Experiment # 5

Lipids

Group Members: Sung Kyun A. Na | Eli T. Gozon | Tisha Alyanna R. Perocho

Year/Section: BSN 1B

Date performed: September 26, 2021 Date Submitted: September 30,

2021

Objective: To study some properties of lipids and reactions used for their identification.

Materials/Apparatus: test tubes, Bunsen burner, test tube holder, transfer pipets, pH
sticks, bond paper, vegetable oil, oil of wintergreen, distilled water, alcohol, dilute HCl,
dilute NaOH, ether, chloroform, acetone, glycerol, lecithin, oleic acid, stearic acid, olive
oil, coconut oil, Hubl’s solution, bile salts

Procedure:

A. Properties of Lipids

1. Solubility

Determine the solubility of vegetable oil in 1 mL of the following solvent:

Observe and tabulate your results

Insoluble
a. Water

Insoluble
b. Dilute NaOH

Soluble
c. Chloroform

Insoluble
d. Dilute HCl

Soluble
e. ether

Soluble
f. acetone
 Insoluble
g. cold alcohol

Insoluble
h. hot alcohol

2. Formation of a Translucent Spot

Place one drop of vegetable oil on a piece of ordinary bond paper. Note the formation of
a semi-transparent spot. Allow the oil to evaporate spontaneously. Does the
translucent spot disappear?

Repeat the same procedure using the oil of wintergreen instead of vegetable oil. Note
the difference in the results below.

Vegetable Oil After the drop of vegetable oil on the

paper the translucent spot is smaller than
the spot of the wintergreen green oil as it
does not disperse immediately. After 30
minutes the translucent spot of the
vegetable oil was still visible and it
increased. Then after an hour it is still
visible but it brightens and is gradually
fading.

Oil of Wintergreen After the drop of the oil of wintergreen on

the paper the translucent spot is slightly
bigger than the drop of the vegetable oil
as it disperse immediately. After 30
minutes, the translucent spot of the
wintergreen oil gently faded away. Then
after an hour the spot totally vanished.

3. Reaction to Litmus Paper

Test the reaction of fresh vegetable oil with red and blue litmus papers previously
moistened with water.

a. What is the reaction?

The litmus paper is an acid-base indicator. The red litmus paper is used to detect
alkaline solutions while the blue litmus paper is used to test for acids when it
 comes in contact with an acidic solution. The reaction we see is the litmus papers
color changing depending on the pH level of the solution.

Allow the oil to stand uncovered until the next laboratory period and test again with
litmus paper.

b. Is there any change in the reaction?

The blue litmus paper didn't experience any immediate reaction after being
applied with vegetable oil but the color changes to red after a day.

The red litmus paper didn't experience any reaction

B. Reactions of Lipids

1. Test for Unsaturation

Use dry test tubes. In each of four test tubes, place separately two drops of the sample
(olive oil, oleic acid, stearic acid, and coconut oil), and add 1 mL of chloroform. Then
add a drop of Hubl’s solution (iodine in mercuric chloride solution,). Cover the test tube
and mix well. If the mixture decolorizes, continue adding Hubl’s solution drop by drop,
shaking well after each drop, until it is no longer decolorized. Use the same dropper for
all samples. Record the number of drops used.

Olive oil 80 drops

Oleic acid 85 drops

Stearic acid 1 drops

Coconut Oil 2 drops

2. Lieberman-Burchard or Acetic Anhydride Reaction

In a clean, dry test tube, dissolve a pinch of the sample separately (cholesterol and bile
salts) in 1 mL of chloroform. Then add five drops of acetic anhydride and a drop of
concentrated H2SO4. Mix. Note the change in color.

a. Cholesterol - the color changed into a deep color of violet after adding the five
drops of acetic anhydride and a drop of conc. H2SO4.
b. Bile salts - the color changed into a golden yellowish color after adding the five
drops of acetic anhydride and a drop of conc. H2SO4.
Results and Observations:

Tests Result

Solubility Based on our observations, the vegetable

oil is insoluble in water, Dilute NaOH,
Dilute HCI, cold alcohol, and hot alcohol
because when the vegetable oil was
added with the mentioned solvents they
are still separated from each other. On
the other hand, the vegetable oil is
soluble in chloroform, acetone, and ether
as it appears that when the vegetable oil
was added with the mentioned solvents
they are combined or incorporated into
one another.

