The Influence of Xylan on Precipitation and Filtration Properties of Lignin

Home , Xylan

DEGREE PROJECT IN CHEMICAL SCIENCE AND ENGINEERING, SECOND CYCLE, 30 CREDITS STOCKHOLM, SWEDEN 2016

A Study in the Context of the LignoBoost Process

HELEN SCHNEIDER AND LYNN SCHNEIDER

KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF CHEMICAL SCIENCE AND ENGINEERING

Master’s thesis 2016:NN

The Inﬂuence of Xylan on Precipitation and Filtration Properties of Lignin

A Study in the Context of the LignoBoost Process

Helen Schneider and Lynn Schneider

This thesis work was a collaboration between the Royal Institute of Technology and Chalmers University. Helen Schneider and Lynn Schneider are KTH students but the thesis work was conducted at Chalmers University.

Department of Chemistry and Chemical Engineering Division of Forest Products and Chemical Engineering Chalmers University of Technology Gothenburg, Sweden 2016 The Inﬂuence of Xylan on Precipitation and Filtration Properties of Lignin A Study in the Context of the LignoBoost Process Helen Schneider and Lynn Schneider

Supervisor: Professor Hans Theliander, Professor in Chemical and Biological Engi- neering, Chalmers University of Technology Examiner: Mikael E. Lindström, Professor at the School of Chemical Sciences and Engineering, KTH Royal Institute of Technology

Master’s Thesis 2016:NN Department of Chemistry and Chemical Engineering Division of Forests Products and Chemical Engineering Chalmers University of Technology SE-412 96 Gothenburg Telephone +46 31 772 1000

Typeset in LATEX Gothenburg, Sweden 2016 iv The Inﬂuence of Xylan on Precipitation and Filtration Properties of Lignin A Study in the Context of the LignoBoost Process Helen Schneider and Lynn Schneider Department of Chemistry and Chemical Engineering Chalmers University of Technology

Abstract

The LignoBoost process is a valuable supplement to the Kraft process. It can increase the pulp production rate of a Kraft mill and it enables lignin separation from black liquor with a high degree of purity. However, residual xylan in black liquor has been observed to increase filtration resistance of lignin during the LignoBoost process. In order to uncover underlying mechanisms, this thesis investigates the potential influence of xylan during lignin precipitation and filtration, which are the two main steps of the LignoBoost process. For this purpose experiments based on a model system were designed. Model liquors consisted of lignin and xylan as the only organic compounds and contained lower salt concentrations (4.2-5.9 wt%) compared to black liquor. Furthermore, reference liquors were prepared without xylan addition. Precipitation mechanisms were studied in the onset precipitation region (i.e. alkaline regime) by in-situ focused beam reflectance measurements (FBRM) during step-by-step acidic precipitation of the model liquor. It was found that the onset precipitation pH does not change with the presence of xylan as all liquors started precipitation around pH 9.15. The filtration process was investigated on model liquors that had been precipitated by fast acidification to acidic regimes (pH 6.5-2.87). The use of FBRM during acid precipitation of model liquors suggested that temperature had a significant influence on the chord length distribution (CLD) of the particles. In all filtration experiments, a decrease in CLD was observed when the temperature was changed from 80 °C to 25 °C. Moreover, this thermal instability of particles seemed to be higher when added xylan was present in the liquor. The investigation of the resulting filer cakes with HPLC showed that xylan was evenly distributed through the cake. Further findings on the influence of xylan were impeded due to variations in ionic strength in the model liquors. It was found that the effect of ionic strength on filtration properties and particle sizes overshadows the effect of xylan. Higher ionic strength was observed to yield a lower filtration resistance, a higher solidosty, larger particles and lower solid surface area, as investigated by filtration measurements, laser diffraction and BET analysis. Finally, xylan was fluorescently tagged (i.e. dyed) with Remazol Brilliant Blue R to investigate xylan position in the lignin- xylan filer cake, using a confocal fluorescence microscope. However, due to the autofluorescence of lignin as well as low emission intensity of the synthesized dyed xylan, xylan could not been tracked within the lignin particle. Nevertheless, valuable insight was gained into the preparation of dyed xylan and the bond stability.

