Characterization of Lipids of Melted Fat, Lecithin, Glycerol and

Vegetable Oils through Qualitative Tests

*Pua Phee, Marie Pamela Celestine E.
Relopez, Maria Charmella P.
San Pedro, Anna Paula R.
So, Ignacius de Charles T.
College of Science, University of Santo Tomas, Espaa Blvd., Manila

Abstract
Lipids are a diverse group of molecules that are hydrophobic and soluble in non-polar solvents. They
function as the major constituents of biological membranes. Lipids c an be classified as saponifiable or
nonsaponifiable. Saponifiable lipids are esters of fatty acids that can undergo saponification (base
hydrolysis of the esters of fats or oils to afford glycerol and the salt of the corresponding fatty acid. This
experiment characterizes saponifiable lipids through four tests. Grease-spot test indicates the presence of
lipids; a filter paper was used and results show that the vegetable oil sample and lecitihin are positive
(translucent spot). Saponification test detects whether the sample has ester bonds . Hydrolysis and
neutralization of the fatty acids through addition of NaOH and dehydration and acidification through
adding HSO yielded bubbles and a precipitate, and an acidic pH tested by a litmus paper if the sample
is positive. The vegetable oil and melted fat tested positive, while the HO tested negative. Acrolein test
detects the presence of glycerols. Upon the addition of KHSO, a dehydrating agent, a pungent odor and
blackening of the mixture indicates a positive result. Unsaturation test determines the degree of saturation
and the presence of double bonds. The reagent bromine-dichloromethane binds to the double bonds of
the unsaturated fatty acids which means that the vegetable oil, melted fat, and glycerol tests as positive.

Introduction
Biological lipids are a chemically diverse group of molecules, the common and
defining feature of which is their insolubility in water. They are generally hydrophobic.
Their functions vary as stored forms of energy in many organisms, phospholipids and
sterols are major structural elements of biological membranes. Other lipids, although
present in relatively small quantities, play crucial roles as enzyme cofactors, electron
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carriers, light-absorbing pigments, hydrophobic anchors for proteins, chaperones to

help membrane proteins fold, and emulsifying agents in the digestive tract, hormones,
and intracellular messengers (Nelson & Cox, 2005). Lipids are non-polar, making them
soluble in non-polar solvents (like dissolves like).
The simplest lipids are the fatty acids, which constitute many complex lipids. They
can be further divided to saturated and unsaturated fatty acids. Saturated fatty acids have
carbons that are saturated with H atoms and only have single bonds, while unsaturated
fatty acids contain one or more double bonds. Many important naturally occurring fatty
acids are unsaturated (Appling, Anthony-Cahill, & Matthews, 2016).
Fats or triacylglycerols are the form of storage of fatty acids in organisms, they are
triesters of fatty acids and glycerol. Those triacylglycerols that are solid are called fats,
while those that are liquid are called oils.
Phospholipids are lipids with phosphate-containing groups. As stated, lipids are
major components of biological membranes, and the major classes include the
glycerophospholipids, sphingolipids and glycosphingolipids (in animals). Glycerolipids are
widespread in plant and bacterial membranes (Appling, Anthony-Cahill, & Matthews,
2016).
Lipids can also be classified as saponifiable or nonsaponifiable. Saponifiable lipids
are esters of fatty acids that can undergo saponification. This includes the triglycerides,
and phospholipids. Nonsaponifiable lipids are lipids that do not contain any fatty acids or
ester linkages. Examples are the steroids, prostaglandins, leukotrienes and terpenes .

