Experiment No.

7 & 9 ISOLATION OF LIPIDS FROM EGG YOLK & DETERMINATION OF ACID VALUE OF FATS

2013-14728; 2013-68117; 2013-14905

Group #5, Biochemistry 34.1 MEJ, Professor Del Rosario
November 9, 2015
I. Abstract
Lipids are relatively non-polar macromolecules that contain hydrocarbons, and have various functions
in biological systems. Examples of lipids include glycerol, phospholipids, natural oils and steroids. In this
experiment, the lipids were extracted from egg yolk sample using liquidliquid extraction, with
chloroform-methanol as the solvent mixture. Using thin layer chromatography and qualitative tests such
as Sudan IV test, Acrolein test, Test for Phosphates, Leibermann-Buchard test and Test for Unsaturation,
the different lipid components of the supernate and precipitate were determined. Results of the TLC
showed that the supernatant contained less polar components, with an R f value of 0.3533, compared to
precipitate (Rf = 0.0667). The qualitative tests showed positive results for both the supernate and
precipitate in the Sudan IV test and Test for Phosphates, while both being negative in the LeibermannBurchard test. In the test for unsaturation, the supernate and precipitate needed 8 and 22 drops
respectively, while only the precipitate showed positive results in the Acrolein test. Another method of
analysis for fatty acids is through the calculation of the acid value of the sample. This can be determined
by the amount of KOH needed to neutralize the free acids present. This experiment determined the acid
value for used commercially available cooking oil, with the results being an average of 0.1683 mg KOH
per gram of fat sample, which is relatively a low acid value. The palm oil analysed, therefore contains only
a small amount of free fatty acids. The obtained acid value can be accounted to the deterioration of the
fats by environmental factors. Both lipid extraction and acid value determination of fats are essential
processes in clinical chemistry, medicine, food technology and various industries.
II. Keywords
Lipids, Acid Value, Liquid-liquid extraction, Fatty acids, Thin layer chromatography
III. Introduction
Lipids are biomolecules which are relatively
nonpolar as compared to water and other
biomolecules, thus their solubility in nonpolar
solvents. They vary greatly in structure and
function, such as cell membrane component and
energy storage (Reusch, 2013).
Since lipids have a wide array of structures, a
unified classification system for lipids is hard to
achieve. However, lipids can be divided into two
groups: polar and nonpolar lipids, relative to each
other. Nonpolar lipids, such as triglycerides, are
less polar than polar lipids because little to no
polar groups are found within the molecule, and
the polar groups available are small as compared
to those of the polar lipids (Hasenhuettl & Hartel,
2008). Thus, nonpolar lipids have less interaction
with water and other polar solvents than polar
ones, allowing them to be easily separated from
polar lipids, and extracted by liquid-liquid
extraction.
Most lipids contain fatty acids, an example of a
polar lipid, in their structures. Compared to other
lipids, fatty acids have simple structures, with a
chain of 10-20 carbon atoms and a carboxylic
group at the end (Figure 1).

Figure 1. An example of a fatty acid.

Source: Classification of lipids (n.d.). Retrieved from
http://www.laney.edu/wp/cheli-fossum/files/2012/01/Classifica
tion-of-Lipids.pdf.

(DPSM, 2013). To determine the amount of free

fatty acids present in a sample, the acid value is
measured.
The acid value of fats measures the amount of
KOH in milligrams required to neutralize the fatty
acids present in one gram of a sample (Analytical
methods to measure the constants of fats and oils,
n.d.). Determination of free fatty acids in a sample
of oil can therefore be achieved through direct
titration with a base.
This experiment aims to successfully isolate
lipids from egg yolk using liquid-liquid extraction,
and confirm the presence of various lipids using
qualitative tests. It also aims to determine the acid
value of a sample of commercially-available oil by
direct titration.
IV. Experimental
A. Isolation of Lipids from Egg Yolk
Twenty milliliters of a solution containg egg
yolk and 1% NaCl solution, in a ratio of 1:3, was
mixed with 60mL of another solution containing
CHCl3 and methanol, in a ratio of 1:2. The
resulting mixture was placed in a separatory
funnel, swirled, and mixed, occasionally releasing
gas and pressure through the valve. The
separatory funnel was then allowed to stand for 30
minutes, or until distinct layers are observable.
The organic layer formed at the bottom of the
funnel was collected and added with acetone until
precipitation stops. The resulting solution was
centrifuged for 10 minutes at 6000rpm.

