Analysis of Lipids

Module Twelve
Analysis of Lipids

Knowledge Acquisition

Introduction

Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules that can be
extracted from tissues by nonpolar solvents. These biomolecules adopt the principle of “like dissolves
like.” Because of their insolubility in aqueous solutions, body lipids are generally found
compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol in white
adipocytes, or transported in plasma in association with protein, as in lipoprotein particles, or on
albumin.

Lipids are a major source of energy for the body, and they also provide the hydrophobic barrier that
permits partitioning of the aqueous contents of cells and subcellular structures. Lipids serve additional
functions in the body, for example, some fat-soluble vitamins have regulatory or coenzyme functions,
and the prostaglandins and steroid hormones play major roles in the control of the body’s homeostasis.

The physical and chemical properties of lipids depend on the presence of carboxyl groups, number of
double bonds, number of hydroxyl groups, and the length of the carbon chain.

What is expected of me from this module?

At the end of this module, you are expected to:
1. Classify lipids as to their physical appearance and properties.
2. Discuss the different chemical reactions that fats undergo.
3. Explain the laboratory procedures that demonstrate chemical properties of fats and their derivatives.
4. Perform laboratory procedures that demonstrate the physical and chemical properties of lipids.

Where do I acquire knowledge to meet expectations?

1. Laboratory Notes 12
2. Video demonstrations

Laboratory Notes 12

Lipids are poorly soluble in water but they dissolve in organic solvents like benzene, chloroform or
dichloromethane (DCM). Their functions are to act as metabolic fuel, as stored forms of energy, and as
components of the cellular membrane. Lipids are classified into fatty acids, triglycerides, phospholipids,
sphingolipids, and steroids.

Fatty acids are the smallest units of lipids. They are further divided into saturated and unsaturated.
Saturated or single-bonded fatty acids are usually solid at room temperature. Unsaturated fatty acids
contain double bonds and are liquid at room temperature. Fatty acids are long chained carboxylic acids
which, when ionizes, cause the formation of H+ and the RCOO- anion. Fatty acids react with NaOH
producing soaps.

The alkali metal salts of fatty acids usually containing 10 to 18 carbon atoms are called soaps. They have
a long nonpolar, oil-soluble hydrocarbon chain at one end, and a water-soluble carboxylate ion at the
other end. This makes soap a good wetting agent and also an emulsifying agent. The ability of soap to
suspend substances that do not dissolve in water makes it a good cleaning agent. However, soaps form
insoluble salts with metal ions like Ca2+ and Mg2+ ions. These ions are present in hard water, forming
precipitates that adhere to sinks and even to clothes. The development of synthetic detergents or syndets
solved the problem of “scum” formation with soap. Syndets also contain fat-soluble groups and water-
soluble groups, but unlike soap they do not from precipitates in hard water.

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Analysis of Lipids

The degree of unsaturation of lipids can be determined by adding bromine mixed with CCl4. The free
bromine attaches itself to the carbons with double bonds. The presence of unsaturation is indicated by
the decolorization of the added bromine.

When enzyme lipase hydrolyzes a compound lipid, one of the products of hydrolysis is glycerol. When
heated with a dehydrating agent such as potassium hydrogen sulfate, KHSO4, glycerol yields acrolein, a
compound with the peculiar, acrid odor of burnt bacon.

Lipids are heterogeneous group of compounds synthesized by the cell and that are sparingly soluble in
water but soluble in nonpolar solvents. It is made up of fatty acids and their naturally existing compounds
and derivatives. There is a variety of classification of lipids, including (a) simple lipids (neutral fats and
waxes); (b) complex lipids (phospholipids, sphingolipids, and glycolipids); (c) derived lipids (steroids,
vitamins, and carotenoids); (d) amphipathic lipids (glycolipids and sphingolipids). Unlike the proteins,
carbohydrates and nucleic acids, lipids are not polymers. Instead, they are small molecules that tend to
associate through noncovalent forces.

