Isolation and Characterization of Complex Lipids From Egg Yolks

BIOCHEMISTRY 601 LABORATORY FORMAL REPORT
Isolation and Characterization of Complex Lipids from Egg Yolks

Peafuerte, Noel S., *Pimentel, Maria Danica B.
Group 20
Department of Chemistry, College of Science
University of Santo Tomas, Espaa, Manila 1008
Abstract. In this experiment, the lipids found in the egg yolks of chicken egg were isolated with organic solvents.
The isolated lipids are then separated into two classes: phosphorylated and non-phosphorylated with the use of
acetone. The two classes of lipids solution were characterized with various chemical tests: Libermann-Burchard,
Krauts, Salkowski, test for phosphate, ninhydrin and Molisch test. The isolation of lipid was successful however the
separation into two classes is not that successful since there were a lot errors observed in the various chemical test.
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. It can be found in most cells and tissues, but rarely exists as
free or uncombined state (Boyer, 2000). They are usually bounded to proteins and
polysaccharides of tissues in complexes of widely varying degrees of stability. Because of their
association with proteins and carbohydrates, it is complicated to extract and to identify the
structure of lipids. Lipids have a wide variety of molecular structures and involved in various
biological functions but have similar properties. Lipids are insoluble in water and ordinary
solvents but soluble in organic solvents. Most lipids are ionic or polar derivatives of hydrocarbon
belonging to the amphiphiles. They have ionic or polar groups which are hydrophilic and nonpolar hydrocarbons which are hydrophobic. The more polar the lipid is the stronger amphiphiles
it is (Cabatit, 1988)
One source of lipid is food. Food contains fats, complex lipids and steroids. Fats are
triglycerides, esters of fatty acids and glycerol. Complex lipids also contain fatty acids but their
alcohol may be either glycerol or sphingosine. The fatty acids also contain other constituents
such as phosphate choline or mono- to oligo- saccharides (Bettelheim, 2001). Complex lipids are
usually more stable than those from simple lipids because of the ionic and polar attractions
involved (Clark, 1977). Sterols are derivatives of a cyclopentanorphenanthrene nucleus or simple
sterid nucleus. The sterid nucleus is a combination of cyclopentane and perhydrophenanthrene
rings (Figure 1). If the compound has one or more hydroxyl groups and no carbonyl or carboxyl
group then it is a sterol. On the other hand, if it has one carbonyl or carboxyl group then it is a
steroid (Cabatit, 1988).
Figure 1. Structure of cyclopentanoperhydrophenanthrene

In this experiment the source of lipids is a chicken egg. In an egg, about 11% by weight is
made up of lipids found in the egg yolk (Todd, 1979). The lipids in the egg yolk are lipoprotein
in the native state (Clark, 1979). Chicken eggs have a consistent composition of its lipids. The
little variation is due to the strain and diet the chicken have. The eggs lipid composition have
approximately 62% if triglycerides, 33% of phospholipids, and 5% cholesterol. The cholesterol is
84% exists as free cholesterol and 16% as cholesterol ester. The phospholipids are 65% lecithin,
20% cephalin and various minor components (Todd, 1979). 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.
Lipids can be classified as saponifiable and non-saponifiable lipids. Saponifiable lipids
are triglycerides, waxes, phospholipids, and sphingolipids which are esters that can be
hydrolyzed under basic conditions. These first types of lipids are derivatives of fatty acids. Nonsaponifiable lipids such as isoprenoids and eicosanoids belong to this type because they are not
esters and cannot be hydrolyzed (Seager and Slabaugh, 2005).
The chickens egg yolk contains saponifiable and non-saponifiable lipids. The
saponifiable lipids presents are lecithin, cephalin, sphingomyelins, cerebrosides, palmitin,
stearin, olein, and small amounts of linoleic acid. The only non-saponifiable and most abundant
lipid in egg yolks is cholesterol. Vitamins A, D, and B complex are also present if the feed fed to
the chicken contains these vitamins. The yolks also contain inorganic substances like sodium and
potassium chlorides, iron, and few amounts of calcium and magnesium phosphates (EspinoCabatit, 1978).