Formation of a Translucent Spot After the drop, the vegetable oil’s

translucent spot is smaller than the spot
of the wintergreen green oil as it does not
disperse immediately whereas the
wintergreen oil’s translucent spot was
bigger than the vegetable oil as it
disperse immediately. After 30 minutes
the translucent spot of the vegetable oil
was still visible and it increased while the
wintergreen oil's translucent spot gently
faded away. Then after an hour, the
vegetable oil’s translucent spot is still
visible but it brightens and is gradually
fading but the oil of the wintergreen totally
vanishes.

The red litmus paper did not undergo any

Reaction to Litmus Paper reaction. The blue litmus paper did not
react to the lipids upon contact but after a
day the color changes to red.

Olive oil (80 drops) and Oleic Acid (85

Test for Unsaturation drops) resulted positive in the test
therefore it is unsaturated while the
stearic acid (1 drop) and coconut oil (2
 drop) means that it tested negative in the
test of unsaturation because it only took
1-2 drops to absorb the color of Iodine
solution.

Lieberman-Burchard or Acetic The bile salt solution changed into a

Anhydride Reaction golden yellowish complex after adding 5
drops of acetic anhydride and a drop of
conc. H2SO4 while cholesterol solution
changed into a deep color of violet after
adding the five drops of acetic anhydride
and a drop of conc. H2SO4.

Discussion:

Solubility

- It's a preliminary test that detects the presence of lipids. The lipid solubility test
determines whether lipids are miscible or immiscible in polar or nonpolar
solvents. It is thus based on the solubility of lipids in various solvents. Lipids are
soluble in non-polar solvents such as chloroform, somewhat soluble in polar
solvents such as ethanol, and immiscible in polar solvents such as water. For a
positive result: Lipids are soluble in a nonpolar solvent, i.e. chloroform and
partially soluble in ethanol which can solubilize upon heating. And for a negative
result: Lipids are insoluble in a polar solvent, i.e. water.

Formation of a Translucent Spot

- A translucent spot test, which is characterized by a translucent and oily spot, is

also a preliminary lipids test. Unlike water, the lipid will not moisten the filter
paper. Because of their greasy texture, the lipids will penetrate the filter paper
and form a greasy or transparent spot. Unlike lipids, the water spot on the paper
will vanish. On a positive result the translucent spot will appear on the filter paper
and on a negative result the translucent spot will not appear on the filter paper.

Reaction to Litmus Paper

The level of concentration of hydrogen in the solution is the indicator of the pH

levels.
 The reaction occurs because of 7-hydroxyphenoxazone when exposed to acids
below pH 4.5 the litmus paper turns red as the pH test solution moves towards
alkaline the hydrogen atoms starts to break off.

Or when the pH approaches neutrality pH 7 more of the indicator molecules lose

this hydrogen atom. Half of the litmus papers molecules will still have hydrogen
and the other half won't. This mixture produces purple shades in the midrange.
Once it reaches alkaline pH 8.3 or higher, all the hydrogen will disassociate and
the litmus paper is now blue.

Test for Unsaturation

The lipids are hydrophobic in nature but can be dissolved in inorganic solvents. The
hubl’s test assesses the unsaturation in a given oil. Iodine upon reacting with
unsaturated fatty acids produces di-hallo abducts. Consumption of more iodine
indicates a higher percentage of unsaturation. Decolorization of alkaline potassium
permanganate is also an indirect measure of unsaturation in fatty acids, where
unsaturated fatty acids undergo incomplete oxidation. This test also detects the
presence of a single or double bond between carbon and lipids samples.

Lieberman-Burchard or Acetic Anhydride Reaction

-The Liebermann–Burchard or acetic anhydride test is used for the detection of

cholesterol. The formation of a green or green-blue colour after a few minutes is
positive. Lieberman–Burchard is a reagent used in a colourimetric test to detect
cholesterol, which gives a deep green colour. This colour begins as a purplish, pink
colour and progresses through to a light green then very dark green colour.

