Fractional Precipitation of Wheat Straw Organosolv Lignin

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Fractional Precipitation of Wheat Straw Organosolv Lignin –Macroscopic Properties and Structural Insights Heiko Lange, Peter Schiffels, Marco Sette, Olena Sevastyanova, and Claudia Crestini ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01475 • Publication Date (Web): 05 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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ACS Sustainable Chemistry & Engineering

Manuscript for publication as Original Research Paper in

ACS Sustainable Chemistry and Engineering

Fractional Precipitation of Wheat Straw Organosolv Lignin –Macroscopic Properties and Structural Insights

Heiko Lange,a,* Peter Schiffels,b Marco Sette,a Olena Sevastyanova,c,d and Claudia Crestinia,*

E-mail: [email protected]; [email protected]

a

University of Rome ‘Tor Vergata’, Department of Chemical Sciences and Technologies, Via della Ricerca Scientifica, 00133 Rome, Italy

b

Fraunhofer-Institut für Fertigungstechnik und Angewandte Materialforschung IFAM, Wiener Straße 12, 28359 Bremen, Germany

c

KTH Royal Institute of Technology, Department of Fibre and Polymer Technology, Stockholm SE-100 44, Sweden

d

KTH Royal Institute of Technology, WWSC Wallenberg Wood Science Center, Stockholm SE-100 44, Sweden

1

ACS Paragon Plus Environment


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ABSTRACT Wheat straw organosolv lignin has been thoroughly characterized with respect to bulk material properties, surface properties and structural characteristics, by means of antioxidant assays, determination of the equilibrium constant in water-octanol partitioning, i.e., logP-determination, optimized gel permeation chromatography, quantitative 31P NMR spectroscopy, quantitative HSQC measurements and XPS studies. The material was subsequently fractionally precipitated based on a binary solvent system comprised of n-hexane and acetone, to yield four fractions that exhibit distinct molecular mass characteristics, while displaying similar structural characteristics, as revealed by the same set of analysis techniques applied to them. Extensive correlation studies underline the versatility of the obtained fractions as higher quality starting materials for lignin valorization approaches, since, e.g., glass transition temperatures, Tg, correlate well with number average molecular weights, Mn, applying the Flory-Fox relation as well as its Ogawa and Loshaek variations.

KEYWORDS organosolv lignin fractional precipitation 31

P NMR

QQ-HSQC HSQC0 logP anti-oxidant activity XPS DSC TGA 2


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INTRODUCTION Almost one third of the mass of lignocellulosic biomass is comprised of polyphenolic oligomers and polymers, which are for the most part lignins.1 Being already present in nature with an myriad of different specifics,2,3 industrial processes aimed at isolating the cellulose and hemi-cellulose parts of various types of lignocellulosic biomass, further modify the structure of the lignin component, which is often obtained as a rather low-quality by-product in biorefinery processes that were optimized with respect to the cellulose components.4,5 Only recently, bigger global players and promising newcomers in the biorefinery business started to view the lignin-containing streams as an additional source for augmented revenues.5 Consequently, different higher quality lignins are available nowadays for potential applications in sectors of material science, functional cosmetics and eventual biomedical devices:6 e.g., the LignoBoost lignin, that is obtained by a novel process for the precipitation of lignin after standard kraft pulping,7 and lignins obtained in industrialized organosolv pulping processes, e.g., Alcell lignin8 and CIMV BioligninTM.9

Any kind of higher value application, however, calls for a rather detailed structural understanding of the technically produced lignin of choice, and the determination of basic thermal characteristics.10, Correlating structural features like, e.g., the abundance of free phenolic residues with observed thermal behaviors have proven to be a powerful tool: thermal properties of an industrial lignin, and thereby its processibility in extruder-based applications could be tuned by modifying the concentration of free phenolic hydroxyl groups.11–15

While the functional groups without doubt play a significant role with respect to any kind of application, the polymer characteristics as such are important, especially the number average molecular weight (Mn) and the polydispersity (PD); most isolated lignins are characterized by a polydispersity that a priori prevents any use in higher value applications, independent of the Mn, which additionally differs significantly depending on the isolation process.4 A rather obvious and 3



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simple way to arrive at lignins that exhibit, at least, an industrially acceptable polydispersity is fractionation of lignin. This idea of fractionating lignins is not new as such, and has been investigated on various samples in the 1980s,16 after initial attempts in the early 1950s;17 recently, however, it re-gained momentum in connection with more specified investigations for industrial applications of lignins. Reported versions include sequential precipitation out of alkaline solutions,18 fractional precipitation of re-dissolved kraft lignin in a gradually changed binary solvent system,19 (sequential) extractions using different solvents18,20–26 or just plain water27 followed by adsorption as well as the fractionation by ultrafiltration of black liquor using ceramic membranes28– 30

as versatile options..

