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Experiment No.
Lipids

Lipids are water – insoluble organic compounds that can be extracted from cell and
tissues by non-polar solvents like ether, chloroform, benzene, etc. Lipids that contain fatty
acid are saponifiable, while those without fatty acid like steroids, are non-saponifiable.
General Procedure:
Prepare 6 test tubes and add 10 drops each of the following tests for lipids
Test Tube Test Solution
1 coconut oil
2 corn oil
3 margarine
4 oleic acid
5 beef tallow
6 pork fats

1. PHYSICAL PROPERTIES
A. Solubility Test
Reagents: chloroform, alcohol, 5% NaOH, 5%HCI, water, ether,

Procedure:
1. Add 1 ml each of the following on all 6 test tubes. Shake well. Observe
A. Chloroform
B. Alcohol
C. Water
D. NaOH
E. HCI
F. Ether
2. Write your results in a tabulated form.
 B. Other Physical Properties:
Reagents: coconut oil, butter, margarine, oleic acid, lanolin, beef tallow.

Procedure:
1. Observe and record the appearance, consistency, color and odor of each of the lipid
samples.
2. Write your answers in tabulated form.

C. Spotting Effect:
Reagents: alcohol, ether, coconut oil

Procedure:
1. Place a drop of coconut oil in a piece of filter paper.
2. On the same spot, place a drop of alcohol. Observe.
3. Repeat the procedure using ether.
Result:
A. oil and alcohol

B. oil and ether

2. CHEMICAL PROPERTIES:
A. Emulsification of Fats and Oils:
Reagents: coconut oil, corn oil, margarine, oleic acid, pork fats, beef tallow.

Procedure:
1. Place three (3) drops of coconut oil into two (2) dry test tubes.
2. Add 3 ml of hot water to each test tube.
3. In the first tube, add 1 ml of soap solution.
4. Shake each test tube vigorously, then let stand and observe.

Result:
Coconut oil: Test Tube 1
 Test Tube 2

Corn oil: Test Tube 1

Test Tube 2

Oleic acid: Test Tube 1

Test Tube 2

Margarine: Test Tube 1

Test Tube 2

Beef Tallow: Test Tube 1

Test Tube 2

Pork Fats: Test Tube 1

Test Tube 2

B. Test for Unsaturation:

Procedure:
1. Test the substance listed below to determine the number of drops of Br2 solution (w/
CCI4 as the solvent) that can be decolorized. The first faint persistence of the color
bromine is the end-point of the reaction. Dissolve approximately 1 ml of each
substance to be tested in 5 ml of CCI4 before adding the bromine solution.
2. Enter the name of the substance tested as well as the amount (drops of bromine
solution) required.

Substance Tested Drops of Bromine solution absorbed

Procedure:
1. Place about one (1) gram of KHSO4 in a dry test tube.
2. Add 10 drops of glycerol.
3. Heat slowly at firs to prevent evolution of SO2.
4. Then heat vigorously and note the odor of acrolein.
5. Repeat the procedure using the other lipid sample and compare the result with
glycerol.
6. Write your result in a tabulated form.

D. Hydrolysis of Fat by Dilute Acids:

Procedure:
1. Place one (1) ml of coconut oil in a test tube.
2. Add two (2) ml of 10%HCI.
3. Place in a water bath for 15 minutes.
4. Pour contents in an evaporating dish and allow to cool at room temperature.
5. Test with blue litmus paper.
6. Identify the fatty acid present.
7. Repeat the procedure using the other lipid samples.
8. Write and record your observations in tabulated form.

Result:
 E. Test for Cholesterol:
Reagents: fatty acids extract from Procedure D, chloroform, conc. sulfuric acid

Procedure:
1. Place a few crystal of cholesterol in a clean dry test tube.
2. Add 2 ml. of chloroform and 5 drops of concentrated sulfuric acid.
3. Observe for the change in color of the solution from bluish red to purple.
4. Repeat the procedure using the fatty acid extract from Procedure D.
5. Observe.

Questions
1) Butter can become rancid as a result of hydrolysis by microorganisms. Which of the
fatty acids are responsible for the bad odor associated with rancidity?

2) Predict the solubility of coconut oil in water, ethanol, and ether.

3) What is the function of soap in dishwashing or washing greasy hands?

