Experiment

36
Carbohydrates

Background

Carbohydrates are polyhydroxy aldehydes, ketones, or compounds that yield

polyhydroxy aldehydes or ketones upon hydrolysis. Rice, potatoes, bread, corn, candy,
and fruits are rich in carbohydrates. A carbohydrate can be classified as a monosaccharide
(glucose or fructose); a disaccharide (sucrose or lactose), which consists of two joined
monosaccharides; or a polysaccharide (starch or cellulose), which consists of thousands of
monosaccharide units linked together. Monosaccharides exist mostly as cyclic structures
containing hemiacetal (or hemiketal) groups. These structures in solutions are in
equilibrium with the corresponding open chain structures bearing aldehyde or ketone
groups. Glucose, blood sugar, is an example of a polyhydroxy aldehyde (Fig. 36.1).

Hemiacetal Hemiacetal
CH2OH CH2OH
CH2OH
O H O
H O H OH
H
H H
H H H
OH H OH H C O OH H
H
HO HO HO

OH H H OH

H OH OH
-D-glucose Open-chain form -D-glucose

Figure 36.1 • The structures of D-glucose.

Disaccharides and polysaccharides exist as cyclic structures containing functional

groups such as hydroxyl groups, acetal (or ketal), and hemiacetal (or hemiketal). Most of
the di-, oligo-, or polysaccharides have two distinct ends. The one end which has a
hemiacetal (or hemiketal) on its terminal is called the reducing end, and the one which
does not contain a hemiacetal (or hemiketal) terminal is the nonreducing end. The name
“reducing” is given because hemiacetals (and to a lesser extent hemiketals) can reduce an
oxidizing agent such as Benedict’s reagent.
Fig. 36.2 is an example:
Figure 36.2
The structure of maltose, Locked ring Unlocked
ring CH2OH CH2OH
a disaccharide. O O
H H
1 4
OH H OH H
HO O OH
Experiment 36 1
 OH OH
Nonreducing end Reducing end

2 Experiment 36
 Not all disaccharides or polysaccharides contain a reducing end. An example
is sucrose, which does not have a hemiacetal (or hemiketal) group on either of its
ends (Fig. 36.3).
Figure 36.3
The structure of sucrose. Acetal group
CH2OH

H C O H CH2OH
O
H H
C C C C
OH H H
HO
C C CH2OH
HO
O
C C

H OH OH H

Polysaccharides, such as amylose or amylopectin, do have a hemiacetal group on one

of their terminal ends, but practically they are nonreducing substances because there is
only one reducing group for every 2,000–10,000 monosaccharidic units. In such a low
concentration, the reducing group does not give a positive test with Benedict’s or
Fehling’s reagent.
On the other hand, when a nonreducing disaccharide (sucrose) or a polysaccharide
such as amylose is hydrolyzed the glycosidic linkages (acetal) are broken and reducing
ends are created. Hydrolyzed sucrose (a mixture of D-glucose and D-fructose) will give a
positive test with Benedict’s or Fehling’s reagent as well as hydrolyzed amylose (a mixture
of glucose and glucose containing oligosaccharides). The hydrolysis of sucrose or amylose
can be achieved by using a strong acid such as HCl or with the aid of biological catalysts
(enzymes).
Starch can form an intense, brilliant, dark blue-, or violet-colored complex with
iodine. The straight chain component of starch, the amylose, gives a blue color while the
branched component, the amylopectin, yields a purple color. In the presence of iodine,
the amylose forms helixes inside of which the iodine molecules assemble as long
polyiodide chains. The helix-forming branches of amylopectin are much shorter than
those of amylose. Therefore, the polyiodide chains are also much shorter in the
amylopectin-iodine complex than in the amylose-iodine complex. The result is a different
color (purple). When starch is hydrolyzed and broken down to small carbohydrate units,
the iodine will not give a dark blue (or purple) color. The iodine test is used in this
experiment to indicate the completion of the hydrolysis.
In this experiment, you will investigate some chemical properties of carbohydrates in
terms of their functional groups.
1. Reducing and nonreducing properties of carbohydrates
a. Aldoses (polyhydroxy aldehydes). All aldoses are reducing sugars because
they contain free aldehyde functional groups. The aldehydes are oxidized by
mild oxidizing agents (e.g., Benedict’s or Fehling’s reagent) to the
corresponding carboxylates. For example,
NaOH
RˆCHO + 2Cu 2+ ¶¶l RˆCOO —Na + + Cu 2Ob
(from Fehling’s reagent) Red precipitate
 b. Ketoses (polyhydroxy ketones). All ketoses are reducing sugars because
they have a ketone functional group next to an alcohol functional group. The
reactivity of this specific ketone (also called a-hydroxyketone) is attributed to its
ability to form an a-hydroxyaldehyde in basic media according to the following
equilibrium equations:

CH2OH CHOH CHO

C O Base Base
C OH H C OH
H C OH
H C OH H C OH

Ketose Enediol Aldose

c. Hemiacetal functional group (potential aldehydes). Carbohydrates with

hemiacetal functional groups can reduce mild oxidizing agents such as
Benedict’s reagent because hemiacetals can easily form aldehydes through
the following equilibrium equation:

H OR O
C
R C H + R OH
R OH

Sucrose is, on the other hand, a nonreducing sugar because it does not contain a
hemiacetal functional group. Although starch has a hemiacetal functional group
at one end of its molecule, it is, however, considered as a nonreducing sugar
because the effect of the hemiacetal group in a very large starch molecule
becomes insignificant to give a positive Benedict’s test.
2. Hydrolysis of acetal groups. Disaccharides and polysaccharides can be converted into
monosaccharides by hydrolysis. The following is an example:

C H22O O6+ C 6H O
12 +H O 12 6

cat aly st
11 ¶ 2¶ l C 6H 12
Lactose Glucose Galactose
(milk sugar)

Objectives

To become familiar with the reducing or nonreducing nature of carbohydrates.

To experience the enzyme-catalyzed and acid-catalyzed hydrolysis of acetal groups.
 Procedure

Reducing or Nonreducing Carbohydrates

Place approximately 2 mL (approximately 40 drops) of Fehling’s solution (20 drops each of

solution part A and solution part B) into each of five labeled tubes. Add 10 drops of each of
the following carbohydrates to the corresponding test tubes as shown in the following
table.

Name of carbohydrate
Test tube no.

1 Glucose Fructose Sucrose Lactose

2 Starch
3
4
5

Place the test tubes in a boiling water bath for 5 min. A 600-mL beaker containing about
200 mL of tap water with a few boiling chips is used as the bath. Record your results on
your Report Sheet. Which of those carbohydrates are reducing carbohydrates?

Hydrolysis of Carbohydrates

Hydrolysis of sucrose (acid versus base catalysis)

Place 3 mL of 2% sucrose solution in each of two labeled test tubes. To the first test tube
(no. 1), add 3 mL of water and 3 drops of dilute sulfuric acid solution (3 M H 2SO4). To the
second test tube (no. 2), add 3 mL of water and 3 drops of dilute sodium hydroxide
solution (3 M NaOH). Heat the test tubes in a boiling water bath for about 5 min. Cool
both solutions to room temperature. To the contents of test tube no. 1, add dilute sodium
hydroxide solution (3 M NaOH) (about 10 drops) until red litmus paper turns blue. Test a
few drops of each of the two solutions (test tube nos. 1 and 2) with Fehling’s reagent as
described before. Record your results on your Report Sheet.

Hydrolysis of starch (enzyme versus acid catalysis)

Place 2 mL of 2% starch solution in each of two labeled test tubes. To the first test tube
(no. 1), add 2 mL of your own saliva. (Use a 10-mL graduated cylinder to collect your
saliva.) To the second test tube (no. 2), add 2 mL of dilute sulfuric acid (3 M H 2SO4). Place
both test tubes in a water bath that has been previously heated to 45°C. Allow the test
tubes with their contents to stand in the warm water bath for 30 min. Transfer a few
drops of each solution into separate depressions of a spot plate or two separately labeled
microtest tubes. (Use two clean, separate medicine droppers for transferring.) To each
sample (in microtest tubes or on a spot plate), add 2 drops of iodine solution. Record the
color of the solutions on your Report Sheet.
Acid catalyzed hydrolysis of starch
Place 5.0 mL of starch solution in a 15 × 150 mm test tube and add 1.0 mL of dilute
sulfuric acid (3 M H2SO4). Mix it by gently shaking the test tube. Heat the solution in a
boiling water bath for about 5 min. Using a clean medicine dropper, transfer about 3
drops of the starch solution into a spot plate or a microtest tube and then add 2 drops of
iodine solution. Observe the color of the solution. If the solution gives a positive test with
iodine solution (the solution should turn blue), continue heating. Transfer about 3 drops
of the boiling solution at 5-min. intervals for an iodine test. (Note: Rinse the medicine
dropper very thoroughly before each test.) When the solution no longer gives a blue color with
iodine solution, stop heating and record the time needed for the completion of hydrolysis.

