Solubility of Softwood Hemicelluloses ... - ACS Publications

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On the solubility of softwood hemicelluloses Saina Kishani, Francisco Vilaplana, Wenyang Xu, Chunlin Xu, and Lars Wågberg Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00088 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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Biomacromolecules

On the solubility of softwood hemicelluloses Saina Kishani*a,b, Francisco Vilaplanab,c, Wenyang Xud, Chunlin Xud, Lars Wågberg*a,b

a

School of Chemical Science and Engineering, Fibre and Polymer Technology, Royal Institute of

Technology, Teknikringen 56-58, SE-10044 Stockholm, Sweden. b

Wallenberg Wood Science Centre (WWSC), Teknikringen 56-58, SE-10044 Stockholm,

Sweden. c

School of Biotechnology, Division of Glycoscience, Royal Institute of Technology, Albanova

University Centre, SE-10691 Stockholm, Sweden. d

Johan Gadolin Process Chemistry Centre, Laboratory of Wood and Paper Chemistry, Åbo

Akademi University, FI-20500, Turku/Åbo Finland.

Abstract

It is demonstrated that the molecular solubility of softwood hemicelluloses is significantly influenced by pre-treatment of the fibers, extraction and downstream processing. To quantify these effects, four hemicellulose samples were extracted from different thermomechanical pulps of Norway spruce. The molecular solubility of the samples was characterized by size and molar

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mass distributions, and the morphology of the molecules was studied using high resolution microscopy techniques. All extracted samples were well dispersed in aqueous media creating transparent dispersions but dynamic light scattering measurements showed that molecular solubility can only be achieved using specific pretreatments and extractions. The procedure yields acetylated galactoglucomannan (AcGGM)-rich hemicelluloses with an average molar mass of 21-35 kDa, and a diameter of 10 nm but also shown that water is a poor solvent for this sample since an association is detected as soon as the concentration is above ca. 20 g/L. These associated hemicellulose dispersions are still absolutely clear on visual inspection, underlining the need for careful measurement when assessing the solubility of wood hemicelluloses.

Keywords: Hemicellulose, Extraction, Solubility, Association

1. Introduction Hemicelluloses are a group of carbohydrates that are closely associated with cellulose and lignin in all higher plants, and they have been shown to be a promising resource for producing films, barriers and hydrogels1-9. These abundant sources of polysaccharides have also received considerable attention during the past decades in a biorefinery context. The focus has been on optimizing the extraction procedures and yield, chemical modification, or on preparing films and coatings for use as different types of barriers, but not so much on understanding the physicochemical properties of the extracted materials. However, to design and develop products to meet the future demands of a sustainable society, and to reduce the use of oil-based bulk plastics, the phase behavior of these materials needs to be investigated more fundamentally since the solution


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properties of the hemicellulose will be of great importance for the properties of films and gels formed from these substances. It has been well established that there is a close correlation between the chemical structure of the polysaccharide films and their barrier properties10. In turn, this means that an understanding of how molecules are organized, i.e. aggregated, or dispersed alone or in combination with different matrices, is essential to tailor the development of future bio-based materials from wood. Since the polysaccharides are usually processed in aqueous solution/dispersion, an understanding of their solubility in water is of crucial importance. In softwoods, O-acetyl-galactoglucomannans (AcGGM) are the predominant hemicelluloses, consisting of a main chain of (1→4)-linked β-D-mannopyranosyl and (1→4)-linked β-Dglucopyranosyl units, with substitutions of (1→6)-linked α-D-galactopyranosyl attached to the mannosyl units11, 12. The hydroxyl groups at the C-2 and C-3 positions of the mannose units in the backbone are partially substituted by O-acetyl groups. The dissolution and preparation of a purified fraction of these hemicelluloses from Norway spruce (Picea abies) and mechanical pulps from Norway spruce have earlier been investigated11, 13, 14. Typically, hemicelluloses are extracted from thermomechanical pulps (TMP) through a hot-water extraction and a series of filtration or ultra-filtration steps. This is not an uncomplicated task since the TMP pulping process creates a complex mixture of dissolved and colloidal substances (DCS) containing different wood components. It has, for example, been shown that the colloidal portion of the DCS contains a significant part of the lipophilic wood extractives lignin and lignans

21

15-20

, wood hemicelluloses,

. The chemical and physical structures of the polysaccharides, i.e. their

composition, degree of branching and degree of substitution, can differ depending on their natural resources, isolation and method of purification22, and the chemical composition will have a profound influence on the solubility of the polysaccharide. Hitherto, the molecular solubility of


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polysaccharides has been poorly characterized, and mostly determined as the turbidity of the dispersion using either ocular inspection, a turbidity measurement or dynamic light scattering (DLS) measurements, where there is a large discrepancy between the molar mass of the materials and the detected hydrodynamic size of the molecules in the solution23. Consequently, little is known about how the extraction and pulp preparation procedures affect the physico-chemical structure, and the subsequent solubility or dispersibility of a softwood polysaccharide. The aim of the present work is to use well-defined separation procedures to investigate the molecular solubility or dispersibility of softwood hemicelluloses, particularly AcGGM. The work is aimed at creating a link between the chemical structure, molar mass and size distribution of these materials, their solubility in water and the structure of the molecules in aqueous media. In this respect, four AcGGM powder samples were prepared using different pulp preparation and extraction procedures. The samples were subjected to particle size and molar mass distribution determinations using dynamic light scattering (DLS), and size exclusion chromatography (SEC) in both dimethyl sulfoxide and aqueous media. In addition, the morphology of the molecules and associated molecular structures were determined using atomic force microscopy (AFM) and cryo-transmission electron microscopy (Cryo-TEM). A careful chemical compositional and structural analysis was carried out by high-pH anion exchange chromatography (HPAEC) and glycosidic linkage analysis by gas chromatography – mass spectrometry (GC-MS).


