Plant Lab Protocols / Methodology for Phenolics
Lignins
Lignins are phenolic polymers present in the cell walls of plants which are responsible together with cellulose, for the stiffness and rigidity of plant stems. Lignins are especially associated with woody plants, since up to 30% of the organic matter of trees consists of lignin. Lignin acts as a physical barrier against invading pathogens.
Principle
Refluxing the sample material with acid detergent solution removes the water-solubles and materials other than the fibrous component. The left-out material is weighed after filtration, dried, treated with 72% H2SO4 and filtered, dried and ashed. The loss of weight on ignition gives the acid detergent lignin.
Materials
» Acid Detergent solution
Dissolve 20g of cetyl trimethyl ammonium bromide in one liter of IN sulphuric acid.
» 72% H2SO4 (w/v)
» Acetone
» Round Bottom Flask and Refluxing Set
» Muffle Furnace
» Sintered Glass Crucible - G2
Procedure
A. Acid Detergent Fibre (ADF)
1.
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Place 1g of powdered sample in a round bottom flask and 100mL of acid detergent solution. Heat to boil in 5 to 10 min. Reduce heat to avoid foaming as boiling begins. Reflux for 1h after the onset of boiling. Adjust boiling to slow, even level.
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2.
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Remove container, swirl and filter the contents through a preweighed sintered glass crucible (G-2) by suction and wash with hot water twice.
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3.
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Wash with acetone and break up the lumps. Repeat acetone washing until the filtrate is colorless.
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4.
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Dry at 100°C for overnight.
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5.
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Weigh after cooling in a desiccator.
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6.
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Express ADF content in percentage i.e., W/S X 100, where W is the weight of the fiber and S is the weight of the sample.
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B. Determination of Acid Detergent Lignin (ADL)
1.
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Transfer ADF to a 100mL beaker with 25-50 mL of 72% sulphuric acid. Add 1g asbestos. Allow it to stand for 3h with intermittent stirring with a glass rod.
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2.
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Dilute the acid with distilled water and filter with preweighed Whatman No. 1 filter paper. Wash the glass rod and the residue several times to get rid of the acid.
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3.
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Dry the filter paper at 100°C and weigh after cooling in a desiccator.
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4.
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Transfer the filter paper to a preweighed silica crucible and ash the filter paper with the content in a muffle furnace at 550°C for about 3h.
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5.
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Cool the crucible in a desiccator and weigh. Calculate the ash content.
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6.
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For blank take 1g asbestos, add 72% H2SO4 and follow the steps 2 to 5.
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Calculation
% ADL =
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Weight of 72% H2SO4 washed fiber
(Test – Asbestos blank)
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–
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Ash
(Test – Asbestos blank)
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x 100
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Weight of sample
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Notes
1.
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For preparing preweighed filter paper, see the note under pectic substances.
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2.
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NDF = Hemicellulose + Cellulose + Lignin + Minerals
ADF = Cellulose + Lignin + Minerals
Hemicellulose = NDF - ADF
Cellulose = ADF - Residue after extraction with 72% H2SO4
Lignin = Residue after extraction with 72% H2SO4 - ash
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3.
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Wet materials can also be used for lignin estimation
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4.
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If the weight loss of asbestos blank on ashing is below 0.002g/g
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References
1. Official Methods of Analysis of the AOAC 12th Edn (1975) p 138.
2. Goering, H D and Van Soest, P J (1975) Forage Fibre Analysis U S Dept of Agriculture Agricultural Research Service Washington DC.
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