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EXTRACTION OF TOTAL LIPIDS FROM CHICKEN EGG YOLK, COLUMN

CHROMATOGRAPHY AND QUALITATIVE TESTS FOR LIPIDS

Maria Feliza C. Abesamis, Marie Em Clarisse P. Acosta, Francheska M. Agustin,


Mary Christelle G. Aquitania and Marilu Jane H. Bagsican
Group 1 2E Medical Technology Biochemistry Laboratory

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.

INTRODUCTION humans find it more economical to use lipids


(fats) instead. Although our bodies do store some
Lipids are substances found in living carbohydrates in the form of glycogen for quick
organisms that are insoluble in water but soluble energy when we need it, energy stored in the
in non polar solvents and solvents of low polarity. form of fats is much more important. The reason
This lack of solubility in water is an important is simply that the burning of fats produces more
property because our body chemistry is so firmly than twice as much energy as burning of an
based on water. Most body constituents including equal weight of carbohydrates.
carbohydrates which are soluble in water. But the
body also needs insoluble compounds for many Composition of Chicken Egg Yolk
purposes, including the separation of
compartments containing aqueous solutions from The yolk makes up about 33% of the
each other, that’s where lipids come in. liquid weight of the egg; it contains
approximately 60 calories, three times the caloric
The water-insolubility of lipids is due to content of the egg white.
the fact that the polar groups they contain are
much smaller than their alkane-like (nonpolar)
One large egg (50 grams in weight, 17
portions. These nonpolar portions provide the
gram yolk) contains approximately: 2.7g protein,
water-repellent, or hydrophobic, property.
210 mg cholesterol, 0.61g carbohydrates and
An important use for lipids, especially in 4.51g total fat. (USDA National Nutrient
animals, is storage of energy. Plant store energy Database)
in form of starch. Animals including
All of the fat soluble vitamins, (A, D, E
and K) are found in the egg yolk. Egg yolks are
one of the few foods naturally containing vitamin the adsorbent. Just as in TLC, there is an
D. equilibrium established between the solute
adsorbed on the silica gel or alumina and the
The composition (by weight) of the most eluting solvent flowing down through the column.
prevalent fatty acids in egg yolk is typically as
follows: Stationary phase (adsorbent)

Unsaturated fatty acids: The stationary phase or adsorbent in


column chromatography is a solid. The most
• Oleic acid 47 % common stationary phase for column
• Linoleic acid 16 % chromatography is silica gel, followed by
• Palmitoleic acid 5 % alumina. Cellulose powder has often been used in
• Linolenic acid 2 % the past. Also possible are ion exchange
chromatography,reversed- phase
Saturated fatty acids: chromatography (RP), affinity chromatography or
expanded bed adsorption (EBA). The stationary
phases are usually finely ground powders or gels
• Palmitic acid 23 %
and/or are microporous for an increased surface,
• Stearic acid 4 %
though in EBA a fluidized bed is used.
• Myristic acid 1 %

Egg yolk is a source of lecithin, an emulsifier


and surfactant. The yellow color is caused by
lutein and zeaxanthin, which are yellow or orange
carotenoids known as xanthophylls.

Figure 1. Chicken egg yolk

Column Chromatography Figure 2. Column Chromatography

Mobile phase (eluent)