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2. Experimental 2.1. Materials and methods Four hemicellulose samples were prepared using different pulp preparation, extraction and purification procedures (Figure 1). The pulp preparation and extraction procedures for the different samples are described in detail below;

2.1.1. Extraction from fiberized wood using hot-water extraction This sample designated GGM1 was kindly provided by Shoaib Azhar (WWSC, KTH, Sweden). The preparation has been described in detail by Azhar et al.24. In brief, chips of Norway spruce were steamed for 5 min and fiberized, i.e. coarsely refined, in a pilot-scale refiner25. Extraction was performed in water, at 60±2 ⁰C and a concentration of 2% (W/W), while stirring with a mechanical stirrer (≈400 rpm) for 3 h. The liquid phase was separated using a fiber cloth sieve under vacuum, followed by vacuum filtration through a glass fiber filter (Whatman GF/A, 1.6 µm) to wash out impurities, e.g. larger particles and colloidal extractives. The filtrate was further concentrated using vacuum evaporation by heating on a water bath to 50 ⁰C. The resulting hemicellulose concentrate was freeze-dried and stored as a powder.

2.1.2. Isolation of hemicelluloses from commercial TMP and refined hand-made chips, using soxhlet and hot-water extraction Two

hemicellulose

samples,

GGM2

and

GGM3,

were

extracted

from

different

thermomechanical pulps (TMPs). For GGM2, a spruce log with a diameter of 26 cm was cut into lengths of 40 cm and sawn into slices with a thickness of 2 cm. These slices were debarked and cleaned, removing all small knots, sticky resins and branches by hand. The wood was chipped


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into chips with dimensions of approximately 3mm×2cm×2.5cm (see Figure 1). The chips were steamed at 125 °C for 20 min at atmospheric pressure, followed by high-pressure heating for 5 min at 100 °C. The steamed chips were passed twice through a pilot-scale refiner (SproutWaldron) with 12” discs at Chalmers University of Technology. The final TMP had a concentration of about ̴ 40 wt%.

The third sample, GGM3 originated from a large-scale commercial TMP production from Norway spruce, provided by Åbo Akademy, Åbo, Finland. After extraction, this sample had a consistency of approximately ̴ 35 wt%. The TMPs were stored at -24 °C before use. Batches of the hand-made and the commercial TMPs were used for the isolation of GGM2 and GGM3, respectively. The extraction was carried out according to the method developed by Willför et al.11. The pulps were freeze-dried and subjected to soxhlet extraction with a mixture of hexane:acetone (9:1) to remove lipophilic extractives and lignin impurities26,

27

. Soxhlet-

extracted TMPs were then submerged in distilled water at 60 ⁰C at a 2% consistency and stirred for 3 h using an overhead stirrer at about 350 min-1. The suspensions were filtered through a paper machine wire (100 mesh) to remove oversized particles. The filtrates were saved, and the pulps were subjected to a second extraction in water under the same condition, i.e. at 60 ⁰C, for 3 h and at 2% consistency. The pH of the filtrates from the hand-made and commercial TMPs were about 5.4 and 5.2, respectively. The filtrates were then centrifuged at 1000 g for 30 min, followed by vacuum filtration through a GF50 glass fiber (Whatman) to remove fines and small particles. The filtrates were then concentrated using vacuum evaporation and heating on a water bath at 40 ⁰C. The concentrates were vacuum filtered using a Whatman nylon membrane filter (0.2 µm pores) to remove colloidal substances which contain mostly extractives18. The samples were then


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Biomacromolecules

precipitated by adding ethanol to the filtrate (8.5:1.5), and leaving the mixture overnight. The supernatants were removed by vacuum and the precipitates were washed twice with pure ethanol, twice with methanol and once with methyl iso-butyl ether (MTBE). The hemicellulose powders, GGM2 and GGM3, were obtained by drying the precipitates in a vacuum desiccator overnight. A schematic representation of the extraction procedure is shown in Figure 1.

2.1.3. Hemicelluloses recovered from TMP water The sample designated GGM4 was extracted from Norway spruce after the first refiner in a Finnish mill, according to Willför et al.2. The isolation was performed by adding a starch-based cationic polymer (Raifix 25035, Raisio Chemicals Oy) to the TMP process water to flocculate and separate colloidal wood resins by filtration2, and passing the material through an XAD-7 resin column (a polyacrylate produced by Rohm and Haas UK) to remove lignin and other aromatic impurities28. Further purification was performed using an Ultrafilter CR200/1 apparatus (Valmet Flootek, Malmö, Sweden) with a membrane cut-off of 20 kDa. After a second purification step, the hemicellulose was dissolved in water and subjected to dialysis using a membrane with 12-14 kDa cut-off. A hemicellulose powder was obtained by vacuum evaporation and freeze-drying13. MTBE and n-hexane were purchased from Merck Millipore (for liquid chromatography). Methanol, ethanol (96%) and acetone were supplied by VWR chemicals.


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Figure 1. Flow sheet for pulp preparation and extraction of hemicellulose samples from Norway spruce.


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2.2. Characterization 2.2.1. Size determination The hydrodynamic size distributions of the hemicelluloses in water were determined using dynamic light scattering (DLS) (Zetasizer, ZEN 3600, Malvern, U.K.). The hydrodynamic diameter (dH) of the samples, in solutions, was calculated from the translational diffusion coefficient using the Stokes-Einstein equation;

= ƞ

[1]

where is the translational diffusion coefficient, is the Boltzmann’s constant, is the absolute temperature, and ƞ is the viscosity. The degree of association and polydispersity were determined from the number distributions evaluated from the autocorrelation functions29. The shape and the decay with time of the autocorrelation function can be used to determine the diffusion coefficient and the mean size of the particles. Hemicellulose samples were dispersed in both Milli-Q water (18.2 MΩ), and DMSO/0.5% (W/W) LiBr and mixed at different concentrations and temperatures in a thermomixture (Eppendorf thermomixture comfort, Germany) for at least 4 h. The size was determined at the preparation temperatures after filtration through a 0.45 µm nylon filter. The refractive index and absorbance of each sample were measured using an automatic refractometer (J457, Rudolph, USA) and UV/Vis spectrophotometer (UV2550, Shimadzu), respectively, and used in the dH calculation. The viscosity values were determined for different eluents at different temperatures.


