Nursing Biochemistry Laboratory (NurBio Lab) 1

Laboratory Manual

Laboratory ProcedureSZX
Carbohydrates I: Common Reactions Activity
3

INTRODUCTION

This activity on carbohydrates will involve two parts: its common and specific reactions.
It will entail two two-hour laboratory periods.

Common Reactions
Carbohydrates are classified according to the
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lBfffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffplexity of their
molecules. The simplest group, called monosaccharides, are the building blocks from
which higher sugars, (dissacharides) and more complex groups (polysaccharides) are
formed. The monosaccharides are sub-classified according to the number of carbon
atoms in their unit chains into trioses, tetroses, pentoses and hexoses; and according to
the position of their carbonyl groups into aldoses and ketoses. Thus, they are sometimes
designated as aldopentoses and ketohexoselllllllllllllllllllllllllllllllllllllllllllls. The ending ose
is generally used in naming carbohydrates and the term sugar refers to those possessing
sweet taste, most of which are monosaccharides and dissacharides.

Only a few classes of carbohydrates have special biological importance. These include
the hexoses: glucose, also known as dextrose or grape sugar, fructose or levulose, and
galactose; and those that are compounds of hexoses, the hexosans.

Glucose is a normal constituent of the blood and tissue fluids of the human body. D-
galactose, also called brain sugar, is found as a cerebroside in the brain and as a
ganglioside in the nerve tissues. It is also the sugar of galactolipids. A glucose
derivative, glucosamine with protein form glycoproteins which are involved in many vital
functions in the body. Certain pentoses are also important. Ribose and deoxyribose are
constituents of nucleic acids and are also present in some enzymes. Trioses and related
oxidation and/or reduction products are involved in the oxidation and synthesis
mechanisms in the body. Structurally, carbohydrates are polyhydroxyketones.
Accordingly, their chemical reactions are characteristic of both the OH and C=O
functional groups.
H
O H O
H OH
-3 H 2O
H OH H O
H OH con c. H 2S O 4

CH2 OH H H
D - ribose furaldehyde or furfural

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 O
H O
H OH - 3 H Lab)
Nursing Biochemistry Laboratory (NurBio 2O 2
HOManual
Laboratory H
co n c . H 2S O H O
H OH 4
H OH O
H OHO
H CH 2
OHH OH -3 H 2O
HO
D - glucose H 5-hydroxymethylfurfural
co n c. H 2S O 4 H O
H OH
H OH
H CH 2 OH
OH
D - glucose 5-hydroxymethylfurfural

With concentrated sulfuric acid, H 2SO4, the polysaccharides and dissacharides are first
hydrolyzed to monosaccharides before they are dehydrated. Pentoses yield furfural
whereas hexoses yield hydroxymethylfurfural which is further degraded into levulinic
acid.

Both furfural and hydroxymethylfurfural condense with a number of compounds to yield

colored products. With -naphthol, furfural derivatives give a purple color, which is the
basis of Molisch’s test, a general test for carbohydrates. With mild oxidizing agents like
Fehling’s, Benedict’s, and Barfoed’s reagents, the alkaline medium of the reagents favor
the oxidation of the CHO group to COOH, while the Cu++ of the soluble Cu complex
is reduced to a brick red Cu2O precipitate.

The disaccharide sucrose is non-reducing because both rings of glucose and fructose are
in the acetal or ketal form. So they cannot be in equilibrium with their aldehyde forms.

H O H OH H OH
H OH OH - O
-
HO H O H HO H O H
HO H
H OH H OH H OH
H OH H OH H OH
CH 2 OH CH 2 OH CH 2 OH
glucose 1,2-enediol fructose

-
O H

H O

HO H
HO H
H OH
H OH
CH2OH

mannose

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Ketoses like fructose give a positive reaction to tests for reducing sugars because of the
Lobry de Buryn-von Eckenstein transformation. The alkaline solution favors the
formation of the enediol and suppresses formation of the ring structure. The outline
reaction below shows the transformation of the hexoses into a common enediol form.
The outline shows the change only in the first 2 carbons of each sugar.

The enediols formed in alkaline medium dilute Ba(OH) 2 decompose readily when heated.
The double bond of the enediol moves down the carbon chain giving rise to a variety of
isomers and eventually to the cleavage of the C=C chain.
H H
H O H OH H
H OH OH
H OH OH H OH
OH
HO H HO H HO
HO
H OH H OH OH
H OH
H OH H OH H OH
H OH
CH 2 OH CH 2 OH CH 2 OH
CH 2 OH

D-glucose D-glucose D-glucose D-glucose

1,2-enediol 2,3-enediol 3,4-enediol

The double bond breaks between 3C and 4C; forming 2 trioses, glycerose and
dihydroxyacetone, which provide the pathway to the oxidation of glucose in the Krebs’s
cycle. In the absence of oxygen, the 3,4-enediol is predominant.

Other decomposition products when the double bond moves further down are
formaldehyde, glycoladehyde, lactic acid, methyl glyoxal and reductone. Some of these
fragments form complex resinous compounds reducing yellow to brownish color. The
property of enediols is the basis of Moore’s test for reducing sugars.

In the Tollen’s test, both aliphatic and aromatic aldehydes are oxidized to carboxylic
acids and the Ag ion is reduced to elemental Ag deposited on the sides of the tube.