These fatty acids are also usually products of

hydrolysis of more complex lipids. Lipids in oil
hydrolyze spontaneously after prolonged storage
Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

Page 1 of 10

The precipitate was collected and dissolved in

5mL methanol. The solution was then labelled P,
and the supernate was labelled S.
Thin Layer Chromatography
A silica gel plate was spotted with the
precipitate from the previous step, P, the
supernate from the last centrifugation, S, and with
suitable standards.
Three milliliters of a methanol-acetone mixture
in a 1:2 v/v ratio was placed in the
chromatographic chamber. The TLC plate was
then allowed to stand inside the chamber until the
solvent line was at about 1cm below the top side
of the plate. The plate was then removed from the
chamber and allowed to dry.
Several iodine crystals were placed in the
chamber, then, after drying, the TLC plate was
returned to the chamber. At the appearance of
distinct orange/brown spots, the plate was
removed, and the distance travelled by the spots
was measured and noted. The respective Rf
values for each sample and for the standards were
then calculated.
Qualitative Tests
SUDAN IV TEST
Ten drops of P and S were placed in separate
test tubes, and a tiny crystal of Sudan IV was
added in each tube. The tubes were shaken and
the results were noted.
ACROLEIN TEST
Ten drops of P and S were placed in separate
test tubes, and a pinch of KHSO4 was added in
each tube. The solutions were then heated gently
for a few seconds by flaming, and placed in a
boiling water bath for five minutes. The resulting
smells of the solutions were noted.
TEST FOR PHOSPHATES
A 0.5mL sample each for P and S was
incinerated in separate porcelain crucibles. After
complete evaporation of the solvents, the residues
obtained were cooled. Three milliliters of distilled
water was added to each of the residues. After
mixing thoroughly, the solutions were filtered, and
the filtrates were obtained. Each filtrate was mixed
with 0.5mL 10% (NH4)2MoO4 and two drops of
concentrated HNO3. The solutions were heated for
two minutes and allowed to stand. The formation
of a yellow precipitate, or lack thereof, was noted.
LEIBERMANN-BURCHARD TEST
Five drops of P and S were placed in separate
test tubes. Ten drops of acetic anhydride and ten
drops of concentrated H2SO4 were mixed with
each of the tubes. The changes in color, or lack
thereof, were noted.
TEST FOR UNSATURATION
Five drops of P and S were placed in separate
test tubes. Hubls solution was then added
dropwise to each tube, while shaking, until the
resulting pink color does not fade. The number of

drops required for decolorization to cease was

recorded for each sample.
B. Determination of Acids Value of Fats
Twenty milliliters of fat sample was titrated
directly using KOH after adding two drops of
phenolphthalein as indicator. The volume of titrant
required to reach the end point was noted.
V. Results and Discussion
Lipids are naturally occurring substances,
arbitrarily grouped together on the basis of their
insolubility in water and solubility in nonpolar
solvents. Due to the fatty acid profile, high oil
soluble vitamin and lecithin content, Egg yolk is
considered a rich source of a variety of
biochemically important compounds such as
proteins and lipids (Minard & West, 2001),
specifically triglycerides (65%), phospholipids (2830%) and cholesterol (4-5%).
Lipids include a wide variety of different
substances, but are commonly subdivided into
several classes based on structural similarities.
TRIACYLGLYCEROLS
The triacylglycerols, (fats and oils) are esters
of glycerol and fatty acids generally formed by a
dehydration reaction as shown below in Figure 2.
Triacylglycerols differ in the types of fatty acids
attached to the glycerol backbone. The fatty acid
always contains an even number of carbon atoms,
commonly ranging between 10-20 carbon atoms
long. The hydrocarbon chain on the fatty acid can
be either saturated (contains only C-C single
bonds), or unsaturated (containing one or more
C=C bonds. Fats are solids, obtained primarily
from animals, and contain a larger proportion of
saturated fats, while oils are liquids obtained
primarily from plants and contain a greater
proportion of unsaturated fatty acids (Properties of
lipids, n.d.).