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2.2.2. Average molar mass (Mw) and molar mass distribution The molar mass distribution of the extracted hemicelluloses was determined using a size exclusion chromatography (SEC) system (Agilent Technologies 1260 Infinity, from PSS, Germany) coupled with a refractive index (RI) detector (G1362A 1260 RID Agilent Technologies), and multi angle laser light scattering (MALLS) detector (SLD 7000 PSS, Germany). The MALLS detector had a 35 mW laser as the light source operating at a wavelength 635 nm with seven scattering angles. The signal at 90⁰ was used to detect aggregates or mass separation in the samples30. The combined light scattering and RI detectors were calibrated using pullulan standards of known molar mass (PSS, Germany). The weight-average molar mass for the samples was investigated in dimethyl sulfoxide solution (DMSO, HPLC grade, SigmaAldrich, Sweden)/0.5% (W/W) LiBr and Water/(0.1M) NaNO3, as eluents. Samples were dissolved in the eluents at a concentration of 2 g/L in small cuvettes and left in a thermo-mixer (Eppendorf thermomixture comfort, Germany) at 60 ⁰C overnight. GGM4 did not show sufficient dispersibility in water/NaNO3, and was not therefore evaluated in this solvent. However, all the samples gave transparent dispersions in DMSO/LiBr eluent. The clear dispersions were transferred to the special SEC vials without filtration, and injected into a PSS GRAM guard column, and two other 100 Å (300 × 8 mm, 10 µm particle size) and 10000 Å (300 × 8 mm, 10 µm particle size) columns (also purchased from PSS, Germany) thermostated at 60 ⁰C. The injection volume was set to 100 µL with a flow rate of 0.5 mL/min. The number-average (Mn) and weight-average (Mw) molar masses were evaluated from the light scattering data using WinGPC UniChrom GPC/SEC software (PSS, Germany). The specific refractive index increment (dn/dc) values were measured separately (Table 1) using an automatic refractometer (J457, Rudolph, USA).


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Biomacromolecules

2.2.3. Charge and zeta potential measurement The cationic demand of the extracted hemicellulose samples was determined by polyelectrolyte titration (PET)31. Dry hemicelluloses were dispersed in MQ water to a concentration of 1 g/L. The pH values of the samples were measured, and titration was performed using polydiallyldimethylammonium chloride (PDADMAC) solution. At least three independent measurements were made, and the cationic demand was calculated from the average amount31 of polyelectrolyte consumed. The zeta potential ξ of the samples was measured in MQ water using a Zatasizer, ZEN 3600, (Malvern Ltd., U.K.). Measurements were performed on 0.5 g/L polysaccharide dispersions prepared at 60 ⁰C with a pH of ca. 4.5 (see Table 2).

2.2.4. Morphology visualization In order to study directly the structure of the molecules and possible aggregates in the samples, two different microscopy techniques were applied. Polysaccharide dispersions with 0.5 g/L consistency in water were observed by atomic force microscopy (AFM). The hemicelluloses being adsorbed onto mica surfaces with an anchoring polymeric surface layer of PAH (polyallylamine hydrochloride with a specific molar mass of 17.5 kDa, Sigma-Aldrich), the mica surfaces being soaked in the dispersions for 20 min, followed by rinsing with MQ water and drying under a nitrogen flow. A multimode 8 AFM (Bruker, Germany) with ScanAsyst-air tips was used for imaging, and DIPimage software (Delft University of Technology, The Netherlands) was used for processing the images. Cryo-transmission electron microscopy (Cryo-TEM, Zeiss EM 902 Ltd. Welwyn. UK) was used for direct imaging of the molecules or aggregates formed at higher concentration, 20 g/L, in aqueous media. The acceleration voltage was 80 keV, and images were recorded digitally with a CCD camera. Specimens were prepared using copper grids (200 mesh) coated with holey


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polymer film according to the method described by Almgren32. About 2 µl of the sample was deposited on the specimen in an improved version of a CEVS (Controlled Environment Vitrification System). The specimen was quenched in liquid ethane quickly and stored under liquid nitrogen until the measurement was made.

2.2.5. Chemical characterization Carbohydrate composition The monosaccharide composition of the extracted hemicelluloses was determined by high-pH anion exchange chromatography equipped with a pulsed amperometric detector (HPAEC-PAD) after acid hydrolysis with 2 M trifluoroacetic acid (TFA) at 120 ⁰C for 3 h. Hydrolysis is milder with TFA acid than with sulphuric acid, and is suitable for analysis of non-crystalline watersoluble polysaccharides33. The monosaccharide components of the hydrolysates were analyzed by HPAEC (IC-3000, Dionex, Sunnyvale, CA, USA) and separation was achieved with a CarboPac PA-1 column (4×250mm) (Thermo Fisher Scientific, USA). The chromatographic system was equilibrated for 7 min using 260 mM sodium hydroxide and for 7 min with 170 mM sodium acetate followed by MQ water for 6 min. The eluent was MQ water with a flow rate of 1 mL/min, and the temperature was 22 ⁰C. A solution of 300 mM sodium hydroxide at a flow rate of 0.5 mL/min was added to the column effluent before PAD detection. Chromeleon 7.1 software was used to process the data. The analysis was duplicated and the results were assessed taking into consideration the standard deviation. Monosaccharide standards (Sigma-Aldrich) were used for determination of the response factors. 4-O-methylglucuronic acid was quantified using the relative response factor provided in the literature34.


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Biomacromolecules

Carbohydrate linkages The glycosidic linkages, including uronic acids, present in the hemicellulose extracts were quantified by gas chromatography-mass spectrometry (GC-MS) as reported by Pettolino et al.35. Activation and carboxyl reduction of the uronic acids was achieved using carbodiimide and sodium borodeuteride, following the method reported by Kim and Carpita36. In brief, the extracts were dispersed in deionized water and the carboxylic groups were activated during 3 h at 30 °C using 500 g/L of carbodiimide in 0.2 M 2-(N-Morpholino)ethanesulfonic acid (MES) buffered at pH 4.75. The carboxylic acids were reduced and labeled using 100 g/L sodium borodeuteride (NaBD4) dissolved in dimethyl sulfoxide (DMSO) overnight at room temperature. The reaction was stopped by the addition of glacial acetic acid and the labeled hemicelluloses were dialyzed (MWCO 3500 Da, 24 h) and freeze-dried. The samples with reduced carboxyl groups were partially methylated in dimethyl sulfoxide (DMSO) using five subsequent additions of iodomethane under alkaline conditions (excess of NaOH) in an inert atmosphere (Ar)37. The partially methylated polysaccharides were hydrolyzed with 2 M TFA at 121 °C for 3 h and further derivatized into permethylated alditol acetates (PMAAs) by reduction with sodium borohydride (NaBH4) followed by acetylation with acetic anhydride and pyridine. The PMAAs were identified and quantified by gas chromatography coupled to an electron impact mass spectrometer (GC-EI/MS) on a SP-2380 capillary column (30 m x 0.25 mm ID, Agilent Technologies) with a temperature ramp of 1 °C/min from 160 to 210 °C. The PMAAs were assigned to the different glycosidic linkages by comparison of the retention times and fragmentation spectra with these of reference polysaccharides. The analysis was performed in triplicate.


