LABORATORY

INTRODUCTION

The term “lipids” applies to a class of compounds that are soluble in organic solvents and insoluble in
water. Lipids contain carbon, hydrogen and oxygen but have far less oxygen proportionally than
carbohydrates.
Lipids are an important part of living cells. Together with carbohydrates and proteins, lipids are the main
constituents of plant and animal cells.
The triesters of fatty acids with glycerol makes up the class of lipids known as fats and oils. The
triglycerides are found in both plants and animals and compose one of the major food groups in our diet.
The steroids are one of the best known and best studied of the lipid groups, and example of which is
cholesterol. Cholesterol is amphiphilic having a polar head, represented by the –OH group and an extensive
nonpolar region made up of fused rings and hydrocarbon tails. The functional group of the cholesterol structure
is the hydroxyl group. Fatty acids are esterified at this position especially under physiological conditions.
Cholesterol and its ester derivatives are abundant in plasma proteins called lipoproteins whose function it is to
transport the cholesterol to peripheral tissue.
DATE: _
DATE ASSIGNED: DATE SUBMITTED:

Activity No.20

PHYSICAL AND CHEMICAL PROPERTIES OF LIPIDS

OBJECTIVES

At the end of the laboratory session, you should be able to

1. determine exactly the solubility of lipids in polar and non-polar solvents
2. perform precisely some tests that determine the presence of lipids in a sample
3. appreciate completely the importance of lipids in the body

MATERIALS

Test tubes Stearic acid Corn oil

Test tube rack Conc. Sulfuric acid Cholesterol powder
Bunsen burner Chloroform Olive oil
Test tube holder acetic anhydride Linseed oil
Test tube brush Chocolate Butter
Pasteur pipet Milk Lard
Watch glass Acetone Glycerol
Porcelain tile Raw egg yolk Potassium bisulfite
Scoring rubric

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For Procedures:
- 3 points is given for every short procedure done that requires an immediate written observation
o 2 points for doing the procedure correctly
o 1 point for the answer
- 1 point is given for items that do not require performing a procedure For
Rationalization
- 3 points is given for every rationalization made
o 3 – all concepts were mentioned and a clear rationalization was made
o 2 – one or 2 concepts were not mentioned and the rationalization is not clear
o 1 – 3 or more concepts were not mentioned and the rationalization is not clear
o 0 – no rationalization was made
For hypothesis/Generalization/Conclusions
- 3 points is given for every hypothesis, generalizations or conclusions made
o 3 – all concepts were mentioned and a clear hypothesis, generalization or conclusion was derived from the activity
o 2 – one or 2 concepts were not mentioned and the hypothesis, generalization or conclusion is not clear
o 1 – 3 or more concepts were not mentioned and the hypothesis, generalization or conclusion is not clear
o 0 – no hypothesis was made

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PROCEDURES AND OBSERVATIONS

Solubility and Physical Appearance: Watch: https://www.youtube.com/watch?v=l2QOi9mZoFc

1. Prepare 6 test tubes and label them properly: Corn oil, linseed oil, lard, butter cholesterol and stearic acid
2. Place 5 drops of corn oil, and linseed oil to the corresponding test tube while a matchstick-head sized portion of
lard, butter, cholesterol and stearic acid will be used.
3. Note their physical state and write your observations on the data sheet below
4. Add 1 mL of water into each of the test tube
5. Shake and note their solubility
6. Write your observations on the data sheet below
7. Repeat procedure “a” and “b”
8. Add 1 mL of petroleum ether into each of the test tube
9. Shake and note their solubility
10. Write your observations on the data sheet below
11. Save the test tubes with ether for the next procedure

 You may want to try this at home by dissolving your mineral oil (baby oil), cooking
oil and lard in water.
pH
1. Prepare 5 sets of blue and red litmus paper and place them side by side on a watch glass or a porcelain
tile
2. Using the test tubes with ether from the procedure on solubility, test the pH of each of the lipids in the test tube
by adding a drop of each into the corresponding litmus paper
3. Record your results

