Name: extraction of lignin with high &#946;-o-4 content by mild ethanol extraction and its effect on the depolymerization yield
Uploaded: 2019-01-07T14:00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Extraction of Lignin with High ÃŽÂ²-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield at JoVE.com

The work was funded by the European Union (Marie Curie ITN 'SuBiCat' PITN-GA-2013-607044, PJD, KB and JGdV), in addition to financial support from the European Research Council, ERC Starting Grant 2015 (CatASus) 638076 (AdS and KB) and the research programme Talent Scheme (Vidi) with project number 723.015.005 (KB), which is partly financed by the Netherlands Organisation for Scientific Research (NWO).

1. Pretreatment of the Walnut Feedstock before Lignin Extraction
<ol>
	<li>Production of cut walnut shells
	<ol>
		<li>Feed the walnut shells to a hammer cutter to fracture the shells. Equip the hammer cutter with a 5 mm sieve at the outlet. Collect the fractured walnut shells in a 1 L glass beaker.</li>
		<li>Feed the fractured shells to a micro hammer cutter to obtain ground shells. Equip the micro hammer cutter with a 2 mm sieve at the outlet. Collect the ground walnut shells in a 1 L glass beaker.</li>
	</ol>
	</li>
	<li>Extraction of fatty acids from ground walnut shells
	<ol>
		<li>Put 150 g of the cut walnut shells in a 500 mL round-bottom flask. Add 200 mL of toluene and a stirring bar to the round-bottom flask.</li>
		<li>Attach a reflux condenser to the round-bottom flask. Heat the mixture at reflux temperature (111 &#730;C) with an oil bath for 2 h with vigorous stirring.</li>
		<li>Stop the heating after 2 h and let the mixture cool down to room temperature by removing it from the oil bath.</li>
		<li>Remove the toluene by filtration (185 mm diameter, 10 &#181;m pore size). Discard the toluene filtrate.</li>
		<li>Remove the toluene residues by heating the walnut shells overnight in a vacuum oven at 80 &#730;C and 50 mbar.</li>
	</ol>
	</li>
	<li>Milling of pre-extracted walnut shells
	<ol>
		<li>Place 7 ZrO2 grinding balls with a 20 mm diameter into a 250 mL grinding bowl made of ZrO2.</li>
		<li>Fill the bowl with 40 g of walnut particles. Add 60 mL of isopropanol to the grinding bowl.</li>
		<li>Perform the grinding of the walnut shells with a rotary ball mill. Grind in 4 cycles of 2 min grinding at 27 x g followed by a 4 min pause. Keep the temperature of the bowl below 80 &#730;C at any time. Perform no more than 3 batches and let the bowl cool down afterwards.</li>
		<li>Collect the finely ground walnut shells into a 500 mL round-bottom flask. Remove the isopropanol by rotary evaporation at 40 &#730;C and 125 mbar.</li>
		<li>Dry the walnut shells overnight in a vacuum oven at 50 &#730;C and 50 mbar.</li>
		<li>Sieve the finely ground walnut shells through a 1 mm sieve. Ground the particles that are too large again with the rotary ball mill.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Wood Feedstocks
<ol>
	<li>Cutting of the wooden planks
	<ol>
		<li>Place the wooden planks under a drill, which is equipped with a flat wood speed drill bit. Collect the wood shavings in a glass beaker.</li>
		<li>Place the wooden shavings in a coffee grinder to cut them into smaller pieces.</li>
	</ol>
	</li>
	<li>Extraction of fatty acids from wood
	<ol>
		<li>Perform the extraction of the fatty acids from wood in precisely the same manner as described for walnut shells in Step 1.2. 