4) What characteristics of soap make it a good emulsifying agent?

5) Give three (3) examples of lipid that are positive in acrolein test. What cause the
positive reaction?

6) What are the products of hydrolysis of fats?

7) What are the conditions and compounds necessary to hydrolyze fats?

Experiment No.
Digestion

Digestion is the process of changing food into simple forms. This process is
catalyzed by enzymes which are secreted by the glands found either within the different
regions of the digestive tube or outside the tube emptying their secretions into the tube
through their respective ducts.

Collection of Saliva

Refer to the experiment on enzymes.

Extraction of Gastric Enzyme:

Turn a pig’s stomach inside out, wash it with water, and strip off the mucous
membrane. Mince the membrane, place it in a clean bottle and completely submerge it
in glycerol. Stir frequently and allow to stand at room temperature for two (2) days.
Decant the glycerol portion into a small flask and set aside.

Extraction of Pancreatic Enzyme:

Remove the fats from a pig’s pancreas and grind them in a meat grinder. Place the
pancreatic tissue in a flask and add 100 ml. of water and 50 ml. of 95% alcohol. Shake
well and allow to stand for two (2) days. Strain the alcoholic extract through cheesecloth.
Test the pH of the extract, and neutralized it.

Preparation of Intestinal Extract:

Scrape the mucous membrane of the washed duodenum and jejunum of a pig’s
intestine. Grind the scrapings with washed sand in a mortar, transfer it to a flask, and
add 50 ml. of 1% NaCl and 5 ml. of toluene. Allow to stand for two (2) days at room
temperature. Shake the mixture frequently. Then strain the mixture with a cheesecloth
and store the extract in a refrigerator.

Digestion of Carbohydrates

1. By Salivary amylase
Reagents: iodine solution, Benedict’s solution.
 Procedure:
1. Place 5 ml. of 1% starch solution in a test tube.
2. Warm in a water bath at 400C, maintaining the temperature throughout the
experiment.
3. Add 1 ml. of saliva to the mixture and mix thoroughly.
4. At one- minute interval, transfer five (5) drops to one depression on the spot plate,
and 5 drops of Benedict’s solution. Continue the test until the starch solution no
longer gives a color reaction with iodine.
5. Treat the remaining mixture in the test tube with 10 drops of Benedict’s solution.
6. Place the test tube in a water bath for three (3) minutes.
7. Observe.

Result:

2. By Pancreatic Amylase:
Reagents: iodine solution, Benedict’s solution

Procedure:
1. Place 5 ml. of starch solution in 2 ml. of pancreatic extract in a test tube.
2. Shake well and place in a water bath at 400C for 30 minutes.
3. Divide the mixture into two (2) equal portions.
4. To the first test tube add a drop of iodine solution and to the second test tube add five
(5) drops of Benedict’s solution.
5. Observe.

Result:

3. By Intestinal Disaccharidase:
Reagents: Benedict’s solution, 1% sucrose, 1% maltose, 1% lactose

Procedure:
1. Place 1 ml of 1% sucrose solution in each of the two test tubes.
2. To test tube 1, add 1 ml of intestinal extract.
3. To test tube 2, add 1 ml of previously boiled intestinal extract.
4. Place the two test tubes in a water bath maintained at 400C for 30 minutes.
5. Add five (5) of Benedict’s solution to the two test tubes. Observe.
6. Repeat the procedure using 1% lactose and 1% maltose.

Result:
 Sucrose with intestinal extract

Sucrose with boiled intestinal extract

Lactose with intestinal extract

Lactose with boiled intestinal extract

Maltose with intestinal extract

Maltose with boiled intestinal extract

Digestion of Proteins:

1. By Gastric Enzyme:
Reagent: 0.4% HCI, 1% Na2CO3, 1% CuSO4, toluene

Procedure:
1. Place the following in four (4) separate test tubes.
A. 5 ml of gastric extract
B.5 ml of 0.4% HCI
A. 5 ml of gastric extract + 3 ml of 0.4% HCI
B. 5 ml of gastric extract + 3 ml of 1% Na2CO3

Add equal slices of coagulated egg white to each tube and place the test tubes in a water
bath at 400C for two (2) hours. Add four (4) drops of toluene to each test tube and store
until next laboratory period. Determine the extent of protein digestion by noting the size
of the coagulated egg white.

