CHEM 137.

2 – BIOCHEMISTRY (LABORATORY)
LABORATORY REPORT

Name: Reynand E. Corpuz Date Submitted: January 22, 2021

Lab Schedule: TTh – 8:00 – 11:00 Date Performed: January 19, 2021

Exercise No. 4
Lipids
I. OBJECTIVES:
At the end of the experiment, the student is expected to:
1. Examine the chemical and physical properties of lipids.
2. To distinguish the differences between saturated and unsaturated oils.
3. Observe the saponification of triacylglycerol in the preparation of soap.
4. Compare the cleaning capacity of different soap solutions.

II. RESULTS AND DISCUSSION:

A. Equations for the formation and hydrolysis of a Triacylglycerol

Fig. 1. Formation and hydrolysis of a triacylglycerol

The formation and hydrolysis reaction of triacylglycerol are shown in the figure above (see Figure
1). For the formation of triglyceride (triacylglycerol), the glycerol must react with free fatty acids through
the process of esterification. Esterification is a process by which an organic acid, usually carboxylic acid
(RCOOH) is reacted with alcohol (ROH) to form an ester (RCOOR) and water. Triglyceride is a triester,
which means three ester linkages are formed due to the reaction of glycerol and three fatty acid. If the
ester linkage is only one (1) it is called monoglyceride (monoacylglycerol) and if the the linkage is two
(2), then it is called diglyceride (diacylglycerol).
 As illustrated in the figure above, the formation of triglycerides is a reversible reaction. Since water
is a reactant in the reverse path, then it is now a hydrolysis reaction of triglycerides. Hydrolysis reaction
is the opposite of the esterification as it uses water to break down the molecule into its simpler components.
The hydrolysis of triglycerides makes the ester linkages in the molecule to break apart therefore, forming
glycerol and three fatty acid as the resulting products. However, the hydrolyzation of triglyceride is not
spontaneous as it needs the presence of acid and heat or with a proper lipase enzyme under biological
condition.

B. Solubility of Lipid Sample on Different Solvents

Table 1. Solubility of lipids (mustard oil) in different solvents

Solvents Solubility (Soluble or Insoluble)

Water Insoluble

Ethanol Soluble

Diethyl ether Soluble

Chloroform Soluble

The solubility of a certain compound depends on the polarity of the compounds itself and the
solvent used. Lipids, which includes steroids, waxes, fats, and oils, are a diverse group of organic
compounds that are related by their solubility characteristics. All the lipids mainly composed of long chain
of hydrocarbon, making them hydrophobic or non-polar in nature. Since lipid is nonpolar, then they will
theoretically only dissolve to non-polar solvents and will not dissolve to polar solvents, according to the
principle ‘like dissolves like’. The lipid used in the experiment was mustard oil, which is a fatty vegetable
oil. It was determined in the experiment that mustard oil is soluble to non-polar solvent such as ethanol,
diethyl ether, and chloroform but is insoluble to water which is a polar solvent. The result was appropriate
because it coincides with the principle ‘like dissolves like’. The absence of a strong intermolecular forces
such as dipole-dipole and hydrogen bonding between the molecules of water and lipid (mustard oil) is the
main reason why polar solvent such as water cannot dissolve lipids.
 C. Test for Unsaturation of Oils
Table 2. Unsaturation test for oils using bromine test
Oils Saturated or Unsaturated

Olive oil Unsaturated

Coconut oil Saturated

Corn oil Unsaturated

Palm oil Unsaturated

Sunflower oil Unsaturated

The bromine test is used to determine the presence of unsaturation such as double bonds and triple
bonds in a certain compound. The concept of the test is that bromine is very easy to polarize by the
double bond or triple bond of an organic compound such as cyclohexene. The now induced dipole of the
bromine is being electrophilically added through the attack of the pi bond, resulting to a formation of a
dibromo compound.

Fig. 2. Electrophilic addition of bromine

In the experiment, the oils underwent unsaturation test using bromine-water and it was found out
that olive, corn, palm, and sunflower oils had positive results as the color of the bromine-water was
disappeared after vigorous shaking, which confirms the presence of unsaturation in the structures of these
oils. These oils therefore determined to be unsaturated hydrocarbons. The electrophilic addition reaction
of bromine to the oils caused the decolorization as the bromine is now being integrated in the lipid
molecule. On the other hand, coconut oil had a negative result in bromine test therefore it is a saturated
hydrocarbon.
 D. Saponification: Formation of Soap
Write the equation for saponification process.

