Extraction of Total Lipids from Chicken Egg Yolk, Column

Chromatography and Qualitative Test for Lipids

Lawrence Saguros, Carmela Sales, Jeanel Samonte, Joseph Santiago, Reiniel Marie S. Sia
Group 7 2K- Medical Technology Biochemistry Laboratory

ABSTRACT
The experiment is to further know lipids, to test the lipid for further experiment; the lipid was extracted from the
chicken egg yolk using HCl. The mixture was then left standing for 5 minutes. The top layer was collected and
subjected to column chromatography. We used Pasteur pipette for column chromatography, it was packed with
dry silica gel and cotton at the bottom of it, the column was washed three times for this experiment. The first
eluent used was the extracted lipids from the chicken egg yolk, the second eluent is 5% CH3OH: DCM and
lastly the DCM: CH3OH: H2O (1:3:1). Eluates for each eluent were collected in separate test tube. Collected
eluates were subjected for the qualitative test for lipids. 5 to 10 drops of elautes were subjected for each
qualitative test.

INTRODUCTION Is a preparative technique used to purify compounds

Lipids are molecules that contain hydrocarbons and
make up the building blocks of the structure and
function of living cells. Lipids are not soluble in
water. They are non-polar and are thus soluble in
nonpolar environment. Lipids are highly reduced
forms of carbon. When metabolized, lipids are
oxidized to release large amounts of energy. They
can be extracted from plants and animals using non
polar solvents such as ether, chloroform and
acetone.
The primary role of lipids in our body is to provide
energy for muscles and body processes. Fat is
energy dense, containing 9 calories per gram,
whereas protein and carbohydrate contain only 4
calories per gram. About half of the fuel your body
needs when at rest or during everyday activity
comes from lipids. If you consume more calories
than you need in a day, the excess energy is stored
as lipids in adipose cells. In between meals and
during exercise your body relies on these fats stores
to provide energy. Lipids are also used to insulate
and protect your body.
Column Chromatography
depending on their polarity or hydrophobicity. In
column chromatography, a mixture of molecules is
separated based on their differentials partitioning
between a mobile phase and a stationary phase. The
different size column could be used for this
technique. The collective term for a set of
laboratory techniques for the separation of mixtures.
It involves passing a mixture dissolved in a "mobile
phase" through a stationary phase, which separates
the analyte to be measured from other molecules in
the mixture based on differential partitioning
between the mobile and stationary phases. Subtle
differences in a compound's partition coefficient
result in differential retention on the stationary
phase and thus changing the separation.
Chromatography may be preparative or analytical.
The purpose of preparative chromatography is to
separate the components of a mixture for further use
(and is thus a form of purification). Analytical
chromatography is done normally with smaller
amounts of material and is for measuring the
relative proportions of analytes in a mixture. The
two are not mutually exclusive. A pasteur pipette is
used as the column and usually 1g of adsorbent (ie
silica/alumina) is used to separate the mixture.

