Benedict'S Test: Qualitative Tests For Carbohydrates

QUALITATIVE TESTS FOR CARBOHYDRATES
Performed by GROUP 1, Biochemistry Laboratory, April 27, 2015

St. Alexius College, Medical Technology Department
- A semi quantitative test.

BENEDICT’S - The color of the precipitate gives a rough estimate of a REDUCING
TEST SUGAR present in the sample.
Carbohydrates with free aldehyde or ketone groups have the ability to
reduce solutions of various metallic ions. Reducing sugars under
alkaline conditions tautomerise and form Enediols which are powerful
Principle reducing agents. They reduce cupric ions to cuprous form and are
themselves converted to sugar acids. The cuprous ions combine with
OH- ions to form yellow cuprous hydroxide which upon heating is
converted to red cuprous oxide.
 Take 5 mL of Benedict’s reagent.
 Add 1 mL of test sample solution
Test Procedure
 Boil over a flame or in a boiling water bath for 2-3 minutes.
 Let the solution cool down.
GREEN COLOR Up to 0.5 G% (+)
GREEN PRECIPITATE 0.5-1.0 G% (++)
Positive Reaction YELLOW PRECIPITATE 1.0-1.5 G% (+++)
ORANGE PRECIPITATE 1.5-2.0 G% (++++)
BRICK RED PRECIPITATE > 2.0 G % (+++++)
EXPERIMENT RESULTS
TEST SAMPLE EXPECTED RESULT DISCUSSION
Galactose POSITIVE Reducing sugar
Glucose POSITIVE Reducing sugar
Fructose POSITIVE Reducing sugar
Sucrose NEGATIVE Not a reducing sugar
Lactose POSITIVE Reducing sugar

- The positive reaction indicates the PRESENCE OF A REDUCING
BARFOED’S MONOSACCHARIDE.
- On prolonged heating disaccharides can also give this test positive.
TEST Hence, the solution should be boiled for 3 minutes only.
Aldoses and ketoses can reduce cupric ions even in acidic conditions.
This test is used to distinguish reducing mono saccharides from
Principle
disaccharides by controlling pH and time of heating. Mono saccharides
react very fast whereas disaccharides react very slowly.
 To 3 mL of Barfoed‘s reagent, add 1 mL of test samples solution
 Keep the test tubes in the boiling water bath for 3 minutes.
Test Procedure
 Cool under running water.
NB: Over-heating should be avoided.
Positive Reaction SCANTY BRICK RED PRECIPITATE
EXPERIMENT RESULTS
NEGATIVE
Galactose but POSITIVE Reducing Disaccharide
if over-heated
Glucose POSITIVE Reducing Monosaccharide
Fructose POSITIVE Reducing Monosaccharide
Sucrose NEGATIVE Non-reducing Disaccharide

NEGATIVE
Lactose but POSITIVE Reducing Disaccharide
if over-heated
- This test is given positive by KETOHEXOSES so it is answered by
fructose, sucrose and other fructose containing carbohydrates.
SELIWANOFF’S - This test distinguishes between glucose and fructose.
- Overheating of the solution should be avoided. Upon continuous
TEST boiling, aldoses get converted to ketoses and give a positive
reaction with Seliwanoff reagent.
Ketohexoses on treatment with hydrochloric acid form 5-hydroxy
Principle methyl furfural which on condensation with resorcinol gives a cherry
red colored complex.
 To 4 mL of Seliwanoff reagent add 1mL of test sample solution
Test Procedure  Boil for 1 minute only.
 Cool the solution.
Positive Reaction CHERRY RED COLOR
EXPERIMENT RESULTS
Galactose NEGATIVE Aldohexose
Glucose NEGATIVE Aldohexose
Fructose POSITIVE Ketohexose
Disaccharide that contains Fructose – a

Sucrose POSITIVE
ketohexose
Disaccharide that do not contain
Lactose NEGATIVE
ketohexose
- This is a sensitive but a non- specific test and is given positive by ALL
MOLLISCH’S TYPES OF CARBOHYDRATES. If the oligosaccharides or
polysaccharides are present they are first hydrolyzed to
TEST monosaccharides which are then dehydrated to give the test
positive.
Carbohydrates when treated with concentrated sulfuric acid undergo
dehydration to give furfural derivatives. These compounds condense
Principle
with Alpha naphthol to form colored products. Pentoses yield furfural
while Hexoses yield 5-Hydroxy methyl furfurals
 Take 4 mL of test sample solution in a clean and dry test tube.
 Add 2 drops of ethanolic Alpha Naphthol (Molisch reagent) and mix.
Positive Reaction
 Incline the test tube and add carefully 5 mL of concentrated Sulfuric
acid along the side of the test tube so as to form 2 layers.
REDDISH VIOLET OR PURPLE COLORED RING AT THE JUNCTION OF
Test Procedure
TWO LIQUIDS
EXPERIMENT RESULTS
Galactose POSITIVE Test sample is a Carbohydrate
Glucose POSITIVE Test sample is a Carbohydrate
Fructose POSITIVE Test sample is a Carbohydrate
Sucrose POSITIVE Test sample is a Carbohydrate
Lactose POSITIVE Test sample is a Carbohydrate

- This is a test for POLYSACCHARIDES
- Amylose- a linear chain component of starch, gives a deep blue
color Amylopectin- a branched chain component of starch, gives a
IODINE purple color
- Glycogen- gives a reddish brown color
TEST - Dextrins- Amylo, Eryhthro and Achrodextrins, formed as
intermediates during hydrolysis of starch give violet, red and no
color
Iodine forms a coordinate complex between the helically coiled
polysaccharide chain and iodine centrally located within the helix due
Principle
to adsorption. The color obtained depends upon the length of the
unbranched or linear chain available for complex formation
 Add 2 drops of Iodine solution (Lugol’s solution) to 1mL of test
sample solution
Test Procedure
 Add 1 mL distilled water
 Stand for 2 minutes
Positive Reaction PURPLE-BLACK
EXPERIMENT RESULTS
Galactose NEGATIVE Not a polysaccharide but a monosaccharide
Glucose NEGATIVE Not a polysaccharide
Fructose NEGATIVE Not a polysaccharide
Sucrose NEGATIVE Not a polysaccharide
Lactose NEGATIVE Not a polysaccharide

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QUALITATIVE TESTS FOR CARBOHYDRATES

Performed by GROUP 1, Biochemistry Laboratory, April 27, 2015

- A semi quantitative test.

Galactose POSITIVE Reducing sugar

Glucose POSITIVE Reducing sugar

Fructose POSITIVE Reducing sugar

Sucrose NEGATIVE Not a reducing sugar

Lactose POSITIVE Reducing sugar

Glucose POSITIVE Reducing Monosaccharide

Fructose POSITIVE Reducing Monosaccharide

Sucrose NEGATIVE Non-reducing Disaccharide

Galactose NEGATIVE Aldohexose

Glucose NEGATIVE Aldohexose

Fructose POSITIVE Ketohexose

Disaccharide that contains Fructose – a

Galactose POSITIVE Test sample is a Carbohydrate

Glucose POSITIVE Test sample is a Carbohydrate

Fructose POSITIVE Test sample is a Carbohydrate

Sucrose POSITIVE Test sample is a Carbohydrate

Lactose POSITIVE Test sample is a Carbohydrate

Galactose NEGATIVE Not a polysaccharide but a monosaccharide

Glucose NEGATIVE Not a polysaccharide

Fructose NEGATIVE Not a polysaccharide

Sucrose NEGATIVE Not a polysaccharide

Lactose NEGATIVE Not a polysaccharide

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