Vegetable oil is a tryglyceride obtained from plants. Castor oil is a vegetable oil
extracted from the seeds of the castor oil plant (Ricinus communis) (Vegetable oil,
2016). It contains ricinoleic acid, an unsaturated fatty acid and has various uses, such as
increasing hair growth, lubricant, and skin cleanser.
This experiment aims to characterize a fat or an oil sample using the grease-spot
test, saponification test, acrolein test, and unsaturation test.
Methodology
A. Grease-Spot Test
A filter paper was obtained and four areas were labelled as castor oil,
lecithin, HO, and dicloromethane. After which a drop of each sample was
placed in the corresponding areas of the filter paper, with the use of Pasteur pipets.
It was then subject to heat by placing it on a hot plate adjusted to its lowest setting.
The translucence of the lipid samples were observed.
B. Saponification Test
Three large-sized test tubes labelled oil, fat, and HO were used for this
test. 8 drops of each sample were placed into their corresponding test tubes. 10
drops of NaOH solution was then added to each tube, which was then placed in a
boiling water bath for 15-20 minutes. After heating, it was cooled to room
temperature, then 5 mL of distilled water was added to the test tubes. The tubes
were stoppered using a cork and the contents were vigorously mixed, and
observations were recorded. The mixtures were acidified with a few drops of
HSO which was checked with blue litmus paper. A glass stirring rod was used to
mix the contents of the tube and the material which collects on top of the solution
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was noted. The pH of the material was recorded using a piece of blue and red
litmus paper, then observations were recorded.
C. Acrolein Test
1 gram of KHSO was weighed and placed in a test tube. 5 drops of the oil
sample or a small piece of solid lipid was placed in the tube. The test tube was
heated over a Bunsen burner for a few minutes, holding it with a test tube holder.
It was allowed to cool while noting any strong odor of acrolein.
D. Unsaturation Test
Three large-sized test tubes labelled oil, fat, and glycerol were used. 3
mL of dichloromethane was placed in each test tube. 10 drops of each sample
were placed into their corresponding test tubes, then the contents were thoroughly
mixed. 5% bromine-dichloromethane solution was added to a 50 mL buret using a
funnel under the fume hood. The initial volume was recorded. Subsequently the
bromine-dichloromethane solution was added drop wise from the buret to each
test tube while stirring until the reddish-brown bromine color first appears. The final
volume of the bromine-dichloromethane solution in the buret was recorded and the
volume of the solution required for the reaction was calculated.
Results and Discussion
A. Grease-Spot Test
The grease-spot test is a general test indicating whether a sample is a lipid
or not. A positive result is a visible, translucent spot left by the sample. In this test,
castor oil and lecithin tested positive. The dichloromethane (DCM) and HO
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samples tested negative because they do not have any lipid components. Fats are
non-volatile, meaning they have high boiling points. In the test, HO and DCM are
evaporated. The lecithin and castor oil showed a translucent spot after placing it in
the hot plate. The translucence observed is because light can be diffracted in the
spot (Experiment 8: Grease Spot Test, 2016). Table 1 shows that all groups
obtained the correct results for each sample.
Table 1. Grease-spot Test.
Group

Vegetable Oil

Lecithin

Dichloromethane

HO

1
Canola Oil
2
Butter
3
Sesame Oil
4
Olive Oil

Spot formed;
translucent
Spot remained

Spot formed; not

translucent
Slight spot remained

No spot; area not

translucent
No spot remained

No spot; area not

translucent
No spot remained

Spot remained

Spot remained

No spot remained

No spot remained

Spot visible,
translucent grease
mark
Translucent grease
mark
Translucent grease
mark

Spot less visible

than olive oil

No spot visible

No spot visible

Translucent grease
mark
Slight translucent
grease mark

Absence of spot

Absence of spot

No visible changes

No visible changes

Spot formed;
translucent
Translucent grease
mark
Spot formed;
Translucent
Translucent spot

Spot slightly seen;

not translucent
Slight translucent
grease mark
Spot formed; Slightly
Translucent
Translucent spot

No spot formed;
evaporated
No visible change

Spot formed, not

translucent
No visible change

No Visible spot

No Visible Spot

No translucent spot

No translucent spot

5
Corn Oil
6
Extra Virgin
Olive Oil
7
Margarine
8
Coconut Oil
9
Castor Oil
10
Palm Oil

B. Saponification Test
Saponification is the base/alkaline hydrolysis of the esters of fats or oils to
afford glycerol and the salt of the corresponding fatty acid. The term literally means
"soap making". The principle of this test is that fats (triglycerides), upon alkaline
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hydrolysis (either with KOH or NaOH) will yield glycerol and potassium or sodium
salts of fatty acids (soap).

