Republic of the Philippines

CEBU TECHNOLOGICAL UNIVERSITY

BIOCHEMISTRY LABORATORY
Experiment No. 5
QUALITATIVE TEST OF LIPIDS

Name: _Pila, Mary Ella Mae C. _ Course/yr/sec.: _BSN I-B_ Date: _May 17, 2021_
Group No. _3_

INTRODUCTION
Lipids are biomolecules that contain hydrocarbons and essentially make up the building blocks
of the structure and the function on living cells. These molecules occurring naturally that can be
classified as fats, waxes, phospholipids and mono-di- triglycerides and more. The main biological
function of lipids includes as structural components of cell membranes, signaling and storing energy. It is
also useful in some industries like food and cosmetics industry. Lipids are insoluble in water but soluble
in organic solvents like chloroform, benzene and ether.
This experimental procedure was basically geared towards understanding the physical and
chemical properties of lipids and how to differentiate them based on their different properties.

PREPARATION
Watch this:
https://www.youtube.com/watch?v=NmphJwjowrQ

1. SOLUBILITY TEST
This test is used to know the solubility of lipids in some solvents. According to polarity feature
lipids are insoluble in polar solvents because lipids are non-polar compounds, thus lipids are soluble
in non-polar solvents like chloroform, benzene, and alcohol.

I. Objective:
To know the solubility of lipids in some solvents.

II. Apparatus
test tubes, test tube rack, test tube holder, dropper

Materials
Clarified butter, vegetable oil, linseed oil, water, alcohol, chloroform

III. Procedures.

• Take three test tubes which contains 5ml of given sample solution to be tested.
• Add 5ml of water to the first test tube, shake and observe the solution.
• To the second test add 5ml of alcohol to each sample solution and heat.
• To the third test add 5ml of chloroform to each sample solution, shake and observe.
• Observe the change in solubility of the given sample.
 Reaction:

Water was first added to the three test tubes that contains 5ml of the given sample solution,
the water settled below the test tube while the given sample formed an oily layer above the surface
of the water. For the alcohol, Bunsen burner was needed, after adding the alcohol to the test tubes
and heated them, the given samples were being slowly solubilized. Lastly, after adding the
chloroform to the test tubes, all of the lipids were solubilized.

IV. Observation and result

Compare the solubility reaction of the 3 solvent (water, alcohol and chloroform) with the given
sample of lipids.
Explain why lipids is insoluble in water.

Lipids are soluble in organic solvents such as the alcohol and chloroform used in the experiment.
For the alcohol, there was a need of using the Bunsen burner, so after adding alcohol to the test
tubes and heating them, the given samples of lipids were partially soluble while for the chloroform,
after adding the chloroform to the test tubes, the given samples of lipids were fully soluble. However,
for the water solvent, the lipids are insoluble in water as there is an oily layer formed above the
surface of the water because lipids are hydrophobic where they are hydrocarbons that include
mostly nonpolar carbon–carbon or carbon–hydrogen bonds. Lipids are also non-polar molecules that
are hydrophobic ("water fearing"), or insoluble in water.

2. TRANSLUCENT SPOT TEST

I. Objectives:

To detect presence of fat and oil.

II. Materials

• Filter paper
• Dropper
• Bunsen burner
• Linseed oil
• Clarified oil
• Vegetable oil

III. Procedure

• Take the sample to be tested, press a little amount on the filter paper.
• Observe that the appearance of greasy spot which indicates the presence of oils or fats.
• Hold the filter paper against the light.
• Observe the translucent spot in the filter paper.
 Reaction:

Fats and oils have higher boiling points so at room temperature they cannot absorb enough
heat to evaporate. When fat or oil is place on a sheet of paper, it diffracts light. The diffracted light
can pass from one side of the paper to another side and produces a translucent spot. Also, if there is
a pungent irritating odor then the presence of fats or oil is confirmed.

IV. Observation and result

In the experiment, few drops of clarified oil was placed in the filter paper and afterwards it was
smeared. Some time later, when placed in front of the lighted candle, a translucent spot then
appears. Thus, the presence of fat or oil is confirmed. The same happens with the vegetable and
linseed oil.

Sample Observation
Clarified Oil Translucent spot appears on the filter paper.
Vegetable Oil Translucent spot appears on the filter paper.
Linseed Oil Translucent spot appears on the filter paper

On unfolding the filter paper, the appearance of translucent spot on the filter paper indicates the
presence of oil or fat.

3. ACROLEIN TEST
It is used to detect the presence of glycerol or fat. When fat is treated strongly in the presence
of a dehydrating agent like potassium bisulphate (KHSO4), the glycerol portion of the molecule is
dehydrated to form an unsaturated aldehyde, acrolein that has a pungent irritating odor.