LIPID PHYSICAL STATE SOLUBILITY IN SOLUBILITY IN Ph (using litmus

WATER ETHER paper)
Corn oil Pale yellow liquid Insoluble Soluble Neutral
Linseed oil Light to dark yellow Insoluble Soluble Base
liquid
Lard Color white solid Insoluble Soluble Acid
Butter Yellow liquid Insoluble Soluble Neutral
Cholesterol White, crystalline insoluble Soluble Acid
solid
Stearic acid Solid, powder Insoluble Soluble Acid

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Make a general statement as to the solubility of lipids in various solvents:

Lipids are soluble in non-polar or weakly polar organic solvents such as ether, chloroform, benzene, and acetone but not in

polar solvents like water. These four solvents are together known as "lipid-solvents."

General tests

Spot test: Watch: https://www.youtube.com/watch?v=wpCtf2aqjs8

1. Prepare 3 test tubes and label them with the substances to be tested: chocolate, milk and cooking oil.
2. Place 5 drops of milk and corn oil to the corresponding test tube. For the chocolate, get a matchstick- head
sized portion.
3. Add 1 mL of acetone to the three test tubes and shake to dissolve the contents
4. Get one drop from the first tube containing corn oil and drop it on a 1/8 sheet of paper that is properly labelled
using pencil. Do the same for the other two test tubes using separate 1/8 sheets of paper.
5. Let the acetone dry
6. Hold the paper against the light and observe.

 Your may do this at home by placing a drop or a smear of oil onto a small piece of clean paper
and letting this stand for 5 minutes. Observe this against the light and take a photo then attach this
below this.

SPOT TEST
Observations:
On a 1/8 sheet of paper, I put a drop of oil and let it dry. After which, putting a drop of water on top of the oil
and upon observing for 5 minutes. The water evaporated, leaving the oil being translucent indicating that there’s a
presence of fat.

Acrolein test: Watch: https://www.youtube.com/watch?v=l2QOi9mZoFc

1. Prepare 4 test tubes and label them with the substances to be tested: Glycerol, corn oil, lard and stearic acid
2. Place 5 drops of glycerol and corn oil to the appropriate test tube while a matchstick-head sized portion is used
for the lard and stearic acid
3. Add a pinch of potassium bisulfate into each of the tube

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 Carefully heat each of the tube until the potassium bisulfate is melted and slightly turns black. Do not overheat
since it would be difficult to clean the test tube afterwards!
4. Waft the odor from the test tube towards your nose. Do not inhale directly and note the odor produced
Observations:

pungent odor produced by the solutions came from the acrolein in the test tube, which is the one that indicates
positive result. It signifies the presence of fat/oil in the solution.

Which of the substances tested did not give a positive result?

None of the solutions showed a negative result.

Why?

It is because in every solution given, tghere is a presence of oil or fat which will give a positive reaction to acrolein.

Test for degree of unsaturation: Watch: https://www.youtube.com/watch?v=_5ObG6fIAdQ

1. Get 4 test tubes and label them with the substances to be tested: Olive oil, corn oil, lard
2. Place 3 mL of chloroform into each of the test tubes
3. Add 5 drops of Lugol’s iodine
4. The solution should turn pink in color due to the presence of Iodine.
5. Get the test tube labelled as olive oil and add olive oil drop by drop until the pink color is discharged. Count the
number of drops. Repeat this procedure for the other test tubes.(melt a small amount of lard in an evaporating
dish, do not boil)
6. Observations:
The least amount of iodine drops is required for olive oil, while the most number is required for lard.

What is the principle behind this test?

The test is based on the addition of halogens such as iodine to the double bonds found in unsaturated fat.
Titrating the iodine released determines the amount of iodine consumed. The iodine content of values is determined.
Halogenation of iodine causes unsaturated fatty acids to become saturated by breaking one of the double bonds and
establishing single bonds with iodine. Iodine will be able to absorb into the unsaturated lipids' weaker double or
triple bonds, making them clear.

