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ABSTRACT
The experiment was about extraction of total lipids from chicken egg yolk, column chromatography of lipids and
qualitative tests for lipids. Total lipids were extracted from the chicken egg yolk using 1M NaCl, isopropyl alcohol and
petroleum ether. The mixture was left standing for five minutes. The lower layer was collected and subjected into
column chromatography. The column used for column chromatography was packed with slurry of 0.5 g silica gel in 4
ml of petroleum ether with tapered end glass wool. For this experiment, the column was washed three times using
different eluents. The first eluent used was of 9:1 mixture of petroleum ether and ethyl ether, the second eluent was
5 ml 5% methanol in dichloromethane and the last eluent was 5ml CH 2Cl2: CH3OH: H2O (1:3:1). Eluates for each
eluent introduced into the column were collected in separate test tubes. Collected eluates were subjected for the
qualitative tests for lipids. 10 drops of eluates were subjected for each qualitative test. The qualitative tests performed
were as follows: test for ester, test for glycerol also known as acrolein test, test for phosphate, test for cholesterol
also known as Liebermann-Burchard test, test for Test for α- amino acids (Ninhydrin Test) and test for lipid
unsaturation with bromine. Test for esters yielded a yellow solution for the first and second eluate and a burgundy
solution for the third eluate. Test for glycerol produced no odor for the three eluates. Test for phosphate produced a
turbid solution for the first eluate, formed white/ slightly yellowish crystals for the second eluate and turbid yellow
with crystals for the third eluate. Test for cholesterol or Liebermann-Burchard test did not show any color change for
the first and third eluate while it produced a green color on the second eluate. Test for Ninhydrin formed a red orange
solution for the first eluate and no color change in the second and third eluates. Triglyceride or triacylglycerol,
cholesterol and phospholipid (Lecithin) were the first second and third eluates identified respectively. In the test for
lipid unsaturation with bromine, 10 drops in the first, second and third eluates, 22 drops in coconul oil, 71 drops in
canola oil, 90 drops in corn oil, 74 drops in olive oil and 8 drops in vegetable oil of 5% of Br 2 in CH2Cl were added
before reddish brown color persisted.
EXPERIMENTAL
The following were used for the test for
A. COMPOUNDS TESTED (SAMPLES glycerol (Acrolein Test): 10 drops of eluate
USED) and pinch amount of KHSO4.
1. Samples to be tested:
The following were used for the test for 2. Column Chromatography of Lipids
cholesterol (Lieberman-Burchard Test): 10
drops of eluate, 0.25 ml CHCl3, 6 drops of Small column was prepared by pouring
acetic anhydride and 2 drops of a slurry of 0.5 g silica gel in 4ml of
concentrated H2SO4. petroleum ether into the glass column
(Pasteur pipette) with a tapered end
plugged with glass wool. The lipid extract
from chicken egg yolk with a volume of 1 ml
8. Test for α- amino acids (Ninhydrin was then introduced into the column, saving
Test) the run-through in a clean test tube. The
column was washed with 5ml 9:1 mixture of
petroleum ether and ethyl ether, collecting
the eluate in the same tube as the run-
The following were used for the test α-
through. The column was again washed with
amino acids (Ninhydrin Test): 10 drops of
the second eluent (5 ml 5% methanol in
eluate and 5-8 drops of ninhydrin reagent.
dichloromethane) the eluate was then
collected in another clean test tube. The
column was washed with the last eluent,
9. Test for Lipid Unsaturation with 5ml CH2Cl2: CH3OH: H2O (1:3:1) and eluate
Bromine was collected in another test tube. The
different eluates culled were saved for
qualitative analysis.
The following were used for the lipid 3. Test for Ester
unsaturation test with bromine: 10 drops of
eluate, 3ml CH2Cl2, 5% Br2 in CH2Cl2, 8 drops
each of coconut, canola, corn and olive oil.
EtOH: 1-BuOH (3:1) with a volume of
0.5 ml was introduced into the 10 drops of
eluate. 2 drops each of 2M hydroxylamine
B. PROCEDURES: hydrochloride and 3M NaOH was
sequentially added and was mixed well. The
samples were allowed to stand for 5
minutes. 2 drops of 6M HCl was added with
1 drop of 5% FeCl3. 6 H2O in 0.1M HCl and
1. Extraction of Total Lipids from
was ensured to be well-mixed. Color was
Chicken Egg Yolk noted. Samples with positive results gave a
burgundy color.