Figure 2: Formation Reaction for Triacylglycerols

Source:http://science.halleyhosting.com/sci/ibbio/chem/notes/c
hpt3/triglyceride1.gif

PHOSPHOLIPDS
Phospholipids,
also
known
as
glycerophosphatides, are widespread type of lipid
which occur in all plant and animal cells as major
structural components of cell membranes. They
play critical roles in the transport of molecules
across membranes, storage and metabolism of
fatty acids, and as activators in the blood clotting
process. Structurally, they are similiar to

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

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triacylglycerols but phospholipids has one of its

fatty acid group replaced with a very polar
phosphate ester group (Minard & West, 2001), as
shown in Figure 3, which is produced by the
esterification of one of the alcohol groups of
glycerol by phosphoric acid molecule rather than
by carboxylic acid molecule (Campbell & Farrell,
2005).

mixture can be a good solvent for extraction of

lipids from egg yolk (Ahn et al., 2006).
The egg yolk is composed of polar and
nonpolar lipids. Lipids are generally waterinsoluble substances that are soluble in non-polar
organic solvents, such as chloroform. Chloroform
is relatively the most nonpolar solvent used in the
experiment. It cannot employ dipole-dipole
interaction because chlorine is too bulky and is
very electron rich, thus it prefers van der Waals
interaction. Chloroform solubilises lipids that are
insoluble in polar solvents such as triglyceride,
cholesterol, phospholipids and glycolipids (Berg, et
al., 2002).

Figure 3. Structure of Phospholipids

Source:http://patentimages.storage.googleapis.com/WO20080
19797A2/imgf000005_0002.png

STEROID
Steroids are lipids containing the core
structure of 17 carbons fused in a ring structure
containing three, six-member rings, and one fivemember ring. The different functionality of steroids
comes from the substituent groups attached to the
core structure.
Cholesterol, a typical example of steroid, is a
major component of cell membranes. An egg yolk
contains about 200 milligrams of cholesterol, much
of it bound as complex lipid. Figure 4 below shows
the structure of cholesterol.

Figure 5. Structure of Chloroform

Source: http://f.tqn.com/y/chemistry/1/S/Q/S/1/Chloroform.jpg

Alcohols, on the other hand, are known to be

good solvents for lipids (Lipid Extraction, n.d.).
Methanol and other alcohol components dissociate
the bonds in the lipoprotein complexes, making the
lipids readily available for chloroform to be
dissolved.
Polar
compound,
such
as
carbohydrates, urea, salts and proteins are
considered as contaminants and are dissolved by
methanol (Murray, et al., 2003).

Figure 4: Structure of Cholesterol

Source:http://blogs.dnalc.org/wp-content/uploads/2012/04
/cholesterol.png

Figure 6. Structure of Methanol

Source:http://d1h8qm6whtl6z3.cloudfront.net/wp-content/up
loads/2014/08/Methanol-structure-300x231.png

Isolation of Lipids from Egg Yolk

There are several methods for the lipid
isolation. One of which is the Liquid-liquid
extraction, which is employed in the experiment to
separate different types of lipids present in egg
yolk sample through the difference in miscibility of
the solution involved.
Liquid egg yolk contains a lot of water and
extraction with non-polar solvents is not efficient
due to difference in solvent and egg yolk polarities.
Polar solvents, such as lower alcohols, denature
egg yolk proteins destroying hydrogen bonds or
electrostatic interaction in protein structure
opening the way to the neutral lipids, what makes
extraction with non-polar solvent possible. Without
protein denaturation, polar solvents will extract
polar membrane-associated lipids from the egg
yolk. However, the combination of polar and nonpolar solvents for better lipid extraction from liquid
egg yolk can be chosen. The methanol/chloroform

The process of lipid extraction from tissues

with methanol/chloroform first was mentioned in
1957 in Folch et al. (as stated in Axelsson &
Gentili, 2014). Folch et al. were the first which
develop the chloroform/methanol/water phase
system (the so-called Folch method) for
extraction of lipids from biological material. The
method is still considered as the classical and
most reliable method for quantitative extraction of
lipids. The method relies on a mixture of
chloroform and methanol in 1:2 ratio, forming a
monophasic solvent system, to extract and
dissolve lipids solution (Abert-VIan et al., 2015).
The addition of chloroform/methanol solvent to
the sample formed three distinguishable layers:
the upper aqueous layer, the middle foamy layer
and the lower yellowish layer. The upper layer is
mainly composed of water, methanol and other
water soluble compounds; the middle emulsion
layer composed of aqueous and organic solution
(Boyer, 2012). A biphasic system is then produced

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

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by the addition of water or saline solution (AbertVIan et al., 2015).

