Nursing Biochemistry Laboratory (NurBio Lab) 1

Laboratory Manual

Laboratory Procedure Activity

Lipids 5

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(H 2C) 7 C C(CH 2)7 C H3C(H 2C) 7 C C(CH 2)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.
Nursing Biochemistry Laboratory (NurBio Lab) 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 40 mL acetone

Iron ring 26 mL ether
10 mL pipette 20 mL 10% alcoholic potassium hydroxide,
Pipette bulb ROH in KOH
Triple beam balance 10 mL 10% hydrochloric acid, HCl
10 mL 2% albumin
9 mL chloroform
6 mL Hanus – iodine solution
6 mL 5% calcium chloride, CaCl2

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

6 mL 5% magnesium chloride, MgCl2

5 mL cottonseed oil
1 mL acetic anhydride
4 mL bromine in carbon tetrachloride,
Br2 in CCl4
3 mL oleic acid
2 mL cottonseed – ethanol mixture
2 mL cottonseed – ether mixture
2 mL carbon tetrachloride, CCl4
2 mL phenolphthalein
2 mL methyl orange
1 mL 95% ethyl alcohol
1 mL benzene
1 mL 5% hydrochloric acid, HCl
1 mL 5% sodium hydroxide, NaOH
1 mL glycerol
1 mL concentrated sulfuric acid, H2SO4
1 g palmitic acid
1 g potassium bisulfate, KHSO4
1 g cholesterol
1 g lecithin
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

1. Pipette 1 mL of the following solvents in separate stoppered vials (or test tubes):
distilled water, ethyl alcohol, ether, chloroform, benzene, 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. Record the time required for the oil to
dissolve.

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.

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

B. Tests for Unsaturation of Fatty Acids

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.

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

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.

D. Test for Rancidity

1. Prepare 6 test tubes or vials. In each tube: 1, 2, 3 – place 5 drops of fresh

coconut oil, and each of tubes 4, 5, 6 – place 5 drops of rancid coconut oil.

2. Test the reaction of fresh coconut oil with 1 – 2 drops phenolphthalein, 1 – 2

drops methyl orange and pH paper (or red and blue litmus paper if pH paper is
not available).

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

E. Saponification

1. Weigh 1.5 g of fresh coconut oil in a dry beaker. Add 10 mL of 10% alcoholic
potassium hydroxide. Cover the beaker with a watch glass.

2. Fill the watch glass with crushed ice. Boil over a water bath until a drop of the
hot solution added to cold water in a test tube does not form globules of fat.

3. Remove the watch glass to drive off the alcohol. Add 20 mL of water to the
gelatinous mass and warm the solution to dissolve the soap. Use the soap
solution for the tests on the properties of soaps.

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

F. Properties of Soap

1. Salting Out

Place 10 mL of the soap solution in a beaker and add table salt gradually with
stirring until no more table salt dissolves.

Remove the solid soap formed from the liquid and wash it with water. Transfer a
small amount of the solid soap into a test tube and shake it with distilled water
to form suds.

2. Formation of Fatty Acids

Place 5 mL of the soap solution in a test tube and add 10% HCl until a
precipitate forms.

3. Insoluble Soaps

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

Observe results. Repeat the test using liquid detergent instead of soap solution.
Observe and compare results with the first part.

G. Liebermann – Burchard Test for Cholesterol

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.

H. 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 DISPOSAL: Dispose of solutions in the proper waste bottles (as

acid or basic wastes, and organic or inorganic wastes).

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

Group No.___ Date Performed :___________

Subject/Section:__________ Instructor’s Initials :___________
Members:
1. _____________________
2. _____________________
3. _____________________

Data Sheet Activity

Lipids 5

Data

A. Solubility Tests

Sample Solvent Used Result

Cottonseed Distilled water

Oil

Ethyl alcohol

Ether

Chloroform

Benzene

5% HCl

5% NaOH

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 101: General and Inorganic Chemistry 8
Laboratory Manual

Mixture Evidence Results (Description)

Cottonseed oil (Attach cut fine bond paper

+ ethyl alcohol here)

Cottonseed oil (Attach cut fine bond paper

+ ether here)

Name of Test Test Reagents Result

Solutions Added
B. Test for Oleic Acid Br2 in CCl4
Unsaturation

Palmitic CHCl3 Hanus

Acid Iodine

Oleic Acid CHCl3 Hanus

Iodine

Cottonseed CHCl3 Hanus

Oil Iodine

C. Acrolein Glycerol KHSO4 (s)

Test

Cottonseed KHSO4 (s)

oil

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 101: General and Inorganic Chemistry 9
Laboratory Manual

Name of Test Test Reagents Result

Solutions Added
D. Fresh Phenolphthalein
Rancidity Test coconut oil

Methyl orange

pH paper

Rancid Phenolphthalein
coconut oil

Methyl orange

pH paper

E. coconut oil 10% KOH

Saponification

F. Properties Test Reagents Result

of Soap Solutions Added

Salting-out Soap
solution

Formation of Soap
Fatty Acids solution

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 101: General and Inorganic Chemistry 10
Laboratory Manual

Name of Test Test Reagents Result

Solutions Added
Insoluble Soap
Soaps solution

Detergent

G. Cholesterol
Liebermann-
Burchard Test

H. 2% Cholesterol
Emulsifying albumin
Action

Cholesterol –
lecithin

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