Conclusion

There were a total of five tests for this experiment which are divided into two categories:
a.) Properties of Lipids and b.) reaction of Lipids. The properties of lipids were further
divided into three test. The first one is a test for solubility of vegetable oil , the water,
Dilute NaOH, Dilute HCI, cold alcohol, and hot alcohol was insoluble in water according
to the experiment while in the chloroform, acetone, and ether, the vegetable oil was able
to combine in these solvents. The second test is about the Formation of a Translucent
Spot in vegetable oil and oil of wintergreen and the results shows that the vegetable
oil’s translucent spot is smaller than the spot of the wintergreen green oil. A translucent
spot will appear on the filter paper indicating a positive result and on a negative result,
the translucent spot will not appear on the filter paper. The third and last test for the
Properties of Lipids includes the Reaction of red and blue Litmus Paper to vegetable oil.
After pouring the vegetable oil on the red litmus paper, there was no reaction however a
change was observed on the blue litmus paper where it turned red overnight which indicates
that the vegetable oil is an acid. Moving on to the next test under the reaction of lipids, we have
test for unsaturation where it involves four substances: olive oil, oleic acid, stearic acid, and
coconut oil. Olive oil (80 drops) and Oleic Acid (85 drops) resulted positive in the test therefore it
is unsaturated while the stearic acid (1 drop) and coconut oil (2 drop) means that it tested
negative in the test of unsaturation because it only took 1-2 drops to absorb the color of Iodine
solution. Lastly, the Lieberman-Burchard or Acetic Anhydride Reaction wherein the bile salt
solution changed into a golden yellowish complex after adding 5 drops of acetic
anhydride and a drop of conc. H2SO4 while cholesterol solution changed into a deep
color of violet after adding the five drops of acetic anhydride and a drop of conc. H2SO4,
which means the cholesterol tested positive in Lieberman-Burchard Test while the bile
salt tested negative on this test. By the end of the laboratory experiment and observation, we
conclude that we successfully identified the lipids by observing and studying the properties
and reactions used for their identification.

Questions:

1. What common characteristics do lipids possess?

- The common characteristics of lipids are they do not dissolve in water as they
are nonpolar molecules and hydrophobic.

2. What is an emulsion?-

-Emulsion, in physical chemistry, is a mixture of two or more liquids in which one

is present as microscopic or ultramicroscopic droplets scattered throughout the
other. To put it another way, an emulsion is a unique form of mixture created by
blending two liquids that ordinarily do not mix. Emulsions are generated from the
component liquids either spontaneously or by mechanical methods, such as
agitation, if the liquids being mixed have no (or extremely limited) mutual
solubility. The word emulsion comes from the Latin word meaning "to milk" (milk
is one example of an emulsion of fat and water). The process of turning a liquid
mixture into an emulsion is called emulsification.

3. How are emulsions stabilized?

Emulsions are stabilized when the mixture is mixed together without separating. You
can add an emulsifier that will bind both the fats and water molecules together.
Hydrocolloids also help achieve stability by increasing the viscosity of the mixture that
keeps globules in suspension. You can also use something that can impart high energy
that can mix and reduce them at a fine globule size upon equally dispersing the mixture
it achieves stable emulsion is achieved.

References

Physical chemistry. (n.d.). Retrieved from

https://www.britannica.com/science/physical-chemistry

Anne Marie Helmenstine, P. D. (2020, January 31). What is an emulsion? definition and
examples. ThoughtCo. Retrieved September 29, 2021, from
https://www.thoughtco.com/definition-of-emulsion-605086.

N, S. (2020, December 31). Qualitative analysis of lipids - definition & methods. Biology
Reader. Retrieved September 29, 2021, from
https://biologyreader.com/qualitative-analysis-of-lipids.html.

Common Acid Base Indicators

Rosann Kozlowski - https://sciencing.com/common-acid-base-indicators-8375206.html

How Does Litmus Paper Work? Not Like Ph Strips?

Patrick-Stephan Logan-Sam Wolfe- Raphdawsons - https://blog.indigoinstruments.com/litmus-paper-test/

“Liebermann–Burchard Test.” Wikipedia, Wikimedia Foundation, 10 Apr. 2019,

https://en.wikipedia.org/wiki/Liebermann–Burchard_test.

https://www.egyankosh.ac.in/bitstream/123456789/68529/1/Experiment-7.pdf