In the frame of an effort aiming at identifying novel valorizations of lignins in material science as well as in biomedical applications, an organosolv lignin was used that was isolated from biomass waste materials abundantly available across Europe, i.e., wheat straw. Wheat straw, while being an abundant source for isolating lignin, represents also one of the more complicated sources, which has been analyzed in numerous studies for decades.26,31–41 As a grass lignin, wheat straw lignins display H-, G- and S-type motifs; rather high amounts of ferulates and coumarates are usually found in connected to the α- and preferentially γ-hydroxyl groups mainly of β-O-4’ interunit bonding motifs. Most findings of this kind have been made using wheat straw milled wood lignins; high amounts of β-O-4’ interunit bonding motifs of up to 75% were reported,40 together with up to 11% of esters.33 We investigated the possibilities of a solubility-based fractional precipitation of a wheat straw organosolv lignin, i.e., the CIMV BioligninTM. Some studies on wheat straw BioligninTM exist, in which it has been purified and was hydrolyzed before use.43 A rather controversial mass analyses-based study on wheat straw BioligninTM exists, in which very regular interunit bonding motif repetitions are claimed.44 The fractional precipitation approach used here is based on re-precipitating the solubilized lignin in an increasingly apolar mixtures of a binary solvent system following a recent pioneering work on 4


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softwood kraft lignin.19 This fractional precipitation was to be done purposefully on the wheat straw BioligninTM as it comes out of the pilot process. We herein report the results of this first solubilitybased fractional precipitation of a non-kraft lignin, and discuss the correlation between structural and their thermal properties delineated for the different fractions.

Figure 1: Structural features of representative lignins: (A) outdated structure of Kraft lignin; (B) ‘generic’ structure of lignosulfonates; (C) updated structure of milled wood lignin and organosolv lignins.

MATERIALS AND METHODS General information Chemicals and solvents were purchased from Sigma-Aldrich in appropriate grades, and were used without further purification if not stated otherwise. Wheat straw organosolv lignin (WS-OSL) was produced via the BioligninTM process in the CIMV (Compagnie Industrielle de la Matière Végétale, 5



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Levallois Perret, France) pilot plant in Pomacle, France, and was kindly provided by CIMV (batch E10-R2).9 The starting lignin and its fractions were oven-dried until a constant weight was achieved prior to analysis.

Fractional precipitation protocol Following the pioneering literature report,19 in a representative run, 100 g wheat straw organosolv lignin were dissolved in 1 L of acetone and stirred for 3 h at ambient temperature. The resulting suspension is filtered under vacuum; the filter cake represents ‘acetone-insoluble organosolv lignin’ (WS AIOL). 250 mL of n-hexane are slowly added over 30 min to the stirred filtrate via a dropping funnel, before the developing suspension is stirred at ambient temperature for 12 h. The resulting pasty precipitation, separated from the 4/1 (v/v) acetone/hexane supernatant by decantation, represents ‘wheat straw acetone-soluble organosolv lignin HEX-250’ (WS ASOL-250). The supernatant was gradually diluted with 750 mL of n-hexane by means of a dropping funnel over a time span of 3 h. Stirring was continued for another 3 h, before the sticky solid precipitating at the walls of the container is isolated by decantation from the 1/1 (v/v) acetone/n-hexane solvent mixture, to isolate ‘wheat straw acetone-soluble organosolv lignin HEX-750’ (WS ASOL-750). Residual wheat straw lignin still in solution in the supernatant is isolated by complete evaporation of the solvents under reduced pressure, and subsequently re-dissolved in 200 mL acetone. The new solution is gradually diluted under constant vigorous stirring with 800 mL of n-hexane over a time span of 30 min using a dropping funnel. Stirring is continued at ambient temperature for 12 h, during which a pasty precipitation is formed at the walls of the reaction vessel. Decanting the 1/4 (v/v) acetone/hexane mixture yields ‘wheat straw acetone-soluble organosolv lignin HEX-800’ (WS ASOL-800). All obtained fractions were oven-dried at 80º C until constant weight.

