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Camaclang, Rosa Magenta

Comia Jasmin
Contreras Charles
De Leon Angelica Luz
*Dela Cruz Kizer
Group 2 2BPH

Lipid molecules include fats, waxes, and fat-soluble vitamins such as A, D, E and K. A column
chromatographic procedure utilizing silica gel is described for separating lipid components of serum
and lipoproteins into individual fractions containing hydrocarbons, cholesterol esters, triglycerides,
cholesterol, free fatty acids and phospholipids. Egg-yolk lecithin has phospholipid classes and
compositions that differ from soybean lecithin and may have unique functional properties. This
experiment determined the components of each eluents. Lipids were based upon their polarity using
column chromatography. The extracted lipids from chicken egg yolk were used in the column
chromatography. The eluents used were 9:1 mixture of petroleum ether:ethyl ether, 5% methanol in
dichloromethane and dichloromethane:methanol:water (1:3:1). The results obtained were analyzed
and it showed that the lipids are eluted by increasingly polar solvents. The lipids present in the crude
extract were triacylglycerol, cholesterol and lecithin. The aim of this experiment is to understand and
to determine the amounts of lipid components in chicken egg yolk. In the end of this experiment we
had founded which lipid component in chicken egg yolk is the most polar among all using column

INTRODUCTION much more important. The reason is

Lipids are substances found in simply that the burning of fats produces
living organisms that are insoluble in more energy than burning of an equal
water but soluble in non polar solvents weight of carbohydrates.
and solvents of low polarity. This lack of
solubility in water is an important property In the experiment, the lipids
because our body chemistry is so firmly present in chicken egg yolk were isolated.
based on water. Most body constituents The egg yolk makes up about 33% of the
including carbohydrates which are soluble liquid weight of the egg and it contains
in water. But the body also needs insoluble approximately 60 calories, three times the
compounds for many purposes, including caloric content of the egg white. All of the
the separation of compartments fat-soluble vitamins (A, D, E, and K) are
containing aqueous solutions from each found in the egg yolk. Egg yolk is one of
other, thats where lipids come in. The the few foods naturally containing vitamin
water-insolubility of lipids is due to the fact D.
that the polar groups they contain are
much smaller than their alkane-like Egg yolk is a source of lecithin. Its
(nonpolar) portions. These non polar yellow color is due
portions provide the water-repellent, or to lutein and zeaxanthin, which are the
hydrophobic, property. An important use yellow or orange carotenoids also known
for lipids, especially in animals, is storage as xanthophylls.
of energy. Plant stores energy in form of
starch. Animals including humans find it The general approach is to extract
more economical to use lipids (fats) total lipids from egg yolk using methanol
instead. Although our bodies do store and chloroform, then separate the lipid
some carbohydrates in the form of fractions, triglyceride, cholesterol, and
glycogen for quick energy when we need phospholipids, by chromatography on
it, energy stored in the form of fats is silica gel. This experiment will investigate
the properties of lipids present in chicken
egg yolks, the lipid components present in The samples were allowed to stand for 5
the crude extract using column minutes. 2 drops of 6M HCl was added
chromatography of the extracted lipids with 1 drop of 5% FeCl3. 6 H2O in 0.1M
from chicken egg yolk. HCl and

The objectives of the experiment II. Test for Glycerol (Acrolein Test)
are as follows: (1) to extract total lipids
from chicken egg yolk, (2) to analyze the A pinch amount of KHSO4 was
lipids present in the crude extract using added to 10 drops of the eluate in the test
column chromatography (3) to identify tube. Test tube was heated in a boiling
lipids present in each of the fractions water bath and odor produced was noted.
using qualitative tests and, (4) to Burned fat odor was observed for positive
determine the degree of unsaturation of test results.
lipids by bromine test.
METHODOLOGY III. Test for Glycerol (Krauts Test)
A. Extraction of Total Lipids from
Chicken Egg Yolk Unfortunately the reagent was not
available so this test was not performed.
The materials we have used were But the steps in performing this specific
test tubes, beaker, stirring rod, Pasteur test were also discussed. First, 3mL of
pipette, hot plate, iron stand and iron Krauts reagent is to be added to 10 drops
clamp. We started the procedures by of the eluate in a test tube. The test tube
extracting total lipids from chicken egg must be warmed before observing any
yolk. We added an equal amount of changes in the appearance of the sample.
ethanol to the egg yolk to increase the
polarity of the organic solvent, and mixed IV. Test for Cholesterol (Liebermann-
it to dehydrate and partially extract the Burchard Test)
polar lipids. We added hexane and then
mixed it again and we had let it stood for Ten drops of eluate was placed in a
5 minutes, until two layers were formed, test tube and 0.25mL of dichloromethane
the fractions of polar and neutral lipids. was added. Six drops of acetic anhydride
We removed the upper polar fraction and and 2 drops of concentrated H2SO4 was
added an equal amount of acetone to added and mixed with the sample. A
further precipitate the polar lipids from greenish color produced after a few
residual neutral ones, especially the minutes indicated the presence of
cholesterol. . When the upper layer was
collected, it was transferred into one V. Test for Lipid Unsaturation with I2
clean test tube and it was used in This portion of the experiment was
Thin-Layer Chromatography and slightly modified. Supposedly, Br2 will be
Column Chromatography. The eluates used instead of I2. But the principle
were used for the qualitative tests for involved here is the same, the only
lipids difference was the reagent used.

