Reactions of lactose & maltose

Gandham. Rajeev

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

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

lactose/maltose solution. Boil it for 2 mints. Observation: Brick red precipitate. Inference: Lactose/maltose 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 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

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 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

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.

Maltosazone

Sunflower shaped crystals

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

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.

Lactosazone

Powderpuff or hedgehog 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.

Lactosuria is seen in 3rd trimester of pregnancy Lactation

Clinical significance

Thank you

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.

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.