LABORATORY EXERCISE
LIPIDS
Introduction
Lipids are amphipathic molecules that encompass fatty acids and derivatives such
as diglycerides, triglycerides, phospholipids, and cholesterol.
Roles in the human body:
Provide energy for the body.
Act as a chemical messenger.
Involved in the maintenance of body temperature.
Involved in membrane layer formation.
Involved in the formation of prostaglandins and mediation of
inflammation.
LEARNING OUTCOMES
At the end of the experiment, the learners should be able to:
Perform lipids from egg yolk.
Perform qualitative analysis for the presence of lipids.
Analyze the results obtained from the experiment.
Discuss the principle behind the chemical reaction of lipids for each chemical test.
Safety Precautions
In this activity, the student will be handling different chemicals, so it is of utmost
importance that safety precautions be practiced.
Extraction of Lipids from Egg Yolk
Principle
Many Phospholipids are insoluble in acetone, and they precipitate out whereas
triglycerides, sterols, and pigments are soluble in acetone.
Egg and egg yolk are very important sources of Lecithin which is a generic term
to designate any group of yellow-brownish fatty substances occurring in animal
and plant tissues, which are amphiphilic – they attract both water and fatty
substances (and so are both hydrophilic and lipophilic) and are used for
smoothing food textures, dissolving powders (emulsifying), homogenizing liquid
mixtures, and repelling sticking materials.
Lecithin can easily be extracted chemically using solvents such as hexane,
ethanol, acetone, petroleum ether, benzene, etc., or extraction can be done
mechanically. It is usually available from sources such as soybeans, eggs, milk,
marine sources, rapeseed, cottonseed, and sunflower. It has low solubility in
water but is an excellent emulsifier.
QUALITATIVE TEST FOR LIPIDS
Acrolein Test
Principle
The acrolein test is used to detect the presence of glycerol or fat. When fat is
treated strongly in the presence of a dehydrating agent like potassium bisulfate
(KHSO4), the glycerol portion of the molecule is dehydrated to form an
unsaturated aldehyde, acrolein that has a pungent irritating odor.
Interpretation of Result
Positive result: If glycerol is present in the sample, it will give a pungent smell.
Negative result: If glycerol is absent in a sample, it will not produce a pungent
smell.
Liebermann-Burchard Test
Test Objective
To detect the presence of cholesterol.
Principle of the Test
A chemical estimation of cholesterol, the cholesterol reacts as a typical
alcohol with strong concentrated acids and the products are colored substances.
Acetic anhydride is used as a solvent and dehydrating agent.
Interpretation of Result
Positive result: It indicates cholesterol in a sample by giving a bluish-green
color to the solution.
Negative result: The color of the solution will not change.
Copper Acetate Test
Principle
This test is used to distinguish between oil or neutral fat and fatty acid saturated
and unsaturated. The copper acetate solution does not react with the oils, while
saturated and unsaturated fatty acids react with copper acetate to form copper
salt. unsaturated fatty acids can only be extracted by petroleum ether.
Interpretation of Result
Unsaturated fatty acid: 2-layer appearance - upper layer color green and the
lower layer color blue.
Saturated fatty acid: 2-layer appearance – appearance of white-blue
precipitate at the bottom of the upper layer.
Spot/Translucent Test
Principle
A translucent spot test is a preliminary test for the lipids, which is characterized
by a translucent and greasy spot. The lipid will not wet the filter paper, unlike
water. The lipids will form a greasy or translucent spot due to their greasy
texture, and penetrate the filter paper. Unlike lipids, the spot of water will
disappear from the paper.
Interpretation of Result
Positive result: A translucent spot will appear on the filter paper.
Negative result: A translucent spot will not appear on the filter paper.
Test for Unsaturation
Principle
An unsaturation test is used to detect the unsaturated fatty acids or double
bonds in a lipid sample. All the neutral fat contains glycerides of fatty acids.
Double bonds are found in the structure of unsaturated fatty acids, which
become saturated by taking up either bromine or iodine. If the lipid contains
more unsaturated fatty acids or more double bonds, it will take more iodine.
Interpretation of Result
Positive result: The pink color will disappear with the addition of unsaturated
fatty acids.
Negative result: The pink color will not disappear.