Two standard lipids were used in this experiment. They are lecithin and cholesterol.
Lecithin is also known as phospatidyl choline. It is present in great quantities in egg yolk, liver
and nervous tissues. Lecithin (Figure 2) is a phospholipid that has choline as a nitrogenous base.
It can exists in alpha or beta form. The -lecithin is asymmetric while the -lecithin is
symmetrical. Egg yolk yields lecithin with arachidonic acid as one of its component fatty acids
(Cabatit, 1988). Its effects on the body are: decreases the blood pressure, slowing of the heart
stimulation of gastric and intestinal peristalsis, and increase of salivary secretions. Cholesterol is
an unsaturated alcohol (Figure 3). It is a sterol and is widely distributed in all cells in the body,
especially the nervous tissues. It can be synthesized from small fragments like acetic acid. It
serves as an insulator in the central nervous system where it exists in its free state. (Cabatit,
1988).
Figure 2. Structure of Lecithin
Figure 3. Structure of Cholesterol
In this experiment, the lipids in egg yolks is isolated and separated into phosphorylated
and non-phosphorylated lipids. The isolated lipids are characterized with various chemical tests.
These tests are Libermann-Burchard test, Salkowski test, test for Phosphate, Krauts test,
Ninhydrin test and Molisch test.
EXPERIMENTAL
I.
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 80mL of a solvent
mixture (CHCl3:CH3OH, 2:1, v/v). The mixture was allowed to stand for 10 minutes. The
mixture was then filtrated through a filter paper. The filtrate was placed in a graduated
cylinder to measure its volume. The filtrate is then placed in a separatory funnel and was
extracted with equal volume of 1% NaCl solution. 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. It was then placed in a
separatory funnel and was extracted with equal volume of 1% NaCl solution. The
organic layer is separated and dried with anhydrous Na 2SO4. The mixture was filtered off
into a clean Erlenmeyer flask. A pinch of hydroquinone was added and transferred into an
evaporating dish. The solution was evaporated to dryness over a beaker of warm water in
the hood. The sticky yellow residue was added with 15mL of acetone and cooled in an
ice bath for about 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 solution is transferred into a test tube and labeled as
non-phosphorylated lipids (NPL). The residue kept earlier is dissolved in 3mL of solvent
mixture and a pinch of hydroquinone was added. The solution is transferred into a test
tube and labeled as phosphorylated lipids (PL).
II.
Characterization of the Isolated Complex Lipids

The isolated lipids were characterized with the following tests. Cholesterol and
lecithin also underwent the following tests to serve as standards.
A. Libermann-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 (H 2SO4)
was carefully added to each test tube and was mixed well. The color produced
was noted.
B. Salkowski test
Ten drops of the lipid solutions and standards were placed separately in
small test tubes. Twenty drops of concentrated H 2SO4 was carefully added down
the side of the tubes to the solutions. The color of the interphase was noted.
C. 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 65C. 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.
D. Krauts test
Ten drops of the lipids solutions and standards were placed separately into
small test tubes. The test tubes were placed in a boiling water bath in the fume
hood to evaporate the solvent. The dried lipid is suspended in 10 drops of distilled
water. In each of the test tube, fifteen drops of Krauts reagent was added. The test
tubes were warmed for about 1 t0 2 minutes. The color of the solution and
precipitate was noted.
E. 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.
F. Molisch test
Ten drops of the lipids solution and standards were placed into separate
small test tubes. The test tubes were placed in a boiling water bath in the fume
hood to evaporate of 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 DISCUSSION

Egg yolk contains lipids such as triglycerides, phospholipids, and cholesterol. The lipids
in the egg are isolated with the use of a solvent mixture which is a mixture of chloroform and
methanol (2:1). This solvent mixture is improvised by Folchs 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. Lipids are also insoluble in water. The solvent mixture
causes the non-lipid components to transfer to the solvent system partly by ionic interactions.
This can also denature proteins. This solvent can extract most of all lipids found in the egg yolk.