Keywords: Xylan, Lignin, Precipitation, FBRM, Filtration, LignoBoost process.

Acknowledgements

First of all, we would like to thank Julie Durruty and Tor Sewring for their super- vision. We are grateful for your input and for the multiple interesting and intensive discussions we have had. We would also like to show gratitude to Hans Theliander for his guidance and keep- ing up good spirit during the thesis work. Furthermore, we want to thank Mikael Lindström for his kind support and uncom- plicated communication over long-distance. For sharing his knowledge and discussions in progress meetings, we thank Tuve Mattsson. We are also grateful that Joanna Wojtasz supported us using HPLC, Maria Gun- narsson helped us using NMR and that Cissi Mattsson was always open for sharing her expertise regarding NMR methods. Last but not least, we enjoyed the open-minded people and the lovely working at- mosphere at the division of Forest Products and Chemical Engineering at Chalmers University.

Gothenburg, June 2016

Helen Schneider & Lynn Schneider

vii

Contents

1 Introduction1 1.1 Background...... 1 1.1.1 The Kraft Process...... 2 1.1.2 The LignoBoost Process...... 3 1.2 Objective...... 5 1.3 Methodology...... 5 1.4 Thesis Outline...... 6

2 Theory7 2.1 Chemistry of Lignin and Xylan...... 7 2.1.1 Lignin...... 7 2.1.2 Xylan...... 9 2.1.3 Ligno-Carbohydrate Complex...... 12 2.2 Precipitation...... 12 2.2.1 Background...... 12 2.2.2 Lignin Precipitation...... 13 2.2.3 Inﬂuences on Lignin Precipitation...... 15 2.3 Filtration...... 18 2.3.1 Background...... 18 2.3.2 Model for Dead-end Filtration...... 19 2.3.3 The Inﬂuence of Xylan in the LignoBoost Process...... 20

3 Experiments 23 3.1 Materials...... 23 3.2 Preparation of Dyed Xylan...... 23 3.3 Precipitation Study...... 25 3.4 Filtration Study...... 25 3.5 Other Characterization Techniques...... 27 3.5.1 Confocal Fluorescent Microscopy...... 27 3.5.2 UV-Vis Spectroscopy...... 28 3.5.3 High Performance Liquid Chromatography...... 29 3.5.4 NMR Spectroscopy...... 30 3.5.5 Focused Beam Reﬂectance Measurements...... 30 3.5.6 Laser Diﬀraction Analysis...... 31 3.5.7 BET Analysis...... 32

ix Contents

4 Results and Discussion 33 4.1 Preparation of Dyed Xylan...... 33 4.1.1 Synthesis Yield...... 33 4.1.2 Substitution Degree...... 34 4.1.3 Bond between Xylan and Dye...... 35 4.2 Precipitation Study...... 36 4.2.1 Particle Formation Dynamics...... 36 4.2.2 Filtration Properties...... 39 4.3 Filtration Study...... 40 4.3.1 Interaction between Lignin and Xylan...... 41 4.3.2 Filtration Properties...... 44 4.3.3 Location of Xylan in Filter Cake...... 46 4.3.3.1 High Pressure Liquid Chromatography...... 46 4.3.3.2 Confocal Fluorescent Microscopy...... 47 4.3.4 Particle Sizes and Shapes...... 49 4.3.4.1 Confocal Fluorescence Microscopy...... 49 4.3.4.2 Focused Beam Reﬂectance Measurement...... 51 4.3.4.3 Laser Diﬀraction...... 55 4.3.4.4 BET Analysis...... 56 4.4 Comparison between Precipitation and Filtration Study...... 57

5 Conclusion 61

6 Recommendations 63

Bibliography 65

A Appendix - Preparation of dyed XylanI A.1 Calculations...... I A.2 Results...... II

