BIOCHEMISTRY

LABORATORY REPORT ON
EXPERIMENT 3: TEST FOR LIPIDS
DMED-1A; GROUP 1: Alborte, T., Amador, A., Amurao, G., Antonio, A., Ayonque, K., Bandoy,
M., Bartolome, C., Basilio, H., Boco, J., Borromeo, K., Buencamino, J., Cabrera, M., Cayari, M.,
Co, J., Corpuz, M., De Torrez, L.

INTRODUCTION

Lipids constitute a large heterogenous group of unrelated physiological and chemical

substances classified together because they are fat-like substances which are insoluble in water
but are soluble in organic solvents such as chloroform, ether, carbon tetrachloride, alcohol, and
benzene. Important lipids include triacyglycerols, cholesterol, and derivatives of arachidonic
acid. Aside from being a rich energy source, lipids play important role as structural components
in practically all plant and animal cells. In the human body, lipids are found mostly in cellular
structures like the cell membrane, in vital organs like the brain and in various tissues like the
nervous tissue.

LABORATORY PROCEDURES

A. Solubility Tests

Solubility test is the preliminary test which detects the presence of all lipids. This test
detects the solubility of lipid in various solvents to check whether it is miscible or
immiscible in polar or non-polar solvents. Principle: Solubility test is based on the property
of lipid to dissolve in different solvents.

 Materials: pipette, stoppered vials, dropper, coupon bond paper

 Solvents: distilled water, ethyl alcohol, ether, chloroform, benzene, 5% HCL, 5%
naOH
 Sample: cottonseed oil

Procedure:

1.
Pipette 1ml of the Add 1-2 drops of
Record the time
required solvents cottonseed oil in
required for the
in separate each vial and
oil to dissolve.
stoppered vials shake thoroughly.
Results:

SAMPLE SOLVENT RESULT

Distilled water Insoluble
Ethyl alcohol Insoluble
Ether Soluble
Cottonseed Oil Choloroform Soluble
Benzene Soluble
5% HCL Insoluble
NaOH Insoluble

Rationale:

The term lipid solubility is also known as lipophilicity, a term usually refers to
refers to the capability of a substance or compound to dissolve in lipids, fats, or oils, lipids.
Lipids are soluble in nonpolar solvents and insoluble in polar solvents. There are more
hydrocarbons in a lipid molecule which are largely hydrophobic than their polar side
making lipids insoluble in organic solvents. On the other hand, lipids are soluble in
nonpolar solvents because of the presence of weak bonds.

2. Prepare the following mixtures:

 Cottonseed oil-ethyl acohol
 Cottonseed oil-ether

On different spots of a
piece of coupon bond
paper, place 3 drops
of each mixture.
Compare the solubility
Allow the solvents to
of the oil in the two
evaporate.
solvents.
 MIXTURE RESULT EXPLANATION
Cottonseed Oil – Ethyl Cottonseed oil residue
Evaporates; miscible
Alcohol remains on the paper
Cottonseed Oil - Ether Evaporates; miscible because lipids do not
evaporate.

Rationale:

When both substances are polar, then they can attract one another - which leads
to mixing (miscibility). In this experiment, both ethyl and ether are polar substances thus
allowing it to attract the polar functional groups of lipids resulting in miscibility.

B. Acrolein Test

Acrolein test is used to detect the presence of glycerol and fat. This test is based
on the “Dehydration reaction”, where the water molecules removed from the glycerol by
the addition of reagent potassium hydrogen sulphate. The reaction between glycerol and
potassium hydrogen sulphate results in the formation of “Acrolein” that is characterized
physically by the release of the pungent smell.

 Materials: 2 test tubes, dropper

 Samples: glycerol, cottonseed oil
 Reagent: KHSO4
 Procedure:

Prepare 2 test tubes. Place in:

Test tube 1: 2 drops of glycerol + a
pinch of KHSO4 Heat each test tube over low flame. Note the odor produced.
Test tube 2: 2 drops of cottonseed oil
+ a pinch of KHSO4

Results:

SAMPLE REAGENT RESULTS

Glycerol KHSO4 Pungent odor
Cottonseed KHSO4 No odor

Rationale:

If glycerol present in the sample it will give a pungent smell due to the formation
of acrolein. If glycerol is absent in a sample, then it will not produce a pungent smell.

C. Test for Rancidity

Rancidity generally is the complete or incomplete oxidation or hydrolysis of fats

and oils when exposed to air, light, or moisture or by bacterial action, resulting in
unpleasant taste and odor. There are three types of rancidity:

1. Hydrolic Rancidity. It occurs when water splits fatty acid chains away from the
glycerol backbone in glycerides.
 2. Oxidative rancidity. It occurs when the double bonds of an unsaturated fatty
acid react chemically with oxygen.
3. Microbial rancidity. It occurs when microorganisms such as bacteria use their
enzymes to break down chemical structures in the fat.

