Reactions of lactose & maltose
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Transcript of Reactions of lactose & maltose
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Reactions of lactose & maltose
Gandham. Rajeev
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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.
Reactions of lactose/maltose
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Molisch’s test
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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.
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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
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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.
Benedict’s test
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Benedict’s test
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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.
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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.
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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
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Benedict’s test
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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
Barfoed’s test
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Barfoed’s test
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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.
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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.
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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.
Osazone test for maltose
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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.
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Maltosazone
Sunflower shaped crystals
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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.
Osazone test for lactose
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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.
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Lactosazone
Powderpuff or hedgehog shaped crystals
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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.
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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.
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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.
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Lactosuria is seen in 3rd trimester of pregnancy Lactation
Clinical significance
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Thank you
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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
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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.
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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.