Experiment 7: Isolation of Lipid from Egg Yolk via Folch Method

2015- 48305, 2015-12972

Group 5, Biochem 34.1, HEJ
November 15, 2017
___________________________________________________________________

I. Abstract
Lipids are biological compounds that can be classified on different basis such as
chemical nature, structural complexity and solubility. Generally, lipids are non-polar due
to their long carbon chains, however, some lipids exhibit polar properties due to the
presence of polar groups thus they can be amphipathic. Lipids are generally soluble in
organic solvents but interact weakly with aqueous solvents. In this experiment, lipid was
isolated from the egg yolk via Folch method, a type of liquid-liquid extraction that uses
chloroform-methanol in a 1:2 ration as the extracting solvent. Extracted lipid was
separated into two one being polar (composed of phospholipids) the other being non-
polar (composed of non-phospholipids) by the addition of acetone. To determine the
composition of each isolate, thin layer chromatography was done using lecithin, oleic
acid, and cholesterol as standards. Rf values for the precipitate, supernate, lecithin, oleic
acid, and cholesterol are 0.25, 0.33, 0.12,0.58 and 0.50 respectively. Based on this
values, it was concluded that the precipitate contains lecithin while the supernate
contains oleic acid and cholesterol. The isolates were also subjected to different
qualitative tests wherein all test results are in line with the theoretical data. For the Sudan
IV test, both isolate tested positive by producing a red color. For the acrolein test, both
tested positive by producing a foul odor. This was visually seen in the formation of the
black mixture when heated further. For the phosphate test, since the precipitate is
composed of phospholipids, it is the only isolate tat tested positive. Lastly for the
Leibermann-Buchard Test for cholesterol, only the supernate tested positive since
between the two isolates, it is the one that contains cholesterol. Thus on the basis of the
qualitative test results, isolation of the lipid from the egg yolk was successful

