The document describes various tests to identify carbohydrates and differentiate between them. Molisch's test uses α-naphthol to detect the presence of carbohydrates. Benedict's test detects reducing sugars using copper sulfate. Barfoed's test distinguishes monosaccharides from disaccharides using copper acetate. Seliwanoff's test uses resorcinol to differentiate between aldoses and ketoses. Phenylhydrazine forms characteristic crystals or osazones that can be used to identify sugars present in urine. Needle-shaped fructosazone crystals formed when fructose is treated with phenylhydrazine indicate its presence.

2.  Experiment:
 Molisch’s test:
 1 ml fructose + 2 or 3 drops of Molisch’s reagent.
 Mix well & add 1-2 ml Conc. Sulphuric acid along the
sides of the test tube without shaking.
 Observation:
 A reddish violet ring at the junction of two liquids.
 Inference:
 Fructose is a carbohydrate.

4.  Composition of Molisch’s reagent:
 1% α- naphthol in 95% ethanol.
 Principle:
 Carbohydrates when treated with Conc.
Sulphuric acid undergo dehydration to give
furfural or furfural derivatives.
 These compounds condense with α- naphthol
to form reddish violet ring.

5.  General test for all carbohydrates
 Excess α- naphthol & impurities in reagent give green
colored ring.
 Molisch’s test is given by carbohydrates with at least 5
carbons.
 Trioses & tetroses do not answer this test.
 Interaction of acid & water produces heat & can cause
charring of carbohydrates (due to precipitation of
carbon) resulting in the formation of black ring.
 Acid should be layered very slowly.

6.  Experiment:
 5 ml Benedict’s reagent + add 8 drops of fructose
solution.
 Boil it for 2 mints.
 Observation:
 Brick red precipitate.
 Inference:
 Fructose is a reducing sugar.

8.  Composition of Benedict’s reagent:
 Copper sulphate – Provides cupric ions
 Sodium carbonate – Provides alkaline medium
 Sodium citrate – Prevents precipitation of cupric
ion (chelating agent)
 Principle:
 Reducing sugars under alkaline condition form
enediols.
 Enediols are powerful reducing agents & unstable.

9.  They decompose to yield a mixture of
aldehydes that reduce cupric ion (Cu2+ ) to
cuprous ion (Cu+) as cuprous hydroxide
(CuOH).
 The cuprous hydroxide during the process of
heating gets converted to different colored
cuprous oxide (Cu2O) precipitate, which
indicates the presence of reducing sugar.

10.  The color of the precipitate gives
approximate % of sugar excreted in urine.
 Identification of reducing sugars such as
glucose, fructose, maltose & lactose.
 Clinical significance:
 0.5% - green precipitate
 1% - yellow precipitate
 1.5% - orange precipitate
 >2% - brick red precipitate

12.  Experiment:
 1 ml fructose + 2 ml of Barfoed’s reagent.
 Mix well & boil it for 1 min.
 Observation:
 Red scum
 Inference:
 Fructose is monosaccharide

14.  Composition of Barfoed’s reagent:
 Copper acetate – Provides cupric ions
 Acetic acid – Provides acidic medium
 Principle:
 It is also a reducing test
 Reduction takes place in acidic medium
 In mild acidic medium reducing sugars undergo
tautomerization to form enediols, which reduce cupric ions
to cuprous ions.
 Cuprous hydroxide is formed, during heating cuprous
hydroxide is converted to cuprous oxide, which gives red
precipitate.

15.  Note:
 Monosaccharides react very fast.
 Reaction with disaccharides is slow.
 This test is used to differentiate between
monosaccharides & disaccharides.
 Higher concentration of disaccharides (5%) give
positive Barfoed’s test.
 Prolonged boiling for 7-12 min may give positive
Barfoed’s test for disaccharides.

16.  Experiment:
 3 ml Seliwanoff’s reagent + 1 ml fructose
solution.
 Mix & boil for 30 sec.
 Cool the test tube & observe.
 Observation:
 Cherry red color.
 Inference:
 Fructose is a ketose.

18.  Composition of Seliwanoff’s reagent:
 Resorcinol in dilute hydrochloric acid.
 Principle:
 Hydrochloric acid in Seliwanoff’s reagent dehydrates the
ketoses to form furfural derivatives, which condense with
resorcinol to form cherry red colored complex.
 Note:
 Used to distinguish between aldoses & ketoses.
 Prolonged boiling may give positive Seliwanoff’s test for
aldoses due to their conversion to keto group by
hydrochloric acid.

19.  Experiment:
 3 ml fructose solution + 1 spatula of
phenylhydrazine hydrochloride + equal amount of
sodium acetate + 2-3 drops of glacial acetic acid.
 Mix well & keep the test tube in boiling water bath
for 20 min.
 Note the formation of yellow crystals.