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Extractives The extractive components were determined using gas chromatography (GC)38, the composition of fatty acids, resin acids, and sterols being analyzed on a HP-11 column (25 m×0.2 mm, 0.11 µm film, Agilent J&W GC columns, USA), using 0.8 mL/min of H2 as the carrier gas. The column temperature was ramped from 120 °C, with 1 min holding, to 320 °C at 6 °C/min, and held for 15 min. The composition of steryl esters (StE) and triglycerides (TG) was analyzed on a HP-1 column (7 m×0.53 mm, Agilent J&W GC columns, USA), with a temperature ramp from 100 °C, with a holding time of 1.5 min, to 340 °C at 12 °C/min, with 5 min holding. Samples were prepared by dispersing the GGM powder in MQ water at 5 g/L. An internal standard, a mixture of 0.02 g/L of heneicosylic acid, betulinol, cholesteryl heptadecanoate, and 1,3-dipalmitoyl-2-oleoyl glycerol in methyl tert-butyl ether (MTBE) and pure MTBE was used to get the extractives into the MTBE phase. The MTBE was then evaporated under a flow of nitrogen and silylation was performed using pyridine, N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA), and TMCS. The silylated samples were injected into the GC (Gas Chromatography Autosystem XL, PerkinElmer, USA) with the above-mentioned columns. Lignin quantification The concentration of Lignin in the extracted samples was determined by ultraviolet spectroscopy (UV/VIS Spectrometer, Lambda 40, PerkinElmer, USA). The lignin component of the samples was extracted using methyl tert-butyl ether (MTBE). The aqueous phase after MTBE extraction was diluted to be suitable for UV absorbance in the 0.3-0.7 range, and transferred to quartz cuvettes. The amount of dissolved lignin was determined using the UV absorption value of the extracted liquid at 280 nm38.


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Degree of acetylation (DSAC) The acetyl content was determined using high-performance liquid chromatography (HPLC), after alkaline hydrolysis (saponification). The acetic acid released was determined according to the method developed by R. Bi et al.39. A weighed portion (7 mg) of the dried hemicellulose powder was dispersed in 0.3 mL of MQ water with 10 µL of propionic acid (1 M), as the internal standard. After the hemicellulose dispersions have been mixed with the internal standard, 1.2 mL sodium hydroxide (0.8 M) was added and the mixture was allowed to react at 60 ⁰C overnight in order to cleave sugar acetate ester linkages. The sample was cooled to room temperature, and neutralized with HCl (37%) followed by centrifugation at 14100 RCF for 30 min at room temperature. The supernatant was filtered through a Teflon filter with 0.2 µm pore size into special HPLC vials. The acetic acid released was quantified by HPLC analysis (DionexThermofisher, CA, USA) equipped with a UV detector (210 nm) and Rezax ROA-Organic acid column (300×7.8 mm, Phenomenex, Torance, CA, USA). The mobile phase was sulphuric acid (2.5 mM) at a flow rate of 0.5 mL/min, and the temperature was 50 ⁰C. The system was calibrated using acetic acid and propionic acid as internal standards. The measurements were run in duplicate, and DSAC was calculated using the Equation:

= (

× %

!"#$ ×%%) '( !"#$ ')×%

[2]

where * is 43 g/mol, and 162 is the molecular weight of the sugar monomer unit (g/mol). The acetyl percentage was calculated as;

%+,-./0 =

!"#$ × !"#$ 12$#3 4567!

× 100

[3]


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where : and :;?@A are the concentrations of acetic acid (mol/L) and polysaccharide in the sample (g/L) respectively.

3. RESULTS and Discussion The solubility of the samples was evaluated by combining size and molar mass distributions with the possible formation of associated structures by microscopy techniques. A useful rule of thumb for estimating the size of a polymer in a theta solvent (for dissolved samples) is that the radius of gyration of the polymer is proportional to the root mean square of the molecular weight8. This has been used here to estimate the molecular solubility by comparing the dimensions determined using DLS with the size of the molecule obtained from the molecular mass determination and the theta solvent size calculation. The purity and molecular structure of the extracted fractions were also determined using chromatography techniques.

3.1. Size determination The autocorrelation function from the DLS measurements was used to estimate the size distribution. The results show broad size distributions for the extracted GGM4, GGM3 and GGM1, respectively, but the distribution for GGM2 was much narrower in both aqueous and DMSO solutions. Figure 2a shows the number distributions of the hemicelluloses dispersed in aqueous media versus diameter (dH) of the polymers. The peak integration of GGM2 reveals that almost 99% of the particles have hydrodynamic diameters below 10 nm, with an average value of 4 nm. For GGM1 almost 75% of the particles were ca. 50 nm in diameter. Samples GGM3 and GGM4 show very broad size distributions from 50 to 400 nm and from 215 to 600 nm,


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respectively. The results were very similar for the samples dissolved in DMSO, the particle size of GGM1, GGM3 and GGM4 being slightly smaller due to better dispersibility (Figure 1S in supporting information). A comparison between molecular mass data and the size data from the DLS measurements showed that associated structures were indeed present. The measured molecular size of GGM2 was in good agreement with that estimated from its molar mass assuming theta solvents. As shown in figure 2, small nano-sized objects may also exist in the samples 1, 3 and 4, although large associated structures predominate in the case of these samples. Figure 2b shows the auto correlation coefficients for the samples, which also provides information about molecular size as well as associated states. The measurements were performed at 0.5 g/L in all cases and all the autocorrelation functions (Figure 2b) show a reliable behavior, i.e. high values at low correlation times and no tailing, whereas GGM2 shows a low intercept and a tailing at longer delay times. There can be several reasons to this behavior including lower dn/dc value (see Table 1) and a poor balance between small molecules and a few larger particles, and a combination of these different factors.

The measurements were therefore repeated at 5 g/L.

