Biochemistry – midterm period Lipids experiment 1.

Molisch test

The term “lipid” refers to both true fats and fat-like substances. These Principle: Molisch test is Specific for all carbohydrates – To
are biomolecules that are insoluble in water but are soluble in organic differentiate between Proteins & Amino Acids (-ve), and Carbohydrates
solvents. (+ve).

Another important function of lipids is the storage energy in the form This test is based on the fact that pentoses and hexoses are
of body fat. The brain and nerve tissues are rich in certain lipids, a fact dehydrated by conc. H2SO4 acid to form furfural or
that indicates the importance of these compounds in the maintenance hydroxymethylfurfural, respectively.
of life.
1. Molisch test
Five groups of lipids:
Result: Reddish Violet ring at the interface - Any Carbohydrate
1. Fats and oils 2. Waxes 3. compound lipids
4. Sterols and steroids 5. Derived lipids No change - Non carbohydrate
General objectives:
At the end of the experiment, the student must be able to: 2. Ninhydrin test
1. Familiarize himself/herself with the different classes of lipids; and
Principle: This test is due to a reaction between a amino group of free
2. Identify each kind of lipid based on the chemical properties of its
amino acid and ninhydrin. Ninhydrin is a powerful oxidizing agent and
hydrolyzed products.
its presence, amino acid undergo oxidative deamination liberating
a. Spotting effect
ammonia, CO2, a corresponding aldehyde and reduced form of
All the lipids are greasy in nature. Therefore the test may be taken as
ninhydrin ( hydrindantin). The NH3 formed from a amino group reacts
group test for lipids.
with another molecule of ninhydrin and is reduced product
PRINCIPLE: the oil does not wet the paper
( hydrindatin) to give a blue substance diketohydrin ( Ruhemanns
b. Solubility
complex).
This test is based on the
Positive compound: Phosphatidylserine
property of solubility of lipids in
organic solvents and insolubility Visible result: blue violet or dark blue solution
in water.
3. Soda lime test
PRINCIPLE: The oil will float on
water because of lesser specific test for amino and amino group// test for acidity and basicity
gravity.
Soda lime positive compound: Sphingomyelin and phosphatidylserine
c. Test for unsaturation (bromine water test)
Principle: deamination and acid-base reaction
PRINCIPLE: All neutral fats contain glycerides of some unsaturated
Visible result:
fatty acids. These unsaturated fatty acids become saturated by taking
- Red litmus -> Blue - Blue Litmus -> Blue
up iodine.
4. Ammonium molybdate test
This test identifies the level of saturation and the number of bonds an
oil, fat, or lipid has. It detects the presence of phosphate group

Visible result: Colorless Test positive compound: Phospholipids

d. Acrolein test Principle: Hydrolysis and double decomposition

It is used to detect the presence of glycerol or fat. When fat is treated Visible results:
strongly in the presence of a dehydrating agent like potassium
bisulphate (KHSO4), the glycerol portion of the molecule is dehydrated yellow precipitate (ammonium phosphomolybdate) insoluble in nitric
to form an unsaturated aldehyde, acrolein that has a pungent acid
irritating odor. lecithin (yellow orange)
Pigs brain (yellow)
e. Extraction of brain lipids
5. Liebermann – burchard test
Brain submerged in Ether
This test is used for the detection of cholesterol. The formation of a
Extracts ceramides, sphingomyelins, choline glycerophospholipids, green or green-blue color after a few minutes is positive.
ethanolamine glycerophospholipids and phosphatidylserines.
Lieberman–Burchard is a reagent used in a colorimetric test to detect
Not all lipids are soluble in Ether, therefore separates polar and non cholesterol, which gives a deep green color.
polar lipids

f. Detection of brain lipids