Reactions of lactose & maltose

Reactions of lactose & maltose

1. Reactions of lactose & maltose Gandham . Rajeev

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. Reactions of lactose/maltose

3. Molisch’s test

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. Note

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. Benedict’s test

7. Benedict’s test

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 (Cu 2+ ) to cuprous ion (Cu + ) as cuprous hydroxide ( CuOH ). The cuprous hydroxide during the process of heating gets converted to different colored cuprous oxide (Cu 2 O) 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 Benedict’s test – semiquantitative test

11. Benedict’s test

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 Barfoed’s test

13. Barfoed’s test

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. Osazone test for maltose

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. Maltosazone 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. Osazone test for lactose

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. Lactosazone 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 3 rd trimester of pregnancy Lactation Clinical significance

26. Thank you

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 Spotters

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 .