Ateneo de Zamboanga University

College of Science and Information Technology

Natural Sciences Department
NURBIO D
______________________________________________________________________________

Name/s: AREVALO, Alessandra L.

BERNARDO, Alexander Scores
LUI, Sharah Gayle P.
Introduction _______
PASCUA, Precious
Objective/s _______
SALIBAY, Chosen Lloyd
Results and Discussion _______
Questions _______
Group No. 6 Conclusion and Insights _______
Section: BSN - D Total _______

Activity No. 6 Lipids

I. Introduction
Lipids are found in living organisms. They are a family of substances that are insoluble in
water, which means they are generally hydrophobic. Lipids play three major roles in human
biochemistry. They store energy within fat cells, they are parts of the membrane that separate
compartments of aqueous solution from each other and they serve as chemical messengers.

Animal fats and plant oils are triglycerides. Triaglycerides are the form of storage of fatty acids
in organisms, they are triesters of fatty acids and glycerol. Those triaglycerides that are solid are
called fats, while those that are liquid are called oils. Phospholipids are lipids that contain
phosphate groups. Lipids are major components of biological elements, and the major classes
include the glycerophospholipids, sphingolipids and glycospingolipids in animals. Glycerolipids
are widespread in plant and bacterial membranes.

Lipids can be classified as saponifable and nonsaponifable. Saponifable lipids are esters of
fatty acids which includes triglycerides and phospholipids, while nonsaponifable lipids are lipids
that do not contain any fatty acids or ester linkages which includes, steroids, prostaglandins,
leukotrienes, and terpenes.

II. Objectives
1. To compare the different tests used in determining lipids.
2. To distinguish the different properties of lipids through the different test performed and
different reagents used.
3. To detect the presence of lipids through the different tests
III. Results and Discussion
A. Solubility Tests

Sample Solvent Used Result

Cottonseed Distilled water Immiscible, polar

Oil (A)
Time: Greater than 5 minutes

Negative Result

Ethyl alcohol Partially immisicible, polar

(B)
Time: Greater than 4 minutes

Negative Result

Ether Immiscible, slightly polar

(C)
Time: 10 seconds

Negative Result

Chloroform Miscible, non-polar

(D)
Time: 2 minutes

Positive Result

5% HCl Immiscible, polar

(D)
Time: Greater than 3 minutes
 Negative Result

5% NaOH Miscible, non-polar

(E)
Time: 3 seconds

Positive Result

In our experiment for the Solubility Test, we first followed the procedure in performing
this experiment, which was to pipette 1mL of the following solvents in separate test tubes: distilled
water, ethul alcohol, ether, chloroform, 5% hydrochloric acid, and 5% sodium hydroxide. From a
pipette, we added 1-2 drops of cottonseed oil in each test tube and shake thoroughly. We then
recorded the time required for the oil to dissolve.

In the experiment, we observed that Distilled Water (A), Ethyl Alcohol (B), Ether (C) and
5% HCL (E) became immiscible, which is a polar solvent, resulting to a negative result. While the
Chloroform (D), and 5% NaOH (F) became miscible, which is a non-polar solvent, resulting to a
positive result in the Solubility Test.

In the Solubility Test, it is the preliminary test which detects the presence of all lipids. This
test detects the solubility of lipids in various solvents to check wherether it is miscible or
immiscible in polar or non-polar solvents. It is based on the property of lipid to dissolve in differet
solvents. Lipids are readily miscible in non-polar solvents like chloroform, partially soluble in a
polar system like ethanol and immiscible in a polar solvent like water. For a positive result, lipids
are soluble in non-polar solvents. A non-polar solvent contains bond atoms with similar
electronegatives. While for a negative result, lipids are insoluble in a polar solvent. A polar solvent
have large dipole movements, they contain bonds between atoms with different electronegatives.
B. Test for Unsaturation

Name of Test Test Reagents Result

Solutions Added

Test for Oleic Acid Br2 in CCl4 It took 3 drops to produce a faint
Unsaturation color of yellow, at around 8 drops
(D) the color yellow became very
noticeable at this point.
There was no presence of orange
color.

Palmitic CHCl3 Haus It took 30 seconds for the color to

Acid Iodine spread.

