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Practical no:6

Practical no:6. Benedict’s Test. Benedict’s test is qualitative and semi-quantitative test for the reducing carbohydrates. Benedict's test is more specific than Fehling’s test. Moreover, in Benedict’s test, uric acid and creatinine do not interfere its results . Reducing sugars.

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Practical no:6

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  1. Practical no:6 Benedict’s Test

  2. Benedict’s test is qualitative and semi-quantitative test for the reducing carbohydrates. Benedict's test is more specific than Fehling’s test. Moreover, in Benedict’s test, uric acid and creatinine do not interfere its results.

  3. Reducing sugars Are those which have potentially active aldehydic or kenotic group in their structures. These sugars have free anomeric carbon. They have ability to give H+ to other substances so can reduce others and themselves get oxidized e.g. glucose, fructose, galactose , maltose, lactose

  4. Reagents Benedict’s qualitative reagent consists of the following: • Copper sulphate • Sodium citrate • Anhydrous sodium carbonate • Benedict’s reagent is prepared by dissolving 173gm of sodium citrate & 90gm of anhydrous sodium carbonate in about 750ml of distilled water. Slightly heat to disslove the contents and filter the solution. Dissolve separately 17.3gm of copper sulfate in about 100ml of water, & then add to the solution of sodium citrate & sodium carbonate with continuous stirring. Finally, the volume is made up to 1000ml with water.

  5. COPPER SULFATE • It supplies cupric ions (Cu++) in solution • SODIUM CARBONATE • It makes the medium alkaline. In alkaline medium the process of reduction is rapid and more complete • SODIUM CITRATE • it prevents the precipitation of cupric ion by forming a loose complex with it.

  6. Principle • The principle is similar to that of the Fehling’s test. The reducing sugars can reduce cupric ions (Cu++) to cuprous ion (Cu+) which is the basis of Benedict’s and Fehling’s tests. Copper sulphate hydrolyses to form cupric hydroxide(the hydroxyl, OH- , group comes from dissociation of water). Cupric hydroxide is reduced of cuprous oxide on heating with a reducing carbohydrate. CuSo4 Cu++ +So4 Cu++ + 2 OH-- Cu (OH)2 CU(OH)2 + Sodium citrate Cu(OH)2 : sodium citrate complex Cu(OH)2 : sodium citrate complex + reducing sugar Cu+ + 2OH + oxidized sugar + Sodium citrate

  7. Cu+ + OH- CuOH 2CuOH Cu2O + H2O Cuprous hydroxide, during the process of heating, is converted to red cuprous oxide, which precipitates immediately. The precipitation of cupric hydroxide is avoided by sodium citrate. In Benedict’s test, alkaline medium is provided by sodium carbonate.

  8. we should note that the ultimate quantity of Cu2 O (red precipitate) produced at the end of reaction depends upon the amount of reducing sugar present in the sample used. We also know that the Benedict's reagent has blue colour . Therefore, the final colour of solution is the mixture of blue colour of Benedict’s reagent with the red colour of precipitated Cu2 o. it may vary form green to brick red depending upon the concentration of reducing sugar up to 2% . If the sample contains reducing sugar more than 2% , the final colour of solution will remain red, because further production of red Cu2O will not effect the already present red colour of solution.

  9. Procedure • Take 5 ml of Benedict's reagent in a test tube. Add 8 drops of the given solution. • Mix and boil over a flame or in a boiling water for 2 minutes. Cool the solution

  10. Interpretation The appearance of a green, yellow, orange or red precipitate indicates that the carbohydrate is a reducing one. This is a semi quantitative test. If solutions are taken in correct proportion and the procedure following strictly, the approximate concentration of the carbohydrate can be judged from the colour of the precipitate.

  11. Colour of the precipitate • Green • Yellow • Orange • Red • Brock Red • Approximate concentration of carbohydrate (gm %) • 0.1-0.5 or ( + ) • 0.5 – 1.0 or ( + + ) • 1.0 – 2.0 or ( + + + ) • 1.5 – 2.0 or ( + + + + ) • Above 2.0

  12. Fallacies This test also gives false positive results in the presence of some non-carbohydrate reducing agents in the sample such as ascorbic acid (vitamin C) or the end products of certain drugs like aspirin (salicylic acid)

  13. Practical applications This is the most widely employed test for the detection of glucose in urine. It is commonly used for the preliminary screening for diabetes mellitus and for monitoring the effect of treatment.

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