		NOTE: No milling of the wood is performed in the ball mill, as the conditions described in Step 1.3 did not result in a reduction of the particle size.</li>
	</ol>
	</li>
</ol>
3. Extraction of High &#946;-O-4 Ethanosolv Lignin
<ol>
	<li>Mild ethanol extraction (Method A)
	<ol>
		<li>Put 25 g of the feedstock in a 500 mL round-bottom flask. Add an 80:20 ethanol/water mixture (200 mL), 4 mL of 37% HCl solution (0.24 M) and a magnetic stirring bar to the round-bottom flask.</li>
		<li>Attach a reflux condenser to the round-bottom flask. Heat the mixture atreflux temperature with an oil bath for 5 h with vigorous stirring.</li>
		<li>Allow the mixture to cool to room temperature by removing it from the oil bath. Filter the mixture (185 mm diameter, 10 &#181;m pore size) and wash the residue with 4 times 25 mL of ethanol.</li>
	</ol>
	</li>
	<li>Work-up and isolation of lignin
	<ol>
		<li>Collect the liquor in a 500 mL round-bottom flask. Concentrate the liquor by rotary evaporation at 40 &#730;C and 150 mbar.</li>
		<li>Dissolve the obtained solid in 30 mL of acetone. Use an ultrasonic bath if the solid does not dissolve completely.</li>
		<li>Precipitate the lignin by adding the mixture to 600 mL of water. If no precipitation occurs, add a small amount of saturated aqueous Na2SO4 solution to flocculate the lignin.</li>
		<li>Collect the lignin by filtration (185 mm diameter, 10 &#181;m pore size). Wash the lignin with 25 mL of water 4 times. Discard the filtrate if no analysis of the hemicellulose fraction is required. If the filtrate is very turbid, add it to a centrifugation tube and collect the (solid) bottom fraction by centrifugation.</li>
		<li>Allow the lignin to air dry overnight. Dry the lignin further in a vacuum oven (overnight at 50 &#730;C and 50 mbar).</li>
		<li>Determine the yield after the lignin is dried overnight in a vacuum oven.</li>
		<li>Determine the lignin extraction efficiency by dividing it with the total lignin content as determined by the Klason method24.</li>
	</ol>
	</li>
	<li>Higher temperature ethanol extraction (Method B)
	<ol>
		<li>Put 15 g of the feedstock in a 250 mL autoclave. Add an 80:20 ethanol/water mixture (120 mL), 2.4 mL of HCl (0.24 M) and a magnetic stirring bar.</li>
		<li>Heat the mixture at 120 &#730;C for 5 h with a stirring speed of 5.2 x g. Cool the mixture afterwards in an ice bath.</li>
		<li>Filter the mixture (185 mm diameter, 10 &#181;m pore size) and wash the residue 4 times with 15 mL of ethanol.</li>
		<li>Perform further work-up and isolation precisely as described in Step 3.2.</li>
	</ol>
	</li>
	<li>Larger scale higher temperature ethanol extraction of walnut shell (Method C*)
	<ol>
		<li>Put 90 g of finely ground walnut shell in a 1 L high pressure autoclave. Add an 80:20 ethanol/water mixture (750 mL) and 6.25 mL of H2SO4 (0.12 M).</li>
		<li>Heat the mixture at 120 &#730;C for 5 h with a stirring speed of 35.8 x g. Cool the mixture back to room temperature by turning on the reactors cooling system after the 5 h reaction time.</li>
		<li>Filter the mixture (185 mm diameter, 10 &#181;m pore size) and wash the residue 4 times with 75 mL of ethanol. 