Lipids exhibit various types of functions. A large fraction of cellular lipids is used to form lipid membranes;
they also serve in storage depot and transport of metabolic fuels. They exhibit structural and insulation
function as cell walls of many bacteria, on leaves of plants and skin of vertebrates.

Lipid structure is generally pictured as having a polar hydrophilic “head” and a nonpolar hydrophobic
hydrocarbon “tail,” thus, lipids are amphipathic. They tend to form surface monolayers, bilayers, micelles,
or vesicles when in contact with water.

Triacylglycerols (Neutral fats)

The major energy reserve of the body are triacylglycerols, with esterified fatty acids. They are classified
as neutral fats and are practically immobilized as major constituents of depot or storage fats in plant and
animal cells especially in the adipose or fatty tissue of vertebrates, where they serve three distinct
functions: (a) energy production (generation of ATP), (b) heat production; “brown fat” of warm blooded
animals, and (c) insulation; thermal insulation in the skin of animals living in cold regions. Neutral fats
have the following structure:

Where R, R’ and R’’, represent the fatty acid chain; if the three are the same, the compound is a simple
glyceride.

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Analysis of Lipids

General Properties of Fats

Physical Properties
1. Neutral fats have a characteristic greasy feel and when brought in contact with a substance like
paper, penetrate through it producing a translucent spot.
2. When pure, neutral fats are odorless, tasteless and colorless, the color of the naturally occurring fats
is due to the pigments dissolved in them.
3. They are insoluble in ordinary solvents but readily dissolve in CHCl3, C6H6, ether, boiling alcohol, etc.
4. They are non-volatile and produce characteristic crystals with definite melting point that serves to
differentiate them from one another.
5. Their congealing or solidifying point is very much lower than the melting point, the latter increasing
proportionately with the increase in molecular weight.
6. Neutral fats containing large amount of unsaturated fatty acids are liquid at room temperature,
hence they are called oils. These oils in the presence of catalyst take up hydrogen and become solid.
7. Fat floats on water because it has a lower specific gravity than the latter. When shaken vigorously
with it, fats are broken up into fine particles forming a temporary emulsion, unless stabilizing or
emulsifying substance is used. Soap, acacia, albumin or bile salts are used as emulsifying agents.
They lower the surface tension of the aqueous phase. An emulsifying substance prevents
coalescence of the fat droplets by adsorbing on their surfaces.

Chemical Properties

1. Hydrolysis
Fats are readily hydrolyzed by acids, enzymes or superheated steam with the liberation of fatty acids
and glycerol.

2. Saponification
If instead of water in the above reaction, an alkali is used, a metallic salt of fatty acids (soap) is formed
and the process is called saponification (alkaline hydrolysis of fats)

Sodium soaps are hard while soaps of potassium are soft. Calcium and magnesium form insoluble
salts. The cleansing power of soap is attributed to its emulsifying property.

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Analysis of Lipids

3. Rancidity
Fats are neutral in reaction, but when exposed to air for some time, they become acidic due to
hydrolysis which results in the liberation of volatile fatty acids. These are subsequently oxidized with
the formation of odoriferous volatile aldehyde and ketones. The presence of an enzyme, lipoxidase
and such agents as heat, light, moisture and bacteria contribute to the rapid onset of rancidity.
Rancidity results in the destruction of the accessory food factors like carotene, vitamin A and vitamin
E. Rancid fat, therefore, is not only unpalatable, but may even be toxic.

4. Hydrogenation

5. Liebermann-Burchardt reaction
When treated with acetic anhydride and concentrated sulfuric acid, a chloroform (or carbon
tetrachloride) solution of sterol gives a characteristic color.

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Analysis of Lipids

6. Salkowski test
Sterols undergo dehydration process in the presence of sulfuric acid to give 3,5-cholestadiene, which
undergo dimerization to bis-cholestadiene.

