Characterization of

Saponifiable lipids
3PSY5

Members
Mercado, Eloisa Belle
Pestano, Julian Terrence
Regino, Paul Andrew R.
Tady, Kristine Claire
Tee, Margarita
Villanueva, Kervin

Introduction
REGINO, Paul Andrew R.
3PSY5

Lipids
Lipids are naturally organic
compounds that are insoluble in
water but soluble in organic
solvents.
Animals: lipids make up most of the
fats in the form of adipose tissues
which helps in insulation and
thermal regulation. Fats are known
to store heat.
Plants: lipids are seen as the waxy

Classification of lipids
Open-chain compounds with polar
heads and long nonpolar tails
1.
2.
3.
4.
5.

Fatty acids
Triacyglycerols
Sphingolipids
Phospholipids
Glycolipids

Fused-ring compounds
1. Steroids

Classification of lipids
On the basis of whether they will
undergo hydrolysis in alkaline
solution:
1. Saponifiable lipids
2. Nonsaponifiable lipids

Fatty Acids
Polar end= the carboxyl group
Nonpolar tail= the hydrocarbon
chain
Fatty acids are amphipathic
They usually have even number of
carbon atoms & the hydrocarbon
chain is unbranched
Two main types: Saturated &
unsaturated

Triacylglycerols
The 3 alcohol groups in glycerol
(glycerol contains 3 hydroxyl
groups) forms ester linkages with
fatty acids.
Function: storage (especially in
animals)

Phospholipids
Type of lipid that contains a
phosphorus atom attached to the
glycerol backbone
They are known to be the main lipid
component of the cell membrane
2 types of phospholipids:
1. Phosphoacylglycerols- glycerol is the
backbone
2. Sphingomyelins- sphingosine instead
of glycerol

Glycolipids
A lipid to which a sugar moiety is
bonded
A carbohydrate is bound to an
alcohol group of a lipid by a
glycosidic linkage
Ceramides are the parent
compounds for glycolipids

Sphingolipids
Do not contain glycerol but instead
they contain sphingosine.
Found both in plants and animals &
they are abundant in the nervous
system

Oils

1. Coconut oil= composed mainly of lauric

acid & is slow to oxidize and is resistant
to rancidification
2. Canola oil= low in saturated fat
3. Olive oil= composed mainly of oleic acid
and palmitic acid and other fatty acids
4. Palm oil= has 30 phenolic compounds &
has high saturated vegetable fats
5. Sesame oil= high proportion of
polyunsaturated linoleic fatty acid
6. Corn oil= high smoke poiny

Four tests for lipids

1. Grease-spot test
2. Saponification test
3. Acrolein test
4. Unsaturation test

Grease-spot test
General test for the presence of
lipids
Positive: oil & lecithin
Negative: water &
dichloromethane

Saponification test
General test for ester bonds
Positive: oil & melted fat
Negative: water

Saponification
Hydrolysis reaction
Hydrolysis using a base such as
sodium hydroxide or potassium
hydroxide
Products are:
1. Glycerol
2. Sodium/Potassium salts- salts are
soaps

Saponifiable lipids
Contains an ester functional group
Can behydrolyzedunder basic
conditions
Includes triacyglycerols,
phospholipids, glycolipids,
sphingolipids and the waxes

Acrolein test
General test for fats or glycerin
Positive result= strong odor

Unsaturation test
General test for the presence of
double bonds and their degree of
saturation

Objectives
to characterize lipids using the
grease-spot test, saponification test,
acrolein test and unsaturation test.

