CLB 11103

SECTION OF BIOENGINEERING TECHNOLOGY

UniKL MICET
BIOLOGY OF CELLS
EXPERIMENT 4: CARBOHYDRATE TESTS
Objective: To introduce students several tests to detect the presence of specific carbohydrate
groups: such as the Benedicts Test to differ reducing and non-reducing sugar, and the Iodine Test
which is used to detect the presence of starch in the specimens.

Introduction
Carbohydrates are classified as polyhydroxy aldehydes or polyhydroxy ketones. Therefore, they
will exhibit chemical properties associated with both alcohols and carbonyl compounds. In the
following series of analyses you will be examining the reactivity of some monosaccharides,
disaccharides and a polysaccharide.
In the Benedicts test, a reducing sugar (a sugar with a free or potentially free, i.e., a cyclic
hemiacetal, aldehyde group) reacts with the blue-colored Cu2+ ion in the presence of base. The
copper (II) ion is reduced to a red-orange Cu2O precipitate whereas the aldehyde group is
oxidized to the carboxylic acid functional group. In addition to all aldose monosaccharides
giving a positive Benedicts test, ketose monosaccharides, though lacking an aldehyde group,
react due to the presence of a hydroxyl group next to the ketone group. Thus -hydroxy ketones
give positive tests. If there is no potential free aldehyde group, i.e., the aldehyde group is tied up
in a glycosidic bond (an acetal bond), the sugar is referred to as a non-reducing sugar.
Starch is composed of two fractions; the linear, helical fraction and the branched amylopectin
fraction. When I2 is inserted into the interior of the amylose fraction, a dark blue color is
observed.
Reagents and apparatuses:

Carbohydrate solutions: Glucose, galactose, fructose, arabinose, maltose, lactose, sucrose,

and starch
Benedicts reagent
Iodine
Test tubes
Water baths

CLB 11103
Procedures:
A) Benedicts test
1. Prepare a boiling water bath and label eight clean small test tubes.
2. In separate test tubes add 1 mL of the Benedicts reagent. To each test tube add 5 drops of the
test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the red Cu 2O precipitate to form; also note if the blue
Benedicts reagent colour disappears.
5. Remove all the tubes after 10 minutes. Did any sugars not produce the red precipitate? Which
are reducing sugars? Which are not?
B) Iodine test
1. Place 3 drops of each test carbohydrate solution in separate wells of a clean spot plate.
2. Add 1 drop of the iodine solution to each test carbohydrate solution.
3. Note and record the colour of each sample.
Did any other solutions besides the starch solution give a positive test?

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APPENDIX
Benedict's solution ( 1 L) (qualitative reagent for glucose). With the aid of heat, dissolve 173 g
of sodium citrate and 100 g of Na2CO3 in 800 ml of water. Filter, if necessary, and dilute to 850
ml. Dissolve 17.3 gof CuSO4.5H2O in 100 ml of water. Pour the latter solution, with constant
stirring, into the carbonate-citrate solution, and make up to 1 liter.
Benedicts Reagent (300 ml): Dissolve 51.9 g C6H5Na3O72H2O sodium citrate dihydrate (MW
= 294.10 g/mol) and 30 g Na2CO3 anhydrous sodium carbonate (MW = 105.99 g/mol) in 200 ml
distilled water with heating. Dissolve 1.73 g CuSO45H2O copper (II) sulfate pentahydrate (MW
= 249.68 g/mol) in 30 ml distilled water. Mix these two solutions slowly, and add distilled water
to produce 300 ml solution.