Lipid Extraction From An

Egg
Experiment 5

BIRUNI UNIVERSITY
FACULTY OF ENGINEERING AND NATURAL SCIENCE
MOLECULAR BIOLOGY AND GENETIC

Biochemistry Lab Report

Lecture : Biochemistry Lab ( CHM213 )
Instructor : Assist. Prof. Dr. Banu Taktak Karaca
Name : Şuara ŞAHİN
Student ID : 160401010
Date : 12.12.2018

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Abstract:
In this experiment there were two different aims. Primary purpose was to determine whether eg yolk or egg
white was containing more lipid content. For this reason Folch method was used. 1 ml of egg white and egg
yolk was diluted with 4 ml saline. After that 2:1 ratio chloroform and metahonol was added and it was
centrifuged. Bottom layer of the centrifuged solution was taken and it was dried in etuve. Dried test tubes
were weighted and the gramage of the white and yolk was recorded. Secondly, the aim was to determine
whether yolk or white was containing cholesterol. For this reason Salkowsky test was used. Already diluted
white and yolk was mixed with sulphuric acid and chloroform. After 5 minute waiting process color change
was observed. The results were recorded.

Introduction:
Lipids are organic compounds that contain hydrocarbons which are the base for the structure and function
of living cells. Lipids are non polar so they are soluble in nonpolar environments though not soluble in water
because water is polar. Fatty acids are “carboxylic acids (or organic acid), often with a long aliphatic tails
(long chains), either saturated or unsaturated.” When a fatty acid is saturated it is an indication that there are
no carbon-carbon double bonds and if the fatty acid is saturated it is an indication that it has at least one
carbon-carbon double bond. The saturated acids have higher melting points than unsaturated acids of
corresponding size. If a fatty acid has more than one double bond then this is an indication that it is a
polyunsaturated fatty acid. Saturated fatty acids have higher melting points due to their ability to bundle
their molecules together therefore leading to a straight rod-like shape. Unsaturated fatty acids on the other
hand have cis-double bond(s) that form a kink in their structure which doesn’t allow them to group their
molecules in straight rod-like shape [1]. (Figure 1.1)

Figure 1.1: Structure of lipids

Lipids are soluble in organic solvents, but slightly soluble or insoluble in water. Existing procedures for the
extraction of lipids from the source material generally involve selective solvent extraction and the starting
material may be subjected to drying prior to extraction. The solubility of the lipids is an important criterion
for extraction from the source material and depends to a large extent on the ratio of the present lipid type and
the non-polar (mainly triacylglycerols) and polar lipids (mainly phospholipids and glycolipids) in the
sample; Therefore, several solvent systems may be considered depending on the sample and its components.
Preferred solvents are usually chloroform/methanol or chloroform/methanol/water, in the case of the Folch
Method or its modified Bligh and Dyer Procedure and sulfuric acid/chloroform in the case of Salkowsky
method [2].

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Egg contains many nutrients. These nutrients are shown in the table (Table 1.1).

Table 1.1: Egg white and yolk nutrients

In this experiment Salkowsky test and Folch method was used to extract lipid content from egg yolk and
egg white.

Materials:
Equipment and materials for Folch Method

• Egg
• Saline
• Methanol
• Chloroform
• Test tubes
• Beakers
• Magnetic stirrer
• 10 ml graduated cylinder

Equipment and materials for Salkowsky Test

• Test tubes
• Olive oil
• Egg yolk
• Egg white
• Distilled Water
• Concentrated Sulphuric acid
• Chloroform
• Pipette

