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Author Topic: Benedict method of estimating the carbohydrate content of a sample.  (Read 8875 times)
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Francis Umeoguaju
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« on: April 10, 2010, 08:57:10 am »



CARBOHYDRATE ASSAY METHOD

This method is used to estimate the reducing sugars such as glucose in sample. It is a direct modification of Fehling's test. Carbohydrates with a free or potentially free or aldehyde or acetone group have reducing  properties in alkaline solution.

In addition, monosaccharide acts as reducing agent in weakly acidic solution. This method of estimation of reducing sugar is based on the ability of an alkaline solution of copper II salt to be reduced to form a copper I salt precipitate. The amount of known sugar concentration that is used to reduce a fixed amount of oxidizing agent (Benedict solution) is used to estimate the reducing concentration of  the unknown sample.

REAGENT AND PREPARATION

   Sodium citrate, Sodium carbonate, Potassium thiocyanate, CuSO4  8.38% (W/V), 5% potassium  ferrocyanide, Anti bumping agent, Na2CO3, 1% standard glucose solution.
   The Benedict reagent is prepared by dissolving 50g of sodium citrate, 18.75g of sodium carbonate and 31.25g of potassium  thiocyanate in 159ml  of hot distilled water. This is mixed with 25ml of an 8.38% (w/v) of CuSO4. 5H2O solution. 1.5ml of a 5% solution of potassium ferrocyanide is then added to the mixture with thorough mixing. The resultant solution is then made up to 250ml by adding distilled water.

PROCEDURE
   
12.5ml of Benedict reagent is measured out into a conical flask, about 3g Na2CO3 was added to keep the reactions mixture alkaline. A few grain  of anti-bumping agent was added and the tube was brought to boil over the flame.
   
The reaction mixture was then titrates with 1% glucose solution with intermittent boiling. The end point is reached when the clear blue solution turns to white precipitate. The volume of the standard sugar solution used up is noted.

The unknown sample is treated in similar manner to obtain the volume of it that is required to give the white precipitate. When small samples are required the sample are introduced into the reaction mixture with micropipettes.
   
   The reducing sugar concentration is determined from this equation.
   Concn standard x Vol of standard = concn samples x Vol of samples
   
Concn sample = (Concn standard x Vol standard)/Vol sample
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