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Acetone-extraction In a representative run, 10 g wheat straw organosolv lignin were extracted for 3 h with 500 mL of acetone by means of a Soxhlet-extractor. The extractives were isolated under reduced pressure and subsequently oven-dried to yield ‘Soxhlet-derived-acetone soluble organosolv lignin’ (WS ASOLSOX). The extracted solids were recuperated, representing ‘Soxhlet-derived acetone-insoluble organosolv lignin (WS AIOL-SOX). The two obtained fractions were oven-dried at 80º C until constant weight.

n-Hexane extraction Approx. 20 g of WS OSL were extracted with 100 mL of n-hexane by means of a Soxhlet-extractor for 24 h, yielding 98.7% of not further Soxhlet-derived hexane-insoluble organosolv lignin (WS HIOL-SOX), and 1.3% of extractives. (The purified wheat straw lignin has not been further analyzed in the course of this work.) An aliquot of the extractives was diluted with an equimolar amount

of

ethyl

acetate,

before

75 µL

of

dry

pyridine

and

75 µL

of

N,O-

bis(trimethylsilyl)trifluoroacetamide were added. After 30 min at room temperature, the mixture was analyzed by gas chromatography coupled with mass spectrometry. Analysis was done using a Shimadzu GCMS QP2010 Ultra equipped with an AOi20 autosampler unit. A SLB®-5ms Capillary GC Column (L × I.D. 30 m × 0.32 mm, df 0.50 µm) was used as stationary phase, ultrapure Helium as the mobile phase. Analysis was done using Shimadzu LabSolutions GCMS Solution software (Version 2.61). The various components were identified by comparison against the NIST11 library.

Optical Microscopy Optical microscopic images were obtained with a Keyence VHX-2000 using lenses VH-Z100 (1001000x) or VH-Z500 (500-5000x) for the different magnifications.

7



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Elemental analysis Elemental analysis were obtained using a Thermo Fisons Carlo Erba Combustion Elemental Analyzer CHNS, calibrated using 2,5-bis(5-tert-butyl-benzoxazol-2-yl)thiophene and operated with the factory-provided Eager300 software.

Determination of logP The equilibrium constant for partitioning of WS OSL and its fractions between distilled water and n-octanol was determined using the shake-flask method. Briefly, the starting material and the fractions were dissolved in n-octanol saturated with distilled water at a concentration of approx.250 µM. An aliquot of these solutions, 5 mL, respectively, was mixed with an equal volume of distilled water saturated with n-octanol, and the biphasic system was mixed by means of wrist shaker for 12 h at room temperature. Equal-volume samples of the shaken organic phase and the starting solution were subsequently taken and analyzed after appropriate dilution by UVspectroscopy at λ = at 517 nm using a Shimadzu UV-1800 spectrophotometer, operated via the manufacturer’s software UV Probe, Version 2.42. Experiments were run in triplicate.

Analysis of antioxidant activity Radical scavenging activity of wheat straw organosolv lignin and the fractions derived thereof against 2, 2’-diphenyl-1-picrylhydrazyl (DPPH)45 was determined using a Shimadzu UV-1800 spectrophotometer, operated via UV Probe, Version 2.42. Approximately 1.0 mg of analyte were accurately weighed and suspended in 1000 µL of spectrophotometric grade DMSO. An initial solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) is prepared at a concentration of 10 mg/mL (25.4 mM), that is subsequently diluted to a working concentration of DPPH in DMSO of 0.04 mg/ml. For each different aliquots from the sample solution (2, 5, 10, 25, 50, 100 µL) were added to 450 µL of diluted solution of DPPH in DMSO, before sample was finally brought to a final volume of 1000 µL with DMSO. Measurements were realised by selecting 300-600 nm as 8


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spectrum range and 0.5 nm as spectral resolution. Antioxidant activity is expressed both in form of IC50-values in mg/L, determined using the linear range of the obtained calibration curve, and antioxidant activity indices46 using λ = 519 nm as standard wavelength for analysis. Measurements were run in triplicate.

Gel permeation chromatography Approx. 5 mg of lignin were acetobrominated for analysis,47 and analyses was performed as detailed before,47 using a Shimadzu instrument consisting of a controller unit (CBM-20A), a pumping unit (LC 20AT), a degasser (DGU-20A3), a column oven (CTO-20AC), a diode array detector (SPD-M20A), and a refractive index detector (RID-10A) ), and controlled by Shimadzu LabSolutions (Version 5.42 SP3). Different set-ups comprising two or three analytical GPC columns (each 7.5 x 30 mm) in series were realized for analyses: in case of two columns Agilent PLgel 5 µm 10000 Å, followed by Agilent PLgel 5 µm 1000 Å, which in case of the three columns are followed by an Agilent PLgel 5 µm 500 Å. HPLC-grade THF (Chromasolv®, Sigma-Aldrich) was used as eluent (0.75 mL min−1, at 40 °C). Standard calibration was performed with polystyrene standards (Sigma Aldrich, MW range 162 – 5 x 106 g mol−1). Analyses were run in duplicate.

