NurBiochemistry (NurBio) 1

Laboratory Manual

Laboratory Procedure Activity

Lipids 4

INTRODUCTION

Lipids constitute a large heterogeneous 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. They are essential constituents of practically all
plant and animal cells. In the human body, lipids are found mostly in the cell
membranes, in the brain and in the nervous tissues.

Fatty acids are constituents of lipids, be they fats or oils, waxes, phospholipids,
cerebrosides, sphingomyelins. However, some so-called lipid derivatives like steroids do
not contain the fatty acid radical although they posses the same physical properties of
fats like their solubility characteristics.

Fatty acids found in nature have even numbers of carbon atoms in the chain. The
double bonds in unsaturated fatty acids are readily attacked by halogens to give addition
products. This is the basis of the iodine number determination, which is a test to detect
the degree of unsaturation of fats and fatty acids. The more unsaturated the fatty acid,
the more double bonds it has and the more halogens it will absorb.

H H H H
O O
H3C(H2C)7 C C(CH2)7 C H3C(H2C)7 C C(CH2)7
OH Br H OH

Due to the presence of double bonds in unsaturated fatty acids, geometric isomerism
leads to cis and trans forms of the acid. The oleic acid structure above is the cis form.

The unsaturated character of oils and their conversion to solid fats is another example of
addition of atoms to the pi-bond. Hydrogenation is of high commercial value as solid fats
are more useful and edible.

Fatty acids do not dissolve in water because they form dimers. Since the partially
negative (-) oxygen and the partially positive (+) hydrogen of ⎯COOH are used in the
dimerization, no group is available for H-bonding with water molecules.
O HO O
H3C CH2 CH3
O OH O

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 2
Laboratory Manual

Fats are hydrolyzed by dilute acids completely to fatty acids and glycerol; by alkalis to
soap and glycerol (saponification); by enzyme lipase into a mixture of fatty acids,
glycerol and glycerides.

The glycerol released can be detected by dehydration. The product formed is acrolein or
propenal which has a pungent irritating odor.

The cleansing power of soap and detergent is due to their emulsifying action and their
ability to reduce surface tension. The soap molecule contains a polar head and a non-
polar tail. The non-polar tail dissolves in the oil droplets, while the polar carboxyl group
interacts with the (-) portion of the aqueous phase. The emulsion is stabilized by the
repelling action of the (-) charges of the oil droplets.

+
-
+
-
oil - + aqueous phase

- +
- +

Fats develop rancid odor and taste when exposed to air at room temperature. The
double bonds of unsaturated fatty acids combine with oxygen of the air to form
peroxides, volatile aldehydes, ketones and acids, responsible for the rancid odor. The
change may be catalyzed by bacteria.

Steroids are derivatives of a parent hydrocarbon compound cyclopentano-

perhydroxyphenantrene. The most common steroid is cholesterol found in the brain,
nerve tissue and gallstones. Both 7-dehydrocholeterol and ergosterol (from ergot rye)
are irradiated by ultraviolet light to vitamin D, the first to vitamin D3 and the second to
vitamin D2.

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Bunsen burner 2 mL ether

Iron ring 2 mL 10% hydrochloric acid, HCl
10 mL pipette 1 mL 2% albumin
Pipette bulb 1 mL 5% calcium chloride, CaCl2
Triple beam balance 2 mL 5% magnesium chloride, MgCl2
2 mL cottonseed oil
2 mL 95% ethyl alcohol
1 mL 5% hydrochloric acid, HCl
1 mL 5% sodium hydroxide, NaOH

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 3
Laboratory Manual

0.5 mL glycerol
0.5 g potassium bisulfate, KHSO4
0.5 g cholesterol
0.5mL lecithin
1 g soap
BRING:
10 g sodium chloride, NaCl
25 mL fresh coconut oil
25 mL rancid oil

PROCEDURE
CAUTION: NEVER smell any reagent, test solution or product formed
! unless instructed to do so!

A. Solubility Tests

SAFETY PRECAUTION:
Always wear safety glasses with side shields, mask, and gloves when
working with chemicals. Make sure to ELIMINATE all ignition sources
(no flares, sparks, or flames) near your work area. Harmful if
swallowed, can cause skin irritation, serious eye damage, corrosion,
and very toxic to aquatic life with long lasting effects. Avoid release
to the environment.

1. Pipette 1 mL of the following solvents in separate stoppered vials (or test

tubes): distilled water, ethyl alcohol, ether, 5% hydrochloric acid, 5% sodium
hydroxide. From a pipette or a dropper, add 1-2 drops of cottonseed oil in
each vial and shake thoroughly. Does each mixture form one uniform phase.

2. On different spots of a piece of coupon bond paper, place 1 – 2 drops of

each of these mixtures: cottonseed – ethyl alcohol and cottonseed – ether.
Be sure the mixtures are well-shaken before dropping. Allow solvents to
evaporate and compare the solubility of the oil in the two solvents.
(NOTE: Please refer to page 6 for the Proper Waste Disposal of used samples)

B. Tests for Unsaturation of Fatty Acids

(SEARCH FOR THEORETICAL RESULTS)

1. To 6 drops of carbon tetrachloride, add 3 drops of oleic acid. Then add

bromine water in carbon tetrachloride drop by drop into the mixture, shaking
vial after each addition. Note the number of drops needed to produce a faint
orange color.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 4
Laboratory Manual

2. Into each of 3 test tubes or stoppered vials, place 2 mL of chloroform. Add to

each test tube 0.2 g of palmitic acid, 4 drops oleic acid, and 4 drops of
cottonseed oil respectively. Shake each tube thoroughly to dissolve the
contents. Add 4 drops of Hanus – iodine solution to each test tube and
record the time in seconds for the color to disappear.

