Method 71b Determination of lignin


• Potassium permanganate, approximately saturated solution - (Note: wear rubber gloves to prevent staining hands.) dissolve 50 g KMnO4 in l l water (solubility = 65 g l-1 at 20°C). Store in a brown glass bottle.

• Buffer solution - dissolve 6.0 g ferric nitrate, Fe(NO3)3.9H2O, and 0.15 g silver nitrate, AgNO3, in water and make up to 100 ml; add this to a solution of 5.0 g potassium acetate in 500 ml glacial acetic acid in a 2-l beaker and stir to mix. Add 400 ml 2-methylpropan-2-ol (tertiary butyl alcohol, (CH3)3COH; this solidifies ^25.5°C, therefore it may need warming to melt before use), and stir to mix. Store in a brown glass bottle.

• Combined permanganate and buffer solution - add two parts by volume saturated KMnO4 solution to one part buffer solution and mix. This will keep for 1 week if refrigerated, therefore only make sufficient for this period's analyses.

• Demineralizing solution - dissolve 50 g oxalic acid ((COOH)2.2H2O) in 700 ml 95% v/v ethanol. Add 50 ml hydrochloric acid (approximately 36% m/m) and 250 ml water and mix.

• Ethanol, approximately 76% v/v - add 200 ml water to 800 ml 95% v/v ethanol.

Procedure. Wear PPE - rubber gloves, face shield and lab coat when handling the permanganate-buffer solution. Place the weighed sintered glass crucibles containing the ADF residue one at a time into a stainless steel or polythene tray containing 2-3 cm cold water (a 400 x 320 x 50 mm tray will hold 48

crucibles). Holding the crucible, immediately add 25 ml of the combined permanganate and buffer solution to each crucible. Adjust the water level so that there is only a small diffusion of permanganate-buffer out of the crucibles. Place a short glass rod (approximately 80 x 4 mm with the ends rounded in a flame) into each crucible, break up the residue mat and stir to ensure thorough contact of the particles with the reagent; leave the rod in the crucible. Allow to stand for approximately 90 min at 20-25°C, and top up with permanganate-buffer as required. The mixture in the crucible should remain purple; if it turns brown, it is exhausted and should be replaced by fresh reagent. Filter the crucibles but do not wash at this time. Place the crucibles in a clean empty tray, half fill with demineralizing solution and stir with the rod to mix. Allow to stand for 5 min, suck dry, and half refill with demineralizing solution, washing down the sides of the crucible. Allow to stand for 20-30 min until the fibrous residue is white. Filter with gentle suction, then remove suction and fill with 80% v/v ethanol, stir thoroughly and suck dry. Repeat this twice more, then wash twice with acetone and suck dry. Allow to air-dry in a fume cupboard until no smell of acetone is detectable, then dry overnight in an oven at 100°C, cool in a desiccator, and weigh.

Calculation. Subtract the weight of the crucible plus fibre after oxidation from the weight of the crucible plus ADF, divide by the initial sample weight and multiply by 100 to obtain the % lignin in the sample DM (air-dry matter for animal feeds).

Notes on the reagents. The permanganate reagent oxidizes and dissolves the lignin and any tannins. The 2-methylpropan-2-ol enhances the wetting of the ADF fibres by the permanganate. The acetic acid in the buffer solution is to neutralize the alkali formed in the oxidation reaction:

The silver nitrate helps preserve the permanganate solution from decomposition. The oxidation reaction is incomplete below 18°C and too vigorous above 25°C. The 90 min oxidation stage is not long enough for the complete removal of lignin from faeces, bark or wood. The demineralizing reagent removes MnO2 from the cellulose fibres. Water must be present to remove the acetic acid and also the MnO2. Ferric nitrate provides the ferric ion which prevents precipitation of manganous oxalate, and potassium acetate prevents free HNO3 formation from acetolysis of the ferric nitrate.

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