1. Several qualitative tests can identify the presence of carbohydrates, including the Molisch test, iodine test, Benedict's test, Barfoed's test, Seliwanoff's test, and osazone tests. 2. The Molisch test uses alpha-naphthol and sulfuric acid to detect the presence of any carbohydrate, producing a purple ring at the interface. Benedict's test uses copper sulfate and citrate in an alkaline solution to detect reducing sugars based on the color of precipitation. 3. The iodine test detects polysaccharides like starch, producing colors from blue to reddish-brown depending on the type. Barfoed's test distinguishes monos

1. QUALITATIVE TESTS FOR CARBOHYDRATES
1
Amit Jha.
Lecturer
UCMS, Bhairahawa

2. 1. Molisch test
2. Iodine test
3. Benedict test
4. Barfoed’s test
5. Seliwanoff’s test
6. Osazone test
2

3. Molisch test
• General test for all carbohydrates.
• Principle:
– Carbohydrates when treated with conc. H2SO4, undergo
dehydration to give furfural derivatives, which
condenses with α-Naphthol to form colored product.
3

4. Molisch test
4
Observation Inference
2mL of carbohydrate solution
was taken in a test tube.
2-3 drops of α-Naphthol was
added to it & was mixed well.
2mL of conc. H2SO4 was then
added along the side wall of tube
Purple colored
ring was seen at
junction of two
liquids.
+ve Molisch Test
[Indicates +nce of
carbohydrate in
given solution]

5. Molisch test
• Reagents required:
– Conc. H2SO4
– α-Naphthol
• Precautions
– α-Naphthol (unstable) should be freshly prepared.
– Excess α-Naphthol leads to formation of green colour.
– Conc. H2SO4 should be added slowly along the side
wall of tube.
5

6. Iodine test
• Test for polysachharides.
– Starch: Blue
– Dextrin: Reddish purple
– Glycogen: Reddish Brown
• Principle:
– Starch when treated with few drops of Iodine solution,
Iodine gets trapped in coiled structure of Amylose to form
Starch-Iodine adsorption complex (deep blue).
6

7. Iodine test
7
Observation Inference
2.0 mL of carbohydrate
solution was taken in a test-tube
2-3 drops of Iodine solution
was added to it.
Heated to 100◦C
Cooled under tap water
Deep Blue color was
observed
Color disappear
[Dissociation of starch-
Iodine complex]
Color reapper due to
reformation of starch
iodine complex
+ve Iodine test
[Indicates +nce of
Starch in given
solution]

8. 8

9. Benedict’s Test
• General test for Reducing sugars
• Reagent’s composition:
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CuSO4 Provide Cu++
Na2CO3 Provide alkaline medium
Na-Citrate Cu++ chelating agent [slowly releases Cu++ ]

10. Benedict’s Test
• Copper reduction test in alkaline medium
• Principle:
– Reducing sugars under alkaline medium, tautomerise
to form enediols (powerful reducing agent), which
reduces Cu++ to Cu+.
CuSO4 → Cu++ + SO4
--
Cu++ + Na-citrate → Cu-Na-citrate complex
Reducing sugar → Enediol
Enediol + Cu++ → Cu+ + sugar acids
Cu+ + OH- → CuOH
2 CuOH → Cu2O (↓)
11

11. Benedict’s Test
12
Blue color -ve
Green colour Trace < 0.5gm%
Green PPT + 0.5-1.0 gm%
Green to yellow PPT ++ 1.0 – 1.5 gm%
Yellow to red PPT +++ 1.5-2.0 gm%
Brick Red PPT ++++ >2.0 gm%
• Final color formed is dependent on amount of reducing
sugars +nt in given sample, thus benedict’s test is known
as Semi-quantitative test.

12. Benedict’s Test
13
Observation Inference
•5.0ml of benedict’s reagent was
taken in a test tube.
•8 Drops of carbohydrate
solution was added to it, mixed
well & boiled for 2 min.
Brick red ppt
was seen
+ve Benedict’s Test
Indicates +nce of
reducing sugars in
given solution.

13. Barfoed’s test
• Copper reduction test in acidic medium
• Used to distinguish reducing monosachharides from
reducing disachharides.
• Reagent composition
– Copper acetate
– Acetic acid (1%)
14

14. Barfoed’s test
• Principle:
– Under controlled condition (temp. & pH), reducing
monosachharides (1-3 min) reduces Cu++ to Cu+ faster
than reducing disachharides (7-12 min).
(CH3COO)2Cu + 2 H20 → 2 CH3COOH + Cu(OH)2
Cu(OH)2 → CuO + H2O
Red. Monosachharides + CuO → Cu2O (↓) + Carboxylic acid
15

15. 16
Observation Inference
•2ml of barfoed’s reagent was
taken in a test tube.
•2ml of sample was added to it
•Was placed in boiling water
bath for exactly 3 min.
•Cooled under tap water.
•Phosphomolybdate was
dropwise added to it till it
become clear.
Red scum was
seen
Deep blue
color was
seen
+ve Barfoed’s’s Test
Indicates +nce of
reducing
monosachharides in
given solution.

16. 17

17. Seliwanoff’s test
• Used to distinguish aldoses from ketoses.
• Reagent composition
• Resorcinol
• HCl (3N)
• Principle
– Ketoses undergo dehydration to give furfural derivatives,
which condenses with resorcinol under acidic condition
to form cherry red colored complex.
– Keto groups are more reactive than aldehyde group.
18

18. Seliwanoff’s test
19

19. 20
Observation Inference
•3ml of seliwanoff’s reagent was
added to 1mL of sample.
•Boiled for 30 sec.
•Cooled under tap water
Cherry red
color was
formed
+ve seliwanoff’s Test
Indicates +nce of
ketose sugar in given
solution.

20. Osazone tests
• Reducing sugars reacts with phenylhydrazine to form
characteristics osazone crystals.
• Principle:
– When reducing sugars are treated with phenyl
hydrazine, 1st phenyl hydrazone is formed which further
reacts with 2 molecules of phenyl hydrazine to form
osazones.
22
Glucose, Fructose, Mannose Fine needle shaped
Galactose Rhombic plate shaped
Lactose Cotton ball (Puff) shaped
Maltose Sun flower shaped

21. 23
3 Molecules
Reducing Sugar
Osazone

22. 24
Observation Inference
•5-10 ml of sample was taken in
a test tube.
•1 spatula of phenyl hydrazine
hydrachloride with one spatula
of Na-acetate & 2-3 drops of
aceitic acid was added to it.
•Was boiled for 20 min.
•Cooled
•Few drops of crystal was kept
on slide, & covered with
coverslip.
Fine needle
shaped crystals
were observed.
Sunflower
shaped crystals
were observed.
Powder puff
shaped crystals
were observed.
+nce of glucose,
fructose & mannose
in given sample.
+nce of Maltose in
given solution
+nce of Lactose in
given solution.

23. 25
Lactosazone
Maltosazone
Glucosazone / Fructosazone / Mannosazone