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Identification of sugars by molisch (1)

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Identification of sugars by molisch (1)

  1. 1. 1. (a) Identification of sugars by Molisch’s test. Molisch’s test is a general test for the identification of all carbohydrates (monosaccharide, disaccharide, and polysaccharide) and glycoprotein. In this test conc. sulphuric acid added to the solution which hydrolyzes the all glycosidic linkage in the sugar molecules (disaccharide, and polysaccharide) to yield monosaccharide, which in the presence of an acid get dehydrated to form furfural and its derivatives. This is very reactive, and condenses with α- naphthol to give a purple or violet colored product. Reaction: Procedure:- Prepareed 5% α-naphthol reagent in ethanol. Added 2 to 3 drops of α-naphthol reagent to 2ml of sugar solution in the test tube. Incline the test tube and very gently added few drops of conc. H2SO4 along the side of test tube. violet colored ring indicates the presence of carbohydrates in the solution. Discussion: Violet rings formed at the junction which indicated the presence of carbohydrates in the sample. 1. (b)Identification of reducing sugar by using Fehling’s test Fehling’s test is a specific test for the identification of reducing sugar. When Fehling’s reagent react with reducing sugar it reduces into brownish-red precipitate Oxidation of carbonyl group take place which give aldonic acid. Formation Brownish-red precipitates indicate the presence of reducing sugar in the sample. Fehling’s reagent consists of two solution Fehling’s solution A (CuSO4) and Fehling’s solution B (KOH+ Na-K tartrate). Mix both solutions before adding the sugar sample.
  2. 2. Reaction: CuSO4 + 2KOH Cu (OH) 2 + K2SO4 ; Cu (OH) 2 CuO + H2O OH O H OH H OH OH H H OH + CuO OH O H OH H OH OH H H OH OH Cu 2O + Procedure: Prepared Fehling’s solution A by dissolving 7.0g CuSO4.7 H2O in distilled water, and then adjusting the total volume to 100ml. For Fehling’s solution B, dissolved 24g of KOH and 34.6 of Na-K tartrate in distilled water and make up the volume to 100ml. mix solution A & B in 1:1 ratio.Added 5-8 drops of sugar samples to 5ml of Fehling’s solution and boil for 2-3 min. Discussion: The precipitate of light brown appeared which show the presence of reducing sugar in the sample. 1. (c) To detect reducing sugar by performing the Osazone test. When phenyl hydrazine reacts with reducing sugars at a boiling temperature, osazone is formed. Phenyl hydrazine reacts with carbonyl compound in neutral or slightly acidic condition to give phenyl hydrazone, which is highly soluble. When hydrazone reacts with further phenyl hydrazine molecules, the condensation products formed are insoluble which precipitate out as crystals. Reaction:
  3. 3. Procedure: Mixed 2g phenyl hydrazine and 3g sodium acetate and transfered 0.3g of this mixture separately in six cleans test tubes.Added 5ml of 1% glucose, fructose, maltose, lactose, sucrose, and distilled water separately into these test tubes.Incubate the tubes in a boiling water bath for 30-45 min.Cool and observed test tubes for the shape of different sugar osazone crystal formed by using a light microscope having magnification of 10x. For fructose and glucose needle shaped crystals of osazone can be observed with unaided eye as well as by using microscope. Maltose and lactose formed flower shaped crystals whereas no osazone formation for sucrose. Disaccharide, namely lactose and maltose yield osazone crystals whish are soluble in hot water, and hence, they separate out as crystal only on cooling. 1. (d)Estimation of reducing sugar by performing Benedict’s test Benedict test is just like Fehling’s test the only difference is that in benedicts test is carried out weak alkaline condition by the sodium carbonates, whereas the reduction reaction in Fehling’s test is brought about in strong alkaline condition by using KOH. The reagent used for Benedict’s test contains CuSO4.7 H2O in alkaline citrate solution. Reducing sugars can reduce the cupric ions into red color cuprous oxide, and turns oxidized into sugar acid. Reaction: Na2CO3 + 2H2O 2NaOH +H2CO3 2NaOH +CuSO4 Cu (OH) 2+ Na2SO4 ; Cu (OH) 2 CuO + H2O D-glucose + 2CuO + Heat D-dluconic acid + Cu2O Procedure: Dissolved 17.3g sodium citrate and 10g sodium carbonate in 85ml of distilled water..Dissolve 1.73g CuSO4.7 H2O in 10ml distilled water and while stirring, add slowly to the alkaline citrate solution.Adjust the total volume to 100ml using distilled water.To the 5ml of the reagent, add 8 drops of the sugar solution. Boil 2-3 min and observe the change. Similarly, for the control, add distilled water to Benedict’s reagent instead of sugar. Discussion: the brown precipitate were formed which indicate the presence of reducing sugar in the sample. 1. (e) Detection of Ketose sugars by performing Seliwanoff’s test. Seliwanoff’s test is a specific test for the detection of ketose sugars namely fructose. Aldose, however, failed to respond to this test. Ketoses undergo dehydration to give
  4. 4. furfural derivatives, which then condense with resorcinol to form a red colored complex. Seliwanoff’s test is responded positively by the sucrose as it gets hydrolyzed to give glucose and fructose due to the action of conc. HCl present in the reagent. Fructose being a keto sugar gives red colored complex when react with Seliwanoff’s reagent. Reaction: Procedure: Prepare Seliwanoff’s reagent by dissolving 50mg resorcinol in 100ml of 3N HCl.Added 8 drop of a sugar sample to 5ml of the reagent and boil for exactly 30 sec. keep a parallel control that contain distilled water instead of a sugar solution.Observe the tube for the presence of a red colored complex, which indicates the presence of a keto sugar. Reference: Lab manual in biochemistry by Arti Nigam.
  5. 5. OrganicCompound water soluble soluble soluble insoluble insoluble insoluble test withpHpaper acidic basic neutral 6MNaOH 6MHCl amines (lowMW) aldehydes ketones alcohols (lowMW) carboxylic acids (lowMW) amines (highMW) carboxylic acids (highMW) aldehydes ketones alcohols (highMW) or alkanes alkenes alkyl halides (loworhighMW)

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