Methodology

Tee, Margarita R.
3PSY5

Grease Spot Test

Tee, Margarita R.
3PSY5

With the use of pencil, four

areas of a filter paper was
labeled vegetable oil, lecithin,
H2O and dichloromethane
apply a drop of the appropriate
substance to the labeled areas
Warm the filter paper

Observations after dropping

and after heating

Tee, Margarita R.
3PSY5

Saponification Test

Tee, Margarita R.
3PSY5

3 large-sized test tubes

labeled oil, fat and
H2O
8 drops of appropriate substance
placed on their corresponding test
tube
Add 10 drops of 3M NaOH on each
test tubes
Place in boiling water bath for 15-20
mins
Place in test tube rack and allow to
cool to room temperature
Tee, Margarita R.
3PSY5

add 5ml of distilled water to

each test tube
Stopper the test tubes and
mix vigorously
Record observations
after shaking whether
an emulsion, micelle or
clear solution
Acidify with few
drops of H2SO4

Tee, Margarita R.
3PSY5

Mix with stirring

rod
Note the color of
the material on
top
record the pH of the
material, either acidic or
basic, using blue and red
litmus paper

Tee, Margarita R.
3PSY5

Acrolein Test

Tee, Margarita R.
3PSY5

1 Kg of KHSO4 in a test
tube
Add 5 drops of an oil sample
or a small piece of solid lipid
Heat over a Bunsen burner
for a few minutes
Allow to cool
Strong odor

Tee, Margarita R.
3PSY5

Unsaturation Test

Tee, Margarita R.
3PSY5

3 large-sized test tubes

labeled oil, fat and
glycerol
Place 3ml of DCM on each test tube
Add 10 drops of appropriate
substance on their corresponding
test tube
Mix thoroughly
Under the fume hood, add dropwise
of 5% bromine-dichloromethane to
each test tube while stirring until
reddish brown color appears

Tee, Margarita R.
3PSY5

Number of drops of brominedichloromethane and the net

number of drops

Tee, Margarita R.
3PSY5

RESULTS
TADY, Kristine Claire D.

GREASE- SPOT TEST: After

dropping
GR
P

OIL

LECITHIN

DCM

HO

Translucent

Opaque

Opaque*

Translucent

Translucent

Opaque

Opaque*

Translucent

Translucent

Opaque

Opaque*

Translucent

Translucent

Opaque

Opaque

Translucent

Translucent

Translucent

Disappeared

Translucent

Translucent

Opaque

Opaque

Translucent

Translucent

Opaque

Opaque

Translucent

Translucent

Opaque

Disappeared

Translucent

Translucent

Opaque

Opaque

Translucent

10

Translucent

Opaque

Opaque

Translucent

11

Translucent

Opaque

Opaque

Translucent

12

Translucent

Opaque

Opaque*

Translucent

GREASE- SPOT TEST: After

heating
GR
P

OIL

LECITHIN

DCM

HO

Transluce
nt

Opaque

Opaque

Opaque
(dried)

Transluce
nt

Opaque

Opaque

Opaque
(dried)

Transluce
nt

Light stain

Disappeare
d

Wrinkles

Transluce
nt

Slight yellow
stain

Opaque

Opaque
(dried)