O O
+
R + 2 Ag(NH 3)2 + 2 OH- 2 Ag + R
+
+ H2O + 3 NH 3
H O NH 4

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Bunsen burner Molisch’s reagent

Iron ring Fehling’s reagents A and B
Iron stand Benedict’s reagent
360C thermometer Barfoed’s reagent
Nylander’s reagent

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Saturated picric acid solution

Tollen’s reagents A and B
1% glucose solution
1% xylose solution
1% lactose solution
1% starch solution
1% furfural solution
1% fructose solution
1% galactose solution
1% maltose solution
1 % sucrose solution
Unknown sugar solution

PROCEDURE

A. Molisch’s Test

1. Put 1 mL of 1% glucose solution in a test tube. Add 1 drop of Molisch reagent

and mix thoroughly. Include the tube and allow 1 mL concentrated sulfuric acid,
H2SO4, to flow down the side of the tube so that layers are formed. Note the
violet color at the junction of the 2 liquids.
2. Repeat the test with 1% solution of xylose, lactose, sucrose, starch,
furfural and unknown sugar solution.

IMPORTANT: The amount of unknown solution is limited. Be sure its use

 is budgeted. No additional amount will be issued.

B. Action of Alkali (DO NOT PERFORM. Just search for theoretical results.)

1. In separate test tubes, place 12 drops each of 1% solution of glucose, fructose,

and galactose. In each tube, add 12 drops of 10% sodium hydroxide solution,
NaOH, solution. Put the tubes in a boiling water bath. Note the color and odor
produced.
2. Repeat the test using the same sugar solution, but using a 1% barium
hydroxide, Ba(OH)2, solution instead of the sodium hydroxide solution as the
alkali. Note the results and compare them with the results of number 1 above.

C. Reduction Tests

Fehling’s Test

1. Dilute 2 mL Fehling’s solution (a mixture of 1 mL Fehling’s A and 1 mL Fehling’s

B) with 8 mL water. Put 1 mL each of the diluted solution in test tubes.

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2. Take one of the test tubes, while heating on a water bath, add 1% glucose
solution drop by drop. Note the result.

3. Repeat the test on the other test tubes, using different 1% sugar solutions of
xylose, fructose, maltose, sucrose, starch, glycogen and an unknown sugar
solution.

Benedict’s Test

1. To 1 mL Benedict’s reagent, add 2 drops of 1% glucose solution in a test

tube. Boil in a water bath for 2 minutes. Allow to cool. Observe the change.

2. Repeat the test on 1% solutions of xylose, fructose, lactose, sucrose and

starch.

Barfoed’s Test

1. Mix 1 mL Barfoed’s reagent with 2 drops of 1% glucose solution. Heat for 30

seconds in a boiling water bath. Allow to stand for 15 minutes before noting the
results.

2. Repeat the tests on 1% solutions of fructose, xylose, lactose, maltose, sucrose

and the unknown sugar solution.

Nylander’s Test (DO NOT PERFORM. Just search for theoretical results.)

1. Mix 15 drops of 1% glucose solution with an equal amount of Nylander’s solution

in a test tube. Heat for 5 minutes in a boiling water bath. Observe.

2. Repeat the test on 1% solutions of fructose, xylose, lactose and maltose.

Picric Acid Test

1. To 1 mL of 1% glucose, add 10 drops saturated picric acid solution and 4 drops

of 10% sodium carbonate, Na2CO3, solution. Warm and note the results.

2. Repeat tests on 1% solutions of fructose, xylose, lactose and maltose.

Tollen’s Test

1. To 1 mL 1% glucose solution, add a few drops of Tollen’s reagent (A and B).

Warm in a water bath. Observe.

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University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 6
Laboratory Manual

CAUTION: The Tollen’s reagent (solution) decomposes on standing and

! yields an explosive precipitate, so it is prepared only when needed. The
excess solution must be immediately discarded in the sink and flushed
with running water.

PROPER DISPOSAL: Dispose of solutions in the proper waste bottles (as

acid or basic wastes, and organic or inorganic wastes).

QUESTIONS

1. Why do all sugars give a positive result for Molisch’s test?

2. What compounds other than -naphthol can be used as a reagent for the test?
3. Why is it important to use freshly prepared Fehling’s reagent?
4. Compare the action of a strong and a weak alkali on a reducing sugar.
5. How are the conditions for Barfoed’s test different from those for Benedict’s test?
6. Account for the black precipitate in Nylander’s test.

Group No.___ Date Performed :___________

Subject/Section:__________ Instructor’s Initials :___________
Members:
1. _____________________
2. _____________________
3. _____________________

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University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 7
Laboratory Manual

Data Sheet
Carbohydrates Part I: Common Reactions Activity
3

Data

A. Molisch Test

Test Result Explanation

Solutions
1% glucose

1% xylose

1% lactose

1% sucrose

1% starch

1% furfural

Unknown
sugar

B. Action of Alkali (Search for Theoretical Results Only)

Alkali Test Result Explanation

Solutions
10% NaOH 1% glucose

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Nursing Biochemistry Laboratory (NurBio Lab) 8
Laboratory Manual

1% fructose

1% galactose

1% 1% glucose
Ba(OH)2

1% fructose

1% galactose

C. Reduction Tests

Name of Test Result Explanation

Test Solutions
Fehling’s 1% glucose
Test

1% xylose

1% fructose

1% lactose

1% maltose

1% sucrose

1% starch

1% glycogen

Unknown

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University, Zamboanga City, Philippines.
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Laboratory Manual

Benedict’s 1% glucose
Test

1% xylose

1% fructose

1% lactose

1% sucrose

1% starch

Barfoed’s 1% glucose
Test

1% fructose

1% xylose

1% lactose

1% maltose

1% sucrose

Unknown
sugar
Name of Test Result Explanation
Test Solutions
Nylander’s 1% glucose
Test

(Search for 1% fructose

Theoretical

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Results
Only) 1% xylose

1% lactose

1% maltose

Picric Acid 1% glucose

Test

1% fructose

1% xylose

1% lactose

1% maltose

Tollen’s 1% glucose
Test

Classification of Unknown: ________________________________________

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.