FT-IR analysis FT-IR spectra were measured on a Perkin Elmer Spectrum 100 FTIR spectrometer. The spectra were acquired in form of potassium bromide pellets as the average of 32 scans between 450 and 4000 cm−1 with a resolution of 4 cm−1.

Quantitative 31P NMR analysis Quantitative

31

P NMR analysis was performed as reported before:48,49 an accurately weighed

amount of lignin (about 30 mg) was phosphitylated using 2-chloro-4,4,5,5-tetramethyl-1,3,2dioxaphospholane (Cl-TMDP) and the spectra were measured on a Bruker 300 MHz 9



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spectrophotometer (256 scans at 20° C). All chemical shifts reported are relative to the reaction product of water with Cl-TMDP, which gives a sharp signal in pyridine/CDCl3 at 132.2 ppm. Quantitative analysis was performed based on previous literature reports.

Quantitative HSQC measurements Approx. 100 mg lignin were acetylated in 2 mL pyridine/acetic anhydride (v/v = 1:1) at 50° C for 48 h as previously reported.50 Spectra were acquired at 303 K with a Bruker Avance 600 spectrometer equipped with a cryoprobe. The sample consisted of 80 mg of acetylated lignin dissolved in 600 µL of DMSO-d6. A matrix consisting of 256 x 2048 points was obtained in eight scans. QQ-HSQC measurements were performed in accordance with the original reference51 as reported before.52 Alternatively, HSQC0 measurements were performed in accordance with the original reference53 as reported before.50 Data processing: NMR data were processed with MestreNova (Version 8.1.1, Mestrelab Research) by using a 60°-shifted square sine-bell apodisation window; after Fourier transformation and phase correction a baseline correction was applied in both dimensions. The final matrix consisted of 1024 x 1024 points, and cross-peaks were integrated with the same software that allows the typical shape of peaks present in the spectrum to be taken into account.

XPS analysis X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Scientific K-Alpha system, equipped with a monochromatic Al Kα (hν = 1486.6 eV) X-ray source. The spot-diameter of the investigated area was typically 0.40 mm. Powdered lignin samples were loosely placed on a sample holder for the analysis. The measurements were made under an ultra-high vacuum of 2 × 10−9 mbar, at room temperature. High-resolution core-level spectra were acquired at pass energy of 40 eV at 0° take-off angle using constant analyzer energy mode (CAE). Survey spectra for each sample were acquired at pass energy of 150 eV. For charge compensation a dual beam Argon/electron source for 10


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ultra-low energy beam neutralization was used. Data processing involved background subtraction and a curve-fitting procedure (a mixed GaussianLorentzian function was employed) based on a least-squares method (Thermo K-Alpha Software). Experimental errors were estimated to be ±0.2 eV for the photoelectron peaks of carbon with FWHM of the C1 component not exceeding 1.7 eV. Charging effects were corrected using the C1s component ascribed to aliphatic / aromatic carbon atoms after deconvolution (component C1, see the discussion below) and taken to be 285.0 eV. The reproducibility of the peak position thus obtained was ±0.2 eV.

Differential scanning calorimetry (DSC) Differential scanning calorimetry was performed using a Mettler Toledo DSC/TGA 1 Calorimeter or a Mettler Toledo DSC 820 Calorimeter. Typical sample amounts of around 2-4 mg were exactly weighted in 40 µL aluminum pans, which were closed with a lid that was centrally punctured to prevent pressure built-up. The following optimized temperature sequence was applied on all samples under a Nitrogen atmosphere (50 mL min−1) if not stated otherwise: 25 °C to 105 °C to 25 °C to 400 °C at a rate of 10 °C/min. Analysis was performed using Mettler Toledo Star1 software. Experiments were run in duplicate or triplicate if not stated otherwise.

Thermogravimetric analysis (TGA) Thermogravimetric analysis under nitrogen atmosphere was performed using a Mettler Toledo DSC/TGA 1 calorimeter. Typical sample amounts of around 4 mg were weighted in oven-dried 70 µL ceramic pans or platinum pans. Exact sample weights were determined automatically using the in-built balance of the calorimeter. The following analysis temperature protocol was used as standard protocol under nitrogen atmosphere (50 mL min−1), and repeated under oxygen atmosphere (50 mL min−1): 25° C to 750° C at a rate of 10° C/min. Analysis was performed using Mettler Toledo Star1 software. Experiments were run in duplicate or triplicate. 11



