Activity Qualitative Tests for Carbohydrates

No. 1 Title of the Activity

Instructions:

1. Watch the video “Classification Tests for Carbohydrates” posted in google

classroom for your reference.
2. All questions and answers should be typewritten. Font: Arial, size 12 and justified
paragraph.
3. Questions in black color and your answers in red color.
4. Submit as pdf file.
5. FILE NAME should be: Act1_MCPH30_SURNAME

I. Overview

A. Tests Bases on the Formation of Furfural and its Derivatives

Sugars are stable to hot dilute mineral acids. But hot sulfuric acid will dehydrate
them into furfural and its derivatives. Pentoses, when heated with concentrated sulfuric
acid, will form fulfuraldehyde, while hexoses will form 5-hydroxymethylfurural.

In the presence of acid, phenolic compounds like alpha-naphtol, orcinol, and

resorcinol will react with furfural or hydroxymethyl furfural to form colored condensation
products. The formation of these colored compounds constitutes a positive test for
carbohydrates.

1. Molisch Test

The Molisch test is the general tests for carbohydrates. The sugars are mixed
with alpha-naphthol. The test tube is inclined, and concentrated sulfuric acid is
added along the side of the tube, causing the formation of a lower layer of acid.
The concentrated sulfuric acid will dehydrate the sugar allowing it to react with
the alcohol forming furfural or hydroxymethylfurfural. Formation of a purple ring at
the interface of the two liquids will indicate the presence of a carbohydrate.

2. Bial’s Orcinol Test

Bial’s test is used to determine the presence of pentoses and nucleotides that
contain pentose sugars. When pentoses are treated with orcinol, furfurals are
formed, and they will yield a blue-green compound in the presence of ferric ions.
The reaction is not specific for pentoses because other compounds like trioses,
uronic acids, and certain heptoses will also give blue or green products.
 Hydroxymethyl furfural is formed from hexoses to give yellow-brown
condensation products.

3. Seliwanoff’s test

This test is used to differentiate ketohexoses from aldohexoses. Ketohexoses

react faster with the solution containing hydrochloric acid and resorcinol than
aldohexoses. The dehydrated ketohexoses form a bright cherry red condensation
product, while the aldohexoses yields only a pale pink coloration, a negative
result. In this test, prolonged heating of samples should be avoided.

B. Tests Based on the Reducing Property of Sugars

Reducing sugars must have a free aldehyde or ketone group. Therefore all
monosaccharides and some disaccharides can reduce oxidizing reagents such as the
cupric ion, dinitrosalicyclic acid, and picric acid.

1. Benedict’s Test

Benedict’s test is a very sensitive test done under mildly alkaline conditions. The
reagent contains CuSO4, Na2CO3, and sodium citrate. The formation of a brick
red precipitate of Cu2O is considered positive. Most aldehydes have the ability to
reduce Benedict’s reagent. Other compounds like formic acid, hydrazobenzene,
phenols, phenylhydrazine, pyrogallol, and uric acid will also give appositive result
in this test.

2. Barfoed’s Test

Barfoed’s reagent contains cupric acetate in diluted acetic acid and is used to
distinguish between monosaccharides, disaccharides, and oligosaccharides.
Barfoed’s reagent oxidizes monosaccharides but not oligosaccharides.
Disaccharides are less easily oxidized but are oxidized if they undergo prolonged
heating, causing hydrolysis of the disaccharides into monosaccharides which will
then give a positive result. The concentration of sugar solutions used in this test
should be approximately the same because the use of more concentrated
disaccharide solution may give a faster reaction than that of a relatively more
dilute monosaccharides solution. Unlike Benedict’s test is carried out under
acidic rather than basic medium.

3. Tollen’s Test

Sugars with aldehyde groups are capable of reducing Tollen’s reagent (an
ammoniacal solution of Silver) to form a gray to black precipitate. If the reaction
is clean and rate of deposition is slow enough, the silver deposits as a silver
mirror.
II. Procedures

1. Molisch’s Test
a. In different sugar solutions with same concentration and quantity, add
drops of Molisch’s reagent. Mix the contents by gently shaking the test
tube.
b. Incline the test tube and cautiously add conc. Sulfuric acid, allowing the
acid to run down the side of the tube. Sulfuric acid is denser than water
and will form a lower layer.
c. Note the color of the ring formed at the junction of the two liquids.

2. Bial’s Orcinol Test

a. In different sugar solutions with same concentration and quantity, add
Bial’s reagent to each test tube.
b. Carefully heat each tube over a Bunsen burner until the solution begins to
boil. Add drops of 10% FeCl3 solution.
c. Note the color of the product formed.

3. Seliwanoff’s Test
a. In different sugar solutions with same concentration and quantity, add
Seliwanoff’s reagent to each test tube.
b. Place the tubes in a water bath filled with boiling water and allow them to
stay there for exactly 1 minute.
c. Note the changes and record which test tube give a positive result in the
shortest time.
d. Contiunue heating and observe the color changes at 1 minute intervals.
Record the time required for a positive test for each sample.

4. Benedict’s Test
a. In different sugar solutions with same concentration and quantity, add
Benedict’s reagent in each test tube.
b. Place all test tubes in a boiling water bath for 2-3 minutes.
c. Observe the color of the solution and note whether precipitate was
formed. A change in color of the solution is not considered a positive
reaction.

5. Barfoed’s Test
a. In different sugar solutions with same concentration and quantity, add
Barfoed’s reagent in each test tube.
b. Place all the tubes in a boiling water bath for 10 minutes. Record color
change.

6. Tollen’s Test
 a. In different sugar solutions with same concentration and quantity, add
Tollen’s reagent in each test tube until brown precipitate of silver oxide
that forms just dissolves.
b. Boil for 5 minutes. Note and Record the color change.

III. Results and Questions

SUBSTANCE VISIBLE RESULT

TESTED
Molisch’s Bial’s Seliwanoff’s Benedict’s Barfoed’s Tollen’s
Test Orcinol Test Test Test Test
Test

Glucose

Xylose

Lactose

Fructose

Sucrose

Starch

Questions:

1. Explain the results for each sugar in Seliwanoff and Barfoed’s test.
2. Will disaccharides and polysaccharides give a positive result for Molisch’s test?
Explain your answer
3. Which of the different carbohydrate tests would give a positive result for maltose?
Describe the color change.
4. Can Seliwanoff’s test be used to distinguish sucrose from fructose? Explain your
answer.
5. Inulin is a polysaccharide composed entirely of fructose units, which tests should
be used to best identify the presence of fructose? Explain your answer.