The obtained

autocorrelation curve at 5 g/L shows a more similar intercept to the other samples and no tailing at longer delay times, but indeed a number distribution equivalent to that at 0.5 g/L (Figure 2a). To further clarify the state of solubility of the GGM2 sample, the DLS experiments were repeated at different concentrations in aqueous media. When the concentration was increased to about 20 g/L, a clear association of the smaller molecules to larger associated structure was detected, as shown by the autocorrelation results for these samples (Figure 2c,d). This indicates that the extracted AcGGM (GGM2) shows a molecular solubility at low concentrations but, as the concentration is increased, the molecules become associated into larger aggregated structures.


17

Biomacromolecules

This indicates that pure water is a poor solvent for the hemicelluloses in GGM2, and this poor solubility of GGMs in water is further supported by the results for the other samples where associated structures were also found at lower concentrations.

a)

b) GGM1(0,5g/L) GGM2(0,5g/L) GGM3(0,5g/L) GGM4(0,5g/L) GGM2(5g/L)

Number(%)

30

GGM1(0.5g/L) GGM2(0.5g/L) GGM3(0.5g/L) GGM4(0.5g/L) GGM2(5g/L)

1,0

Correlation coefficient

35

25 20 15 10

0,8 0,6 0,4 0,2

5 0,0 0,1

0 1

10

100

1000

1

10

100

1000

10000

Time(µs)

Size(d.nm)

c)

d) GGM2(5g/L) GGM2(10g/L) GGM2(20g/L)

30 25 20 15 10

GGM2(5g/L) GGM2(10g/L) GGM2(20g/L)

1,0


35

Number(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0,8 0,6 0,4 0,2

5 0 0,1

1

10

100

0,0 0,1

1000

1

10

100

1000 10000 100000

Time(µs)

Size(d.nm)

Figure 2. Size distribution of the polysaccharides extracted in water at 60 ⁰C. Number distributions versus hydrodynamic diameter (dH) (a) for the different GGMs and (c) for GGM2 at different concentrations. Auto correlation functions for (b) the different GGM samples and (d) for the GGM2 sample at different concentrations.


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Biomacromolecules

3.2. Molar mass distributions The average molar mass and molar mass distributions of the extracted hemicelluloses were assessed using DMSO and water-based SEC systems, with pullulan as a linear standard. The combination of MALLS and IR detectors was also used to obtain further insight into the state of association of the molecules. Figure 3 shows overlay profiles for all the samples consisting of the refractive index (RI) (Red) and light scattering (LS) (Blue) signals as functions of the apparent molar mass estimated from the pullulan calibration. The RI signals indicate that all the samples dissolved in DMSO/LiBr (Figure 3 a,b,c,d) had the main peak at an average molar mass of about Mw≈ 25 kDa, but the curves for the GGM1 and GGM4 samples show shoulders after the main peak at higher molar masses. The LS signals in the latter samples are divided into two peaks. The separation is based on the hydrodynamic volumes from two populations of molecules/associated structures. The large increase in polydispersity index (Table 1) is also in agreement with the bimodal light scattering signals. The existence of a fraction with a larger molar mass indicates the existence of associated structures in the samples40. This was also observed to some extent in the GGM3 sample, but, GGM2 possessed only a main peak collected with the RI detector with a negligible signal from the LS detector. The results were similar for the samples dispersed in aqueous media (Figure 2S, supporting information), but the average-molar masses of the samples were higher in aqueous media than in DMSO solution probably because DMSO is a better solvent for these polymers. These results were also consistent with the DLS results. Table 1 summarizes the weight-average molar masses of the hemicelluloses in aqueous and DMSO media.


19

Biomacromolecules

a)

b) 0,040

0,040

0,0030

0,030

0,004

0,025 0,020

0,003

0,015 0,002 0,010 0,001

0,005

0,000 2,0

0,000 2,5

3,0

3,5

4,0

4,5

5,0

5,5

6,0

6,5

0,035

0,0025

0,030

0,0020

0,025 0,020

0,0015

0,015 0,0010 0,010 0,0005

0,005

0,0000 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0

0,000

Light Scattering(90°)

0,005

Differential Weight Fraction

0,035

Light Scattering (90°)


0,006

LogMw

Log Mw

c)

d) 0,040

0,040

0,0030

0,030

0,0020

0,025 0,020

0,0015

0,015 0,0010 0,010 0,0005

0,005

0,0000 2,0

0,000 2,5

3,0

3,5

4,0

4,5

5,0

5,5

6,0


0,0025


0,035

0,035

0,0025

0,030

0,0020

0,025 0,020

0,0015

0,015 0,0010 0,010 0,0005


0,0030


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

0,005

0,000 0,0000 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5

6,5

LogMw

LogMw

Figure 3. Molecular mass distributions of the hemicelluloses dispersed in the DMSO/0.5% (W/W)LiBr solvent; a) GGM1, b) GGM2, c) GGM3 and d) GGM4. The red and blue peaks represent RI and LS signals (from MALLS evaluation), respectively. The X-axis values are calculated from the apparent molar mass estimated from the pullulan calibration.

Samples

dn/dc(mL/g) Mn(kDa) Mw(kDa) PDI in DMSO

in DMSO

in DMSO

in

dn/dc(mL/g) Mn(kDa)

Mw(kDa) PDI

in water

in water

in water

in water

DMSO

GGM1

0.096

18.246

64.415

3.53

0.172

27.967

85.278

3.05

GGM2

0.068

12.701

21.891

1.72

0.136

21.376

35.426

1.65

GGM3

0.074

16.683

28.527

1.71

0.228

30.992

52.886

1.70

GGM4

0.096

19.537

66.718

3.41

Not dissolved

Not dissolved*

Not dissolved*

Not dissolved


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Biomacromolecules

Table 1. Summary of the molar mass distribution measurements shown as refractive index increment, average molar mass and polydispersity index (PDI) of the extracted hemicelluloses in DMSO and in water-based systems. * These values have been analyzed earlier13, but with the current methodology GGM4 could not be dissolved. Floating particles in the dispersions were observed by ocular inspection.

3.3. Charge and zeta potential measurements Table 2 shows the cationic demand and zeta potential data for the hemicellulose samples. The negative charges and pH values were similar for all the samples. The negative charge can be due to pectic substances (anionic polysaccharides), charged hemicellulose and the occurrence of oxidized lignin structures in the extracted hemicellulose samples, which was supported by carbohydrate composition/linkage analysis presented in the next sections. The polyelectrolyte titration data show that the materials had a considerable charge but the values are low compared to earlier published results41.The zeta potential values are also very low, indicating that the charge of the extracted GGM samples was low at the pH of the purified samples. With the current techniques it is not possible to determine how the charged molecules are associated with the AcGGMs, but the zeta potential data show only single peaks, indicating that the charged molecules are associated with the GGMs.