(A)

Oleic Acid CHCl3 Haus It took 40 seconds for the color to

Iodine spread.
(B)

Cottonseed CHCl3 Haus It took 40 seconds of the color to

Oil Iodine spread.

(C)

In our experiment for the Tests for Unsaturation of Fatty Acids, we first followed the
procedure in performing this experiment, 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. Then, into each of 3 test tubes, we placed 2 mL of chloroform. Next we added
to each test tube 0.2 g of palmitic acid, 4 drops of oleic acid, and 4 drops of cottonseed oil
respectively. We shaked each test tube thorougly, then we added 4 drops of Hanus – iodine solution
to each test tube. Afterwards, we recorded all results, and time in seconds for the color to disappear.

In the experiment, for the Oleic Acid, it took around 3 drops of Br2 in CCl4 to have a faint
color of yellow, and then on the 8th drop, the color yellow in the solution became more noticeable.
For the Palmitic Acid, it took around 30 seconds for the color to spread. Then for the Oleic Acid,
it took around 40 seconds for the color to spread. Lastly, for the Cottonseed oil, it took around 40
seconds for the color to spread. All solutions produced a color of pink which resulted to a positive
result.

In the Test for Unsaturation, it is used to detect the presence of unsaturated fatty acids or
the amount of double bond in a liquid sample. All the neutral fat contains glycerides or fatty acids.
The double bond found in the structure of unsaturated fatty acids which becomes saturated by
either taking up bromine or iodine. If the lipids are more unsaturated fatty acids or more double
bonds, that means it will take more iodine. For a positive result, the outcome will be a pink color
that will disappear by the addition of unsaturated fatty acids. While for the negative result, the
outcome is that the pink color will not disappear.
C. Acrolein Test
Name of Test Test Reagents Result
Solutions Added

Acrolein Test Glycerol KHSO4 (s) The solution turned into brown-red
after being heated into a low flame.
(A)
A strong pungent smell was
produced.

Positive Result

Cottonseed KHSO4 (s) The solution became transparent in

oil color after being heated into a low
flame.
(B)
No strong pungent smell was
produced.

Negative Result

In our experiment for the acrolein test, we first followed the procedure, which was to
prepare two test tubes. For the first test tube, it was to place 2 drops of glycerol and a pinch of
KHSO4, while for the second test tube, place 2 drops of cottonseed oil and a pinch of KHSO4. We
heated each test tube over a low flame, and then we recorded the order that was produced from the
solutions.
 In the results that we recorded from the experiment, we noticed that in the first test tube,
after heating it resulted to a color of brown-red and it produced a pungent smell. While for the
second test tube, the color became transparent, and there was no pungent smell that was produced.
Between the two test tubes, the first test tube which contains glycerol and potassium bisulfated
resulted to positive, wherein there is a presence of glycerol and fats, since it produced a pungent
smell compared to the second test tube.
In the Acrolein Test, it is used to detect the presence of glycerol and fats. This test is based
on the “Dehydration Reaction”, where the water molecules removed from the glycerol by the
addition of reagent potassium hydrogen sulphate. The reaction between glycerol and potassium
hydrogen sulphate results in the formation of “Acrolein” that is characterized physically by the
release of the pungent smell.
D. Rancid Test
Test Reagents Result
Name of Test Solutions Added

Rancidity Test Fresh Phenolphthalein The color became light pink, a bit of
coconut oil purple.
(A)

Methyl orange The color became light orange.

(B)

pH paper The litmus paper became

transparent.
(C)

Rancid Phenolphthalein The color became dull yellow, a bit

coconut oil of brown.
(D)

Methyl orange The color resulted to a golden

yellow.
(E)

pH paper The blue pH paper reacted violet, the

red remained
(F)
 In our experiment for the Rancidity Test, we first followed the procedure in performing
this experiment, which was to prepare six test tubes, three test tubes for the coconut oil, and three
test tubes for for the rancid oil. On the first test tube, we placed 5 drops of coconut oil, and 5 drops
of rancid oil. Next, we tested the reaction of fresh coconut oil with 1-2 drops phenolphthalein, 1-
2 drops methyl orange, and red and blue litmus paper. Then, we did the same reaction test with
rancid oil. Afterwards, we recorded the results from the tests.
In the first test with fresh coconut oil, when mixed with the reagents, there was no oxidation
in the outcome. For the phenolpthalein, it became pink which means that the oil was basic. Then
for the methyl orange, it resulted with a color of light yellow, which resulted that the coconut oil
was still basic. Lastly, for the litmus paper, both resulted to becoming transparent. While for the
second test with the rancid oil, the pheolphthalein resulted light yellow which signfies as pH 6-7
which is near to basic. Then for the methyl orange, it turned golden yellow which means the
solution increased in acidity. And lastly, for the litmus paper, the blue litmus paper turned purple
as a result of it being acidic, while the red litmus paper remained as it is.
Rancidity is a term generally used to denote a condition of unpleasant odors and flavours
in food resulting from the deterioration in the fat or oil portion of a food. In the Rancidity Test, in
knowing whether an oil is rancid, it is by the development of unpleasant smells in fats and oils
that are often accompanied by the changes in their texture and appearance.
E. Saponification

Test Reagents Result

Name of Test Solutions Added

Saponification coconut oil 10% KOH The whole procedure took 25

minutes. The result of the coconut oil
became clear, an oil at the top.

In doing our experiment for the Saponification test, we first followed the procedure, which
was to weigh 1.5 g of fresh coconut oil in a dry beaker, then we added 10 mL of 10% alcoholic
potassium hydroxide. Then, we covered the beaker with a watch glass. Afterwards, we fill yje
watch glass with crush ice, boil it over a water bath until a drop of the hot solution aaded to cold
water in a test tube does not form globules of fat. Then, we removed the watch glass to drive off
the alcohol, next we added 20 mL of water to the gelatinous mass and warm the solution to dissolve
the soap. Afterwards, we observed and recorded the results.
In doing the Saphonification test, we followed the procedure that took around 25 minutes
in order to produce the outcome of a froth that appeared in the test tube signifying that it was a
positive result.
In the Saponification test, it is a test based on the “Saponification reaction”, where the
triglycerides of lipid react with an alkali NaOH (Sodium Chloride) and produce soap and
glycerol in the presence of ethanol. This reaction also refers to as “Alkaline hydrolysis of
ethers”. For a positive result, a froth must appear in the test tube. While for the negative result,
there is an absence of froth in the test tube.
F. Properties of Soap

Name of Test Test Reagents Result

Solutions Added
The NaOH (Salt) diffused on the test
Salting Out Soap tube when mixed with the solution
Solution from the previous experiment. Some
floated at the top of the test tube.
(A) There was a formation of crystals;
white substance
The solution became cloudy, and
Formation of Soap there was a formation of bubbles in
Fatty Acids Solution the test tube

(B)

Soap 5% Calcium There was a formation of white suds

solution Chloride that looked cloudy
Insoluble
Soaps (C)

Soap 5% Magnesium There was a formation of white suds

Solution Chloride that looked cloudy

(D)

Detergent 5% Calcium There was a formation of white suds

Chloride that looked cloudy
(E)

Detergent 5% Magnesium It produced white precipitation, suds

Chloride were formed
(F)

In doing our experiment for the Properties of Soap, we first followed the procedure
for Salting Out, Formation of Fatty Acids, and Insoluble Soaps. For the Salting out, we placed 10
mL of the soap solution in a beaker and added table salt gradually with stirring until no more table
salt dissolves. Then we removed the solid soap formed from the liquid and washed it with water.
We transferred a small amount of solid soap into a test tube and shaked it with distilled water to
form suds. Second, for the Formation of Fatty Acids, we first placed 5 mL of the soap solution in
a test tube and added 10% HCl until a precipitate forms. Lastly, for Insoluble soaps, we placed 2
test tubes, each containing 4 mL of distilled water and 1 mL soap solution. To test tube 1, we added
5 drops CaCl2 solution. To test tube 2, we added 5 drops 5% MgCl2 solution. We observed the
results and repeated the test using liquid detergent instead of soap solution. We observed and
recorded the results, afterwards, we compared it with the results of the first part.
For the Salting Out, it is used to investigate the effect of a NaCl on soap solubility. It is to
get the soap out of the solution by salting out, when added sodium chloride (NaCl – Salt) to the
solution until saturation; separated soap in the form of insoluble and floats above the surface. The
NaCl solution provides Na+ and Cl- ions that bond to the polar water molecules and help separate
the water from the soap. This process is called salting out the soap. For Insoluble soaps, it is used
to investigate the effect of different cations on soap solubility. Working calcium, magnesium, lead
or iron ions to the deposition of soap and make it insoluble in water, where some of these ions
replace the sodium or potassium ions are present in the soap. Due to the hard water, it contain
significant quantities of Ca2+, Mg2+, and some Fe3+ that react with the charged ends of the soaps
to form insoluble salts of fatty acids. The insoluble salts of fatty acids Mg2+ and that Ca2+ and Mg2+
form with soap anions cause white precipitate to form.
 According to Wikipedia, due to their opposite polarity, water by itself cannot penetrate
grease or oil. However, when grease or oil (non-polar hydrocarbons) are mixed with a soap-water
solution, the soap molecules work as a "bridge" between polar water molecules and non-polar oil
molecules. Soap molecules are amphipathic and thus have both properties of non-polar and polar
at opposite ends of the molecule. The oil is a pure hydrocarbon so it is non-polar. The non-polar
hydrocarbon tail of the soap dissolves into the oil. That leaves the polar carboxylate ion of the soap
molecules are sticking out of the oil droplets, the surface of each oil droplet is negatively charged.
As a result, the oil droplets repel each other and remain suspended in solution (this is called an
emulsion) to be washed away by a stream of water. The outside of the droplet is also coated with
a layer of water molecules. This is also similar to a micelle which works with the same principles-
the center of the micelle would contain the oil.
F. Emulsifying Action of Lecithin

Name of Test Test Reagents Result

Solutions Added
Emulsifying 2% albumin Cholesterol Turned white in color.
Action No odor was produced after the two
(A) solutions were mixed.
Layers were formed; soluble in
albumin.
Negative Result
Cholesterol – Produced light brown in color.
lecithin No strong odor was produced
There was no layer formed, and
(B) there was a mixture between the
cholesterol and albumin.
Positive Result
In our experiment for the test of Emulsifying Action of Lecithin, we first followed
the procedure in performing this experiment, in each of two test tubes, then we placed 5 drops of
2% albumin solution. To the test tube 1 – we added a pinch of cholesterol and shaked the tube.
While to test tube 2 – we added a pinch of cholesterol and five drops of lecithin. We then compared
and recorded the results.
 In our experiment for the Emulsifying Action of Lecithin, in the first experiment for the
2% albumin and cholesterol, layers were formed, it was soluble in albumin. While for the second
experiment for the 2% albumin and cholesterol – lecithin, there was no layer formed and it resulted
to a mixture for the cholesterol – lecithin and albumin. In the first experiment, it resulted to a
negative result, while for the second experiment, it was a positive result.

In the Emulsification Test, it is used to detect the presence of lipids. Emulsification is the
process which stabilizes the water and oil emulsion, by the help of emulsifying agents. The lipid
or oil in water appears on the top of the water because of the high surface tension of water which
gets together to form a separate layer. The emulsifying agents emulsify the lipid by which the lipid
appears as the tiny droplets suspended in the solution. For a positive result, it gives a permanent
or stable emulsion of lipid and water. While for the negative result, the oil in water emulsion will
form at the top, due to the high surface tension of water.

Questions
1. Why are fatty acids insoluble in water?
Fatty acids are insoluble in water since there are more hydrocarbons which
are more hydrophobic than the carboxyl group which is soluble in water. The longer
the chain of the fatty acid becomes, the more soluble it gets.

2. Explain why the cis-form is the predominant configuration of unsaturated

fatty acids?

Cis-form configuration is more predominant in unsaturated fatty acid

since most of the fatty acids are in liquid form, and most of these liquid form
unsaturated fatty acids has this configuration.

