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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.
INTRODUCTION
Lipids are substances found in living
organisms that are insoluble in water but soluble
in non polar solvents and solvents of low polarity.
This lack of solubility in water is an important
property because our body chemistry is so firmly
based on water. Most body constituents including
carbohydrates which are soluble in water. But the
body also needs insoluble compounds for many
purposes,
including
the
separation
of
compartments containing aqueous solutions from
each other, thats where lipids come in.
The water-insolubility of lipids is due to
the fact that the polar groups they contain are
much smaller than their alkane-like (nonpolar)
portions. These nonpolar portions provide the
water-repellent, or hydrophobic, property.
An important use for lipids, especially in
animals, is storage of energy. Plant store energy
in form of starch. Animals including
Oleic acid 47 %
Linoleic acid 16 %
Palmitoleic acid 5 %
Linolenic acid 2 %
Palmitic acid 23 %
Stearic acid 4 %
Myristic acid 1 %
EXPERIMENTAL
2. Extraction of total
chicken egg yolk
lipids
from
(Lieberman-
B.
PROCEDURES:
1. Extraction of Total Lipids from
Chicken Egg Yolk
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 the diluted egg
yolk was mixed with 3ml isopropyl alcohol in
a separate clean test tube. Petroleum ether
with a volume of 2 ml was then added. It
was covered with rubber stopper and was
ensured to be well mixed. The mixture was
then allowed to stand for 5 minutes until
two layers were formed. After 5 minutes,
the lower layer was collected and was
transferred to another clean test tube. Twodimensional thin layer chromatography
(TLC) and column chromatography (CC)
was performed using the lower layer.
Table 1. Summarized Positive results for each Qualitative Test for Lipids
Chemical Test
Positive Result
Ester
Burgundy color
Phosphate
Greenish color
Glycerol
Phosphate
Liebermann
Ninhydrin
1st
eluate
Yellow
solution
No
odor
Turbid solution
No color
change
Red orange
solution
2nd
eluate
Yellow
solution
No
odor
White crystals
slightly
yellowish
Green
No color
3rd eluate
Burgundy
solution
No
odor
Turbid yellow
with crystals
negative
No color
Chemical Test
Ester
nd
on
the
10
22
71
90
74
Vegetable oil
Olive oil
Corn oil
Canola oil
Coconut oil
3rd
1st
2nd
10
BOOKS:
Bettelheim,F.A., March,J. (1990). Introduction to
organic and biochemistry. Philadelphia:
Saunders College.
Heftman, E. (1967). Chromatography. New York:
Reinhold Publishing Corporation
WEBSITES:
10
REFERENCES:
eluate: cholesterol
# of
drops of
bromine
triglyceride/triacylglycerol
Eluate