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IMBAO, Ma. Rio, JAMORABON, Jasmine Mary A., LAI, Kurt Raven T., LIM, Beatrice Andrea G.,
LIWAG, Danvel C.
Group 5 2C Biochemistry General Biochemistry Laboratory
ABSTRACT
The experiment was done to identify the characteristics of lipids through qualitative
analysis. Lipids are molecules that contain hydrocarbons and make up the building blocks of
the structure and function of living cells. Examples of lipids include fats, oils, waxes, certain
vitamins, hormones and most of the non-protein membrane of cells. To attain the objectives
of the experiment, total lipids were extracted from chicken egg yolk using ethanol and
hexane. Two-Dimensional Thin-Layer Chromatography Analysis was performed for further
separation of liquids through adsorption using mixtures of organic solvents in varying
proportion. These mixtures separate phospholipids based on the head group polarity and
charge. The extracted polar lipids were then subjected to different qualitative tests, which
were Test for Ester, Acrolein Test, Krauts Test, Liebermann-Burchard Test, and Test for Lipid
Unsaturation with Bromine. Lipids are determined by isolation and the ability to purify the
substance. Due to the way the substances interact with the matrix in different ways we are
able to separate them. Substances which interact strongly with the matrix but not with the
solvent will move very slowly and those soluble in the solvent will dissolve easily and be
carried along the solvent.
INTRODUCTION
Lipids are large and diverse group of important use for lipids, especially in
naturally occurring organic compounds animals, is storage of energy. Plant stores
that are related by their solubility in energy in form of starch. Animals
nonpolar organic solvents. Lipids have including humans find it more economical
mainly hydrocarbons in their composition to use lipids (fats) instead. Although our
and are highly reduced forms of carbon. bodies do store some carbohydrates in the
When metabolized, lipids are oxidized to form of glycogen for quick energy when
release large amounts of energy and thus we need it, energy stored in the form of
are useful to living organisms. Lipids are fats is much more important. The reason
not soluble in water. They are non-polar is simply that the burning of fats produces
and are thus soluble in nonpolar more energy than burning of an equal
environments like in choloroform but not weight of carbohydrates.
soluble in polar environments like water.
A particularly frequent approach is to
Lipids are often subsequently separated
obtain information on the various
into groups through the use of
phospholipid components of the lipid
chromatography. Thin layer
extract under investigation. This can be
chromatography has traditionally been the
achieved easily and efficiently by
chromatography of choice.
subjecting a sample to thin-layer
Organic solvents are employed to chromatography (TLC). With TLC in the
separate lipids from proteins, adsorption mode, the principal application
carbohydrates and water soluble in lipid analysis is for the separation of
metabolites. The lipid composition of cells different lipid classes from animal and
and membranes can differ significantly plant tissues.
with respect to lipid group. These nonpolar
portions provide the water-repellent, or
hydrophobic, property. An The objectives of the experiment are as
follows: (1) To extract total lipids from
egg yolk , (2) To analyze lipids present in One (1) mL of the sample solution
the crude extract , (3) TLC, (4) to (starch) in a test tube was added with 5 drops of
determine the degree of unsaturation of Molisch's reagent (5% -naphthol in 95%
lipids by Bromine test.
ethanol). Then, two (2) mL of concentrated
H2S04 was carefully poured down the side of the
EXPERIMENTAL tube until a layer is formed. The color at the
junction of the two liquids was observed.
A. Samples used
3. I2 Reaction
The egg yolk was separated from then
chicken egg and its volume was A few drops of 0.01M I2 was added to
determined. It was then diluted with 2 the solution. The mixture was warmed in a water
volumes of the hexane: ethanol mixture, bath and any change in color was observed.
anhydrous Na2SO4. Subsequently, the mixture was cooled, and any
change was also observed.
For the Two-Dimensional Thin-Layer
Chromatography Analysis of Lipid the 4. Enzymatic Hydrolysis
following compounds were used: 65: 25:
4 (v/v/v) petroleum ether: methanol: The 10 mL portion of the isolated
water and 65: 25: 4 (v/v/v) petroleum carbohydrate was placed in a beaker. 2.3 mL of
ether: methanol: ammonium hydroxide. saliva was added which was prepared by rinsing
the mouth with warm water for a minute and
In the Qualitative Tests for Lipids the collecting the washings in a beaker. The solution
following compounds were also used: was allowed to stand for 30 minutes and the any
ethanol, 2M NH2OH, HCl, 3M NaOH, changes in its viscosity were noted.
FeCl3, KHSO4, dicholoromethane and Br2.
The solution was introduced into a
B. Procedure dialyzing bag and the bag was suspended
overnight in a small flask filled with 50 mL distilled
1. Extraction of Total Lipids water. The solution was removed and the
dialyzing bag was discarded.
from Chicken Egg Yolk.
The materials we have used were The solution was concentrated inside the
testtubes, beaker, stirring rod, Pasteur flask using an open flame to a volume of 10 mL.
pipette, hotplate, iron stand and iron The presence of reducing sugars in the
clamp. We started theprocedures by hydrolysate was checked by performing the
extracting total lipids from chickenegg Benedict's test.
yolk. We added an equal amount of
ethanol tothe egg yolk to increase the 4. Thin Layer Chromatography
polarity of the organicsolvent, and mixed
it to dehydrate and partiallyextract the The solvent system with the amount of
polar lipids. We added hexane and 40 mL was placed in a developing chamber, was
thenmixed it again and we had let it stood covered with an inverted watch glass, and was
for 5 minutes,until two layers were equilibrated for 10 minutes. A pencil line across
formed, the fractions of polarand neutral one end of the TLC plate was lightly drawn, about
lipids. We removed the upper polarfraction 2 cm from the bottom. Equidistant points along
and added an equal amount of acetone the origin were marked for the standards and
tofurther precipitate the polar lipids from enzymatic hydrolysates. The standards were
residualneutral ones, especially the applied five times and the hydrolysates ten times
cholesterol. using the capillary tube and was dried after every
application.
2. Molisch's Test
The TLC plate was placed inside the colored complexes with I2. The partially-
developing chamber and was covered and hydrolyzed starch, glycogen forms red-brown or
allowed to be developed until the solvent is about brown-blue colored complex. [7]
1cm from the top of the TLC plate. After
development, the chromatoplate was removed Molisch's test is a sensitive, general test
and the solvent front was marked with a pencil. for all carbohydrates. It detects or confirms the
presence of a carbohydrate in a given solution.
The chromatoplate was air-dried and The principle involved is that carbohydrates form
sprayed with the visualizing agent. It was placed furfural derivatives (pentoses) upon
inside the oven and was heated at 100 - dehydration(or 5-hydroxymethylfurfural for
hexoses) by H2SO4, which form colored
150 for 10 minutes. The colored spots compounds when condensed with ethanolic
were encircled with a pencil and the Rf values of
-naphthol (Molisch's reagent).
each spot on the chromatoplate was computed.
CONCLUSION