Chemicals and Equipment

Bunsen burner
Medicine droppers
Microtest tubes or a white spot plate
Boiling chips
Fehling’s reagent
3 M NaOH
2% starch solution
2% sucrose
2% fructose
2% glucose
2% lactose
3 M H2SO4
0.01 M iodine in KI
 Glodove, Ariane Kyle B. BAC 09/12/21
NAME SECTION DATE

N/A``
PARTNER GRADE

Experiment 36

PRE-LAB QUESTIONS
1. Circle and label the hemiacetal functional group and the acetal functional group in the
following carbohydrates:
a. sucrose b. lactose
CH2OH CH2OH H OH
C CH2OH C C C

O O
H H O
HO O OH OH
H H H H
C C C C C C C C
O H HO H H
OH CH OH OH H H H
HO H
H
2
C C C C C C C O
H OH OH H OH CH2OH

2. Which carbohydrate in question 1 is a reducing sugar?

In the question 1, the reducing sugar is the lactose because the cyclic hemiacetal of the D-glucopyranose unit is in
equilibrium with its open-chain form and can be oxidized to a carboxyl group.
Experiment 36 379
3. Write an equation to indicate the formation of the products resulting from the
dilute acid hydrolysis of each of the following carbohydrates.
(a) maltose

(b) sucrose
 Glodove, Ariane Kyle B. BAC 09/12/21
NAME SECTION DATE

N/A
PARTNER GRADE

Experiment 36

REPORT SHEET
Reducing or nonreducing carbohydrates

Reducing or nonreducing
Test tube no. Substance Observation carbohydrates
It reduces the copper (II) to form red Reducing carbohydrates
1 Glucose
precipitates of cuprous oxide.

2 Fructose
No precipitate formed Non-reducing carbohydrates
3 Sucrose
It reduces the copper (II) to form red Reducing carbohydrates
4 Lactose precipitates of cuprous oxide.
No precipitates forms Non-reducing carbohydrates
5 Starch

Hydrolysis of carbohydrates

Hydrolysis of sucrose (acid versus base catalysis)

Fehling’s reagent
Sample Condition of hydrolysis Observation (positive or negative)

1 Acidic (H2SO4)

2 Basic (NaOH)

Hydrolysis of starch (enzyme versus acid catalysis)

Iodine test
Sample Condition of hydrolysis Observation (Positive or negative)

1 Enzymatic (saliva)

2 Acidic (H2SO4)

Experiment 36 381
 Acid catalyzed hydrolysis of starch

Heating time Iodine test

Sample no. (min.) (Positive or negative)
1 5

2 10

3 15

4 20

5 25

6 30

POST-LAB QUESTIONS
1. Fructose is a ketose. Ketones usually do not give a positive reaction with
Fehling’s reagent. Why is fructose oxidized by Fehling’s reagent? (Consult your
textbook.)

Tollen's, Fehling's, and Benedict's reagents are mild oxidizing reagents that easily oxidize aldehydes. Ketones lack
such reducing properties and therefore cannot be oxidized under the same conditions. Fructose, on the other hand, reduces
such reagents even when they do not contain an aldehyde group. This is because the reagents are basic solutions, and under
basic conditions, fructose is easily isomerized to a mixture of aldoses. Fehling's reagent is also used to distinguish between
water soluble aldehydes and ketone function groups. Fructose, a monosaccharide, will test positive due to the base in the
reagent's conversion of aldoses.

2. What chemical test is performed to indicate the completion of the hydrolysis of starch
by acid?

The chemical test can be perform as an indication of the hydrolysis of starch by acid through the iodine test .

3. Which hydrolysis of starch is faster: the acid or the enzyme catalyzed reaction?

Hydrolysis of starch is faster with the enzyme catalyzed reaction because it becomes negative with iodine and positive with
Benedict’s reagent but it changes in color.

4. Amylose gives a positive iodine test and a negative test with Fehling’s reagent. On
the basis of the structure of amylose, explain the results of the two tests.

Amylose reacts with iodine to form starch - tried iodide complex, which results in a deep blue color.
This color results from the formation of a starch-iodine complex. The reducing sugar is determined
using Fehling's test. As a result, all reducing monosaccharide sugars pass Fehling's test.
Polysachharides are all non-reducing in nature. As a result, they produce negative results for amylose.
Amylose has a single reducing end. As a result, it is undetectable because the concentration is too low.
As a result, the test was negative.