Nitrobenzene Oxidation Method Research Articles

Lignin Analysis by HPLC and FTIR: Spectra Deconvolution and S/G Ratio Determination.

Fourier transform infrared spectroscopy (FTIR) is a simple nondestructive technique that allows the user to obtain quick and accurate information about the structure of the constituents of wood. Spectra deconvolution is a computational technique, complementary to FTIR analysis, which improves the resolution of overlapped or unobserved bands in the raw spectra. High performance liquid chromatography (HPLC) is an analytical technique useful to determine the ratio of the lignin monomers obtained by the alkaline nitrobenzene oxidation method. Furthermore, lignin content has been commonly determined by wet chemical methods; Klason lignin determination is a quick and accessible method. Here, we detail the procedures for chemical analysis of the wood lignin using these techniques. Additionally, the deconvolution process of FTIR spectra for the determination of the S/G ratio, in lignin isolated by this or other methods, is explained in detail.

Syringyl ratio and its relation to the erythro ratio of β-O-4-structure in leaf cell walls

In case of wood cell walls, there is a very clear relationship between the aromatic structure of lignin and the stereo configuration of β-O-4-structures; the syringyl ratio (S ratio, syringyl/(syringyl + guaiacyl)) obtained by the nitrobenzene oxidation method is linearly correlated to the erythro ratio (E ratio) of β-O-4 structure (erythro/(erythro + threo)) obtained by the ozonation method. In this study, 21 leaf samples and 9 stem samples were analyzed to examine whether such relationships can be found in the lignin of leaf and stem cell wall samples. Although some correlation between the S and E ratios was found in leaf and stem cell wall samples, the correlation was not as clear as in the case of woods (correlation coefficient, R2= 0.988 for 21woods and R2= 0.653 for all the leaf, stem and wood samples). In addition, the E ratios of some leaf samples were lower than 0.5, which is the lowest limit of the E-ratio for wood lignin. It was demonstrated that wood, stem and leaf samples are roughly distinguished by the yield of ozonation and nitrobenzene oxidation products from the sample (not from lignin), but not by the apparent lignin content obtained by the Klason method. The presence of syringyl nuclei in the leaves of Ginkgo, which is classified as gymnosperm, was confirmed by nitrobenzene oxidation.

Breaking the Limit of Lignin Monomer Production via Cleavage of Interunit Carbon–Carbon Linkages

Summary Conversion of lignin into monocyclic hydrocarbons as commodity chemicals and drop-in fuels is a highly desirable target for biorefineries. However, this is severely hindered by the presence of stable interunit carbon–carbon linkages in native lignin and those formed during lignin extraction. Herein, we report a new multifunctional catalyst, Ru/NbOPO4, that achieves the first example of catalytic cleavage of both interunit C–C and C–O bonds in one-pot lignin conversions to yield 124%–153% of monocyclic hydrocarbons, which is 1.2–1.5 times the yields obtained from the established nitrobenzene oxidation method. This catalyst also exhibits high stability and selectivity (up to 68%) to monocyclic arenes over repeated cycles. The mechanism of the activation and cleavage of 5–5 C–C bonds in biphenyl, as a lignin model adopting the most robust C–C linkages, has been revealed via in situ inelastic neutron scattering coupled with modeling. This study breaks the conventional theoretical limit on lignin monomer production.

Development and validation of a sensitive UPLC-MS/MS instrumentation and alkaline nitrobenzene oxidation method for the determination of lignin monomers in wheat straw.

A method to determine the lignin monomers (p-hydroxybenzaldehyde, vanillin and syringaldehyde) in plant cell wall of wheat internode was developed and validated using a high-throughput nitrobenzene oxidation step and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantification. UPLC analyses were carried out using an reversed phase C18 column (ACQUITY UPLC BEH, 1.7μm, 2.1×100mm) and gradient elution with water and acetonitrile. This method was completely validated in terms of analyzing speed, linearity, sensitivity, limits of detection (LODs) and limits of quantification (LOQs).The three lignin monomers were successfully separated within 6min and only 2min were required to regain its equilibrium. The method linearity with regression coefficients values (R2) greater than 0.997. Additionally, LODs ranged from 0.21 to 0.89μgL-1 and LOQs ranged from 0.69 to 2.95μgL-1. The applicability of this analytical approach for determining the three lignin monomers was confirmed by the successful analysis of real samples of wheat stem internodes. The nitrobenzene oxidation method was used for the analysis of lignin monomers. We have optimized the treatment temperature (170°C, 1h) and realized the high-throughput using the microwave digestion instrument. Recovery of this extraction method ranged from 68.4% to 77.7%. The analysis result showed that the guaiacyl unit (G) was the major component of lignin and there was a higher content of the syringyl unit (S) than that of the hydroxybenzyl unit (H).