After letting the solution stand for 30 minutes,
the organic layer was observed to be separated
from the aqueous layer, resulting into 2 layers as
shown in Figure 7. The bottom organic layer
displayed a golden yellow clear liquid while the
upper aqueous layer appeared as a pale yellow,
semi-clear liquid.

separated through centrifugation process (Cristie,

2011).

Figure 8. Structure of Acetone

Source:http://www.sigmaaldrich.com/content/dam/sigma-al
drich/structure2/194/mfcd00008765.eps/_jcr_content/renditions
/mfcd00008765-medium.png

The precipitate from the extraction process is

therefore composed mainly of phospholipids, while
the supernatant liquid is composed of triglycerides,
cholesterol and glycolipids.

Figure 7. Liquid-liquid Extraction Set-up

Source:https://upload.wikimedia.org/wikipedia/commons/thumb/
d/d1/Liquid_liquid_extraction.png/220px-Liquid_liquid_extracti
on.png

The addition of NaCl to the egg yolk helps

desolubilize the proteins in the sample through
salting out process (Wang, et al., 2000). This leads
to the migration of polar compounds along with the
methanol into an upper aqueous phase and
leaving the lipids in the lower chloroform phase
(Abert-VIan et al., 2015). The separation is
employed by the differences in density, with the
aqueous layer having the lowest and the
chloroform-lipid layer having the highest density
(Boyer, 2012).
In the extraction of lipids of other biological
samples such as the myelin sheath, the
chloroform/methanol ratio of 1:2 could be
maintained. Myelin sheaths are cholesterolenriched nerve cell insulators. The methanol must
have a higher volume than the chloroform
component to maximize the amount of lipids
extracted from the tissue without lowering the
water content (Rumsby, et al., 1966).
The solvent-sample ratio greatly influences the
per cent yield of the extraction process. A lower
ratio may form two phases which may lower the
interaction between the polar and non-polar
solvent and the membrane lipids. A higher ratio
might not bring any harm to the extraction process,
but the extraction of polar lipid might be lower due
to lower water content (Wang, et al. 2003).
A polar acetone solvent, as shown in Figure 8,
was added to the organic bottom layer extracted
through liquid-liquid extraction until precipitation
ceases. This further facilitates the solubilisation
between polar and non-polar lipids. Simple lipids
and glycolipids dissolve readily in acetone, while
phospholipids do not due to the presence of the
phosphate groups. The addition of acetone
employs dipole-dipole interaction and precipitates
the phospholipids, making them easier to be

Thin Layer Chromatography

Thin Layer Chromatography (TLC) is a
technique used to analyse the components of a
mixture through separation by determining the
number and identity of components in a mixture,
as well as the purity of the compound through the
process of elution. The mobile phase consisting of
a volatile organic solvent or mixture of solvent is
eluted through a stationary phase, as shown in
Figure 9, via capillary action (Thin Layer
Chromatography, n.d.).

Figure 9. Thin-Layer Chromatography Process (Boyer, 2012)

The mobile phase moves the sample up the

stationary phase at different rates. In the
experiment, the methanol-acetone solvent was
used. Methanol dissolves polar substances, while
acetone can dissolve both polar and non-polar
substances. The solubility of the compounds in the
eluting solvent and the ability of the solvent to be
absorbed on the stationary phase affect how fast
they travel through the TLC plate. Too high solvent
affinity to the absorbent may cause the samples to
move up close to the solvent front without
separation, while too low solvent affinity may result
to the slow movement of the components without
enough separation. The stationary phase, on the
other hand, is made from silica (SiO2) supported
on a metal foil. It shows maximum selectivity on
the types of compounds being separated so that
the differences in the elution rates will be large
enough for effective separation (Thin Layer
Chromatography, n.d.).
The chromatography chamber, as shown in
Figure 10, must be saturated with the solvent
vapour before the start of the elution process. The
methanol-acetone solvent mixture used in TLC are
highly volatile thus, evaporation is spontaneous.

