Characterization of saponifiable lipids present in different vegetable oils

Abad, Matthew*; Abeleda, Ara Patricia; Arce, Christine Grace; Bautista, Kenny
College of Science
University of Santo Tomas
Espaa Blvd., Manila

Abstract
Different vegetable oil samples (Jolly soya oil, Golden Fiesta palm oil, Minola coconut oil, Colorado
sunflower oil, Jolly canola oil, Hojiblanca extra virgin olive oil, Doa Elena pure olive oil, Baguio pure
vegetable oil) were characterized using the saponification test, unsaturation test, and grease-spot test. All the oil
samples and lecithin tested positive for the grease-spot test. Oil and Fat samples tested positive for the saponification
test as well as in the unsaturation test. For the unsaturation test, the oils showed a higher degree of unsaturation
compared to the fat sample.

Introduction
A diverse group of compounds, lipids, are defined primarily by their insolubility in water
and solubility in organic solvents, such as chloroform or acetone (Campbell & Farrell, 2012).
Lipids perform different biological functions due to their diverse chemistry. Example of which
are the fats and oils, which are the principal stored forms of energy in different organisms, and
the phospholipids and sterols, which perform structural roles in biological membranes. Based on
their chemical nature, there are two main groups of lipids namely the open chain compounds (ex.
fatty acids, sphingolipids, glycolipids, etc.) and the fused-ring compounds (ex. the steroid,
cholesterol).

Figure 1. Saponification
http://cronodon.com/sitebuilder/images/Saponification2-600x198.jpg

Lipids can also be subdivided as either saponifiable or non-saponifiable. From the words
itself, saponifiable lipids are those that undergo saponification, while non-saponifiable are those
that do not. Saponification is a reaction in which the ester linkages of triacylglycerols are
hydrolysed under basic conditions, such as when sodium hydroxide is used. This reactions
produce glycerol and the fatty acid salts. Fatty acids are composed of a carboxyl group together
with a hydrocarbon chain and are said to be amphipathic. These fatty acids can either be
saturated, having only C-C single bonds, and unsaturated, those having C-C double bonds
In the experiment, different vegetable oil samples were used.

Vegetable oils are

triacylglycerols usually extracted from different plants, primarily from the seeds. These oils are
utilized in human culture primarily for culinary purposes. Examples of which are canola oil from
rapeseeds, coconut oil from the coconut palm, and corn oil from the germ of a corn among
others.
Characterization of oil samples is done by using different tests. Grease-spot tests and
Saponification tests gives a positive result for the presence of triacylglycerols. Acrolein tests
indicate the presence of glycerols. Lastly, Unsaturation tests differentiate saturated from
unsaturated fatty acids by indicating the presence of C-C double bonds.
In this experiment, the objective was to characterize a fat or oil sample using the different
tests mentioned, namely, grease-spot test, saponification test, acrolein test, and unsaturation test.

Methodology

Grease-spot test. A piece of filter paper was obtained. Using a pencil, four (4) areas of the
filter paper was labeled as 'vegetable oil', 'lecithin', 'water', and 'dichloromethane'. Using a
pasteur pipet, a drop of the appropriate substance was applied to each labeled area. The filter
paper was then warmed by placing it on a hot plate adjusted to the lowest setting. Observations
were recorded.
Saponification test. Three (3) large test tubes were labeled as 'oil', 'fat', and 'water'. Eight
(8) drops of the substance was placed into each of their respective test tubes. After which, ten
(10) drops of 3M NaOH were added to each test tube. The test tubes were placed in a boiling
water bath with constant agitation. After 15-20 mins in the water bath, the test tubes were then
removed, placed in a test tube rack, and were allowed to cool to room temperature. Five (5) mL
of distilled water was added to each test tube. The test tubes were stoppered and the contents
were mixed vigorously. After 15 seconds, observations were recorded. The solutions were
acidified by adding a few drops of concentrated H2SO4 into each test tube and mixed using a
stirring rod. The material which collects on top of the solution was noted. A piece of red and blue
litmus paper was then dipped into this material and the pH for the three test tubes were recorded.
Acrolein test. One (1) gram of KHSO4 was weighed and placed in a test tube. Five (5)
drops of the oil sample were added to the test tube. The test tube was heated over a bunsen
burner for a few minutes, allowed to cool, and the presence of a strong odor of acrolein was
noted.
Unsaturation test. Three (3) large sized test tubes were labeled as 'oil', 'fat', and 'glycerol'.
Three (3) mL of dichloromethane was placed in each test tube. Ten (10) drops of the respective
substances were then added to each test tube and mixed thoroughly. Under the fume hood, 5%

bromine-dichloromethane solution was added dropwise to each tube until a reddish brown
bromine color appears. The number of drops was noted.

