The nitrobenzene oxidation

Nitrobenzene oxidation involves the treatment of cell wall material with sodium hydroxide containing nitrobenzene. The method was developed by Freudenberg (1939). Nitrobenzene oxidation degrades the phenylpropane structure from a C6-C3 unit to a C6-C1 unit. After incubation the soluble fraction is acidified and extracted with ether. The degradation products -p-hydroxybenzaldehyde (4.13), vanillin (4.14), and syringaldehyde (4.15), and the corresponding acids p-hydroxybenzoic acid (4.16), vanillic acid (4.17), and syringic acid (4.18) - can be separated on an HPLC column to determine the lignin composition. Alternatively, gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) can be used.

The lignin composition can be expressed as the S/V ratio, whereby the 'V' (vanillin (4.14), vanillic acid (4.17)) reflects guaiacyl residues and the 'S' (syringaldehyde (4.15), syringic acid (4.18)) reflects syringyl residues. (Monties, 1989). Nitrobenzene oxidation does not affect the aromatic C-C bonds, but only the bonds from the so-called uncondensed units, so that the estimates of lignin composition can be somewhat biased (Lewis and Yamamoto, 1990). In grasses these methods cannot distinguish between aromatic compounds derived from lignin and from p-coumaric and ferulic acid esterified to the cell wall (Lapierre, 1993), so that this method is seldom used for the analysis of lignin from grasses.

An example of the use of the nitrobenzene oxidation to elucidate differences in lignin subunit composition between wild-type and mutant Arabidopsis plants can be found in Chapple et al. (1992). They dried and ground stem tissue of Arabidopsis and extracted 50 mg with methanol (three times 1 mL) and deionized water (twice, 1 mL) at 60°C. Esterified phenolics r f

OCH3 H3CO'

OCH3 H3CO'

OCH3

OCH3

OCH3 H3CO

OCH3 H3CO

OCH3

OCH3

were saponified in 1 M NaOH (37°C, 60 min.), after which the tissue was rinsed twice and resuspended in water. Sodium hydroxide solution was added to an aliquot representing 20 mg of dry tissue so that the final concentration was 2 M NaOH in a volume of 980 ^L. Nitrobenzene (20 ^L) was added to this suspension, followed by a 2-hour incubation at 160°C. After cooling the sample, a 250-^L aliquot was added to 750 ^L 1 M acetic acid to neutralize the sample. A 100-^L aliquot was then reduced (addition of 1 mL 2% (w/v) sodium borohydride in DMSO; 90 min. at 40°C), acetylated to increase the volatility (4 mL of acetic anhydride plus 250 ^L of 1-methylimidazole as catalyst; 10 min. at room temperature), and extracted in methylene chloride (CH2Cl2). The break-down products of the lignin were subsequently analyzed on a GC with an FID, using p-hydroxybenzaldehyde (4.13), vanillin (4.14), 5-hydroxyvanillin, and syringaldehyde (4.15) as reference compounds.

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