University of Santo Tomas

College of Nursing
S.Y. 2013-2014

Experiment No. 2

LIPIDS

Group Members:
Ilao, Ariel M.
Jimenez, Patricia Addie G.
Kitajima, Erika O.
Lachica, Mary Joyce T.
1NUR-3, Group No. 5
I. Introduction

The term “lipid” refers to both true fats and fat-like substances. This is a biomolecule

that is soluble in organic solvents and insoluble in water. The water insolubility is due

to the fact that the polar portion of their molecule is smaller than the non polar

portion. Lipids are molecules that can be extracted from plants and animals by low

polarity solvents such as ether, chloroform, or even acetone. The main biological

functions of lipids include storing energy, signaling, and acting as structural

components of cell membranes. Fats (and the fatty acids from which they are made)

belong to this group, as do oils, waxes, and steroids.

Lipids can be divided into five groups: 1) fats and oils; 2) waxes: 3) compound lipids;

4) sterols and steroids; 5) derived lipids.

A. SPOTTING EFFECT

Spotting effect may sometimes be called as ‘Greasy spot test’ to determine if the

lipid-like material contains lipids. The positive visual result is the presence of a

translucent spot because of the lipid content. The principle behind the results is that

most grease of fat have higher boiling point so they are non-volatile therefore the

grease cannot absorb enough heat for it to vaporize. When the liquid is inside the

paper, it diffracts light and then results to a translucent spot.

Procedure:

1. Draw 5 circles in a filter paper using pencil

2. Label samples with vegetable oil and 4 other suspected lipid-like

material

3. Put 1 drop of each sample within each of the labeled circular areas

4. Allow the spots to dry

5. Hold the filter paper against the light and note the presence of

translucent spots

6. Record observations

Test for: Presence of lipids

Positive visual result: Translucent spot

Observation:

Samples With (+) or without (-) translucent spot

Vegetable oil With translucent spot

Hair wax With translucent spot (minimal)

Hair shine Without translucent spot

Omega pain reliever With translucent spot

Butter With translucent spot

Interpretation of results:

1. Vegetable Oil (+)

- It is nonvolatile, it does not easily vaporize into gas.

- Liseen oil, an ester of tri-basic glycerin and unsaturated fatty acids.

- Oil is classified under lipids therefore it yielded a positive visual result.

2. Hair wax (+)

- Beeswax was present in the sample.

- Wax is classified under lipids therefore it yielded a positive visual

result.

3. Hair shine (-)

- Hair shine gave a negative result for it does not contain any lipids.

4. Omega pain reliever (+)

- Oleic acid and other types of oil were present in the sample.

- Oleic acid is a fatty acid and is under lipids therefore it yielded a

positive visual result.

5. Butter (+)

- Vegetable oil was present in the sample.

- Oil is classified under lipids therefore it yielded a positive visual result.

 What property of lipids is detected by this test?

- Non-polarity of lipids

 Are the results of this test conclusive?

 - No, because not all lipids are created alike.

- Lipids containing glycerol or sphingosine will leave the said spot,

however those not containing either glycerol or sphingosine will not

leave a grease spot.

Left to right

Top - Butter, Omega pain reliever,

Hair Treatment

Bottom - Hair wax, Vegetable oil

B. SOLUBILITY

Solubility of organic compounds is often summarized by the phrase “like

dissolves like". This means that molecules with many polar groups are more soluble

in polar solvents, and molecules with few or no polar groups are more soluble in

nonpolar solvents.

Lipids are generally insoluble towards polar compounds such as water, but

soluble towards organic compounds such as petroleum ether, benzene and mineral

oil. This is because lipids have a nonpolar tails.

Procedure:

1. 4 clean dry test tubes

2. Label: water, methylene chloride, ether and toluene

3. Put 1 ml of these solvents into each respective containers

4. In each tube, add 3 drops of vegetable oil and mix thoroughly

5. Compare solubility of vegetable oil in the different solvents

 6. Repeat procedure but instead use a drop of lecithin from a soft gel

capsule.

Test for: The solubility of the suspected lipid-containing samples

Observation:

Methylene
Sample Water Ether Toluene
chloride

Vegetable
Immiscible Miscible Miscible Miscible
oil

Lecithin Immiscible Miscible Miscible Miscible

Interpretation of results:

Linseed Oil
(Vegetable oil)

Lecithin

Solubility of vegetable oil in different solvents:

1. Water is a polar compound and vegetable is nonpolar. So they do not

follow rule “like dissolves like”.

2. Methylene chloride is slightly polar, not as much polar as water but it still

mixed with vegetable oil.

3. Ether is non polar and insoluble in water. The rule “like dissolves like”

applies.

4. Toluene is non polar so vegetable oil has the ability to dissolve in it. The

rule “like dissolves like” applies.

Solubility of lecithin in different solvents:

Lecithin, because of its polar and nonpolar ends, can act as an interface

between organic and aqueous environments in the body.