Materials Reagents
1. Beaker 1. Clarified butter
2. Test Tubes 2. Potassium bisulphate
3. Test Tube Rack 3. Cholesterol
4. Test Tube Holder 4. Chloroform
5. Filter Paper 5. Acetic anhydride
6. Funnel 6. Concentrated sulfuric acid
7. Petri dish 7. Oleic acid
8. Graduated cylinder 8. Petroleum ether
9. Stirring Rod 9. Vegetable oil
10. Tripod 10. Linseed oil
11. Centrifuge 11. Olive oil
12. Bunsen burner 12. Sunflower oil
13. Dropper 13. Palm oil
14. Filter paper 14. Corn oil
15. Light source 15. Coconut oil
16. Acetate
17. Ethanol
18. Copper acetate solution
Extraction of Lipids from Egg Yolk
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1. Collect egg yolk from 2 eggs in a beaker and mix well.
2. Add 50 ml of cold acetone mix with a glass rod and allow to stand for 15
min.
3. Collect the precipitate by centrifugation for 5 min at 4000 rpm.
4. Wash the precipitate with a sufficient quantity of acetone till the
supernatant is clear and colorless.
5. Extract the precipitate with about 100 ml of chloroform-methanol mixture
(2:1) for 3-4 hours at room temperature.
6. Filter and collect the filtrate (extract).
7. Evaporate the extract to dryness under a stream of nitrogen or at room
temperature.
8. Dissolve the residue in a small volume of petroleum ether (10-15 ml) and
re-precipitate the phospholipid with the addition of cold acetone (about 50
ml) and then allow the precipitate to settle down at the bottom After this
collect the precipitate of phospholipid
9. Dry under vacuum and store in the dark in a minimum volume of
chloroform-methanol system or as solid.
Qualitative Test for Lipids
Acrolein test
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1. Get 3 test tubes and label them with clarified butter, vegetable oil, and
linseed oil. Add 2mL of each to their respective tube.
2. Add a small amount of Potassium bisulfate crystals to each tube.
3. Heat the mixture directly on a burner.
4. Observe the formation of a gas and odor that will be produced.
5. Record this as your observation.
Liebermann-Burchard Test
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1. Get a test tube with 2 ml of cholesterol dissolved in chloroform.
2. Add 2 mL of acetic anhydride to the cholesterol solution and mix completely.
3. Add 5 drops of concentrated sulfuric acid to the previous mixture by tilting
the test tube and dropping on the side of the test tube then gently mix
the solution.
4. Observe for a bluish green color.
5. Record this as your observation.
Copper Acetate Test
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1. Get 2 test tubes and label them with olive oil and oleic acid. Add 2mL of
each to their respective tube
2. Add petroleum ether to both test tubes
3. Shake test tubes to mix the content
4. Add copper acetate solution to both test tubes
5. Shake vigorously both test tubes
6. Observe for blue-green color
7. Record this as your observation.
Spot/Translucent Test
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1. Prepare a filter paper and drop the Clarified butter on it then press
another filter paper on top of the other
2. Hold the filter paper against a light source
3. Then repeat the process with Vegetable oil and Linseed oil
4. Record your observations.
Test for Unsaturation
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1. Prepare 5 test tubes and label with Olive oil, Coconut oil, Corn oil, Palm oil
and Sunflower oil. Add 1mL of each to their respective tube
2. Add few drops of Bromine Water to each tube one by one and make sure
to replace the lid of the container
3. Shake each test tube vigorously
4. Note if the color of Bromine water disappeared.
5. Record your observation.
References
Berroya, A. C., Cruz, M. B. – Books Atbp. Publishing Corp. Laboratory Manual in
Biochemistry (3rd Edition).
Go, J. C., Kamantigue, E. C., National University - Manila, College of Education,
Arts and Sciences, Department of Mathematics and Sciences, College of
Allied Health, & Department of Pharmacy. (n.d.). Biochemistry Laboratory
Manual (1st Edition). National University - Manila.
TOTAL SCORE:
LABORATORY REPORT
Lipids
Name: Carreoon, Akeysha Mae Date: 09/14/23
Frane, Hanna Kriscel
Group No.: 8
Lopez, Jose Manuel F.
Pineda, John Cyril
Course, Year & Section: BSMT, 2nd Year – MED224
Acrolein Test
Lipid Visible Result
Clarified Butter A pungent irritating odor of acrolein
Vegetable oil A pungent irritating odor of acrolein
Linseed oil A pungent irritating odor of acrolein
Interpretation of Result: The Acrolein Test is used to determine the presence of
glycerol or fat. A strong, harsh, and unpleasant odor is present to identify whether the
sample contains glycerol. A negative result, on the other hand, will not emit any pungent
odor. Thus, the experiment confirmed the existence of glycerol in all three compounds,
as evident by the fact that all three substances had a distinct odor.
