Experiment 6: Characterization of Saponifiable Lipids

Abstract:
Saponifiable lipids are lipids that contain a long chain carboxylic (of fatty) acid,
that are linked to an alcoholic functional group through an ester linkage. In this
experiment four different test are done to a fat or oil sample in order to
characterize saponifiable lipids. The first test is the grease-spot which requires a
filter paper as a medium to check the spot that the lipid leaves at room
temperature. The samples were subjected into a saponification test through
alkaline hydrolysis that yielded to the formation of salts of fatty acids
called soaps. After that is the acrolein test is used to determine the presence of
glycerin in a fat. The last test is the unsaturation test pinpoints the level of
saturation and the number of bonds an oil, fat or lipid sample has. The lesser
bromine absorbed by the sample means it is more saturated than samples
that absorbed great amount of bromine.
Keywords: Saponifiable lipids, acrolein, saturated, unsaturation
Introduction
Lipids are an exceedingly diverse group of biologically important materials that
are distinguished by solubility. A lipid is a member of a group of compounds that are
poorly soluble in water but are soluble in organic solvents like benzene or chloroform
which has low polarity. Lipids have a non-polar nature because a large portion of the
molecule contains only carbon and hydrogen. If the structure had significant amounts of
oxygen or nitrogen, the substance would be more polar and hence more soluble in
water (Moore & Langley, 2011). Their biological function is to act as metabolic fuel, as
stored forms of energy, and as components of the cellular membrane. Many lipids work

like soaps and detergents, like soaps, lipids have a polar region and a nonpolar region
which is usually a fatty acid. Figure 1 shows a representation of a soaps structure.

FIG.

Soap forms a

micelle in water in

which the

nonpolar region

(hydrophobic)

of the different

molecules merge and leave the polar region (hydrophilic) on the outside are exposed to
water (Moore &Langler, 2011).
Lipids are classified into fatty acids, triglycerides, sphingolipids, and steroids.
Fatty acids are the smallest portions of the lipids. They are further categorized into
saturated and unsaturated. Saturated or single-bonded fatty acids are typically solid at
room temperature while unsaturated fatty acids which contains a carbon-carbon double
bond are liquid at room temperature. As stated earlier, fatty acids are long-chained
carboxylic acids which, when ionized, cause the formation of H + and the RCOO- anion,
hence when fatty acids react with NaOH it yields to soaps.

When lipids are heated with a dehydrating agent such as KHSO 4, the glycerol
portion of the molecule is dehydrated to form the unsaturated aldehyde, acrolein
(CH2=CH-CHO), a compound with the peculiar, acrid odor of burnt grease.
Unsaturation means having the capacity to still dissolve more substance and this
can be measured by finding out the degree of unsaturation of lipids. It is identified by
adding bromine mixed with dicholoromethane (DCM) to the samples. The presence of
instaurations is indicated by the decolorization of the added bromine.
Methodology
A. Grease-spot test
A pencil was used to label 4 areas of the paper as olive oil, lecithin, H 2o and
dichloromethane (DCM). Pasteur pipets were used to apply to each labeled area of
the filter a drop of the appropriate substance, and then the filter paper was warmed by
placing it on a hot plate that was adjusted to its lowest setting or by placing it on a watch
glass over boiling water. After 5 minutes on the hot plate the observation were described
and recorded.
B. Saponification test
3 large-sized test tubes were labeled as olive oil, fat and water, and then 8
drops of each sample were placed in their corresponding test tubes. 10 drops of 3M
NaOH solution were added to each test tube. The solution was then was heated in a
boiling water bath for 15 20 minutes. The test tubes were cooled down to room
temperature before 5mL of distilled water were added to each test tube. A stopper was

placed on each test tube as the mixed were mixed vigorously. Observations before
acidification were recorded. With a few drops of H 2SO4 the solution were acidified, it was
then mixed by a stirring rod and any material on the top of the solution was noted.
Pieces of red and blue litmus paper were dipped into this material and pH was
recorded, it may be acidic or basic.
C. Acrolien test
A gram of KHSO4 was placed in a test tube. Then, 5 drops of the oil sample was
added. Using a test tubes holder he mixture was heated over the Bunsen burner for a
few minutes. After heating, the mixture was allowed to cool, and the appearance and
odor of the acrolein was described.
D. Unsaturation test
3 large-sized test tubes were labeled as olive oil, fat and glycerol. Then, 3mL
of DCM was added to each tube, after that 10 drops of each sample were added to their
corresponding test tubes. The contents were mixed thoroughly. Under the fume hood,
funnel was used to add 5% bromine-dichloromethane solution to 50mL buret. The initial
volume was recorded. To each tube bromine-dichloromethane was added slowly and
carefully just until it failed to be decolorized, stirring it after each addition. The final
volume of the bromine-dichloromethane solution in the buret and the number of drops
used was recorded.