Some of the extracted lipids form lipid-proteins complexes (Clark, 1977). The solvent mixture
dissociates the lipid-protein complexes in plasma membrane in the yolk but the solvent mixture
has a tendency to dissolve some non-lipid molecules such as proteins (Boyer, 2000).
Phospholipids are polar lipids which is why polar solvent is used to extract this. The solvent
mixture is also chosen since it is inexpensive, have relatively low boiling point, non-toxic and
nonflammable. When the solvent mixture is mixed with egg yolk, it was allowed to stand and
filtered off. The residue is the denatured proteins while the filtrate contains the solvent mixture
and the extracted lipids.
The filtrate is placed in the separatory funnel which will be extracted with 1% sodium
chloride solution (NaCl). Sodium chloride is an inorganic salt. Inorganic salts in aqueous
solution extract the lipids that are non-covalently attached to proteins or carbohydrate. The
sodium chloride solution extracts the non-lipid components present in the filtrate. This inorganic
solution disturbs the noncovalent bond between proteins and lipids so that the lipids will be the
only one to be extracted (Switzer and Garrity, 1999). Multiple extractions were done for more
efficient extraction of lipids. If single extraction was done, some of the lipids may remain in the
aqueous layer. The solvent mixture is less dense than water which is why it is in the bottom layer.
The aqueous layer consists of the water soluble components in the sample while the organic
contains the lipids. The organic layer is added with anhydrous sodium sulfate (Na 2SO4). 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.
The filtrate is added with hydroquinone. Hydroquinone is an anti-oxidant. It is necessary
for an anti-oxidant to be added since lipids can auto-oxidize upon exposure to air or sunlight. If
lipids are exposed to air or sunlight, the unsaturated fatty acid chain of lipid would react with
oxygen and cleave double bonds forming aldehydes or if further exposed, it will form carboxylic
acids (Scheve, 1984). Hydroquinone is first oxidized before the lipid is oxidized. Only a small
amount of hydroquinone is needed. If too much is added, it may serve as impurities. 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. This process is done under the hood since the solvent may
be harmful if it is inhaled.
The residue left from the evaporation of the solvent is added with cold acetone. Acetone
is used to separate the phospholipids from the non-phosphorylated lipids. Acetone is provides a
mild but rapid method of dehydrating tissues and as the water content decreases the acetone
extracts fats, sterols and other simple lipids. Complex lipids are relatively insoluble to acetone
and are converted to friable powder (Clark, 1977). Phospholipids are more polar than nonphosphorylated lipids. Acetone extracts the non-phosphorylated lipids since acetone extracts
non-polar and hydrophobic lipids (Boyer, 2000). The solution is filtered off. The residue contains
the phospholipids while the non-phosphorylated is in the filtrate.
The residue is mixed with solvent mixture. It is done to make the lipid into a solution and
hydroquinone was added so that the sample wont oxidize when it is kept. This serves as the
phospholipids solution. To the filtrate, it is again evaporated in the hood then added with solvent
mixture and hydroquinone. This solution serves as the non-phosphorylated lipid solution. The
appearance of the isolated lipid solution was noted (Table 1).
Table 1. The appearances of the isolated lipid solutions obtained in the experiment.
SAMPLE
Non- Phosphorylated Lipids (NPL)
Phospholipids (PL)
OBSERVATION
Orange- yellow solution
Yellow solution
The isolated lipid solution as well as two standards, namely cholesterol and lecithin,
underwent various chemical tests to determine its properties. These chemical tests are
Libermann-Burchard test, Salkowski test, test for Phosphate, Krauts test, Ninhydrin test and
Molisch test. These tests are color reactions.
The Libermann-Burchard test is a specific test for the detection of cholesterol,
unsaturated steroids or sterol. It is also known as acetic anhydride test since this tests uses acetic
anhydride. If cholesterol is present, a deep green color will be observed. The color begins as
purplish-pink color and progresses through a light green then to a very dark 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). The acetic anhydride is for the acetylation of the hydroxyl group
of cholesterol located at c-3 then when it is reacted with concentrated sulfuric acid, it undergoes
sulfonation and the addition of unsaturation yielding polyenes, aromatic steroids and
rearrangement of cholesterol molecule. This gives the intense color observed in the experiment.