 Materials: 6 tubes/vials, dropper

 Samples: fresh coconut oil, rancid coconut oil
 Reagents: phenolphthalein, methyl orange, pH paper

Procedure:

1. Prepare 6 tubes or vials:

 In each of the test tubes # 1, 2, 3, place 5 drops of fresh coconut oil
 In each of the test tubes 4, 5, 6, place 5 drops of rancid coconut oil

2. Test the reaction of fresh coconut oil with:

 Phenolphthalein
 Methyl orange
 pH paper

3. Do the same reaction tests with the rancid coconut oil.

SAMPLE REAGENT REACTION

Phenolphtalein No Reaction; Colorless
Fresh Coconut Oil Methyl Orange Orange Color
pH Paper No Reaction
Phenolphtalein No Reaction; Colorless
Rancid Coconut Oil Methyl Orange Orange Color
pH Paper No Reaction
 Rationale:

The test yielded a basic result, thus, there is an oxidation or in this test, an
oxidative rancidity that occurred.

D. Liebermann-Burchard Test for Cholesterol

The Liebermann–Burchard or acetic anhydride test is used for the detection of

cholesterol.

 Materials: evaporating dish, dropper

 Sample: crystals of cholesterol
 Reagents: chloroform, acetic anhydride, concentrated H2SO4

Procedure:

Place a few Add 2 ml of

crystals of chloroform and Add 2-3 drops of
Note the color
cholesterol in a 10 drops of acetic concentrated
changes.
dry evaporating anhydryde. Mix H2SO4 and shake.
dish. thoroughly.

Result:

SAMPLE REAGENT REACTION

Chloroform, Acetic
Crystals of Cholesterol Purple to Blue in Color
Anhydryde, H2SO4

Rationale:

Positive results of this test will yield to purple, blue, or emerald green color. Since
this is a general test for sterols, it is positive to cholesterol and non-polar lipids due to the
condensation of acetic anhydride with C-3-OH and C5 double bonds.
QUESTIONS

1. Why are fatty acids insoluble in water?

Fatty acids are insoluble in water because there are more hydrocarbons which are
more hydrophobic than the carboxyl group which is soluble in water. The longer the chain
of fatty acid, the more insoluble it gets.

2. Explain why the cis- form is more predominant configuration of unsaturated fatty acids.

Cis- form configuration is more predominant in unsaturated fatty acids since most
of the fatty acids are in liquid form, and most of these liquid form unsaturated fatty acids
has this configuration.

3. Why is the Acrolein Test the general test for fats?

Acrolein test is used to detect the presence of glycerol or fat. When fat is treated
strongly in the presence of a dehydrating agent like potassium bisulphate (KHSO4), the
glycerol portion of the molecule is dehydrated to form an unsaturated aldehyde, acrolein
that has a pungent irritating odor.

Acrolein test is used because acrolein is the end product when a substance that
contains fat reacts with the reagent, potassium bisulfate, and heat. If acrolein is
produced, it indicates that there is a presence of fats in the substance.

4. What type of rancidity occurs in vegetable shortening and how can it be prevented?

The type of rancidity that occurs in vegetable shortenings is the oxidative

rancidity. Oxidative rancidity is associated with the degradation by oxygen in the air. The
double bonds of an unsaturated fatty acid can be cleaved by free-radical reactions
involving molecular oxygen.

Oxidation primarily occurs with unsaturated fats. In oxidation rancidity, the

oxygen molecules interact with molecules of the oil which causes damage or changes to
the substance. To prevent this from happening, vegetable shortenings must be stored in
a dark, cool place where it is less exposed to oxygen.

5. Explain the cleansing action of detergents.

Detergents are primarily made up of surfactants, or “surface active agents” of

which reduce the surface tension of water by adsorbing at the common boundary
between liquid and gas or one liquid and another. Surfactants form into aggregates
known as micelles, formations of amphiphilic lipids.
 The cleansing action of detergents is caused by its molecular structure. The
hydrophobic part of the molecule is attracted to other hydrophobic substances such as
oils, and the like. The hydrophilic part then attaches itself to water, thus, when it is rinsed
away with water, the hydrophobic part that is attached is rinsed as well.

Detergents act to clean dishes in the same manner as they break down cell
membranes. Detergents bind to grease (lipids), which allows the grease particles to be
washed away with water.

6. Write the structure of the parent compound of cholesterol.

7. Explain the cooperative solvent effect of lecithin and albumin.

Lecithin and albumin are good emulsifiers because they both have a polar and
nonpolar portion which helps reduce the immiscibility of two substances. When these
two solvents work together, these substances are able to hold polar substances through
their hydrophilic molecules thus making the immiscibility of two substances possible.