Characterization of Saponifiable Lipids (Melted

Fat, Lecithin, and Plant Oils) Using Grease-Spot,

Saponification, Acrolein, and Unsaturation Tests
Experiment #06
BUHAYO, Sophia Joan
CUAN, Lindane Camille
ENRIQUEZ, Vergel Jigs

Lipids
A heterogeneous class of naturally occurring organic compounds
Classified together on the basis of common solubility properties
Insoluble in water but soluble in nonpolar and organic solvents
Amphipathic in nature
Substances such as oils, fats and wax
May be saponifiable or non-saponifiable

Biological Functions of Lipids

1. Energy storage

2. Vitamins
3. Components of the cell membrane

4. Thermal insulators
5. Electrical insulators

6. Provides shape and contour to the body

Classification of Lipids
1. Simple Lipids or Homolipids
2. Complex Lipids or Heterolipids

3. Derived Lipids

Classification of Lipids
1. Simple Lipids or Homolipids
- are the esters of fatty acids with various alcohols.
a. Fats and Oils (triglycerides and triacylglycerols) are esters of fatty
acids with a trihydroxy alcohol, glycerol.
b. Simple Triglycerides are one in which three fatty acids radicles are
similar or are of the same type.

Classification of Lipids
c. Mixed Triglycerides are one in which the three fatty acids radicles are
different from each other.

d. Waxes are the esters of fatty acids with high molecular weight
monohydroxy alcohols.

Classification of Lipids
2. Compound Lipids or Heterolipids
- are esters of fatty acids with alcohol and possess additional groups also.
a. Glycolipids are the compounds of fatty acids with carbohydrates and
contain nitrogen but no phosphoric acid.
b.Phospholipids or Phosphatids are compound containing fatty acids and
glycerol in addition to a phosphoric acid, nitrogen bases and other

substituents. They usually possess one hydrophilic head and two nonpolar tails. They are called polar lipids and are amphipathic in nature.

Classification of Lipids
Phospholipids can be phosphoglycerides, phosphoinositides and
phosphosphingosides.

a. Phosphoglycerides are major phospholipids found in membranes. They

contain fatty acid molecules which are esterified to hydroxyl groups of
glycerol. The glycerol group also forms an ester linkage with phosphoric

acid. Example: Lecithin and cephalins.

b.Phosphoinositides
c. Phosphosphingosides

Classification of Lipids
Phospholipids can be phosphoglycerides, phosphoinositides and
phosphosphingosides.

a. Phosphoglycerides are major phospholipids found in membranes. They

contain fatty acid molecules which are esterified to hydroxyl groups of
glycerol. The glycerol group also forms an ester linkage with phosphoric

acid. Example: Lecithin and cephalins.

b.Phosphoinositides
c. Phosphosphingosides

Classification of Lipids
3. Derived Lipids
-are the substances derived from simple and compound lipids by hydrolysis.

a. Steroids do not contain fatty acids, they are nonsaponifiable, and are not
hydrolyzed on heating.
b. Terpenes in majority are found in plants such as natural rubber, gern oil,
etc.
c. Carotenoids are tetraterpenes. Due to the presence of many conjugated
double bonds, they are colored red or yellow.

Classification of Lipids
1. Saponifiable Lipids are lipids with an ester functional group, that can be
hydrolyzed under basic conditions. These include triglycerides,

phospholipids, glycolipids, sphingolipids, and the waxes. These lipids are

known as simple lipids.

2. Non- Saponifiable Lipids are lipids that cannot be hydrolyzed

Examples: Terpenes , Steroids , Fat-soluble Vitamins

Grease-Spot Test
Tests the presence of lipids
Reagents: H2O, DCM, LECITHIN, OIL SAMPLE

Saponification Test
Tests the presence of ester bonds
Reagents: 3M NaOH, H2SO4

Acrolein Test
Tests the presence of glycerol
Reagents: KHSO4 (Dehyrdrating Reagent)

Unsaturation Test (Bromine Test)

Tests the presence of double bonds and degree of

saturation
Reagents: DCM AND 5% Br-DCM

Objective
To characterize saponifiable lipids using
Grease-spot test, Saponification test, Acrolein
Test, and Unsaturation test.