Results and Discussion

Table I. Grease-Spot Test
Water
Absence of
grease spot; paper
slightly crumpled
Paper crumpled
after drying

Dichloromethane

Filter paper light

yellow,
translucent

Lecithin
Grease spot
smaller than soya
oil; yellow
Smaller
translucent to
light yellow spot
Filter paper light
yellow, slightly
translucent

Filter paper dry

and crumpled

Filter paper dry

4
(coconut
oil)
5
(sunflowe
r oil)
6
(canola
oil)
7
(extra
virgin
olive oil)
8
(palm oil)

Colorless
translucent

Yellowish
translucent

Wrinkled paper

No visible change

Yellow pigment

Yellow pigment

Clear, dried

dried

Colorless almost
transparent spot

Light brownyellow spot

Wrinkled spot

No spot observed

Light yellow
spot; translucent

Light yellow
spot; less
wrinkled

No color

No spot observed

Presence of
grease spot

Absence of
grease spot

Absence of
grease spot

9
(pure
olive oil)

Light yellowish
colored spot;
filter paper
translucent

No color; filter
paper wrinkled

No color; no spot
observed

10
(vegetable
oil)

Big spot

Grease spot not

prominent
compared to palm
oil
Light yellowish
colored spot;
filter paper
slightly
translucent
Yellow stain

Wrinkled

None

Group #
1
(soya oil)

Oil
Presence of
grease spot

2
(palm oil)

Big grease spot

on the filter paper

3
(soya oil)

Absence of
grease spot
Spotted part was
gone

All of the groups were able to observe grease on the area dropped with oil and lecithin
and no spot for the water and dichloromethane. The grease spot test, tests positive for the
presence of triacylglycerols in lipids. The principle behind this test lies on the high boiling points
of most fats and oils. Dichloromethane and water, having considerably lower boiling points as
that of oil and lecithin, are able to absorb enough heat to vaporize. This is not the case for oils
having a boiling point approximately above 300 C, thus leaving a grease spot on the filter paper.
Because of this, the trapped liquid in the filter paper diffracts light giving the oil and lecithin
with a translucent appearance.

Table II. Saponification test

Group #
1
(soya oil)

Observations

Oil

Fat

Water

Observation 1:
after mixing

UL: white bubbles

LL: clear light
yellow soln
UL: turbid grey
soln
LL: turbid white
soln
Acidic

UL: yellow bubbles

LL: turbid white
soln
UL: yellow ppt
LL: turbid white
soln

Clear colorless soln

Acidic

Acidic

Turbid white soln

with bubbles on top

UL: yellow soln

LL:Turbid light
yellow solution

Clear light yellow

soln

Observation 2: after
acidifying
pH of material on
top
Observation 1:
after mixing
Observation 2: after
acidifying
pH of material on
top
Observation 1:
after mixing
Observation 2: after
acidifying

Cloudy white soln

on top
Acidic

Yellow ppt
Acidic

Clear soln; no
material on top
Acidic

Cloudy white soln

White ppt

Cloudy light yellow

soln
Yellow ppt

Clear light yellow

soln
Slightly turbid soln

Acidic

Acidic

Acidic

White soln

Yellow soln

Clear colorless soln

White cloudy soln

w/ white material

Clear colorless soln

pH of material on
top

Acidic

Pale yellow cloudy

soln w/ yellow
material
Acidic

Observation 2: after
acidifying

2
(palm oil)

3
(soya oil)

4
(coconut oil)

pH of material on
top
Observation 1:
after mixing

Clear colorless soln

acidic

5
(sunflower oil)

Observation 1:
after mixing
Observation 2: after
acidifying

6
(canola oil)

pH of material on
top
Observation 1:
after mixing
Observation 2: after
acidifying

