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Lignin is a biopolymer that resists degradation because of its phenolic aromatic character and highly branched structure. Lignin is one of the most abundant aromatic molecules on earth. The valuable aromatic compounds may be produced from lignin. In pulp and paper mills only 2 % of lignin is used commercially in the production of valuable aromatic compounds like Vanillin etc. The valorization of lignin depends on an understanding of degradation processes and products. In the present investigation, attempts have been made to understand the oxidation products of Anthocephalus indicus wood dust and unbleached pulps lignin obtained under optimum conditions of pulping using alkaline nitrobenzene as an oxidation agent. Eight compounds i.e P-hydroxy benzoic acid, Vanillic acid, Syringic acid, p-hydroxybenzaldehyde, vanillin, Syringaldehyde, Acetovanillone, Acetosyringone. were identified with the help of High-Performance Liquid Chromatography (HPLC).

Pulp and paper mills treat lignin as a waste product and most of the lignin is incinerated for heat energy. The under-utilization of lignin is due to a lack of knowledge about degradation products and the challenge of separating a mixture of these degradation products. Present investigation is attempts have been made to understand the oxidative degradation products of lignin. This may help in the extraction of some commercially high-valve aromatic compounds from lignin.

Lignin is the most abundant aromatic molecule and the second largest renewable source of carbon on the earth (Kirk et al 1987, Rodrigues Pinto et al 2011). More than 50 million tons per year of lignin is produced by the pulp and paper industries (Busch 2006). Lignin has huge economic potential as a source of high commercial valve aromatic compounds, for example, the production of vanillin by alkaline oxidation of lignin (Fargues et al 2019, Silva et al 2009, Chan et al 2020, Schutyser et al 2018). Lignin oxidation converts lignin to aldehydes such as vanillin and syringaldehyde (Pinto et, al 2013, Araújo et al 2010, Strassberger et al 2014). The full-scale valorization and exploitation of lignin as a source of valuable aromatic compounds should be possible by understanding the different oxidative products.(Beckham et al 2016, Busse et al 2017).

Wood Dust:

Anthocephalus indicus belongs to the family Rubiaceae. It is a large deciduous tree commonly known as Kadam. Anthocephalus indicus logs were debarked and chipped and chips were screened. Screened chips were converted to dust in a laboratory disintegrator. Dust passing through 40 mesh and retained over 60 mesh was taken for experimentation.

Pulping:

Anthocephalus indicus logs were debarked and chipped and chips were screened. Screened chips (15-30 x 10-20 x 2-3 mm) were taken for pulping experiments. Pulping experiments were carried out in an air pulping bath unit consisting of six bombs of 2.5 liter capacity using 12 and  14, % active alkali as Na₂O at 23.0 % sulphidity level. The bath ratio 1: 4 maximum temperature, 170^oC was kept constant in all the cases. Room temperature to 100^oC temperature was raised in 90 minutes followed by a 10^oC rise in 15 minutes to 170^oC and kept at maximum temperature for 60 minutes. Pulping schedule corresponds to H-factor, 1110. Cooked material was washed with hot water, fiberized in a laboratory disintegrator, and screened over a flat laboratory screen having 0.25 mm slots.

Alkaline nitrobenzene oxidation:

Wood dust and unbleached pulps obtained under optimum conditions of pulping were subjected to alkaline nitrobenzene oxidation adopting the procedure of Gee et al (1968) under the following conditions

Lignin,g	0.10
Maximum temperature oC	170
Time to raise the temperature to 170 oC, min	60
Time at 170 oC, min	90
Nitrobenzene, ml	0.8
NaOH (2N),ml	6.0

Purification of nitrobenzene oxidation products:

Nitrobenzene Oxidation products were extracted with diethyl ether (5 X 20ml). The solvent fraction was washed with sodium hydroxide solution (1N, 2 X 25ml) and added to the fraction obtained after removing nitrobenzene and its reduction products. Combined sodium hydroxide solution and original solution were mixed and acidified to pH about 2 with hydrochloric acid (5 N)followed by extraction with dichloromethane ( 4 X 50 ml)and diethyl ether ( 4 X 50 ml), both the solvent fractions were mixed together. The combined solvent fraction was dried over anhydrous sodium sulphate, reduced to a small volume in a rotary evaporator under reduced pressure, then transferred to a dry vial and reduced to near dryness under a nitrogen atmosphere.

