Identification of Unknown carbohydrates

1. Qualitative Analysis
for
Carbohydrates

2. Objective
• To study the properties of carbohydrates
• To identify the unknown carbohydrate by the carrying
out series of the chemical reactions.

3. Carbohydrates
•Defined as polyhydroxy aldehydes or ketones or
compounds which yield these on hydrolysis.
•General formula : Cn(H2O)n.
•Also called as “saccharides”.

4. Classification of Carbohydrates

5. Why to study the tests for their detection?
Glucose in urine in diabetes mellitus
Fructose in urine in fructosuria
Galactose in urine galactosemia

6. REACTION DUE TO
HYDROXYL GROUP:
Dehydration
(eg. Molisch test, Seliwanoff’s
test)
REACTION DUE TO
CARBONYL GROUP:
Reduction
(eg. Benedict’s test , Barfoed’s
test)
Condensation
(eg. Osazone formation )

7. MOLISCH TEST
TEST OBSERVATION INFERENCE
[General test for
carbohydrates]
Take 2 ml of
Carbohydrate solution
+ 2 - 3 drops of α-
naphthol, mix + equal
volume (2 ml) of
concentrated sulphuric
acid (H2SO4)along the
walls of the tube
carefully.
A purple or violet
colour ring is observed
at the junction of two
layers.
This test is specific for
carbohydrates.
Carbohydrates
undergo dehydration
when treated with
sulphuric acid & forms
furfurals. These
furfurals condense
with α-naphthol to
form coloured
compound.

9. • α
α-
napthol

11. IODINE TEST
TEST OBSERVATION INFERENCE
[Test for
polysaccharides]
2ml of
carbohydrate
solution + 2-3 drops
of iodine solution
mix well.
Blue colour observed This test is specific for
polysaccharides. Iodine
forms a deep blue
colour complex with
Polysaccharides

12. Adsorption occurs
of iodine on starch
molecules.

14. BENEDICT’S TEST
TEST OBSERVATION INFERENCE
[Test for Reducing
sugars]
5 ml of Benedict’s
qualitative reagent. Boil
for 20 seconds Add 8
drops (0.5 ml) of
carbohydrate solution
and boil for2 minutes.
Brick red precipitate is
observed
This test detects the
presence of reducing
sugars. Reducing sugars
under alkaline condition
forms Enediols, which
reduce cupric ions (Cu2+)
to cuprous ions. Cuprous
ions (Cu+) form cuprous
hydroxide (CuOH).
Cuprous hydroxide during
the process of heating is
converted to red coloured
cuprous oxide (Cu2O).

18. Reducing
sugar

20. Benedict’s Reagent composition
• Copper sulphate: Dissociate to give sufficient cupric ions.
• Sodium citrate: keeps cupric hydroxide in solution without
getting precipitated
• Sodium carbonate: make the pH of the medium alkaline.

24. BARFOED’S TEST
TEST OBSERVATION INFERENCE
[Test for
monosaccharides]
2 ml of Carbohydrate
solution + 2 ml of
Barfoed’s reagent and
mix. Boil for 1 min
Red scum is observed
(Scanty Red Precipitate
is seen at the bottom)
Examine the
precipitate after 5-10
minutes.
This test is specific for
monosaccharides.
Monosaccharides
under acidic
conditions reduce
cupric ions to red
coloured cuprous
oxide.

26. Monosaccharides
Red scum
precipitates at
the bottom

28. Barfoed’s Reagent composition
Copper acetate in acetic acid

29. Seliwanoff’s test
TEST OBSERVATION INFERENCE
[Test for ketoses]
2 ml Carbohydrate
solution + 2 ml of
Seliwanoff’s reagent
and mix. Boil strongly
for 30 seconds only
Cherry red colour is
observed
This test is specific for
ketose sugars [Mainly
for fructose]. The
reaction depends on
formation of furfural
from fructose and its
condensation with
resorcinol to form red
colour complex

32. INVERSION TEST
TEST OBSERVATION INFERENCE
[Test for non-reducing
sugars]
2 ml of sucrose solution +
5 drops of concentrated
HCl, Boil for 2 min. Cool.
Neutralize with 6 drops of
40% NaOH. Check using
red litmus paper. Perform
Benedict’s test with this
neutralized solution as
follows: solution + 3 ml of
Benedict’s reagent. Boil for
2 min over a low flame.
Sucrose on hydrolysis
gives glucose and
fructose. These reduce
cupric ions to cuprous
ions and a red
precipitate of cuprous
oxide is formed.
Red precipitate is observed

33. HYDROLYSIS TEST
TEST OBSERVATION INFERENCE
Step-1: Perform
Benedict’s Test with OS.
Step-2: 5 ml OS + 2
drops of conc. HCl . Boil
for 2 mins. Cool.
 Make alkaline with 5
drops of 40% NaOH.
From this solution
perform Benedict’s test
Step-1 Benedict’s test is
negative/ weakly
positive
Step-2 Benedict’s test is
positive
Starch present (weak
Benedict’s test with OS
is due to free reducing
groups at end of starch
molecules.)