1. Water is a polar compound and with lecithin having polar and nonpolar

ends, lecithin did not dissolve in it.

2. Methylene chloride is a nonpolar substance and most of the lecithin

dissolved in it even though it has polar ends

3. Ether is a nonpolar substance and most of the lecithin dissolved in it even

though it has polar ends

4. Toluene is a nonpolar substance and most of the lecithin dissolved in it

even though it has polar ends

C. Test for Unsaturation (Bromine Water Test)

Objective: To test for unsaturation of lipids through a change in color or bromine

water.
Positive Visible Result: Discoloration or a colorless solution
Reagent: Bromine Water (Orange Liquid)

Procedures:
1. Put 1 mL of vegetable oil in testube A

2. Dissolve lcithin gel in 1 mL of methylene chloride in testube B

3. Add 1 mL of Bromine water on both testubes

4. Mix and observe the following reactions

Results:
Samples Color Change in Bromine Water

Vegetable Oil 2 Layers: a) White (Top Layer)

b) Colorless (Bottom Layer)

Lecithin 2 Layers: a) White (Top Layer)

b) Yellow (Bottom Layer)

Observations:
The vegetable oil and lecithin formed two layers. For vegetable oil, the top layer is
white then the bottom layer is now colorless. For lecithin the top layer is white then
the bottom layer is yellow.

Study Questions:
1. What is the principle involved in the Bromine water test? What is its purpose?
 The principle involved in this test is Halogenation by addition. It is a test for
unsaturated lipids.

2. Provide the type of equation used in the bromine water test.

Br – Br

3. Show the sample equation involved in the reaction between Bromine water
and your sample/s which gave a positive result.

CH2 – O – C – (CH2)16 – CH3

CH2 – O – C – (CH2)16 – CH3 + Br – Br

CH2 – O – C – (CH2)16 – CH = C(CH2)7 – CH3

CH2 – O – C – (CH2)16 – CH3

CH2 – O – C – (CH2)16 – CH3

O Br Br

CH2 – O – C – (CH2)16 – C – C(CH2)7 – CH3

 (Vegetable Oil)

CH3 O

CH3 – N – CH3 – CH2 – O – P – O – CH2 O

CH3 O CH – O – C – (CH2)16 – CH3

CH2 – O – C – (CH2)7 – CH = C(CH2)7 –

CH3
+ Br – Br

CH3 O

CH3 – N – CH3 – CH2 – O – P – O – CH2 O

CH3 O CH – O – C – (CH2)16 – CH3

O Br Br

CH2 – O – C – (CH2)7 – C – C(CH2)7 –

CH3
(Lecithin)
4. Provide a possible explanation for the difference in results between the
vegetable oil and lecithin.

Phospholipids such as lecithin have a polar or charge portion and a non polar
portion consisting of the long chain fatty acids with the same molecule. The
charge portion of these molecules will mix with the water and the non polar
will repel the water but mixes with lipids. These phospholipids will form small
globules in water that makes the water look opaque and milky. When lipids
 mix with water, this is knows as emulsifications the mixture is known as
emulsion.

D. ACROLEIN TEST

Objective: test for the presence of glycerine or fats

Positive (+) Visible Result: Burnt fat odor

Procedure:

1. Prepare 3 clean and dry test tubes.

2. To the 1st test tube, add a drop of glycerol; to the 2nd tube, a drop of

cooking oil and to the 3rd tube, a drop of lecithin from the soft gel capsule.

3. Mix pinch-size amount of powdered potassium bisulfate to all the tubes.

4. Heat the mixture gently over a small flame.

5. Note the odor and record your observations.

Results:

sample Odor of the vapor

Glycerol No odor

Cooking Oil Burnt fat odor

Lecithin Burnt fat odor

Study Questions:

1. What is the principle involved in the Acrolein test? What is its purpose?

- Dehydration

2. Show the sample equation involved in the reaction (a) glycerol and the KHSO 4

CH2OH CH2
 CHOH KHSO4 CH + 2H2O

CH2OH CHO

(Glycerol)

3. What is the compound responsible for your observation?

- acrolein

4. What is the purpose of Potassium Bisulfate used?

- It acts as a dehydrating agent. Whenever fat is heated in the presence of a

dehydrating agent, the fat molecule shed its glycerol in the form of the

unsaturated aldehyde - acrolein. It was used in the first world war as a

chemical weapon.
E. Extraction of Brain Lipids

Lipids

Glycolipid

Phospholipids Sterol

Added Reagent Extracted type of lipid

Ether (NP) Lipid

Acetone (P) Phospholipid and Sterol

Hot 95% ethanol Glycolipid

F. Detection of brain lipids

F.1 Molisch’s Test

Purpose: To test the presence of carbohydrate in lipids

Principle involved: Dehydration and condensation

Reagent: H2SO4 and α-naphthol

Purpose of the reagent: Sulfuric acid (H2SO4) was used as dehydrating

agent and α-naphthol as condensing agent.

