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Lab Activity 3 Carbohydrates Reactions

Lab Activity 3 Carbohydrates Reactions. Alaa S Baraka Islamic university of Gaza Feb 2013. Content. Formation of osazons Test for individual carbohydrates: Bial's (Orcinol) Test for pentoses Aniline Acetate Test for Pentoses Seliwanoff's (Resorcinol) Test. Formation of osazones.

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Lab Activity 3 Carbohydrates Reactions

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  1. Lab Activity 3Carbohydrates Reactions Alaa S Baraka Islamic university of Gaza Feb 2013

  2. Content • Formation of osazons • Test for individual carbohydrates: • Bial's (Orcinol) Test for pentoses • Aniline Acetate Test for Pentoses • Seliwanoff's (Resorcinol) Test

  3. Formation of osazones • Osazones are formed when the sugars(monosaccharides) react with a compound known as phenylhydrazine • These sugars are reducing ones which have either a free aldehyde or a ketone group to react with the phenylhydrazine.

  4. Introduction • Phenylhydrazine is the chemical compound with the formula C6H5NHNH2

  5. General Reaction of osazons formation • This reaction is complete in 3 step and consume 3 moles of phenylhydrazine • During reaction with monosaccharides, additional phenyl hydrazine is consumed in oxidizing the adjacent OH-group to carbonyl group which then forms a second phenyl hydrazone. • Such bisphenyl hydrazones are calledosazones.

  6. Osazone formation involves hydrazone formation at C-1 of an aldose (or C-2 of a ketose) and oxidation of C-2 (or C-1) of an alcohol group to a ketone (or an aldehyde). The new carbonyl group is also converted to a hydrazone.

  7. General Reaction of osazons formation

  8. osazones • a crystalline compound with a sharp melting point will be obtained • Since only C1 & C2 of a saccharide are involved in osazones, sugars with the same configuration at the remaining carbon atom gives the same osazone. • D-fructose and D-mannose give the same osazone as D-glucose • seldom used for identification; we now use HPLC or mass spectrometry

  9. osazones • Depending on the time required to form the insoluble yellow osazone, different sugars can be classified into the following: Mannose: 1-5 min Fructose: 2 min Glucose:5 min Xylose: 7 min Arabinose: 10 min Galactose: 20 min Maltose osazone soluble in hot water

  10. Application • For identifying sugars esp. Reducing sugars. • Osazones are used as dyes

  11. Experiment 1: Formation of Osazones(important in determining sugar structure) Reagents: • 1% solutions of glucose, fructose, maltose, mannose, and xylose • Phenyl hydrazine mixture (2 parts phenyl hydrazine hydrochloride are mixed with 3 parts sodium acetate).

  12. Procedure • To 300 mg of phenyl hydrazine mixture add 5 ml of the tested solution, • Shake well, and heat on a boiling water bath for 30 – 45 min. • Allow the tubes to cool slowly (not under tap) and examine the crystals microscopically, draw the shapes of the crystalls

  13. Results: Viewed under the microscope: Glucosazone

  14. Viewed under the microscope: Fructosazone

  15. Viewed under the microscope: Galactosazone

  16. Xylosazone: Viewed under the microscope

  17. Tests for Individual Carbohydrates

  18. 1. Bial's (Orcinol) Test for pentoses( for the detection of pentoses) • Principle • Pentoses are converted to furfural by this reagent, which formsa blue greencolor with orcinol

  19. Reagents • Set up 1 % solutions of: • xylose, • arabinose, • glucose, • fructose, • maltose. • Bial's reagent (0.1 % orcinol in concentrated HCl containing 0.1 % FeCl3.6H2O).

  20. Procedure • 1. Add about 2 ml of 1% xylose, glucose, fructose, maltose, in test tubes. • 2. Add 3 ml of Bial's reagent to each tube and mix well. • 3. Carefully heat in boiling water bath • A blue-green color indicates a positive result. Prolonged heating of some hexoses yields hydroxymethyl furfural which also reacts with orcinol to give colored complexes.

  21. Aniline Acetate Test(for Pentoses) • The furfural produced by the reaction of HCl on pentoses forms a bright red color with aniline acetate in a test paper held over the mouth of the reaction flask.

  22. Reagents • 1% solution of glucose, xylose • Aniline solution is prepared as follows: Shake 5 ml of aniline with 5 ml of water and add 5 ml of glacial acetic acid to adjust clear the emulsion.

  23. Procedure • Place 2.5 ml of solution to be tested and 10 ml water in 250 ml Erlenmeyer flask. • Add 10 ml of conc. HCl and boil gently for about 1 min. • Cease heating, hold a filter paper moistened with a few drops of aniline acetate over the mouth of the flask. • Bright red color on a filter paper indicates a positive result.

  24. Seliwanoff's (Resorcinol) Test(used for detection of Ketoses) • Principle • Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as sucrose) form a cherry-red condensation product. Other sugars (e.g. aldose) may produce yellow to faint pink colors.

  25. Reagents • Set up 1 % solution of: • glucose, • sucrose, • fructose, • lactose, • maltose. • Seliwanoff's reagent (0.5 % resorcinol in 3N HCl).

  26. Procedure • Add about 2 ml of Seliwanoff's reagent to each labeled test tube. • Add 1 ml of sugar solution to the test tubes and mix well. • Place all the test tubes in the boiling water bath at and heat for 3 min after the water begins to boil again. • A positive result is indicated by the formation of • A red color with or without the separation of a brown-red precipitate.

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