A recently developed method based on acetylbromide cleavage is 'Derivatization Followed by Reductive Cleavage' (DFRC; Lu and Ralph, 1997). This method uses acetylbromide to acetylate alcohols and phenols in lignin (4.26). In addition, the a-carbon is brominated (4.27). Cleavage of the P-O-4 linkage is catalyzed by zinc. The monomeric residues (4.28) that are formed are acetylated to yield diacetylated monolignols (4.29) and separated using gas chromatography. The DFRC reaction is presented in Figure 4-5.
DFRC is achieved by heating the sample in 2.5 mL acetyl bromide with HCl, followed by removal of the reagent by evaporation. The residue that remains is dissolved in 2.5 mL of dioxane/acetic acid/H2O (5:4:1). To this solution is added 50 mg zinc dust, to catalyze the cleavage of P-bromo-ethers. After stirring for 30 min. the zinc is removed by filtration through glass wool. Dichloromethane (10 mL) is then added to the filtrate along with 10 mL saturated NH4Cl solution, and the internal standard tetracosane is added. After mixing, phase separation, and removal of the organic phase, the aqueous phase is extracted twice more in dichloromethane. The combined dichloromethane extracts are then combined and dried. The resulting residue is dissolved in 1.5 mL dichloromethane, acetylated with 0.2 mL acetic anhydride and 0.2 mL pyridine. Ethyl acetate (2.5 mL) is added after 90 min. and the mixture is evaporated. Ethanol is added during the evaporation to get rid of the pyridine. Pyridine-free samples are dissolved in 200 ^L dichloromethane and a 1-2 ^L is injected in a gas chromatograph for analysis using the reference compounds p-acetoxycinnamyl acetate (4.29a), coniferyl diacetate (4.29b), and sinapyl diacetate (4.29c) for peak identification.
Advantages of the DFRC method over thioacidolysis include the better yield, simpler and milder reaction conditions, and the fact that esters are not cleaved. Especially in the analysis of lignin in grasses, with large amounts of esterified p-coumaric acid (1.13), this is an important consideration.
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