3. What type of rancidity occurs in vegetable shortenings? How can it be

prevented?
The type of rancidity that occurs in vegetable shortenings is the oxidation
rancidity. In this ‘oxidation rancidity’, the oxygen molecules interact with
molecules of the oil which causes damage or changes to the substance. The easiest
 way to prevent this from happening is to to store these vegetable shortenings in a
dark, cool place where it is less exposed to oxygen.

4. Show the structure of the parent compound of cholesterol.

5. Explain the cooperative effect of lecithin and albumin.

Lecithin and albumin are good emulsifiers because they both have a non-
polar and a polar portion which helps reduce the immiscibility of two substances.
When they work hand-in-hand, they are able to hold polar substances through their
hydrophilic molecules, thus making the immiscibility of the two substances
positive.

IV. Conclusion and Insights

Lipids are fats that are important part of cells and sources of energy. Lipids are fat-like
substances for they contain a large heterogenous group of unrelated physiological and chemical
substances classified together which also observes the rule “like dissolves like”. The polar end of
the fatty acid is carboxyl group (R-COOH) which helps with the polarity of the lipids. The
hydrocarbon chains are the reason why lipids are nonpolar meaning it makes them insoluble in
polar solvents like water.

The different test performed in the lipids determined some of its properties. The first test
is the solubility test, the test for presence of lipid and if it is miscible and immiscible in polar or
nonpolar solvents. It showed that distilled water, Ethyl alcohol, Ether and 5% HCL became
immiscible, which is a polar solvent, resulting to a negative result. Thus, the dissolving process of
different solvents is based on the property of lipid. The test for unsaturation of fatty acids
determined the varying number of double bonds that each fatty acid (lipid) contain. It showed that
the Oleic acid took 8th drop for the color yellow in the solution to be more visible or noticeable.
The Palmitic acid, it took only 30 seconds to spread; however, 40 seconds it took for the color to
spread in Oleic Acid and Cottonseed oil. Thus, all the solutions resulted to positive because it
produces a color of pink. In the acrolein test, the test tube 1 (2 drops of glycerol and a pinch of
KHSO4), a color of brown-red and with a pungent smell that indicates a positive result. Rancidity
test is the test to determine the rancidity of the oil is. It showed that phenolphthalein became pink
that indicates the oil was basic. The methyl orange showed color of light yellow that indicates oil
was still basic. Lastly, it showed that both became transparent. For the second test, phenolphthalein
resulted light yellow and indicates near to basic and for the methyl orange it resulted to golden
yellow that indicates that solution has increase in acidity. Saponification is where the triglycerides
of lipid react with an alkali NaOH (Sodium Chloride) and produce soap and glycerol in the
presence of ethanol. It resulted that the procedure that took around 25 minutes in order to produce
the outcome of a froth that appeared in the test tube signifying that it was a positive result. Salting
out is a purification method that relies on the solubility of the protein. It is to get the soap out of
the solution by salting out, when added sodium chloride (NaCl – Salt) to the solution until
saturation. For Insoluble soaps, it is used to investigate the effect of different cations on soap
solubility. Lastly, the emulsification test is use to detect the presence of lipids. The first experiment
showed negative result, while the second experiment showed a positive result because there was
no layer formed on top.
 V. Referrences

Lab Activity 5 Lipids. (n.d.). Retrieved July 24, 2019, from https://studylib.net/doc/9790812/lab-
activity-5-lipids

Lipids Chemlab Report. (n.d.). Retrieved July 24, 2019, from

https://www.scribd.com/doc/90427181/Lipids-Chemlab-Report

Memije-Cruz, L. (2018, April 25). Qualitative and quantitative tests for lipids. Retrieved July 24,
2019, from https://www.slideshare.net/memijecruz/qualitative-and-quantitative-tests-for-lipids

N, S. (2019, May 28). What is Qualitative Analysis of Lipids? definition & methods. Retrieved July
24, 2019, from https://biologyreader.com/qualitative-analysis-of-lipids.html#DichromateTest

Qualitative and Quantitative Tests for Lipids. (2015, October 16). Retrieved July 24, 2019, from
http://www.biologydiscussion.com/lipids/tests/qualitative-and-quantitative-tests-for-lipids/13050

Wikibooks. (2019). Structural Biochemistry/Lipids/Soap. Retrieved July 24, 2019 from

https://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Soap