I. Objective

To detect the presence of glycerol or fat.

II. Materials

• Test tube, test tube rack, test tube holder and Bunsen burner
• potassium bisulfate
• lipid sample

III. Procedures

• Take the sample to be tested in a test tube.

• Add few crystals of potassium bisulfate to it.
• Heat the mixture and observe the change in odor.
• If there is pungent irritating odor then the presence of fate or oil is confirmed.
 Reaction:

A pungent irritating odor or smell of acrolein confirms the presence of fat or oil. It is produced
after heating the mixture of the test sample and the potassium bisulfate.

IV. Observation and result

What is the name of the compound formed when fats or oils reacts with potassium bisulfate?

The compound formed is unsaturated aldehyde, acrolein (CH2=CH-CHO). When a fat is heated
strongly in the presence of a dehydrating agent such as potassium bisulfate, the glycerol portion of
the molecule is dehydrated to form this compound. Acrolein has the odor peculiar to burnt cooking
grease. When the test samples were heated (Refined oil, Desi ghee and Vegetable ghee), all produced
a pungent irritating odor that confirms the presence of fats in the samples.

4. HUBLE’S TEST
This test is used to detect the degree of unsaturation in oil or fat. Huble's reagent reacts with
alcoholic solution of iodine that contains some mercuric chloride. During the reaction, the violet
color of iodine fades away if the oil or fat is unsaturated.

I. Objectives

To detect the degree of unsaturation of in oil or fat.

II. Materials
• Test tubes and test tube holder
• Test tube stand
• Bunsen burner
• Hubl’s Reagent ( Mix equal volumes of 7% mercury chloride in alcohol with 5% solution
of iodine in 96% of alcohol).
• Chloroform
• Linseed oil
• Cotton oil

III. Procedure
• Place 3 ml of cotton oil and 3 ml of linseed oil in a test tube
• Add 2 ml of chloroform in each solution and shake
• Add 2 ml of Hubl’s reagent in each solution and observe.

Reaction:

Huble’s test detects the degree of unsaturation in oil or fat. The Huble’s reagent reacts with
alcoholic solution of iodine that contains some mercuric chloride. During the reaction, the violet color
of iodine fades away if the oil or fat is unsaturated. If the oil or fat is saturated, the violet color of
iodine does not fade away.
 IV. Observation and result
Differentiate between saturated and unsaturated fats.

In the experiment it can be observed that, upon pouring chloroform and Huble’s reagent in
both test tubes containing different oils or fats (specifically Cotton seed oil in test tube A and Linseed
oil in test tube B), the unsaturated fat or oil can be distinguished when the violet color of iodine in
the Huble’s reagent fades away. As a result, we were able to determine that test tube B containing
Linseed Oil is unsaturated while test tube A containing Cotton Seed Oil is saturated as the violet
color of the iodine in Huble’s reagent does not fade away. The test also indicates that linseed oil is
more unsaturated than the cotton seed oil.

5. SUDAN III TEST

Watch this: https://www.youtube.com/watch?v=NfP0fvFK618

Sudan III is a red fat-soluble dye that is utilized in the identification of the presence of lipids,
triglycerides and lipoproteins. Sudan III reacts with the lipids or triglycerides to stain red in color.

I. Objective
To identify the presence of lipids, triglycerides and lipoproteins.

II. Materials
• Test tube, test tube rack,
• Water
• Mustard oil
• Sudan 3 reagent

III. Procedure
• Take 2 ml of water in a test tube
• Add 2 ml of mustard oil and shake
• Add 2 drops of Sudan 3 and shake
• Observe the color of the upper and lower layer of the solution.

Reaction:

When the Sudan III reagent was placed inside the water and mustard oil mixture and was shook,
the layers separated out and the lipid layer had a red stain and the water layer remained uncolored.

