Professional Documents
Culture Documents
Expt. 3 Analysis of Lipids
Expt. 3 Analysis of Lipids
YB2C
1. Leslie Jane Jala Section: YB2
2. Shimea Pagurayan Date Submitted: October 12,
2020
V. EMULCIFICATION TEST
Dissolve in 1 ml CHCl3
mix
CHCl3
test tube
𝜶 – C28H48O2 Vitamin E
0.05%
1 mL 1 mL
Water Bath
80℃
10 min.
Observe
change of color
V. Emulsification Test
Microscope
Explain
Porcelain crucible
Filter
Filtrate
+ 1 mL 5% (NH4)2MoO4
Heat
Observe
Yellow solution
ANALYSIS
Introduction
Lipids are organic compounds that contain hydrogen, carbon and oxygen atoms
which form the framework for the structure and function of living cells (BYJU, 2020).
They are immensely valuable and important in the biological world as they fill multiple
roles in the cells and maintaining the health of all Earth’s organisms (Basic Biology,
2020). In order to study lipids, numerous experiments were conducted to identify
various reactions of lipids, to test its solubility and to analyze its physical and chemical
properties. Initially, to determine the presence of double bonds in the lipid samples,
the test for unsaturation was completed (Calhoun,2020). To estimate the cholesterol
available in the samples, the Liebermann-Burchard Test was used (Delos Reyes,
2020). To detect the presence of vitamins A and E, the Carr-Price reaction (Parker,
1941) and Modified Furter-Meyer test (Bencze, 1957) were used respectively. Then
the emulsification test was done to detect lipids in sample using wet chemistry
(Cassiday, 2018). And lastly, the phosphate test was completed in order to determine
the presence of free phosphate in acidic solution (Chawla, 2014). Thus, understanding
the properties and reactions of lipids is important in any study of biology because they
play important functions such as energy storage, insulation, cellular communication,
protection and lastly, its most important role is as building blocks of cellular
membranes (Basic Biology, 2020).
Discussion
I. Test for Unsaturation
The test for unsaturation is used to detect the presence of double bonds in the
lipid sample (Calhoun, 2020). A lipid is said to be saturated if there are only single
bonds between neighboring carbons in the hydrocarbon chain and the lipid is
saturated with hydrogens (Boundless, 2020). While a lipid is unsaturated when the
hydrocarbon chain contains double bonds (Boundless, 2020). In the experiment, two
drops of the sample (olive oil, oleic acid, stearic acid, and coconut oil) and 1 mL of
CHCl3 were mixed. Lipids are all insoluble in polar solvents like water as it is
hydrophobic but highly soluble in the non-polar or weakly polar organic solvents
including ether, chloroform, benzene and acetone (Hunt, 2020). Hence the use of
chloroform in the experiment. Then Hubl’s solution (I2 in HgCl2) is added drop by drop
until the color of the sample solution is the same as Hubl’s solution. In the result, olive
oil had fifty-one (51) drops of Hubl’s solution, oleic acid had eleven (11) drops, stearic
acid had five (5) drops, and coconut oil had thirty-one (31) drops of Hubl’s solution.
The principle of the test is that unsaturated fatty acids become saturated as the
unsaturated fatty acids absorb the iodine present in Hubl’s solution at the double bond
until all the doubles bonds are saturated with iodine (Calhoun, 2020). The reaction
goes as follow:
R-CH=CH-R’ + I2 → R-CHI-CHI-R’
Thus, the more double bonds the lipid contains, the more drops of the reagent
are needed in order to produce de-coloration thus indicating the degree of lipid
unsaturation (Calhoun,2020). In the test samples, olive oil proved to be the most
unsaturated followed by coconut oil, oleic acid and lastly, stearic acid. This result is as
expected as olive oil consist mostly of unsaturated fat with double bonds, with 14%
saturated fat, 11% polyunsaturated fat and 73% monounsaturated fat (Leech, 2018).
