Extraction, Fractionation and Identification of

Brain Lipids
OBJECTIVES
Discuss the pig's brain lipid extraction
Explain how triglycerides are isolated and saponified
Define principle behind the tests performed on triglyceride
Explain how lecithin and cholesterol are isolated
Describe the principle behind the tests performed on lecithin
Define the principle behind the tests performed on cholesterol

LIPIDS
Lipids are one of the essential biomolecules needed by the body.
composed of heterogeneous group which includes fats, oils, steroids, waxes, and
phospholipids and sphingolipids
fraction of any biological materials that is insoluble in water but soluble in one or more
solvents of low polarity
PRIMARY FUNCTIONS OF LIPIDS
source of energy
component of cellular structure especially the cell membrane
Adipose tissue cushions and protects internal organs such as the heart and kidney
Procedure:
30 grams of pigs brain
(homogenized in ether-alcohol solution)
TOTAL LIPID EXTRACT


2/3 portion 1/3 portion
Isolation of Triglycerides Lecithin-Cholesterol Isolation



20 ml portion 30 ml portion
Test on Soaps A. Test on Fatty Acids A. Test on Lecithin
1. Salting Out 1. Solubility 1. Acrolein
2. Insoluble Salts 2. Translucent Spot 2. Iodine Absorption
3. Surface Tension 3. Iodine Absorption 3. Phosphorus

B. Test on Glycerol B. Test on Cholesterol
1. Solubility 1. Liebermann-
2. Translucent Spot Burchard Test
3. Acrolein 2. Salkowski Test
4. Benedicts Test
30 ml Portion
Test on Soap

Salting Out Insoluble Soaps Surface Tension
5 ml of 5 ml 5 ml 5 ml 5 ml Soap 5 ml Solution
Soap Solution Distilled Water Solution 2% NaCl








Add Add Add Add
NaCl CaCl
2
MgSO
4
Sulfur Powder




Precipitate Precipitate Precipitate




Add Add Add Observe
Water Water Water






Observe Observe Observe
Saponification - A chemical reaction in which an ester is heated with an alkali to make soap
Soap - is a salt of compound fatty acid



INTERMOLECULAR FORCES
Hydrogen Bond
Ionic Bond
Covalent Bond

TESTS ON SOAP
Salting out
Insoluble salts
Surface Tension Test

SALTING OUT
Purification method that utilizes reduced solubility of molecules in solution of very high
ionic strength
Soaps are easily precipitated by concentrated salt solution
It is an effect based on the electrolyte to non-electrolyte interaction
Test Obtained Result Expected Result
Salting Out The soap has precipitated and
dissolved
White precipitate which
dissolves in water



INSOLUBLE SOAPS
They are formed when calcium or magnesium react with higher fatty acids of soaps
Form an insoluble white curd like precipitate of magnesium or calcium salts


Test Obtained Result Expected Result
Insoluble Soaps

CaCl
2
and MgSO
4
formed
white precipitates and upon
contact with water, they did
not dissolve

The soaps have precipitated
and are insoluble

Surface Tension
the force with which the surface molecules attract each other.
Water is known to have high surface tension due to the hydrogen bonds present
Test Obtained Result Expected Result
Surface Tension Test
a. In distilled water

The sulfur powder floated

Floats

b. Soap Solution The sulfur powder slowly
descended

Descends
c. 2% NaCl The sulfur powder floats Floats

SIGNIFICANCE OF SURFACE TENSION

Bile salts
Bile salts lowers surface tension of fat droplets

Hays test
based on the surface tension
employed for detecting the presence of bile salts in urine
(+) if sulfur powder sprinkled settles down
Indication for Jaundice

DEFINITION OF FATTY ACID AND GLYCEROL

Fatty acid - consists of long carbon chain with a carboxylic at one end
short chain saturated, long chain saturated, unsaturated

Glycerol - three hydroxyl groups that are responsible for its solubility in water
glycerol backbone is central to all lipids known as triglycerides
Fatty Acid Glycerol
Solubility

Solubility

Translucent spot test Translucent spot test
Iodine Absorption Test
Acrolein Test
Benedicts Test

SOLUBILITY of FATTY ACID
Fatty acids have non polar properties, they dissolve easily in a non polar solvent.
They are insoluble in water because they are composed primarily of long chains of
hydrocarbons.




Short-chain saturated
fatty acids
Long-chain saturated
fatty acids
Unsaturated fatty
acids
Solubility in Water Limited Solubility Insoluble Insoluble


SOLUBILITY OF GLYCEROL
Glycerol contains three hydroxyl or alcohol group making it soluble in water.
Hydroxyl or alcohol groups are hydrophilic and are responsible the solubility of glycerol
in water.

TRANSLUCENT SPOT TEST in FATTY ACID and GLYCEROL
The test is used to determine whether or not the lipid contains a glycerol or sphingosine.
Spot of grease or fat cannnot absorb enough heat to evaporate, so when the liquid is
inside other sheet of paper, it diffracts light.

