Embudo, Helen Gail Chemical Biology

III
BSBIO2-A
QUALITATIVE TESTS FOR CARBOHYDRATES
Activity 2

Instruction:

From the video presentation on the qualitative test for carbohydrates, provide a schematic diagram for the
Molish, Benedict’s, Fehling’s, BArfoed’s, Seliwanoff’s and Iodine tests methodologies in determining
presence of carbohydrates. Also, give the basic principle for each test.

MOLISH TEST

Molisch test is a general test used to detect the presence of carbohydrates by using concentrated sulfuric
acid as the dehydrating acid. This acid dehydrates carbohydrates and some compounds with
carbohydrates in combined form, distinguishing carbohydrates from non-carbohydrates. The dehydration
products of carbohydrates, furfural or 5-hydroxymethylfurfural result from the reaction of sulfuric acid
with pentoses or hexoses. These products condense with α-napthol to yield a purple condensation product.

Laboratory Precautions:

Caution: Molisch reagent contains concentrated sulfuric acid, which is toxic and corrosive. It can
cause severe burns. Prevent eye, skin, clothing, and combustible material contact. Avoid ingesting
the substance. If you spill any reagent or acid, immediately notify your laboratory instructor
Caution: You will be adding aqueous solutions to concentrated sulfuric acid. You must be
extremely careful while performing this step.
Caution: In the test, you will place the test tubes containing the reaction solutions in a boiling-
water bath. Be careful that you do not come in contact with the steam or the hot apparatus, and that
you do not knock over the bath.
Note: Do not place your thumb over the open end of a test tube when mixing its contents

Procedures:

Make sure to first label the test

tubes properly using a marking
pen

Do not place thumb over the

open end of the test tube when
mixing its contents
BENEDICT’S TEST

Benedict’s test is a general test for aldehydes and alpha hydroxyl ketones. It uses a mixture of copper (II)
sulfate, sodium citrate, sodium carbonate, I a mildly basic solution. It can be used to detect the presence
of reducing sugars in any given sample.

A reducing sugar is any sugar that, in a solution, has an 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; like in the case of sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the
presence of alkali causes extensive enolization especially at high pH and temperature. 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+, Ag+ to Ag and so forth.

A positive test with Benedict's reagent is shown by a color change from clear blue to brick-red with a
precipitate

Laboratory Precautions:
Caution: Benedict’s reagent is toxic. If you spill any of the solution on yourself or on the bench,
immediately notify your laboratory instructor.
Caution: In the test, you will place the test tubes containing the reaction solutions in a boiling-
water bath. Be careful that you do not come in contact with the steam or the hot apparatus, and that
you do not knock over the bath.
Note: Do not place your thumb over the open end of a test tube when mixing its contents

Procedures:

If the solution remained clear

blue after immersing it into the
water bath for 30 minutes, it
indicates that the reagent is not
contaminated so proceed to the
next procedures or to the
carbohydrate test.

In this procedure, make sure to

use separate droppers for
transferring each of the
carbohydrate sample into the test
tube with Benedict’s solution to
prevent variation of results
FEHLING’S TEST

Fehling's solution is a chemical reagent used to differentiate between water-soluble carbohydrate and

ketone functional groups, and as a test for reducing sugars and non-reducing sugars. In contrast to
Benedict’s solution which contains copper (II) citrate, Fehling's solution particularly Fehling B solution is
a colorless solution of aqueous potassium sodium tartrate (also known as Rochelle salt) made strongly
alkali with sodium hydroxide

Fehling's solution is prepared by combining two separate solutions: Fehling's A, which is a deep blue
aqueous solution of copper (II) sulfate, and Fehling's B, which is a colorless solution of aqueous
potassium sodium tartrate

The appearance of a reddish-brown precipitate indicates a positive result and the presence of reducing
sugars. The absence of the reddish precipitate or the appearance of deep blue color indicates a
negative result and lack of reducing sugars.

Laboratory Precautions:

Caution: Causes eye and skin burns. Harmful if swallowed. May cause severe respiratory tract
irritation with possible burns. May cause severe digestive tract irritation with possible burns.
Caution: In the test, you will place the test tubes containing the reaction solutions in a boiling-
water bath. Be careful that you do not come in contact with the steam or the hot apparatus, and that
you do not knock over the bath.
Note: Do not place your thumb over the open end of a test tube when mixing its contents

Procedures:

Make sure to first label the test

tubes properly using a marking
pen.

If the solution remained blue

after immersing it into the water
bath for 30 minutes, it indicates
that the reagent is not
contaminated so proceed to the
next procedures or to the
carbohydrate test.

In this procedure, make sure to

use separate droppers for
transferring each of the
carbohydrate sample into the test
tube with Fehling’s solution to
prevent variation of results
BARFOED’S TEST

Barfoed's reagent is used to detect the presence of reducing monosaccharides in the presence of
disaccharides. The reagent, here, uses copper ions to detect reducing sugars in an acidic solution. This test
differs from other tests that detect reducing sugars both mono- or disaccharides.