Liebermann-Burchard Test
Visible Result Visible Result
Addition of Acetic anhydride Addition of Concentrated sulfuric acid
No Color Change Blue Green
Interpreation of Result: The Liebermann-Burchard Test is simply used to detect the
presence of cholesterol. A positive result will yield to a color change of greenish or blue-
green color indicating the presence of cholesterol, while a negative result won’t have
any color change. The experiment showcased a change of color from clear to blue-green
color as seen in the results which confirms the presence of cholesterol. However, a little
concentration of cholesterol was added that’s why there was no color change when
adding Acetic anhydride.
Copper Acetate Test
Lipid Visible Result
Olive oil The upper layer color is green, and the
lower layer color is blue.
Appearance of white-blue precipitate at
Oleic oil
the bottom of the upper layer.
Interpretation of Results: The Copper Acetate Test is a qualitative test used to
distinguish between oil or neutral fat and fatty acid saturated and unsaturated. If the
upper layer of the test tube turns green-blue, it indicates the presence of unsaturated
fatty acids, while a white-blue precipitate at the bottom of the upper layer indicates the
presence of saturated fatty acids.
Spot / Translucent Test
Lipid Visible Result
A translucent spot appears on the filter
Clarified Butter paper.
A translucent spot appears on the filter
Vegetable oil paper.
A translucent spot appears on the filter
Linseed oil paper.
Interpretation of Results: The Spot/Translucent Test is a preliminary test for lipids,
which is characterized by a translucent and greasy spot. A positive result is indicated by
the appearance of a translucent spot on the filter paper, while a negative result is
indicated by the absence of a translucent spot.
Test for Unsaturation
Lipid Visible Result
The color of bromine water has disappeared
Olive oil showing the oil is unsaturated
The color of bromine water has not
Coconut oil completely disappeared.
The color of bromine water has disappeared
Corn oil completely and showing the oil is
unsaturated
It has decolorized completely showing
Palm oil unsaturated fatty acid in the oil
The color of bromine water has disappeared
Sunflower oil completely showing the presence of
unsaturated bond
Interpretation of Results:
Positive result: The pink color will disappear with the addition of unsaturated fatty acids.
Negative result: The pink color will not disappear.
Post Laboratory Questions
Complete the table.
Test for Positive Result Negative result
used to detect the It will give a It will not produce a
Acrolein Test presence of glycerol pungent smell pungent smell
or fat.
A chemical It indicated The color of the
estimation of cholesterol in a solution will not
Liebermann –
cholesterol sample by giving change
Burchard Test
bluish green color or
solution
used to distinguish Upper layer color Appearance of
between oil or green and lower white-blue
Copper Acetate Test neutral fat and fatty layer color blue precipitate at the
acid saturated and bottom of the upper
unsaturated. layer
preliminary test for Spot will appear on Spot will not appear
the lipids, which is the filter paper on the filter paper
Spot/Translucent Test characterized by a
translucent and
greasy spot.
Test for used to detect the The pink color will The pink color will
unsaturated fatty disappear with the not disappear
acids or double addition of
Unsaturation
bonds in a lipid unsaturated fatty
sample. acids
Grading Rubrics
Rate the Student's performance by checking the appropriate box using the
following criteria:
Rate Description
4 – Excellent The student carries our procedures efficiently,
systematically, and independently.
Appropriate behavior is observed at all times.
The student exhibits strong knowledge about the
corresponding core competency.
3 – Satisfactory The student carries out procedures efficiently and
systematically but requires minimal
guidance and supervision.
Appropriate behavior is observed most of the time.
The student exhibits moderate knowledge about the
corresponding core competency.
2 – Fair The student carries out procedures efficiently and
systematically but requires close
guidance and supervision.
Appropriate behavior is seldom observed.
The student exhibits minimal knowledge about the
corresponding core competency.
1 – Poor The student carries out procedures inefficiently,
unsystematically even under close
guidance and supervision.
Appropriate behavior is never observed.
The student does not exhibit any knowledge about the
corresponding core competency.
RATE
CRITERIA
4 3 2 1
Skill Assessment
1. The chemical reactions involved in each chemical
test were illustrated/explained correctly.
2. The actual results were interpreted correctly.
Post Laboratory Questions
1. The student were able to answer the post
laboratory questions correctly.
Total Score
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