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RESULTS AND DISCUSSION Fractional precipitation protocol Wheat straw organosolv lignin (WS OSL) was isolated by CIMV within their pilot plant for the BioligninTM process was chosen as starting material. The CIMV organosolv process consists in a fractionation of lignocellulosic materials based on a mixture of acetic acid/formic acid/water (55:30:15, w/w/w) during 3.5 h at 105°C under atmospheric pressure.9 The lignin is separated from the extraction by water-induced gradual precipitation and subsequent filtration. The fractional precipitation chosen in this paper is based on the fractional precipitation approach published by Argyropoulos et coll. in 2014 for Lignoboost kraft lignin.19 This solubility-based fractionation on the basis of a binary system of solvents that can be readily recuperated via simple distillation was deemed superior with respect to principal alternative of a sequential washing-out of fractions using a series of solvents as demonstrated on Lignoboost kraft lignin by Lawoko et coll. Ultrafiltration was sorted out as option due to its technological requirements, since a more easily applicable process was desired. The optimized fractional precipitation protocol starts with dissolving the WS OSL in acetone at a rather low concentration of 1 g per 10 mL. The fractional precipitation protocol is depicted in detail in Figure 2. Despite the low concentration, WS OSL proofed to be rather insoluble for the most part, resulting in approx. 80% acetone insoluble material, isolated as WS AIOL. The soluble part is gradually precipitated by lowering the polarity of the solution via the addition of n-hexane so, that three more WS OSL fractions, i.e., acetone soluble WS ASOL-250, acetone soluble WS ASOL750 and acetone soluble WS ASOL-800, are obtained at acetone/n-hexane ratios of 4/1, 1/1 and 1/4, respectively. Summed-up yields of the soluble fractions are low, accounting for maximum 20% of the starting material (Table 1, Figure 2).

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Figure 2: Flow chart illustrating the fractional precipitation of wheat straw organosolv lignin (WS OSL). AIOL – acetone insoluble organosolv lignin; ASOL – acetone soluble organosolv lignin; for further details, see Materials and Methods section. Yields are the average of three representative runs.

Table 1: Bulk data for solubility-based fractions of wheat straw organosolv lignin. lignin / lignin fraction WS OSL f

WS AIOL WS ASOL-250 WS ASOL-750 WS ASOL-800 WS AIOL-SOX WS ASOL-SOX

yield a [%]

C9-formula b

C9 MW [Da]

---

C9H7.1O0.6(OCH3)1.2

162

80 4.9 10 4.4 64 36

C9H7.3O3.4(OCH3)1.3 C9H6.7O2.8(OCH3)1.3 C9H7.6O2.5(OCH3)1.4 C9H9.1O2.2(OCH3)1.3 C9H6.1O4.0(OCH3)1.3 h C9H7.0O2.6(OCH3)1.3 h

210 200 197 193 220 199

Mn c [Da] 860 f (920 g) 1100 1800 1100 610 610 f 660

Mw c [Da] 5000 f (4600 g) 23600 7700 3900 3500 10800 f 5000

PDI c 5.9 f (5.0 g) 21 4.3 3.5 5.7 18 f 7.7

logP d

antiox. activity e

−0.22

2.0 (9)

0.54 0.24 0.18 1.48 0.89 0.20

1.4 (13) 2,0 (9) 2.0 (9) 1.5 (12) n.d. i 1.5 (12)

a: average of three representative runs. b: based on elemental analysis in combination with H/G/S-ratio as delineated by quantitative HSQC measurements. c: determined by gel permeation chromatography (THF-based system as described by Lange et al.47 d: determined using n-octanol and water. e: numbers represent antioxidant activity indices (AAIs); numbers in brackets represent IC50 values in mg/L; data obtained in DMSO. f: sample not completely soluble nor after acetobromination, nor acetylation, and neither by using 10% (v/v) dioxane in THF. g: values for acetobrominated sample dissolved in 10% (v/v) dioxane in THF. h: H/G/S-ratio estimated based on the similarity of these fractions with WS AIOL and combined WS ASOL fractions, respectively. i: sample was not fully soluble in various solvent systems.

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In a second, better scalable and thus user-friendly approach with respect to the simpler generation of a fraction of WS OSL that exhibits superior solubility characteristics, WS OSL was simply extracted with acetone for 3 h using a Soxhlet extractor, yielding two fractions, i.e., WS AIOLSOX and (Figure 2). Most obviously, Soxhlet-derived WS ASOL-SOX can be seen as combination of WS ASOL-250, WS ASOL-750 and WS ASOL-800; an interpretation subsequently underlined by WS ASOL-SOX the detailed analysis obtained for WS ASOL-SOX in comparison to the nonSoxhlet-derived fractions.