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

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The saturation of the atmosphere inside the

chamber prevents the solvent from the stationary
phase to evaporate even before the elution
process is complete (Libal, n.d.).

Figure 10. A typical chamber for Thin-layer Chromatography

(Boyer, 2012)

However, some compounds may not be

readily visible after a chromatography process.
Chemical processes are used to make the marks
visible for clear determination of distance travelled.
Some of these development processes include the
use of a development chamber with a saturated
iodine vapor. The vapour present inside the
chamber complexes with the double bonds in
organic compounds on the plate to form visible
brown marks. This developing technique is
reversible and the iodine marks will disappear
upon reheating of the plate (Boyer, 2012).
Upon detection of separated compounds, the
Rf value (retention factor) of each samples was
calculated using the formula:

the attraction between the SiO2 molecules of the

plate and the polar molecules of the compound
(Boyer, 2012). Thus, it can be said that the
supernatant
liquid
sample,
composed
of
triglycerides, cholesterol and glycolipids are less
polar than that of the precipitate which is
composed mainly of phospholipids.
Qualitative Tests
In order to verify the presence of specific types
of lipids in the isolates, the supernatant liquid
sample and precipitate were subjected to
qualitative analysis. The tests include Sudan IV
Test, Acrolein Test, Test for Phosphates,
Leibermann-Burchard Test, and Test for
Unsaturation.
SUDAN IV Test
Sudan IV (C24H2ON4O) is a red nonpolar, lipidsoluble dye used in the staining process of lipids,
triglycerides and lypoproteins. Upon addition to a
solution of lipids and water, the dye will mix into
the lipid layer and produce a red colored layer
(Testing for Biologically Important Molecules, n.d).
Polar compounds and aqueous solutions, on the
other hand, will not interact with the nonpolar
Sudan IV stain.

Source: Biochemistry 34.1 Laboratory Manual, UP Manila

The supernatant liquid and the precipitate from

the first part of the experiment, together with a
standard palm oil solution, were subjected to thin
layer chromatography. The distance travelled by
each sample was measure and their respective Rf
values were computed.
Sample computations of the Rf value

Figure 11. Sudan IV Test: Positive result (left) and negative

result (right)
Source:http://emp.byui.edu/wellerg/Molecules%20of%20the%2
0Cell%20Lab/instruction/Molecules%20of%20the%20Cell%20I
nstructions.html

Results were obtained for each sample of lipid

isolation and were tabulated in Table 1.

The Sudan IV test was done on both the

supernatant liquid and precipitate, and positive
results were observed in both samples (Table 2)
indicating the presence of necessary lipids such as
triglycerides and lypoproteins to produce a red
color with the Sudan IV.

Table 1. Rf values of the different samples subjected to TLC

Table 2. Sudan IV Test Results

Samples
S
P
Standard
Solvent

dtravelled
2.65
0.5
5.7
7.5

Rf
0.3533
0.0667
0.7600
---

The results of this experiment shows that the

standard palm oil has the greatest Rf value
(Rf=0.7600), followed by supernatant liquid sample
(Rf =0.3533), and the precipitate having the least
Rf value (Rf =0.0667).
The Rf values are always greater than zero
and less than one. Less polar compounds have
higher values than high polar compounds due to

Test
Compounds
S
P

Experimental
Results
+++
++

Theoretical
Results
+++
-

However, the precipitate sample must

theoretically not test positive for the test, as it
should no longer contain tryglycerides or
lypoproteins, which should have readily dissolved
in the acetone added in the sample. The positive
(++) result of the precipitate sample suggests that
there are remaining triglycerides in the
precipitates, although in smaller amounts
compared to that of the supernatant liquid sample.

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

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ACROLEIN Test
The Acrolein test is a general test for the
presence of glycerol or fats in a molecule. The
principle behind the acrolein test is a specific
chemical reaction. When heated strongly in the
presence of a dehydrating agent, potassium
bisulfate (KHSO4), the fat undergoes hydrolysis
forming glycerol which is then dehydrated to form
the simplest unsaturated aldehyde, acrolein
(CH2=CHCHO), which has a characteristic
irritating odor that can be compared with burnt
grease (Ballou et al., 2005).

