The document describes several tests used to identify carbohydrates and sugars: - Molisch's test uses α-naphthol to detect the presence of carbohydrates, forming a reddish-violet ring. - Benedict's test uses copper sulfate to detect reducing sugars, forming different colored precipitates. - Barfoed's test distinguishes monosaccharides from disaccharides by their reaction speed. - Phenylhydrazine forms characteristic crystals (osazones) with different sugars that can be viewed under a microscope.

2.  Experiment:
 Molisch’s test:
 1 ml lactose/maltose + 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:
 Lactose/maltose 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
lactose/maltose solution.
 Boil it for 2 mints.
 Observation:
 Brick red precipitate.
 Inference:
 Lactose/maltose 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 lactose/maltose + 2 ml of Barfoed’s
reagent.
 Mix well & boil it for 1 min.
 Observation:
 Red scum is not seen
 Inference:
 Lactose/maltose is a disaccharide

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 maltose 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.

17.  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:
 Sunflower shaped crystals.
 Inference:
 Maltose forms maltosazone.

18. Sunflower shaped crystals

19.  Experiment:
 3 ml lactose 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:
 Powderpuff or hedgehog shaped crystals.
 Inference:
 Lactose forms lactosazone.

21. Powderpuff or hedgehog 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.  Lactosuria is seen in
 3rd trimester of pregnancy
 Lactation

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.  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.