		NOTE: Using multiple filters saves a lot of time.</li>
		<li>Collect the liquors in 2 equal batches. Perform the work-up and isolation as described as in Step 3.2 with double amount of the solvents for both batches.</li>
	</ol>
	</li>
	<li>Control experiments of Step 3.1. (Method A*) and Step 3.3. (Method B*) (optional)
	<ol>
		<li>Put the exact some materials as described in Step 3.1.1 but replace the HCl solution with 1.67 mL of H2SO4 (0.12M). The rest of Step 3.1-3.2 is identical to Method A.</li>
		<li>Put the exact some materials as described in Step 3.3.1 but replace the HCl solution with 1.0 mL of H2SO4 (0.12M). The rest of Step 3.3 is identical to Method B.</li>
	</ol>
	</li>
</ol>
4. De-etherification of the Lignin (optional)
<ol>
	<li>Dissolve 1000 mg of lignin in a 24 mL of 1:1 1,4-dioxane/water mixture in a 100 mL round-bottom flask. Add 1 mL of a 37% HCl solution to the mixture.</li>
	<li>Add a stirring bar and attach a reflux condenser to the round-bottom flask. Heat the mixture to 100 &#730;C with an oil bath for 5 h with vigorous stirring.</li>
	<li>Allow the mixture to cool down to room temperature by removing it from the oil bath. Add the mixture to 160 mL of water to precipitate the lignin.</li>
	<li>Collect the lignin by filtration (185 mm diameter, 10 &#181;m pore size) and wash the lignin with 25 mL of water 2 times. Allow the lignin to air dry overnight. Dry the lignin further in a vacuum oven (overnight at 50 &#730;C and 50 mbar).</li>
</ol>
5. Analysis of Lignin
<ol>
	<li>Two-dimensional nuclear magnetic resonance (2D-NMR) analysis
	<ol>
		<li>Dissolve 60 mg of dried lignin in 0.7 mL of d6-acetone. Add a few drops of D2O if the lignin does not fully dissolve. Put the mixture in an NMR-tube and take a 2D proton heteronuclear single quantum coherence spectra (HSQC) with an NMR spectrometer with the following parameters: (11, -1), (160, -10), nt = 4, ni = 51220.</li>
		<li>Analyze the obtained HSQC spectra. Adjust the spectra by manual phase corrections on both axis until all signals are positive, as this is especially important along the horizontal (f2) axis. Perform no baseline corrections. The positions of all the linkages are given in Step 5.1.3 and 5.1.6.</li>
		<li>Integrate the signals in the aromatic region that correspond to the three different aromatic units (proton numbering as per Figure 4). These signals are in the region [(Proton range)(Carbon range)]: 
		S2/6:&#160; &#160; &#160; &#160; &#160; &#160; &#160;&#8239;&#8239; [(6.48-6.90)(104-109)] 
		S&#39;2/6:&#160; &#160; &#160; &#160; &#160; &#160;&#8239; &#8239;&#8239;&#8239;[(7.17-7.50)(105-109)] 
		Scondensed:&#160; &#160; &#160; &#160;[(6.35-6.65)(106-109)] 
		G2:&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;[(6.78-7.14)(111.5-116)] 
		G5:&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;[(6.48-7.06)(115-120.5)] 
		G6:&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;[(6.65-6.96)(120.5-124.5)] 
		H2/6:&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;[(7.05-7.29)(128.5-133)] 
		Note: H3/5 overlaps with the G5 signal, and it is assumed that H2/6 has the same intensity as H3/5. The signal for condensed G overlaps with G5. If no (or hardly any) G2 and G6 signals are present, this indicates that full condensation of G has occurred.</li>
		<li>Calculate the amount of aromatic units with the formula: 
		Total aromatic = (((S2/6 + S&#39;2/6)/ 2) + Scondensed) + ((G2 + G5 + G6- H2/6) / 3) + (H2/6 / 2)</li>
		<li>Calculate the percentage of G, H and S units with the following formulas: 
		Ratio S = (((S2/6 + S&#39;2/6)/ 2) + Scondensed): total aromatic x 100% 
		Ratio G = ((G2 + G5 + G6- H2/6) / 3) : total aromatic x 100% 
		Ratio H = (H2/6 / 2) : total aromatic x 100%</li>
		<li>Integrate the signals in the aliphatic region signals that correspond to the &#946;-O-4, &#946;-&#946; and &#946;-5 linkages and Hibbert Ketones. These are in the region [(proton range)(carbon range)]: 
		&#946;-O-4&#945; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;[(4.76-5.10)(73-77.5)] 