7. Test for Unsaturation

Palmitic acid is a saturated fatty acid. Oleic acid is monoenoic (monounsaturated) fatty acid. When
unsaturated fatty acids are treated with halogens like bromine, they add across the double bond. As
halogens are added to the double bonds, the color of the halogen disappears.

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Analysis of Lipids

Knowledge Application
Let us apply the concepts you have learned from the previous sections.

General Instructions
(For experiment results, experiment questions, and critical thinking questions)
1. Create an answer sheet by using Microsoft Word (or another application with the same function).
2. Type your answers on the created answer sheet using Calibri or Arial with font size of 11 or 12.
3. Type you name, year and section, and date of submission at the top of the answer sheet.
4. Copy the experiment questions and/or critical thinking on the answer sheet. Follow the sequence
of questions in this module. Use proper headings to identify which experiment the questions
belong to. When typing your answers, please leave a space between your answer and the
question.
5. Write your answers on the table instead of typing them. Take pictures of the tables with your
answers. Paste the pictures on the answer sheet. Follow the sequence of tables in this module.
Use proper headings to identify which experiment the tables belong to.
6. After answering all questions and tables, save the answer sheet as a PDF file using this format:
II(section)_Last Name_First Name_Module(number).pdf; for example,
IIB_Barber_Johnna_Grace_Module1.pdf
7. Send the answer sheet as email attachment to treblanation1976@idc.edu.ph.
8. For numbers requiring you to draw, draw on a separate piece of bond paper, label the necessary
parts, and take a picture of your drawing. Send the pictures as an attachment together with your
answer sheet.
9. Please make sure that your answer sheet has the correct format. Answer sheets with incorrect
format will not be checked.
10. This is NOT a group work. Please DO NOT COPY FROM YOUR CLASSMATE. Identical answers for
essay questions will automatically be disqualified.
11. For questions requiring computation, always show your solution.

Laboratory Activity 14: Physical and Chemical Properties of Lipids

Materials
1. Beaker
2. Crucible
3. Erlenmeyer flask
4. Flask with stopper
5. Graduated cylinder
6. Heating set-up
7. Medicine dropper
8. Test tubes
9. Water bath
10. Burette

Reagents
1. Lipid samples (olive oil, coconut oil, lard, and butter)
2. Ether
3. Phenolphthalein solution
4. Solid KHSO4
5. 20% NaOH
6. 15% Potassium iodide solution
7. NaCl solution
8. Concentrated HCl
9. Acetic anhydride
10. Carbon tetrachloride
11. Concentrated H2SO4

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Analysis of Lipids

12. Anhydrous CHCl3

13. Alcoholic KOH
14. 0.5M HCl
15. Hanus iodine solution
16. 0.1M Na2S2O3
17. 2% starch

Procedures for Qualitative Tests

Video demonstration from YouTube

Module 12 - Video demonstration 35 - Qualitative Analysis of Oil and Fats - MeitY OLabs

A. Test for Unsaturation

1. Add 1 mL of each lipid sample into 4 separate test tubes.
2. Add 5 mL of ether and 3 drops of 15% KI solution to each lipid sample.
3. Shake the mixtures and note the changes in KI solution.

Video demonstration from YouTube

Module 12 - Video demonstration 36 - Testing for Unsaturation in Oils

B. Acrolein Test
1. Place a small amount of olive oil into the crucible and add 0.5 g of solid KHSO4.
2. Heat and note the odor.
3. Repeat steps 1 and 2 using coconut oil.

C. Saponification
1. Add a few drops of olive oil into a test tube.
2. Add 6 mL of 20% NaOH and mix.
3. Keep in a boiling water bath for 20 minutes. Stir occasionally and cool.
4. Divide the clear solution into 3 test tubes.
5. To the first test tube, add 10 mL of water, and then shake vigorously.
6. Add 2 mL of phenolphthalein solution. Observe any change in color.
7. To the second test tube, add 2 mL of NaCl solution. Note the precipitate formed.
8. To the third test tube, add concentrated HCl drop by drop with mixing. Note the scum forming
at the top.