The objectives of the experiment, first to extract
lipids from chicken egg yolk, second to analyse the
lipid from the column chromatography extract and
lastly to identify the distinct lipids present using
qualitative test.
EXPERIMENTAL
I. Extraction of Total Lipids from Chicken
Egg Yolk
A. Materials / Samples Used
Chicken egg
Graduated Cylinder
Hexane: ethanol mixture
Beaker
Anhydrous NaSO4
Test tube
B. Procedure
1. Separate the egg yolk from the egg
white and measure the volume of the
egg yolk.
2. Add two volumes of the Hexane:
ethanol mixture (2:1 ratio)
3. Mix the solution thoroughly and let it
stand for 5 minutes. This will extract
the polar and neutral lipids.
4. Collect the lipid estract and transfer
it to a clean beaker.
5. Add anhydrous Na2SO4 to the lipid
extract to remove the residual water.
6. Decant the extract on a clean test
tube.
II. Column Chromatography of Lipids
A. Materials/ Samples Used
Egg Yolk Extract
Test tube
Dry silica gel
5% CH3OH: DCM
DCM: CH3OH: H2O
B. Procedure
1. Prepare the column by pouring dry
silica gel into the Pasteur pipette
with the tapered end plugged with
cotton.
2. Wash the column with 10 mL of the
petroleum ether.
3. Pour 1 mL of the lipid extract into
the column, saving the run-through
in a clean test tube.
4. Wash the column with 5 mL of the
petroleum ether: ethyl ether (9:1) and
collect the eluate in a clean test tube.
5. Wash the column with the second
eluent (5 mL of the 5% methanol in
dichloromethane) and collect the
eluate in the second test tube.
6. Finally, wash the column with the
last eluent, 5 mL of the DCM:
CH3OH: H2O (1:3:1) and collect the
eluate in the third test tube.
7. Save the eluates for qualitative
analyses.
III.Qualitative Test for Lipids
A. Materials/ Samples Used
B. Procedure
Test for Ester
1. Place 10 drops of eluate in separate test
tubes.
2. Add 0.5 mL of the ethanol: 1-butanol
(3:1).
3. Sequentially add 2 drops of each of the
2 M NH2OH HCl and 3 M Naoh.
4. Mix well and let the samples stand for 5
minutes.
5. Add 2 drops of the 6 M HCl and 1 drop
of the 5% FeCl3 6H2O in the 0.1 M HCl.
Mix well. The samples with ester will yurn
burgundy.
Test for Glycerol (Acrolein
Test)
1. Add a pinch amount of KHSO4 to 10
drops of the eluate in a test tube.
2. Gently heat the test tube in a Bunsen
burner, continuously shaking the test tube
from side to side. Note the odor produced. A
burn fat odor indicates the presence of
glycerol.
Test for Choline (Krauts Test)
1. Add 3 mL of Krauts reagent to 10 drops
of the eluate in a test tube.
2. Warm the test tube and observe for any
changes.
 Test for Phosphate (Ascorbic
Acid Method)
1. Add 3 mL of the 6 M HNO3 to 20 drops
of eluate in a test tube.
2. Boil the mixture in boiling water bath
for 5 minutes.
3. Add 3 mL of the 6 M NaOH to the test
tube containing the eluate and mix.
4. Get 2 mL of the neutralized solution
from the test tube and transfer it to another
clean test tube.
5. Add 3 mL of the molybdate reagent to
the neutralized solution. Mix.
6. Heat the mixture in a boiling water bath
for 5 minutes.
7. Cool the mixture to room temperature.
8. Add 10 drops of the ascorbic acid
solution (freshly prepared). Mix.
9. Let the solution stand for 20 minutes.
The purple colour of the mixture indicates
the presence of phosphate.
Test for Cholesterol
(Liebermann-Burchard Test)
1. Place 10 drops of eluate in a test tube.
2. Add 0.25 mL of the dichloromethane.
3. Add 6 drops of the acetic anhydride and
2 drops of the conc. H2SO4. A greenish
color produced after a few minutes indicate
the
Eluates Components
1st eluate Triacylglycerol
2nd
eluate Cholesterol
3rd eluate Lecithin
presence of cholesterol.
RESULTS AND DISCUSSION
Extraction of Total Lipids from Chicken Egg Yolk.
Lipids are soluble in organic solvents. The
extraction of lipids includes particular dissolvable
extraction and the beginning material might be
subjected to drying before extraction.
Dissolvability of lipids is an essential model for
their extraction from source material and
depends intensely on the sort of lipid present,
and the extent of nonpolar (chiefly
triacylglycerols) and polar lipids (basically
phospholipids and glycolipids) in the example; in
this manner, a few dissolvable frameworks may
be considered, contingent upon the kind of test
and its segment. The solvents of decision are
normally hexane, chloroform, methanol or
chloroform, methanol or water.
The basic lipids incorporate waxes, fats, and
oils. These compounds are basically like each
other in light of the fact that they comprise of
alcohols joined with long organic acids known as
unsaturated fats. Waxes are constructed of a
single molecule each of alcohol and acid while
fats and oils contain three fatty acid molecules for
each alcohol molecule. Fats are recognized from
oils in that the previous are solids and the latter,
fluids.
Lipoproteins are made out of proteins and lipid.
There are no less than four gatherings of
lipoproteins present in plasma: High-thickness
lipoproteins (HDL), low-thickness lipoproteins
(LDL), low thickness lipoproteins (VLDL), and
chlyomicrons. The diverse densities allude to the
relative measures of lipid and protein. The higher
