Group 1

Abuan, Michaila Gwen

Adviento, Mikaela Jemea
Angnged, Jamaica Ces
Antolin, Chelsea Marie
Cabradilla, John Harold

Activity No. 20
PHYSICAL AND CHEMICAL PROPERTIES OF LIPIDS

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 water Solubility in pH (using litmus

ether paper)
Corn oil Liquid Immiscible Miscible No change in color

Linseed oil Liquid Immiscible Miscible No change in color

Lard Solid Insoluble Soluble Blue litmus paper

turns red
Red litmus paper
remains red

Butter Solid Insoluble Soluble No change in color

Cholesterol Solid Insoluble Soluble No change in color

Stearic acid Solid Insoluble Soluble Blue litmus paper
turns red
Red litmus paper
remains red

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

Lipids are insoluble in polar solvents like water but highly soluble in the non-polar or weakly polar organic
solvents such as ether, chloroform, benzene, and acetone.

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.

• You 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 below this.

SPOT TEST

Observations:
 They all look translucent to start with because they’re wet so it is important to leave them to dry for a few
minutes and the chocolate contains 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
4. Carefully heat each of the tubes until the potassium bisulfate is melted and slightly turns black. Do not
overheat since it would be difficult to clean the test tube afterwards!
5. Waft the odor from the test tube towards your nose. Do not inhale directly and note the odor produced

Observations:
When oil or fat is treated strongly in presence of a dehydrating agent like potassium bisulphate, the glycerol
portion of the molecule is dehydrated to form an unsaturated aldehyde, acrolein which has a pungent
irritating odor.

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

The substances tested that didn’t give a positive result is cholesterol.

Why?
Further heating results in polymerization of acrolein, which is indicated by the slight blackening of the
reaction mixture. Both the pungent smell and the black color indicate the presence of glycerol, and thereby
fat and/or lecithin. Cholesterol gives a negative acrolein test.

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:
When the solution was mixed in the olive oil, the solution disappeared due to the presence of unsaturation.
In the corn oil, after the solution was mixed into the corn oil, complete discoloration of the solution has
taken place and it shows unsaturation. Lastly, when the solution was mixed with the lard, the pink color of
the solution disappears indicating that lard is unsaturated,
What is the principle behind this test?
The principle behind the reaction is the addition of halogens like iodine to the double bonds present in the
unsaturated fat. Furthermore, the most important application of the iodine value is to determine the amount
of unsaturation contained in fatty acids. This unsaturation is in the form of double bonds which react with
iodine compounds. The higher the iodine value, the more unsaturated fatty acid bonds are present in a fat.

Which of the substances is the most unsaturated?

Corn oil
 Which of the substances is the least unsaturated?
Lard

Cholesterol

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

Liebermann-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
6. Immediately record and document your observations. Documentations should be passed together with
the questions for research.
On the egg yolk, it took a few seconds for the light green color to appear which indicates that it is a
positive result for this test. On the other test tube which contains cholesterol, when the concentrated
sulfuric acid was added into the side of the test tube, the dark green color settled at the bottom of the
test tube.
Download a photo of the positive result of 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.
 Brick-red color is formed indicating the presence of cholesterol.
Download a photo of the positive result of this test:

QUESTIONS FOR RESEARCH

1. Differentiate fats from oils.
One difference between fats and oils is that at room temperature fats are solid whereas oils are
liquid. Fats and oils are both made of one part called glycerol attached to three other parts called
fatty acids. Glycerol is a thick liquid with many industrial uses. The fatty acids can vary and this
makes different kinds of fats and oils.
2. What are glycolipids, phospholipids and sphingolipids? State their importance in the body
Glycolipids are sugar-containing lipids. Phospholipids are lipids containing phosphorus.
Sphingolipids are phospholipids or glycolipids that contain the unsaturated amino alcohol
sphingosine rather than glycerol. The main function of glycolipids in the body is to serve as
recognition sites for cell to cell interactions. Phospholipids regulate cellular processes related to
growth, synaptic transmission and immune surveillance. While sphingolipids in digestive system
are responsible for numerous important physiological and pathological processes. In the
membrane of gut epithelial cells, sphingolipids provide structural integrity, regulate absorption of
some nutrients, and act as receptors for many microbial antigens and their toxins.
3. What is the importance of cholesterol in the body?
We need a small amount of blood cholesterol because the body uses it to build the structure of
cell membranes; make hormones like oestrogen, testosterone and adrenal hormones; help your
metabolism work efficiently, for example, cholesterol is essential for your body to produce vitamin
D; and produce bile acids, which help the body digest fat and absorb important nutrients.
4. What is rancidity? What are the factors that bring about rancidity?
When food containing fat and oil come in contact with surrounding oxygen and these auto-oxidation
leads to bad smell and change in taste, the whole process is said to be rancidity.
Factors Affecting Rancidity Includes:
1. Oxidation - Oxygen is eight times more soluble in fats than in water and it is the oxidation resulting
from this exposure that is the primary cause of rancidity. Oxidation primarily occurs with unsaturated fats
by a free radical-mediated process.
2. Hydrolysis - Triglycerides react with water under appropriate condition to form diglycerides and free
fatty acid residues. Diglycerides later combine with water to form monoglycerides and fatty acids. Finally
 the monoglycerides completely hydrolysed to form glycerol and fatty acids. This process is called
hydrolytic rancidity.
3. Presence of Microorganisms – Microbial Lipase: Certain microorganisms can produce the hydrolytic
enzyme called lipase, which directly interferes the hydrolysis of triglcerides and produce glycerols and
fatty acid. These fatty acids undergo auto- oxidation to form rancid. The microbial lipase requires suitable
pH and other conditions for its activity upon fats and oil.
4. Presence of Unsaturation in Fatty Acid Chain - When a fatty substance is exposed to air, its
unsaturated components are converted into hydroperoxides, which break down into volatile aldehydes,
esters, alcohols, ketones, and hydrocarbons, some of which have disagreeable odours.
5. Polyunsaturation:
The more polyunsaturated a fat is, the faster it will go rancid. Vegetable oils have to become several
times more rancid than animal fats. Presence of polyunsaturation in oils and fats makes them more
susceptible to rancidity than monosaturated and other types of saturated fatty acids.
6. Chemical Structure of Oils and Fats - If oils and fats are chemically more complex and consists more
number of double bond, more number of carboxyl or hydroxyl groups, then the chances of become
rancid is high.
7. Temperature and pH - These are the important factor which influences the food items rich in fat and
oils become rancid. Suitable temperature and alkaline pH are required for the hydrolytic action of
microbial lipase. Temperature and pH indirectly influence the auto-oxidation and hydrolysis.
8. Heat and Light - Presence of heat and light accelerate the rate of reaction of fats with oxygen, i.e.,
heat accelerates auto-oxidation.

4. What is the Sudan III test?

Sudan III is used to identify the presence of lipids in liquids. It will stain fat cells red. Fats are
hydrophobic organic molecules that are rich in energy due to their high density of C-H bonds. They
can be detected by the Sudan Test, which relies on hydrophobic interactions between Sudan III dye
and lipids. Sudan III dissolved in ethanol is allowed to interact with the lipids bound to a filter, then
when the filter is washed with water the water will not permit Sudan III bound to the lipids to escape.
Consequently, spots containing lipids will appear orange against a pink background.
5. What test can be used to differentiate saturated from unsaturated fatty acids? How is it done and what
is the positive result?
Iodine test is used for distinguishing between saturated and unsaturated fatty acids as well as between
oils and fats. In performing the test, a known excess of iodine, usually in the form of iodine
monochloride, is allowed to react with a known weight of the oil, fat, or wax, and then the amount of
iodine remaining unreacted is determined by titration. As for the result, if the iodine changes from brown
to clear the lipid is unsaturated. If the iodine does not change colors the lipid is saturated.
6. 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?
The advantages of this is that the muscles, lungs and heart pick up these fatty acids, break them apart,
and use the energy stored in the bonds to execute their activities. While the disadvantages of reserved
fats can cause cholesterol that build up in the arteries. Saturated fats raise your LDL cholesterol. High
LDL cholesterol increases your risk for heart disease and stroke.
References:

Amrita.olabs.edu.in,. (2015). Qualitative Analysis of Oils and

Fats.amrita.olabs.edu.in/?sub=73&brch=8&sim=210&cnt=1

Better Health. (n.d). Cholesterol.

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

Byjus. (2021, March 22). General Data Protection Regulation(GDPR) Guidelines BYJU’S. BYJUS.
https://byjus.com/chemistry/difference-between-fats-and-oils/

Kurek, K. (2013, September 5). Metabolism, Physiological Role, and Clinical Implications of Sphingolipids in
Gastrointestinal Tract. Hindawi. https://www.hindawi.com/journals/bmri/2013/908907/

Libretexts. (2020, August 11). 15.4: Membranes and Membrane Lipids. Chemistry LibreTexts.
https://chem.libretexts.org/Courses/Sacramento_City_College/SCC%3A_Chem_309_-
_General_Organic_and_Biochemistry_(Bennett)/Text/15%3A_Lipids/15.4%3A_Membranes_and_Membrane_L
ipids

Manisha, S. (n.d.). Rancidity of Food: Introduction, Types, Factors and Prevention of Rancidity | Food Chemistry
| Biotechnology. https://www.biotechnologynotes.com/food-biotechnology/food-chemistry/rancidity-of-food-
introduction-types-factors-and-prevention-of-rancidity-food-chemistry-biotechnology/14100

Salkowski’s test: Part 2 (Identification of Cholesterol). (2013, May 12). [Video]. YouTube.
https://www.youtube.com/watch?v=uHDOzBzcDu8

University of Calgary. (n.d.). Lipids. https://www.chem.ucalgary.ca/courses/350/Carey5th/Carey.html

Staffweb. (n.d). Biological Molecules. https://staffweb.wilkes.edu/william.terzaghi/BIO-121/labs/lab2.html

Science Project. (n.d). Good and Bad Lipids. https://www.education.com/science-fair/article/lipid-testing/
Britannica. (n.d). Iodine Value. https://www.britannica.com/science/iodine-value
Dr. Mag. (2013, May 13). Libermann-Burchard's test: Part 2 (Identification of Cholesterol) [Video].
https://www.youtube.com/watch?v=hc7cauZHNK8
Singh, C. (2020, November 23). Experiment No. 8 -Test for unsaturation of oils [Video].
https://www.youtube.com/watch?v=zKZIlQm3emo
GROUP MEMBERS CONTRIBUTIONS

ABUAN, Michaila Gwen Absent/Cannot be reached

ADVIENTO, Mikaela Jemea Researcher for:

- Salkowski test

QFR 1 & 2

ANGNGED, Jamaica Ces Researcher for:

- Spot Test
- Acrolein Test

QFR 7

ANTOLIN, Chelsea Marie Researcher for:

- Test for degree of unsaturation
- Leibermann-Burchard Test

QFR 5 & 6

CABRADILLA, John Harold Researcher for:

- Solubility and Physical Appearance
- pH

QFR 3 & 4