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  Section: Plant Lab Protocols
 
 
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Methodology for Carbohydrates

 
     
 
Phenol suplhuric acid method for total carbohydrate

The phenol sulphuric acid method to estimate total carbohydrates is described below:




Principle

In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms a green colored product with phenol and has absorption maximum at 490nm.




Materials
Phenol 5%: Redistilled (reagent grade) phenol (50g) dissolved in water and diluted to one liter.
Sulphuric acid 96% reagent grade.
Standard Glucose: Stock – 100mg in 100mL of water. Working standard – 10mL of stock diluted to 100mL with distilled water.


Procedure

1.

Weigh 100mg of the sample into a boiling tube.

2.

Hydrolyse by keeping it in boiling water bath for 3 hours with 5mL of 2.5 N-HCl and cool to room temperature.

3.

Neutralise it with solid sodium carbonate until the effervescence ceases.

4.

Make up the volume to 100mL and centrifuge.

5.

Pipette out 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard into a series of test tube.

6.

Pipette out 0.1 and 0.2mL of the sample solution in two separate test tubes. Make up the volume in each tube to 1mL with water.

7.

Set a blank with 1mL of water.

8.

Add 1mL of phenol solution to each tube.

9.

Add 5mL of 96% sulphuric acid to each tube and shake well.

10.

After 10min shake the content in the tubes and place in a water bath at 25-30°C for 20min.

11.

Read the color at 490nm.

12.

Calculate the amount of total carbohydrate present in the sample solution using the standard graph.






Calculation

Absorbance corresponds to 0.1mL of the test = ‘x’ mg of glucose

100mL of the sample solution contains =

‘x’

X 100mg of glucose

0.1


                                                                       =   % of total carbohydrate present





Readings

1. Dubois, M, Gilles, K A, Hamilton, J K, Rebers, P A and Smith, F (1956) Anal Chem 26 350.
2. Krishnaveni, S, Theymoli Balasubramanian and Sadasivam, S (1984) Food Chem 15 229.

 
     
 
 
     




     
 
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