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).
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.
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 1 of the above solutions or a mixture of 2 of the above
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 that can be dehydrated to furfural or hydroxymethylfurfural
in the presence of H2SO4. 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 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 H2SO4 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. Benedict’s Test for Reducing Sugars
Alkaline solutions of copper are reduced by sugars that have 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 CuCO3 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
The 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 55°C 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 FeCl3. 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
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.
Test 7. Iodine Test for Starch and Glycogen
The use of Lugol’s iodine reagent 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 produce a brownblue
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 produces 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.