Materials
A.Grease Spot Test

Oil Sample
Lecithin (0.1 g/mL dichloromethane)

Dichloromethane
Filter paper

Materials
B.Saponification Test

Oil Sample
Melted fat

3M NaOH

Materials
C.Acrolein Test

Oil Sample or Fat

KHSO4

Materials
D.Unsaturation Test
Oil Sample
Melted fat
Glycerol
Dichloromethane
5% bromine-dichloromethane solution

50-mL buret

Methodology
A.Grease Spot Test

Filter paper
- divide into four regions; label
each region into oil sample,
lecithin, dichloromethane,
and HO
+ 1 drop of the appropriate
substance on each region using
Pasteur pipette
- warm on hot plate for 1 minute
- record observations

Methodology

3 Large Test Tubes

-

label each test tube with oil sample, fat, and HO

8 drops of the appropriate substance on their respective test

tubes using Pasteur pipette

10 drops of 3M NaOH solution to each test tube

place the test tubes in boiling water bath for 15-20 minutes

remove the test tubes and cool to room temperature

5 mL of distilled water to each test tubes; place a stopper

and vigorously shake the test tubes to mix the contents

few drops of HSO to each test tube; check acidity using

blue litmus paper

mix the solutions using a stirring rod and note the material
collected on top of the solutions

check and record the pH of the collected material using red

and blue litmus paper

B.Saponification Test

Methodology
C.Acrolein Test

Test Tube

1 gram of KHSO

5 drops of an oil sample or a

small piece of lipid

heat over Bunsen burner for a

few minutes

cool test tube and note the

formation of the strong odor of
Acrolein

Methodology
D.Unsaturation Test

3 Large Test Tubes

-

label each test tube with oil sample, fat, and glycerol

3 mL of dichloromethane in each test tube

10 drops of the appropriate substance on their respective

test tubes

mix the contents thoroughly

5% of bromine-dichloromethane solution to a 50-mL buret

using a funnel under the fume hood

record initial volume of the solution

Add the bromine-dichloromethane dropwise from the buret

to each test tube while stirring until reddish-brown bromine
color appear

record the final volume of the bromine-dichloromethane

solution in the buret and calculate the volume of solution
required for the reaction

Results and Discussion

Grease - Spot Test

Grease-Spot Test
Reagents: H2O, DCM, LECITHIN, OIL SAMPLE
Test for: Presence of Lipids
Principle: Determination of high boiling point
Positive Results: Translucent grease marks
Positive for: Oil Samples/ Lecithin

Results and Discussion

Group
(Oil Sample)

Oil Sample

Lecithin

Dichloromethane

HO

1
Canola oil

Spot formed;
translucent

Spot formed; not

translucent

No spot; area not

translucent

No spot; area
not translucent

2
Butter

Spot remained

Slight spot
remained

No spot remained

No spot
remained

3
Sesame oil

Spot remained

Spot remained

No spot remained

No spot
remained

4
Olive oil

Spot visible,
translucent grease
mark

Spot less visible

than olive oil

No spot visible

No spot visible

5
Corn oil

Translucent
grease mark

Translucent
grease mark

Absence of spot

Absence of spot

Results and Discussion

Group
(Oil Sample)