7
(extra virgin
olive oil)

pH of material on
top
Observation 1:
after mixing

Observation 2: after
acidifying

8
(palm oil)

9
(pure olive oil)

10
(vegetable oil)

pH of material on
top
Observation 1:
after mixing
Observation 2: after
acidifying
pH of material on
top
Observation 1:
after mixing
Observation 2: after
acidifying
pH of material on
top
Observation 1:
after mixing
Observation 2: after
acidifying
pH of material on
top

UL: white soln w/

bubbles
LL: turbid colorless
soln
White turbid
solution with turbid
white UL and turbid
LL
Acidic

UL: yellow soln w/

bubbles
LL:Turbid light
yellow soln
White turbid soln
with yellow ppt on
top

Clear light yellow

soln

Acidic

Acidic

UL: white soln w/

bubbles on top
LL: turbid white
soln
Turbid white soln
w/ white mass on
top
Acidic

UL: yellow soln w/

bubbles on top
LL: turbid white
soln
Turbid white soln
w/ yellow mass on
top
Acidic

Clear colorless soln

UL: white
emulsion
Interphase: yellow
soln
LL: turbid soln
UL: yellow ppt
LL: turbid white
soln
Acidic

UL: white
emulsion
Interphase: yellow
opaque soln
LL: turbid soln
UL: yellow ppt
LL: turbid white
soln
Acidic

UL: white emulsion

LL: turbid yellow
soln

Turbid beige soln

w/ bubbles on top

Turbid light yellow

soln w/ yellow ppt
on top
Turbid white soln

Clear light yellow

soln few bubbles
on top
Clear colorless soln

--no data--

--no data-Clear colorless soln

Turbid white soln

Turbid yellowish
soln
Yellow ppt

Acidic

Acidic

Acidic

UL: white
suspension
LL: turbid solution
UL: white soln
LL: turbid soln
Acidic

UL: yellow
suspension
LL: turbid soln
UL: yellow solution
LL: Turbid soln
Acidic

Clear colorless soln

Turbid white soln

w/ white ppt on top
--no data-Turbid white soln

Clear colorless soln

Clear colorless soln

Acidic

Clear yellowish
soln
Acidic

Clear colorless soln

Clear colorless
soln
Acidic

When triacylglycerols are subjected to alkaline hydrolysis, the reaction yields glycerol and
the fatty acid salts or soaps. By making use of 3M NaOH, the basic condition is achieved and the
reaction is made to proceed. The formation of bubbles indicates the presence of the soaps in the
form fatty acid salts. By acidifying the soap furthermore, this produces the fatty acid derivatives
of the samples that precipitate on top of the solution.

Figure 2. Saponification
http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/sapon/sapon.gif

The
saponification value is a representation of the number of milligrams of potassium hydroxide that
is required to saponify 1 gram of fat under specific conditions. It is a measure of the chain length
of al the fatty acids present in a sample. A low saponification value indicates a long fatty acid
chain because they have relatively fewer number of carboxlylic acid groups per unit mass of the
sample. Table III lists the saponification number of some oil samples.
Table III. Saponification Number of Some Oils
Oil Type
Saponification Number
Coconut Oil
252
Olive Oil
194.6
Sunflower Oil
193
Palm Oil
142
Acrolein Test is a qualitative test for the presence of glycerol. When a sample containing
a glycerol component is heated together with potassium bisulphate, it will yield molecules of
water together with an aldehyde the acrolein. Acrolein has a characteristic acrid odour, which

makes it easy to be detected. This test is based on the principle of oxidative dehydration, with the
KHSO4 acting as a dehydrating agent.