the ratio, the higher the protein to lipid
proportion. LDLs transport cholesterol to cells and
deposit excess cholesterol in the blood vessels,
which increases the risk of arteriosclerosis. HDLs,
however, transport cholesterol from the tissues to
the liver where it is excreted, lowering the risk
of arteriosclerosis. A high HDL to total
cholesterol ratio is the best indication of
decreased risk of arteriosclerosis. HDL can be
influenced by such things as heredity, sex, age,
and physical activity. Smoking and obesity have
been shown to decrease plasma HDL levels.
The composition of chicken egg yolk makes up
about 33% of the liquid weight of the egg; it
contains approximately 60 calories, three times
the caloric content of the egg white.
Figure1. Components of Eluates
Chromatography of lipids using a glass column
filled with a suitable material is a common and
useful method for fractionation of lipid classes
either on an analytical or a semi-preparative
scale. The retention results in a variety of
mechanisms including hydrogen bonding, Van der
Waals' forces and also ionic bonding. The solid
phase is relatively polar (normal
chromatography) and the more polar the lipid,
the more strongly is it adsorbed. Thus, the lipids
are eluted by increasingly polar solvents. Lecithin
is the most polar among the three eluates, next
is cholesterol and last is triacylglycerol.
The first eluate was triacylglycerol or triglyceride.
They are an ester of three fatty acids and
glycerol. It is the most concentrated source of
energy in the human body and are stored in
subcutaneous fat deposits it contributes to
insulation. Triglycerides float on water, and solid
or liquid at normal room temperatures.
Independent studies of biosynthesis of fatty
acid and glycerol components of glycerolipids
exhibited that 3H-leucine was mainly consumed
in synthesis of glycerol moiety of phospholipids
and triacylglycerols, whereas 14C-acetate was
utilized in synthesis of fatty acids. Ethanol
activated most distinctly the synthesis of glycerol
moiety as compared with the synthesis of
triacylglycerol fatty acids. Ethanol activated more
effectively esterification of fatty acids with
formation of triacylglycerols as compared with
phospholipids. Incorporation of the label into
glycerol molecule occurred in response to
activation of glycero-glyconeogenesis by ethanol.
The second eluate was cholesterol. The eluent
was 5 mL 5% methanol in dichloromethane.
Cholesterol is naturally produced by the body
and is a combination of lipid (fat) and steroid. It
is a building block for cell membranes and for
hormones like estrogen and testosterone.
Majority of the body's cholesterol is produced by
the liver, while the rest comes from the food we
ate.
Cholesterol is only one of several lipids
circulating in our blood stream. Its components,
Triglycerides are an additional form of fat
circulating in the blood. Cholesterol and
Triglycerides cannot dissolve in water due to
being lipids, or fats. Because our blood is
comprised primarily of water, for Cholesterol and
Triglycerides to circulate through your blood, the
Cholesterol and Triglycerides must be carried by
protein packages.
The third eluate was lecithin. The eluent was 5
mL dichloromethane:methanol:water (1:3:1).
Lecithin is a lipid that consists mostly of choline,
and inositol, phosphorus, and linoleic acid.
Lecithin helps to prevent arteriosclerosis,
improves brain function, protects against
cardiovascular disease and many other benefits.
This nutrient is essential to every living cell in the
human body.
One of the different elements of lecithin is to
keep cholesterol in line. Its capacity to emulsify
oils and hold them in arrangement assumes a
noteworthy part in anticipating nerve stone
development. Together with bile and bile salts, it
contains the three noteworthy constituents of
bile. Bile is generally comprised of fats, which
lecithin keeps in fluid shape so as to keep irritate
stones from framing. Then again, cholesterol
holds a sensitive adjust with the bile salts. In the
event that the adjust is tipped on either side, the
outcome could stone arrangement. By holding
cholesterol in line, lecithin forestalls stone
development.
As a component of the enzyme lecithin
cholesterol acyl tranferase, the compound is said
to help in the metabolism of cholesterol to its by
products. As mentioned earlier, this substance is
also called phosphatidylcholine and is an
excellent source of choline. Much of the medical
benefits of lecithin, particularly on high
cholesterol-related conditions have been
attributed to the presence of choline.
We have concluded that the lipids extracted
from the chicken egg yolk are separated based
on its differences in solubility. Morover, it is
important to properly handle the reagents
properly to avoid contamination. Therefore, it is
necessary to keep track of the column
chromatography until the last drop drops.
REFERENCES
http://healthyeating.sfgate.com/lipids-used-
body-8282.html
http://www.utsc.utoronto.ca/webapps/chemistry
online/production/column.php
https://www.scribd.com/doc/29391261/Extractio
n-of-Total-Lipids-from-Chicken-Egg-Yolk-
Column-Chromatography-and-Qualitative-
Tests-for-Lipids