LIPIDS

Group Number: ______1_________ Year/Section: ______BN-1B___

Date Performed: ______________ Date Submitted: ___________
Members: ___________________
___________________
___________________

Objective: To study some properties of lipids and reactions that are used in their identification.

Materials/Apparatus: Tripod, wire gauze, graduated cylinder, 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, KHSO4, glycerol, lecithin,
oleic acid, stearic acid, olive oil, coconut oil, Hubl’s solution, bile salts, conc. H2SO4, Acetic
anhydride

Procedure:

A. Properties of Lipids

1. Solubility

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

a. Water e. cold alcohol

b. Dilute HCl f. hot alcohol
c. Dilute NaOH g. ether
d. Chloroform h. acetone

Tabulate your results.

WASTE DISPOSAL - ORGANIC, HALOGENATED (for all samples a-h)

Sample Solubility of Oil

Water Insoluble
Dilute HCl Insoluble
Dilute NaOH Slightly soluble
Chloroform Soluble
Cold Alcohol Insoluble
Hot Alcohol Slightly soluble
Ether Soluble
Acetone Slightly soluble
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 to evaporate spontaneously. Does the translucent spot
disappear?

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

For this test, there was the formation of a translucent or greasy spot made by both vegetable oil
and wintergreen. After a while, the greasy spot is still visible on the bondpaper with the
vegetable oil. On the other hand, there is no more spot found on the bondpaper with
wintergreen and is only left with the stain.

NO WASTE GENERATED FOR THIS PROCEDURE

3. Reaction to Litmus Paper

Test the reaction of fresh vegetable oil with red and blue litmus papers previously
moistened with water. What is the reaction? Allow the oil to stand uncovered until the
nest laboratory period and test again with litmus paper. Is there any change in the
reaction?

NO WASTE GENERATED FOR THIS PROCEDURE

The color of red and blue litmus paper that was previously moistened with water stays the same
after dropping the vegetable oil on it since the oil is neutral. After setting it aside and exposing it
to the air, it likely went oxidation. We tested it again at the end of the period, the blue litmus
paper turned red, indicating mild acidic reaction, and the red litmus paper stays the same.
B. Reactions of Lipids

1. Acrolein Test

Put a pinch of KHSO4 crystals and 2 drops of the sample (glycerol, vegetable oil, lecithin, and
oleic acid) separately in four dry test tubes. Heat gently at first and then vigorously. Note the
odor produced. To what is it due? Write the chemical reactions involved in this test.

WASTE DISPOSAL - ORGANIC, NONHALOGENATED

The Acrolein test is used for detection of glycerol or any compound which may have glycerol in
it. When half a spatula pinch potassium bisulfate (KHSO4) and few drops of the sample
(glycerol, vegetable oil, lecithin, or oleic acid) are heated in a dry test tube, suddenly, there is a
pungent, irritating smell like burnt fat is observed. This colourless and oily liquid has an odor
due to the formation of acrolein (CH2 = CH-CHO) because KHSO4 acts as a dehydrating agent
and water molecules in glycerol are omitted. It has been established by this experiment that
when glycerol and other glycerol like substances such as vegetable oil and lecithin are heated
they produce acrolein. However, oleic acid, which is all fatty acid without glycerol, does not
produce this odor.
2. Test for Unsaturation

Use dry test tubes. In each of four test tubes, place separately 2 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.

WASTE DISPOSAL - METAL, INORGANIC

We had to use 2-4 drops of Hubl’s solution per sample only. Subjecting olive oil and oleic acid to
decolorization means that both of them are unsaturated while stearic acidshows minimal
reaction and coconut oil moderate reaction.
3. Emulsification Test

Prepare the following mixtures in separate test tubes:

Test tube 1: 1 mL olive oil + 5 drops of 0.1% bile salts

Test tube 2: 1 mL olive oil + 5 drops of water and a tiny crystal of cholesterol

Test tube 3: 1 mL olive oil + 5 drops of 1% aqueous lecithin

Shake each mixture very well. Compare results. Examine a drop of each mixture under the
microscope right after shaking. Sketch.