Alkaline nitrobenzene oxidation products were characterized using high-performance liquid chromatography. Analysis was performed on Perkin Elmer (USA) model -235 High-Performance liquid Chromatograph equipped with the programmable LC binary pump and Perkin- Elmer UV diode array programmable detector model-235.

Elution Media:

Several binary mixtures of solvents viz., 0.5 N acetic acid and acetonitrile, 0.5 N acetic acid and n-propanol, 0.5 N acetic acid and butanol and 0.5 N acetic acid and methanol in the different ratio were tried for the analysis. On the basis of the resolution achieved, in each case, 0.5 N acetic acid and acetonitrile were considered to be the best for the separation of oxidation products, among the solvent system, tried. Analysis was performed using different ratios of acetic acid and acetonitrile ranging from (50:50 v/v) to ( 95:5v/v). The flow rate varied from 0.4 to 2.2 ml/min. The optimum separation was achieved at 0.5ml/min elution rate and 85:15v/v ratio of 0.5 N  acetic acid and acetonitrile.  Reverse phase ecosphere sphere C₁₈ column of 30mm followed by 150 mm, in series having 12% loading of bonded monomer, particle size 5µm of spherical shape is used. The guard column was fitted in series prior to the C₁₈ column. The average pore size of the column was 80 A^o units corresponding to a plate number of 100,150/m². Oxidation products were scanned at 225,280 and 290 nm. On the basis of results obtained for the resolution pattern, scanning wavelength 280nm was considered to be optimum for the resolution of phenolic carbonyl and carboxylic compounds under investigation.

Quantitative estimation of oxidation products:

An equal amount of authentic sample (~0.01 g) of all the identified compounds was taken and dissolved in 100 ml of the elution media, 0.5 N acetic acid: acetonitrile ( 35:015 v/v). From the prepared stock solution 5, 10, 15, and 20 ml solution was taken in the volumetric flask (25 ml) and further diluted to 25 ml to have solutions of different concentrations of the authentic samples. 20 microlitres of each diluted solution were injected into the chromatograph and the peak area in each case was calculated. The Peak area for an equal amount of a single compound in different diluted solutions was computed from the values. (Table  1)

Table 1 High-Performance liquid Chromatography of authentic samples at different concentrations

Wood dust and unbleached pulps were subjected to alkaline nitrobenzene oxidation studies.  The identity of eight compounds was established by taking advantage of their higher polarity / better partition coefficient in the polar solvent like of acetic acid and acetonitrile (Higuchi et al 1967, Chang et al 1971 ). The order of elution was P-hydroxy benzoic acid, Vanillic acid, Syringic acid, p-hydroxybenzaldehyde, vanillin, Syringaldehyde, Acetovanillone, Acetosyringone. From the elution pattern, it is clear that the comparatively more or less the same, the low molecular weight compound was eluted first as expected in reverse phase chromatography using polar elution media. 

Table 2 Nitrobenzene oxidation products of wood dust and unbleached pulps of Anthocephalus indicus 

Alkaline nitrobenzene oxidation products of Anthocephalus indicus wood dust and unbleached pulps

Data recorded in Table -2  and 3 for the relative percentage, moles, and relative ratio of nitrobenzene oxidation products of wood dust and unbleached pulps revealed that the formation of all three aldehydes increases considerably in unbleached pulps over wood dust, while the formation of acids decreased. The relative molar ratio of Vanillin: Syringaldehyde : P- hydroxyl benzaldehyde for wood dust was 6.711: 20.032: 3.523 and increased to 15.520: 21.951: 8.943 and  11.678: 27.632: 9.046 respectively in unbleached pulps produced using 12 % and 14% alkali during pulping. The improvement in the relative mole of aldehydes in unbleached pulps over wood dust may be due to the formation of a higher amount of α-β double bond conjugated and phenyl nuclei having a free hydroxyl group at para position of propyl chain (Alder et al 1964, Anderson et al 2016) and presence of higher amount of α carboxyl group having capacity of forming enolic groups with the contribution of neighboring groups in the phenyl nuclei having a free p-hydroxyl group. The free phenolic hydroxyl group present at para position delocalizes its dipole, and the electron-withdrawing nature of  >C=O group prohibits electron transfer through quinine methide with the formation of enol structure and finally breaking down to aldehydes. This may also be the reason for the increase in Acetovanillone and Acetosyringone in unbleached pulps.

