PRACTICAL NO PHENYL HYDRAZINE TEST OSAZONE TEST TO IDENTIFY REDUCING SUGAR IN A GIVEN SOLUTION. • This test is for reducing carbohydrates. • Reducung disaccharides and many monosaccharides can be identified with the formation of characteristic Osazone crystals’ • Hence this test is also known as Osazone formation test. REAGENTS: • Phenyl hydrazine hydrochloride • Sodium acetate • Glacial acetic acid • Osazone mixture is prepared by mixing thoroughly one part of phenyl hydrazine hydrochloride and two parts of sodium acetate by weight. PRINCIPAL: • When the reducing carbohydrates are treated with phenyl hydrazine at 100 C and pH 4.3 a series of reaction takes place resulting in the formation of osazones of respective carbohydrates. The general reaction is • Glucose + 3 phenylhydrazine molecules Glucosazone + NH3 +H2O • A constant temperature is provided by the boiling water bath . • A constant pH is provided by the buffer pair of sodium acetate and acetic acid. • The reaction involves only first and second carbon atoms of the reducing carbohydrate. • Since glucose and fructose differ only with respect to the 1st and 2nd carbon atoms they form the same osazones. Mannose will also form the same osazone. Galactose differs from glucose at carbon number 4,therefore its crystal shape is different. • Sucrose and starch do not form crystals, because they are non reducing sugars. They can form crystals only if they are first hydrolyzed into their components. PROCEDURE • Take 5ml each of glucose,fructose,galactose,maltose and lactose solutions in correspondingly label test tubes. • Add about 0.3gm of osazone mixture and 3 drops of glacial acetic acid in all the test tubes. • Put all test tubes in boiling water bath. • Observe each test tube after every 5 minutes till crystals appear. • Note in each test tube how much time (in minutes) is being taken by the crystals to appear. • If no crystal appears after 30 minutes,remove these tubes from the water bath and allow them to cool spontaneously. • Observe after every 5 minutes till crystals appear upto 25 min. • With the help of a glass rod,take out some crystals on a glass slide, cover them with a cover slip and observe under a microscope. INTERPRETATION • Yellow crystals will appear in the tubes containing glucose and fructose within 10 min of heating. • Galactose gives crystals within 20 min of heating. • Maltose gives crystals within 10-15 min of cooling. • Lactose gives crystals within 20-25 min of cooling. • Generally monosaccharides give crystals on heating and all disaccharides give crystals on cooling. • The osazones of different carbohydrates differ in their time of formation,solubility,melting point and crystalline structure. • Carbohydrates osazone solubility shape of crystals glucose glucosazone insoluble bundle of grass mannose mannosazone insoluble bundle of grass fructose fructosazone insoluble bundle of grass galactose galactosazone insoluble needle shaped maltose maltosazone soluble sunflower shaped lactose lactosazone soluble puff ball shaped PRECAUTIONS • Reagents should be taken in correct proportion. • Cooling should always be spontaneous,preferably in the water bath itself. • Before boiling it should be noted that the solution is not too concentrated,because crystals will not be formed in very concentrated solution.