Figure 1. Principle of saponification.

Triacylglycerols are nonpolar, hydrophobic, insoluble and water and is
bound by ester bonds (Estimation of Saponification Value, 2016). In the
experiment, upon the addition of NaOH, a strong base, all samples show turbid
solutions. The NaOH serves to neutralize the fatty acids. After heating and shaking
vigorously, the presence of bubbles and a precipitate should form (positive).
Almost all groups obtained the same observation in the vegetable oils and melted
fats. HSO was used to acidify and as a dehydrating agent. Upon its addition, the
melted fat or oil floats on top of the solution. With the use of litmus paper, its pH is
recorded and it should test positive (acidic) in the melted fat and oil and negative
on the HO, however, human error and mishandling of glass wares may have
caused this result. Saponification test yields positives for lipids that can undergo
alkaline hydrolysis, as well as those with ester bonds. Results are tabulated below:

Table 2. Saponification Test of oil samples.

Group
1
Canola Oil
2
Butter
3
Sesame Oil
4
Olive Oil
5
Corn Oil
6
Extra Virgin
Olive Oil
7
Margarine
8
Coconut Oil
9
Castor Oil
10
Palm Oil

After Heating & Shaking

Bubbles formed, cloudy solution

After Acidification
White substance on top

pH
Acidic

Cloudy white with bubble formation

White cloudy ring at the surface

Acidic

Lesser bubble formation on the top

compared to fat; cloudy solution
Formation of few bubbles

Light yellow top layer; white

solution
Whitish material formed in the top
of the whitish solution
White turbid solution with yellow
material
Yellow top layer in a turbid solution

Acidic

Light yellow, thick solution

Turbid w/ light yellow layer on top

Acidic

Appearance of bubbles and

precipitate on top
Slightly turbid, w/ bubbles

Appearance of bubbles and

precipitate on top
White layer on top

Acidic

White liquid w/ bubbles

White layer on top

Acidic

After Acidification
Yellow substance on top

pH
Acidic

Yellow ring at the surface

Yellow top layer; cloudy solution

Acidic
Acidic

Yellowish material formed in top

of a white opaque solution
White turbid solution with yellow
ppt.

Acidic

Yellow layer on top of turbid

solution
Yellow layer on top
Appearance of bubbles and
precipitate on top
Yellow layer on top
Yellow layer on top

Acidic

Upper layer: yellow, lower layer:

white liquid, bubble formation
Presence of bubbles and yellow top
layer in transparent solution.

Acidic
Acidic
Acidic

Acidic

Table 3. Saponification Test of Melted Fat.

Group
1
2
3
4
5

6
7
8
9
10

After Heating & Shaking

Cloudy, yellowish solution. bubbles
formed
Cloudy yellow with bubble formation
Greater bubble formation at the top,
suspended white particles in the soln.
Formation of bubbles in the upper
layer
Upper layer: yellow ppt., lower layer:
yellow turbid solution, bubble
formation
Turbid solution with the presence of
bubbles and a yellow top layer
Yellow-orange w/ oil precipitate
Appearance of bubbles and
precipitate on top
Slightly yellow turbid soln w/ bubbles
Light yellow liquid w/ yellow bubbles
on top
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Acidic

Acidic
Acidic
Acidic
Acidic

Table 4. Saponification Test of HO.