Table 3 Relative percentage, moles and molar ratio of nitrobenzene  oxidation products of wood dust and unbleached pulps of Anthocephalus indicus 

Quantitatively, the relative molar ratio of Vanillic acid : Syringic acid: P-hydroxyl benzoic acid in wood dust was 20.805:19.966: 24.161 and decreased to 10.081 :10.081: 21.789 and 8.553: 9.704: 18.750 in unbleached pulp produced using 12 % and 14 % alkali during pulping ( Table -3).

Table 4  Ratio of various units of nitrobenzene  oxidation products of wood dust and unbleached pulps of Anthocephalus indicus 

Particulars	Wood Dust	Unbleached pulp produced using 12 % alkali during pulping	Unbleached pulp produced using 14 % alkali during pulping
Total Syringyl/ Total guaiacyl units	1.421	1.184	1.540
Total Syringyl/ Total p-hydroxyphenyl  units	1.533	1.222	1.640
Total guaiacyl/ Total p-hydroxyphenyl  units	1.079	1.032	1.065
Total Syringaldehyde/ Total vanillin units	3.025	1.752	2.366
Total Syringaldehyde/ Total p-hydroxy benzaldehyde units	5.763	2.455	3.055
Total vanillin / Total p-hydroxy benzaldehyde units	1.905	1.400	1.291

The Syringaldehyde: Vanillin molar ratio in wood dust was 3.025: 1.000 and 7.752:1.000, 2.366 : 1.000 for unbleached pulp produced using 12 % and 14 % alkali during pulping. While the total Syringyl: total guaiacyl units was 1.421:1.000 for wood dust and 1.184:1.000 and 1.540: 1.000 in the unbleached pulp after delignification. Total Syringaldehyde: total p-hydroxy benzaldehyde units was 5.763:1.000 for wood dust and decreased to 2.455: 1.000 and 3.055: 1.000 for unbleached pulps. Total Syringyl: total p-hydroxyphenyl units were 1.533: 1.000 for wood dust and decreased to 1.222: 1.000 and 1.640: 1.000 in unbleached pulps producing using 12 % and 14 % alkali during pulping. While the Vanllin : p-hydroxyphenyl ratio was 1.905:1.000 for wood dust and increased to 1.400: 1.000 and 1.905: 1.000 in unbleached pulps. The ratio of total guaiacyl :  Total p-hydroxyphenyl units for wood dust was 1.0079: 1.000 and decreased to 1.032; 1.000 and 1.065: 1.000 for unbleached pulps.

Particulars	Guaiacyl : Syringyl : p-hydroxyphenyl	Vanillin : Syringaldehyde : p-hydroxy    benzaldehyde
Wood Dust	0.704    :  1.000     :    0.652	0.330    :               1.000          :    0.174
Unbleached pulp produced using 12 % alkali during pulping	0.844    :  1.000     :    0.818	0.570   :               1.000          :    0.407
Unbleached pulp produced using 14 % alkali during pulping	0.649    :  1.000     :    0.610	0.423    :               1.000          :    0.327

Table 5: Molar ratio of various units of nitrobenzene  oxidation products of wood dust and unbleached pulps of Anthocephalus indicus 

The molar ratio of the aldehyde was 0.330: 1.000: 0.179  for Vanillin: Syringaldehyde: p-hydroxy benzaldehyde units in wood dust and 0.570: 1.000:  0.407 and 0.423; 1.000: 0.327 for unbleached pulps. The ratio of total Guaiacyl: Syringyl: p-hydroxyphenyl units was 0.704: 1.000: 0652 for wood dust as against 0.844: 1.000: 0.818 and 0.649: 1.000: 0.610 for unbleached pulps isolated for pulps producing using 12 % and 14% alkali during pulping (Table – 5). The values indicated that perhaps during the course of delignification, the syringyl units suffered more degradation than the other two units. However, under the severe condition of delignification, the degradation of other units is also increased.

Oxidative degradation and quantitative estimation of nitrobenzene oxidation products of Anthocephalus indicus lignin present in its wood dust and unbleached pulps revealed that though the aldehyde forming structure was slightly increased in pulp lignins over all the relative ratio of Guaiacyl: Syringyl: p-hydroxyphenyl unit remained almost comparable except a little higher ratio of Guaiacyl and p-hydroxyphenyl moiety for the pulp lignin isolated from pulp produced using 14 % alkali during pulping.

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