While, coconut oil consists of high concentration of saturated fat with more than 90%
saturated fatty acid and low content of unsaturated fatty acids (Lal, Kumar and Indira,
2003). Consequently, oleic acid is a monounsaturated omega 9 fatty acid found in
animal and vegetables (Choulis, 2011). Lastly steric acid is a fully saturated fatty acid
with no carbon-carbon double bonds (Wong, 2020).
CH3 CH3
L-B reagent
H 2SO 4/HOAc
HO HOO2S
In the experiment, only cholesterol gave a blue color which indicates a positive
result. This is expected as this test is used to detect the presence of cholesterol. Bile
salt gave a negative result as the solution turned into a milky white color indicating the
absence of cholesterol. This is expected as bile salt does not contain cholesterol but
are synthesized in the hepatocytes from cholesterol (Bodewes, Wouthuyzen-Bakker,
& Verkade, 2014).
The Carr-Price test is used to detect the presence of retinol (vitamin A) (Parker,
1941). First 1 mL of chloroform is placed in a dry test tube and 2 drops of the sample
(cod liver oil, coconut oil, and olive oil) is added. Vitamin A is a fat-soluble vitamin so
they are not soluble in water but are soluble in organic solvents like chloroform
(Arnarson, 2017). Then it is cooled in an ice bath. This is because the temperature of
which the reaction is carried affects both intensity of the color and its duration (Parker,
1941). At low temperatures the color is less intense, but fades less rapidly (Parker,
1941). Then 2 mL of cold saturated chloroform solution of SbCl3 was added. When
vitamin A is reacted with antimony trichloride (a Lewis acid) in chloroform, an unstable
transient blue color which lasts for about ten seconds is developed which may be
quantified spectrometrically (λmax 620 nm) (Liaaen-Jensen & Kildahl-Andersen, 2008).
But spectrometry was not done in the experiment.
Vitamin A
It is generally accepted that the amount of blue color produced by the reaction
of vitamin A with antimony trichloride is proportional to the amount of vitamin A present
(Koehn & Sherman, 1939). In the experiment only cod liver oil elicited a positive result
that gave a blue color at the top layer of the solution but then faded into a transparent
gray color. This is expected as cod liver oil has high concentrations of vitamin A and
vitamin D (Raman, 2017) Coconut oil and olive oil gave a brown and yellow colored
solution respectively which is a negative result for the presence of vitamin A. This is
expected as coconut oil contains vitamin E, but little to no other vitamins or minerals
(Butler & Brazier, 2019) while olive oil contains modest amounts of vitamins E and K
but not vitamin A (Gunnars, 2019).
CH3
H3C O
CH3
CH3
Alpha Tocopherol
(Vitamin E)
Free radical
CH3
O
CH3 CH3 CH3 CH3 CH3
H3C O CH3
CH3
Tocopheroxyl Free Radical
(Oxidized Vitammin E)
V. Emulsification Test
Hydrogen Bond
H H
δ−
H C C O H δ+
H H δ+ H O H
δ−
When oil and water, which are immiscible, are shaken together, the oil is broken
up into very tiny droplets which are dispersed in water (Quadro, 2020). This is known
as oil in water emulsion (Quadro, 2020). The water molecules due to the high surface
tensions have a tendency to come together and form separate layers (Quadro, 2020).
So, the oil in water emulsion is unstable in the presence of substances that lower the
surface tension of water. (Quadro, 2020). The tendency of the water molecule to
coalesce is decreased and the emulsion becomes stable (Quadro, 2020).