B Appendix - Precipitation StudyV B.1 Results...... V

C Appendix - Filtration StudyVII C.1 Calculations...... VII C.2 Results...... VIII

x Abbreviations

B Batch BL Black Liquor CFM Confocal Fluorescence Microscopy CLD Chord Length Distribution DR Dissolution/ Re-precipitation DLCA Diffusion Limited Cluster (Colloid)-Cluster (Colloid) Aggregation dX dyed Xylan (dye used is Remazol Brilliant Blue R (RBBR)) F Filtration FBRM Focused Beam Reflectance Measurement GGM Galacto glucomannan HexA Hexenuronic Acid HPLC High Performance Liquid Chromatography KL Kraft Lignin L Lignin LCC Lignin-Carbohydrate Complexes LDA Laser diffraction analysis L-dX Lignin-dyed Xylan L-(d)X Lignin-dyed Xylan and Lignin-Xylan L-X Lignin-Xylan L-(d)X Lignin-Xylan as well as Lignin-dyed Xylan M Mixing MeGlcA Methylglucuronic Acid MW Molecular Weight NMR Nuclear Magnetic Resonance P Precipitation PSD Particle Size Distribution RBBR Remazol Brilliant Blue R RLCA Reaction Limited Cluster (Colloid)-Cluster (Colloid) Aggregation S Synthesis UV-vis Ultraviolet-visible spectroscopy

1 Introduction

1.1 Background

In times of global warming and economic global competition, the concept of a biorefinery shows great promise for the forest product industry by enhancing both envi- ronmental sustainability and revenues. In a biorefinery, biomass is utilized to the greatest extent. The biorefinery converts biomass into bioenergy and a variety of bio-based-products, in analogy to established oil refineries. An advantage of the biorefinery is that it can rely on a wide and sustainable choice of raw materials. Furthermore, biomass already offers a wide range of functionalized building blocks, as opposed to oil based products which have to be functionalized in further processes. However, the compositional variety of biomass also poses a great challenge. It complicates separation of compounds and results in the need for a relatively larger range of processing technologies, where most of these technologies are still at a pre- commercial stage (Kamm & Gruber, 2006). Pulp and paper mills can be considered ideal sites for biorefineries by providing untapped potentials for the utilization of raw material. The principal task of pulp and paper mills is the processing of wood into pulp, which is a lignocellulosic fibrous material that finds its main application in the paper and board production. The dominating pulping process today is known as the Kraft process and produces a pulp which consists almost entirely of the cellulose fraction in wood. Modern kraft pulp mills therefore only use about 45-50 % of the raw wood material to produce bleached pulp (Wallmo, 2008). The other major wood components, namely lignin and hemicellulose are seperated to different degrees from the pulp. In todays pulp and paper industry, separated compounds are predominantly used for heat recovery. However, effective separation, specifically of lignin would be a great benefit. Firstly, lignin is a valuable material in itself and can be considered as the only abundant and renewable source of aromatics found on earth (Ek et al. , 2009). Re- search is currently aiming for high value products from lignin such as marcomolecular- derived products like carbon fibres, activated carbon, binders and polyurethane foams. Especially lignin-based carbon fibres would be promising for structural applications (e.g. airplanes) as they provide high strength at low weight and would be an alternative to commercial carbon fibres, made out of oil-based polyacrylnitrile (Kadla et al. , 2002). A second advantage of lignin separation has to do with todays pulping process. In traditional Kraft mills, separated compounds are burned in the recovery boiler for recovery of cooking chemicals and the generation of heat and electricity. The recovery boiler is typically the single most expensive component in

1 1. Introduction the Kraft process, accounting for almost 20 % of the total investment of a new mill (Brewster, 2007). As it is typically operated close to its maximum limit, its size is the deciding factor on the process throughput of the mill. As lignin has a com- parably high heating value, the separation of lignin would enable a higher process throughput of the mill as more cooking chemicals could be burnt and recovered. Ac- cording to Höglund & Otterbeck(2004) the amount of lignin that can be extracted without disturbing the Kraft process is estimated to be around 0.8-1.2 million tones annually in Sweden. As pointed out there is good reason to investigate eﬀective lignin separation in pulp and paper mills. The following chapters will therefore trace the route of lignin through the Kraft process and discuss challenges of current lignin separation processes.