20.  Allow the test tube to cool under tap water.
 Takeout the crystals with the help of glass rod,
mount on a glass slide using cover slip & observe
under microscope (both low & high power).
 Observation:
 Needle/broom stick shaped crystals.
 Inference:
 Fructose forms fructosazone.

21.  Needle/broom stick
shaped crystals

22.  Principle:
 When reducing sugars are treated with phenylhydrazine,
first phenylhydrazones (soluble) are formed.
 On heating, these hydrazones further react with
phenylhydrazine to form sugar osazones (insoluble).
 Non-reducing sugars like sucrose do not form an osazone.
 Significance:
 For distinguishing different reducing sugars in urine
 E.g. condition of glycosuri/lactosuria.
 This is the only test to differentiate between maltose &
lactose.

23.  Note:
 Only reducing sugars, which have free aldehyde or
keto group will form osazones.
 Glucose, fructose & mannose form identical
osazones.
 These sugars differ only C1 & C2.
 Osazone formation involves C1 & C2.
 The difference between these sugars at C1 & C2 are
masked during osazone formation.

24.  Glucosazone/fructosazone crystals are formed
during boiling itself.
 Maltosazone & lactosazone crystals are formed only
on cooling.
 Osazone of these sugars are soluble in hot solution.

25.  Fructosuria is seen in
 Hereditary fructose intolerance
 Deficiency of fructokinase

27.  Molisch’s reagent:
 It is used to detect the presence of carbohydrates.
 This reagent is prepared by dissolving 1% α-
naphthol in 95% ethanol
 Benedict’s reagent:
 Used to detect reducing sugar.
 Composed of copper sulphate, sodium citrate &
sodium carbonate
 Used in semiquantitative estimation of glucose in
urine

28.  Barfoed’s reagent:
 Used to distinguish between
monosaccharides & reducing disaccharides
 Composed of copper acetate & glacial acetic
acid
 Seliwanoff’s reagent:
 Used to distinguish between aldose & ketose
 Prepared by dissolving resorcinol in dilute
hydrochloric acid.

29.  Experiment:
 3 ml fructose solution + 1 spatula of
phenylhydrazine hydrochloride + equal amount of
sodium acetate + 2-3 drops of glacial acetic acid.
 Mix well & keep the test tube in boiling water bath
for 20 min.
 Note the formation of yellow crystals.

30.  Allow the test tube to cool under tap water.
 Takeout the crystals with the help of glass rod,
mount on a glass slide using cover slip & observe
under microscope (both low & high power).
 Observation:
 Needle/broom stick shaped crystals.
 Inference:
 Fructose forms glucosazone.

31.  Needle/broom stick
shaped crystals

32.  Principle:
 When reducing sugars are treated with phenylhydrazine,
first phenylhydrazones (soluble) are formed.
 On heating, these hydrazones further react with
phenylhydrazine to form sugar osazones (insoluble).
 Non-reducing sugars like sucrose do not form an osazone.
 Significance:
 For distinguishing different reducing sugars in urine
 E.g. condition of glycosuri/lactosuria.
 This is the only test to differentiate between maltose &
lactose.

33.  Note:
 Only reducing sugars, which have free aldehyde or
keto group will form osazones.
 Glucose, fructose & mannose form identical
osazones.
 These sugars differ only C1 & C2.
 Osazone formation involves C1 & C2.
 The difference between these sugars at C1 & C2 are
masked during osazone formation.

34.  Glucosazone/fructosazone crystals are formed
during boiling itself.
 Maltosazone & lactosazone crystals are formed only
on cooling.
 Osazone of these sugars are soluble in hot solution.

35. H-C=O
I
H-C-OH
I
R
Glucose
+ H2N-NH-C6H5
Phenylhydrazine
H-C=N-NH-C6H5
I
H-C-OH
I
R
Glucohydrazone
H-C=N-NH-C6H5
I
H-C=N-NH-C6H5
I
R
Glucosazone
H2N-NH-C6H5

36. Glucose + Phenyl hydrazine Glucose phenyl hydrazone
+ Phenyl hydrazine
Deoxy sugarGlucosazone
+ Phenyl hydrazine
H2O
- H2O
- NH3
- C6H5NH2

37.  Glucosazone/fructosazone
 These broom shaped, yellow colored crystals are
that of monosaccharides, i.e., glucose & fructose
termed as glucosazone/ fructosazone, respectively.
 They are obtained when a solution of glucose or
fructose is heated with phenylhydrazine and sodium
acetate mixture.
 Useful in diagnosis of glycosuria/fructosuria.