In the experiment, test tube 1 formed a light yellowish solution and when
observed under the microscope tiny droplets can be seen, thus an emulsion had
formed and a positive result for lipids was observed. This is expected as bile salt act
as an emulsifier because of their hydrophilic head that is attracted to water molecules
and hydrophobic tail that is attracted to lipid molecules (Pathwayz, 2020). In test tube
2, a yellow solution formed and the presence of big circular droplets can be observed
under the microscope. This means that although there is a positive result for lipids,
emulsion did not occur as cholesterol destabilizes an oil in water emulsion (Restaurant
Business Online, 2006). Lastly, in test tube 3, a milky white solution formed and small
droplets was observed under the microscope, thus an emulsion had formed and a
positive result for lipids was observed. This is expected as lecithin is a great emulsifier,
just like bile salt, it is a molecule with one end that is hydrophobic and another that is
hydrophilic (Gavigan, 2020). Among the three test tubes, test tube 3 had formed the
smallest droplets thus lecithin is the best emulsifier.
In the last experiment, the lipid sample used is lecithin and the reagents are
concentrated nitric acid (HNO3) and ammonium molybdate ((NH4)2MoO4). The
phosphate test began with the incineration of the samples. A small amount of lecithin
is incinerated in a porcelain crucible. The reason for this is that incineration helps to
oxidize the ester bonds of lipids that contain phosphate (Chawla, 2014). In the
process of doing so, water and carbon dioxide are evaporated leaving a phosphate
residue (Chawla, 2014). The crucible is then allowed to cool down and the residue is
mixed with 5 mL distilled water. After incineration, distilled water was added to
separate the non-lipid contaminants from the residue (Chawla, 2014). The solution
was then filtered discarding the residue containing the contaminants (Chawla, 2014).
After that, 1 mL of the filtrate is measured and placed in a test tube. Then, 1 mL of 5%
Ammonium Molybdate ((NH4)2MoO4) and 3 drops of concentrated nitric acid (HNO3)
were added. The mixture is the heated. The presence of free phosphate in the filtrate
can be detected using ammonium molbydate as long as the phosphate is in an acidic
environment forming a yellow precipitate. This is the reason why nitric acid was added.
The equation below shows how the free phosphate reacts with ammonium molybdate
in the presence of nitric acid.
K3PO4 + 3(NH4)2MoO4 2(NH4)3PO4 + 3K2MoO4
Ammonium Molybdate Potassium molybdate
H2MoO4 MoO3 + H 2O
Conclusion
In conclusion, the purpose of this experiment was to conduct multiple tests in
order to analyze the properties and reactions of lipids. In the test for unsaturation, it
detects the presence of double bonds by adding Hubl’s solution. The lipid samples
were olive oil, oleic acid, stearic acid and coconut oil. In this test, olive oil proved to be
the most unsaturated followed by coconut oil, oleic acid and lastly stearic acid. In the
acetic anhydride reaction, which is a test for the presence of sterol. Cholesterol
indicated and proved positivity in the test showing a blue solution upon adding of the
reagents. In the Carr-Price reaction which detects the presence of vitamin A and
related carotenoids. Cod liver oil indicated and proved a positive result which formed
a layer of blue and yellow solution. In the modified Furter-Meyer test which detects the
presence of tocopherols. The 𝛼-tocopherol and vitamin E gave a positive result of
strong bronze-red solution. In the emulsification test that detects the presence of lipids
which are polar and non-polar groups. Test tubes 1 and 3 gave a positive result which
was the formation of small droplets that can be observed under the microscope
indicating that emulsion was formed. It was also concluded that lecithin is the best
emulsifier out of the three as it has the smallest droplets formed. In the last experiment
which is test for phosphate, lecithin was used as the sample. Through the incineration,
filtration and addition of 5% (NH4)2MoO4 and 3 drops of concentrated HNO3, the
experiment showed a yellow precipitation which indicated a positive outcome. Overall,
these tests are highly important in biological study as lipids play vital roles in the body
and its structure drive the formation of important biological elements, membranes, lipid
droplets and lipoproteins (Ubhi, 2016).
References
Butler, N. & Brazier, Y. (2019). What to know about coconut oil. Medical News Today.
