reactions of fructose
TRANSCRIPT
Reactions of Fructose
Gandham. Rajeev
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.
Reactions of Fructose
Molisch’s test
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.
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.
Note
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.
Benedict’s test
Benedict’s test
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.
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.
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
Benedict’s test – semiquantitative test
Benedict’s test
Experiment: 1 ml fructose + 2 ml of Barfoed’s reagent. Mix well & boil it for 1 min. Observation: Red scum Inference: Fructose is monosaccharide
Barfoed’s test
Barfoed’s test
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.
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.
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.
Seliwanoff’s test
Seliwanoff’s test
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.
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.
Osazone test for fructose
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.
Fructosazone
Needle/broom stick shaped crystals
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.
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.
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.
Fructosuria is seen in Hereditary fructose intolerance Deficiency of fructokinase
Clinical significance
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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
Spotters
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.
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.
Osazone test for fructose
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.
Fructosazone
Needle/broom stick shaped crystals
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.
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.
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.
H-C=OI
H-C-OHIR
Glucose
+ H2N-NH-C6H5
Phenylhydrazine
H-C=N-NH-C6H5 I
H-C-OH I R
Glucohydrazone H-C=N-NH-
C6H5I
H-C=N-NH-C6H5
IR
Glucosazone
H2N-NH-C6H5
Osazone formation
Glucose + Phenyl hydrazine Glucose phenyl hydrazone
+ Phenyl hydrazine
Deoxy sugarGlucosazone+ Phenyl hydrazine
H2O
Osazone formation - H2O
- NH3- C6H5NH2
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.
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