Saponification is the reaction that involves the conversion of lipid into soap. The reaction is
somewhat similar to hydrolysis as it also produces glycerol, but the difference is that in saponification,
the triglyceride was reacted with strong base NaOH to produce glycerol and sodium carboxylate which
is the soap molecule. The addition of the NaOH induces a nucleophilic attack from the hydroxide ions
to the ester group of the triglyceride. The leaving group then is removed, and deprotonation occurs to
form the carboxylate salt at the end. Soaps are carboxylate salts of fatty acids with a long hydrocarbon
chain. The by-product glycerol is both a humectant (attracts moisture from the air) and an emollient
(soothes or softens the skin).

E. Cleaning Capacity of Soaps

Tests A B C

Solubility (Soluble/Insoluble) Soluble Soluble Insoluble

Yes Yes Yes

Presence of foam (Yes/No) (Maximum (Medium length of (Maximum length
length of lather) lather) of lather)

Cleaning action
Effective Effective (Partially) Ineffective
(Effective/Ineffective)

The last experiment was to determine the effect of ions in the solubility, foaming and cleaning
capacity of soaps. The soap used in each sample was the same and they only differ on the quality of
water the soap was dissolved into. The first test tube is a solution of soap and distilled water (soft water).
 On the other hand, the second solution contains soap dissolved in well water (hard water). While the last
solution contains soap and distilled water with dissolved CaCl 2 (very hard water). Based on the result, it
was observed that soap is soluble and dissolved completely in distilled and well water. The reason for
the solubility of soap in distilled and water is due to the low presence of ionic species suspended in these
waters. On the other hand, it was observed that scum or precipitate was produced as soap was dissolved
in the distilled water with dissolved CaCl2. This is because in distilled water with dissolved CaCl 2, the
Ca2+ ion reacts with soap to form calcium salt of soap which is insoluble in water making to precipitate
out of solution. The reaction was shown below.
COONa + CaCl2 → (RCOO)2Ca + 2 NaCl
The foaming capacity of the soap solution were also tested by shaking the solution in a vigorous
manner. It was observed that the soap solution in the test tube A produces maximum length of lather, the
solution B produces smaller length of lather, and the solution C produces the shortest (minimum) length
of lather among the three.
The cleansing capacity of each soap solution were also tested through assessing its ability to
remove stain from the cloth. It was observed that the ink spot on the cloth dipped in solution A, B and C
has almost disappeared, partially disappeared, and not disappeared at all, respectively. Therefore,
hardness of water affects the cleansing capability of water because suspended ions can react with the
soap molecules. Soap has a polar and nonpolar end which can encapsulates oily material by forming
micelles making it soluble to water. The long nonpolar tail of soap and detergent molecules attracts the
oil and surrounds it in a way that all of the tails are inside the sphere while the charged head faces the
exterior surface which can be dissolved by water. This is why soap can clean effectively because dirt,
ink and grease are oily in nature.

III. CONCLUSIONS
In the experiment, it was determined that esterification of glycerol and fatty acid produces
triglyceride and water while the reverse reaction, which is the hydrolysis, needs the presence of acid
and heat to proceed in reverse path. In the second part, the mustard oil was observed to be soluble in
ethanol, diethyl ether, and chloroform but was insoluble in water. Moreover, it was determined that
olive, corn, palm, and sunflower oils are unsaturated oils while coconut oil is a saturated one. It was
also determined that in saponification, triglyceride is added with strong base to produce glycerol and
soap molecules. Furthermore, it was also observed that the hardness of water affects the solubility,
foaming capacity, and cleaning action of the soap. The solution of soap and distilled water is observed
to be homogenous (soluble) and has the highest foaming and cleaning capacity. The solution of soap
and well water was also observed to be homogenous (soluble) and has moderate foaming and cleaning
capacity. Lastly, the solution of soap and distilled water with dissolved CaCl 2 was observed to be
heterogenous (precipitate) and has the lowest foaming and cleansing capacity.