Group
After Heating & Shaking
After Acidification
1
Bubbles formed, clear yellowish solution
No formation on top
2
Clear solution w/ little bubble formation
Clear solution
3
Least bubble formation at upper layer
Clear solution
4
No bubbles formed
Clear Solution
5
Transparent liquid
Transparent liquid
6
Transparent Solution
Clear solution w/o top layer
7
Light yellow liquid
Clear solution
8
No bubbles and precipitate on top
No bubbles and precipitate on top
9
Transparent solution w/ bubbles
W/ slightly soluble material
10
Clear liquid
Clear Solution

pH
Acidic
Acidic
Acidic
Acidic
Acidic
Acidic
Acidic
Acidic
Acidic
Acidic

The quantitative part of the saponification test but was not performed in the
experiment involves calculating the saponification number of value of the fat. The
saponification number is also called the Koettstorfer number. It is the weight (in
milligrams) of potassium or sodium hydroxide required to neutralize the fatty acids
resulting from the complete hydrolysis of 1 g of fat (Estimation of Saponification
Value, 2016). The higher saponification number, the lower the molecular weight
of the lipid. Listed below are the saponification numbers of the vegetable oils used
by the groups.
Table 5. Saponification numbers of oil samples.
Oil Sample
Canola Oil
Butter
Sesame Oil
Olive Oil
Corn Oil
Extra Virgin Olive Oil
Margarine
Coconut Oil
Castor Oil
Palm Oil

Saponification Number
173
188-195
184-196
188-193
184-196
248-265
177-185
190-205

The two remaining tests (acrolein and unsaturation test) were not performed in the
experiment, however, the principles will be discussed.
C. Acrolein Test
This test detects the presence of fats/glycerols. Its principle is the oxidation
and dehydration reaction due to the exposure to heat (Sottrup-Jensen, 2007).
Acrolein is an unsaturated aldehyde formed when glycerol reacts with potassium
hydrogen sulfate (KHSO). As stated previously, glycerol is the product of the
hydrolysis of a lipid. The reagent KHSO then serves as a dehydrating agent. Too
much heating would also cause the blackening of the mixture, and a positive result
of this test would be the pungent odor of the acrolein.
D. Unsaturation Test
As the name suggests, this test determines the degree of saturation as well
as the presence of double bonds of the lipid. As mentioned in the introductions,
saturated fatty acids only have single bonds while unsaturated fatty acids contain
double bonds. The reagent used, bromine-dichloromethane, contains a halogen
component. This halogen reacts with unsaturated fatty acids (Qualitative and
Quantitative Tests for Lipids, 2016). A reddish-brown bromine color appears,
indicating the presence of double bonds. Therefore, the vegetable oil, melted fat,
and glycerol would test positive. The degree of saturation of the sample could be
determined by the amount of bromine taken up by the lipid sample, which is why
the initial and final volumes should be recorded. Furthermore, iodine can also
substitute bromine in this test

Conclusion
Saponifiable lipids were characterized in the experiment through two tests. The
grease-spot test show visible positive result in the vegetable oil and lecithin.
Saponification test show the formation of soaps by base hydrolysis of the esters of fats
or oils to afford glycerol and the salt of the corresponding fatty acid. The presence of
bubbles and precipitate and an acidic pH resulted from the vegetable oil and melted fat,
indicating that it is positive.
References
Appling, D.R., Anthony-Cahill, S.J., & Mathews, C.K. (2016) Biochemistry: Concepts
and connections. Boston: Pearson Education Unlimited.
Experiment 8: Grease Spot Test - lungtp.com. (n.d.). Retrieved October 24, 2016, from
http://www.lungtp.com/biochem/e_bcdxb.html
Nelson, D., & Cox, M. M. (2005). Lehninger Principles of Biochemistry. New York: W.H.
Freeman and.
Qualitative and Quantitative Tests for Lipids. (n.d.). Retrieved October 24, 2016, from
http://www.biologydiscussion.com/lipids/tests/qualitative-and-quantitative-testsfor-lipids/13050
Sottrup-Jensen, L. (2007). General biochemistry: Instructions for laboratory exercises.
Aarhus: Department of Molecular Biology, University of Aarhus.
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oil

MedLibrary.org.

(n.d.).

Retrieved

http://medlibrary.org/medwiki/Vegetable_oil
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October

23,

2016,

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vlab.amrita.edu,. (2011). Estimation of Saponification Value of Fats/Oils. Retrieved

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