CARBOHYDRATES - QUALITATIVE TESTS  

Carbohydrates are a class of organic molecules with the general chemical formula C n(H2O)n.  These
compounds are literally carbon hydrates.  Only the monomeric form of these compounds, the
monosaccharides, fit this description precisely.  Two monosaccharides can be polymerized together
through a glycosidic linkage to form a disaccharide.  When a few monosaccharide molecules are
polymerized together, the result is an oligosaccharide.  A polysaccharide is an extensive polymer of
carbohydrate monomers.        

The monosaccharide glucose is our primary energy source.  The function of the polysaccharides
starch (plants) and glycogen (animals) is to store glucose in a readily accessible form, as well as to
lower the osmotic potential of internal fluids.  Some polysaccharides serve a structural role in living
organisms.  The glucose polymer cellulose is a major component of plant cell walls.  Chitin, a polymer
of N-acetylglucosamine, is a major structural component of the exoskeleton of insects and
crustaceans.  Hyaluronic acid and chondroitin sulfate occurs in the connective tissues of animals,
especially in cartilage.  Oligosaccharide side chains of glycoproteins may also serve as signals for
intracellular sorting of the protein (i.e. mannose-6-phosphate signal designating lysosomal enzymes).

Several qualitative tests have been devised to detect members of this biologically significant class of
compounds.  These tests will utilize a test reagent that will yield a color change after reacting with
specific functional groups of the compounds being tested.   The following exercises are reactions that
can detect the presence or absence of carbohydrates in test solutions.  They range in specificity to
the very general (i.e. Molisch test for carbohydrates) to the very specific (i.e. mucic acid test for
galactose).   

Learning Objectives   

Upon completion of the following exercises, the student should be able to:   

1. Define the term qualitative tests for chemical compounds.   

2. Describe the basis of qualitative tests for monosaccharides, disaccharides, and polysaccharides.   

3. Use the following qualitative tests to distinguish specific sugars from each other.   

Pre-Laboratory Exercise     

You are given solutions containing:  fructose, glucose, lactose, galactose, ribose, ribulose, sucrose,
and starch.  Devise a scheme by which you can systematically identify these compounds.   

Procedure   

Perform the following qualitative tests on 0.2 M solutions (unless otherwise stated) of starch, sucrose,
glucose, lactose, galactose, ribose, and ribulose.  Use the scheme you devised in the prelab section
to identify an unknown solution.  The unknown will be one of the above solutions or a mixture of two
of the above solutions.   

Test 1.  Molisch Test for Carbohydrates        

The Molisch test is a general test for the presence of carbohydrates.  Molisch reagent is a solution
of alpha-naphthol in 95% ethanol.  This test is useful for identifying any compound which can be
dehydrated to furfural or hydroxymethylfurfural in the presence of H 2SO4.  Furfural is derived from the
dehydration of pentoses and pentosans, while hydroxymethylfurfural is produced from hexoses and
hexosans.  Oligosaccharides and polysaccharides are hydrolyzed to yield their repeating monomers
by the acid.  The alpha-naphthol reacts with the cyclic aldehydes to form purple colored condensation
products.  Although this test will detect compounds other than carbohydrates (i.e. glycoproteins), a
negative result indicates the ABSENCE of carbohydrates.                 

Method:  Add 2 drops of Molisch reagent to 2 ml of the sugar solution and mix thoroughly.  Incline
the tube, and GENTLY pour 5 ml of concentrated H 2SO4 down the side of the test tube.  A purple
color at the interface of the sugar and acid indicates a positive test.  Disregard a green color if it
appears.   

Test 2.  Benedicts's Test for Reducing Sugars        

Alkaline solutions of copper are reduced by sugars having a free aldehyde or ketone group, with the
formation of colored cuprous oxide.  Benedict's solution is composed of copper sulfate, sodium
carbonate, and sodium citrate (pH 10.5).  The citrate will form soluble complex ions with Cu++,
preventing the precipitation of CuCO 3 in alkaline solutions.                   