Retrieved from October 11, 2020 from
https://www.medicalnewstoday.com/articles/282857
Calhoun, M. (2020) Good and Bad Lipids. Retrieved October 9, 2020 from
https://www.education.com/science-fair/article/lipid-testing/
Cassiday, L. (2018). Emulsions: making oil and water mix. AOCS. Retrieved October
10, 2002 from https://www.aocs.org/stay-informed/inform-
magazine/featured-articles/emulsions-making-oil-and-water-mix-april-
2014?SSO=True
Choulis, N. (2011). Chapter 49 - Miscellaneous drugs, materials, medical devices, and
techniques. In Side Effects of Drugs Annual, Vol. 33, pp. 1009-1029.
Retrieved from doi:https://doi.org/10.1016/B978-0-444-53741-6.00049-0
Delos Reyes, C. (2020). Liebermann-Burchard Test. Retrieved October 10, 2020 from
https://www.coursehero.com/u/file/47949595/LIUBERMANN-BURCHARD-
TEXTdocx/
Gavigan, F. (2020). Why is lecithin a good emulcifier? Live Strong. Retrieved October
10, 2020 from https://www.livestrong.com/article/276580/why-is-lecithin-a-
good-emulsifier/
Gunnars, K. (2019). Why Extra Virgin Olive Oil is the Healthiest Fat on Earth.
Healthline. Retrieved October 11, 2020 from
https://www.healthline.com/nutrition/extra-virgin-olive-oil
Koehn, C. & Sherman W. (1940). The determination of vitamin A and carotene with
the photoelectric colorimeter. J. Biol. Chem. 132: pg. 527-538. Retrieved from
http://m.jbc.org/content/132/2/527
Lal, J., Kumar, C., & Indira, M. (2003). Coconut palm. Encyclopedia of Food Sciences
And Nutrition, Second Edition, pp 1464-1475. Retrieved from
https://doi.org/10/1016/B0-12-227055-X/00263-7
Leech, J. (2018). 11 Proven benefits of olive oil. Healthline. Retrieved October 9, 2020
from https://www.healthline.com/nutrition/11-proven-benefits-of-olive-oil
Liaaen-Jensen, S. & Kildahl-Andersen, G. (2008) Blue carotenoids. ARKIVOC, (vi),
pp. 5-25. Retrieved from ISBN 1551-7012
Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals, antioxidants and
functional foods: Impact on human health. Pharmacognosy reviews, 4(8),
118–126. https://doi.org/10.4103/0973-7847.70902
Lopez, A. (2018). Experiment 5: Lipid Isolation from Brain Tissues. Retrieved from
https://www.scribd.com/document/387340960/experiment
National Library of Medicine (2020) Vitamin E. Retrieved October 10, 2020 from
htts://pubchem.ncbi.nlm.nih.govr/compound/Vitamin-E
Parker, W. (1941). Studies on vitamin A and carotene. McGill University Library, pp.
13-21. Retrieved from scholarship.mcgill.ca
Pathwayz (2020). Liver and Gallbladder. Retrieved October 10, 2020 from
https://www.pathwayz.org/Tree/Plain/LIVER+%26+GALLBLADDER
Restaurant Business Online (2006). Emulsifiers and the egg. Retrieved October 10,
2020 from
https://www.google.com/amp/s/www.restaurantbusinessonline.com/amp/em
ulsifiers-egg
Sujatha, K. B., Priyanka, A., Pavithra, S., & Manoj, K. (2019). Non-enzymatic
antioxidant defense system in plants. Pharmacognosy.
http://www.phytojournal.com/archives/?year=2019&vol=8&issue=2S&ArticleI
d=7741
Thompson, T. (2020). Lipid biochemistry. Retrieved October 9, 2020 from
https://www.britannica.com/science/lipid
Ubhi, B. (2016). Why study lipids. Sciex. Retrieved October 12, 2020 from
https://sciex.com/community/blogs/blogs/why-study-lipids