C. Acrolein test

SAFETY PRECAUTION:
When handling potassium hydrogen sulfate (KHSO4), wear safety glasses
with side shields, mask, and gloves. Avoid excessive dust exposure as it
can cause skin and eye damage, corrosion, or irritation.

1. Prepare 2 test tubes, place:

test tube 1 – 2 drops glycerol + a pinch of KHSO4
test tube 2 – 2 drops cottonseed oil + pinch of KHSO4
2. Heat each tube over a low flame. Note the odor produced.
(NOTE: Please refer to page 6 for the Proper Waste Disposal of used samples)

D. Test for Rancidity

1. Prepare 2 vials. In vial 1, put about 5 mL rancid coconut oil. In vial 2, put 5
mL of fresh coconut oil.

2. Test the reaction of rancid and fresh coconut oil samples with red and blue
litmus paper, separately.

3. Repeat the test using pH paper instead of litmus paper.

(NOTE: Please refer to page 6 for the Proper Waste Disposal of used samples)

E. Properties of Soap
1. Use a white soap bar for this test. Grate some of the soap and transfer a
small amount into a test tube, add distilled water, seal with a rubber stopper
and shake it to form suds.

• Formation of Fatty Acids

SAFETY PRECAUTION:
Always wear safety glasses with side shields, mask, and gloves when
working with hydrochloric acid. It can cause skin irritation, serious eye
damage, corrosion, or irritation.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 5
Laboratory Manual

2. Place 5 mL of the soap solution in a test tube and add 10% HCl until
precipitate form. Describe the precipitate formed.

• Insoluble Soaps

SAFETY PRECAUTION:
Always wear safety glasses with side shields, mask, and gloves when
working with chemicals.

3. Place 2 test tubes, each containing 4 mL distilled water and 1 mL soap

solution. To test tube 1 – add 5 drops 5% CaCl2 solution. To test tube 2 –
add 5 drops 5% MgCl2 solution

4. Observe results. Repeat the test using liquid detergent instead of soap
solution. Observe and compare results with the first part.
(NOTE: Please refer to page 6 for the Proper Waste Disposal of used samples)

F. Liebermann – Burchard Test for Cholesterol

(SEARCH FOR THEORETICAL RESULTS)

1. Place a few crystals of cholesterol in a dry evaporating dish. Add 2 mL of

chloroform and 10 drops of acetic anhydride. Mix thoroughly.

2. Add 2-3 drops of concentrated sulfuric acid and shake. Note the color
changes during the first few minutes.

G. Emulsifying Action of Lecithin

1. In each of 2 test tubes, place 5 drops of 2% albumin solution. To test tube 1

– add a pinch of cholesterol and shake the tube. To test tube 2 – add a pinch
of cholesterol and 5 drops of lecithin. Shake the tube. Compare the results.

PROPER WASTE DISPOSAL

Dispose of solutions in the proper waste bottles (as acid or basic wastes, and
organic or inorganic wastes).

Do not dispose of solutions with 5% hydrochloric acid, 5% sodium hydroxide

down the sink or discarding in regular trash containers. Instead, dispose 5%
hydrochloric acid in “Hazardous Acid Waste” and 5% sodium hydroxide in
“Hazardous Base Waste”.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 6
Laboratory Manual

QUESTIONS

1. Why are fatty acids insoluble in water?

2. Explain why the cis-form is the predominant configuration of unsaturated fatty
acids?
3. What type of rancidity occurs in vegetable shortenings? How can it be
prevented?
4. Show the structure of the parent compound of cholesterol.
5. Explain the cooperative effect of lecithin and albumin.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 7
Laboratory Manual

Name :_______________________________ Subject/Section :___________

Course/Year:_____________ Date Performed :___________
ID Number:______________

Data Sheet Activity

Lipids 4

Data

A. Solubility Tests

Test Sample Reagents Added Observation

Distilled water
95% ethyl alcohol

Cottonseed oil Ether

5% HCl solution

5% NaOH solution

Test Sample Reagent Added Observation

ethyl alcohol

Cottonseed oil ether

B. Tests Unsaturation (SEARCH FOR THEORETICAL RESULTS)

Test Sample Reagent Added Observation

Oleic acid Br2 in CCl4

Palmitic acid
CHCl3 Hanus Iodine
Oleic acid

Cottonseed Oil

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
NurBiochemistry (NurBio) 8
Laboratory Manual

C. Acrolein Test

Test Sample Reagent Added Observation

Glycerol KHSO4 (s)

Cottonseed oil KHSO4 (s)

D. Rancidity Test

Test Sample Red & Blue Litmus Paper pH Meter

Rancid coconut oil

Fresh coconut oil

E. Properties of Soap

Test Sample Reagent Added Observation

Soap solution 10% HCl solution

CaCl2 solution
Soap solution
MgCl2 solution

Detergent CaCl2 solution

solution MgCl2 solution

F. Liebermann-Burchard Test (SEARCH FOR THEORETICAL RESULTS)

Test Sample Reagent Added Observation

Chloroform + acetic
Cholesterol anhydride + sulfuric
acid

G. Emulsifying Action

Test Sample Reagent Added Observation

Cholesterol
2% albumin
solution Cholesterol + lecithin

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.