Table IV. Unsaturation Test

Figure 3. Acrolein Oxidative Dehydration

http://www.cfs.purdue.edu/fn/fn453/Image20.gif

Group #

Sample

Bromine-Dichloromethane
# of Drops
Net # of Drops

1
(soya oil)

Glycerol
Fat
Oil
Glycerol
Fat
Oil
Glycerol
Fat
Oil
Glycerol
Fat
Oil
Glycerol
Fat
Oil
Glycerol
Fat
Oil
Glycerol
Fat
Oil

2
5
49
2
5
36
2
5
50
1
4
6
2
5
65
2
4
40
2
4
39

3
47
3
34
3
48
3
5
3
63
2
38
2
37

Glycerol
Fat
Oil
Glycerol
Fat
Oil
Glycerol
Fat
Oil

2
5
45
2
5
43
2
5
6

3
43
3
41
3
4

2
(palm oil)
3
(soya oil)
4
(coconut oil)
5
(sunflower oil)
6
(canola oil)
7
(extra virgin olive
oil)
8
(palm oil)
9
(pure olive oil)
10
(vegetable oil)

Unsaturation tests differentiate between saturated and unsaturated faty acids and oils and
fats. Halogens are involved in unsaturation tests. Halogens like iodine and bromine will add

across double bonds, thus resulting in the decolorization of the iodine or bromine solution
indicating the presence of unsaturated fatty acids. The more degree of unsaturation, the sample
decolourizes more of the bromine-dichloromethane solution. Glycerol, having no double bonds,
serves as a control for the experiment. Between fats and oils, oils exhibit a lower degree of
unsaturation, thus having a lesser number of C-C double bonds. Among the different oil samples
used, the amount of drop of 5% bromine-dichloromethane is tabulated in Table V in increasing
number of drops.

Table V. Bromine Number Ranking

Group #

Type of Oil

Net # of drops

Group 10

Vegetable Oil (made from coconut) (Baguio)

Group 4

Coconut Oil (Minola)

Group 2

Palm Oil (Golden Fiesta)

34

Group 7

Extra Virgin Olive Oil (Hojiblanca)

37

Group 6

Canola Oil (Jolly)

38

Group 9

Pure Olive Oil (Doa Elena)

41

Group 8

Palm Oil (Golden Fiesta)

43

Group 1

Soya Oil (Jolly)

47

Group 3

Soya Oil (Jolly)

48

Group 5

Sunflower Oil (Colorado)

63

Bromine number/ Iodine Number is the amount of bromine/iodine in grams absorbed by

100 grams (3.5 oz) of a sample. The number indicates the degree of unsaturation. The greater the
number, the greater degree of unsaturation. The ideal iodine number for these types of oils is
summarized in Table VI. Although this table present the iodine number, this data can be used in
relation with the bromine number since the same principle is applied. The data gathered follows
that of the ideal iodine number. One difference observed is the data gathered by group 8. Palm oil
should fall under that of canola and the olive oil. One possible source of error could be that not
enough stirring was done for every every drop of 5% bromine-dichloromethane made.
Table VI. Ideal Iodine Number of Different Oils
Oil Type

Iodine Number

Coconut Oil

7-12

Palm Oil

44-51

Olive Oil

80-88

Canola Oil

105-120

Soya Oil

120-136

Sunflower Oil

125-144

Conclusion
The

different

oil

and

fat

samples

were

characterized

using

the

different

tests.

Grease-spot tests, a test for the presence of triacylglycerols, gave positive results for all the oil
samples and lecithin. Saponification test, which also detects the presence of triacylglycerols,
gave positive results for the oil samples and the fat sample indicated by the production of
bubbles upon base hydrolysis. Lastly, unsaturation test differentiates saturated fatty acids from
unsaturated fatty acids such as the fat and oil sample, wherein between the two, the oil sample
showed a higher degree of unsaturation as to that of the fat sample.

References

Lehninger. (2008). Lehninger principles of biochemistry. New York : W.H. Freeman.

Ferrier, D. R. (2014). Biochemistry. Philadelphia: Lippincott Williams & Wilkins.
Campbell, M. K., & Farrell, S. O. (2012). Biochemistry (7th ed.). Belmont, CA: Brooks/Cole
Cengage Learning.
Adithya, M., Sankar, A., & Peter, V. (2013). Determination of Saponification, Acid and Ester
Values;Percentage of Free Fatty Acids and Glycerol in someSelected edible Oils: Calculation of
concentration of Lye Needed to Prepare soap from These Oils. Scholars Association of Kerala,
Ruby Villa, Kottayam, Kerala , 3 (3), 220-224.

Milczanowski, S. (2012, January 19). Saponification: The preparation of Soap . Retrieved April
7, 2015, from Florida State College at Jacksonville:
http://web.fscj.edu/Milczanowski/eleven/Soap.pdf