If water is present, the test doesnt show the deep green color instead it shows a red to dark red
solution (Espino-Cabatitt, 1978). 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. This
test is performed in the standard and sample solutions and the color is noted (Table 2). In the
experiment, the cholesterol produced a deep blue green solution which expected since this test is
specifically for this. In the lecithin sample, red-violet solution is observed since some water
maybe present in the sample. In the non-phosphorylated lipid, a gray-violet solution is observed
that is a negative result. In the phosphorylated lipid, a dark brown solution is observed which is
negative for this test but it indicates that water is present in the sample. There is an error in this
10
test since in the non-phosphorylated lipid solution it should be positive. Cholesterol is a nonphosphorylated lipid; it should have been present in the non-phosphorylated lipid solution.
The second test performed is the Salkowski test. This test is similar to LibermannBurchard test since it is a specific test for cholesterol. This taste is named after Leopold
Salkowski, a German physiological chemist. In this test, only sulfuric acid was added in a
chloroform solution of the samples. In the presence of the acid, dehydration occurs forming a
bisteroid which gives a red color interphase (Espino-Cabatit, 1988). The red interphase can also
be blue. This interphase is observed in the chloroform layer while in the acid layer a green
fluorescence. This test is done in all the samples and the color of interphase was noted (Table 2).
In the cholesterol sample, a red interphase is observed which is expected. In the lecithin sample,
a red interphase was also observed. In the non-phosphorylated and phosphorylated lipids, both
showed dark-brown interphase which indicates that they are positive for cholesterol. In the
lecithin, there was an error since it should have been negative. It is clear that lecithin is not
cholesterol. In the phosphorylated lipid, there is also an error since it should have been negative.
Cholesterol is a non-phosphorylated lipid.
The third test done is Krauts test. Krauts test is a modification of Dragendroffs test
which is a test for alkaloids, pseudo alkaloids, and false alkaloids. The reagent used in this
experiment is Krauts reagent which is bismuth subnitrate with potassium iodide in 3M nitric
acid. When Krauts reagent reacts with a phospholipid, complexation involving the
phosphorylated lipid occurs. This reaction gives a brick red precipitate. It also determines the
presence of choline. Choline with bismuth potassium iodide undergoes a complexation reaction
which also gives a brick red precipitate. This test is done in the samples and the color of the
solution and its precipitate is noted (Table 2). In the cholesterol standard, a red orange solution
11
and brown precipitate is observed since cholesterol is a false alkaloid. The cholesterol standard is
a positive result. In the lecithin sample, a red orange precipitate was observed which is negative
for this test. In the non-phosphorylated sample and phosphorylated sample, a red orange solution
is observed as well as brown precipitate. Both of these samples are positive for false alkaloids or
cholesterol. There is an error in the phosphorylated sample since cholesterol should not be
present in this sample.
The fourth test is the test for phosphate. It is a test that determines the presence of
phospholipids. The samples were mixed with fusion mixture, a mixture of potassium nitrate and
sodium carbonate (3:1), and combusted under an open flame. This is done to remove all carbon
components and retain the phosphate in addition with water. The solution is also acidified with
3M nitric acid. The solution is acidified so that the ammonium hydroxide is converted to
phosphate since the nitrogen is liberated. The solution is heated till 65C. After heating, 2.5%
ammonium molybdate is added which reacts with phosphate to form ammonium phosphate
molybdate which gives a light yellow to yellow green solution. The color is due to the oxidation
of phosphate forming yellow to green solution depending on the concentration (Espino-Cabatit,
1978). This test is done on the samples and standards. The color of the solutions was noted. In
the sample of cholesterol and non-phosphorylated lipid, they are clear solution with black or
brown precipitate, this precipitate maybe residue of carbon. These samples are negative for the
presence of phosphate group or are not phospholipids. In the sample or lecithin and
phosphorylated, a light yellow solution is observed which is a positive test and indicates that the
samples contain a phosphate group or phospholipids.
Ninhydrin test is a chemical test that detects ammonia, primary and secondary amines.
