CHY 47.

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Biochemistry Laboratory

Exercise 4
Test for Carbohydrates

Objectives:
1. To characterize carbohydrates on the basis of various chemical reactions
2. To hydrolyze sucrose with acid

Theoretical Background:

Carbohydrates are most abundant organic compounds found in living organisms and are composed of carbon,
hydrogen and oxygen. Carbohydrates act as the primary source to provide energy for functioning of living
organisms. These are called carbohydrates because they can be considered as hydrates of carbon. Most of them
have the general formula Cx(H2O)y. Carbohydrates are polyhydroxy aldehydes and ketones or substances that
hydrolyze to yield polyhydroxy aldehydes and ketones (Figure 1).

Figure 1. Structure of aldose and ketose

Carbohydrates or saccharides are divided into four chemical groups: monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Monosaccharides are those carbohydrates that cannot be hydrolyzed into
simpler carbohydrates. They may be classified as trioses, tetroses, pentoses, hexoses, or heptoses, depending
upon the number of carbon atoms; and as aldoses or ketoses depending upon whether they have an aldehyde
or ketone group (Table 1). The commonly occurring monosaccharides includes glucose, fructose, galactose, and
ribose.

Monosaccharides exist mostly as cyclic structures containing hemiacetal or hemiketal groups (Figure 2). 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. As shown in Figure 2, the end
that has a hemiacetal for aldoses (glucose) or hemiketal for ketoses (fructose) on its terminal is called the
reducing end, and the one that 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 Fehling’s reagent and Benedict’s reagent and has an unlocked ring.

Table 1. Monosaccharides

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 Figure 2. The structure of D-glucose

The two monosaccharides combined together to form disaccharides which include sucrose, lactose and maltose
(Table 2). Oligosaccharides are condensation products of three to ten monosaccharides. Starch and cellulose fall
into the category of polysaccharides which are condensation products of more than ten monosaccharide units;
which may be linear or branched polymers (Table 3). Disaccharides such as maltose (Figure 3) and
polysaccharides exist as cyclic structures containing functional groups such as hydroxyl groups, acetal or ketal
groups, and hemiacetal or hemiketal, thus, are considered reducing sugars. However, not all disaccharides or
polysaccharides contain a reducing end. An example is sucrose (Figure 4), which does not have a hemiacetal or
hemiketal group on either of its ends.

Figure 3. The structure of maltose.

Figure 4. The structure of sucrose

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 Table 2. Disaccharides

Table 3. Polysaccharides

Qualitative Tests for Carbohydrates

Molisch Test

Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing carbohydrates
in a combined form based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde
(furfural or hydroxymethylfurfural). These products condense with -naphthol to form purple condensation
product (Figure 5). Molisch reagent is prepared by dissolving -naphthol in ethanol.

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 Figure 5. Reaction of glucose with Molisch reagent

Iodine test

This test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an aqueous solution of
potassium iodide) reacts with starch producing an intense, brilliant, dark-blue or violet-colored color. The color
obtained depends upon the length of the unbranched or linear chain available for complex formation. Iodine
forms a coordinate complex between the helically coiled polysaccharide chain and iodine centrally located
within the helix due to adsorption (Figure 6). The iodine reagent is a solution of potassium iodide with iodine in
water.

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 Figure 6. Starch-iodine complex

Test for Reducing Sugars

A reducing sugar is any sugar that, in a solution, has free aldehyde or a ketone group. The enolization of sugars
under alkaline conditions is an important consideration in reduction tests. The ability of a sugar to reduce
alkaline test reagents depends on the availability of an aldehyde or keto group for reduction reactions. A
number of sugars especially disaccharides or polysaccharides have glycosidic linkages which involve bonding a
carbohydrate (sugar) molecule to another one, and hence there is no reducing group on the sugar such as in the
case of sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the presence of base causes
extensive enolization especially at high pH and temperature (Figure 7). This leads to a higher susceptibility to
oxidation reactions than at neutral or acidic pH. These sugars, therefore, become potential agents capable of
reducing Cu+2 to Cu+1, Ag+1 to Ag. Most commonly used tests for detection of reducing sugars are Fehling’s Test,
Benedict’s Test and Barfoed’s Test.

