Chemical Reactions

Carbohydrate Qualitative Analysis: Foundation

Lab
 2012, Sharmaine S. Cady
East Stroudsburg University

Skills to build:

 Doing microscale reactions

 Observing evidence of a chemical reaction
 Using chemical reactions to identify a carbohydrate
 Identifying reducing sugars
 Identifying aldoses and ketoses
 Identifying pentoses

Introduction

Along with proteins and fats, carbohydrates, or sugars, are one of the three main
classes of food used to sustain life. They are essential components of all living
organisms and constitute the most abundant class of biological molecules. The
classification carbohydrate stems from the general molecular formula for
monosaccharides, (C·H2O)n where n  3, which implies that these compounds are
hydrates of carbon. However, carbohydrates are not true hydrates in the chemical sense.
Carbohydrates, chemically, are polyhydroxy aldehydes (CHO) or ketones (C=O) or
compounds which upon hydrolysis yield these compounds. Note that each carbon in a
polyhydroxy aldehyde or ketone structure, except for the carbonyl functional group (in
yellow), bears a hydroxyl (OH) functional group (in green). Polyhydroxy aldehydes and
ketones with the same number of carbons are structural isomers of each other.
H

C O CH2OH
H C OH C O
H C OH H C OH
H C OH H C OH
CH2OH CH2OH
polyhydroxy aldehyde polyhydroxy ketone
 Carbohydrate Qualitative Analysis

Carbohydrates may be classified based upon the number of polyhydroxy

aldehyde or ketone (saccharide) units found in their structure. The basic carbohydrate
unit is a single polyhydroxy aldehyde or ketone, or monosaccharide. A disaccharide can
be viewed as two monosaccharides from which water is removed to join the units. The
bond that forms between the two sugar units is known as a glycosidic bond or linkage.
Oligosaccharides consist of 3-10 sugar units covalently joined together, and
polysaccharides are large biomolecular polymers of covalently bonded monosaccharide
units.

Monosaccharides

Monosaccharides are further classified based upon the carbonyl functional group
present and the total number of carbon atoms in the structure. The polyhydroxy
aldehyde on the previous page contains five carbons and is classified as an aldopentose,
where the prefixes aldo- and pent- indicate the aldehyde functional group and five carbon
atoms, respectively. The polyhydroxy ketone structure is a ketopentose, where keto-
indicates a ketone functional group.
Monosaccharides may be represented by either a Fischer or Haworth projection.
Fischer projections show the open chain form of the sugar, and Haworth projections
show the cyclic form of the sugar. In a Fischer projection, a chiral carbon may be
indicated by a pair of intersecting lines . In the Haworth projection, the chain of
carbons forms a ring structure. The Fischer and Haworth projections for glucose are
given below. The designation D indicates the stereoisomer where the hydroxyl group
(green) at the highest numbered chiral carbon appears on the right in the Fischer
projection. The  symbol indicates the stereoisomer in which the hydroxyl group (blue)
lies below the plane of the ring for the lowest numbered carbon in the ring (the anomeric
carbon). When this hydroxyl group is shown above the plane, the designation is . Note
that this hydroxyl group is formed from the carbonyl group during the ring closing
reaction. Only the cyclic form is found in the solid state, while the linear form exists only
at a low concentration in equilibrium with the  and  cyclic forms in solution.

H
1
C O
2
H C OH 6
3
CH2OH
HO C H
H 5 O H
4
H C OH H H 1
4 OH
5
H C OH
OH 3 2 OH
6
CH2OH H OH

D-glucose -D-glucose

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 Carbohydrate Qualitative Analysis

The  cyclic structures of galactose, fructose, and ribose, are given below.

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

H OH H OH H OH
H OH OH H OH OH

D-galactose D-fructose D-ribose

Disaccharides

Disaccharides are carbohydrates which upon hydrolysis (reaction with water) yield
two monosaccharide structures. The most abundant disaccharide is sucrose, which is
hydrolyzed to D-glucose and D-fructose. The two sugar units are covalently joined
through an oxygen atom at carbons 1 and 2 on glucose and fructose, respectively. The
covalent link between the two sugar units is referred to as an (12)

CH2OH CH2OH

H H O H
O H
H H H 1
H OH
OH
OH OH OH
-H2O H OH
H OH glycosidic bond
O
CH2OH CH2OH
OH
O O
H HO HO 2
H
H CH2OH H CH2OH
OH H OH H

sucrose

glycosidic linkage. The structures of the disaccharides maltose and lactose are given
below. Maltose contains two D-glucose units joined by an (1 4) glycosidic bond,
while lactose consists of D-galactose and D-glucose joined by a (14) linkage. The
glycosidic linkages are destroyed when disaccharides undergo hydrolysis back to the
individual monosaccharides.

