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
membranes or in diverse ones such as
Lipids are biological compounds that egg yolks and the human nervous
occurs frequently in nature and are best system (Campbell,2013).
defined based on their solubility. Lipids
being composed of long carbon chains Together with carbohydrates the
are generally insoluble in water but main function of lipids is for energy
readily soluble in organic solvents storage and for structural purposes being
(Campbell & Farell, 2013). However, an important component of cell
some lipids contain both polar and non- membranes. Other functions of lipids are
polar groups thus they can also be source of vitamins A D E and K which are
amphpatic (Garett & Grisham, 2013). fat soluble and act as insulators
Lipids can be found in simple settings protecting the body from cold
such as animal, plant and microbial temperatures thus contributing to the
body temperature regulation. They also of the middle layer. Acetone was added
participate in intra- and intercellular to the collected layer until precipitate
signaling (TutorVista, 2017). ceases. It was then subjected to
centrifugation at 6000 rpm for 10 minutes
There are different categories in separating the precipitate from the
classifying lipids. According to (Campbell supernate and collecting both.
and Farell, 2013) lipids can be classified
into two groups based on their chemical Thin Layer Chromatography
nature. The first group includes open-
chained compounds with polar head and The TLC chamber was first saturated
long non-polar tails while the second with the mobile phase methanol-acetone
group is composed of fused-ring solvent in a 1:2 ratio. An appropriate
compounds. (Zubeda & Zarin, 2012) silica gel was obtained and was spotted.
classifies lipids based on their structural Afterwards it was placed inside the
complexity and biological functions. saturated chamber. The TLC plate was
Lipids classified under structural taken out and was air dried. To visualize
complexity can still be grouped into the movement of the spots, the plate was
whether they are simple lipids, saturated with iodine vapor.
compound lipids or derivative lipids.
Lipids classified under biological Qualitative Tests
functions can also be grouped into
whether they are structural lipids, storage SUDAN IV TEST
lipids or precursor lipids. While,
(BiologyExams4U, 2017) classifies lipids Ten (10) drops of the precipitate
into 4 groups fatty acids, glyceride and the supernate was placed in
lipids, non-glyceride lipids and complex separate test tubes. To each test tube,
lipids -based on their solubility in non- tiny crystal of Sudan IV was added. After
polar solvents. shaking the test tubes, results were
noted.
The objective if this experiment is to
use liquid-liquid extraction in isolating the ACROLEIN TEST
lipid present in the egg yolk and to
subject the isolated lipid in different Ten (10) drops of the precipitate and
qualitative tests. the superante were placed in separate
test tubes. To each test tube, a pinch of
IV. Methodology KHSO4 was added and was subjected to
gentle heating. After wards, it was placed
Isolation of Lipids from Egg Yolk in a boiling water bath. After the water
bath, the smell of each test tube was
Egg yolk was combine with 1% NaCl noted.
in a 1:3 ratio. Afterwards, it was mixed
with a 1:3 ratio of chloroform methanol TEST FOR PHOSHATES
solvent in a separatory funnel. After
mixing, the solution was left to stand for In separate crucibles, 0.5mL of the
30 minutes. The organic bottom layer precipitate and the supernate was
was collected as well as a small portion incinerated making sure that no liquid is
left. Distilled water was then added to the responsible for the separation of the
residue. After wards, the solution was lipids from the non-lipid contaminants.
filtered discarding the residue and Chloroform being a non-polar solvent will
collecting the filtrate. To the filtrate, weakly interact with polar components
0.5mL of 10% ammonium molybdate and thus it will be in the phase wherein there
2 drops conc. HNO3 was added. It was is less water and non-lipid contaminant
heated and was allowed to stand after and more lipid content. The other
cooling. Formation of yellow precipitate component which is methanol is polar so
was observed. it strongly interacts with polar
components thus it will be in the phase
LEIBERMANN-BURHCARD TEST wherein there is more water and non-lipid
contaminant and less lipid content.
Five (5) drops of P and S were placed Basically the isolation process can be
in separate test tubes. To each test tube, divided into two phases; the methanol
10 drops acetic anhydride and conc. phase which contains all the water and
H2SO4 was added. After mixing, color of non-lipid contaminants and the
solution was noted. chloroform phase which essentially
contain pure lipids from the sample (Bligh
TEST FOR UNSATURATION & Dryer, 1957). Another purpose of
adding the salt was to increase the
Hubls solution was added drop by amount of lipids that will be isolated.
drop to 5 drops of the precipitate and According to (Folch etal, 1957), there are
supernate placed in separate test tubes. still lipids in the methanol phase since
While adding, shaking was done and the there are amphipathic lipids. The lipids in
number of drops until the color of the methanol phase exist as salts and are
solution did not change anymore was in the dissociated form. Without the
recorded. presence of additional salt, the amount of
the isolated lipids cannot be maximized
V. Results and Discussion since the dissociated forms are in
equilibrium with the solvent mixture. But
Isolation of Lipids from the Egg Yolk with the presence of the additional salt,
there would be an equilibrium shift
The isolation process began with 1% shifting the lipids in the methanol phase
sodium chloride (NaCl) being added to to the chloroform phase. The added salt
the lipid source which is egg yolk. This is together with water and the non-lipid
to initiate the salting out of the proteins contaminants will remain in the methanol
present resulting in an isolated lipid of phase while all the lipid contents will be
high purity (Nelson & Cox, 2012). To in the methanol phase.
extract the lipid content of the egg yolk,
liquid-liquid was used wherein the The egg yolk-salt mixture was
solvent is a mixture of chloroform and combine with the solvent mixture in a
methanol in a 1:2 ratio. This solvent separatory funnel where it was mixed
mixture was used because it is regarded thoroughly while opening the valve once
to extract the most amount of lipid when in a while to let the accumulated pressure
compared to other solvent mixtures. The out. Afterwards, it was left to stand for 30
components of the solvent mixture will be minutes. As seen in figure 1, instead of
forming just two layers, it formed three. The technique used in lipid isolation is
The top most layer is the methanol phase known as Folch method. It is the first
containing all the polar contaminants plus technique used to isolate lipids using
the NaCl. The bottom most layer is the chloroform water-methanol solvent in
chloroform phase containing all the lipids. an 8:4:3 ratio. According to (Bligh &
What corresponds to the middle layer is Dryer, 1957) the above ration of
the emulsion formed due to the mixing chloroform to ethanol is only applicable to
done. The middle layer is composed of samples wherein the water content is
amphipathic lipids (Wang, Sunwoo, greater than the lipid content such as in
Cherian, & Sim, 2000) 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.
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 of colorimetric test used to determine the
precipitate. This is the reason why nitric presence of cholesterol. The hydroxyl
acid was added. The equation below group of cholesterol reacts with the
shows how the free phosphate reacts reagents resulting in the increase in the
with ammonium molybdate in the conjugation and unsaturation of the fused
presence of nitric acid (Identifying Lipids rings in cholesterol. The effect of this
Using Chemical Tests, n.d.). 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
HPO42(aq) + 12MoO42(aq) + 3NH4+(aq) + sample can be computed by subjecting
23H3O+(aq) the result of the test to a
(NH4)3[P(Mo3O10)4](yellow,s) + 35 H2O(l) spectrophotometry though it was not
done in this experiment (Identifying
Lipids Using Chemical Tests, n.d.).
This test was done for both the Reaction of cholesterol with the reagents
precipitate and the supernate isolate. is shown below.
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.
VI. Conclusion and Recommendation Folch, J. et al, A Simple Method for the
Isolation and Purification of Total Lipids
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