Tests for Carbohydrates (Known Samples)
Molisch Test:
Two drops of 5% alpha-naphthol /thymol alcoholic solution are added to about 3 ml of
carbohydrate solution. Mix and then concentrated H2SO4 is added gently to the mixture
(along the side of the test tube) so as to form a lower layer of acid.
Observation.: A deep purple ring appears at the surface of contact.
Inference.: Carbohydrates are present. Furfural or furfural derivatives formed by
carbohydrates under dehydrating action of strong sulphuric acid, react with
alpha-naphthol/thymol. It then forms a condensation product with naphthol/thymol that
appears as deep purple ring at the junction of acid and carbohydrate layers. It indicated
some carbohydrate in present in the test solution
Tests for Glucose
BENEDICT’S TEST: It is used as a simple test for reducing sugars eg. glucose,
fructose.
Procedure: 1 ml. of test solution was taken in a test tube and 3 ml of Benedict’s
qualitative reagent was added into it. Then the mixture was heated over flame just for
30secs. Then it was allowed to cool under tap water.
Obs.: A brick red ppt was found in the solution.
Inf.: Glucose is present. Reducing sugars like glucose under alkaline condition
tautomerise and form enediols. Enediols are powerful reducing agents. Benedict's
Solution contains copper sulphate, sodium carbonate, sodium citrate etc. The enediols
reduce cupric ions (Cu2+) to cuprous form (Cu+), which is precipitated as red coloured
cuprous oxide and made change in color of the reaction mixture. This is the basis of
Benedict’s test.
FEHLING’S TEST:
At first Fehling’s reagent is prepared by mixing equal volumes of Fehling’s reagent A
and reagent B. Then 3 ml of test sample and 4 ml of Fehling’s reagent are mixed and
the mixture is boiled, then cooled.
Obs.: First a yellowish ppt appears which then turns red as become coarser.
Inf.: Glucose is present. Reducing sugars like glucose under alkaline condition
tautomerise and form enediols. Enediols are powerful reducing agents. Fehling’s
Solution contains cupric sulfate, KOH and Rochelle’s salt. The enediols reduce cupric
ions (Cu2+) to cuprous form (Cu+), which is precipitated as yellow CuOH or red
coloured cuprous oxide Cu2 O and made change in color of the reaction mixture. It
indicates presence of reducing sugar in the test sample.
BARFOED'S TEST:
Barfoed's test is a chemical test used for detecting the presence of monosaccharide. It
is based on the reduction of copper (II) acetate to copper(I) oxide (Cu2O), which forms a
brick-red precipitate.
Procedure: 1 ml. of test solution was taken in a test tube and 3 ml of Barfoed’s reagent
is added into it. Then the mixture was heated over flame just for 30secs. Then it was
allowed to cool.
Obs.: A brick red ppt was found at the bottom of the test tube.
Inf.: Reducing monosaccharide- glucose is present. Reducing sugars under
alkaline condition tautomerise and form enediols. Enediols are powerful reducing
agents. Barfoed's reagent contains 0.33 molar solution of neutral copper acetate in
1% acetic acid solution. The enediols cause the reduction of copper(II) acetate to
copper(I) oxide (Cu2O), which forms a brick-red precipitate.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
SELIWANOFF’S TEST
Procedure: 1 ml. of test solution was taken in a test tube and 3 ml of Seliwanoff’s
reagent was added into it. Then the mixture was heated over flame just for 30secs.
Then it was allowed to cool.
Obs.: No change was observed.
Inf.: Glucose being aldose sugar does not respond to this test. Seliwanoff’s test is
a chemical test which distinguishes between aldose and ketose sugars.
Tests for Fructose
Benedict’s test : Same as glucose
Barfoed's test : Same as glucose
SELIWANOFF’S TEST
Procedure: 1 ml. of test solution was taken in a test tube and 3 ml of Seliwanoff’s
reagent was added into it. Then the mixture was heated over flame just for 30secs.
Then it was allowed to cool.
Obs.: Cherry red colour observed.
Inf.: Reducing ketose monosaccharide, i.e fructose is present. Seliwanoff’s reagent
consists of resorcinol and concentrated hydrochloric acid. Keto sugars get dried out in
presence of concentrated acids and form furfurals which react with resorcinol in
Seliwanoff reagent to yield a cherry-red hue complex.
Tests for Maltose
Benedict’s test : Same as glucose
Barfoed's test :
Procedure: Same as glucose
Obs.: No change found
Inf.: Maltose being disaccharide shows no reaction with Barfoed’s reagent.
SELIWANOFF’S TEST
Procedure: Same as glucose
Obs.: No change found
Inf.: Reducing disaccharide Maltose is present. Maltose being aldose sugar do
not form furfurals with Seliwanoff’s reagent.
Tests for Sucrose
BENEDICT’S TEST : Procedure: Same as glucose
Obs.: No change was observed.
Inf.: Sucrose consists of glucose and fructose in which the reducing groups are blocked
by 1,2-glucosidic bonds. As a result it is unable to form 1,2-enediols. Thus cannot
reduce cupric ions (Cu2+) to cuprous form of Benedict’s reagent, so no red ppt is formed.
BARFOED'S TEST: Procedure: Same as glucose
Obs.: No change was observed.
Inf.: Sucrose being disaccharide does not react with Barfoed’s reagent.
SELIWANOFF’S TEST
Procedure: 1 ml. of test solution was taken in a test tube and 3 ml of Seliwanoff’s
reagent was added into it. Then the mixture was heated over flame just for 30secs.
Then it was allowed to cool.
Obs.: Cherry red colour observed.
Inf.: Sucrose is present. On heating sucrose produces glucose and fructose.
Seliwanoff’s reagent consists of resorcinol and concentrated hydrochloric acid. This
ketose fructose get dried out in presence of concentrated acids and form furfurals
which react with resorcinol in Seliwanoff’s reagent to yield a cherry-red hue complex.
Acid hydrolysis test:
Procedure: Three drops of concentrated H2SO4 is added to about 6 ml of test
carbohydrate solution and mixed, then boiled gently for 3 mins. Then it is cooled and
neutralized carefully with solid Na2CO3 until no further effervescence occurs. Then with
this solution Benedict’s test is performed.
Obs.: A brick red ppt was found in the solution
Conclusion: Sucrose is hydrolysed by acid into glucose and fructose which form
enediols in alkaline solution present in Benedict’s solution. The enediols reduce cupric
ions (Cu2+) to cuprous form (Cu+), which is precipitated as red coloured cuprous oxide
and made change in color of the reaction mixture. So sucrose is present in the test
sample.
Tests for starch
IODINE TEST
Procedure: one to two drops of dilute iodine solution is added to about 3 ml of test
carbohydrate solution .
Obs.: A dark blue colouration was found in the solution which disappears on
heating and reappear on cooling.
Conclusion: Blue colour formation occurs due to formation of iodine-polysaccharide
adsorption complex. Heating dissociates the coloured complex making the solution
colourless while cooling again join them making blue colour again.