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 Carbohydrate Qualitative Analysis

CH2OH
H O H
CH2OH CH2OH CH2OH H
4 H
OH
H O H H O H OH O O OH
H H H H
H 1 4 OH H 1
OH OH H OH
O OH
OH H H
H OH H OH H OH

maltose lactose

Polysaccharides

Polysaccharides consist of large numbers of monosaccharides joined by

glycosidic linkages. When the monosaccharide units are all identical, the molecule is
referred to as a homopolysaccharide. If the monosaccharide units differ, the molecule is
classified a heteropolysaccharide. Starch is a homopolysaccharide synthesized by plants
for the storage of -D-glucose units and serves as a source of carbohydrates in animal
diets. Plant starch occurs as a mixture of two different polymeric structural units. -
amylose is a linear polymer of several thousand glucose units joined by (14)
glycosidic bonds. Amylopectin is a branched polymer with glucose connected in linear
chains by (14) bonds and by (16) bonds at the branch points which occur on the
average every 24 to 30 glucose units. Containing up to a million glucose residues,
amylopectin is the larger of the two polymeric structures. Glycogen, the storage
polysaccharide for glucose in animals and humans is structurally similar to amylopectin
except that branching occurs every 8 to 12 glucose residues.

CH2OH CH2OH CH2OH CH2OH

H O H H O H H O H H O H
H 1 4H H H
O O H O H O H O H O
O O O

H O H O H O H O
amylose

CH2OH CH2OH
H O H H O H
H 1 4 H 1
O O H O H
O
O
H O H O
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CH2OH CH2OH CH2 CH2OH
H O H H O H H O H H O H
H 1 4H H H
O O H O H O H O H O
O O O

H O H O H O H O
amylopectin

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 Carbohydrate Qualitative Analysis

Carbohydrate Chemical Reactions

The chemical reactions of carbohydrates is largely that of the hydroxy and

carbonyl groups. The aldehyde and -hydroxy ketone (a ketone that has an OH on a
carbon next to the carbonyl group) structural units in sugars undergo mild oxidation to
carboxylic acids. In solution, the ring structure of sugars are able to open at the anomeric
carbons to form the open-chain aldehyde or -hydroxy ketone form, which undergoes
oxidation in the presence of a mild oxidizing agent. Such sugars are referred to as
reducing sugars. However, when the anomeric ring carbon is involved in a glycosidic
linkage, the ring is locked in place and cannot open for oxidation to occur. All
monosaccharides are reducing sugars since no glycosidic linkages exist in their
structures. The disaccharides maltose and lactose are reducing sugars since only one of
the C1 anomeric carbons is involved in a glycosidic linkage, while sucrose is a
nonreducing sugar since both the C1 and C2 anomeric carbons participate in the
glycosidic linkage. Starch and glycogen are also nonreducing sugars because of their
size.

Molisch Test

The Molisch test is used to determine the presence of carbohydrates, regardless

of structure. The Molisch reagent provides the condensation reagent, -naphthol in
alcohol. Concentrated sulfuric acid acts as the dehydrating reagent. It also serves as a
catalyst for the hydrolysis of di- and polysaccharides. The presence of a purple ring at

-naphthol
OH O

O
CH2OH
H conc. H C
O H2SO4 O -naphthol O [O] O
H H
-2 H2O H C C
H OH
OH OH
ribose furfural
colorless
dehydration product
OH OH
purple

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 Carbohydrate Qualitative Analysis

the interface between the carbohydrate solution and concentrated sulfuric acid is a
positive indication of a carbohydrate structure. Monosaccharides react more quickly than
di- and polysaccharides, which must slowly undergo hydrolysis before they react with the
Molisch reagent.

Benedict's Test

Benedict's reagent reacts with reducing sugars to form inorganic precipitates

which are readily detected by visual observation. In the reaction between a reducing
sugar and Benedict's reagent, copper(II) ion is reduced to copper(I) by the aldehyde
functional group:

H OH
C O C O
H C OH Cu2+ / OH- H C OH
(blue)
HO C H HO C H + Cu2O (red precipitate)

H C OH H C OH
H C OH H C OH
CH2OH CH2OH
glucose gluconic acid

The formation of a red to orange precipitate indicates the presence of a reducing sugar.