Figure 7. Keto- and enol tautomerism in sugars and oxidation of aldoses

a. Benedict’s Test

Carbohydrates with free aldehyde or ketone groups have the ability to reduce solutions of Cu+2 ions
to to Cu+1 form and are oxidize to sugar acids. The Cu+1 ions combine with OH- ions to form yellow
cuprous hydroxide which upon heating is converted to red cuprous oxide (Figure 8). Benedict‘s test is a
semi quantitative test. The color of the precipitate gives a rough estimate of a reducing sugar present in
the sample (Table 4). Benedict's reagent is a chemical reagent and complex mixture of sodium
carbonate, sodium citrate and copper(II) sulfate pentahydrate.

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 O H O OH
C C
H C OH H C OH
HO C H 2+ HO C H
+ Cu + Cu2O(s)
H C OH H C OH
H C OH H C OH
CH 2OH CH 2OH

D-Glucose D-Gluconic acid

Figure 8. Oxidation of aldose by Benedict’s reagent

Table 4. Rough estimate of a reducing sugar present in the sample by Benedict’s reaction

Color Estimated %
Green color Up to 0.5 %

Green precipitate 0.5-1.0 %

Yellow precipitate 1.0-1.5 %

Orange precipitate 1.5-2.0 %

Brick red precipitate Greater than 2.0 %

b. Fehling’s Test

Fehling’s solution is used to determine the presence of reducing sugars and aldehydes. Fehling’s
test uses two separate solutions which are labelled as Fehling’s A and Fehling’s B. Fehling’s A is a
solution containing copper(II) sulfate, which is blue. Fehling’s B is a clear liquid consisting of potassium
sodium tartrate (Rochelle salt) and a strong alkali, usually sodium hydroxide.

D-Glucose D-Gluconic acid

Figure 9. Oxidation of aldose by Fehling’s reagent

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 c. Barfoed’s Test

This test is used to distinguish reducing monosaccharides from disaccharides by controlling pH

and time of heating. Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced
so that is can only be reduced by monosaccharides but not less powerful reducing sugars (Figure
10). Disaccharides may also react with this reagent, but the reaction is much slower when compared
to monosaccharides.

An aldose carboxylic acid cuprous oxide

Figure 10. Oxidation of aldose by Barfoed’s reagent

Seliwanoff’s Test

This test distinguishes aldose from ketose sugars. Ketoses are distinguished from aldoses via their ketone or
aldehyde functionality. If the sugar contains a ketone group, it is a ketose and if it contains an aldehyde
group, it is an aldose. This test is based on the fact that, when heated, ketoses are more rapidly dehydrated
than aldoses. Ketohexoses on treatment with hydrochloric acid form 5-hydroxy methyl furfural which on
condensation with resorcinol gives a cherry red colored complex (Figure 10). Seliwanoff’s reagent consists
of resorcinol and concentrated hydrochloric acid.

Figure 10. Reaction of fructose with Seliwanoff’s reagent

Bial’s Test

This test differentiates pentoses from hexoses. The components of Bial’s reagent are orcinol, HCl, and ferric
chloride. In this test, the pentose is dehydrated to form furfural and the solution turns blue green and a
precipitate may form (Figure 11). Hexoses generally react to form brown products.

Figure 11. Reaction of fructose with Bial’s reagent

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Hydrolysis Test for Sucrose

Sucrose is a non-reducing disaccharide so it does not reduce the Cu++ solution (Bendict's and Fehling's test)
because the glycosidic bond is formed between the two hemiacetal bonds. So there is no free aldehydic or
ketonic group to give positive reducing properties. Sucrose on hydrolysis with HCl solution is converted to
glucose and fructose, invert sugars which are reducing sugars (Figure 12). The presence of these two
monosaccharides can be confirmed by Benedict’s and Seliwanoff Tests.

Figure 12. Acid hydrolysis of sucrose

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Procedure
(Virtual Activity)

A. Test Samples

a. Starch and water mixture

b. 2% aqueous solution of the following:

 Glucose  Arabinose
 Fructose  Maltose
 Xylose  Sucrose
 Galactose  Lactose

Note: Refer to the datasheet for the sample/s to be used for a particular test.