		&#946;&#39;-O-4&#945;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;[(4.44-4.84)(81.5-86)] 
		&#946;-O-4&#946; and &#946;&#39;-O-4&#946;&#160; &#160; &#160; [(4.03-4.48)(85-90.5)] 
		&#946;-O-4&#947; and &#946;&#39;-O-4&#947;&#160; &#160; &#160; &#160;[(3.10-4.00)(58.5-62)] 
		&#946;-5&#945;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; [(5.42-5.63)(88-92)] 
		&#946;-5&#946;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; [(3.36-3.56)(53-54.5)] 
		&#946;-5&#947;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; [(3.50-4.00)(62-64.5)] 
		&#946;-&#946;&#945;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; [(4.59-4.77)(86.5-89.5)] 
		&#946;-&#946;&#946;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; [(2.98-3.20)(55.5-59)] 
		&#946;-&#946;&#947;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; [(3.75-3.96)(72.5-76)] and [(4.10-4.31)(72.5-76)] 
		HK&#947;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;&#8239;&#160; &#160;[(4.20-4.30)(66-68)] 
		Note: The &#946;-protons of the &#946;-O-4 and &#946;&#39;-O-4 linkages overlap. The structural motifs of these linkages are given in Figure 1.</li>
		<li>The total number of linkages per 100 C9 units are all based on the signal of the &#945; proton of the linkages. Calculate the total number of linkages with the following formulas: 
		&#946;-O-4 linkages = (&#946;-O-4&#945; + &#946;&#39;-O-4&#945;) / total aromatic x 100 
		# &#946;-5 linkages = &#946;-5&#945; / total aromatic x 100 
		&#946;-&#946;linkages = &#946;-&#946;&#945; / total aromatic x 100</li>
	</ol>
	</li>
	<li>Gel permeation chromatography (GPC) analysis
	<ol>
		<li>Dissolve 10 mg of dried lignin in 1 mL of tetrahydrofuran (THF) (with a drop of toluene as the internal standard). Filter this mixture through a 0.45 &#181;m syringe filter into an autosampler vial with a reduced volume inlet of 0.3 mL. Close the autosampler vial with a cap.</li>
		<li>Inject 20 &#181;L of the sample into a THF GPC. Process the obtained data.</li>
		<li>Correct the obtained signal for the reference signal (toluene). Select the elution volume for the correct range (~200-10000 Da). Calculate mass distribution by the software.</li>
	</ol>
	</li>
</ol>
6. Depolymerization of Lignins to Phenolic 2-Arylmethyl-1,3-Dioxolanes (Acetals)
<ol>
	<li>Place 50 mg of dried lignin in a 20 mL microwave vial as the reaction vessel equipped with a magnetic stirrer. Add 0.85 mL of 1,4-dioxane, 50 &#181;L of ethylene glycol in 1,4-dioxane (0.54 mL/mL) and 50 &#181;L of octadecane (internal standard) in 1,4-dioxane (26 mg/mL).</li>
	<li>Close the reaction vessel and heat the solution to 140 &#176;C while stirring at 3.8 x g.</li>
	<li>When the reaction vessel reaches 140 &#176;C, add 50 &#181;L of Fe(III)OTf3 in 1,4-dioxane (0.1 g/mL).</li>
	<li>Stir the reactor for 15 min.</li>
	<li>Cool the reactor to room temperature and remove the depolymerization liquid as described in Step 7.1.</li>
</ol>
7. Work up and Analysis of Depolymerization Mixtures
<ol>
	<li>Filter the liquid over Celite (permeability: 2.60-6-50 darcy; particle size: 150 mesh Tyler; sieve retain (140 M US): 2.0-25.0%) and collect in a 2 mL centrifuge tube.</li>
	<li>Concentrate the liquid overnight at 35 &#176;C in a rotational vacuum concentrator.</li>
	<li>Extract the final oil/solid with the following procedure:
	<ol>
		<li>Suspended and swell the residue in 0.15 mL of dichloromethane (DCM) by extensive mixing (by vortex), 15 min of sonication and 30 min in automatic wheel.</li>
		<li>Centrifuge the samples for up to 10 s using a minispin tabletop centrifuge to ensure that the liquid is at the bottom of the tube (centrifugation speed: 671 x g).</li>
		<li>Add 0.75 mL of toluene and mix extensively (by vortex and 10 min sonication).</li>
		<li>Centrifuge the samples for up to 10 s using a minispin tabletop centrifuge (centrifugation speed: 671 x g) to ensure that the liquid is at the bottom of the tube.</li>
		<li>Separate the light organic liquid from the solid or thick oily residue and filter this liquid over a plug of Celite and collect in a glass vial. 