Lignin Analysis by HPLC and FTIR.

Fourier transform infrared spectroscopy (FTIR) is a simple non-destructive technique which allows the user to obtain quick and accurate information about the structure of the constituents of wood. High performance liquid chromatography (HPLC) is an analytical technique useful to determine the ratio of the lignin monomers obtained by the alkaline nitrobenzene oxidation method. Furthermore, lignin content has been commonly determined by wet chemical methods; Klason lignin determination is a quick and accessible method. Here, we detail the procedures for chemical analysis of the wood lignin using these techniques.

Revisiting alkaline nitrobenzene oxidation: quantitative evaluation of biphenyl structures in cedar wood lignin (Cryptomeria japonica) by a modified nitrobenzene oxidation method

Abstract A modified alkaline nitrobenzene oxidation (NBO) method was developed to enable the analysis of the biphenyl structures of cedar wood lignin. The most essential point of the process is a modified work-up process in pyridine and a prolonged gas chromatography analysis of the silylated products. By applying this mean to cedar wood meal, a novel biphenyl product, dehydrovanillin-vanillic acid (1-carboxy-1′-formyl-4,4′-dihydroxy-3,3′-dimethoxy-5,5′-biphenyl), was detected together with known products, dehydrodivanillin and dehydrodivanillic acid. The highest total yield of biphenyl products was detected when NBO was carried out for 2–4 h at 170°C. The work-up procedure was slightly modified so that the biphenyl products can be quantified. The NBO conversion rates of biphenyl linkages of lignin were also examined with a biphenyl-type lignin model compound, and this gave rise to 75% biphenyl-type NBO degradation products. Under the same condition, the total yield of vanillin and vanillic acid (VA) from a non-condensed-type β-O-4 model compound was 89%. Because the latter did not exhibit any peak of the biphenyl products, it can be concluded that all the biphenyl products obtained by NBO (0.17 mmol g-1) were from the lignin of the native wood. It was calculated that at least 6.7 of 100 phenylpropanoid units of cedar lignin were involved in biphenyl structures even if the NBO conversion rate was not taken into consideration. The synthesis and analytical data of 11 lignin model compounds is described.

Isolation and characterization of lignin from the oak wood bioethanol production residue for adhesives

Lignin was isolated from the residue of bioethanol production with oak wood via alkaline and catalyzed organosolv treatments at ambient temperature to improve the purity of lignin for the materials application. The isolated lignins were analyzed for their chemical composition by nitrobenzene oxidation method and their functionality was characterized via wet chemistry method, element analysis, 1H NMR, GPC and FTIR-ATR. The isolated lignin by acid catalyzed organosolv treatment (Acid-OSL) contained a higher lignin content, aromatic proton, phenolic hydroxyl group and a lower nitrogen content that is more reactive towards chemical modification. The lignin-based adhesives were prepared and the bond strength was measured to evaluate the enhanced reactivity of lignin by the isolation. Two steps of phenolation and methylolation were applied for the modification of the isolated lignins and their tensile strengths were evaluated for the use as an adhesive. The acid catalyzed organosolv lignin-based adhesives had comparable bond strength to phenol-formaldehyde adhesives. The analysis of lignin-based adhesives by FTIR-ATR and TGA showed structural similarity to phenol adhesive. The results demonstrate that the reactivity of lignin was enhanced by isolation from hardwood bioethanol production residues at ambient temperature and it could be used in a value-added application to produce lignin-based adhesives.

