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Galactose detection qualitatively Osazone Test To 0.5 g of phenylhydrazine hydrochloride add 0.

1 g of sodium acetate and 10 drops of glacial acetic acid. To this mixture add 5 mL of test solution and heat on a boiling water bath for about half an hour. Allow the tube to cool slowly and examine the crystals under a microscope. Glucose, fructose and mannose produce needle-shaped yellow osazone crystals, whereas lactosazone is mushroomshaped/ Badminton ball, powder puff shaped crystals. Different osazones show crystals of different shapes. Maltose produces flower-shaped crystals.

PHENYL HYDRAZINE TEST(OSAZONE TEST) This is a test for reducing carbohydrate, reducing dissacharides and many monosaccharides to be identified with the formation of osazone crystals.Hence this test is known as osazone formation test. REAGENTS : 1)Phenyl hydrazine hydrochloride 2) Sodium acetate 3)Glacial acetic acid Osazone mixture is prepared by mixing thoroughly one part on phenyl hydrazine hydrochloride and two parts of sodim acetate by weight. PROCEDURE: 1)Take 5ml each of glucose, fructose, maltose and lactose solution in different test tubes and label them. Add about 0.3g of osazone mixture and 3 drops of glacial acetic acid in all test tubes 2)Put all test tubes n boiling water bath, observe each tube after every 5 mins till crystals appear. Note in each test tube how much time in min is been taken by the crystals to appear If no crystals appear after 30 min remove these test tube from water bath and allow them to cool spontaneously, observe after every 5 mins till crystals appear upto 25 min. With the help of a glass rod take out some crystals on a glass slide, cover them with cover slip and observe under microscope. INTERPRETATION: Yellow crystals in the test tubes containing glucose and fructose within 10 mins of heating, Galactose will give crystals within 20 mins of heating Maltose will give crystals within 10-15 min of cooling Lactose will give crystals within 20-25 mins of cooling Generally monosaccharide give crystals on heating and all diasaccharide give crystals on cooling. PRINCIPLE:

When the reducing carbohydrate are treated with phenyl hydrazine at 100c and PH 4.3. A series of reactions take place resulting in formation of osazone of respective carbohydrate Glucose + 3 phenyl hydrazine mol Glucosazone + NH3 + H2O A constant temp is provided by boiling water. A constant PH is provided by buffer pair of sodium acetate and aceticacid. There action involves only 1st and 2nd carbon atoms of reducing carbohydrate. Since the glucose and fructosediffer with respect to 1st and 2nd carbon atom. They form the same osazone. Mannose will form the same osazone.Galactose differ from glucose at carbon no 4.Therefore its crystal shape is different. Sucrose and starch dont formcrystals because they are non-reducing sugar. They can form crystals only when they are first hydrolyzed. PRECAUTIONS: 1)Reagents should be taken in correct proportion 2)Cooling should always be spontaneous preferably in water bath itself 3) Before boiling 4)It should be noted that the solution is not too concentrated because crystals will not form a very concentrated solution