		NOTE: This procedure for suspension/washing is repeated three times, and in the last extraction, 0.5 mL of toluene is used.</li>
	</ol>
	</li>
	<li>Concentrate the combined organic phases by rotary evaporation (40 &#730;C, 20 mbar).</li>
	<li>Dissolve the oily residue in 1 mL of DCM for gas chromatography flame ionization detector (GC-FID) analysis.</li>
	<li>Perform GC-FID using a GC equipped with an FID detector and use helium as the carrier gas. Standard settings: 1 &#181;L injection, a split ration of 50:1, a helium flow of 0.95 mL/min. Equip the GC apparatus with a HP5 column (30 m x 0.25 mm x 0.25 &#181;m) and run with a temperature profile which starts with a 5 min 60 &#176;C isotherm. Follow-up by a 10 &#176;C/min ramp for 20 min to 260 &#176;C. Hold this temperature for 20 min.</li>
	<li>Integrate the peaks in the spectra manually. The retention times of the peaks are as following: octadecane (21.4 min), H-acetal (19.5 min), G-acetal (20.8 min), S-acetal (23.4 min). Use the obtained values in Step 7.8 to perform the quantification.</li>
	<li>Perform the quantification of G acetal, which is based on a calibration curve with a standard compound isolated using an internal standard (octadecane). 
	NOTE: Calibration curve: <img alt="Equation 1" src="/files/ftp_upload/58575/58575eq1.jpg" />&#160;(R2=0,9991) 
	Yield G-acetal:&#160;<img alt="Equation 2" src="/files/ftp_upload/58575/58575eq2.jpg" /> 
	Based on previous results9,21,23 a response factor was estimated for H and S acetal as 2.19 and 1.82, respectively.</li>
</ol>

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+model+compounds+for+understanding+acid-catalyzed+lignin+depolymerization:+Identification+of+renewable+aromatics+and+a+lignin-derived+solvent.">Advanced model compounds for understanding acid-catalyzed lignin depolymerization: Identification of renewable aromatics and a lignin-derived solvent.</a> Journal of the American Chemical Society. 138 (28), 8900-8911 (2016).</li><li>Shuai, L., Saha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+high-yield+lignin+monomer+production.">Towards high-yield lignin monomer production.</a> Green Chemistry. 19 (16), 3752-3758 (2017).</li><li>Rinaldi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paving+the+Way+for+Lignin+Valorisation:+Recent+Advances+in+Bioengineering,+Biorefining+and+Catalysis.">Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis.</a> Angewandte Chemie International Edition. 55 (29), 8167-8215 (2016).</li><li>Schutyser, W., Renders, T., van den Bosch, S., Koelewijn, S. F., Beckham, G. T., Sels, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemicals+from+lignin:+an+interplay+of+lignocellulose+fractionation,+depolymerization,+and+upgrading.">Chemicals from lignin: an interplay of lignocellulose fractionation, depolymerization, and upgrading.</a> Chemical Society Reviews. 47 (3), 852-908 (2018).</li><li>Constant, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+structure+and+composition+of+technical+lignins:+a+comparative+characterization+study.">New insights into the structure and composition of technical lignins: a comparative characterization study.</a> Green Chemistry. 18 (9), 2651-2665 (2016).</li><li>Smit, A., Huijgen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+fractionation+of+lignocellulose+in+herbaceous+biomass+and+hardwood+using+a+mild+acetone+organosolv+process.">Effective fractionation of lignocellulose in herbaceous biomass and hardwood using a mild acetone organosolv process.</a> Green Chemistry. 19 (22), 5505-5514 (2017).</li><li>Lancefield, C. S., Panovic, I., Deuss, P. J., Barta, K., Westwood, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-treatment+of+lignocellulosic+feedstocks+using+biorenewable+alcohols:+towards+complete+biomass+valorization.">Pre-treatment of lignocellulosic feedstocks using biorenewable alcohols: towards complete biomass valorization.</a> Green Chemistry. 19 (1), 202-214 (2017).</li><li>Deuss, P. J., Lancefield, C. S., Narani, A., de Vries, J. G., Westwood, N. J., Barta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+acetals+from+lignins+of+varying+compositions+via+iron+(III)+triflate+catalysed+depolymerization.">Phenolic acetals from lignins of varying compositions via iron (III) triflate catalysed depolymerization.