S/G ratio and lignin structure among Eucalyptus hybrids determined by Py-GC/MS and nitrobenzene oxidation

The ratio among syringyl (S) and guaiacyl (G) lignin substructures is increasingly used as criteria in the selection of Eucalyptus species to pulping processes. The S/G distribution of several Brazilian Eucalyptus species and their crossings was determined by analytical pyrolysis-GC/MS (Py-GC/MS) and alkaline nitrobenzene oxidation directly from wood fibers. The ratio varied among the Eucalyptus hybrids. In Py-GC/MS, the S/G ratio is calculated by summing up the proportions of S and G type lignin pyrolysis products with various 1–3 carbon containing side chain structures. Py-GC/MS revealed that the distribution of the lignin side chain structures varied among the species. Similar trends were seen both in the G and in the S series. The result was verified with principal component analysis (PCA), which also revealed that similar side chains structures in the G and S units are correlated with each other. That PCA result confirmed that all G and S type degradation products should be included in the calculation of S/G ratios instead of selected products. S/G ratios determined by Py-GC/MS and alkaline nitrobenzene oxidation were close to each other, but not equal. Lower values were obtained by Py-GC/MS. The differences between the two methods are probably related the degradation mechanism of lignin with Py-GC/MS and nitrobenzene oxidation methods. Based on these results, it is not possible to say which of the methods is more reliable. However, both methods can well be used to compare the S/G ratios of lignin among samples.

Characterization of lignin-derived products from Japanese beech wood as treated by two-step semi-flow hot-compressed water

Japanese beech (Fagus crenata) wood was treated by two-step semi-flow hot-compressed water (the first stage: 230 °C/10 MPa/15 min, the second stage: 270 °C/10 MPa/15 min), and produced lignin-derived products in the hot-compressed water-soluble portions at the first and second stages, and the final residue of the second stage was characterized with alkaline nitrobenzene oxidation method and gel permeation chromatographic analysis. As a result, the lignin-derived products at the first stage, where hemicellulose was also decomposed, consisted of lignin-based monomers and dimers and oligomers/polymers in the water-soluble portion. A large part of the oligomers/polymers was, however, recovered as the precipitate during 12 h setting after hot-compressed water treatment. By the analysis of nitrobenzene oxidation products, there were relatively higher contents of ether-type lignin in the precipitate at the first stage than in original beech wood. Since the ether linkages of lignin are more preferentially cleaved by this hot-compressed water, lignin-based polymeric fractions were flowed out from the porous cell walls from which hemicellulose was removed. On the other hand, at the second stage condensed-type lignin remained in the precipitate and residue. Based on these results, decomposition behavior of lignin in Japanese beech wood as treated by the two-step semi-flow hot-compressed water was discussed regarding the topochemistry of lignin structure.

Characterization of Jatropha curcas lignins

Lignins of Jatropha curcas organs were qualitatively and quantitatively characterized by thioglycolic acid, thioacidolysis, and nitrobenzene oxidation methods. The lignin content of the seed coat was 49.4%, and was the highest among various organs of the plant, while the stem had 15.9% lignin, within the range of usual lignin contents of angiosperm trees. Lignin aromatic components of all organs were composed of guaiacyl and syringyl units. Nitrobenzene oxidation indicated that the ratios of syringyl to guaiacyl lignins in the fruit coat and stem were higher than those in other organs. This study provides basic data for the total utilization of J. curcas wood.

Occurrence of guaiacyl/p-hydroxyphenyl lignin in Arabidopsis thaliana T87 cells

Arabidopsis thaliana is the most widely used model plant in the area of plant biosciences, and the cell line T87 is one of the most commonly used lines in Arabidopsis cell cultures. The characteristics of lignins in cultured cells often differ from those of lignins in intact plants. To date, nothing is known about the lignins of T87 cells. In this study, we characterized lignins of T87 cells using six analytical methods; phloroglucinol staining, thioacidolysis, nitrobenzene oxidation, alkaline hydrolysis, and Klason and acetyl bromide methods. These analyses clearly showed that lignins of T87 cells are composed of guaiacyl and p-hydroxyphenyl units, and are quite different from lignin found in the Arabidopsis inflorescence stem, which is composed of guaiacyl and syringyl units.

Determination of Eucalyptus spp lignin S/ G ratio: A comparison between methods

The syringyl/guaiacyl ratio was determined for six different Eucalyptus spp. wood clones cultivated in four regions in Brazil. The determinants were made by pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) and the results were compared with those obtained by alkaline nitrobenzene oxidation method. The S/ G ratios were obtained considering all the identified lignin derivatives in the pyrograms and also using two groups of markers. The first group of markers consisted of guaiacol, 4-methylguaiacol, 4-vinylguaiacol, trans-isoeugenol, syringol, 4-methylsyringol, 4-vinylsyringol and trans-4-propenylsyringol compounds as markers. The second group included guaiacol, 4-methylguaiacol, 4-vinylguaiacol, vanillin, 4-ethylsyringol, 4-vinylsyringol, syringaldehyde, syringylacetone and trans-4-propenylsyringol. It was observed from the statistical analysis that the values of S/ G obtained by Py–GC–MS using the two groups of markers did not differ significantly from those obtained by nitrobenzene oxidation method.