1.1.1 The Kraft Process

The Kraft process is the dominating pulping process worldwide and makes use of chemical pulping for liberating the fibres from the wood matrix. The key step here is the degradation and dissolution of the lignin fraction in wood, as lignin can be roughly described as the glue which is holding the fibres together. Kraft pulping relies on alkaline conditions and high temperatures, which facilitate the degradation of lignin. In alkaline conditions, lignin is cleaved and equipped with phenolic groups which results in an increase in lignin solubility. The separation of lignin from the pulp increases the pulp strength and is one advantage of Kraft pulping over other processes such as mechanical pulping or semi-chemical pulping. A disadvantage of the process is a lower yield compared to other methods since the lignin fraction is removed. Additionally, not only lignin is degraded during alkaline cooking but also some cellulose and hemicellulose get dissolved. After chemical pulping is completed, the pulp consists almost entirely of undis- solved cellulose fibres and can be separated from the dissolved compounds (Gellerst- edt, 2004). The dissolved compounds involve dissolved organic materials, inorganic materials and spent cooking chemicals; the mixture is generally known as black liqour (BL). The composition of BL varies due to feedstock and process; common composition ranges can be seen in table 1.1.

2 1. Introduction

Table 1.1: Approximate composition of black liquor for elements with a concentration above 1 g/kg dry black liquor. (Ek et al. , 2009)

Element Amount Organic material Amount (weight %) (weight %) Carbon 34-39 Lignin 29-45 Hydrogen 3-5 Hydroxy acids 25-35 Oxygen 33-38 Extractives 3-5 Sodium 17-25 Formic acid ∼5 Sulfur 3-7 Acetic acid ∼3 Potassium 0.1-2 Methanol ∼ 1 Chlorine 0.2-2 Nitrogen 0.05-0.2

BL is concentrated in an evaporation plant and combusted in the recovery boiler for recovery of cooking chemicals by removing the salt smelt after combustion. In this way salts can be reused for chemical pulping, which closes the recovery cycle (Brännvall, 2004). Furthermore, the recovery boiler generates electricity and heat. A simpliﬁed scheme of the Kraft process is given in ﬁgure 1.1.

Figure 1.1: Simpliﬁed scheme of the Kraft process.

1.1.2 The LignoBoost Process Lignin can be extracted from BL using acid precipitation or ultrafiltration. Acid precipitation is more common as it provides higher lignin yield at lower costs. (Uloth & Wearing, 1989) Nevertheless, ultrafiltration can be beneficial before conducting acid precipitation to decrease the filtration resistance when processing e.g. hardwood (Wallmo et al. , 2009). The advantage of ultrafiltration is that no temperature and pH adjustment is needed but it is still less cost effective (Jönsson & Wallberg, 2009). In the traditional acid precipitation process, BL is acidified by decreasing its pH to about 10. After acidification of the BL, filtration and washing are applied. However, the traditional lignin precipitation and separation from BL often shows complete or partial plugging of the filter cake. Partial plugging results in the de-

3 1. Introduction velopment of preferred flow channels, which prevents uniform washing throughout the cake and leads to high impurities in the extracted lignin. Furthermore, partial plugging results in a decrease of flow, necessitating larger filter areas. The plugging issues originate from changes in lignin solubility due to large differences between the pH and ionic strength of the liquor in the cake and the washing liquid (Öh- man, 2006), (Jönsson & Wallberg, 2009). In the first washing step, the pH is still high due to buffering capacity of lignin while the ionic strength has already drastically decreased due to salt ions being washed out by acidic wash water, see figure 1.2. The high pH combined with lowered ionic strength in the filter cake leads to re-dissolution of lignin, resulting in complete or partial plugging of the filter cake.

Figure 1.2: Effect of varying amount of wash water on filtrate pH (left y-axis, solid line, incoming wash water pH 1.05, 20 °C); on the right y-axis sodium and lignin concentration in the filtrate (Öhman, 2006)

The LignoBoost process is an improved version of traditional acid precipitation because it drastically reduces the plugging issues, resulting in a higher purity of the extracted lignin. (Öhman, 2006). The LignoBoost process separates lignin from BL by precipitation, using acid and a two stage filtration/washing system, as shown in figure 1.3. A part of the BL stream is redirected from the pulping plant and acidified with CO2 to pH 9-10 at 60 °C - 80 °C. In the next step, precipitated lignin is filtered with a chamber press filter (see first "Filtration" in figure 1.3). In contrast to the traditional acidification process, the lignin filter cake is then redispersed with the acidic filtrate (see “Resuspension" in figure 1.3). The pH and temperature of the cake re-slurry are adjusted to approximately the same conditions as in the final washing liquor (i.e. pH 2-4). In this way, large differences in pH and ionic strength between liquor and washing liquid are avoided during the final washing stage. Therefore, lignin re-dissolution and consequent plugging of the filter cake