Which of the substances is the most unsaturated?

Olive oil
Which of the substances is the least unsaturated?
In comparison to the two substances, lard is the least unsaturated due to its high saturated content.

Cholesterol

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Caution: sulfuric acid is corrosive, be sure to wear goggles and gloves for this activity.

Leibermann-Burchard Test
1. Prepare 2 test tubes and label with the substances to be tested for: cholesterol and egg yolk
2. Place a pinch of cholesterol and a small amount of raw egg yolk in the appropriate test tube
3. Dissolve this in 0.5 mL of chloroform
4. Add 5 – 6 drops of acetic anhydride to each tube with gentle mixing
5. Carefully add 0.5 mL of concentrated sulfuric acid down the side of the test tube without mixing

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 6. Immediately record and document your observations. Documentations should be passed together with the
questions for research.
When acetic anhydride and concentrated sulfuric acid are introduced to cholesterol, a dark green color results, indicating that
cholesterol is present, whereas when acetic anhydride and concentrated sulfuric acid are added to egg yolk, a light green hue
results, indicating that cholesterol is present.
Download a photo of the positive result for this test:

Salkowski test
1. Prepare 2 test tubes and label with the substances to be tested for: cholesterol and egg yolk
2. Place a pinch of cholesterol and a small amount of raw egg yolk in the appropriate test tube
3. Dissolve this in 0.5 mL of chloroform
4. Add 0.5 mL of concentrated sulfuric acid
5. Record and document your observations. Documentations should be passed together with the questions
for research.
To detect cholesterol in a solution, the Salkowski test is utilized. The solution turns bluish red and changes to
violet red when a solution contains cholesterol and the sulfuric acid turns red with a green color.

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 Download a photo for the positive result of this test

QUESTIONS FOR RESEARCH

Scoring Rubric
10 points for the whole research paper
o 10 – outstanding – no mistakes identified
o 8 – excellent – 2 - 3 mistakes or missing concepts identified
o 6 – Good – 4 - 5 mistakes or missing concepts identified

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 o 4 – Fair – 6 -7 mistakes or missing concepts identified
o 2 – Poor – 8 or more mistakes or missing concepts identified
o 0 – No research paper was submitted

1. Differentiate fats from oils.

 The main difference between Fats and Oils is that, fats are usually derived from animals, whereas oils are
usually derived from plants. In simple terms, fats are animal fats whereas oils are vegetable oils. The other
difference is fats tend to be solids at room temperature; on the other hand, oils tend to be liquid at room
temperature.

2. What are glycolipids, phospholipids and sphingolipids? State their importance in the body
 Glycolipids – it is essential in providing stability of the plasma membrane. Furthermore, they are also
associated with cell to cell interactions, e.g. cell adhesion to form a tissue. They also facilitate cellular
recognition, which is important in immunologic functions.

 Phospholipids - It regulates the permeability of the membrane. It helps by preventing the accumulation of fats
in the liver. It plays a major role in the transportation and removal of cholesterol from the cells. They act as
surfactants in the respiratory system and are also involved in the coagulation of blood cells.

 Sphingolipids – they are enriched in the Central Nervous System (CNS) and display multiple biological
functions. They participate in tissue development, cell recognition and adhesion, and act as receptors for toxins.

 The body uses cholesterol to build the structure of cell membranes, make hormones such as oestrogen,
testosterone, and adrenal hormones, it helps the metabolism work efficiently, and produces bile acids, which
help the body digest fat and absorb important nutrients.

3. What is the importance of cholesterol in the body?

4. What is rancidity? What are the factors that bring about rancidity?
 An unpleasant aroma or flavor is formed by aerial oxidation of unsaturated fat found in meals and other
items. Rancidity is caused by a combination of elements including time, temperature, light, air, exposed
surface, moisture, nitrogenous organic material, and metal traces.