21


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Samples

Zeta Potential PET (mV) (µeq/g)

pH

GGM1

-9±1

250±20

4.72±0.01

GGM2

-7±1

244± 6

4.64±0.02

GGM3

-9±1

227± 7

4.43±0.02

GGM4

-18±1

248 ±8

4.54±0.03

Table 2. Zeta potential, cationic demand and pH values for the GGM samples.

3.4. Morphology The morphology of the hemicelluloses was investigated at low and high concentrations using AFM and Cryo-TEM techniques, respectively. AFM images (Figure 4) show the existence of adsorbed nano-particles on model mica surfaces at the low concentration (0.5 g/L). The width of the adsorbed structures in samples GGM1, GGM3 and GGM4 varies considerably, and they are probably formed by association of the smaller structures which are probably the AcGGM polymers. The inhomogeneous size distribution of the clusters indicates high polydispersity, which is in agreement with both the DLS results and the molecular mass distribution curves. A more homogeneous size distribution was observed for the GGM2 sample. There was a slight flattening of all the samples due to the adsorption on the model surface and drying before the AFM analysis.


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Biomacromolecules

Figure 4. AFM images of a) GGM1, b) GGM2, c) GGM3 and d) GGM4 samples adsorbed onto model mica surfaces pretreated with PAH (PolyAllyamine Hydrochloride) and dried before the AFM analysis. A Cryo-TEM study of the state of solubility of the samples at a higher concentration (20 g/L) showed the association of smaller spherical hemicellulose molecules to larger fractal structures,


23


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which is again in close agreement with the DLS and the AFM measurements. In addition, some fibril-like submicron impurities were observed in samples GGM1 and GGM4. The micro-fibrils fragments present in the suspensions, designated as fines, could have an important effect on the stability since they have a remarkable specific surface area that could attract hemicelluloses that are on the verge of being associated. These fibril-like impurities were removed by filtration (0.45 µm nylon filter) before the DLS and AFM studies.

Figure 5. Cryo-TEM images of the hemicelluloses at high concentration (20 g/L) a) GGM1, b)


24

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Biomacromolecules

GGM2, c) GGM3 and d) GGM4 (images were taken at different magnifications to represent the micro-fibrils and aggregates).

3.5. Chemical characterization Table 3 shows the results from the chemical characterization techniques used to determine the chemical structures of the hemicelluloses. The results show that the extracted polysaccharides consist of neutral (mostly O-acetyl-galactoglucomannans), and pectic polysaccharides (galacturonans) with low amounts of xylan42. Mannose, glucose and galactose (in a ratio of 4:11.8:06-0.9) are the main components and this strongly suggests the presence of galactoglucomannan, considering previously reported values

1, 12, 43, 44

. However, the amounts of

oligo- or polysaccharides released from the samples depend on the pulp preparation and extraction methods. A glycosidic linkage analysis (Table 3, Table S1, supporting information) shows that the purity of the GGM samples ranges from 83 – 89 %mol. Pectic polysaccharides consisting of galacturonic acid, arabinose, galactose and rhamnose45 are present in different ratios in the samples, i.e. GGM3 and GGM4 have a significant pectin content (14-16% measured with

the

monosaccharide

and

linkage

analysis,

Table

3

and

Table

S1),

and

arabinoglucuronoxylan (AGX), the second most abundant hemicellulose in softwoods, can be also detected in different amounts in the different hemicelluloses, being most significant in GGM2. Samples GGM1 and GGM4 have a significant abundance on 4-Glcp (Table 3), probably due to the higher content of unsubstituted Glc(4-Glcp) in the GGM backbone, but also due to cellulose contaminations (micro-fibrils) in such fractions. GGM and cellulose share β-(1→4)Glcp linkages in their polymeric backbone that are indistinguishable by glycosidic linkage analysis. It has been reported that β-(1→4)-Glcp glycosidic linkages are stiffer in their


25


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intramolecular conformation than their β-(1→4)-Manp counterparts46. This could naturally contribute to an overall rigid macromolecular conformation in these GGM fractions and affect their molecular solubility. Sample GGM2 has a larger ratio of branching points to unsubstituted units in the GGM backbone (bp:u, Table 3) than the other GGM fractions, arising from higher proportion of 4,6-Manp units in this sample. This greater abundance of substitutions could lead to an overall increased molecular flexibility, preventing the association of GGM individual molecules due to steric hindrances imposed by the higher amount of substitutions/side chains and hence contributing to an improved molecular solubility. This influence of the side chains is naturally based on the inherent assumption that these units have a good solubility in used solvent, in this case water.

Yields (% pulp)a

GGM1

GGM2

GGM3

GGM4

6

5.8

7

5

Compositional analysis (mg/g) Ara (mg/g) b

16.41 (±0.64)

10.95 (±2.52)

15.69 (±0.25)

23.98 (±0.40)

Rha (mg/g) b

4.28 (±0.17)

2.64 (±0.48)

2.41 (±0.13)

7.58 (±0.59)

Xyl (mg/g) b

4.89 (±0.28)

28.36 (±6.53)

3.28 (±0.33)

4.60 (±0.12)

GlcA (mg/g) b

2.70 (±3.12)

2.16 (±2.49)

5.14 (±5.93)

5.28 (±6.10)

GalA (mg/g) b

17.97 (±4.16)

40.66 (±13.83)

9.56 (±1.29)

22.59 (±4.62)

Man (mg/g) b

421.29 (±16.02)

464.85 (±96.96)

433.14 (±7.26)

393.12 (±7.42)

Gal (mg/g) b

66.94 (±2.26)

79.97 (±16.66)

92.23 (±1.96)

86.64 (±0.80)

Glc (mg/g) b

189.66 (±6.01)

118.93 (±23.65)

102.66 (±2.41)

183.85 (±2.33)

mGlcA (mg/g) b

5.10 (±1.09)

4.68 (±0.59)

11.29 (±1.84)

9.93 (±2.43)

Extractives (mg/g)c 0.3 (±0.11)

0.4 (±0.06)