4 1. Introduction during the final washing stage are prevented. Finally, the re-slurry is filtered and then displacement washing is applied (see “Filtration & washing” in figure 1.3) (Öhman, 2006). The re-slurrying step in the LignoBoost process allows to efficiently extract lignin of high purity because plugging of the filter is avoided. Furthermore, with the LignoBoost process a higher lignin yield can be obtained because no lignin is redissolved during washing. In a nutshell, the LignoBoost process requires a lower filter area, lower volume of acidic washing water and a higher lignin yield and purity (i.e. lower ash and carbohydrate content in the lignin) can be obtained. (Öhman, 2006)

Figure 1.3: A scheme of the LignoBoost process stages used for isolation of kraft lignin. (Kalogiannis et al. , 2015)

1.2 Objective

In the Lignoboost process careful pH adjustments lead to precipitation of lignin from the BL and enable filtration for the separation of lignin. However, it has been observed that the presence of hemicelluloses, in particular of xylan in BL increases the filtration resistance and thereby decreases filtration performance (Wallmo et al. , 2009). A better understanding of lignin-xylan interactions during the LignoBoost process is therefore necessary. This thesis investigates the influence of possible interactions between xylan and lignin during the precipitation and filtration process and the resulting influence on filtration properties.

1.3 Methodology

The influence of xylan on the precipitation mechanism and on filtration properties is studied in two different sets of experiments. Model liquors are used in all experiments, since BL is a very complex mixture, making it easier to isolate the effect of

5 1. Introduction different variables. Model liquors are limited on lignin and xylan in their organic material and are minimized in their salt content compared to BL. In order to investigate the effect of xylan, reference liquors of lignin only are made for comparison. In the following, experiments with lignin only are denoted with L while experiments where xylan was added are labeled with L-X. To our knowledge, precipitation mechanisms of lignin-xylan mixtures have not been studied. Therefore, the first set of experiments focus on the particle formation dynamics of lignin-xylan in the alkaline regime. For this purpose, focused beam reflectance measurements (FBRM) are performed in-situ, which allow the monitoring of particle formation dynamics, including the precipitation onset. In a second step, the precipitation samples were filtered in order to link information on particle formation dynamics to the respective filtration resistance. Previous filtration experiments of lignin-xylan mixtures have shown an increase in filtration resistance and a decreases in solidosity (Durruty et al., not published). In order to understand this effect, the location of xylan in the filter cake might be elucidating. Therefore, a second set of experiments has been designed to study the filtration process. For tracking the xylan in the lignin-xylan filter cake, xylan was tagged with a fluorescent dye and the filter cake was investigated using a confocal fluorescence microscope (CFM). The preparation of dyed xylan (dX) is discussed separately as it involves an elaborate procedure and dX was observed to feature different properties that are important for further interpretation of results. The location of xylan within the filter cake obtained after filtration experiments is furthermore investigated by performing high performance liquid chromatography (HPLC) measurements on the filter cake. The particle size and shapes is also investigated by making use of the obtained CFM images and by using laser diffraction analysis (LDA), BET measurements and FBRM. As FBRM is used in-situ during particle formation, its data is also utilized to study the effect of temperature on particle formation dynamics.

1.4 Thesis Outline

The thesis starts with an introduction (Chapter 1) into the pulp and paper industry and the scope of the LignoBoost process. Chapter 2 provides relevant theory on the chemistry of lignin and xylan and on the precipitation and filtration processes. The chapter also includes relevant research findings on the topics. In chapter 3 includes experimental procedures and conditions as well as a description on the used analysis techniques. The experimental work is divided into the preparation of dyed xylan, the precipitation study and the filtration study. Chapter 4 provides results and discussion for the preparation of dyed xylan, the precipitation study and the filtration study. A final discussion, relating the precipitation and the filtration study is also included in this chapter. The respective conclusions for the three parts are drawn in chapter 5 while recommendations for further research are given in chapter 7.