</a> Green Chemistry. 19 (12), 2774-2782 (2017).</li><li>Bauer, S., Sorek, H., Mitchell, V. D., Ib&#225;&#241;ez, A. B., Wemmber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Miscanthus+giganteus+Lignin+Isolated+by+Ethanol+Organosolv+Process+under+Reflux+Condition.">Characterization of Miscanthus giganteus Lignin Isolated by Ethanol Organosolv Process under Reflux Condition.</a> Journal of Agricultural and Food Chemistry. 60 (33), 8203-8212 (2012).</li><li>Deuss, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+triflates+for+the+production+of+aromatics+from+lignin.">Metal triflates for the production of aromatics from lignin.</a> ChemSusChem. 9 (2), 2974-2981 (2016).</li><li>Nicholson, D. J., Leavit, A. T., Francis, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-stage+Klason+method+for+more+accurate+determinations+of+hardwood+lignin+content.">A three-stage Klason method for more accurate determinations of hardwood lignin content.</a> Cellulose Chemistry and Technology. 48 (1-2), 53-59 (2014).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+lignocellulose+conversion+with+integrated+catalyst+recycling+yielding+valuable+aromatics+and+fuels.">Complete lignocellulose conversion with integrated catalyst recycling yielding valuable aromatics and fuels.</a> Nature catalysis. 1, 82-92 (2018).</li><li>Lancefield, C. S., Ojo, O. S., Tran, F., Westwood, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Functionalized+Phenolic+Monomers+through+Selective+Oxidation+and+C-O+Bond+Cleavage+of+the+&#946;-O-4+Linkages+in+Lignin.">Isolation of Functionalized Phenolic Monomers through Selective Oxidation and C-O Bond Cleavage of the &#946;-O-4 Linkages in Lignin.</a> Angewandte Chemie International Edition. 54 (1), 258-262 (2015).</li><li>Rahimi, A., Ulbrich, A., Coon, J. J., Stahl, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formic-acid-induced+depolymerization+of+oxidized+lignin+to+aromatics.">Formic-acid-induced depolymerization of oxidized lignin to aromatics.</a> Nature. 515, 249-252 (2014).</li><li>Bosque, I., Magallanes, G., Rigoulet, M., K&#228;rk&#228;s, M. D., Stephenson, C. R. 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The authors report no conflict of interest

The results from the extractions at different conditions and from different biomass sources reveal how the optimal conditions for lignin extraction with a relatively high content of &#946;-O-4 linkages can vary depending on the source. For example, walnut extraction at harsher conditions (method B) provides a three-fold increase in the yield with a near retention of the amount of &#946;-O-4 units, while for beech and cedar the yield increases but is accompanied by a significant drop in the amount of &#946;-O-4 units. On the other hand, for pine, the harsher extraction conditions provide very little benefit in yield and also lead to a lignin with a very low amount of &#946;-O-4 units. This means that typically some form of optimization has to be involved to get the correct balance between the lignin yield and quality in the form of retention of the amount of &#946;-O-4 units in the obtained lignin material.
The large increase in the Mw of the lignin material obtained from harsher extraction conditions demonstrates that under these conditions larger fragments can be extracted providing higher yields. However, at these conditions, additional fragmentation occurs, providing additional lower molecular weight material and thus increasing the polydispersity as can be clearly seen in the GPC graphs of walnut (Figure 5a) and cedar (Figure 5d) in the form of the signal at ~500 Da.
HSQC NMR is an important informative tool to provide comparative data on the quality of different lignins. It should be noted that in this procedure a standard HSQC experiment is performed, this is great for obtaining comparative data but is not necessarily quantitative due to differences in relaxation times. The high amount of linkages displayed for some lignins in Table 1 are overestimated. Quantitative HSQC experiments provide better results but cost significantly more NMR time, although alternatives exist29. In our experience, the numbers in Table 1 should be divided by a factor of a 1.3 to better reflect the actual amount of &#946;-O-4 units per 100 aromatic units.