II. Keywords: Egg yolk, lipids, Folch Method, TLC, Liquid-liquid extraction

III. Introduction
Lipids are biological compounds that
occurs frequently in nature and are best
defined based on their solubility. Lipids
being composed of long carbon chains
are generally insoluble in water but
readily soluble in organic solvents
(Campbell & Farell, 2013). However,
some lipids contain both polar and non-
polar groups thus they can also be
amphpatic (Garett & Grisham, 2013).
Lipids can be found in simple settings
such as animal, plant and microbial
membranes or in diverse ones such as
egg yolks and the human nervous
system (Campbell,2013).
Together with carbohydrates the
main function of lipids is for energy
storage and for structural purposes being
an important component of cell
membranes. Other functions of lipids are
source of vitamins A D E and K which are
fat soluble and act as insulators
protecting the body from cold
temperatures thus contributing to the
body temperature regulation. They also
participate in intra- and intercellular
signaling (TutorVista, 2017).
There are different categories in
classifying lipids. According to (Campbell
and Farell, 2013) lipids can be classified
into two groups based on their chemical
nature. The first group includes open-
chained compounds with polar head and
long non-polar tails while the second
group is composed of fused-ring
compounds. (Zubeda & Zarin, 2012)
classifies lipids based on their structural
complexity and biological functions.
Lipids classified under structural
complexity can still be grouped into
whether they are simple lipids,
compound lipids or derivative lipids.
Lipids classified under biological
functions can also be grouped into
whether they are structural lipids, storage
lipids or precursor lipids. While,
(BiologyExams4U, 2017) classifies lipids
into 4 groups fatty acids, glyceride
lipids, non-glyceride lipids and complex
lipids -based on their solubility in non-
polar solvents.
The objective if this experiment is to
use liquid-liquid extraction in isolating the
lipid present in the egg yolk and to
subject the isolated lipid in different
qualitative tests.
IV. Methodology
Isolation of Lipids from Egg Yolk
Egg yolk was combine with 1% NaCl
in a 1:3 ratio. Afterwards, it was mixed
with a 1:3 ratio of chloroform methanol
solvent in a separatory funnel. After
mixing, the solution was left to stand for
30 minutes. The organic bottom layer
was collected as well as a small portion
of the middle layer. Acetone was added
to the collected layer until precipitate
ceases. It was then subjected to
centrifugation at 6000 rpm for 10 minutes
separating the precipitate from the
supernate and collecting both.
Thin Layer Chromatography
The TLC chamber was first saturated
with the mobile phase methanol-acetone
solvent in a 1:2 ratio. An appropriate
silica gel was obtained and was spotted.
Afterwards it was placed inside the
saturated chamber. The TLC plate was
taken out and was air dried. To visualize
the movement of the spots, the plate was
saturated with iodine vapor.
Qualitative Tests
SUDAN IV TEST
Ten (10) drops of the precipitate
and the supernate was placed in
separate test tubes. To each test tube,
tiny crystal of Sudan IV was added. After
shaking the test tubes, results were
noted.
ACROLEIN TEST
Ten (10) drops of the precipitate and
the superante were placed in separate
test tubes. To each test tube, a pinch of
KHSO4 was added and was subjected to
gentle heating. After wards, it was placed
in a boiling water bath. After the water
bath, the smell of each test tube was
noted.
TEST FOR PHOSHATES
In separate crucibles, 0.5mL of the
precipitate and the supernate was
incinerated making sure that no liquid is
left. Distilled water was then added to the
residue. After wards, the solution was
filtered discarding the residue and
collecting the filtrate. To the filtrate,
0.5mL of 10% ammonium molybdate and
2 drops conc. HNO3 was added. It was
heated and was allowed to stand after
cooling. Formation of yellow precipitate
was observed.
LEIBERMANN-BURHCARD TEST
Five (5) drops of P and S were placed
in separate test tubes. To each test tube,
10 drops acetic anhydride and conc.
H2SO4 was added. After mixing, color of
solution was noted.
TEST FOR UNSATURATION
Hubls solution was added drop by
drop to 5 drops of the precipitate and
supernate placed in separate test tubes.
While adding, shaking was done and the
number of drops until the color of
solution did not change anymore was
recorded.
V. Results and Discussion
Isolation of Lipids from the Egg Yolk
The isolation process began with 1%