Microscale alkaline nitrobenzene oxidation method for high-throughput determination of lignin aromatic components

The nitrobenzene oxidation method is widely used for structural analysis of lignin. However, the conventional nitrobenzene oxidation method has several drawbacks including the requirement of a sizeable amount of sample material and the rather slow completion of the reaction process. In this paper, we describe a microscale nitrobenzene oxidation method using deuterium-labeled vanillin and syringaldehyde as internal standards. Using this method, we show that the microscale nitrobenzene oxidation method realized high-throughput determination of lignin components using a small amount of sample, i.e. 10 mg per reaction, and high reproducibility. Due to the small sample sizes, the oxidation reaction and extraction steps of a number of samples can be completed in parallel, thus enabling us to process more than 40 samples per day.

Comparative study of lignins isolated from Alfa grass ( Stipa tenacissima L.)

Soda lignin, dioxane lignin and milled lignin were isolated from Alfa grass ( Stipa tenacissima L.). The physico-chemical characterization of three different lignins: one industrial lignin precipitated from soda spent liquor and two lignin preparations isolated under laboratory conditions from Alfa grass (also know as Esparto grass) was performed. The structures of lignins were studied by three non-destructive (FT-IR, solid state 13C NMR and UV/visible spectroscopy) and two destructive (nitrobenzene oxidation and thermogravimetric analysis) methods. Elemental analysis and the methoxyl content determination were performed in order to determine the C 9 formulae for the studied lignins. The total antioxidant capacity of the studied lignins has been determined and compared to commercial antioxidants commonly used in thermoplastic industry.

Lignin content versus syringyl to guaiacyl ratio amongst poplars

Two oxidation techniques that afford high yields of monomers and dimers were used to more accurately estimate the syringyl to guaiacyl (S:G) ratio of hardwood lignins. Permanganate oxidation of the woodmeal after a CuO pre-hydrolysis step gave poor results and this was attributed to preferential oxidation and degradation of syringyl nuclei by CuO. However, this procedure did provide a good estimate of the percentages of both S and G phenylpropane (C 9) units that were uncondensed. When the total S and G products from nitrobenzene oxidation (NBO) of the uncondensed fractions were corrected, credible S:G ratios were obtained. These ratios were in good agreement with results from KMnO 4 oxidation of dissolved kraft lignin without CuO pre-hydrolysis. The corrected NBO method was used to determine the S:G ratio of 13 poplars, and the values ranged from 1.01 to 1.68. Unlike results from other investigations, an excellent linear correlation ( R 2 = 0.846) was obtained for a decreasing lignin content (28% to 16.5%) with an increase in the S:G ratio.

Comparison between analytical pyrolysis and nitrobenzene oxidation for determination of syringyl/guaiacyl ratio in Eucalyptus spp. lignin

Pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS) was applied to measure the lignin syringyl/guaiacyl (S/G) ratio in E. dunni, E. grandis, E. nitens, E. urograndis, and E. urophylla woods. A total of 41 compounds were identified, of which 11 were derived from carbohydrates and 30 from lignins. The S/G ratio was calculated on the basis of the areas of peaks recorded in the pyrograms and compared with the results obtained by alkaline nitrobenzene oxidation. The values of S/G found by pyrolysis were similar for all the species using the compounds guaiacol, 4-methylguaiacol, 4-vinylguaiacol, vanillin, 4-ethylsyringol, 4-vinylsyringol, homosyringaldehyde, acetosyringone, and syringylacetone, as lignin markers. The selected markers were efficient for the deter-mination of S/G ratio in eucalyptus wood by Py-GC-MS. The Py-GC-MS technique produced results that are comparable to the nitrobenzene oxidation method, with the advantage of requiring small wood samples and a short analysis time.