B. Qualitative Tests for Lipids Ten drops of each eluate was placed in
different test tubes. Aside from the three
eluates, 10 drops of Canola oil was also
I. Test for Ester used. I2 was added dropwise in every test
tube until a desired color was achieved. In
EtOH: 1-BuOH (3:1) with a volume this case, it should be light yellow or
of colorless. The color of each sample should
0.5 ml was introduced into the 10 drops of be the same, if not; the colors should be
eluate. 2 drops each of 2M hydroxylamine close enough to each other
Hydrochloride and 3M NaOH was
sequentially added and was mixed well. RESULTS AND DISCUSSIONS
used as a nucleophile that displaces a
Table 1: Actual Results of halide ion in an SN2 reaction.
Qualitative Tests for Lipids
With Acid hydrolysis using sulphuric
acid and water (equilibrium reaction), the
Chemic Ester Glycerol Lieberman
ester splits into a carboxylic acid and
al n-Burchard alcohol, protons are donated from the
Test acid. The solution can be then distilled and
1st Yellow No odor Blue green the remaining acid can be checked using
eluate solution solution UV indicator. Positive results for the test
2nd Yellow No odor Green for ester yields a burgundy color. Based on
eluate solution solution Table 1, the first and second eluate yielded
3 rd
Burgundy Burnt fat Colorless yellow solution which is a negative result
for ester while the third eluate gave a
eluate color odor solution
burgundy solution which is a positive
result and shows the presence of ester.
Table 1 shows the actual results for each
qualitative performed in lipids. The Test for Glycerol (Acrolein Test)
principles or mechanisms behind each
qualitative test are as follows: Acrolein test is a test for the
presence of glycerin or fats. A sample is
Test for Ester heated with potassium bisulfate, and
acrolein is released if the test is positive.
Esterification is the general name for a When a fat is heated strongly in the
chemical reaction in which two reactants presence of a dehydrating agent such as
(typically an alcohol and an acid) form an KHSO4, the glycerol portion of the
ester as the reaction product. molecule is dehydrated to form the
unsaturated aldehyde, acrolein (CH2=CH-
Esters are common in organic CHO), which has the peculiar odor of burnt
chemistry and biological materials, and grease.
often have a characteristic pleasant, fruity
odor. This leads to their extensive use in Based on the results that were
the fragrance and flavor industry. Ester culled (Table 1), the first second and third
bonds are also found in many polymers. eluate did not produce any odor hence
indicates the absence of glycerol for each
Esterification is a reversible eluates.
reaction. Hydrolysisliterally "water
splitting"involves adding water and a Test for cholesterol (Lieberman-
catalyst (commonly NaOH) to an ester to Burchard Test)
get the sodium salt of the carboxylic acid
and alcohol. As a result of this
The Lieberman-Burchard or acetic
reversibility, many esterification reactions
anhydride test is used for the detection of
are equilibrium reactions and therefore
cholesterol. The formation of a green or
need to be driven to completion according
greenblue color after a few minutes is
to Le Chatelier's principle. Esterifications
positive. Lieberman-Burchard is a reagent
are among the simplest and most often
used in a colorimetric test to detect
performed organic transformations.
cholesterol, which gives a deep green
color. This color begins as a purplish, pink
The most common esterification
color and progresses through to a light
processes involve nucleophilic acyl
green then very dark green color. The
substitution where the carbonyl compound
color is due to the hydroxyl group (-OH) of
is used as an electrophile and is attacked
cholesterol reacting with the reagents and
by a nucleophilic alcohol. However, other
increasing the conjugation of the un-
processes are possible; esterification by
saturation in the adjacent fused ring.
alkylation reverses the roles of "classic"
carbonyl chemistry: a carboxylate anion is
Based on the results that were FROM BOOKS:
culled (Table 1), the first and third eluate
did not produce any change in color. The Barreto , M.C. (2005). Lipid extraction and
second eluate produced a greenish color cholesterol quantification:A simple
which indicated the presence of protocol. Journal of Chemical Education
cholesterol. 82(1), 103-104.

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