Tollens's Test

The Tollens's test also involves a mild oxidizing agent. In the Tollens's test,
silver(I) ion is reduced to metallic silver. The formation of a silver mirror on the inside of
the test tube indicates the presence of a reducing sugar. The Tollens's reagent must be
freshly prepared to produce the proper results.

H OH
C O C O
H C OH H C OH
Ag(NH3)2+
HO C H HO C H + Ag (mirror)

H C OH H C OH
H C OH H C OH
CH2OH CH2OH
glucose gluconic acid

Barfoed's Test

Barfoed's reagent also uses the reduction of copper(II) ion to red Cu2O as an
indication of a reducing sugar. However, it not as reactive as Benedict's reagent, and the

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 Carbohydrate Qualitative Analysis

rate at which the red precipitate forms can be used to distinguish between mono-
saccharides and disaccharides. The appearance of red Cu2O within 2-3 minutes is a
positive test for a monosaccharide; disaccharides produce the precipitate in
approximately 10 minutes.

Bial's Test

The ability of the hydroxy groups to undergo dehydration reactions allows for
identification of certain aldoses and ketoses. Concentrated acid is used to dehydrate the
sugars, which produce colorless dehydration products. These dehydration products then
react with a condensation reagent to form colored condensation products, which offer a
visual means of positive identification. Figure 1 shows two condensation reagents
commonly used to test for sugars.

OH OH

H3C OH OH

orcinol resorcinol

Figure 1. Condensation Reagents for Bial's and Seliwanoff's Tests

Bial's test is used to distinguish a pentose from a hexose structure. The

dehydrating agent is concentrated HCl, and the condensation reagent is orcinol in the
presence of iron(III) chloride. Pentoses undergo dehydration to furfural, a cyclic
aldehyde, which further reacts with orcinol to give a blue-colored condensation product.

O
CH2OH
H conc. H C
O O orcinol
HCl
H H
blue condensation product
-2 H2O
H OH
OH OH
ribose furfural

All other colored products are negative indicators for the presence of a pentose.

Seliwanoff Test

The Seliwanoff test differentiates between ketohexoses and aldohexoses. It

also uses concentrated HCl for the dehydrating agent, but the condensation agent is
resorcinol. Ketohexoses are dehydrated to 5-hydroxymethylfurfural, which undergoes
condensation with resorcinol within two minutes to form a red-colored product.

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 Carbohydrate Qualitative Analysis

H
CH2OH CH2OH
CH2OH conc. C O
O O
HCl resorcinol
H HO red condensation product
H OH -2 H2O

OH H
fructose 5-hydroxymethylfurfural

The appearance of a peach color is not a positive test for a ketohexose.

In this experiment, the above tests will be performed on glucose, ribose, fructose,
galactose, sucrose, xylose, maltose, and lactose. The rates of reaction with the different
reagents and the visual characteristics (color, intensity, amount of precipitate) of the
products formed will be noted for comparison to an unknown carbohydrate sample. The
flow chart may be used to identify the unknown carbohydrate as to class and structure.

mono- or disaccharide

Barfoed's test

red ppt ~ 10 min red ppt. 2-3 min

disaccharide monosaccharide
Benedict's test Bial's test

no ppt. red ppt. other colors blue

nonreducing reducing hexose pentose

disaccharide disaccharide Seliwanoff test
( ribose
( sucrose ) ( lactose xylose )
maltose ) red other

ketohexose aldohexose
( fructose ) ( glucose, galactose )

Figure 2. Flow chart for unknown carbohydrate

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 Carbohydrate Qualitative Analysis

Experimental Methods and Materials

Safety considerations

Wear suitable protective clothing, gloves, and eye/face protection!

BENEDICT'S REAGENT
eye, skin, and respiratory irritant
harmful if swallowed; may cause gastrointestinal discomfort

BARFOED'S REAGENT
harmful if swallowed; may cause gastrointestinal discomfort
corrosive to skin and eyes

BIAL'S REAGENT
corrosive to skin and eyes; harmful if ingested

SELIWANOFF REAGENT
corrosive to skin and eyes; harmful if ingested

AMMONIUM HYDROXIDE
contact may cause tissue damage to skin, eyes, mucous membrane,
gastrointestinal & respiratory mucosa

SILVER NITRATE
damage to the eyes and can burn skin
inhalation can irritate respiratory passages and mucous membranes
ingestion can cause severe abdominal pain and gastroenteritis, that may be fatal