Column chromatography is one of the
most useful methods for the separation and
purification of both solids and liquids when The mobile phase or eluent is either a
carrying out small-scale experiments. Column pure solvent or a mixture of different solvents. It
chromatography is another solid-liquid technique is chosen so that the retention factor value of the
in which the two phases are a solid (stationary compound of interest is roughly around 0.2 - 0.3
phase) and a liquid (moving phase). The theory in order to minimize the time and the amount of
of column chromatography is analogous to that of eluent to run the chromatography. The eluent
thin-layer chromatography. The most common has also been chosen so that the different
adsorbents - silica gel and alumina - are the compounds can be separated effectively. The
same ones used in TLC. The sample is dissolved eluent is optimized in small scale pretests, often
in a small quantity of solvent (the eluent) and using thin layer chromatography (TLC) with the
applied to the top of the column. The eluent, same stationary phase.
instead of rising by capillary action up a thin
layer, flows down through the column filled with
A faster flow rate of the eluent minimizes 2. Extraction of total lipids from
the time required to run a column and thereby chicken egg yolk
minimizes diffusion, resulting in a better
separation, see Van Deemter's equation. A simple
laboratory column runs by gravity flow. The flow
rate of such a column can be increased by The following were used for the
extending the fresh eluent filled column above Extraction of total lipids from chicken egg
the top of the stationary phase or decreased by yolk: Chicken egg yolk, 5 volumes of 1M
the tap controls. Better flow rates can be NaCl, 3ml isopropyl alcohol and 2ml
achieved by using a pump or by using petroleum ether.
compressed gas (e.g. air, nitrogen, or argon) to
push the solvent through the column (flash
column chromatography).
3. Column Chromatography of Lipids
The particle size of the stationary phase is
generally finer in flash column chromatography
than in gravity column chromatography. For The following were used for the column
example, one of the most widely used silica gel chromatography of lipids: extracted total
grades in the former technique is mesh 230 – lipids from chicken egg yolk, 0.5 g silica gel,
400 (40 – 63 µm), while the latter technique 4ml of petroleum ether, 5ml of 9:1 mixture
typically requires mesh 70 – 230 (63 – 200 µm) of petroleum ether and ethyl ether, 5 ml
silica gel. 5% methanol in dichloromethane, and 5ml
CH2Cl2: CH3OH: H2O (1:3:1).
A spreadsheet that assists in the
successful development of flash columns has
been developed. The spreadsheet estimates the
retention volume and band volume of analytes,
the fraction numbers expected to contain each
4. Test for Ester
analyte, and the resolution between adjacent
peaks. This information allows users to select
optimal parameters for preparative-scale
separations before the flash column itself is The following were used for the test for
attempted. ester: EtOH: 0.5 ml of 1-BuOH (3:1) with
10 drops of eluate, EtOH: 0.5 ml of 1-BuOH
The objectives of the experiment are as (3:1), 2 drops each of 2M hydroxylamine
follows: (1) to extract total lipids from chicken hydrochloride and 3M NaOH, 2 drops of 6M
egg yolk, (2) to analyze the lipids present in the HCl, 1 drop of 5% FeCl3. 6 H2O in 0.1M HCl.
crude extract using column chromatography (3)
to identify lipids present in each of the fractions
using qualitative tests and, (4) to determine the
degree of unsaturation of lipids by bromine test. 5. Test for Glycerol (Acrolein Test)

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 samples were subjected 6. Test for Phosphate


to test using column chromatography and
qualitative test for lipids: Chicken egg yolk,
Coconut oil, Canola oil, Corn oil, Olive Oil,
Vegetable oil, Cholesterol and Lecithin. The following were used for the test for
phosphate: 10 drops of eluate, 0.5 ml of
10% Mg(NO3)2, 6H2O in 95% EtOH,
Mg(NO3)2, 0.5 ml of 2M HCL, 0.5ml of 6% minutes until two layers were formed. After
(NH4)2MoO4 and 4M HNO3. 5 minutes, the lower layer was collected
and was transferred to another clean test
tube. Two-dimensional thin layer
chromatography (TLC) and column
7. Test for Cholesterol (Lieberman- chromatography (CC) was performed using
Burchard Test) the lower layer.

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.

2M HCL with a volume of 0.5ml was


added and was mixed to dissolve the solid
residue. Test tube was then heated in a
boiling water bath for 5 minutes. 0.5ml of
6% (NH4)2MoO4 was added with 4M HNO3.
Color change was noted. Positive results for
this test were formation of a yellow color
followed by formation of a fine yellow
precipitate. This indicated the presence of
phosphate.

6. Test for Cholesterol (Lieberman-


Burchard Test)

A volume of 0.25 ml CHCl3 was


introduced into the 10 drops of eluate. 6
drops of acetic anhydride was then added
with 2 drops of concentrated H2SO4 and well
mixed. Color was noted. Positive results
produced a greenish color which indicated
the presence of cholesterol.