sodium chloride (NaCl) being added to
the lipid source which is egg yolk. This is
to initiate the salting out of the proteins
present resulting in an isolated lipid of
high purity (Nelson & Cox, 2012). To
extract the lipid content of the egg yolk,
liquid-liquid was used wherein the
solvent is a mixture of chloroform and
methanol in a 1:2 ratio. This solvent
mixture was used because it is regarded
to extract the most amount of lipid when
compared to other solvent mixtures. The
components of the solvent mixture will be
responsible for the separation of the
lipids from the non-lipid contaminants.
Chloroform being a non-polar solvent will
weakly interact with polar components
thus it will be in the phase wherein there
is less water and non-lipid contaminant
and more lipid content. The other
component which is methanol is polar so
it strongly interacts with polar
components thus it will be in the phase
wherein there is more water and non-lipid
contaminant and less lipid content.
Basically the isolation process can be
divided into two phases; the methanol
phase which contains all the water and
non-lipid contaminants and the
chloroform phase which essentially
contain pure lipids from the sample (Bligh
& Dryer, 1957). Another purpose of
adding the salt was to increase the
amount of lipids that will be isolated.
According to (Folch etal, 1957), there are
still lipids in the methanol phase since
there are amphipathic lipids. The lipids in
the methanol phase exist as salts and are
in the dissociated form. Without the
presence of additional salt, the amount of
the isolated lipids cannot be maximized
since the dissociated forms are in
equilibrium with the solvent mixture. But
with the presence of the additional salt,
there would be an equilibrium shift
shifting the lipids in the methanol phase
to the chloroform phase. The added salt
together with water and the non-lipid
contaminants will remain in the methanol
phase while all the lipid contents will be
in the methanol phase.
The egg yolk-salt mixture was
combine with the solvent mixture in a
separatory funnel where it was mixed
thoroughly while opening the valve once
in a while to let the accumulated pressure
out. Afterwards, it was left to stand for 30
minutes. As seen in figure 1, instead of
forming just two layers, it formed three.
The top most layer is the methanol phase
containing all the polar contaminants plus
the NaCl. The bottom most layer is the
chloroform phase containing all the lipids.
What corresponds to the middle layer is
the emulsion formed due to the mixing
done. The middle layer is composed of
amphipathic lipids (Wang, Sunwoo,
Cherian, & Sim, 2000)
A
B Subjecting
C determination of the components and the
Figure 1 Layers formed after letting the mixture stand.
A) Methanol phase containing all polar contaminants;
B) Emulsion layer due to mixing of amphipathic lipids;
C) Chloroform phase containing lipids.
After collecting the lipid isolates,
acetone was added until precipitation
stops or a the original color turns light
yellow. Addition of acetone allows the
separation of phosphorylated lipids and
non-phosphorylated lipids present in the
sample. Since acetone is non polar, it
mixes with the non-phosphorylated lipids
which are also non-polar precipitating out
the phosphorylated lipids which are polar
(Hills, 1988). The solution was subjected
to centrifugation wherein the supernatant
was successfully separated from the
precipitate afterwards.
The technique used in lipid isolation is
known as Folch method. It is the first
technique used to isolate lipids using
chloroform water-methanol solvent in
an 8:4:3 ratio. According to (Bligh &
Dryer, 1957) the above ration of
chloroform to ethanol is only applicable to
samples wherein the water content is
greater than the lipid content such as in
egg yolk. In the case of a sample such as
myelin sheath wherein the lipid content is
greater than the water content, a larger
amount of chloroform is needed. Thus,
the ratio above will be adjusted
increasing the amount of chloroform
content in the solvent mixture.
Thin Layer Chromatography
the Isolates to a
chromatography enables the
identity of the isolates as well as its
purity. The type of chromatography was
a normal phase thin layer
chromatography wherein the mobile
phase used is 1:2 methanol-acetone
solvent while the stationary phase used
was silica gel plate. How this
chromatography will work is that as the
solvent will carry up the stationary phase
in which the lipid carried highest will be
the least polar.
Since the samples used were colorless,