Macroscopic analysis and chemical composition Analysis by Optical Microscopy The various fractions were first analyzed by optical microscopy, and representative images are given in Figure 3. As it can be delineated from the images, the fractions result in optically very different materials. Especially the acetone soluble WS ASOL fractions obtained upon increasing levels of hexane exhibit a character that optically and haptics-wise resemble of hardened resins. Acetone insoluble WS AIOL and WS AIOL-SOX appear charcoal-like, and especially WS AIOLSOX was re-isolated in form of very recalcitrant brick-like material.

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Figure 3: Optical microscopy images of the fractions obtained in the solubility-based fractional precipitation of wheat straw (Magnification: 500x) organosolv lignin: (A) WS-OSL; (B) WS AIOL; (C) WS ASOL-250; (D) WS ASOL750; (E) WS ASOL-800; (F) WS AIOL-SOX; (G) WS ASOL-SOX.

Determination of Elemental Composition Elemental analysis of the solids revealed traces of nitrogen in the starting material, as well as in all fractions (Table 1). It has to be speculated at this point, that these nitrogen impurities originate from residual protein content in the wheat straw used for pulping, or represent inorganic nitrogen sources originating from fertilizer residues with which the wheat straw used for pulping was contaminated. Interesting enough, both elemental analysis and X-ray photoelectron spectroscopy (XPS) data (vide infra) suggest that not all the nitrogen impurities are removed through the fractionation process. Normalized for the nitrogen impurity, elemental compositions were translated into standard C9formulas for the different fractions, which are given in Table 1 together with the molecular weight of the average monomers. The relatively high amount of impurities in the starting material is reflected in the very low theoretical molecular weight found for WS OSL. Overall results suggest, 15



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however, that the differences in elemental compositions of the fractions are much less significant than one might expect based on the solvent system used for fractional precipitation; this finding was subsequently underlined by the more detailed structural analysis.

Determination of hydrophobic extractives In order to gain further insight into the nature of the impurities, especially in order to understand the amount of fatty acid impurities that could explain the results of the elemental analysis of the starting material, WS OSL was continuously extracted with n-hexane for 24 h. Analysis of the extractives by gas chromatography coupled with mass spectrometry (GC-MS) reveals the presence of a series of saturated and unsaturated fatty acids, as well as steroids (Table 2). The overall quantity of isolated extractives was, however, rather low (1.3%).

Table 2: Identified n-hexane extractives from WS OSL. identified extractive a,b

amount [%]

hexadecanoic acid (E)-9-octadecenoic acid tetradecanoic acid β-sitosterol (Z,Z)-9,12-octadecadienoic acid octacosane octadecanoic acid campesterol docosanoic acid tetracosanoic acid stigmasterol stigmasterol acetate eicosanoic acid

17 5.8 4.8 3.9 3.4 3.2 2.8 2.5 2.1 2.0 1.7 1.1 0.8

a: analysis was done after silylation of free hydroxyl groups with N,O-bis(trimethylsilyl)trifluoroacetamide. b: extractives with a relative abundance of >0.5% are listed.

Molecular weight determination The overall effect of the fractionation in terms of obtaining batches of lignin with different distinct number average molecular weights is delineable from a comparison of chromatograms obtained by gel permeation chromatography of acetobrominated samples in THF (Figure 4, Table 1). These 16


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GPC traces graphically clearly indicate that the fractions are characterized by different Mn and Mw; most noteworthy, peaks of maximum absorbance of the different fractions appear as shoulders in the chromatogram of the parent WS OSL. Numbers for Mn for the acetone insoluble WS AIOL and the WS AIOL-SOX fractions appear surprisingly low, but have to interpreted with caution since only the soluble part is reflected; most noteworthy, considerable amounts of the sample in form of insoluble precipitates had to be filtered out by means of a syringe filter prior to injection into the GPC set-up after standard derivatization via acetobromination. The two fractions remained also largely insoluble after acetylation, and when submerged in DMSO at concentrations as low as 2 mg per mL. While Mn is largely following the trend that could be expected based on reported findings, 19,24 Mw seems to indicate that the samples remain being highly polydispers. While being enriched in a bigger amount of oligomers exhibiting a narrower molecular weight range, i.e., a small PDI, but are, at the same time, significantly polluted by larger oligomers or polymers, causing Mw, and hence also the PDI, to be out of scope. In light of the chosen fractional precipitation approach, it has to be speculated that these higher order oligomers and polymers must exhibit solubility characteristics that resemble those of the ‘smaller’ bulk material of the fraction.