7. Test for α- amino acids (Ninhydrin


Test)

In a clean test tube, 5-8 drops of


ninhydrin reagent was introduced into the
10 drops of eluate. It was subjected into
heat in the boiling water bath for 15-20
secs. The color produced was observed.
Positive results yielded a formation of blue
violet color which indicated the presence of
α- amino acids.

8. Test for Lipid Unsaturation with


Bromine

A volume of 3ml CH2Cl2 was added into


10 drops of eluate was placed in a test
RESULTS AND DISCUSSION:

Table 1. Summarized Positive results for each Qualitative Test for Lipids

Chemical Test Positive Result

Ester Burgundy color

Glycerol (Acrolein Test) Burned fat odor

Phosphate Fine yellow precipitate

Cholesterol (Liebermann- Greenish color


Burchard Test)

α- amino acids (Ninhydrin Blue violet color


Test)
Table 2. Actual Results for each Qualitative Test for Lipids:

Glycerol

Phosphate

Liebermann

Ninhydrin
Ester
Chemical Test

1st Yellow No Turbid solution No color Red orange


eluate solution odor change solution

2nd Yellow No White crystals Green No color


eluate solution odor slightly
yellowish

3rd eluate Burgundy No Turbid yellow negative No color


solution odor with crystals
Table 1 shows the positive results for each Acrolein test is a test for the presence of
qualitative test performed on lipids while Table 2 glycerin or fats. A sample is heated with
shows the actual results for each qualitative potassium bisulfate, and acrolein is released if
the test is positive. When a fat is heated strongly
performed in lipids. The principles or mechanisms
in the presence of a dehydrating agent such as
behind each qualitative test are as follows: KHSO4, the glycerol portion of the molecule is
dehydrated to form the unsaturated aldehyde,
Test for Ester acrolein (CH2=CH-CHO), which has the peculiar
odor of burnt grease.
Esterification is the general name for a
chemical reaction in which two reactants Based on the results that were culled
(typically an alcohol and an acid) form an ester (Table 2), the first second and third eluate did
as the reaction product. Esters are common in not produce any odor hence indicates the
organic chemistry and biological materials, and absence of glycerol for each eluates.
often have a characteristic pleasant, fruity odor.
This leads to their extensive use in the fragrance
Test for Phosphate
and flavor industry. Ester bonds are also found in
The presence of free phosphate in acidic
many polymers.
solution can be detected by adding a molybdate
to the solution. The equation below illustrates the
Esterification is a reversible reaction. pertinent reaction between phosphate and
Hydrolysis—literally "water splitting"—involves ammonium molybdate solution in the presence of
adding water and a catalyst (commonly NaOH) to nitric acid (HNO3).
an ester to get the sodium salt of the carboxylic
acid and alcohol. As a result of this reversibility, HPO42-(aq) + 12 MoO42-(aq) + 3 NH4+(aq) + 23
many esterification reactions are equilibrium H3O+(aq) -> (NH4)3[P(Mo3O10)4] (yellow)+35
reactions and therefore need to be driven to H2O(l)
completion according to Le Chatelier's principle.
Esterifications are among the simplest and most Yellow precipitate results from the
often performed organic transformations. reaction in the mixture. When lipids containing
phosphate groups in their structures are added to
The most common esterification processes strong acid solution such as the solution used,
involve nucleophilic acyl substitution where the the lipid hydrolyses, producing free phosphate,
carbonyl compound is used as an electrophile and forming a yellow precipitate.
is attacked by a nucleophilic alcohol. However,
Based on the results that were culled
other processes are possible; esterification by
alkylation reverses the roles of "classic" carbonyl (Table 2), the first eluate produced a turbid
chemistry: a carboxylate anion is used as a solution, the second eluate produced white
nucleophile that displaces a halide ion in an SN2 crystals which is slightly yellowish and the third
reaction. eluate produced turbid yellow crystals.

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.

3rd eluate: phospholipid (Lecithin)

WEBSITES:

Table 3 Actual Results for Lipid


Unsaturation with Bromine: Analysis of Lipids Egg Yolk and Milk
http://faculty.mansfield.edu/bganong/bioch
Eluate
Coconut oil

Canola oil

Corn oil

Olive oil

Vegetable oil

emistry/liptlc2.htm

Retrieved: March 8, 2010


2nd

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