TRANSLUCENT SPOT TEST in FATTY ACID
The volatility of fatty acids will also depend whether the fatty acids are saturated or
unsaturated and if saturated whether it is a short-chain, medium-chain or long-chain fatty
acids.
Test Obtained Result Expected Result
Transluscent Spot Test on
Fatty Acid
There is a transluscent spot
(yellowish) that remains
There is a presence of
translucent spot and it
remains even after long
standing.
Transluscent Spot Test on
Glycerol
Presence of long standing
transluscent spot
There is a presence of
transluscent spot that
evaporates

IODINE ABSORPTION TEST of FATTY ACID
To test the presence of unsaturated fatty acids
The fatty acid residues may differ in chain length and be distinguished by the difference
in intensity of color due to the absorption of iodine.
Elemental iodine dissolves easily in most organic solvents.

IODINE ABSORPTION TEST
Halogens ( I, Br ) will add across the double bonds and thus the decolorization of an
iodine or bromine solution will indicate the presence of unsaturated fatty acids.

Test Obtained Result Expected Result
Iodine Absorption Test Iodine solution
decolorized
(clear pink)
Decolorized


ACROLEIN TEST on GLYCEROL
This reaction is utilized to determine the presence of glycerin in fat.
Heating lipids with Potassium bisulphate will result to the formation of Acrolein.
When glycerol is heated with Potassium bisulphate or KHSO4, dehydration occurs and
aldehyde Acrolein formed which has a characteristic pungent odor.








Test Obtained Result Expected Result
Acrolein test on Glycerol Burned fat odor Produces pungent odor

BENEDICTS TEST on GLYCEROL
The Benedict's test provides quantitative result for reducing sugars.
The reagent contains blue copper (III) ions which are reduced to copper (I) ions. These
are precipitated as red copper (I) oxide which is insoluble in water.
Change in color indicates that the solution is POSITIVE for reducing sugar.
Test Obtained Result Expected Result
Benedict's test on Glycerol Observable color change
from blue to brick red upon
standing
Presence of brick red color

COMPARISON OF RESULTS ON FATTY ACIDS AND GLYCEROL
Fatty Acid Glycerol
Solubility In water - Insoluble
In CHCl3 -
Soluble

Solubility

In water - Soluble
In CHCl3 - Insoluble

Transluscent Spot
Test

Transluscent Spot
that remains even
after long standing

Transluscent
Spot Test

Transluscent Spot that
evaporates

Iodine Absorption
Test

Iodine Solution
decolorized from
orange brown clear
pink
Not applicable

Iodine Absorption Tesy Acrolein Test Produces a pungent odor
or burned fat
Not Applicable

Benedict's Test

presence of brick red
color


CH2 OH CH2

CH OH CH + 2H2O

CH2 OH CHO

Glycerol Acrolein
Heat

KHSO4

LECITHIN
Functions of Lecithin
A dietary precursor of choline
Makes up most of the composition of the lipid bilayer of the cell membrane.
Most abundant lipid in lung surfactant

TESTS ON LECITHIN
Acrolein Test
Iodine Absorption Test
Test on Phosphorus

ACROLEIN TEST
Purpose:
used to test for the presence of glycerol
Principle:
Glycerol decomposes slowly at a boiling temperature.
When a fat is heated strongly in the presence of a dehydrating agent such as potasium
bisulfate (KHSO4). The glycerol portion of the molecule is dehydrated to form the
aldehyde Acrolein
Acrolein, an aldehyde is easily recognized by its penetrating and irritating odor.

Test Obtained Result Expected Result
Acrolein test on Glycerol Burned fat odor Produces pungent odor

IODINE ABSORPTION
Purpose:
test for unsaturated fatty acids that would cause decolorization of the solution.
Principle:
Halogens such as Iodine will interact with the double bonds of the unsaturated fatty acids
thus becoming saturated
a change in color will indicate that it contains unsaturated fatty acids which reacted to
iodine through halogenation reaction.

TEST FOR PHOSPHORUS

Phosphatidyl choline + ammonium molybate = ammonium phophomolybdate
Lecithin is a phosphlipid, when it reacts with ammonium molybdate under concentrated
Nitric Acid (HNO3) the positive result of yellow crystals is expected.


Results for tests of Lecithin
TEST Obtained Result Expected Result
Acrolein test It produces an unpleasant odor
(pungent)
Pungent
Iodine absorption test Pinkish color of precipitate
region upon dropping of
iodine
(decolorization of iodine)
Decolorization of iodine
Test for phosphorus Yellow precipitate formed and
formation of crystals upon
cooling
Yellow precipitate formed and
formation of crystals upon
cooling

Cholesterol
It is a steroid alcohol and is a constituent of all animal cells
Cholesterol plays an important part in the body. Its functions are as follows:
- Hormone Production
- Vitamin D Production
- Bile Production
- Cell Membrane support

Salkowskis Test
Purpose:
Test for: Cholesterol
Principle:
H
2
SO
4
was added on the lipid sample. The oxidative property of H
2
SO
4
triggers the
formation of additional double bonds between two cholesterol molecules.
A red disulphonic acid of bicholestadien is formed


Liebermann-Burchard Test:
A test for unsaturated steroids

Liebermann-Burchard Reaction:
- Acetic anhydride reacts with cholesterol in a chloroform solution to produce a characteristic
blue-green color.

Liebermann-Burchard Reaction


+
H2SO
4
-
H2O
3,5-cholestadiene








Results on Cholesterol Tests
Test Obtained result Expected result
Salkowski test CHCl3: cherry red color
Acid layer: clear
CHCl3: Cherry red color
Acid layer: fluorescent
greem
Liebermann-Burchard
reaction
Formation of sulfur precipitate at the bottom

Green color