Observation:

A. Test tube 1

B. Test tube 2

C. Test tube 3

D. Test tube 4
Place 1 ml. of the supernatant fluid from each test tube in separate tubes. Neutralize test
tube B and C with solid Na2CO3. In all the test tubes, add 1 ml. of 1% NaOH and two (2)
drops of 1% CuSO4.
Result:

Test tube A

Test tube B

Test tube C

Test tube D

2. By Pancreatic Enzyme:
Reagents: 0.4% HCI, 1% Na2CO3

Procedure:
1. Place the following in four (4) different test tubes.
A. 5 ml of gastric extract
B. 5 ml of 0.4% HCI
C. 5 ml of gastric extract + 3 ml of 0.4% HCI
D. 5 ml of gastric extract + 3 ml of 1% Na2CO3

Place equal sizes of coagulated egg white in each test tube and place them in a water bath
at 400C for one and a half hours. Add three (3) drops of toluene to all test tubes and set
aside until the next laboratory period. Then add 1 ml of CUSO4 to all mixtures.

Result:

Test tube A

Test tube B

Test tube C

Test tube D
3. By Intestinal Enzymes:
Reagents: 1% Na2CO3, 0.4% HCI

Procedure:
1. Prepare four (4) test tubes as follows:
A. 5 ml of water
B. 5 ml of intestinal extract + 1 ml of 1% Na2CO3
C. 5 ml of boiled intestinal extract + 1ml of 1% Na2CO3
D. 5 ml of boiled intestinal extract + 1 ml of 0.4% HCI

Add 1 ml of milk to each test tube and mix thoroughly. Divide the contents of the four
(4) test tubes equally and set aside the remaining four test tubes as control tubes. Place
the test solutions in a water bath at 400C for one hour. Add 1 ml of CUSO4 for both
incubated tubes and the control tubes.

Result:
Incubated tubes:
Test tube A

Test tube B

Test tube C

Test tube D

Control Tubes:

Test tube A

Test tube B

Test tube C

Test tube D

4. Intestinal Digestion by Proteases:

Procedure:
1. Prepare four(4) test tube as follows:
A. 5 ml of 1% peptone
 B. 5 ml of 1% peptone
C. 5 ml of 1% albumin
D. 5 ml of 1% casein solution

2. Prepare another set of four (4) test tubes containing the above solution and make these
as the control tubes.
3. Add three (3) drops of 1% Na2CO3 to 10 ml of the intestinal extract.
4. Add two (2) ml of the alkaline extract to test tube A, C, and D. Boil the remaining
extract and add to test tube B.
5. Place all test tubes in a water bath at 400C for one and a half hours.
6. Add 1 ml of CUSO4 in all the mixtures.

Result:
Incubated tubes:
Test tube A

Test tube B

Test tube C

Test tube D

Control tubes:

Test tube A

Test tube B

Test tube C

Test tube D

Digestion of Lipids:
Reagents: 0.05N NaOH, phenolphthalein

Procedure:
1. Place the following in each of the four (4) test tubes
A. 1 ml coconut oil + 4 ml pancreatic extract + 5 ml of H2O
B. 1 ml coconut oil + 9 ml of water
C. 1 ml coconut oil + 2 ml bile + 7 ml of H2O
D. 1 ml coconut oil + 2 ml bile + 4 ml pancreatic extract + 3 ml of H2O
2. Shake the test tubes well and place in a water bath at 400C for one and a half hours.
3. Add a drop of phenolphthalein to each test tube.
4. Add drop by drop of 0.05 N NaOH using a syringe until the solution turns to a faint
pink color.
5. Record the amount of 0.05 N NaOH use for each test tube.

Result:

Test tube A

Test tube B

Test tube C

Test tube D

Questions:

1. Give the effect of the following:

A. Boiling the enzyme on its activity

B. Bile on fat digestion

C. HCI on gastric digestion

2. Proteolytic enzymes are secreted as proenzymes. What is the significance of this?

3. Discuss briefly the activation of:

A. Pepsinogen

B. Trypsinogen

C. Chymotrypsinogen

4. In these experiments on digestive enzymes, what factors influenced the rate of