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  Section: Plant Lab Protocols
 
 
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Methodology for Carbohydrates

 
     
 
Estimation of cellulose
 
Cellulose, a major structural polysaccharide in plants, is the most abundant organic compound in nature, and is composed of glucose units joined together in the form of the repeating units of the disaccharide cellobiose with numerous cross linkages. It is also a major component in many of the farm wastes.
 




Principle

Cellulose undergoes acetolysis with acetic/nitric reagent forming acetylated cellodextrins wich gets dissolved and hydrolyzed to form glucose molecules on treatment with 67% H2SO4. This glucose molecule is dehydrated to form hydroxymethyl furfural which forms green colored product with anthrone and the color intensity is measured at 630nm.
 



Materials

Acetic/Nitric Reagent: Mix 150ml of 80% acetic acid and 15mL of concentrated nitric acid.
Anthrone: Dissolve 200mg anthrone in 100mL of ice-cold 95% sulphuric acid. Prepare fresh and chill for 2h before use.
67% sulphuric acid
 
Procedure
1.
Add 3mL acetic/nitric reagent to a known amount (0.5g or 1g) of the sample in a test tube and mix in a vortex mixture.
2.
Place the tube in a water bath at 100°C for 30min.
3.
Cool and then centrifuge the contents for 15-20min.
4.
Discard the supernatant.
5.
Wash the residue with distilled water.
6.
Add 10mL of 67% sulphuric acid and allow it to stand for 1h.
7.
Dilute 1mL of the above solution to 100mL.
8.
To 1mL of this diluted solution, add 10mL of anthrone reagent and mix well.
9.
Heat the tubes in boiling water bath for 10min.
10.
Cool and measure the color at 630nm.
11.
Set a blank with anthrone reagent and distilled water.
12.
Take 100mg cellulose in a test tube and proceed from step No. 6 for standard. Instead of just taking 1mL of the diluted solution (Step 7) take a series of volumes (say 0.4 to 2mL corresponding to 40-200mg of cellulose) and develop the color.




Calculation

Draw the standard graph and calculate the amount of cellulose in the sample.




Reading

1. Updegroff, D M (1969) Anal Biochem 32 420.

 
     
 
 
     




     
 
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