When phospholipids are hydrolyzed in acidic

medium, both the fatty acid ester bonds and the
phosphate ester bonds are broken and free fatty
acids and inorganic phosphate are released
reaction (Koolman & Roehm, 2005).
The supernatant of the lipid isolation is
theoretically negative for the test for phosphates,
because the phosphates must be present only in
the precipitate, since the acetone cannot dissolve
the phosphate groups. However the experimental
results (Table 4), show positive results for both the
supernatant and precipitate samples. The false
positive results may be accounted to the probable
presence of phosphate impurities in the
supernatant sample.
Table 4. Test for Phosphates Results

Figure 12. Acrolein Test Reaction

Source:http://image.slidesharecdn.com/activity5abiochemreport
-130513015903-phpapp01/95/activity-5-a-biochem-report-14638.jpg?cb

Experimental data, as shown in Table 3,

shows that the precipitate produced burnt grease
odor upon the completion of reaction, indicating
the presence of glycerol, while the supernatant
showed negative results.
Table 3. Acrolein Test Results

Test
Compounds
S
P

Experimental
Results
++

Theoretical
Results
+

Test for PHOSPHATES

The oxidation of fatty acids and the formation
of water and carbon dioxide by fatty acid
metabolism yields a reduced coenzyme used for
the production of ATP. The test for phosphates
uses the same principle by incinerating the
samples to destroy organic parts, evaporating
them as carbon dioxide and water, leaving solid
inorganic phosphate residue. The inorganic
phosphate
residue
was
separated
from
contaminants and other nonpolar lipid components
by dissolving the residue in water. Components
not soluble in water were discarded (Koolman &
Roehm, 2005).
When lipids containing phosphate groups in
their structures are added to a strong acid solution,
the lipid hydrolyses, producing free phosphate.
The presence of free phosphate in a solution was
then detected by adding ammonium molybdate
and nitric acid to the filtrate to produce yellow
ammonium
molybdo-phosphate
precipitates,
according to the reaction (Koolman & Roehm,
2005):
2

HPO4 (aq)+ 12MoO4 (aq)+ 3 NH4 (aq)+ 23 H3O

(NH4)3[P(Mo3O10)4] (yellow,s) + 35 H2O(l)

+
(aq)

Test
Compounds
S
P

Experimental
Results
+
+

Theoretical
Results
+

LIEBERMANN-BURCHARD Test
The LiebermanBurchard is a reagent used in
a colorimetric test to detect the presence of
cholesterol. A positive result for the test is
observed when the solution begins as a purplish,
pink colour and finally turns into a bluish-green
solution. The colour is due to the hydroxyl group (OH) of cholesterol reacting with acetic anhydride
(used as solvent and a dehydrating agent), and
sulfuric acid (used as dehydrating and oxidizing
agent), thereby increasing the conjugation in the
adjacent fused ring, as shown in Figure 13.
Cholesterol reacts with acetic anhydride and
sulfuric acid to yield pentoenylic cation, a highly
conjugated compound whose maximum light
absorption is at 620 nm wavelength. This accounts
for the purple or pink color observed initially at the
addition of the reagents. Further reaction with
sulfuric acid produces cholestahexone sulfuric
acid. The color of the final solution is green due to
the maximum absorption of the compound at 410
nm wavelength (Burke et al., 1974).

Figure 13. Liebermann-Burchard Reaction

Source:https://upload.wikimedia.org/wikipedia/commons/thumb/
8/83/Liebermann-Burchard.svg/600px-Liebermann-Burchard.
svg.png

Experimental results, as shown in Table 5,

suggest the negative result, therefore the absence
of cholesterol for both the supernatant and

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

Page 6 of 10

precipitate sample. However, the supernatant

sample from lipid isolation must theoretically
contain cholesterol, since it can readily dissolve in
acetone.
Table 5. Liebermann-Burchard Test Result

Test
Compounds
S
P

Experimental
Results
-

Theoretical
Results
+
-

Test for UNSATURATION

In order to determine the degree of
unsaturation of the fatty acids in the lipid sample,
an alcoholic solution of iodine containing some
mercuric chloride called Hubls iodine reagent, was
added to the samples until a change in color was
observed, The test works in line with the principle
that unsaturated fatty acids become saturated by
taking up iodine and if it contains more
unsaturated fatty acids, it will take up more iodine,
and thus producing a color change in the solution.

