EXPERIMENT 5A LIPIDS

ISOLATION OF
LIPIDS
Group 5 Presentation
OBJECTIVES
- To extract three different precipitates from the
brain tissue of pig’s brain that was subjected to
cold acetone overnight using different reagents
with different solubility
- To identify the identity of the three precipitates
by the qualitative tests.
DEFINITION OF TERMS
1. Fatty acids- molecules that have carboxylic acids at
the polar end and long hydrocarbon chain at the nonpolar
tail
2. Simple lipid- organic compounds which are esters of
fatty acids with various alcohols
3. Compound lipid- esters of fatty acids that contains
additional group aside from alcohols and fatty acids
4. Derive lipid- derivatives obtained from hydrolysis of
simple lipids and compound lipids
7. Glycerol- simple three- carbon compound that contains
three hydroxyl groups
8. Amphipathic- molecule that has polar, water- soluble
LIPIDS
- Heterogeneous class of naturally occurring
organic compounds classified together on the
basis of common solubility properties
- Hydrophobic
- Highly insoluble to water but is soluble in
aprotic organic solvents.
- Functions mainly as a cell membrane component,
energy storage and insulation, and signaling
molecules.
THE BRAIN
- high content of lipid compared to all other tissues of
the body
- Contains variety of lipids
- Complex lipids such as the phosphatides, cerebrosides,
and sphingosides contain fatty acid components of the C-
16 to C-24 carbon chains
- also contains 17% grams of fat and 4% of saturated fat
- The steroids alcohol, cholesterol, occurs abundantly in
the brain.
- Brain tissues are rich in saponifiable and non-
saponifiable lipids which are important to life processes
- Main lipids are: cerebroside, phospholipids, cholesterol
 3 MAIN LIPIDS IN
CEREBROSIDES
THE BRAIN
- Under glycosphingolipids
- found in nerve and brain CHOLESTEROL
cells, primarily in cell - Type of sterol
membranes - serves as the biosynthetic
- characterized by a precursor needed to
carbohydrate bounded synthesize steroid
glycosidically to the hormones, bile acids, and
alcohol group of
PHOSPHOLIPIDS a lipid. vitamin D.

- main component of the cell - highly hydrophobic

membrane
- phospholipid is made up of
two fatty acid tails and a
phosphate group head, which
are connected by third
molecule, glycerol
 DISCUSSION OF RESULTS
- addition of cold acetone to the brain
tissue overnight for the brain tissues to
settle.
- Decanted and labeled the extract “Acetone
extract”
- Acetone extract was evaporated into a
steam bath leading to a dry residue
- Hot 95% alcohol was added
- White dried crystals
- soluble to acetone and hot alcohol but is
not soluble in ether.
 DISCUSSION OF RESULTS
- Addition of 15 ml cold ether in residue 1
- Decanted and evaporated
- Added 20 ml of acetone
- Crystal- like, yellow precipitate
- Soluble to ether and hot alcohol
 DISCUSSION OF RESULTS
- addition of 10 ml boiling 95% ethyl
alcohol
- Ensure to have a good precipitate
- soluble in both ether and hot alcohol but
is not soluble to acetone.
ACROLEIN TEST
Used to detect glycerin in a fat

PRINCIPLE:
By heating the fat sample in the presence of potassium
bisulfate (KHSO4), which acts as a dehydrating agent, acrolein
(C3H4O, or CH2=CH-CHO) is formed and can easily be detected by
its odor. Whenever fat is heated in the presence of a
dehydrating agent, the fat molecule will shed its glycerol in
the form of the unsaturated aldehyde - acrolein.
 ACROLEIN TEST
PRECIPITATE 1: FAINT ROTTEN EGG WHITE
SMELL
PRECIPITATE 2: VERY STRONG SMELL OF
SAMPALOC
PRECIPITATE 3: VERY FAINT SMELL OF WHITE
EGG
TEST FOR UNSATURATION
Used to detect the presence of
unsaturated fatty acids or the amount of
double bond in a lipid sample.
PRINCIPLES:
The unsaturated fatty acids absorb iodine at the double
bonds until all the double bonds are saturated with iodine.
Hence the amount of iodine required to impart its color to the
solution is a measure of the degree of the fatty acids.
TEST FOR UNSATURATION
PRECIPITATE 1: ONE DROP OF HANUS
REAGENT
PRECIPITATE 2: ONE DROP OF HANUS
REAGENT
PRECIPITATE 3: 3 DROPS OF HANUS
REAGENT
TEST FOR PHOSPHATE
Used to detect the presence of phosphate
group
PRINCIPLES:
When lipids containing phosphate groups in their
structures are added to a strong acid solution, the lipid
hydrolyses, producing free phosphate. The purpose of the
reagents is to break the bonds in order to reveal the free
phosphate group. When a solution containing phosphate ions is
heated with a solution of ammonium molybdate [(NH4)2MoO4] and
dilute nitric acid a bright yellow precipitate of ammonium
phosphomolybdate [(NH4)3PO4 • 12MoO4] is formed. The yellow
TEST FOR PHOSPHATE
PRECIPITATE 1: WHITE PRECIPITATE
PRECIPITATE 2: YELLOW PRECIPITATE
PRECIPITATE 3: WHITE PRECIPITATE
EMULSIFICATION TEST
used to detect the presence of polar and nonpolar
groups in lipids, specifically in bile and lecithin