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Method:
In this experiment two different method were used which were Folch method and Salkowsky method.
Firstly Folch method was used. Folch method purpose was to determine whether the yolk or the white of an
egg has more lipid content. At first, test tubes 1-6 were weighed out and marked. Later on, egg was cracked
open and the yolk was placed into a 100 ml beaker while the white was placed into another 100 ml beaker.
Each part was homogenized by using a magneticstirrer.1 ml of the yolk was measured out into test tube 1 by
using the 10 ml graduated cylinder. This process was repeated with the white and placed into test tube 2.
Both of them were diluted with 4 ml of saline and they were mixed with vortex.1 ml of each of two diluted
solutions was measured out, the yolk was placed into the test tube 3 and white was placed into test tube 4.1
ml of methanol and 2 ml of chloroform were added into both of the tubes 3 and 4 and they were mixed with
vortex. The tubes were centrifuged at 5.000 rpm for two minutes to separate the lipid contents of the egg
components. While the tubes were centrifuging empty test tubes 5 and 6 was weighted out. After that, the
bottom layer which was composed of lipids and chloroform was placed into test tube 5 (coming from test
tube 3- the yolk) and test tube 6 (coming from test tube 4- the white). Tube 5 and 6 were etuved to dry out
the mixture and the tubes were weighed out to determine the mass of lipids in 1 ml of egg white and egg
yolk.
Secondly, Salkowsky method was used to analyze cholesterol in a given sample. Four test tubes were named
as positive control (olive oil) , negative control (distilled water), sample 1 (egg yolk), sample 2 (egg white).
1 ml of the sample was replaced into their named tubes. Then 1ml of chloroform and 10 drops of
concentrated sulfuric acid was added into each test tube. Test tubes were mixed with vortex and the color
change for each tube was recorded.

Result:
In Folch method chloroform and methanol was used in 2:1 ratio to extract lipid from the egg yolk and egg
white. Purpose of this method was to determine whether egg white or the egg yolk had more lipid
components. The egg yolk and the egg white was diluted with saline (Figure 3.1).Chloroform and methanol
was added into egg yolk and egg white in 2:1 ratio and they were centrifuged for two minutes (Figure 3.2).

Figure 3.1: Egg yolk and egg white after Figure 3.2: Egg yolk and egg white after
diluting with saline centrifuge with chloroform and ethanol

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Empty test tube 5 and 6 was measured. Bottom layer which was composed of lipid and chloroform was
placed into test tube five and six (Figure 3.3) and they were etuved to dry out (Figure 3.4).

White test Yolk test

tube 6 tube 5

Figure 3.3: Bottom layer of centrifuged egg yolk and egg

white composed of lipid and chloroform
Figure 3.4: Etuve process of test tube 5 and 6
After drying process test tube 5 and 6 was measured to determine which component had more lipid content.
Empty test tube 5: 8.52 gram Empty test tube 6: 8.48 gram
Filled test tube 5 (yolk): 8.58 gram Filled test tube 6 (white): 8.48 gram

It had shown that egg yolk sample used in the experiment was containing 0.06 gram lipid content. On the
other hand the egg white was not contained lipid content.
In Salkowsky test chloroform and sulfuric acid mixture was used to dye cholesterol. Purpose of the test was
to determine whether egg yolk or egg white had higher cholesterol. For this purpose olive oil was used as
positive control and distilled water was used as negative control. 1 ml of egg yolk and egg white was placed
into test tubes and mixed with chloroform and sulfuric acid mixture. After 5 minutes color changes were
observed (Figure 3.5)

Negative control Egg white

group water

Egg yolk
Positive control
group olive oil

Figure 3.5: Color change after Salkowsky test

After the Salkowsky test it was observed that both egg yolk and egg white contains cholesterol. Also it has
shown that egg yolk contains more cholesterol than egg white.

