CSA-B BIOCHEMISTRY LAB MANUAL

Experiment No. 2
Hydrolysis of Carbohydrates

I. OBJECTIVES

1. To determine the results when polysaccharides such as starch and cellulose are
hydrolyzed to simple sugars by the action of strong acids or by enzyme activity (saliva).
2. To relate the hydrolysis reaction observed in the experiment with digestion of starchy
food.

II. INTRODUCTION

Polysaccharides such as starch and cellulose are hydrolyzed to simple sugars either by
the action of strong acids or by enzyme activity as in digestion.

III. MATERIALS AND APPARATUS

12 mL iodine solution 1 water bath

10 mL 6N HCl 1 125mL Erlenmeyer flask
12 mL 6N NaOH 1 beaker
12 mL phenolphthalein indicator solution 1 hot plate
10 mL 0.2M NaCl 1 spot plate
10 buffer solution 2 test tube holder
10 mL Fehling’s solution A 1 stirring rod
10 mL Fehling’s solution B Litmus paper
2 g starch Test tubes

IV. PROCEDURE

Preparation of Starch Paste

1. Mix 1 g of starch with 5 mL of water on a beaker.
2. Pour this mixture with constant stirring into 100 mL of boiling water until the solution
is clear.
3. Allow to cool.

Preparation of 1% Starch Solution

1. Prepare 1 g of starch.
2. Add 99 mL of water. Stir until solution is homogenous.

A. ACID HYDROLYSIS
1. Conduct Fehling’s test to undiluted starch paste.
2. To 50 mL of undiluted starch paste in a beaker, add 1 mL of 6N HCl.
3. Heat in a boiling water bath.
4. At 2 minutes intervals, take 1 mL aliquot of the solution and add 1 drop of iodine
solution.
5. When the mixture no longer produces a blue color with iodine, cool and neutralize
with 6N HCl the remaining starch paste in the beaker.
6. Conduct Fehling’s Test.

Fehling’s Test
1. Transfer 8-10 drops of solution to be tested in a small test tube.
2. In a separate test tube, mix 1 mL of Fehling’s solution A with 1 mL of Fehling’s
solution B. Add this mixture to the other test tube.
3. Heat for a few minutes in a boiling water bath. Observe.
CSA-B BIOCHEMISTRY LAB MANUAL

B. ENZYME HYDROLYSIS

Preparation of Amylase
1. Rinse your mouth several times with water.
2. Wash your mouth with around 20 mL of warm water for 1 minute.
3. Collect the washings in a beaker. This will serve as a dilute solution of amylase for
the following experiments.

Enzymatic Hydrolysis of Starch

1. Add 1 drop of iodine solution and 2 mL of starch paste to a test tube containing 2 mL
of amylase.
2. Mix and allow mixture to stand until blue color disappears. Record time.
3. When blue color disappears, set aside for 1 hour and test the solution with Fehling’s
reagent.

Influence of pH
1. Set up 8 numbered test tubes containing 5 mL of 1% starch solution, 1 mL of 0.2 M
NaCl and 1 mL buffer solution.
2. Place all tubes in a large beaker containing water heated to about 37°C.
3. After 15 minutes, add 2 mL of amylase to each of the 8 experimental tubes rapidly,
mix well and note the time.
4. Immediately and at 2 minutes interval, remove 1 drop from each of the tubes into a
spot plate on which drops of iodine solution have been placed.
5. Repeat the withdrawal of the samples from each tube and note for each tube the
time (in minutes) at which the blue color is no longer obtained. Record your
observation.
 CSA-B BIOCHEMISTRY LAB MANUAL

Name: ___________________________________ Rating: _______________________

Group No.: __________ Date Performed: _______________

Experiment No. 3
Hydrolysis of Carbohydrates

A. ACID HYDROLYSIS

TEST RESULTS

Fehling’s test for starch sol’n before addition of HCl

Fehling’s test for starch sol’n after addition of HCl

B. ENZYMATIC HYDROLYSIS

TEST RESULTS

Fehling’s test for starch sol’n before addition of

enzyme (saliva)

Fehling’s test for starch sol’n 1 hour after addition of

enzyme (saliva)

Other observation referring to viscosity of starch

sol’n after addition of enzyme

C. INFLUENCE OF PH ON AMYLASE REACTION RATE

TUBE NUMBER TIME (MINUTES)

1

3
4

5
6

8
CSA-B BIOCHEMISTRY LAB MANUAL

1. What is the purpose of adding iodine solution to starch?

2. What is the function of amylase in the hydrolysis of starch? Which test in the experiment
confirms its action on starch? Explain.

3. How do you relate this hydrolysis reaction with digestion of starchy food?

CONCLUSION: