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BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012

Score:

Experiment 6
Analysis of Lipids
Name: Ana Margarita L. Baytion
Groupmates: Marla C. Basa
Joann H. Justiniane

I.

Date Performed: August 2,


2016
Instructors Signature:

Objectives
1. To detect presence of fats or glycerin in Acrolein test
2. To indicates the amount of presence of double bonds in the lipid sample
through unsaturation test
3. To determine the different properties of lipids through the results of each tests

II.

Chemicals
Oleic acid
CHCl3
Hubls solution
Glycerol
KHSO4

III.

Apparatus/Materials/Equipment
Test tubes
Dropper
Water bath
Hot plate
Stirring rod

IV.

Schematic Diagram of the Procedure


Please write on a short bond paper.

V.

Summary of Theory

Lipids are chemically heterogeneous mixtures. The only common property


they have is their insolubility in water. We can test for the presence of various lipids
by analyzing their chemical constituents. Foods contain a variety of lipids, most
important among them are fats, complex lipids, and steroids. Fats are triglycerides,
esters of fatty acids and glycerol. Complex lipids also contain fatty acids, but their
alcohol may be either glycerol or sphingosine. They also contain other constituents
such as phosphate, choline, or ethanolamine or mono- to oligo-saccharides. An
important representative of this group is lecithin, a glycerophospholipid, containing
fatty acids, glycerol, phosphate, and choline. The most important steroid in foods is
cholesterol. Different foods contain different proportions of these three groups of
lipids
Acrolein test is a chemical test for the presence of glycerin or fats. Acrolein is
the simplest unsaturated aldehyde. It is produced widely but is most often
immediately reacted with other products due to its instability and toxicity. It has a
piercing, disagreeable, acrid smell similar to that of burning fat, it can be produced by
No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

heating glycerol to 280 Celsius or in this case, by reacting potassium bisulfate with
glycerol. 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 therefore fat and/orlecithin
Fatty acids in animal fats are usually saturated, whereas those in vegetable
oils are generally unsaturated. Halogens (I, Br) will add across the double bonds and
thus the decolorization of an iodine or bromine solution will indicate the presence of
unsaturated fatty acids. Iodine test is used for distinguish between saturated and
unsaturated fatty acids as well as between oils and fats. The unsaturated fatty acids
absorb iodine at the double bonds until all the double bonds are saturated with
iodine. Hence the amount of iodine required to impart its color to the solution is a
measure of the degree of the fatty acids. In this test, Hubls solution (I2 in HgCl2) is
used to determine the unsaturation by measuring the iodine absorption in the
reaction. One application of the iodine number is the determination of the amount of
unsaturation contained in fatty acids. This unsaturation is in the form of double bonds
which react with iodine compounds. If the substance reacted and the color
disappeared in a shorter time, this means that the fatty acid contain lesser double
bonds, thus, the degree of unsaturation is lesser.
The presence of free phosphate in acidic solution can be detected by adding a
molybdate to the solution. Equation illustrates the pertinent reaction between
phosphate and ammonium molybdate solution in the presence of nitric acid (HNO 3).
When lipids containing phosphate groups in their structures are added to a strong
acid solution such as the solution used here, the lipid hydrolyses, producing free
phosphate. The free phosphate then reacts as in Equation, forming a yellow
precipitate.
HPO42(aq) + 12MoO42(aq) + 3 NH4+(aq) + 23 H3O+(aq)
H2O(l)

(NH4)3[P(Mo3O10)4] (yellow,s) + 35

The emulsion test the procedure is for the sample to be suspended in ethanol,
allowing lipids present to dissolve (lipids are soluble in alcohols). Emulsification is
permanent and complete in the presence of emulsifying agent. The important
emulsifying agents are bile salts, proteins, soaps, mono- and diglycerides.
Emulsification is important in the processes of fat digestion in the intestine.
Emulsifying agents lower surface tension of the liquid. (Parul Kumar; 2013) When oil
and water, which are immiscible, are shaken together, the oil is broken up into very
tiny droplets which are dispersed in water. This is known as oil in water emulsion. The
water molecule due to the high surface tensions has a tendency to come together
and form a separate layer. this is why the oil and water emulsion is unstable in the
presence of substances that lower the surface tension of water. Since bile salts cause
the greatest decrease in surface tension they are best emulsifying agents
LiebermannBurchard Test for Cholesterol is used in the estimation of blood
cholesterol. Cholesterol produces a characteristic green color when it is mixed with
the LiebermannBurchard reagent, a mixture of acetic anhydride and sulfuric acid.
The change in color may be gradual, initially pink, then blue-purple, and finally deep
green.The LiebermannBurchard 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. The colour is due to the hydroxyl group (-OH) of cholesterol reacting with the
reagents and increasing the conjugation of the un-saturation in the adjacent fused
No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

ring. the cholesterol is react as a typical alcohol with a strong concentrated acids and
the product are colored substances. Acetic anhydride are used as solvent and
dehydrating agents, and the sulfuric acid is used as dehydrating and oxidizing agent.