0.4 (±0.06)

0.6 (±0.02)

Lignin (mg/g) d

0.057 (±0.0002)

0.046 (±0.0010)

0.145 (±0.0109)

0.158 (±0.0017)


26

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Biomacromolecules

Degree of 0.24 (±0.03) acetylation (DSAC)

0.23 (±0.04)

0.26 (±0.04)

0.21 (±0.021)

e

Polysaccharide composition by glycosidic linkage analysis (%mol)f Total (GGM)g

Mannan 89.41 (0.46)

86.98 (1.16)

85.02 (0.15)

82.71 (0.63)

t-Manp

0.93 (0.09)

1.35 (0.07)

1.00 (0.02)

0.78 (0.02)

4-Manp

53.80 (0.46)

55.14 (0.39)

60.34 (0.37)

49.86 (0.39)

4,6-Manp

2.81 (0.05)

4.82 (0.12)

2.62 (0.10)

2.40 (0.03)

t-Glcp

1.23 (0.05)

1.64 (0.32)

1.66 (0.21)

1.88 (0.12)

4-Glcp

23.18 (0.15)

12.62 (0.40)

12.64 (0.87)

21.63 (0.73)

4,6-Glcp

1.49 (0.02)

1.42 (0.08)

0.86 (0.05)

1.29 (0.01)

Xylan 0.95 (0.09)

4.29 (0.26)

0.56 (0.04)

0.75 (0.08)

Total Pectin (RG, 8.66 (0.11) AG)h

7.70 (0.58)

14.71 (0.40)

16.07 (0.43)

bp:ui

0.09

0.05

0.05

Total (AGX)g

0.05

Table 3. Yields and chemical compositions of isolated hemicelluloses, the amount of extractives and the amount of lignin in the different GGM samples. The table also contains information about the degree of acetylation and glycosidic linkage analysis of the isolated GGMs. a

Extraction yields are calculated gravimetrically.

b

Monosaccharide content is calculated by HPAEC-PAD after TFA hydrolysis.

c

Extractives were calculated by GC analysis.

d

Lignin content was calculated after methyl tert-butyl ether (MTBE) extraction and analysis by UV spectroscopy. e

The degree of acetylation is calculated after saponification and HPLC analysis.

f

The polysaccharide composition is calculated using the results from glycosidic linkage analysis (Table S1, supporting information).


27


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g

Total Mannan is calculated from the linkages in the galactoglucomannan (GGM) backbone: tManp + 4- Manp + 4-Glcp + 2×4,6-Manp + 2×4,6-Glcp. h

Total Xylan is calculated from the linkages in the arabinoglucuronoxylan (AGX) backbone: tXylp + 4- Xylp + 2×2/3,4-Xylp. i

Total Pectin is calculated from the linkages in the rhamnogalacturonan (RG) and arabinogalactan (AG) backbone. j

The branching point (bp) versus unsubstituted (u) backbone ratio (bp:u) is calculated from the substituted units (4,6-Manp + 4,6-Glcp) and unsubstituted units (4-Manp + 4-Glcp) in GGM.

Extractives and Lignin Extractives constitute a very small fraction of the materials and consist of a large number of individual chemical substances including both lipophilic and hydrophilic types, such as, fatty acids, resin acids, and sterols22. The amount and composition of extractives vary in different parts of the tree, such as stem, branches, roots, bark, and needles22,

47

. In the mechanical pulping

process, lipophilic extractives are released together with dissolved substances and constitute the main part of the colloidal substances (DCS)48. The colloidal fraction of the DCS is both electrostatically and sterically stabilized49, 50. It has been suggested that the steric stabilization originates from dissolved wood polymers present on the surface of the colloidal particles

20, 51

. It has also

been suggested that dissolved polysaccharides are adsorbed onto the dispersed hydrophobic extractives, forming stable colloids52. In the present work, great care has been taken to remove all the extractive impurities. Since only 0.3 to 0.6 mg/g extractives were present in the samples, the treatments have been successful. The initial association of AcGGM with extractives dispersed in water

49, 50

may affect the final solubility of the hemicelluloses, but this is beyond the scope of

the present investigation. Since 15-25% of the wood is composed of lignin, it is also necessary to quantify how much of this component can be isolated in the polysaccharide samples. The quantitative lignin data,


28

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Biomacromolecules

determined by UV-spectroscopy, are also shown in Table 3. The results show very low concentrations of aromatic residues in the samples, indicating that these compounds have played no role in the solubility of the extracted hemicelluloses.

Degree of acetylation An important structural feature of acetyl-galactoglucomannans is the presence of acetyl groups in the C-2 and C-3 positions of the mannose units1,

53

AcGGM, determined by NMR and titration methods12,

. The degree of acetylation of native

53

, has been reported to be 0.24-0.32.

Since the degree of acetyl substitution is very critical for the solubility in water4, the objective of this study was to extract an unaffected galactoglucomannan. The average degree of acetylation of the different hemicelluloses is reported in Table 3. The results are almost the same for all the samples and within the limit for native AcGGM, which was one of the major objectives of the work, i.e. to extract a representative AcGGM sample from spruce. The AcGGM is easily deacetylated by increasing the pH, and buffered solutions with a pH of 8-8.5 are enough to deacetylate the GGM.

4. Solubility of galactoglucomannans in water Hemicellulose powders extracted with different procedures showed different size and molar mass distributions in aqueous media even though the solutions were fully transparent on ocular inspection. The sample extracted from coarsely refined wood, with no pretreatment or soxhlet extraction (GGM1) showed a broad particle size distribution and associated structures up to 700 nm in diameter. The average molar mass was 64 kDa in DMSO and 85 kDa in water. Samples