SUGARS
may cause eye irritation

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 Carbohydrate Qualitative Analysis

Molisch Test

Clean and thoroughly rinse with distilled water five test tubes. Label one for
each of the five known carbohydrates. Place 10 drops of each sugar solution in its
labeled test tube. Add 2 drops of the Molisch reagent to each test tube. Mix
thoroughly with a glass stir rod. Be sure to clean the stir rod after each test tube. Hold
the test tube at a 45 angle, and slowly and carefully add 20 drops of concentrated
H2SO4 down the side of the test tube. The more dense acid will form a layer on the
bottom of the test tube. Place in a rack and note the time for the appearance of a red
to purple ring at the interface of the two layers. Aldopentoses, ketopentoses, and
ketohexoses react more rapidly than aldohexoses and disaccharides. Record your
results on your lab report.

Benedict’s Test

Clean and thoroughly rinse with distilled water five test tubes. Label one for
each of the five known carbohydrates. Place 5 drops of each solution in its labeled
test tube. Add 20 drops of Benedict's reagent to each solution. Mix thoroughly.
Place all test tubes in boiling water at the same time. Note the time for the
appearance of a red to reddish orange precipitate. After 1 minute remove the test
tubes and place in a rack. Record your results.

Barfoed's Test

Prepare a boiling water bath(add boiling chips to prevent bumping). Clean and
thoroughly rinse with distilled water five test tubes. Label one for each of the five
known carbohydrates. Place 5 drops of each solution in its labeled test tube. Add
20 drops of the Barfoed's reagent to each test tube. Mix thoroughly. Place all test
tubes in the boiling water at the same time. Note the time for the appearance of a red
precipitate and remove the test tube. After 10 minutes remove all the test tubes and
place in a rack. Record your results.

Bial's Test

Clean and thoroughly rinse with distilled water five test tubes. Label one for
each of the five known carbohydrates. Place 5 drops of each solution in its labeled
test tube. Add 20 drops of Bial's reagent to each test tube. Mix thoroughly. Place
all test tubes in boiling water at the same time. Remove after 1 minute. A dark blue
color indicates a pentose. Record your results.

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 Carbohydrate Qualitative Analysis

Seliwanoff Test

Clean and thoroughly rinse with distilled water five test tubes. Label one for
each of the five known carbohydrates. Place 5 drops of each solution in its labeled
test tube. Add 20 drops of Seliwanoff reagent to each solution. Mix thoroughly.
Place all test tubes in boiling water at the same time. Note the time for the
appearance of a cherry red color. Poly- and disaccharides hydrolyze slowly in the
presence of acid and should take more time to give a positive test. After two minutes
remove from the water and place in a rack. Record your results.

Tollens's Test

Clean and thoroughly rinse with distilled water three test tubes. Label one for
glucose, one for sucrose, and one for lactose. Place 20 drops of 5% silver nitrate
into a 100-mL beaker. Add 1 drop of 3 M NaOH to the beaker. Continue to add 2%
ammonia with stirring to the beaker until the brownish precipitate of silver oxide just
disappears. Divide the solution equally among the three labeled test tubes. Add 5
drops of each sugar solution to its labeled test tube. Do not mix. Place the test
tubes in the boiling water bath for 2 minutes. Record your results.

Identifying an Unknown Carbohydrate

Obtain an unknown from your instructor and record its number on your lab
report. Using the flow chart in Figure 2, determine the identity of your unknown. Do
only those tests which are necessary to clearly establish the identity of the unknown.
The possible unknowns are glucose, xylose, fructose, lactose, or sucrose.

References

Hendrickson, C. H.; Byrd, L. C.; Hunter, N. W. A Laboratory Manual for General,

Organic, and Biochemistry, 3rd ed.; McGraw-Hill: Boston, 2001, pp 333-342.
Pasto, D. J.; Johnson, C.R.; Miller, M. J. Experiments and Techniques in Organic
Chemistry; Prentice Hall: Upper Saddle River, NJ, 1992, p 336.
Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G. Introduction to Organic
Laboratory Techniques: A Small Scale Approach, 2nd ed.; Brooks/Cole: Belmont, CA,
2005, pp 444-450.

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 Carbohydrate Qualitative Analysis

Laboratory Report

How accurate are the tests in identifying and classifying carbohydrates? Is your
unknown a mono- or disaccharide? Is it a reducing or non-reducing sugar?

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