BIOCHEMISTRY LABORATORY 1

Laboratory Activity
INMalaluan

GENERAL REACTIONS OF CARBOHYDRATE

Carbohydrates are vital sources of energy for both plants and animals. They also serve as
skeletal structure for plants and as storage for chemical energy of both plants and animals.

In terms of chemical structure, carbohydrates are derivatives of aldehydes and ketones. Their
interesting chemical reactions are due to such functional groups.

Any material containing carbohydrates yields positive result in the Molisch Test. This test is
based on the dehydration of monosaccharide by concentrated sulfuric acid to form furfural derivatives
and the subsequent reaction with alpha napthol to form colored complexes. A similar reaction can be
observed through Anthrone test which is also based on the formation of furfural derivatives. Anthrone
is a keto form of 9-hydroxy-anthracene which, when dissolved in concentrated sulfuric acid and mixed
with carbohydrates, forms a blue or green color.

Carbohydrates with free aldehyde or ketone groups have reducing properties. They are oxidized
by alkaline solutions of cupric sulfate (Fehlings and Benedicts Reagents) producing a reddish brown
precipitate because of the reduction of cupric to cuprous ions. Barfoeds reagent, a solution of cupric
acetate in a weak acetic acid, serves as another test as it is acted upon by reducing monosaccharides but
not by reducing disaccharides.

The simplest carbohydrates called monosaccharides consist of only one saccharide unit and thus
cannot be hydrolyzed. The most abundant in the group is glucose. Other common monosaccharides are
fructose, galactose, ribose, and deoxyribose. Disaccharides consist of monosaccharide units joined by a
glycosidic linkage. Common disaccharides include sucrose, lactose, and maltose. Polysaccharides, the
most complex of all carbohydrates, composed of many saccharide units, can be hydrolyzed by enzymes
or by heating with dilute strong acids to yield monosaccharides.

OBJECTIVES:

1. To describe the properties and structures of carbohydrates in general.

2. To differentiate reducing sugars from non-reducing sugars

MATERIALS

Glucose lactose 1% AgNO3

Sucrose Molisch Reagent 6 N NaOH
Starch Benedicts Reagent Ba(OH)2
maltose Ammonia solution Conc. H2SO4
fructose Phenolphthalein Dil. HCl
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Laboratory Activity
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PROCEDURE

A. REACTION WITH ACIDS

1. Molisch Test

a. To 1ml of 1% glucose, add 2 drops of Molisch reagent. Mix thoroughly.

b. Incline the test tube and allow 1 ml of conc. H2SO4 acid to flow down the side of the test
tube. Do not shake. Note the color formed at the junction of the two layers.
c. Repeat the procedure using 1% fructose, 1% sucrose, 1% starch, 1% maltose and 1%
Lactose solutions.
(Q1) Did all the samples give the same result? Account for the results observed.

B. REDUCING PROPERTY

1. Tollens Test

a. Preparation of Tollens reagent.

To 10 ml of 1% AgNO3 solution, add 2 drop of 6M NaOH. Add ammonia solution
dropwise, shaking after each addition until the precipitate initially formed is dissolved.
Avoid adding more than what is necessary to dissolve the precipitate.
b. Place 6 drops of 1% glucose solution in a clean test tube and add 2 ml of Tollens reagent.
Mix well. Allow to stand for 5 minutes. If no result is obtained, warm in a water bath for
exactly 2 minutes.
c. Repeat the procedure using 1% fructose, 1% sucrose, 1% starch, 1% maltose and 1%
lactose solutions

2. Benedict Test

a. Place 1 ml of Benedicts reagent in a test tube and add 5 drops of 1% glucose solution.
b. Heat in a boiling water bath. Note the color produced.
c. Repeat the procedure using 1% fructose, 1% sucrose, 1% starch, 1% maltose and 1%
lactose solutions.

(Q2) Which compounds did not give positive result to Benedicts test? Explain why?

3. Fehling Test
a. Mix 1 mL of Fehlings solution A and B in a test tube and add a ten drops of the glucose
solution.
b. Place the test tube in a boiling water bath and note your observations
c. Repeat the procedure using 1% fructose, 1% sucrose, 1% starch, 1% maltose and 1%
lactose solutions.

C. HYDROLYSIS
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Laboratory Activity
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a. Prepare the rice solution by placing a spatula of cooked rice in a mortar and pounding
using a pestle. Add 10 ml of distilled water. Stir.
b. Put 2 ml of the rice solution in a test tube, add 1 ml of 2.0 M HCl and heat in a water
bath for 15 minutes to hydrolyze.
c. Neutralize the resulting mixture with about 1 mL of 2.0M NaOH.
d. Add 2 mL of Benedicts reagent. Heat in a water bath for 2 minutes.

(Q3) Was there a change in color? Is a positive result obtained?

e. In another test tube, mix 2 ml of the rice solution (from step 1) and 1ml of Benedicts
reagent. Heat in a water bath for 2 minutes.

(Q4) Was there a change in a color? Is a positive result obtained?

(Q5) What do you think is the purpose of adding HCl to the rice and heating the mixture in a
water bath?
(Q6) What are the products of hydrolysis of rice?

GENERAL REACTIONS OF CARBOHYDRATE

REPORT SHEET

Table 1. Reaction of Sugars with Molisch Reagent

SAMPLE SUGAR Observation Result
(positive or negative)

Glucose

Sucrose

Maltose

Lactose
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Starch

Table 2. Reaction of Sugars with Tollens Reagent

SAMPLE SUGAR Observation Result
(positive or negative)

Glucose

Sucrose

Maltose

Lactose

Starch

Table 3. Reaction of Sugars with Benedicts Reagent

SAMPLE SUGAR Observation Result
(positive or negative)

Glucose

Sucrose
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Maltose

Lactose

Starch

Table 4. Reaction of Sugars with Fehlings Reagent

SAMPLE SUGAR Observation Result
(positive or negative)

Glucose

Sucrose

Maltose

Lactose

Starch

Table 5. Hydrolysis
SAMPLE Observation Benedicts Test
(positive or negative)
Rice solution + HCl
+ NaOH +
Benedicts Reagent

Rice solution +
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Laboratory Activity
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Benedicts Reagent