Video demonstrations from YouTube

Module 12 - Video demonstration 37 - Saponification - The Process of Making Soap - MeitY OLabs
Module 12 - Video demonstration 38 - Saponification Test for Lipids by Solution Pharmacy

D. Liebermann-Burchardt Test
1. Place 0.5 mL of olive oil into a test tube.
2. Add 3 mL CCl4, 0.5 mL concentrated H2SO4, and 0.5 mL acetic anhydride.
3. Shake gently and allow to stand for a few minutes.
4. Note the colors produced.
5. Repeat the procedure using 0.5 g of lard.

Video demonstrations from YouTube

Module 12 - Video demonstration 39 - Liebermann-Burchard Test 1
Module 12 - Video demonstration 40 - Liebermann-Burchard Test 2

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Analysis of Lipids

E. Salkowski Test
1. Dissolve a few milligrams of butter in 3 mL anhydrous CHCl3.
2. Add an equal volume of concentrated H2SO4 and shake the tube gently.
3. Let the liquid layers separate and observe any color change at the interface of the two layers.

Procedures for Quantitative Tests

A. Saponification Number
1. Transfer 1 g of lard into a 250 mL Erlenmeyer flask containing 4 mL of solvent mixture (1:1
ethanol/ether)
2. Dissolve the lard in 50 mL of 0.1 mL alcoholic KOH and boil for 30 minutes.
3. Cool and titrate with 0.5M HCl using phenolphthalein as indicator until the faint pink disappears.
4. Perform a blank determination using alcoholic KOH solution.
Subtract the amount of 0.5M HCl obtained during the determination of the sample from the
amount obtained in the blank to obtain the number of mL of 0.5M HCl equivalent to the KOH
used in the saponification of the sample.
5. Calculate as the saponification number, which is the number of milligrams of KOH required to
saponify 1 g of lard.

Saponification number (SV) = ([(B - S) x N] / W) x 56.1

Where: SV = saponification value

B = mL of 0.5M HCl required to titrate blank
S = mL of 0.5M HCl required to titrate sample
N = normality of HCl (equivalent to molarity of HCl)
W = weight of sample in grams
56.1 = molecular weight of KOH

Video demonstration from YouTube

Module 12 - Video demonstration 41 - Estimation of Saponification Value of Oils & Fats

B. Iodine Number
1. Transfer 1 g of lard into 250 mL Erlenmeyer flask.
2. Dissolve the sample in 10 mL CCl4.
3. Add 25 mL Hanus iodine solution and allow to stand for 30 minutes with occasional shaking.
4. Add 10 mL 15% KI solution and shake thoroughly.
5. Add 100 mL of freshly-boiled and cooled water, washing down any free iodine that may be found
in the stopper.
6. Add 2 mL of 2% starch solution.
7. Titrate mixture with 0.1M Na2S2O3 until the yellowish-brown color of the solution disappears.
8. Run blank determinations using Hanus iodine solution without the fat substance.
9. Calculate the grams of iodide by 100 g of the fat. This is the Hanus iodine number.

Iodine number (IV) = [(B - S) x N x 12.69] / mass of sample

Where: IV = iodine value

B = mL of 0.1M Na2S2O3 required to titrate blank
S = mL of 0.1M Na2S2O3 required to titrate sample
N = normality of Na2S2O3
12.69 = amount of g of iodine contained in 1 L of 0.1N iodine

Video demonstration from YouTube

Module 12 - Video demonstration 42 - Estimation of Iodine Value of Fats and Oils

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Analysis of Lipids

Results for Laboratory 14 (copy the tables below on your answer sheet)

TEST FOR UNSATURATION

Describe a positive test for unsaturation. Paste two pictures below showing the original
color when the KI or bromine water was
immediately added and the final color after
mixing was done.

What is responsible for the color change in KI or

bromine water after mixing?

ACROLEIN TEST
Describe a positive acrolein test. Paste a picture below of the acrolein test set-up.

Name the major products formed.

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Analysis of Lipids

SAPONIFICATION
Describe the appearance of the soap formed. Paste a picture below of a positive result for each
test tube in the written procedure.

Based on the written procedure, what was

formed in the hydrolytic process in the first test
tube?

Based on the written procedure, what does the

scum on the top layer of the third test tube
indicate?

Based on the written procedure, what was

formed after the addition of HCl in the third test
tube?

LIEBERMANN-BURCHARDT REACTION
Describe a positive Liebermann-Burchardt test. Paste a picture below of a positive result

What is the effect of the presence of water in the

test?

What is the purpose of running this test? What

does it test for?

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Analysis of Lipids

SALKOWSKI TEST
Describe a positive Salkowski test. Paste a picture below of a positive result

To get a positive result, what would the liquid

layer at the lower portion of the solution exhibit?

The upper chloroform/carbon tetrachloride layer

develops _________.

SAPONIFICATION NUMBER
Look for a sample computation in the Internet or any chemistry book for the saponification number
activity. Show your computation and fill out the necessary information below
Lipid sample used
Weight of sample
Molarity or normality of HCl used
Volume of HCl used in blank determination
Volume of HCl used in actual determination
Saponification value
Computation

IODINE NUMBER
Look for a sample computation in the Internet or any chemistry book for the iodine number activity.
Show your computation and fill out the necessary information below
Lipid sample used
Weight of sample
Molarity or normality of Na2S2O3 used (or any
equivalent substance based on example)
Volume of Na2S2O3 used in blank determination
Volume of Na2S2O3 used in actual determination
Iodine number or value
Computation

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Name of Student:
Date Performed: Instructor’s Signature:

PERFORMANCE RUBRICS

Availability of Experimental Write-Up, and Assembly of Materials and Reagents,

Complete and Incomplete Unable to Score
on-time and/or late perform
(5) (2) (0)
Personnel Protective Equipment and Safety
Complete and properly Incomplete or improperly No personnel protective Score
worn PPEs, safety worn PPE, or safety equipment and safety
protocols followed protocols not followed protocols not followed
(5) (2) (0)
Performance Efficiency
Instructions properly Instructions not properly Instructions not properly Score
followed; able to finish followed or unable to followed and unable to
on time finish on time finish on time
(5) (2) (0)
Performance Accuracy
More than 90 percent of 60 to 89 percent of results Less than 60 percent of Score
results are correct are correct results are correct
(5) (3) (0)
Waste Disposal, Return of Materials and Reagents, and Overall Cleanliness
Waste properly disposed; Waste not properly Waste not properly Score
M&R properly returned; disposed, M&R not disposed, M&R not
cleanliness was observed returned, or cleanliness returned, and cleanliness
during and after the was not observed during or was not observed during or
exercise after the exercise after the exercise
(5) (2) (0)
Score/25 points

ORAL EXAMINATION
5 0 Score
Question 1 x1
Question 2 x2
Question 3 x2
Score / 25 points

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Knowledge Assessment
You will be given a scheduled quiz to assess the knowledge you have acquired from this learning activity.

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Analysis of Lipids

References
Chua-Suba, Sally M. Laboratory Manual in Biochemistry (2011). Revised edition. C&E Publishing., Inc.

Devlin, Thomas M. Textbook of Biochemistry with Clinical Correlations (2011). Seventh edition. John Wiley
& Sons, Inc.

Ferrier, Denise R. Lippincott’s Illustrated Reviews: Biochemistry (2017). Seventh edition. Wolters Kluwer.

McKee, Trudy, McKee, James R., Biochemistry: An introduction (1999). 2nd edition. McGraw-Hill.

Nelson, David L., and Cox, Michael M. Lehninger Principles of Biochemistry (2008). W.H. Freeman and
Company.

Rodwell, Victor W., Bender, David A., Botham, Kathleen M., Kennelly, Peter J., Weil, P. Anthony. Harper’s
Illustrated Biochemistry (2018). Thirty-first edition. McGraw-Hill Education.

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