complex lipids are

relatively insoluble to acetone and are converted to friable powder (Clark, 1977). Phospholipids are more

polar than non-phosphorylated lipids. Acetone extracts the non-phosphorylated lipids since acetone

extracts non-polar and hydrophobic lipids (Boyer, 2000). It is the solvent of choice as compared to

alcohols since it is less reactive to the lipid components. The composition of the phosphorylated lipids

and the nonphosphrylated lipids can be seen in Table 1.

The isolated lipid solutions as well as three standards, namely cholesterol, lecithin, and

galactocereberoside were subjected to different color reaction tests to detect specific groups found in the
different lipid types present in the sample.. These chemical tests are Libermann-Burchard test, Salkowski

test, test for Phosphate, Kraut’s test, Ninhydrin test and Molisch test.

The Libermann-Burchard test is a specific test for the detection of sterols with unsaturation in the carbon

5 and carbon 6 of the fused rings like cholesterol (fig.1). The positive result for this test is the formation

of an initial blue or red solution which was immediately converted to emerald green solution. The color is

due to the hydroxyl group (-OH) of cholesterol reacting with the acetic anhydride and concentrated

sulfuric acid and the increasing conjugation of the unsaturation in the adjacent fused ring (Bettelheim,

2001). This test is not only used as a qualitative test but also as a quantitative test. The concentration of

cholesterol is determined by the intensity of the color. The result of the experiment is shown in Table 2. It

can be seen that only Cholesterol yielded the positive color result which is emerald green while the rest

are negative. Theoretically, non-phosphorylated lipid solution should also show a positive result but due

to some errors made by the group they have failed to produce the correct and positive result.

Figure 1

The next test that was performed was the Salkowski test also a specific test
Boyer, R. Modern experimental biochemistry. San Francisco : Addison-Wesley. (2000). Pages 303-305.

Clark, J. M. Experimental biochemistry. San Francisco : W. H. Freeman, (1977). Pages 47-48.

Domodoran, G. (2011). Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publisher (P)

Ltd.

Mine, Y. (2008). Egg Bioscience and Biotechnology. USA: John Wiley and Sons Inc.