PRINCIPLES:
Oil or liquid fat becomes finely divided and is dispersed
in water when shaken with water to form emulsification.
Emulsification is permanent and complete in the presence of
emulsifying agent. The important emulsifying agents are bile
salts, proteins, soaps, mono- and diglycerides. Emulsifying
agents lower surface tension of the liquid.
LIEBERMANN- BURCHARD TEST
used to detect the presence of sterols

PRINCIPLES:
Emerald green color indicates positivity to the test.
Formation of a green or green-blue color after a few minutes is
positive. Ideally, the cholesterol solution gives a nice
reaction, the coconut oil should show no significant color
change, and the lard gives a weak reaction.
LIEBERMANN- BURCHARD TEST
PRECIPITATE 1: VERY LIGHT GREEN
COLOR
PRECIPITATE 2: VERY LIGHT GREEN
COLOR
PRECIPITATE 3: BLUE- GREEN MIXTURE
MOLISCH TEST
general test for all carbohydrates

PRINCIPLES:
carbohydrates when reacted with conc. H2SO4 get
dehydrated to form furfural and its derivatives. Positive test
will give purple (violet red) colored complex.
 MOLISCH TEST
PRECIPITATE 1: YELLOW SOLUTION
PRECIPITATE 2: PURPLE SOLUTION IN
THE MIDDLE OF THE SOLUTION

PRECIPITATE 3: YELLOW SOLUTION

RESULTS AND DISCUSSION
Precipitate 1- Cerebroside

Precipitate 2- Phospholipids

Precipitate 3- Cholesterol
PRECIPITATE 1:
CEREBROSIDE
a. Cerebrosides have polar properties which in turn when
subjected to the addition of acetone which is also a
polar reagent, it readily dissolved cerebroside from the
brain tissue.

b. It was then furthered supported by the facts that

Precipitate 1 have tested the following tests and adhered to
what are the properties of the cerebroside.
PRECIPITATE 1:
CEREBROSIDE
i. It tested negative to the acrolein’s test since cerebrosides do
not have glycerol in the backbone.
ii. It only needed one drop of hanus reagent since it only has one
double bond it the sphingosine.
iii. It tested negative to the Phosphorus test yielding to white
precipitate since it has no phosphorus.
iv.Emulsification test
v. In Acetic Anhydride Test, precipitate 1 tested negative since it has
no sterol present in the cerebroside.
vi.And most of all, it tested positive to Molisch test showing that
there is a carbohydrate in the cerebroside which is true since
PRECIPITATE 2: PHOSPHOLIPIDS

a. Phospholipids have nonpolar properties since it has a

hydrophobic tail which in turn when a nonpolar reagent like
ether was added to it, it dissolved phospholipids plus the
addition of alcohol purified the precipitate.

b. It was then furthered supported by the facts that

Precipitate 2 have tested the following tests and adhered to what
are the properties of the phospholipids.
PRECIPITATE 2: PHOSPHOLIPIDS
i. It tested positive to the acrolein’s test since it has a glycerol
backbone that in turn reacts to have a pungent odor.
ii. Precipitate 2 only needed one drop of hanus reagent since the
phospholipids may contain only one double bond in the alkyl chain on
the hydrophobic tail.
iii. It is the only precipitate that tested to Phosphorus test
yielding to yellow precipitate since it has phosphorus in the
hydrophilic head.
iv. Emulsification Test
v. In Acetic Anhydride Test, precipitate 1 tested negative since it
has no sterol present in the phospholipid.
vi. It then tested negative to Molisch test since it has no
PRECIPITATE 3: CHOLESTEROL

a. Cholesterol is heavily a hydrophobic molecule thus it is only

a natural that it can be dissolved in ether.

b. It was then furthered supported by the facts that

Precipitate 3 have tested the following tests and adhered to what
are the properties of the cholesterol.
PRECIPITATE 3: CHOLESTEROL
i. It tested negative to the acrolein’s test since it don’t
have a glycerol backbone.
ii. Precipitate 3 only needed one drop of hanus reagent since
the cholesterol since it only have one double bond in its
structure.
iii. It tested negative since it yielded white precipitate
which showed that there is no phosphorus in the precipitate.
iv. Emulsification Test
v. In acetic anhydride test, it is the only positive
precipitate which exhibited a blue green mixture since
cholesterol has a sterol.
CONCLUSION
- Lipids have different solubility properties which helped to
cluster their types together
- Differences in solubility helped to isolate the three
precipitate and with the help of the various tests
- Precipitate 1: Cerebroside because it is positive to Molisch
test
- Precipitate 2: Phospholipids because it is positive to test
for phosphorous
- Precipitate 3: Cholesterol because it is positive to
Liebermann- Burchard test
REFERENCES
http://www.uobabylon.edu.iq/eprints/publication_1_8265
_904.pdf
https://www.biochemden.com/color-reactions-of-
lipids/#4_A_TEST_FOR_UNSATURATION