Lipids can be tested qualitatively through solubility, spotting, acrolein, saponification, Liebermann-Burchard, and Salkowski tests. The solubility test uses organic solvents to test solubility. The spotting test examines greasiness. Acrolein detects glycerol. Saponification produces soap through hydrolysis. Liebermann-Burchard identifies sterols like cholesterol by color change. Salkowski uses sulfuric acid to detect cholesterol through layer color changes. These tests characterize lipids by properties like solubility, structure, and chemical reactions.

1. QUALITATIVE AND
QUANTITATIVE TESTS FOR
LIPIDS.
Prof. Liwayway Memije-Cruz

2. Lipids
 A large number of heterogeneous compounds
are referred to as lipids including fats, steroids,
waxes, and related compounds, which are
related more by their physical than their
chemical properties.
 They have the common property of being:
1. relatively insoluble in water
2. soluble in polar solvents such as ether
and chloroform.

3. Solubility Test
Spotting Test
Acrolein Test
Saponification Test
Liebermann-Burchard Reaction for
Steroids
Salkowski’s Test (H2SO4 Test)
Qualitative and Quantitative Tests
for Lipids

4. Lipids are water-insoluble organic compounds that can be
extracted from cells and tissues by non-polar solvents like ether,
chloroform, benzene, etc. Lipids that contain fatty acids are
saponifiable, while those without fatty acids like steroids are non-
saponifiable which do not produce fatty acid nor alcohol. Some
common lipids are the oil, fats, wax, steroid, hormones and
vitamins like A, D, E and K. In the body, lipids are converted to
fatty acid and glycerol by bile then by steapsin, or lipase.
In the laboratory, fats and oil may be tested qualitative to
determine their characteristics structure like acrolein, an
unsaturated aldehyde, cholesterol, a sterol and the polar and on
polar group of saponified fatty acid.
Overview

5. Solubility Test
 The test is based on the
property of solubility of lipids
in organic solvents and
insolubility in water.
 PRINCIPLE: The oil will float
on water because of lesser
specific gravity.
 TEST: Take 3ml of solvents in
each test tube and add 5
drops of sample. For water
and ethanol, it is insoluble
and for chloroform and ether,
it is soluble and hence the
given sample is lipid.

6. Spotting Effect
 All the lipids are greasy in
nature. Therefore the test
may be taken as group test
for lipids.
 PRINCIPLE: The oil does not
wet the paper.
 TEST: Take 3ml of ether in a
test tube and dissolve 5 drops
of oil in tit. Put a drop of the
solution on the filter paper
and let it dry. A translucent
spot on the filter paper was
observed and this indicates
the greasy character of the
lipid.

7. Acrolein Test
 used to detect the
presence of glycerol
or fat. When fat is
treated strongly in the
presence of a
dehydrating agent like
potassium bisulphate
(KHSO4), the glycerol
portion of the
molecule is
dehydrated to form an
unsaturated
aldehyde, acrolein
that has a pungent
irritating odor.

8. Saponification-The process of Making
Soap
 The Theory
Soaps are made from fats
and oils or their fatty
acids. The saponification
reaction is exothermic in
nature, because heat is
liberated during the
process. The soap formed
remains in suspension
form in the mixture. Soap
is precipitated as a solid
from the suspension by
adding common salt to
the suspension. This
process is called Salting
out of Soap.

9. Liebermann-Burchard Test (Test for Sterol)
 This test is used for the detection of
cholesterol. The formation of a
green or green-blue color after a
few minutes is positive.
 Lieberman–Burchard is a reagent
used in a colorimetric test to detect
cholesterol, which gives a deep
green color. This color begins as a
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 reacting with the
reagents and increasing the
conjugation of the un-saturation in
the adjacent fused ring. Since this
test uses acetic anhydride and
sulfuric acid as reagents, caution
must be exercised so as not to

10. Salkowski Test: Test for
Cholesterol
 Dissolve cholesterol
in 2 ml of chloroform
in dry test tube. Add
equal amount of
con. H2SO4. Shake
gently. The upper
layer turns red and
the sulphuric acid
layer shows a yellow
colour with a green
fluorescence.

11. References:
 https://www.biochemden.com/color-reactions-
of-lipids/
 http://www.biologydiscussion.com/lipids/tests/q
ualitative-and-quantitative-tests-for-
lipids/13050
 https://steemit.com/steemiteducation/@chamu
diliyanage/chemical-basis-of-life-part-2-let-s-
learn-series-14-biology