As previously mentioned, the reported results point out how finding optimum conditions can vary depending on the source in order to obtain the maximum monomer yields. For instance, when walnut is used as the starting material, the overall total acetals yield increases around two times if harsher conditions (method B) are employed for lignin extraction. However, this is mainly due to the great difference in lignin extraction yield, without affecting the &#946;-O-4 content. Differently, when pine is used milder extraction conditions (method A) are preferable. In fact, lignin extraction results in very similar yields in the two cases but harsher conditions cause a drop in &#946;-O-4 units (especially non-etherified &#946;-O-4 linkages) which can be the reason for such a low monomer yield, as indicated in the previous paragraph. A significant loss of non-etherified &#946;-O-4 linkages can be observed as well in the cases of beech and cedar if conditions (method B) are applied for extraction which possibly leads to a lower monomers yield. However, the overall acetal yield does not differ that much depending on the extraction conditions. In fact, an approximate two-fold increase in lignin extraction yield is observed for both biomass sources switching from methods A to B&#160;which compensates for the roughly two-fold decrease in monomer yield.

For the chemical industry to become sustainable, renewable feedstocks such as lignocellulosic biomass should be used as alternative to current dominant fossil ones1. However, to make the use of such feedstocks economically viable, high-value applications should be sought for all its contents. Lignocellulosic biomass can contain about 30 wt% of the lignin that is an aromatic biopolymer for which currently only a few applications have been developed beyond its use as low-value fuel2. Therefore, the methodology towards potentially increased-value aromatic components is of major interest to ensure the success of future biorefineries.
Recent research has focused heavily on the development of novel methodologies for the selective cleavage of the most abundant &#946;-O-4 linkage (Figure 1a) in lignin to obtain specific aromatic, typically phenolic, monomers3,4,5,6.&#160;For example, the application of acids between 80 &#176;C to 180 &#176;C is very effective in cleaving the &#946;-O-4 linkage forming aldehyde and ketone fragments7,8. Our groups and others have recently demonstrated that acidolysis combined with the methodologies to stabilize and trap reactive fragments is extremely potent to obtain phenolic monomers with specific chemical motifs9,10,11,12. Of these, in particular acetal trapping of reactive aldehydes with alcohols to obtain phenolic 2-arylmethyl-1,3-dioxolanes (acetals) proved powerful due to its relative simple application and the retention of the highly functionalized nature of the lignin monomers (Figure 1b)13,14. These acetals are obtained from the depolymerization in a ratio that relates to the distribution of the H, G and S monomers present in the parent lignin feedstock.
Acid catalyzed depolymerization, like many of the most elegant methodologies developed, are relatively mild and do not cleave stronger C-C bonds that occur in lignin15. However, C-C bonds become especially abundant when harsh lignocellulose fractionation conditions are applied due to the condensation of reactive fragments released from the cleavage of weaker C-O bonds16,17. The loss of &#946;-O-4 content from the biomass processing method is clearly demonstrated by the analysis of a range of technical lignins, which were shown to only retain up to 6 &#946;-O-4 linkages per 100 aromatic units18, while in lignocellulose these numbers range from 45 to 90 linkages per 100 aromatic units depending on the source16. Turning to milder extraction conditions, lignins can be obtained with the linkage distributions that better reflect the natural lignin. Nevertheless, this demands a trade-off between the extraction efficiency and the quality of the obtained lignin material17. This also has precedence in organosolv extraction of lignin, which is a popular method to fractionate lignin. Many variations of this process exist, with the methods employing different temperatures, acid content, extraction times and solvents. Here, the extraction severity has a direct impact on the obtained lignin structure and thus its suitability for further valorization19,20,21. For example, organosolv lignin produced by the ethanol based Alcell process, operated for 5 years at demonstration scale, had relatively low amount of &#946;-O-4 linkages left as it was operated at relatively high temperature to ensure efficient delignification in order to obtain high quality carbohydrates for bioethanol production. Nevertheless, biobased solvents with little environmental impact like ethanol are preferred and thus the extraction methods that result in lignins of higher value are of interest. Alcoholic solvents are of particular interest as in addition to being the extraction medium they also incorporate into the lignin structure, for example, &#946;&#39;-O-4 (Figure 1a)22, which partly &#34;protects&#34; the structure from undesired cleavage. A suitable method would potentially be first to obtain lignin with a high &#946;-O-4 content and in a sequential step to remove the remainder of the lignin to access high value cellulose.