17



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Figure 4: Comparison of GPC traces of fractions of wheat straw organosolv lignin. The offset of the chromatogram of WS OSL is due to the multiplication of the normalized experimental values by a factor of 1.5.

Evaluation of Lipophilicity/Hydrophilicity Variations Hydro- and lipophilicity of WS OSL and the delineated fractions were estimated by means of their water-octanol partitioning coefficients in form logP. Original WS OSL exhibits a low negative logP value of −0.22, and thus macroscopically displays a rather hydrophilic character. However, given that the batch under analysis exhibits impurities in terms of protein residues, etc. and given that the sample did not fully dissolve, this hydrophilicity-indicating value has rather to be seen as an artefact, probably mainly reflecting the partitioning behavior of the better soluble impurities. Acetone insoluble WS AIOL seems to be rather hydrophilic, which is in accordance with the fact that this fraction did not dissolve in the polar acetone. The logP values found for the acetone soluble fractions WS ASOL-250 and WS ASOL-750 exhibit similar values indicating a rather lipophilic character, that is, however, lower than that of acetone insoluble WS AIOL. This lipophilic character can be explained, given that these fractions were soluble in polar solvent, and then obtained by adding apolar n-hexane to the acetone. The value for acetone soluble WS ASOL-800 18


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fits the expected pattern as well: a value of 1.48 marks this fraction as the most hydrophilic one. The delineated values for WS AIOL-SOX and WS ASOL-SOX do sustain the just discussed picture regarding the hydrophilicity/lipophilicity of the wheat straw lignin under analysis.

Determination of Antioxidant Activity One of the main beneficial characteristics of natural polyphenols is their anti-oxidant activity. This activity is usually mainly attributed to the number of free phenols present in the lignin. Since the chosen fractional precipitation is based on polarity changes of the solvent systems from which the fractions are precipitated, different fractions might exhibit different amounts of free phenolic groups, and hence might display different anti-oxidant activities. Fractions of KL isolated from a binary solvent system yielded fractions exhibiting distinctive differences in anti-oxidant activities.54 Using the DPPH-based assay for the determination of the antioxidant activity, it was necessary to work in DMSO as solvent system, since this was the only inert solvent in which WS OSL and its fractions with exception of WS AIOL-SOX could be dissolved in at reasonable concentrations; the more standard solvent system of 10% water in 1,4-dioxane was not suitable for achieving complete dissolution. The results are displayed in Table 1 in form of antioxidant activity indices (AAI) and IC50-values; it must be concluded that there are moderate differences between the fractions: interpreting the AAI-number as detailed in subscript e) of Table 1, unfractionated WS OSL as well as acetone soluble fractions WS ASOL-250 and WS ASOL-750 display very strong antioxidant activities (all AAI > 2), whereas still strong antioxidant activities were found for acetone soluble fractions WS ASOL-800 and WS ASOL-SOX (AAI = 1.5), as well as for acetone insoluble WS AIOL, which exhibits the weakest antioxidant activity with a strong AAI of 1.4. As a note of caution, it should be explicitly stated here, that every solvent has a certain effect on the oxidation potential of a substance, eventually favoring or disfavoring ceratin steps of the underlying mechanism. As such, the absolute values of antioxidant activity determined in this study for this lignin might not be easily comparable to other reported data, but they allow a direct comparison of 19



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the antioxidant activity of the parent lignin with its fractions. Noteworthy, the findings of strong to very strong antioxidant activities in unfractionated WS OSL as well as in all fractions was indirectly confirmed when different fractions were used in the production of chitin-lignin electrospun films for wound-healing purposes: a series of films differing only in the nature of the lignin fraction showed similar characteristics in cell-based in vitro studies elucidating the effect on the concentration of cell-markers indicative of oxidative stress.55

Structural characterization of WS OSL Analyses of the bulk material of the fractions with respect to overall lipophilicity/hydrophilicity and antioxidant activity do not support the idea that significant structural differences between the fractions were obtained by gradual precipitation from a binary solvent system increasing apolarity; only the average molecular weights differs, and exhibits a trend that could have been expected based on the physico-chemical properties and literature knowledge. In order to verify that the fractions differ mainly in oligomer/polymer lengths and only to a significantly lesser extend in composition of these chains, detailed structural features of WS OSL and the fractions obtained thereof were to be elucidated.

FT-IR Analysis An initial FT-IR analyses allowed for initial identification of structural motifs expected for the WS OSL, and subsequently trackable in the delineated fractions. The findings, reported in detail in the Supporting Information, confirm the qualitative similarity of the batch under study with previous productions.43 The FT-IR spectra for the different fractions do not differ drastically from the spectrum of WS OSL. This finding fits the similarity of the fractions as delineated by the other analytical techniques used (vide infra).