The egg yolk was separated from the
chicken egg and its volume was
determined. It was then diluted with 5
volumes of 1M NaCl. After dilution, 2ml of 4. Test for Glycerol (Acrolein Test)
the diluted egg yolk was mixed with 3ml
isopropyl alcohol in a separate clean test A pinch amount of KHSO4 was added
tube. Petroleum ether with a volume of 2 ml to 10 drops of the eluate in the test tube.
was then added. It was covered with rubber Test tube was heated in a boiling water bath
stopper and was ensured to be well mixed. and odor produced was noted. Burned fat
The mixture was then allowed to stand for 5 odor was observed for positive test results.
tube. The solution was ensured to be well
mixed. 5% of Br2 were added drop wise
5. Test for Phosphate with CH2Cl2 into the test tube. Solution was
shaken after each addition until reddish
A volume of 0.5 ml of 10% Mg(NO3)2 brown color persisted. Number of drops was
with 6H2O in 95% EtOH was added with 10 noted upon the addition of 5% Br2 in CH2Cl2.
drops of eluate. The test tube was placed in Procedure was repeated and result was
a boiling water bath until the solvent was compared with the following: 8 drops each
evaporated and the Mg(NO3)2 was of coconut, canola, corn and olive oil.
decomposed. The test tube was removed
from the bath when the white residue was
formed and brown gas stopped evolving.
Table 1. Summarized Positive results for each Qualitative Test for Lipids
Glycerol
Phosphate
Liebermann
Ninhydrin
Ester
Chemical Test
Acid hydrolysis using sulphuric acid and Test for cholesterol (Lieberman-
water (equilibrium reaction). The ester splits into Burchard Test)
a carboxylic acid and alcohol, protons are
donated from the acid. The solution can then be
distilled and the remaining acid can be checked The Lieberman-Burchard or acetic
using UV indicator. anhydride test is used for the detection of
cholesterol. The formation of a green or green-
blue color after a few minutes is positive.
Positive results for the test for ester yields
a burgundy color. Based on Table 2, the first and
second eluate yielded yellow solution which is a Lieberman-Burchard is a reagent used in a
negative result for ester while the third eluate colorimetric test to detect cholesterol, which
gave a burgundy solution which is a positive gives a deep green color. This color begins as a
result and shows the presence of ester. purplish, pink color and progresses through to a
light green then very dark green color. The color
is due to the hydroxyl group (-OH) of cholesterol
Test for Glycerol (Acrolein Test) reacting with the reagents and increasing the
conjugation of the un-saturation in the adjacent bromine it absorbs. Based on the results that
fused ring. were culled (table 3), The order from the most
unsaturated to least unsaturated are as follows:
Based on the results that were culled Corn Oil, Olive Oil, Canola Oil, Coconut Oil, 1st 2nd
(Table 2), the first and third eluate did not and 3rd eluates (which are on the same level),
produce any change in color. The second eluate and finally Vegetable Oil.
produced a greenish color which indicated the
presence of cholesterol. Possible sources of errors for the
experiment were the use of incorrect or wrong
Test for α- amino acids Ninhydrin Test reagents and the lack of precision and accuracy
in measuring samples or reagents.
The principle involved in this test is
Oxidative deamination followed by REFERENCES:
decarboxylation. It is used to detect the presence
of α- amino acids. Positive result for this test is BOOKS:
Blue-violet solution.
Bettelheim,F.A., March,J. (1990). Introduction to
Based on the results that were culled organic and biochemistry. Philadelphia:
(Table 2), the first eluate produced a red orange Saunders College.
solution while the second and third eluate did not
produced any change in color. Heftman, E. (1967). Chromatography. New York:
Reinhold Publishing Corporation
The eluates identified based on the
chemical test performed are as follows: Lehninger, A.L. (2008). Legninger Principles of
Biochemistry. New York: W.H. Freeman.
1st eluate: triglyceride/triacylglycerol
McKee. (2003). Biochemistry: The Molecular
2nd
eluate: cholesterol Basis of Life. Boston: McGraw-Hill.
WEBSITES:
Canola oil
Corn oil
Olive oil
Vegetable oil
emistry/liptlc2.htm
3rd
1st
Column Chromatography
http://orgchem.colorado.edu/hndbksupport/
# of 10 10 10 22 71 90 74 8 colchrom/colchrom.html
drops of
bromine Retrieved: March 8, 2010
Lipid Library
Table 3 shows the actual results for lipid
unsaturation with bromine. The test for http://lipidlibrary.aocs.org/Lipids/whatlip/in
unsaturation with bromine identifies the level of dex.htm#def
saturation and the number of bonds an oil, fat or
lipid has. The more unsaturated, multi-bonded, Retrieved: March 8, 2010
the lipid is, the more it absorbs bromine. The less