a staining step would be applied. There
are two ways to stain. The first is using
UV light.Due to the indicators present in
TLC plates, it provides a green color
when put under a UV light of wavelength
254 nm ( Thin Layer Chromatography,
n.d.) However, since not all samples
used in TLC are UV active, another
method of staining will be done. This
second way makes use of iodine vapor.
The TLC plate are saturated by the iodine
vapor. This allow the iodine to form a
complex with the unsaturated and
aromatic compounds producing a brown
color (Thin Layer Chromatography,
2015). The latter way of staining was
used in this experiment.
After visualizing the spots, the Rf value
can now be obtained by obtaining the
distance travelled by the spots. The Rf
values of the isolates can be compared
with the Rf value of the standards.
Standards used in this experiment are
Oleic Acid, Lecithin and Cholesterol.
Figure 2 shows the result of the TLC.
Figure 2 Iodine Stained TLC
Using the equation below, the Rf values
of the isolates as well as the standards
were obtained. Data is shown in table _.
Sample computation:
1.5
Precipitate Rf = = 0.25
6
Table 1 Data Table for TLC result
Sample Distance Rf Value
Travelled
Precipitate 1.5 cm 0.25
Supernate 2 cm 0.33
Lecithin 1 cm 0.12
Oleic Acid 3.5 0.58
Cholesterol 3 0.50
As expected the supernate which is
the less polar of the isolates travelled
farther than the more polar precipitate. Of
the standards used, Oleic acid travelled
the farthest next is cholesterol then
lecithin travelled the least.
Oleic acid is a monounsaturated
omega-9 fatty acid and is considered to
be the most widely distributed and
abundant fatty acid in nature (PubChem,
n.d.). As seen in figure 3, oleic acid is
composed of a very long chain of
hydrocarbons. This is the reason why its
Rf value is very high. Comparison of its
Rf with the Rf of the isolates would show
that it is nearer to the Rf of the superante
than the precipitate. Thus it can be said
that a small amount of oleic acid is
present in the supernate.
Figure 3. Oleic Acid retrieved from
https://pubchem.ncbi.nlm.nih.gov/compound/oleic_aci
d#section=2D-Structure
As shown in figure 4 cholesterol is
composed of 3 regions, a hydrocarbon
tail, a mid-part hydrocarbon rings and a
hydroxyl head (Masterjohn, 2013). The
hydroxyl head give cholesterol its weak
polar property while the hydrocarbons
give it its non-polar property making it
amphipathic thus making it less non-
polar when compared to oleic acid.
Comparison of its Rf value with the
isolates showed that it is closer to the Rf
of the supernate making it also a
component of it.
Figure 4. Cholesterol Structure retrieved from
http://www.cholesterol-and-
health.com/cholesterol_structure.html
Phosphatidylcholine which is
commonly known as lecithin is another
standard used. Lecithin as shown in
figure 5, consists of glycerol, 2 fatty
acids, a phosphate group and choline.
The glycerol serves as the back bone of
the lecithin molecule. The 2 fatty acids
are connected to carbon 1 and 2 while
the phosphate group is connected to
carbon 3 of glycerol. The choline in turn
is connected to the phosphate group
(Lecithin, n.d.). The presence of the 2
fatty acids and choline which are all polar
contributes to the small value of its Rf
value. Comparison of the Rf value of
Figure 5. Lecithin structure retrieved from
http://science.jrank.org/pages/3884/Lecithin.html
lecithin with the two isolates showed that
it is much closer to the Rf value of the
precipitate thus making it a component.
Qualitative Tests
Sudan IV
Sudan IV is a reagent that contains fat
soluble dyes. When added to lipid
containing samples, the dye reacts with
the lipids producing a red color. Sudan IV
is used to determine the presence of
lipids wherein if tested negative, there will
be no formation of red precipitate
(Qualitative Test of Lipids II, n.d.).
Figure 6. Sudan IV Structure retrieved from
http://fac.ksu.edu.sa/sites/default/files/Qualitative%20t
est%20of%20Lipids%20II.pdf
Figure 7 shows the result of the
isolates being subjected to Sudan IV test.
It is seen that both of them produced a
red color with the supernate being darker
than the precipitate. This is in line with
the theoretical data. Since both isolate
contain lipids, both should also test
positive.
 After subjecting the isolates to
acrolein test, both gave off a foul odor
thus testing positive. This is in line with
the theoretical result. Figure 9 also
shows the polymerization of acrolein in
the precipitate indicated by the black
mixture. A similar image is seen for the
supernate.

