The nitrobenzene oxidation

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 Chappie 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 

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Nitrobenzene oxidation was carried out by adding 50 mg of dry soda lignin into a mixture of 7 mL of 2 M NaOH and 4 ttiL of nitrobenzene in a 15 ttiL steel autoclave. Then, the antoclave was heated to 165°C for 3 hours in a preheated thermostat oil bath. After the autoclave was cooled to room temperature, the mixture was then transferred to a liqnid-hquid extractor for continuous extraction with chloroform (5 x 20 mL) in order to remove any nitrobenzene reduction product and excess nitrobenzene. The oxidation mixtnre was then acidified by concentrated HCl to pH 3 and further extracted with chloroform (5x15 mL). The solvent from the second chloroform solution was then removed using a rotary evaporator at 40°C under reduced pressure in order to obtain the nitrobenzene oxidation mixture. The mixture was then dissolved into dicloromethane and made up to 10 luL. This mixture was then used as a stock solution for high performance liquid chromatography (HPLC) analysis [6]. 

The nitrobenzene oxidation mixture was analyzed using the HPLC method. 0.2 mL of the stock solution was pipetted into a 25 mL volumetric flask and acetonitril-water (1 2 vA ) was added to it. About 20 gL of the sample solution was next injected into the HPLC system (Shimatzu) equipped with a Hypersil bond C,g coluitm (particle size 5 gL, 25 x 4.6 mm i.d.) to quantitatively determine the vanillin component while another component was determined qualitatively. Acetonitril-water (1 8) containing 1% acetic acid was used as an eluent with a flow rate of 2 tuL/min. The eluent was then monitored with an UV (ultraviolet) detector at 280 ran [6]. 

The nitrobenzene oxidation product was used in this process [13]. The mixture was dissolved into acetone and made up to 10 mL. Next, the mixture was heated to 60°C for 10 minutes using a hot plate. The precipitate was then filtered and washed using... 

Enough heat is generated by the reaction to keep the suspension at boiling point. It is essential to have an excess of iron metal to ensure complete decomposition of the nitrobenzene. The details of the reaction mechanism are not fully understood, but it is presumed that the nitrobenzene oxidizes Ee to Ee which is then hydrolysed. The acid is released by hydrolysis and pigment formation and dissolves the metallic iron and thus renews the supply of Ee " no additional acid other than that produced dur-... 

The reaction mechanism for nitrobenzene oxidation of lignin is still not well understood Thus, although nitrobenzene m an alkaline solution has been usually regarded as two-electron-transfer oxidant (Chang and Allan 1971), recent investigations on the nitrobenzene oxidation of lignins and related compounds indicate that nitrobenzene m alkaline solution acts as an one-electron-transfer oxidant (Schultz and Templeton 1986)... 

Weigh accurately about 200 mg of a dned, extracted wood meal (40-60 mesh) with known moisture and extractives contents or 50 mg of a dned, purified lignin preparation The specimen, 7ml of 2M NaOH, and 0 4ml of freshly distilled nitrobenzene are placed m a 10-ml stainless steel bomb (Note 1) The bomb is sealed tightly with a screw cap fitted with a Teflon gasket and heated at 170°C for 2 5h m an electrically preheated (170°C), thermostated aluminum block with six holes to accommodate the cylindrically shaped bombs or in a thermostated oil bath The bomb is shaken occasionally and, after the heating period, is removed from the heater and cooled with ice water The oxidation mixture is transferred to a liquid-liquid extractor, and extracted continuously with CHCI, for 4h to remove nitrobenzene reduction products and any excess nitrobenzene The oxidation mixture is acidified to pH 3-4 with cone HC1, and further extracted continuously with CHCI, for 48 h The solvent from the second CHCI, solution is removed at 40°C under reduced pressure to obtain the nitrobenzene oxidation mixture The mixture is then dissolved m 5 ml of CH2C12 and transferred to a 10-ml volumetric flask The solution is made up to 10 ml... 

The yield of the nitrobenzene oxidation products is calculated in mmol/ 100 mg of lignin (or Klason lignin in lignocellulose samples) from Eq (2) and m mol % from Eq (3)... 

As mentioned previously (Chap. 2.2.2.4.1), the major purpose of the GC-MS analysis of a nitrobenzene or cupric oxide oxidation mixture is to verify the identity of the oxidation products established previously by GC or HPLC analysis, and to elucidate the structure of unknown constituents. For example, GC-MS analysis of the nitrobenzene oxidation mixture of milled bamboo lignin from Phyllostachys pubescence showed unequivocally that compounds (l)-(3) in the total ion chromatogram of the oxidation mixture (Fig. 6.2.2) are indeed p-hydroxybenzaldehyde, vanillin, and syringaldehyde, respectively (Tai et al. 1990) (see Chap. 9.1 for a discussion of the GC-MS technique). In addition, the unknown compound in the chromatogram was identified as p-hydroxyazobenzene (15) (Fig. 6.2.1), one of the phenolic reduction products of nitrobenzene. 

Leopold B, Malmstrom IL (1952) Studies on lignin IV Investigation on the nitrobenzene oxidation products of lignin from different woods by paper partition chromatography Acta Chem Scand 6 49-54... 

Schultz TP, Templeton MC (1986) Proposed mechanism for the nitrobenzene oxidation of lignin Holzforschung 40 93-97... 

Table I. Pseudo First-Order Rate Constants and Activation Parameters for the Nitrobenzene Oxidation of Selected Compounds (7)...

The relative rate data of Table II were subjected to Hammett plots of log kx/kh versus o and o+. In the nitrobenzene oxidations, a better correlation was found with +(p+ = -0.48, r = -0.994). The preferred dependence on <7+ was also found in the copper(II) oxidations when the kinetic point for the methyl... 

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