Figure 14. Reaction with the Hubls Reagent

Source:http://www.assignmentpoint.com/wp-content/uploads/
2013/12/reaction1.jpg

The experimental results (Table 6) show that

the supernatant sample reacted and employed a
color change in a smaller amount of the Hubls
reagent, thereby showing that the sample contain
less double bonds, and thus a smaller degree of
unsaturation. The precipitate sample, on the other
hand, requires a higher amount of the reagent
before a color change. Hence, it contains a higher
degree of unsaturation.

The acid value indicates the proportion of free

fatty acid present in oil or fat and may be defined
as the number of milligrams of potassium
hydroxide (KOH) required to neutralize the free
fatty acid in one gram of oil or fat (Determination of
acid number of edible oil, n.d.). Acid value can
help determine the purity of the oil or fat. A high
acid value thus indicates that the oil is old and
rancid.
Fat Solvent
The dissolution of fat/oil samples is done
through the use of a fat solvent, which is generally
composed of the mixture of an absolute alcohol
like ethanol, and a nonpolar compound such as
diethyl ether (Thomas, n.d.).

Figure 15. Structure of Diethyl ether

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/struc
ture0/009/mfcd00011646.eps/_jcr_content/renditions/mfcd0001
1646-medium.png

Ethanol, shown in Figure 16, has both polar

and nonpolar end, making it able to dissolve both
polarities. Lipids and fatty acids are arranged with
the hydrophobic parts pointing inwards while the
hydrophilic parts are exposed in the aqueous
environment. The ethanol then dissolves the
exposed hydrophilic part so that inner hydrophobic
parts will be dissolved by the nonpolar diethyl
ether solvent (Thomas, n.d.).

Figure 15. Structure of Ethanol

Source:https://upload.wikimedia.org/wikipedia/commons/6/6f/Et
hanol_flat_structure.png

The fat sample used for this experiment was

used palm oil. Upon its titration with potassium
hydroxide, the acid value was calculated using the
formula:

Table 6. Test or Unsaturation Results

Test
Compounds
S
P

Experimental
Results
(no. of drops)
8
22

Determination of Acid Value of Fats

Fat may become rancid after long storage. It
may be hydrolysed by different microorganisms,
thus breaking its ester bonds. This then leads to
the formation of free fatty acids making the
solution acidic or rancid. The amount of free fatty
acid present in the oil then indicates the age and
the quality of that oil (Determination of acid
number of edible oil, n.d.). In order to analyse this,
the acid value of the fat sample was determined.

Source: Biochemistry 34.1 Laboratory Manual, UP Manila

where VKOH is the volume of KOH used in the

titration, MKOH is the concentration of KOH used
and MWKOH is the molecular weight of KOH (56.11
g/mol).
Sample computations for Acid Value

mg KOH per g of palm oil

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

Page 7 of 10

After the acid values of the samples were

determined, the results were tabulated. Table 7
shows the results of the experiment.
Table 7. Determination of Acid Value of Palm Oil

VKOH at

Acid Value

equivalence point
(mL)

Fat
Sample

Trial 1
Trial 2
Trial 3

0.3
0.3
0.3
Average

From the experimental results, it can be said

that the used palm oil analysed has a relatively low
acid value. Therefore, the palm oil contains only a
small amount of free fatty acids and may then
suggest that the oil is not yet old and rancid.
Aside from the acid value, the quality of fats
may be determines using other constants or
values, such as the Saponification Value, Ester
Value, Hydroxyl Value and Iodine Value (Assays,
2005).
Saponification Value
The saponification value (Is) is the amount in
milligrams of KOH needed to saponify the ester
and neutralize the free acids in 1 gram of sample.
The procedure in the determination of the
saponification value includes performing a blank
test similar to back titration (Assays, 2005). The
saponification value is calculated using the
formula:

Source:http://lib.njutcm.edu.cn/yaodian/ep/EP5.0/02_methods_
of_analysis/2.5.__assays/2.5.0%20Assays.pdf

where a is the volume in mL of 0.5M

ethanolic KOH consumed in the blank test while b
is the volume in mL of 0.5M ethanolic KOH
consumed in the actual test.
Iodine Value
Lastly, the Iodine Value (II) is the amount in
grams of halogen, particularly Iodine, that can be
consumed (in certain conditions) by 100 grams of
sample. Iodine values are often tabulated in
tables, as in Table 8 (Assays, 2005).
Table 8. Unsaturated Fat Levels in various Foo Ingredients

Source:http://nationalhogfarmer.com/site-files/nationalhogfarme
r.com/files/uploads/Keeping%20Iodine%20table1.jpg

VI. Conclusion

Source:http://www.ffcr.or.jp/zaidan/ffcrhome.nsf/7bd44c20b0dc
562649256502001b65e9/146fd852cd5e269049256f32001a133
e/$file/b11.pdf

where a is the volume (mL) of 0.5 M

hydrochloric acid (HCl) used in the blank test and
b is the volume (mL) of 0.5 M HCl consumed in the
assay (General Tests, n.d.).
Ester Value
The ester value (IE) is the amount of KOH in
milligrams needed to saponify the esters present
in 1 g of substance (Assays, 2005). The IE is
calculated using the saponification value IS and
acid value IA as follows:
Ester Value, IE = IS - IA
Source:http://lib.njutcm.edu.cn/yaodian/ep/EP5.0/02_methods_
of_analysis/2.5.__assays/2.5.0%20Assays.pdf

Hydroxyl Value
The Hydroxyl value (IOH) expresses the
amount of KOH in milligrams required to neutralize
the acid combined by acylation in 1 g of the
substance (Assays, 2005), which is calculated
using the formula:

Lipids are major macromolecules that include

relatively non-polar compounds and serve as
storage for energy in biological systems. One way
of extracting water-insoluble lipids from a sample is
through liquid-liquid extraction, a method that
utilizes the solubility of the lipid in a polar solvent,
in this experiment being chloroform-methanol
solvent mixture. The supernate and precipitate
from the extracted sample then underwent TLC
and several qualitative tests.
Results of the TLC showed that supernatant
has a greater Rf value (0.3533) compared to that of
the precipitate (Rf = 0.0667). This means that that
precipitate is the more polar isolate, since they
have more interaction with the stationary phase of
the TLC (silia gel).
The qualitative tests aided the identification of
lipids present in the isolates: glycerol for the
Acrolein test, triglycerides for the Sudan IV test,
phospholipids for test of Phosphates, cholesterol
for the Leibermann-Burchard test, and unsaturated
fatty acids for test for Unsaturation. The results
suggest that supernate and precipitate samples
contain triglycerides, phosphates, unsaturated fatty
but not cholesterol. The precipitate, in addition,
contains glycerol.
Another method of qualitative analysis for fatty
acids is through the calculation of the acid value of
the sample. In this experiment, this value was

Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats

Page 8 of 10

determined by the amount of KOH required to

neutralize the fatty acids present in the process of
titration.
The results gave an acid value of 0.1683 for
the used cooking oil which is a relatively low acid
value. Therefore, the palm oil contains only a small
amount of free fatty acids and may then suggest
that the oil is not yet old and rancid. Although a low
acid value was calculated, the values still suggests
low purity for the sample and the possible
deterioration of the free fatty acids.
For greater accuracy in the isolation of lipids, it
is suggested that the collection of the sample from
the liquid-liquid extraction be done carefully. In the
TLC, it should also be made sure that the samples
spots do not touch one another.
As for the determination of acid values, it is
recommended to have the proper fat solvent ratio
to better extract the fats from the sample. It is also
important to use fresh samples to isolate from, in
order to more accurately determine the acid value
of the fats.
VII. References
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Campbell, M. & Farrell, S. (2012). Biochemistry,

7th Edition. Brooks/Cole, 20 Davis Drive,
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College of arts and sciences, Department of
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Khaira, G. & Kot, A. (n.d.) Henderson-Hasselbalch
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Lipids: Qualitative tests of lipids (n.d.) Retrieved
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peripheral nerve tissue 1. University of

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