29


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extracted from commercial TMPs, after soxhlet extraction with a mixture of hexane and acetone, (GGM3), had a lower molar mass, 28 kDa in DMSO and 52 kDa in water, respectively. It was also shown that separation of the extractives using an organic solvent before hot-water extraction can successfully prevent the extensive formation of large associated structures with a large molar mass. This was shown for GGM1 at 0.5 g/L, but DLS shows that this sample still had a broad size distribution with some large associated structures up to 700 nm. Hemicelluloses recovered from TMP effluent, using different filtration and ultrafiltration steps (GGM4) had the largest molar mass (66 kDa in DMSO) and the largest associated structures (up to 1000 nm). A controlled separation procedure in the case of GGM2 gave a sample with an acceptable molecular solubility at low concentrations, i.e. below ca. 20 g/L, where polymers with a molar mass of 21-35 kDa dissolved with an average hydrodynamic radius of 4 nm, indicating that pure water is basically a theta solvent for the isolated GGM2. The larger hydrodynamic diameter of the GGM2 sample at higher concentrations supports the finding that water is a theta solvent for this polymer, since the associated structures found with both the DLS and the Cryo-TEM investigation indicate a phase transition with increasing polymer concentration, as can be expected for a polymer with poor solubility54. The Cryo-TEM study showed that the highest concentrated GGM2 sample (20 g/L) had associated structures with an average diameter of 200 nm, which is in reasonable agreement with the results of the DLS study. A detailed inspection of the Cryo-TEM images shows smaller structures in the larger associated structures, indicating a clustering as the concentration is increased. As expected, the association of the small molecules in the GGM2 sample was found to be a reversible process, suggesting that this is indeed simply a phase transition. The associated structures are able to dissociate by dilution of the concentrated samples as shown in Figure 6.


30

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The dissociation process was found to be slower than the association process, i.e. leaving the sample (5 g/L) in a thermo-mixture at 60 ⁰C overnight did not lead to complete dissociation of the clusters. Dilution to 0.5 g/L, and mixing overnight was needed to revert to the initial state.

a)

b) GGM2(20g/L) GGM2(5g/L) GGM2(0.5g/L)

30 25 20 15 10

0,8 0,6 0,4 0,2

5 0 0,1

GGM2(20g/L) GGM2(5g/L) GGM2(0.5g/L)

1,0


35

Number(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

10

100

1000

0,0 0,1

1

Size(d.nm)

10

100

1000 10000 100000

Time(µs)

Figure 6. Size distributions (a) and auto correlation curves (b) for GGM2 at three different concentrations.

It is important to note that the chemical analysis showed that all the hemicelluloses consisted mainly of AcGGM with small amounts of impurities. For example, GGM2, which showed a somewhat greater solubility, contained more xylose and galacturonic acid than the other samples. The linkage analysis also showed a marked increase in substituted units and more branches in GGM2 than in the other samples, which may have a steric effect preventing association and enhancing molecular solubility (Table 3). GGM1 and GGM4 had a higher concentration of 4Glcp or cellulose “contamination” in the form of micro-fibrils, which was also supported by the Cryo-TEM images of these samples. The negative charges of the samples are also caused by impurities. All the samples showed a low zeta potential at low pH values, which were the natural


31


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pHs of the extracted samples and were kept the same to avoid the risk of changing the main molecular structures of the extracted samples. Any of these differences in the chemical or physical structures of the hemicelluloses can naturally be a driving force, either alone or in combination with other factors, for increasing or decreasing the molecular solubility. Nevertheless, all the samples showed a poor to very poor solubility in water. This is perhaps the most important result of the present investigation. It will have a larger impact on the properties of films and gels made from these hemicelluloses since most of the films and gels are formed by concentrating aqueous solutions of the hemicelluloses. The results also have a very important implication for the molar mass determinations on these materials. In order to use SEC with certain calibration standards, it is essential that the polymers are molecularly soluble in order to be able to use the calibration standards. If the polymers are not completely soluble, the molar mass determinations are unreliable. The results in Figure 3 are indeed close to this limit.

5. Conclusions The results of the present work suggest that appropriate pulp preparation, fiber pretreatment and extraction procedures are of crucial importance for the molecular solubility of the extracted hemicelluloses. All the hot-water extracted samples were found to disperse well in liquid media and to create transparent dispersions with no visible macroscopic particles. However, the molecular solubility was found to be different for each sample. For the first time, to the knowledge of the authors, the molecular solubility of the softwood hemicelluloses was evaluated in relation to pulp preparation and extraction procedures.


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Biomacromolecules

The sample extracted from coarsely refined wood, without pretreatment or soxhlet extraction (GGM1) showed a broad particle size distribution with aggregates up to 700 nm in diameter. The average molar mass was determined to be 64 kDa in DMSO and 85 kDa in water. After soxhlet extraction with a mixture of hexane and acetone, sample from commercial TMPs (GGM3) showed smaller molar masses, 28 kDa in DMSO and 52 kDa in water. Removal of extractives in the organic solvents before the hot-water extraction can prevent the formation of large aggregates with higher molar masses, as was seen in GGM1. However, DLS showed that there were still a broad size distribution and large aggregates up to 700 nm. Hemicelluloses recovered from TMP effluent, using different filtration and ultrafiltration steps (GGM4) had the largest molar mass (66 kDa in DMSO) and the largest aggregates (up to 1000 nm). A controlled separation procedure leads to good molecular solubility by producing hemicelluloses with an average molar mass of 21-35 kDa, and nano-particles with an average diameter of 4 nm. However, this is only valid at low concentrations. Increasing the concentration leads to association of the dispersed nanoparticles. These fundamental findings are of great importance for developing a more explicit path for extracting soluble hemicelluloses from spruce, and for the further improvement of products made from these biopolymers.


33


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Supporting Information Size determination in DMSO-based system using DLS. Molar mass distribution in aqueous system investigated by SEC-MALLS. Glycosidic linkage analysis of the different GGM fractions obtained from GC-MS study.

Author information Corresponding authors *

Email: [email protected]

*

Email: [email protected]

Author contribution The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. Acknowledgements The Wallenberg Wood Science Centre (WWSC) is gratefully acknowledged for financial support. The authors would also like to thank Tommy Friberg at Chalmers University, Gothenburg, Sweden for excellent help and support in the experimental procedure for refining pulp. And Jonny Eriksson at Uppsala University, Uppsala, Sweden is gratefully acknowledged for the help in the Cryo-TEM imaging.


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(54) Holmberg, K.; Jönsson, B.; Kronberg, B.; Lindman, B., Surfactants and polymers in aqueous solution. Wiley Online Library: 2002.

Table of Contents graphic

Water is a poor solvent for carefully extracted AcGGM from spruce, since an increase in concentration initiates a phase separation of the polymer.


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Water is a poor solvent for carefully extracted AcGGM from spruce, since an increase in concentration initiates a phase separation of the polymer. 35x15mm (300 x 300 DPI)


Solubility of Softwood Hemicelluloses ... - ACS Publications

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