Barfoed’s test uses copper (II) ions in a slightly acidic medium. Reducing monosaccharides are oxidized
by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide within
three minutes. Reducing disaccharides undergo the same reaction, but do so at a slower rate.

Laboratory Preautions:

Caution: Barfoed’s reagent is corrosive and an irritant. If you spill any of the solution on yourself
or on the bench, immediately notify your laboratory instructor.
Caution: In the test, you will place the test tubes containing the reaction solutions in a boiling-
water bath. Be careful that you do not come in contact with the steam or the hot apparatus, and that
you do not knock over the bath.
Note: Do not place your thumb over the open end of a test tube when mixing its contents

Procedures:

Make sure that test tubes were

labelled properly before
transferring solutions and reagents
of any kind to the test tubes.

Make sure that the test tubes are

standing upright or in proper
position to avoid pouring out of
contents

Note: A red precipitate before 5

min have elapsed indicates a
positive result for a reducing
monosaccharide. If a red
precipitate appears at about 10-15
mins, the sample is a reducing
disaccharide.
SELIWANOFF’S TEST

Seliwanoff’s test is a chemical test which distinguishes between aldose and ketose sugars. If the sugar

contains a ketone group, it is a ketose. If a sugar contains an aldehyde group, it is an aldose. This test
relies on the principle that, when heated, ketoses are more rapidly dehydrated than aldoses. It is named
after Theodor Seliwanoff, the chemist that devised the test. When added to a solution containing ketoses,
a red color is formed rapidly indicating a positive test. When added to a solution containing aldoses, a
slower forming light pink is observed instead.
The reagents consist of resorcinol and concentrated hydrochloric acid:

 The acid hydrolysis of polysaccharide and oligosaccharide ketoses yields simpler sugars followed
by furfural.[1]
 The dehydrated ketose then reacts with two equivalents of resorcinol in a series of condensation
reactions to produce a molecule with a deep cherry red color.
 Aldoses may react slightly to produce a faint pink color.
Fructose and sucrose are two common sugars which give a positive test. Sucrose gives a positive test as it
is a disaccharide consisting of fructose and glucose.
Generally, 6M HCl is used to run this test. Ketose get dehydrated faster and hence they give the test
faster. Aldoses react very slowly and give faint colors.
Laboratory Precautions:

Caution: Seliwanoff’’s reagent is corrosive and an irritant. If you spill any of the solution on
yourself or on the bench, immediately notify your laboratory instructor.

Caution: In the test, you will place the test tubes containing the reaction solutions in a boiling-
water bath. Be careful that you do not come in contact with the steam or the hot apparatus, and that
you do not knock over the bath.

Procedures:

Make sure that the test tubes are

standing upright or in proper
position to avoid pouring out of
contents
IODINE TEST

Iodine test is a chemical test used to distinguish mono- or disaccharides from certain polysaccharides like
amylase, dextrin, and glycogen. This test has a variation termed starch-iodine test that is performed to
indicate the presence of glucose made by plants in the leaves.
Principle of Iodine Test
 Iodine test is based on the fact that polyiodide ions form colored adsorption complex with helical
chains of glucose residue of amylase (blue-black), dextrin (black), or glycogen (reddish-brown).
 Monosaccharides, disaccharides, and branched polysaccharides like cellulose remain colorless.
Amylopectin produces an orange-yellow hue.
 The reagent used in the iodine test is Lugol’s iodine, which is an aqueous solution of elemental
iodine and potassium iodide.
 Iodine on its own is insoluble in water. Addition of potassium iodine results in a reversible
reaction of the iodine ion with iodine to form a triiodide ion, which further reacts with an iodine
molecule to form a pentaiodide ion.
 Bench iodine solution appears brown, whereas, the iodide, triiodide, and pentaiodide ion are
colorless.
 It is observed that the helix (coil or spring) structure of the glucose chain is the key to this test.
 Further, the resulting color depends on the length of the glucose chains.
 The triiodide and pentaiodide ions formed are linear and slip inside the helix structure.
 It is believed that the transfer of charge between the helix and the polyiodide ions results in
changes in the spacing of the energy levels, which can absorb visible light, giving the complex its
color.
 The intensity of the color decreases with the increase in temperature and the presence of water-
miscible organic compounds like ethanol.
 On heating, the blue color amylase-iodine complex dissociates but is formed again on cooling
because the helical structure is disrupted; thereby amylose loses its iodine binding capacity and the
blue color.
 The blue color reappears on cooling due to the recovery of iodine binding capacity due to
regaining of the helical structure.

Laboratory Precautions:

Caution: Iodine is highly toxic, corrosive, and irritant. If you spill any of the solution on yourself or on the
bench, immediately notify your laboratory instructor.
Caution: In the test, you will place the test tubes containing the reaction solutions in a boiling-water bath. Be
careful that you do not come in contact with the steam or the hot apparatus, and that you do not knock over
the bath.

Procedures:

Make sure that the test tubes

are standing upright or in
proper position to avoid
pouring out of contents