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Discussion:
Purpose of this experiment was to determine whether egg yolk or egg white had more lipid content and
which one had more cholesterol. In order to control accuracy of the experiment nutrients of the egg white
and egg yolk table was taken from reference experiments (Table 4.1).

cholesterol

Table 4.1: Egg nutrients table

Folch method is a technique that is used for extracting lipid content from a sample. In this experiment
Folch method was used to determine whether the yolk or white of an egg had more lipid content. In Folch
method chloroform and methanol was used in 2:1 ratio. Chloroform (CHCl3), which has a weak dipole
moment, is only weakly drawn into the water and is therefore present in substances that are mostly
lubricated in the phase with less water and consequently more lipids. Evidence proves that only 5,5% of
water can be dissolved in the chloroform phase. On the other hand, more polar methanol (CH3OH) may
attract more water and non-lubricated substances than chloroform and therefore can be combined in a single
phase. Basically, the isolation system can be divided into two phases: the methanol phase containing almost
all non-combustible materials and a small amount of lipids, and the chloroform phase consisting essentially
of pure lipids from the sample. Since the main purpose of this step is to isolate the lipids found in the sample
at high efficiency, the amount of lipid remaining in the methanol phase should also be isolated. The isolation
process was carried out with saline which was a diluted NaCl solution [4]. After egg white and egg yolk was
separated they were mixed with 2:1 chloroform-methanol and 4 ml saline. They were centrifuged and the
bottom part of the mixtures were taken and placed into measured tubes. Due to density of the lipids, they
were in the bottom layer of the tubes. The test tubes were etuved to dry out. Dried test tubes were measured
again and it showed that egg yolk was containing lipid and the egg white was not. According to egg nutrient
table (Table 4.1) egg white contains 0.1 dram lipid in total. Considering the experiment was done in a 1 ml
egg white percentage of the lipid it had contained was too small to measure. Because of this reason it was
detected by the weigher.
Salkowsky test is technique that is used for determining cholesterol in a sample. In this experiment it was
used to determine whether egg yolk or egg white had more cholesterol. In Salkowsky test, sulfuric acid and
chloroform was used. Sulfuric acid (H2SO4) acts as a dehydrating agent which can either cause the two
cholesterol molecules to fuse or form an additional unsaturation [5]. The reaction can be detected by the
formation of reddish purple interphase. Egg white and egg yolk diluted with the saline were added to
sulfuric acid and chloroform mixture in two different test tubes. They were put into a vortex to ensure even
distribution. After mixing process in both white and yolk reddish purple color were observed. While egg
yolk contains 1.6 grams of cholesterol and white does not contain cholesterol (Table 4.1). In this the
experiment had given correct result for yolk but in correct result for white. According to the reference table,
there should had been no discoloration in the egg white. Reason for color change could have caused by
mistaken separation of yolk and white. Which means there could been yolk inside the white.

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In future experiments in order to determine better results more precision scales can use and separation
process can done with more cautiously.

In conclusion it was observed that Folch method carried out lipid content with 4:2:1 ratio of saline,
chloroform and methanol. In this experiment egg white and egg yolk lipid content was calculated. At the end
it was shown that egg yolk contains higher lipid content than egg white. Secondly, it was observed that
Salkowsky test color cholesterol red with reaction of breaking steroid bond with sulfiric acid. In this
experiment it was observed that both egg yolk and egg white contains cholesterol. The experiment was
resulted incorrectly for white.

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Reference:
1. Chem.ucla.edu. (2018). [online] Available at: http://www.chem.ucla.edu/~harding/notes/notes_14C_lipids.pdf
[Accessed 21 Dec. 2018].
2. Genesandgenomes.yolasite.com. (2018). [online] Available at:
http://www.genesandgenomes.yolasite.com/resources/extraction%20of%20lipids%20using%20soxlet%20met
hod.PDF [Accessed 21 Dec. 2018].
3. Juicedmuscle.com. (2018). Egg Nutrition: Yolk vs. White | JuicedMuscle.com. [online] Available at:
https://juicedmuscle.com/jmblog/content/egg-nutrition-yolk-vs-white [Accessed 21 Dec. 2018].
4. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from
animal tissues. J Biol Chem. 1957;1:497–509. [PubMed]
5. Roechlau P, Bernt E, Gruber W. Kinetics of the cholesterol, sulfuric acid reaction. A fast kinetic method for
serum cholesterol. Clin Chem Clin Biochem. 1974;12:403–8.

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