Transluce
nt

Translucent

No stain

Disappeared

Transluce
nt

Opaque

Opaque

Wrinkles

Transluce
nt

Opaque

Disappeare
d

Dried

SAPONIFICATION TEST: After

shaking
GR
P

OIL

MELTED FAT

HO

Bubbles with white

interphase; cloudy
solution

Bubbles with yellow

interphase; cloudy
solution

Light yellow
soln

Emulsion

Micelle

Light yellow
liquid

Murky yellow emulsion

Cloudy yellow micelle

Light brown
soln

Yellow cloudy liquid

Cloudy white liquid

Colorless
Solution

Emulsion

Micelle

Colorless
Solution

Bubbles formed

No bubbles formed

Clear Solution

Emulsion

Micelle

Clear Solution

White foam on top

White and yellow foam

on top

Faint brown
soln

SAPONIFICATION TEST: Material

on top
GR
P

OIL

MELTED FAT

HO

Dirty white
liquid

Yellow solids

None

White ppt

Yellow ppt

None

Bubbles

Yellow spots

Bubbles

Bubbles yellow layer

Bubbles, white
film

Bubbles, yellow
powder

None

Light yellow

Yellow

None

Cloudy white

Yellow

Colorless

White
substance

Yellow formation

Colorless

White ppt

Vivid yellow ppt

None

10

Dirty white
liquid

Yellow solids

None

SAPONIFICATION TEST: Acid or

Base
GR
P

OIL

MELTED FAT

HO

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

Acid

10

Acid

Acid

Acid

11

Acid

Acid

12

Acid

Acid

Acid

UNSATURATION TEST: Br in
DCM
GR
P

GLYCEROL

OIL

MELTED FAT

21

155

25

100

41

11

66

20

135

60

22

95

27

16

20

60

25

20

18

10

30

130

29

11

58

50

12

25

289

22

26

UNSATURATION TEST: Net # of

drops
GR
P

GLYCEROL

OIL

MELTED FAT

42

80

25

134

75

16

20

130

17

58

18

110

40

22

73

44

10

100

-1

11

50

42-

12

-3

Discussion
REPORTER: Terrence Julian B. Pestao

PESTAO,

Grease Spot Test

A simple test in determining the
presence of lipids

Reagents: DCM, H2O, Lecithin, Oil

sample
Principle: Vaporization of volatile
substances
Visible Result: Translucent Stains
Positive results in: Oil Sample
and Lecithin
PESTAO,

Grease Spot Test

Volatility
Unsaturation
Number of Carbons

PESTAO,

 Triglyceride

PESTAO,

 LECITHIN

PESTAO,

Saponification Test
Involves saponification hydrolysis using a
strong base
Lipids (fat, oil)

Glycerol and Soap

PESTAO,

Saponification Test

PESTAO,

PESTAO,

Saponification Test
Reagents: NaOH (strong
base), H2SO4
Principle: base hydrolysis
Visible Result: Formation of
bubbles / precipitate
Positive results in: Melted
Fat and Oil
PESTAO,

Saponification Number/ Value

(a.k.a. Koettstorfers Number)
is the needed mg of NaOH (or KOH) to neutralize
the fatty acids from the hydrolyzed 1g of the lipid
Can give notions regarding the structure of fatty
acids
Molecular weight
Chain Length

The higher the saponification number (),

the lower the molecular weight ()
(more fatty acids are freed per gram of fat after
hydrolysis)

PESTAO,

PESTAO,

References
Retrived:http://www.florinag.ch/index.php?
sid=OV15cjStGNEbKLsL3baR56UxgZ1755qvyPOlQReb&c5p=49
5&c5l=en. (November 26, 2015).
Campbell, M.K. & Farrell, S.O. (2012/. Biochemistry. (7th ed.).
California, USA: Brooks/Cole, Cengage Learning.

Conclusion
Villanueva, Kervin B.

Objectives
To characterize a fat or an oil sample
using the grease-spot test,
saponification test, acrolein test, and
unsaturation test.

The groups were able to

characterize a fat or an oil sample
using the grease-spot test,
saponification test, acrolein test,
and unsaturation test.

The grease-spot test as a general

test for the presence of lipids, would
give a positive result in both the oil
sample and lecithin but a negative
result in water and
DCM(Dichloromethane).

The saponification test as a test for

the presence of ester bonds, would
give a positive result in both the oil
sample and melted fat but a negative
result in water.

The unsaturation test as a test for the

presence of double bonds and the
degree of saturation, would have
different number of drops of brominedicholoromethane in unsaturating the
glycerol, oil and melted fat until they
colorize to a red-brown solution.

The acrolein test as a test for the

presence of glycerol, would give a
positive result in lecithin.

Sources of Error
inaccurate measurement of the reagents
used
observational error

References
Book References
Boyer, R. (2006). Concepts in Biochemistry,3rdedition.
John Wiley
and Sons(Asia) Pte.Ltd
Campbell, M. (2015). Biochemistry. Cengange Publishing
(Singapore).
Legaspi, G. (2009). Essentials of Biochemistry
Laboratory. UST
Publishing (Philippines).
Menguito, C., et al. (2010). Basic Laboratory
Experiments in
Biochemistry. College of Science,
University of Santo
Tomas.
Vance, D. (2008). Biochemistry of lipids, lipoproteins
and
membranes. Elsevier B.V.
Online References
Caballero, B. MD, PhD (2006) Lipids. Retrieved on