IV. Observation and result

In detecting the presence of lipids using the Sudan III Test, the Sudan III reagent was added in a
test tube with water and mustard oil mixture. The indication of a positive result from this test is a
red-stained layer and that the layers are separated out. In a test tube, water and mustard oil was
mixed by shaking them. Then, the Sudan III reagent was added to the mixture and was shaken. After
shaking, the layers separated out and the lipid layer became red-stained. The water layer, on the
other hand, remained the same. It was still colorless. It met the principles on how to detect a positive
 result from the Sudan III test. Therefore, the test gave a positive result in detecting the presence of
lipids.
Name of test
1 Solubility Test
2 Translucent spot test
3 Acrolein Test
4 Huble’s test
Summary of the qualitative tests of Lipids table:
Sample lipids Observation
Clarified butter, The sample of lipids
vegetable oil, dissolves in organic
linseed oil solvents such as the
alcohol and the
chloroform. However,
water was unable to
dissolve the sample of
lipids.
Linseed oil, Upon few drops of
Clarified oil, and each the sample lipids
vegetable oil (Linseed oil, Clarified oil,
and Vegetable oil) on
each of the filter paper,
all have revealed a
translucent spot.
Refined oil, Desi There was a pungent
ghee, and irritating odor that was
Vegetable ghee detected when the test
samples were heated.
Cotton seed oil When chloroform and
and Linseed oil Huble’s reagent is
poured in test tube A
with cotton seed oil, the
violet color of iodine did
not fade away which
indicate that it is
saturated. On the other
hand, test tube B with
linseed oil, the violet
Result / Inference
Lipids are soluble in
non-polar solvents like
chloroform, benzene, and
alcohol. But are also non-
polar molecules that are
hydrophobic ("water
fearing"), or insoluble in
water.
The experiment showed
that the Linseed oil,
clarified oil, and vegetable
oil has presence of fat or
oil.
Upon heating the
sample lipids with the
presence of a dehydrating
agent, potassium
bisulfate, the glycerol
portion of the molecule
was dehydrated, thus,
forming the unsaturated
aldehyde, acrolein.
Moreover, through the
detection of the pungent
irritating smell, the
presence of fats in the
samples was confirmed.
The test indicate that
linseed oil is more
unsaturated than the
cotton seed oil

5 Sudan III Test
color of iodine fade
away which indicate
that it is unsaturated
Water and The test tube was The water and mustard
mustard oil added with water and oil mixture that was
mustard oil, then was added with Sudan III
shaken. It was then reagent showed a positive
added with Sudan III result where it indicates
reagent, then was that there is presence of
shaken again. It lipids.
produced a red-stained
lipid layer and a
colorless water layer
which indicated a
positive result.

Conclusion

In the final analysis, a lipid is any of various organic compounds that is insoluble in water. It includes
fats, waxes, oils, hormones, and certain components of membranes and function as energy-storage
molecules and chemical messengers. It comes in four types: fatty acids, glycerides, non-glycerides, and
complex lipids. To understand the physical and chemical properties of lipids and how to differentiate them
based on their different properties, we have a few experiments here. For the first experiment which is the
Solubility test, lipids are soluble in non-polar solvents like chloroform, benzene, and alcohol, but are
hydrophobic ("water-fearing"), or insoluble in water. In the Translucent spot test, which is the second test,
the three oils revealed a translucent spot which means these oils have fats or oils. Fats and oils have higher
boiling points, so at room temperature, they cannot absorb enough heat to evaporate. When fat or oil is
placed on a sheet of paper, it diffracts light. The following test, the Acrolein test proved to us that there
is a presence of fats in all the sample lipids because of the pungent irritating odor detected upon heating
them with the presence of a dehydrating agent which is the sodium bisulfate so the glycerol portion of
the molecule was dehydrated forming the unsaturated aldehyde acrolein. The next test is the Huble’s
which we were able to indicate that linseed oil was more unsaturated than cotton seed oil. This was
determined because the violet color of iodine did not fade and when the chloroform and Huble’s reagent
was poured into the test tube with cotton seed oil which means or indicates saturated. The last test was
the Sudan III Test wherein it resulted after the water was added with mustard oil; both didn’t mix because
oil is less dense than water and insoluble in water. Then, as the layers separate, the lipid layer is seen to
be stained red, whereas the water layer remained uncolored. Thus, this indicates the presence of lipids.
Lipids are important in biological systems because they form the cell membrane, a mechanical barrier that
divides a cell from the external environment. Non-polar lipids, such as triglycerides, are used for energy
storage and fuel. Polar lipids, which can form a barrier with an external water environment, are used in
membranes. Polar lipids include glycerophospholipids and sphingolipids. Fatty acids are important
components of all of these lipids. They are important dietary constituents not only because of their high
energy value but also because of the fat-soluble vitamins and the essential fatty acids contained in the fat
of natural foods. Qualitative analysis of lipids has a significant contribution in identifying the adulteration
of edible oils and hence has a vital role in the industry, as well as in the health sector.
References:

https://www.academia.edu/37659221/Experiment_4_Lipids_doc

https://www.youtube.com/watch?v=NmphJwjowrQ

https://www.youtube.com/watch?v=5BBYBRWzsLA

https://www.youtube.com/watch?v=l2QOi9mZoFc&t=36s

http://amrita.olabs.edu.in/?sub=73&brch=8&sim=210&cnt=1

amrita.olabs.edu.in,. (2015). Qualitative Analysis of Oils and Fats. Retrieved 10 May 2021, from
amrita.olabs.edu.in/?sub=73&brch=8&sim=210&cnt=1