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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 - 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 Br2 in CH2Cl were added before reddish brown color persisted.
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, 1 st 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 - 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 - 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.
Canola oil
Corn oil
Olive oil
Vegetable oil
colchrom/colchrom.html
Retrieved: March 8, 2010
3rd
2nd
1st
Lipid Library
http://lipidlibrary.aocs.org/Lipids/whatlip/in
dex.htm#def
# of 10 10 10 22 71 90 74 8 Retrieved: March 8, 2010
drops of
bromine