Analyses by Quantitative 31P NMR and Quantitative HSQC Methods In order to gain more detailed insights, quantitative

31

P NMR spectroscopic analyses as well as 20


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quantitative (2D) 1H-13C HSQC measurements were performed, the latter in form of both quick quantitative HSQC (QQ-HSQC),51,52 as well as in form of HSQC0.53 It has been shown before that both quantitative HSQC approaches deliver comparable data.50 Unfortunately, analysis of the AIOL fraction suffers also in this context from low solubilities under the necessarily heavily standardized analysis protocols; data obtained and displayed for the acetone insoluble WS AIOL fraction represent only the soluble part. The Soxhlet-derived fractions were excluded from this detailed structural analysis, since WS AIOL-SOX showed unsatisfying solubilities just as WS AIOL, and since acetone soluble WS ASOL-SOX can be seen as a combination of acetone soluble fractions WS ASOL-250, WS ASOL-750 and WS ASOL-800. The 31P NMR spectra, as well as the HSQCspectra obtained for WS OSL and its ASOL fractions are available in the supplementary material.

Analyses of the

31

P NMR spectra were performed based on the ppm-regimes of different, with 2-

chloro-4,4,5,5-tetramethyl-1,3,2-dioxophospholane

in situ

phosphitylated

hydroxyl

(OH-)

groups.48,56 Also the HSQC cross peaks were identified based on previous literature reports on this matter;10,33,40 functional groups were not only identified on the basis of the existence of their most indicative cross peak, but were considered only in case all theoretically expectable HSQC crosspeaks stemming from this very group could be identified. Quantification was either done using well resolved and isolated representative peaks, or via averaging over two or more signals of a given interunit bonding motif in case the cross peaks were partly overlapped with other signals. The latter approach bears by trend the risk of over- or underestimating the abundance of a given motif, but in light of the purity of the starting material, it was decided that this approach would yield more reliable data overall. Results obtained by quantitative

31

P NMR were correlated with those delineated via quantitative

HSQC analysis, by converting the ‘mmol/g’ obtained results of the

31

P NMR measurements into

abundances in ‘% C9 units’, using the molecular weight of the average C9 monomers of each fraction (Table 1). The combined results for the acetone soluble fractions WS ASOL-250, WS 21



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ASOL-750 and WS ASOL-800 were set in comparison to the starting material WS OSL, and are listed in detail in Table 3.

22


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Table 3: Structural data obtained for WS OSL and solubility-based fractions thereof. quantities in % C9 for wheat straw and derived fractions a,b structural feature total aromatics H-type G-type S-type ratio H/G/S aromatics internal motifs ox. β-O-4' H-type ox. β-O-4' der. G-type ox. β-O-4' der. S-type β-O-4' β-5' β-β' β-1' d 5-5' free esterified e 4-O-5' free esterified 5-5'/β-O-4', a-O-4''(DBDO) β-O-4', α-O-5' (BD) β-1', α-O-α’ (SD) β-O-4', α-O-4' aryl-enol-ether total internal motifs terminal motifs aliphatic OH e free esterified H-type phenolic OH e free esterified e G-type phenolic OH free esterified S-type phenolic OH e free esterified ratio H/G/S phenolics

OSL c

ASOL750 d

ASOL250 d

AIOL d

ASOL800 d

ASOLSOX d

8.4 63.0 28.6

2.8 63.8 33.4

4.4 57.1 38.5

3.8 57.2 39.0

3.5 62.5 34.0

n.d. n.d. n.d.

1 / 7.5 / 3.4

1 / 23 / 12

1 / 13 / 8.8

1 / 15 / 10

1 / 18 / 10

n.d.

0.8 1.5 2.6 22.2 4.7 3.0 1.4

0.7 2.3 1.2 21.7 7.0 3.5 3.0

2.1 4.3 2.5 32.1 5.7 5.9 3.1

1.4 4.2 2.7 26.7 7.0 5.2 2.5

1.1 4.3 2.3 11.4 8.9 3.3 3.4

n.d. n.d. n.d. n.d. n.d. n.d. n.d.

0.2 (0.01) 0.2 (0.01)

0.3 (0.01) n.d.

0.4 (0.02) n.d.

0.2 (0.01) n.d.

0.9 (0.05) 0 (< 0.01)

1.1 (0.06) n.d.

4.3 (0.27) 0.6 (0.04)

Fractional Precipitation of Wheat Straw Organosolv Lignin

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