Results and Discussions:

In the Grease- spot test, samples of oil and some water are smeared onto a
piece of filter paper. After sometime, the water smear would become not translucent.
But the smear of oil would keep translucent for a long time. This is because most
grease or fat (oil and lecithin) are not as evaporative as water and dichloromethane
because they have higher boiling points. In room temperature, the spot dichloromethane
can absorb enough heat and evaporate because it is a volatile chemical. However, the
spot of the olive oil and lecithin can never absorb enough heat even when subjected to
hot-plate heating to hasten the evaporation process and appears as translucent.
Table 1. Grease- spot test results
Sample

Observation after filter paper was warmed

Olive oil

Heavy mark was left after heating

(Filippo Berio

Translucent, colorless spot

Lecithin

Light mark was left after heating

Spot with a pale yellowish color

H2O

No mark was left after heating

DCM

No mark was left after heating

Saponification is a process of hydrolysis of oils or fat with alkaline and result in

glycerol and salts of fatty acids (soap). Saponifiable substances are those that can be
converted into soap. The process of saponification is useful in the separation of
saponifiable materials from unsaponified (which are soluble in lipid). Positive result of

this test is the formation of bubbles on top of the solution Figure shows the alkaline
hydrolysis of triglyceride to yield glycerol and sodium palmitate and the addition of
3H3O+ that yields the fatty acid.

Fig. 2 alkaline hydrolysis of triglyceride

Sodium hydroxide (NaOH) is a caustic base in this experiment but potassium
hydroxide can also be used. A hard soap is formed if NaOH is used, whereas when
KOH is used, a soft soap is formed. Vegetable oils and animal fats are fatty esters in the
form of triglycerides. The base, 3M NaOH causes the ester bond to break and that
releases the fatty acid and glycerol. Saponification is the number of milligrams of NaOH
required to neutralize the fatty acids resulting from the complete hydrolysis of 1g of fat.
The saponification value gives an indication of the nature of the fatty acids constituent of
fat and thus, depends on the average molecular weight of the fatty acids constituent of
fat. The greater the molecular weight (the longer the carbon chain), the smaller the
number of fatty acids is liberated per gram of fat hydrolyzed and therefore, the smaller
the saponification number and vice versa.
Table 2. Saponification test results

Olive oil

Formation of bubbles
on top; dirty white
turbid soln

Pale yellow turbid soln

(acidic)

Melted fat

Formation of bubbles
on top; Yellow turbid
soln

Yellow turbid soln

(acidic)

H2 O

Clear colorless
solution

Clear colorless solution

Acrolein is a compound formed by dehydration of glycerol, basically its presence

indicates the occurrence of a glyceride ester that are usually a triglyceride (fat or oil).
When the enzyme lipase hydrolyzes a compound lipid, one of the products of the
hydrolysis is glycerol. When heated with the dehydrating agent (KHSO4), the glycerol
portion of the molecule is dehydrated to yield the unsaturated aldehyde, acrolein
(CH2=CH-CHO), a compound which has the peculiar odor of burnt grease.
The fatty acid residues may differ according to the extent of present unsaturated
bonds in the hydrocarbon chain. This is usually measured by the iodine number but in
this experiment bromine is used instead of iodine because iodine adds less readily than
bromine across the double bonds in an unsaturated bond. The extent of unsaturated
bonds can be demonstrated by adding a halogen solution (bromine-DCM) that
decolorizes the sample. The free bromine then attaches itself to the double bonded
carbons; the presence of unsaturation is indicated by the decolorization of the added
bromine. The more double bonds a fat contains, the more iodine is required for the
addition reaction; thus, a high bromine number means a high degree of unsaturation.
The test indicated that animal fats are saturated and vegetable fats are unsaturated.

Table 3. Unsaturation test results

Number of drops of
Br2-DCM added

Net Number of
drops of
Br2-DCM
added

Glycer
ol

Fats

12

Olive
Oil

121

116

Conclusions:
The 4 different tests (grease-spot test, saponification test, acrolein test and
unsaturation test) where the samples are subjected to able to characterized saponifiable
lipids.
There are two general types of lipids. Simple lipids such as cholesterol and other
steroids do not have the ester linkages and cannot be hydrolyzed and complex lipids
which include fats, oils, and waxes that contain ester linkages that can be hydrolyzed to
smaller molecules via saponification test.
Due to the fact that the cis C=C bond in the hydrocarbon chains, which reduces the
extent of association of the molecules unsaturated fatty acids have lower melting points
and boiling points than saturated fatty acids.

The high melting points of the solid animal fats are associated to the fact that the
saturated fats have straight chains and the molecules can pack together more closely
than vegetable oils which has a greater portion of unsaturated fatty acids.

Reference:
Books

Bettelheim, F. (2013). Laboratory Experiments for Introduction to General, Organic and

Biohemistry. Belmont, Calif. : Brooks/ Cole.
Campbell, M. & Farrell, S. (2012). Biochemistry (7th ed.) Belmont: CA: Brooks/Cole
Cengage Learning.
Moore, J. &Langley R. (2011). Biochemistry For Dummies. Indianapolis, Indiana: Wiley
Publishing, Inc.
Murray, R., Daryl K. Garner & Victor W. Rodwell. Harpers Illustrated Biochemistry. New
York:McGraw-Hill, 2006.
Seager, S. (2011). Organic and Biochemistry for Today. Australia: Brooks/ Cole
Cengage Learning.
Internet Sources
(2012, 10). Saponifiable Lipids Fr. StudyMode.com. Retrieved September 24, 2014 from
http://www.studymode.com/course-notes/Saponifiable-Lipids-Fr-1118432.html
Lipids. . Retrieved September 24, 2014 from
http://www.chem.latech.edu/~deddy/chem121/Lipids.htm