Oil Sample

Lecithin

Dichloromethane

HO

6
Extra Virgin Olive
oil

Translucent
grease mark

Slight translucent
grease mark

No visible changes

No visible
changes

7
Margarine

Spot formed;
translucent

Spot slightly seen;

not translucent

No spot formed;
evaporated

Spot formed, not

translucent

8
Coconut oil

Translucent
grease mark

Slight translucent
grease mark

No visible change

No visible
change

9
Castor oil

Spot formed;
Translucent

Spot formed;
Slightly
Translucent

No Visible spot

No Visible spot

10
Palm oil

Translucent spot

Translucent spot

No translucent spot

No translucent
spot

Results and Discussion

Saponification Test

Saponification Test
Reagents: 3M NaOH, H2SO4
Test for: Presence of Ester Bonds
Principle: Hydrolysis of ester linkages under basic
conditions to form glycerol and fatty acid salts.
Positive Results: Presence of Bubbles and Precipitates
Positive for: Oil Samples and Melted Fats

Saponification Test
The Saponification Test is positive for lipids
that can undergo base hydrolysis and has
ester linkages like triglyceride waxes.

Formation of Soap
After the addition of NaOH and shaking of the oil/fat, the
presence of bubbles and precipitate are on top of the
solution.

This indicates the formation of fatty acid salts which are

also known as soaps

Saponification Number
Defined as the number of milligrams of potassium and
sodium hydroxide required to neutralize the fatty
acids in 1 gram of fat.
Also known as the Koettstorfer Number.

Gives information concerning the character of the fatty

acids of the fat.
Inversely proportional to the molecular weight of the
fat/oil.

Saponification Number
OILS

SAPONIFICATION NUMBER

Rapeseed/ Canola Oil

170-179

Olive

185-196

Corn

188-193

Palm

196-205

Coconut

246-260

Results and Discussion

Saponification Test
of Oil Samples

Results and Discussion

Group
(Oil Sample)

After Heating & Shaking

After Acidification

pH

1
Canola oil

Bubbles formed, cloudy

solution

White substance on top

Acidic

2
Butter

Cloudy white with bubble

formation

White cloudy ring at the surface

Acidic

3
Sesame oil

Lesser bubble formation on the

top compared to fat; cloudy
solution

Light yellow top layer; white

solution

Acidic

4
Olive oil

Formation of few bubbles

Whitish material formed in the top

of the whitish solution

Acidic

5
Corn oil

Upper layer: yellow, lower layer:

white liquid, bubble formation

White turbid solution with yellow

material

Acidic

Results and Discussion

Group
(Oil Sample)

After Heating & Shaking

After Acidification

pH

Presence of bubbles and yellow

top layer in transparent solution

Yellow top layer in a turbid

solution

Acidic

7
Margarine

Light yellow, thick solution

Turbid with light yellow layer on

top

Acidic

8
Coconut oil

Appearance of bubbles and

precipitate on top

Appearance of bubbles and

precipitate on top

Acidic

9
Castor oil

Slightly turbid with bubbles

White layer on top

Acidic

10
Palm oil

White liquid with bubbles

White layer on top

Acidic

6
Extra Virgin Olive
oil

Results and Discussion

Saponification of
Melted Fat

Results and Discussion

Group
(Oil Sample)

After Heating & Shaking

After Acidification

pH

1
Canola oil

Cloudy, yellowish solution; bubbles

formed

Yellow substance on top

Acidic

2
Butter

Cloudy yellow with bubble formation

Yellow ring at the surface

Acidic

3
Sesame oil

Greater bubble formation at the top,

suspended white particles in the solution

Yellow top layer; cloudy

solution

Acidic

4
Olive oil

Formation of bubbles in the upper layer

Yellowish material formed

in top of a white opaque
solution

Acidic

5
Corn oil

Upper layer: yellow ppt.; lower layer:

yellow turbid solution, bubble formation

White turbid solution with

yellow ppt

Acidic

Results and Discussion

Group
(Oil Sample)

