Acetylbromide lignin

The acetyl bromide procedure was developed by Johnson et al. (1961) to determine lignin content in small samples from woody species. This method uses acetyl bromide (4.10) to acetylate unbound hydroxyl groups in the lignin (4.11), while the hydroxyl group on the a-carbon is substituted with a bromine group. The substituted lignin derivative (4.12) is soluble under acidic conditions, and its concentration can be measured with a spectrophotometer at 280 nm.

Figure 4-3. Reaction of acetyl bromide with lignin. Adapted from Hatfield and Fukushima (2005).

Figure 4-3. Reaction of acetyl bromide with lignin. Adapted from Hatfield and Fukushima (2005).

Acetyl bromide lignin is obtained from isolated cell walls. These can be obtained by extraction of ground woody samples in ethanol and chloroform.

The isolated cell walls or the dioxane lignin are dissolved by incubation in 2.5 mL of 25% (w/v) acetyl bromide in glacial acetic acid at 70°C for 30 min. The lignin is extracted by adding the dissolved cell walls to a flask containing 10 mL 2 M sodium hydroxide and 12 mL glacial acetic acid. A 1.75-mL volume of 0.5 M hydroxylamine is then added, and the total volume is brought up to 50 mL with acetic acid. The lignin content is determined spectrophotometrically by measuring the absorbance at 280 nm.

Modifications have been made to the original protocol in order to apply this method to herbaceous species. Iiyama and Wallis (1990) added 100 ^L perchloric acid (HClO4) to improve the dissolution of wall material. This decreased the overall time required for the procedure and made the use of hydroxylamine unnecessary.

Hatfield et al. (1999) reexamined the different protocols used for the determination of lignin content in herbaceous species, using alfalfa stems and corn rind as representative samples. They performed the initial incubation at 50°C for up to 4 h., at 60°C for up to 2 h., or at 70°C for up to 1 h., and with or without 100 ^L perchloric acid. They concluded that the addition of perchloric acid indeed raises the overall absorbance values, but that this increase is the result of oligomeric xylan-degradation products that also absorb light in the 250-300-nm range. In order to avoid excessive xylan degradation, they recommended performing the extraction in acetyl bromide at 50°C for a period of 2-4 h, in the absence of perchloric acid.

As for any spectrophotometric method, a standard curve needs to be made. Since lignin is very heterogeneous in nature (variation in subunit composition and interunit linkages within and between species), finding a good standard is not trivial. Fukushima and Hatfield (2001) recommended using lignin extracted with dioxane-HCl, rather than isolated cell walls, because it contains little protein and polysaccharide residues. Isolating dioxane lignin requires adding 100 mL of acidified dioxane (90 mL dioxane and 10 mL 2 N HCl) to 5 g dry cell wall material. The flask is then connected to a reflux condenser, nitrogen gas is blown onto the liquid surface, and the solution is refluxed for 30 min. under nitrogen gas. The solution is then cooled and filtered through a Whatman GF/C glass fiber filter, and collected in a flask. The residue on the filter is washed in 20 mL 96% dioxane, and then added to the filtrate. In order to neutralize the solution, 4 g sodium bicarbonate (NaHCO3) is added to the flask, followed by a 3-min. incubation on a rotary shaker until the pH was neutral. The solution is filtered through a 0.45-^m nylon membrane, and subsequently concentrated to 10-15 mL under reduced pressure on a rotary evaporator using a waterbath set at 40°C. The solution is then added drop-wise to a 250-mL centrifuge bottle containing 200 mL of rapidly stirring distilled water. Any insoluble residue remaining in the flask is washed with 2.0 mL 96% dioxane solution and added drop-wise to the water. The drop-wise addition to the water should result in the precipitation of the lignin, which can be stimulated by the addition of 2 g anhydrous Na2SO4. The lignin precipitate can be collected by centrifugation (9000g for 20 min.). The pellet is then dissolved in 100% dioxane, filtered through a 0.45-^m nylon membrane, and added drop-wise to 200 mL stirring anhydrous ether in a centrifuge bottle. The lignin precipitates and is collected by centrifuging at 9000g for 15 min. at 0°C. In order to remove all hydrophobic non-lignin contaminants, the lignin pellet is again dissolved in dioxane, filtered, and added to ether. The ether is then removed and 60 mL petroleum ether is added to wash the lignin. After removal of the petroleum ether, the lignin residue is freeze dried.

Lignin content determined with the acetyl bromide method using dioxin lignin as a standard, showed the best correlation with in vitro dry matter digestibility for a diverse set of forage samples (Fukushima and Hatfield, 2004). Even though negative correlations between lignin content and digestibility were identified for the other methods, including Klason lignin and acid detergent lignin, the correlation coefficients were not as high. Fukushima and Hatfield (2004) pointed out, however, that it is important to calculate the lignin content of a given sample based on the standard derived from that same type of sample.

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