WASTE DISPOSAL - ORGANIC, NONHALOGENATED

Mixture under Emulsification After Shaking Under microscope

microscope

1. Small and moderately distributed

droplets.

Moderate
 2. Large and unevenly distributed
droplets.

Weak

3. Very small, evenly distributed

droplets.

Strong

4. 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 5 drops of acetic anhydride and a drop of conc. H2SO4. Mix. Note
the change in color.

WASTE DISPOSAL - ORGANIC, HALOGENATED

Lieberman Burchard test is a kind of chemical test for the identification of cholesterol.
When a little of cholesterol is soluble in chloroform, to this a little of acetic anhydride and a
drop of concentrated sulphuric acid is added, a sharp color change takes place. The solution
goes from shades of blue into green and then towards the emerald green which identifies
cholesterol. Such change of color is ascribed to the interaction of cholesterol with reagents with
the subsequent formation of a complex that has certain ability to absorb visible light.
Nonetheless, different structural reasons, as bile salts do not contain sterol nucleus, typical for
cholesterol, do not cause this color change.

Questions:

1. What common characteristics do lipids possess?

The majority of lipids share the traits of hydrophobic or amphipathic organic compounds,
having hydrocarbon chains as their constituents, and being non-polar molecules—that is,
incapable of dissolving in water. On the other hand, they dissolve in organic or non-polar
solvents like ether, benzene, and chloroform. Furthermore, lipids release a lot of energy per
mass unit due to their high energy density.

2. What is an emulsion?

In an emulsion, one liquid (the dispersed phase) is distributed throughout the other (the
continuous phase) of a mixture of two or more immiscible phases (hydrophilic and
hydrophobic). Additionally, it is a particular kind of colloid, or mixture, in which minute particles
are distributed throughout another substance without dissolving.

3. How are emulsions stabilized?

Emulsions are stabilized through emulsifiers. Emulsifiers reduce the interfacial tension between
the two liquids that comprise an emulsion, increasing its stability. These compounds have the
ability to stabilize emulsions by increasing the viscosity of the continuous phase to decrease the
contacts between oil droplets.

Conclusion:

To summarize, the experiment showed how lipids may be observed, their reactions, and
behaviors, which is the goal of the experiment as it sought to define lipids and how they work.
This was proven through observing that lipids dissolved in butter whereas they did not dissolve
in polar solvents like water and hydrochloric acid but dissolved in nonpolar solvents like
chloroform and ether. The positive outcome of the translucent spot test was because lipids
were greasy and when using the litmus paper test, it was found that lipids were neutral but can
turn slightly acidic when oxidized.

A positive Acrolein test means that the samples, such as vegetable oil and lecithin,
contain glycerol because of the characteristic smell of acrolein. The test for unsaturation
showed the existence of the double-covalent bonds found in olive oil and oleic acid where more
amount of Hubl’s solution was used to decolorize them than what was used for the saturated
lipids. The emulsification test illustrated the action of bile salts and lecithin in stabilizing the oil
emulsions that define digestive and food processes. Third and last, the Lieberman-Burchard test
produced a desired color change pointing to the detection of cholesterol thus providing a
different color from that of bile salts.

These findings combine together to illustrate the variability in the physical and chemical
characteristics of lipids, the structural complexity of lipids, and how they are involved in
biological and industrial activities.

References:

Liu, Y., Wu, Q., Zhang, J., Yan, W., & Mao, X. (2024). Food emulsions stabilized by proteins and

emulsifiers: A review of the mechanistic explorations. International Journal of Biological

Macromolecules, 261, 129795. https://doi.org/10.1016/j.ijbiomac.2024.129795

Piacentini, E., Figoli, A., Giorno, L., & Drioli, E. (2010). Membrane emulsification. In Elsevier

eBooks (pp. 47–78). https://doi.org/10.1016/b978-0-08-093250-7.00024-4