5. What is the Sudan III test?

 Sudan III Stain is a fat detection stain that can be employed in feces, urine, and tissues. Patients with fat in
their stool (steatorrhea) may have a link to pancreatic illness or other fat-absorption disorders.

6. What test can be used to differentiate saturated from unsaturated fatty acids. How is it done and what is the
positive result?
 Saturated and unsaturated fatty acids, as well as oils and fats, are differentiated using the iodine test. Iodine
is used to determine if a lipid is saturated or unsaturated. The lipid is unsaturated if the iodine goes from
brown to clear. The lipid is saturated if the iodine does not change color. Iodine is added to the unsaturated
lipid to determine the degree of lipid saturation.

7. If the body cannot use carbohydrates as the source of energy, it will start using the fat reserves. What are the
advantages and disadvantages of this?
 One of the advantages of using fat as fuel storage in the body rather than carbohydrates is that fat
molecules release more energy than carbohydrates when utilized by the body. Moreover, the storage of
carbohydrates increases blood sugar and insulin level in the body. Because fats are such an efficient form
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 of energy, the body stores any excess energy as fat. The body deposits excess fat in the abdomen (omental
fat) and under the skin (subcutaneous fat) to use when it needs more energy. The body may also deposit
excess fat in blood vessels and within organs, where it can block blood flow and damage organs, often
causing serious disorders.

 Headaches, weariness, weakness, difficulty concentrating, nausea, constipation, foul breath, and vitamin
and mineral shortages are all symptoms of a carbohydrate-deficient diet.

References:

Rancidity. Rancidity - an overview | ScienceDirect Topics. (n.d.). Retrieved November 17, 2021, from
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/rancidity.

Oxford languages and google - english. Oxford Languages. (n.d.). Retrieved November 11, 2021, from
https://languages.oup.com/google-dictionary-en/.

Indiastone.com. Sudan Red Counter Tops. (n.d.). Retrieved November 17, 2021, from http://indiastone.com/sudan-
red-counter-tops-eoztt.biz.

University of Babylon. (n.d.). Retrieved November 17, 2021, from

https://repository.uobabylon.edu.iq/2010_2011/4_3213_96.doc.

Education.com. (2010, November 6). Good and bad lipids: Science project. Science project | Education.com.
Retrieved November 17, 2021, from https://www.education.com/science-fair/article/lipid-testing/.

Al-Noor, T.. (2019). Re: What are the different between Fats and oils?. Retrieved from:
https://www.researchgate.net/post/What_are_the_different_between_Fats_and_oils/5ca7afa2d7141b723956deda/
citation/download.

Biologyonline (2021). Glycolipid. Retrieved from https://www.biologyonline.com/dictionary/glycolipid

BYJU’s Biology (n.d.). Phospholipid. Retrieved from https://byjus.com/biology/phospholipid/

Dasgupta, S., & Ray, S. K. (2017). Diverse Biological Functions of Sphingolipids in the CNS: Ceramide and
Sphingosine Regulate Myelination in Developing Brain but Stimulate Demyelination during Pathogenesis of
Multiple Sclerosis. Journal of neurology and psychology. Retrieved from https://doi.org/10.13188/2332-
3469.1000035

Better Health Channel (n.d.). Cholesterol. Retrieved from

https://www.betterhealth.vic.gov.au/health/conditionsandtreatments/cholesterol

study.com (n.d.). What are two advantages using fat over carbohydrates for fuel storage in the body. Retrieved
from https://study.com/academy/answer/what-are-two-advantages-of-using-fat-over-carbohydrates-for-fuel-
storage-in-the-body.html

Youdim, A. (2019). Carbohydrates, Proteins, and Fats. Retrieved from

https://www.merckmanuals.com/home/disorders-of-nutrition/overview-of-nutrition/carbohydrates-proteins-and-
fats

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