This test indicates if an amino acid is attached to the lipids. Most amino acid reacts with
12
ninhydrin except proline and hydroxyproline. It is an oxidative deamination and decarboxylation

reaction of the free serine group with ninhydrin in ethanol upon heating would yield a blue violet
solution (Espino-Cabatit, 1988). The reagent ninhydrin is a strong oxidizing agent and reacts
with the -amino acids. The color produce is purple. This test is only positive for cephalin
because it is the only lipid with the free serine group. The solution was heated to catalyze the
reactions. This test is done on the samples and standards. The color of the solution was noted
(Table 2). In the cholesterol sample, a colorless solution is observed. This indicates that
cholesterol doesnt contain cephalin. In the lecithin test, a purple solution is observed which
indicates the present of cephalin. In the non-phosphorylated lipids, a red solution was observed
which indicates that no presence of cephalin. In the phosphorylated lipid, a purple solution was
observed which indicates the presence of cephalin.
Molisch test is chemical test that indicates the presence of carbohydrates or sugars. It is
named after an Austrian botanist named Hans Molisch. This test is very sensitive and can detect
the presence of carbohydrates in dilute solution as low as 0.001% that will give a definite
positive result (Espino-Cabatit, 1975). It is the hydrolysis of the glycosidic bonds present in the
sample to convert them into monosaccharides then it is converted to furfurals. Then the furfural
or its derivatives is condensed with two molecule of phenol from the Molisch reagent. Molisch
reagent is -naphthol (Figure 4) dissolved in ethanol. The final product would be red or purple
colored interphases. This test was done in the sample and the color of the interphase was noted
(Table 2). In the cholesterol sample, the interphase was colorless which indicates that cholesterol
is not a carbohydrate or sugar is present. In the lecithin sample, a purple interphase was observed
which means this sample contains carbohydrates or sugars. In the non-phosphorylated and
13
phosphorylated sample, a brown interphase was observed. This could be a positive result since
the color brown is near to the color red which a positive result is.
Figure 4 Structure of -naphthol.

Table 2. The results in the various chemical tests performed in the standards and samples.
OBSERVATION
TESTS
STANDARDS
Cholesterol
Lecithin
Liebermann
- Burchard
Blue-green solution
Red-violet
solution
Salkowski
Red interphase
Red interphase
Krauts
Red-orange
solution and brown
precipitate
Red-orange
solution and
orange
precipitate
Phosphate
Clear solution and

brown precipitate
Light yellow
solution
Colorless solution
Colorless
interphase
Purple solution
Purple
interphase
Ninhydrin
Molisch
SAMPLES
Nonphosphorylated
Gray-violet
solution
Dark-brown
interphase
Phosphorylated
Dark brown
solution
Dark-brown
interphase
Red-orange
Red-orange
solution and
solution and
brown precipitate brown precipitate
Clear solution
and black
precipitate
Red solution
Brown
interphase
Light yellow
solution
Purple solution
Brown interphase
CONCLUSION
The isolation of lipids from chickens egg yolk was successful. On the other hand, the
separation into two classes was not that reliable. The two classes were not separately properly
which is why some of the tests have errors. The possible sources of errors are human errors,
technique, the reagent (e.g. the acetone might not be cold enough), time pressure, etc.
14
REFERENCES
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Boyer, R. Modern experimental biochemistry. San Francisco : Addison-Wesley. (2000). Pages
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Cabatit, B. E. Biochemistry. 9th Ed. Manila: UST Press.(1978).
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Clark, J. M. Experimental biochemistry. San Francisco : W. H. Freeman, (1977). Pages 47-48.
Karlson, P.(1963) .Introduction to modern biochemistry. New York : Academic Press
Lehninger, A. L. Biochemistry : the molecular basis of cell structure and function. New York :
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McKee, T. and McKee, J.R. (2009). Biochemistry: The Molecular Basis of Life. 4th Ed.
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McGilvery, R.W. Biochemical concepts. Philadelphia : Saunders. (1975). Page 499.
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BIOCHEMISTRY 601 LABORATORY FORMAL REPORT