After Heating & Shaking

After Acidification

pH

6
Extra Virgin Olive
oil

Turbid solution with the

presence of bubbles and a
yellow top layer

Yellow layer on top of turbid

solution

Acidic

7
Margarine

Yellow-orange with oil

precipitate

Yellow layer on top

Acidic

8
Coconut oil

Appearance of bubbles and

precipitate on top

Appearance of bubbles and

precipitate on top

Acidic

9
Castor oil

Slightly yellow turbid solution

with bubbles

Yellow layer on top

Acidic

10
Palm oil

Light yellow liquid with yellow

bubbles on top

Yellow layer on top

Acidic

Results and Discussion

Saponification of
Water

Results and Discussion

Group
(Oil Sample)

After Heating & Shaking

After Acidification

pH

1
Canola oil

Bubbles formed, clear yellowish

solution

No formation on top

Acidic

2
Butter

Clear solution with little bubble

formation

Clear solution

Acidic

3
Sesame oil

Least bubble formation at upper layer

Clear solution

Acidic

4
Olive oil

No bubbles formed

Clear Solution

Acidic

5
Corn oil

Transparent liquid

Transparent liquid

Acidic

Results and Discussion

Group
(Oil Sample)

After Heating & Shaking

After Acidification

pH

Transparent solution

Clear solution without top layer

Acidic

7
Margarine

Light yellow liquid

Clear solution

Acidic

8
Coconut oil

No bubbles and precipitate on

top

No bubbles and precipitate on

top

Acidic

9
Castor oil

Transparent solution with

bubbles

With slightly soluble material

Acidic

10
Palm oil

Clear liquid

Clear solution

Acidic

6
Extra Virgin Olive
oil

Results and Discussion

Acrolein Test

Acrolein Test
Reagents: KHSO4 (Dehyrdrating Reagent)
Test for: Presence of Glycerol
Principle: Oxidation, Dehydration with Heat
Positive Results: Blackening of Reaction Mixture and
Pungent Smell
Positive for: Phosphoglycerin (Lecithin)

Acrolein Test
Upon heating the oil sample with potassium bisulfate (dehydration), it
would yield acrolein (unsaturated aldehyde).
Further heating would result to the polymerization of the acrolein,
which is indicated by the blackening of the reaction mixture.
Both the pungent smell and the black color indicate the presence of
glycerol.

Acrolein Test

Results and Discussion

Unsaturation Test
(Bromine Test)

Unsaturation Test (Bromine Test)

1. Saturated fat
Saturated fats contain only single bonds within the carbon chain. Saturated
fats are of animal origin and are usually present in solid form.
2. Unsaturated fat

Unsaturated fats contain double bonds within the carbon chain. Unsaturated
fat is found in fish like salmon and tuna, nuts, seeds etc

Unsaturation Test (Bromine Test)

Reagents: DCM AND 5% Br-DCM
Test for: Presence of Double Bonds and Degree of
Saturation

Principle: Halogenation of alkenes via Bromination.

Positive Results: Colorization of solution
Positive for: Oil Samples, glycerol and melted fat.

Bromine Number
Bromine number is the amount of bromine
in grams absorbed by 100 grams (3.5 oz) of
a sample. The number indicates the degree
of unsaturation. The Bromine Number is
useful as a measure of aliphatic
unsaturation in gasoline samples.

Conclusion
The characterization of saponifiable lipids were carried out through four
main tests.

First, the Grease-Spot test that indicates the high boiling

points of both lecithin and oil samples. Second, the saponification test
confirmed the presence of ester linkages in the oil samples and melted
fat. Third, the acrolein test affirmed that the phosphoglycerins indeed
possess glycerol. Lastly, the unsaturation test determined the presence of
double bonds as well as the degree of saturation of oil samples and
melted fats.

References
Appling, D., Anthony-Cahill, S. and Mathews, C. (2016). Biochemistry: Concepts
and Connections. Essex, England: Pearson Education Limited.
Boyer, R. (2009). Biochemistry Laboratory: Modern Theory and Techniques.
San Francisco, CA.: Benjamin Cummings.

Prasad, D.S.K. (2010) Biochemistry of Lipids. New Delhi, India: Discovery

Publishing House PVT. LTD.