34. OSAZONE FORMATION
TEST OBSERVATION INFERENCE
2 ml of phenyl hydrazine
solution + 2 ml of
carbohydrate solution. Mix.
Keep in boiling water bath for
20 minutes. At the end
remove the tubes from the
bath. Allow them to cool at
lab temperature. Mount the
crystals on a slide cover with
cover slip and observe
Yellow crystals are formed
after the contents cool down.
Reducing sugars react with
phenyl hydrazine to form
osazones of characteristic
shape. Osazones of
disaccharides are soluble
when hot and hence
crystallise only after cooling
Glucose, Fructose, Mannose
shows fine needle, broom
like structure,
Maltose shows Hedge hog
shape
Lactose shows Cotton ball,
Powder puff shape

35. Phenyl hydrazine in acetic acid, when boiled with reducing sugar
form phenyl hydrazones followed by the formation of osazone.
The first two carbons (C1 and C2) are involved in this reaction.
The sugar that differs in their configuration on these two carbons
gives the same type of osazone, since the difference is masked
by binding with phenyl hydrazine.

38. NEEDLE SHAPE / BROOM LIKE
GLUCOSE / FRUCTOSE / MANNOSE

39. SUNFLOWER SHAPE/HEDGE HOG
SHAPE
MALTOSE

40. COTTON BALL / POWDER PUFF SHAPE
LACTOSE

41. REACTION FOR GLUCOSE
Sr.
No.
TEST OBSERVATION INFERENCE
1 Molisch Test A purple or violet colour ring is
observed at the junction of two layers.
specific for carbohydrates. Or
Given sample is carbohydrate/
Carbohydrate Present
2 Iodine Test No colour change observed Polysaccharides absent.
Mono or disaccharide may be present.
3 Benedict’s Test Brick red precipitate is observed Reducing sugar is present.
(Glucose/Fructose/Lactose/Maltose)
4 Barfoed’s Test Red Scum precipitates observed at the
bottom
Monosaccharides are present.
(Glucose/ Fructose)
5 Seliwanoff’s Test No colour change observed Ketose sugar is absent.
(Fructose absent)
6 Osazone Test Needle shape crystal are seen Glucose is present.

42. REACTION FOR FRUCTOSE
Sr.
No.
TEST OBSERVATION INFERENCE
1 Molisch Test A purple or violet colour ring is
observed at the junction of two layers.
specific for carbohydrates. Or
Given sample is carbohydrate/
Carbohydrate Present
2 Iodine Test No colour change observed Polysaccharides absent.
Mono or disaccharide may be present.
3 Benedict’s Test Brick red precipitate is observed Reducing sugar is present.
(Glucose/Fructose/Lactose/Maltose)
4 Barfoed’s Test Red Scum precipitates observed at the
bottom
Monosacharides are present.
(Glucose/ Fructose)
5 Seliwanoff’s Test Cherry red colour is observed Ketose sugar is present.
(Fructose present)
6 Osazone Test Needle shaple crystal are seen fructose is present.

43. REACTION FOR LACTOSE/ MALTOSE
Sr.
No.
TEST OBSERVATION INFERENCE
1 Molisch Test A purple or violet colour ring is
observed at the junction of two layers.
specific for carbohydrates. Or
Given sample is carbohydrate/
Carbohydrate Present
2 Iodine Test No colour change observed Polysaccharides absent.
Mono or disaccharide may be present.
3 Benedict’s Test Brick red precipitate is observed Reducing sugar is present.
(Glucose/Fructose/Lactose/Maltose)
4 Barfoed’s Test No change observed. Monosacharides are absent .
Diasaccharide may be present
(Lactose/ Maltose)
5 Seliwanoff’s Test No colour change observed Ketose sugar is absent.
(Fructose absent)
6 Osazone Test Powder puff crystal are seen
Sun flower shape crystal are seen
Lactose is present.
Maltose is present

44. REACTION FOR SUCROSE
Sr.
No.
TEST OBSERVATION INFERENCE
1 Molisch Test A purple or violet colour ring is
observed at the junction of two layers.
specific for carbohydrates. Or
Given sample is carbohydrate/
Carbohydrate Present
2 Iodine Test No colour change observed Polysaccharides absent.
Mono or disaccharide may be present.
3 Benedict’s Test No change is observed Reducing sugar is absent .
(Sucrose )
4 Inversion Test Red precipitate is observed Sucrose present.
Sucrose on hydrolysis gives glucose
and fructose. These reduce cupric ions
to cuprous ions and a red precipitate
of cuprous oxide is formed.

45. REACTION FOR STARCH
Sr.
No.
TEST OBSERVATION INFERENCE
1 Molisch Test A purple or violet colour ring is
observed at the junction of two layers.
specific for carbohydrates. Or
Given sample is carbohydrate/
Carbohydrate Present
2 Iodine Test Blue colour observed Polysaccharides present.
3 Benedict’s Test No change is observed Reducing sugar is absent .
4 Hydrolysis Test Red precipitate is observed
Benedicts test positive.
Starch present (weak Benedict’s test
with OS is due to free reducing groups
at end of starch molecules.)

46. Unknown identification Scheme