Equation:
α-naphthol

+ H2SO4
Positive visible result: Violet or brown ring

Compound responsible for visible result:

Group of lipid identified in this test: Glycolipids*

* Glycolipids are lipids with a carbohydrate attached.

Compounds that will give a + VR:

Cerebroside

Ganglioside
F.2 Ninhydrin Test

Purpose: Test for the presence of free α-amino group

Principle involved: Oxidative decarboxylation, deamination and

condensation

* Decarboxylation- removal of CO2

* Deamination- removal of amine (-NH2)

Reagent: 2,2-dihydroxyindane-1,3-dione (Triketohydrindene Hydrate)

Purpose of reagent: removal of amine group

Equation:
 Steps:

Positive visible result: Deep blue/Purple solution (Ruhemann’s Purple)

Compound responsible for visible result: diketohydrin

Group of lipid identified in this test: Phospholipid

Account for the precaution with Ninhydrin reagent: The Ninhydrin

reagent will stain the skin blue because of amino acid on the skin and cannot

be immediately washed off completely if it comes in contact with the skin.

Ninhydrin is most commonly used to detect fingerprints.

F.3 Soda-Lime Test

Objective: To determine if the sample is acidic or basic with the soda lime.

Positive Visible Result: Change in color of the Litmus Paper

Reagent: Soda-Lime

Procedures:

1. Add a pinch amount of Soda-Lime to Residue B.

2. Heat Gently (Water Bath)

3. Moisten the litmus paper then note the change in color.

Observations:

After giving the litmus paper contact with the mixed reagent, the red litmus paper

turned blue.

Study Questions:

1. What is the principle involved in the Soda-Lime test? What is its purpose?

The principle involved is Deamination, a process of removing amine group

(NH2) from an organic compound or molecule. This is a test to detect N-

containing lipids or amino group.

2. What is the purpose of the reagent used?

The Soda-Lime acts as the Deaminating agent. It absorbs CO2 and H2O

vapor. To detect N-containing lipids or amino group.

3. Show the equation involved in the reaction between Soda-Lime and your

sample/s which gave a positive result

NH3 + H20 = NH4OH(vapor)

4. What is the compound responsible for your observation?

NH4OH is the compound responsible for the PVR. This is a basic compound

that is why the red litmus paper turned blue.

5. What group of lipids was identified by this test?

 The group identified is the Sphingolipids which is a class of lipid found in

biological membranes.

F.4 Ammonium Molybdate Test

Objective: to test for the presence of the phosphate group.

Positive (+) Visible Result: Yellow solution

Procedure:

1. Add 1.0 ml of 6N nitric acid to a portion of Residue B.

2. Heat in a boiling water bath for 5 minutes

3. Add 1.0 ml of the ammonium molybdate olution.

4. Continue heating for another 5minutes.

5. Note the change in color of the solution.

6. Record your observations.

7. Repeat the test using instead the contents of a lecithin soft gel capsule.

8. Record your observations.

Results:

sample Observation

Residue B Yellow with a yellowish solid

Lecithin Clear Yellow with a yellowish brown

colloid

Study Questions:

1. What is the principle involved in the Ammonium Molybdate test?

- Dehydration and Double Decomposition

2. What is the purpose of the reagent used?

- HNO3 serves as the dehydrating agent;

 3. What is the compound responsible for your observation?

- Ammonium Phosphomolybdate

4. What group of lipids was identified by the test?

- Phospholipids

F5. LIEBERMANN BURCHARD TEST

Procedure:

1. Prepare 2 clean and DRY test tubes

2. In the 1st tube, dissolve residue C in 1.0 ml of methylene chloride. In the

2nd tube, place 1.0 ml of vegetable oil.

3. Add 5 drops of acetic anhydride followed by 3 drops of conc. sulfuric acid

into both tubes. Mix thoroughly

4. Record observation

Test for: Cholesterol

Principle: Oxidation

Observation:

Samples Color of solution

Residue C Blue – green solution

Vegetable oil Emerald green solution

 What is the purpose of the reagents?

- Acetic anhydride is responsible for the color blue-green solution.

- Conc. Sulfuric Acid is responsible for the emerald green solution.

 Show the equation involved in the reaction:

 - Conc. Sulfuric Acid (H2SO4)

- When (H2SO4) is added to cholesterol, a water molecule is

removed from (hydroxyl group) C3 of cholesterol molecule, and it is

oxidized to form 3,5 – cholestadiene. This product is then converted

to a green coloration indicating a positive result.

- Acetic anhydride (CH3CO)

- (CH3CO) reacts with cholesterol in a chloroform solution to produce

a blue green solution.

 What compounds are responsible for the positive visible result?

- Acetic anhydride (CH3CO)

- Conc. Sulfuric Acid (H2SO4)

 What group of lipids were identified by the test?

- Steroids/cholesterol

 Why must one take the precaution of using only very dry containers and

equipment?

- Because water might alter the outcome of the given experiment. It may

react with the corrosive reagents used therefore the result may not be

accurate.
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