In this manuscript, we describe a straight forward and highly reproducible procedure for the extraction of high &#946;-O-4 lignin by mild ethanol extraction. Depending on the biomass source, this can lead to relatively high extraction efficiency and yield. Procedures for the characterization of the obtained lignin are provided as well as how to &#34;deprotect&#34; the etherified &#946;&#39;-O-420. Additionally, an assessment procedure is presented for the potential of these lignins in selective depolymerization procedures that rely on selective cleavage of the &#946;-O-4 linkages. This assessment is performed using the iron(III) triflate catalyzed depolymerization in the presence of ethylene glycol to obtain phenolic 2-arylmethyl-1,3-dioxolanes23 which demonstrates the link between &#946;-O-4 content in the lignin material and the monomer yields21. The results show the balance between high lignin extraction efficiency and the potential of the obtained lignin to be depolymerized to specific aromatic monomers.

<table border="0" cellpadding="0" cellspacing="0" style="width:952px;" width="953">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Materials</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Iron (III) triflate</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td>708801-1G</td>
			<td>purity: 90%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Octadecane</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td>0652-100G</td>
			<td>purity: 99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Celite</td>
			<td style="width:164px;">Alfa Aesar</td>
			<td>H33152.0B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Silica Gel</td>
			<td style="width:164px;">SiliCycle</td>
			<td>R12030B-1KG</td>
			<td>P60 40-63 &#956;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dichloromethane</td>
			<td style="width:164px;">Macron Fine Chemicals</td>
			<td>6779-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Walnut shells</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pine wood</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cedar wood</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Beech wood</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol</td>
			<td style="width:164px;">JT Baker Chemicals</td>
			<td style="width:119px;">00832000001</td>
			<td>Ethanol absolute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isopropanol</td>
			<td style="width:164px;">Acros Organics</td>
			<td>149320025</td>
			<td>99.5+% extra pure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acetone</td>
			<td style="width:164px;">Macron Fine Chemicals</td>
			<td>2440-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tetrahydrofuran</td>
			<td style="width:164px;">Boom B.V.</td>
			<td>164240025</td>
			<td>stabilized with BHT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Toluene</td>
			<td style="width:164px;">Macron Fine Chemicals</td>
			<td>8608-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Water</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td>Demi water from the internal supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">1,4-Dioxane</td>
			<td style="width:164px;">Acros Organics</td>
			<td>408820010</td>
			<td>99+% extra pure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Hydrochloric acid</td>
			<td style="width:164px;">Acros Organics</td>
			<td>124620026</td>
			<td>37% solution in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sulfuric acid</td>
			<td style="width:164px;">Boom B.V.</td>
			<td>760519081000</td>
			<td>95-97%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acetone-d6</td>
			<td style="width:164px;">Acros Organics</td>
			<td>325320500</td>
			<td>99.8 atom% D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Deuterium oxide</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:119px;">151882-100G</td>
			<td>99.9 atom% D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Filters</td>
			<td style="width:164px;">Munktell</td>
			<td style="width:119px;">400303185</td>
			<td>185 mm diameter, 10 &#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Magnetic stirring bars</td>
			<td style="width:164px;">VWR</td>
			<td style="width:119px;">442-4525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe filter</td>
			<td style="width:164px;">Sartorius</td>
			<td style="width:119px;">17559-Q</td>
			<td>0.45 &#956;m filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Autosampler vial (2 mL)</td>
			<td style="width:164px;">Brown</td>
			<td style="width:119px;">151123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Reduced volume inlet (0.3 mL)</td>
			<td style="width:164px;">Brown</td>
			<td style="width:119px;">150820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Autosampler caps (11 mm)</td>
			<td style="width:164px;">Brown</td>
			<td style="width:119px;">151216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Autosampler vial crimper</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Oil bath</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringes (1 mL)</td>
			<td style="width:164px;">Henke Sass Wolf</td>
			<td style="width:119px;">4010-200V0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Heating block-4 positions</td>
			<td style="width:164px;">IKA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Micro tubes 2 ml</td>
			<td style="width:164px;">Sarstedt</td>
			<td style="width:119px;">72691</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Crimp seals-20 mm</td>
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extraction of lignin with high &#946;-o-4 content by mild ethanol extraction and its effect on the depolymerization yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Watch this Scientific Journal Video about Extraction of Lignin with High ÃŽÂ²-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield at JoVE.com

Extraction of Lignin with High ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

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