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 .

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 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 Spot formed; Spot formed; not No spot; area not No spot; area not
Canola Oil translucent translucent translucent translucent
2 Spot remained Slight spot remained No spot remained No spot remained
Butter
3 Spot remained Spot remained No spot remained No spot remained
Sesame Oil
4 Spot visible, Spot less visible No spot visible No spot visible
Olive Oil translucent grease than olive oil
mark
5 Translucent grease Translucent grease Absence of spot Absence of spot
Corn Oil mark mark
6 Translucent grease Slight translucent No visible changes No visible changes
Extra Virgin mark grease mark
Olive Oil
7 Spot formed; Spot slightly seen; No spot formed; Spot formed, not
Margarine translucent not translucent evaporated translucent
8 Translucent grease Slight translucent No visible change No visible change
Coconut Oil mark grease mark
9 Spot formed; Spot formed; Slightly No Visible spot No Visible Spot
Castor Oil Translucent Translucent
10 Translucent spot Translucent spot No translucent spot No translucent spot
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:

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 Table 2. Saponification Test of oil samples.
Group After Heating & Shaking After Acidification pH
1 Bubbles formed, cloudy solution White substance on top Acidic
Canola Oil
2 Cloudy white with bubble formation White cloudy ring at the surface Acidic
Butter
3 Lesser bubble formation on the top Light yellow top layer; white Acidic
Sesame Oil compared to fat; cloudy solution solution
4 Formation of few bubbles Whitish material formed in the top Acidic
Olive Oil of the whitish solution
5 Upper layer: yellow, lower layer: White turbid solution with yellow Acidic
Corn Oil white liquid, bubble formation material
6 Presence of bubbles and yellow top Yellow top layer in a turbid solution Acidic
Extra Virgin layer in transparent solution.
Olive Oil
7 Light yellow, thick solution Turbid w/ light yellow layer on top Acidic
Margarine
8 Appearance of bubbles and Appearance of bubbles and Acidic
Coconut Oil precipitate on top precipitate on top
9 Slightly turbid, w/ bubbles White layer on top Acidic
Castor Oil
10 White liquid w/ bubbles White layer on top Acidic
Palm Oil

Table 3. Saponification Test of Melted Fat.

Group After Heating & Shaking After Acidification pH
1 Cloudy, yellowish solution. bubbles Yellow substance on top Acidic
formed
2 Cloudy yellow with bubble formation Yellow ring at the surface Acidic
3 Greater bubble formation at the top, Yellow top layer; cloudy solution Acidic
suspended white particles in the soln.
4 Formation of bubbles in the upper Yellowish material formed in top Acidic
layer of a white opaque solution
5 Upper layer: yellow ppt., lower layer: White turbid solution with yellow Acidic
yellow turbid solution, bubble ppt.
formation
6 Turbid solution with the presence of Yellow layer on top of turbid Acidic
bubbles and a yellow top layer solution
7 Yellow-orange w/ oil precipitate Yellow layer on top Acidic
8 Appearance of bubbles and Appearance of bubbles and Acidic
precipitate on top precipitate on top
9 Slightly yellow turbid soln w/ bubbles Yellow layer on top Acidic
10 Light yellow liquid w/ yellow bubbles Yellow layer on top Acidic
on top

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 Table 4. Saponification Test of HO.
Group After Heating & Shaking After Acidification pH
1 Bubbles formed, clear yellowish solution No formation on top Acidic
2 Clear solution w/ little bubble formation Clear solution Acidic
3 Least bubble formation at upper layer Clear solution Acidic
4 No bubbles formed Clear Solution Acidic
5 Transparent liquid Transparent liquid Acidic
6 Transparent Solution Clear solution w/o top layer Acidic
7 Light yellow liquid Clear solution Acidic
8 No bubbles and precipitate on top No bubbles and precipitate on top Acidic
9 Transparent solution w/ bubbles W/ slightly soluble material Acidic
10 Clear liquid Clear Solution 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 Saponification Number
Canola Oil 173
Butter
Sesame Oil 188-195
Olive Oil 184-196
Corn Oil 188-193
Extra Virgin Olive Oil 184-196
Margarine
Coconut Oil 248-265
Castor Oil 177-185
Palm Oil 190-205

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 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

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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-tests-

for-lipids/13050

Sottrup-Jensen, L. (2007). General biochemistry: Instructions for laboratory exercises.

Aarhus: Department of Molecular Biology, University of Aarhus.

Vegetable oil - MedLibrary.org. (n.d.). Retrieved October 23, 2016, from

http://medlibrary.org/medwiki/Vegetable_oil

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

October 24, 2016, from vlab.amrita.edu/?sub=3&brch=63&sim=688&cnt=1

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