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In this article we will discuss about the principle, requirements and procedure for estimation of cellulose.
Cellulose is the major component in plants. It is composed of glucose molecules joined together in the form of repeating units of cellobiose with several cross linkages.
Principle:
With acetic/nitric reagents cellulose undergoes acetolysis forming acetylated cellod-extrins. These cellodextrins get dissolved and hydro- lysed to form glucose molecules when treated with 67% H2SO4. These glucose molecules are dehydrated to form hydroxy methyl furfural that develops a green coloured product with an throne.
Requirements:
Reagents:
1. Sulphuric acid 67%.
2. Acetic/nitric reagent:
150 ml of 80% acetic acid and 15 ml of concentrated HNO3.
3. Anthrone reagent:
200 mg of anthrone in 100 ml concentrated H2SO4. Prepare fresh and chill for 2 hours before use.
4. Test tubes and stand.
5. Water bath (100°C).
6. Centrifuge.
7. Spectronic 20.
8. Plant material,
9. Vortex mixer.
10. Standard cellulose.
11. Distilled water.
Procedure:
Preparation of standard:
1. Take 100 mg of cellulose in a test tube and add 10 ml of 67% H2S04 and allow it to stand for one hour.
Preparation of sample:
2. Mix in a vortex mixer 0.5 g of leaves and 3.0 ml acetic/nitric reagent.
3. Place the tubes in water bath at 100°C for 30 minutes. Cool and centrifuge at 1200 rpm for 15-20 minutes.
4. Discard supernatant, wash the residue with distilled water and add 10 ml of 67% H2SO4 and allow it to stand for one hour.
5. Dilute 1.0 ml of the solution to 100 ml with distilled water.
6. Take 1 ml of this diluted solution and a series of volumes 0.4, 0.6, 0.8, 1.0 and 2.0 ml (corresponding to 40-200 µg of cellulose) of the standard in tubes along with a blank of distilled water.
7. Add 10 ml of anthrone reagent to all the tubes, mix well and keep them in boiling water bath for 10 minutes.
8. Cool and measure the colour at 630 nm.
9. Plot a graph of standard and find out the concentration of Cellulose in the plant sample.