Carr-Price Test is chemical test to detect the presence of vitamin A and related
carotinoids.Chloroform is used to dissolve vitamin A, it soluble in the organic solvent
as chloroform.SbCl3 (antimony trichloride) will react with vitamin A to produce
molecul which have blue color in the solution.
The Modified Furter-Meyer test is used to detect the presence of tocopherols by
giving a bronze-red solution.
VI.
Observations
A. Acrolein Test
Table A.1
Sample
Glycerol
Oleic acid

Glycerol + KHSO4 after heating

Sample + KHSO4 after heating


Insoluble; pungent odor
Wax-like odor

Oleic acid + KHSO4 after


heating

B. Test for Unsaturation


Table B.1
Sample

No. of drops of Hubls solution

Oleic acid

7 drops

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

Oleic acid + 1 drop


Hubls solution

Oleic acid + 1 drop


Hubls solution after
shaking

Oleic acid + 7 drops


Hubls solution

VII. Analysis
In the Acrolein Test the available fatty acid samples for the experiment were
glycerol and oleic acid, it was heated strongly with the presence of potassium
bisulfate (KHSO4) which serves as a dehydrating agent. Such as portion of the
molecule is then dehydrated to form the unsaturated aldehyde, acrolein (CH2=CH
CHO), which has the odor peculiar to burnt cooking grease.

Glycerol and oleic acid gave a positive result for acrolein test since both
inhibited a pungent odor specifically called the burnt grease odor which is the
characteristic for this test. The principle behind the acrolein test is a specific chemical
reaction (illustrated above). This reaction is utilized to determine the presence of
glycerin in a fat. By heating the fat sample in the presence of potassium bisulfate
(KHSO4), which acts as a dehydrating agent, acrolein is formed and can easily be
detected by its odor. Whenever fat is heated in the presence of a dehydrating agent,
the molecule will shed its glycerol in the form of the unsaturated aldehyde acrolein.
All neutral fats contain glycerides of some unsaturated fatty acids. These
unsaturated fatty acids become saturated by taking up iodine. If the fat contains
more unsaturated fatty acids, it will take up more iodine.
it should be remembered that more the number of drops required to discharge
the pink color, the less is the unsaturation. The unsaturated fatty acids absorb iodine
at the double bonds until all the double bonds are saturated with iodine. Hence
the amount of iodine required to impart its color to the solution is a measure of the
No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

degree of the fatty acids. From the results obtained, it shows that it took 7 drops for
oleic acid to be saturated.
VIII.
Conclusion
In Acrolein test both Glycerol and Oleic acid are positive since both fatty acids
inhibited the pungent odor/ grease like odor that easily tells us that acrolein is
present. Since the characteristic of this test is the presence of the grease like odor by
acrolein. This test responds to glycerol free or linked as an ester. This explains why
both samples are positive for this test.
From the obtained results from table B.1 it took drops for oleic acid to have
the same color as the Hubls solution. Since we could not compare the iodine
absorption of oleic acid from other samples we could not compare the degree of
unsaturation as well if the fatty acid is saturated of the following samples: olive oil,
oleic acid, coconut oil, and stearic acid because of the lack of chemicals. If all of the
sample are performed for the unsaturation test this would most likely be the result:
Olive oil>Oleic acid>Coconut oil>Stearic acid
Therefore, stearic acid has a lesser time required, while olive oil has the
highest.It took a while for olive oil and oleic acid to change since there are more
unsaturated bonds inthese groups of lipids.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

IX. References
J. Ramos (2 September 2012 ). Identifying Lipids Using Chemical tests. Retrieved from
https://www.scribd.com/doc/104684644/Acrolein-Test
amrita.olabs.edu.in,. (2015). Qualitative Analysis of Oils and Fats. Retrieved from
amrita.olabs.edu.in/?sub=73&brch=8&sim=210&cnt=1
P. Kumar (2013). Qualitative and Quantitative Tests for Lipids. Retrieved from
http://www.biologydiscussion.com/lipids/tests/qualitative-and-quantitative-tests-forlipids/13050

Qualtative
Analysis
of
Lipids
(9
August
2011 ).
Retrieved
https://www.scribd.com/doc/61939357/Qualtative-Analysis-of-Lipids

from

J.
R
Roque
(19
May
2012 ).
Reaction
of
https://www.scribd.com/doc/94115612/Reaction-of-Lipids

from

Lipids.

Retrieved

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.