Method:  Add 1 ml of the solution to be tested to 5 ml of Benedict's solution, and shake each tube. 
Place the tube in a boiling water bath and heat for 3 minutes.  Remove the tubes from the heat and
allow them to cool.  Formation of a green, red, or yellow precipitate is a positive test for reducing
sugars.   

Test 3.  Barfoed's Test for Monosaccharides        

This reaction will detect reducing monosaccharides in the presence of disaccharides.   This reagent
uses copper ions to detect reducing sugars in an acidic solution.  Barfoed's reagent is copper acetate
in dilute acetic acid (pH 4.6).  Look for the same color changes as in Benedict's test.   

Method:  Add 1 ml of the solution to be tested to 3 ml of freshly prepared Barfoed's reagent.  Place
test tubes into a boiling water bath and heat for 3 minutes.  Remove the tubes from the bath and
allow to cool.  Formation of a green, red, or yellow precipitate is a positive test for reducing
monosaccharides.  Do not heat the tubes longer than 3 minutes, as a positive test can be obtained
with disaccharides if they are heated long enough.   

Test 4. Lasker and Enkelwitz Test for Ketoses        T

he Lasker and Enkelwitz test utilizes Benedict's solution, although the reaction is carried out at a
much lower temperature. The color changes that are seen during this test are the same as with
Benedict's solution.  Use DILUTE sugar solutions with this test (0.02 M).   

Method:  Add 1 ml of the solution to be tested to 5 ml of Benedict's solution to a test tube and mix
well.  The test tube is heated in a 55oC water bath for 10-20 minutes.   Ketopentoses demonstrate a
positive reaction within 10 minutes, while ketohexoses take about 20 minutes to react.   Aldoses do
not react positively with this test.   

Test 5.  Bial's Test for Pentoses        

Bial's reagent uses orcinol, HCl, and FeCl 3.  Orcinol forms colored condensation products with furfural
generated by the dehydration of pentoses and pentosans.  It is necessary to use DILUTE sugar
solutions with this test (0.02 M).   
Method:  Add 2 ml of the solution to be tested to 5 ml of Bial's reagent.  Gently heat the tube to
boiling.  Allow the tube to cool.  Formation of a green colored solution or precipitate denotes a
positive reaction.   

Test 6.  Mucic Acid Test for Galactose      

Oxidation of most monosaccharides by nitric acid yields soluble dicarboxylic acids.  However,
oxidation of galactose yields an insoluble mucic acid.  Lactose will also yield a mucic acid, due to
hydrolysis of the glycosidic linkage between its glucose and galactose subunits.   

Method:  Add 1 ml of concentrated nitric acid to 5 ml of the solution to be tested and mix well.  Heat
on a boiling water bath until the volume of the solution is reduced to about 1 ml.  Remove the mixture
from the water bath and let it cool at room temperature overnight.  The presence of insoluble crystals
in the bottom of the tube indicates the presence of mucic acid.  CAUTION:  PERFORM THE
REACTION UNDER A FUME HOOD.   

Test 7.  Iodine Test for Starch and Glycogen        

The use of Lugol's iodine reagent (IKI) is useful to distinguish starch and glycogen from other
polysaccharides.  Lugol's iodine yields a blue-black color in the presence of starch.  Glycogen reacts
with Lugol's reagent to give a brown-blue color.   Other polysaccharides and monosaccharides yield
no color change; the test solution remains the characteristic brown-yellow of the reagent.  It is
thought that starch and glycogen form helical coils.  Iodine atoms can then fit into the helices to form
a starch-iodine or glycogen-iodine complex.  Starch in the form of amylose and amylopectin has less
branches than glycogen.  This means that the helices of starch are longer than glycogen, therefore
binding more iodine atoms.  The result is that the color produced by a starch-iodine complex is more
intense than that obtained with a glycogen-iodine complex.   

Method:  Add 2-3 drops of Lugol's iodine solution to 5 ml of solution to be tested.  Starch gives a
blue-black color.  A positive test for glycogen is a brown-blue color.  A negative test is the brown-
yellow color of the test reagent.