B. Tests

a. Molisch Test (General test for carbohydrates)

1. Add 2 drops of the Molisch reagent to 2 mL of the sample solution in a test tube.
2. Carefully pour 1-2 mL of conc. H2SO4 down the side of the test tube.
3. Observe. A positive (+) result is indicated by the formation of a violet ring at the interphase
of the two layers.

b. Iodine Test (Test for complex carbohydrates/polysaccharide)

1. Add 3 drops of iodine solution to 1 mL of each sample solution.
2. Observe. A positive (+) result is indicated by dark blue coloration.

c. Benedict’s Test (Test for reducing sugars)

1. Add 2 mL of the Benedict’s solution to 1 mL of the sample.
2. Heat in a boiling water bath for 2-5 minutes.
3. Observe. A positive (+) result is indicated by the formation of brick-red precipitates.

d. Barfoed’s Test (Test to differentiate reducing monosaccharides from reducing disaccharides)

1. Add 2 mL of Barfoed’s reagent to 1 mL of the sample solution.
2. Heat in a boiling water bath for 3-4 minutes.
3. Observe. Formation of brick red precipitate indicates presence of monosaccharide reducing
sugars.
4. If no precipitates is observed after heating for 3-4 minutes, extend heating for 10 more
minutes and observe again. Formation of brick red precipitate indicates presence of disaccharide
reducing sugars.

e. Seliwanoff’s Test (Test to detect ketoses)

1. Add 2 mL of Seliwanoff’s reagent to 1 mL of the sample solution.
2. Heat in a boiling water bath for 30 seconds.
3. Observe. A deep cherry red color indicates presence of ketoses.

f. Bial’s Orcinol Test (Test to distinguish pentoses from hhexoses)

1. Add 2 mL of Bial’s reagent to 1 mL of the sample solution.
2. Heat in a boiling water bath until a color change is observed. Formation of a blue green
precipitate indicates presence of pentoses. A brown product indicates presence of hexoses.

g. Hydrolysis of Sucrose
1. Add 5 mL of sucrose solution into two separate test tubes.
2. Add 5 drops of conc. HCl to one test tube.
3. Heat both tubes in boiling water bath for 10 minutes.
4. Cool and neutralize with diluted NaOH (use litmus paper).
5. Test both solutions for the presence of reducing sugar with Benedict’s Test.

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 CHY 47.1
Biochemistry Laboratory

Exercise 4
Test for Carbohydrates
Activity Sheet

Name:__________________________________________

Table 1. Molisch’s Test

Sample Observation* Inference**
Glucose
Lactose
Sucrose
Starch
* violet ring or no observable result
** positive or negative for carbohydrates

Table 2. Iodine Test

Sample Observation* Inference**
Glucose
Lactose
Sucrose
Starch
* blue color or no observable result
** positive or negative for complex carbohydrates/polysaccharides

Table 3. Benedict’s Test

Sample Observation* Inference**
Glucose
Fructose
Sucrose
Maltose
* brick red precipitates or no observable result
** positive or negative for reducing sugar

Table 4. Barfoed’s Test

Observation*
Sample Inference**
After 3 minutes After 10 minutes
Glucose
Fructose
Maltose
Lactose
* brick red precipitates or no observable result
** brick precipitate formation within 3 minutes - positive for reducing monosaccharides
** delayed formation of brick precipitate formation (after 10 minutes) - positive for reducing disaccharides

Table 5. Seliwanoff’s Test

Sample Observation* Inference**
Glucose
Fructose
* cherry red coloration or no observable result
** positive or negative for ketoses

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Table 6. Bial’s Orcinol Test
Sample Observation* Inference**
Glucose
Fructose
Xylose
Arabinose
* blue green coloration or brown coloration
** blue green coloration - positive for pentoses
** brown coloration - positive for hexoses

Table 7. Hydrolysis of Sucrose

Sample Observation with Benedict’s reagent* Inference**
Without HCl
With HCl
* brick red precipitates or no observable result
** hydrolysis occurred (presence of reducing sugars) or no hydrolysis happened

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