Structural characterization of residual lignins isolated with cyanamide‐activated hydrogen peroxide from various organosolvs pretreated wheat straw

AbstractThe posttreatment of various organosolvs pretreated wheat straw with cyanamide‐activated hydrogen peroxide was studied. About 44–80% of the total residual lignin and 38–85% of the total residual hemicelluloses were released or degraded during the posttreatment with 1.8% H2O2–0.18% cyanamide at 50°C under pH 10.0 for 4 h from different aqueous organic acids or alcohols pretreated straw. The seven degraded residual lignin preparations were subjected to a comprehensive physicochemical and structural characterization by UV, FTIR, and 1H and 13NMR spectroscopy, and GPC. The nitrobenzene oxidation method was also applied to the in situ lignins. It was found that the seven residual lignin preparations contained large amounts of noncondensed syringyl and guaiacyl units, together with fewer noncondensed p‐hydroxyphenyl units, esterified p‐coumaric acid, and mainly etherified ferulic acid. All of the lignin fractions are free of associated polysaccharides and had molecular‐average weights ranging between 2980 and 3820 g mol−1. Analysis of these low molecular weight degradation products revealed an oxidation of residual lignin had occurred. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Inhomogeneities in the Chemical Structure of Sugarcane Bagasse Lignin

Sequential treatments of dewaxed bagasse with distilled water, 0.5 M NaOH, 0.5, 1.0, 1.5, 2.0, and 3.0% H(2)O(2) at pH 11.5, and 2 M NaOH at 55 degrees C for 2 h solubilized 2.8, 52.5, 14.9, 3.3, 5.5, 5.0, 2.8, and 2.2% of the original lignin, respectively. The eight isolated lignin fractions were subjected to a comprehensive structural characterization by UV, FT-IR, and (1)H and (13)C NMR spectroscopies and thermal analysis. The nitrobenzene oxidation method was also applied to the in situ lignins. The seven lignin fractions, isolated successively with alkali and alkaline peroxide, were all SGH-type lignins, with a small amount of esterified p-coumaric acid and mainly etherified ferulic acid. No significant differences were found in the weight-average molecular weights (1680-2220 g/mol) of the seven alkali and alkaline peroxide dissolved lignins. However, the first four lignin fractions, isolated with 0.5 M NaOH and 0.5, 1.0, and 1.5% H(2)O(2) at pH 11.5, were rich in syringyl units and contained large amounts of noncondensed ether structures, whereas the last three lignin fractions, isolated sequentially with 2.0 and 3.0% H(2)O(2) at pH 11.5 and 2 M NaOH at 55 degrees C for 2 h, had a higher degree on condensation and were rich in guaiacyl lignins.

Changes in lignin content of leaf litters during mulching

Alkaline nitrobenzene oxidation, ozonation and methoxyl content determinations were applied to decomposing leaf litter of Ginkgo biloba L., Cinnamomum camphora sieb., Zelkova serrata Makino and Firmiana simplex W. F. Wight, respectively, during mulching to investigate the properties and estimate changes in lignin composition and content . Since the Klason lignin residue originated from components highly resistant to degradation by acid, the methoxyl content of Klason residue was used to estimate the lignin content of leaf litter. Quantitative analysis of presumed lignin-derived fragments, by use of alkaline nitrobenzene oxidation and ozonation methods, suggested that the estimated lignin content approximates that of the real lignin content of leaves, which is greatly overestimated by the Klason procedure. The estimated lignin contents ranged from 3.9 to 10.0% while the Klason lignan residue varied from 37.1 to 46.7% in un-mulched leaf litter. The absolute amounts of the measured lignin somewhat decreased during mulching, while the structure of lignin remaining in leaf litters after mulching was considered not to be very different from its original structure.

Determination of nitrobenzene oxidation products by GC and1H-NMR spectroscopy using 5-iodovanillin as a new internal standard

The nitrobenzene oxidation method was modified to obtain more reproducible data and more structural information about lignin, not only by gas chromatography (GC) but also by proton nuclear magnetic resonance (1H-NMR) spectroscopy for quantitative determination of the oxidation products and to simplify the procedures. The nitrobenzene oxidation mixture was directly extracted after acidification without preextraction of by-products. The direct extraction made the extractive step easy and gave reproducible data. 5-Iodovanillin was selected as a new internal standard. The reason for this selection was that 5-iodovanillin did not exist in the nitrobenzene oxidation products from any plant species and had an aldehyde group whose peak did not overlap with the other aldehyde peaks on an1H-NMR spectrum. Thus, the use of 5-iodovanillin enabled us to quantifyp-hydroxybenzaldehyde, vanillin, and syringaldehyde in oxidation products on the basis of1H-NMR analysis as well as GC. Furthermore, more information about the condensed structure of lignin was derived by comparing the1H-NMR and GC analyses.