Figure 7. Sudan IV test result. Supernate (left)

and Precipitate (right) both tested positive.

Acrolein Test
Acrolein test is used to determine the
presence of glycerol and its fatty acid
esters. A positive test would give off a
foul odor while a negative would produce
no odor or no change in odor. The
principle behind this is the dehydration of
glycerol to acrolein in the presence of
heat and a dehydrating agent. When
glycerol or its fatty acid ester is heated
Figure 9 Acrolein test result for the
with a dehydrating agent such as Precipitate
potassium bisulfate, it is dehydratedd to
form the simplest unsaturated aldehyde
Phosphate Test
acrolein (Qualitative Test for Lipids II,
n.d.). In the case of fatty acids, they are The phosphate test began with the
first hydrolyzed to form glycerol before incineration of the samples. The reason
being dehydrated. Acrolein is the reason for this is that incineration helps oxidize
for the fould odor since it is characterized the ester bonds of lipids that contain
by a rancid smell. When further heated, phosphate. In the process of doing so,
the acrolein becomes polymerized water and carbon dioxide are evaporated
indicated by the blackening of the mixture leaving a phosphate residue (Chawla,
(Stevens & Maier, 2008). 2014). After incineration, distilled water
was added to separate the non -lipid
contaminants from the residue. The
solution was then filtered discarding the
Figure 8 Acrolein formation from glycerol retrieved from residue containing the contaminants.
http://fac.ksu.edu.sa/sites/default/files/Qualitative%20test%2
0of%20Lipids%20II.pdf The presence of free phosphate in the
filtrate can be detected using ammonium
molybdate as long as the phosphate is in
an acidic environment forming a yellow
precipitate. This is the reason why nitric
acid was added. The equation below
shows how the free phosphate reacts
with ammonium molybdate in the
presence of nitric acid (Identifying Lipids
Using Chemical Tests, n.d.).
HPO42(aq) + 12MoO42(aq) + 3NH4+(aq) +
23H3O+(aq)
(NH4)3[P(Mo3O10)4](yellow,s) + 35 H2O(l)
This test was done for both the
precipitate and the supernate isolate.
Figure 10 shows the result of test. As
seen in the figure, only the precipitate
tested positive and the supernate did not.
The reason for this is that phospholipids
are only present in the precipitate and not
in the supernate. This result is in line with
the theoretical data.
Figure 30 Phosphate Test Result. Precipitate
(left) tested postive while supernate (right) tested
negative
Leibermann-Burchard Test
Leibermann-Burchard Test also
known as acetic anhydride test is a type
of colorimetric test used to determine the
presence of cholesterol. The hydroxyl
group of cholesterol reacts with the
reagents resulting in the increase in the
conjugation and unsaturation of the fused
rings in cholesterol. The effect of this
reaction is the formation of a colored
solution which at first is color pink after a
while it transitions to light green then to
dark green color after some time. The
concentration of the cholesterol in the
sample can be computed by subjecting
the result of the test to a
spectrophotometry though it was not
done in this experiment (Identifying
Lipids Using Chemical Tests, n.d.).
Reaction of cholesterol with the reagents
is shown below.
Figure 11 Leibermann-Burchard reaction retrieved
from
https://www.researchgate.net/profile/Joseph_Chidiac/
post/What_is_result_of_interaction_of_oil_with_sulfuri
c_acid_and_with_ammonium_molybdate/attachment/
59d61e9c79197b807797d067/AS:279436512514052
@1443634264285/download/Identifying+Lipids+Using
+Chemical+Tests.docx
Figure 12 below shows the result when
the isolates where subjected to the test.
It is shown in this figure that only the
supernate tested positive. The reason for
this can be traced back to the separtion
fo the polar and non polar lipids by the
addtion of acetone. Since cholesterol is a
non-polar lipd, it should be in in the
supernate and is not precipitated out.

Figure 12 Leibermann-Burchard test result.
Supernate (left) tested positive while precipitate
(right) tested negative
Test for unsaturation
Lipid unsaturation can also be tested.
When iodine or bromine is added to a
sample containing an unsaturated lipid,
the iodine or bromine will undergo
decolorization thus indicating the
presence of an unsaturated lipid. If the
lipid present is saturated, then
decolorization will not happen
(Identifying Lipids Using Chemical Tests,
n.d.). This test was not performed in this
experiment.
Other test that can be done are the
acetate test which can differentiate
neutral fats from fatty acids by reacting it
with acetate and Kreis-Kerr test to
determine the type of rancidity of the
sample by reacting it with 1%
Phloroglucinol. Other tests for
cholesterol such as Salkowski and Zak
test can also be done (Identifying Lipids
Using Chemical Tests, n.d).
VI. Conclusion and Recommendation
Based on the results of the qualitative
tests, isolation of lipid via Folch method
is very effective. The qualitative results of
the isolate are all in line with the expected
data. The only test that was quite
erroneous was the TLC. The Rf values of
the isolates should be much closer to the
standards which they contain.
It is recommended that other tests
such as test for unsaturation, copper
acetate test, kries-kerr test, Salkowski
test and Zaks Test.
VII References
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eph_Chidiac/post/What_is_result_of_int
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59d61e9c79197b807797d067/AS:27943
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