6 2 Theory

2.1 Chemistry of Lignin and Xylan

2.1.1 Lignin Lignin is a wood constituent mostly found in cell walls and it is the most abundant aromatic natural polymer. The lignin content in wood varies from 26-32 % depending on the tree type e.g. softwood usually shows higher lignin contents compared to hardwood. Lignin content may vary within a single tree. In the plants and trees, lignin serves as a glue, holding the fibers together and providing mechanical strength to the tissue. Furthermore, lignin prevents water from being absorbed into the cell wall due to its hydrophobic nature and thus lignin also plays an important role for the water transport in the plants and trees. Lignin is a three-dimensional, branched bio-macromolecule mostly composed of randomly cross-linked phenylpropane units. The main building units of lignin (i.e. monolignols) are: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignols differ by their number of methoxy groups attached to the ring, see figure 2.1.

Figure 2.1: Monolignol monomer units: (a) p-coumaryl alcohol, (b) coniferyl alcohol and (c) sinapyl alcohol (Amarasekara, 2013)

Softwood contains almost only coniferyl alcohol while hardwood contains mainly sinapyl alcohol. Native lignin is still diﬃcult to directly study since the isolation of lignin modiﬁes its chemical structure. Therefore, the exact structure of lignin has not been determined yet (Norgren & Edlund, 2014). The monomers in the lignin network are covalently bond by approximately two third ether (C-O-C) and one third carbon-carbon (C-C) bonds. The type of linkage between the monolignols for

7 2. Theory hardwood and softwood lignin are specified in table 2.1 and figure 2.2. The most common linkages between the phenylpropane units are the β-O-4 bonds (figure 2.2).

Table 2.1: Proportions of diﬀerent type of linkages, connecting the phenylpropane units in lignin (Sjöström, 1993)

Percent of the total linkages Linkage type Dimer structure Softwood Hardwood

β-O-4 Arylglycerol-β-aryl ether 50 60 α-O-4 Noncyclic benzyl aryl ether 2-8 7 β-5 Phenylcoumaran 9-12 6 5-5 Biphenyl 10-11 5 4-O-5 Diaryl ether 4 7 β-1 1,2-Diaryl propane 7 7 β-β Linked through side chains 2 3

Figure 2.2: Structural formula of lignin, showing diﬀernt type of linkages (Deng et al. , 2015)

The most common functional groups in native lignin are hydroxyl (phenolic and aliphatic), methoxyl, carbonyl and carboxyl groups. The occurrence of the bond type and functional group varies depending on the wood species. The functional groups in lignin strongly inﬂuence its interaction mechanisms. By way of example, the solubility of lignin in alkaline solution originates from the deprotonation of the hydroxyl and carboxylic groups (Ek et al. , 2009).

Lignin during the Kraft and LignoBoost Process The conditions and chemicals used in the pulping process strongly aﬀect the structure of lignin. Lignin separated in the pulp and paper process is called technical lignin (e.g. kraft lignin (KL)) and has to be distinguished from native lignin. The

8 2. Theory chemical treatment in the pulping process causes bond cleavage of different lignin monomers and of covalent linkages between monomers. Therefore, pulping conditions lead to a decrease in molecular weight (MW) and the formation of differently sized lignin fragments (i.e. polydispersity of lignin). The predominant reaction in Kraft pulping is the cleavage of the β-O-4 bonds and the formation of phenolic groups. The dissociation of phenolic groups increases the solubility of KL in alkaline solution (Norgren & Lindström, 2000). KL is more hydrophilic compared to native lignin because of higher amounts of phenolic groups and a decrease in MW. The main functional groups present in KL are phenolic hydroxyl, carboxyl, methoxyl and sulfonate groups but their respective content depends on the specific pulping conditions (Norgren & Edlund, 2014). An example of a possible fragment softwood kraft lignin is shown in figure 2.3. Usually, KL has a weight-averaged MW between 1–5 kDa and a glass transition temperature of 124-174 °C(Amarasekara, 2013).

Figure 2.3: Molecular structure of fragment softwood kraft lignin (Norgren & Edlund, 2014)

2.1.2 Xylan Hemicellulose is a class of polysaccharides in wood that differ from cellulose. Con- trary to cellulose, hemicelluloses typically feature a structure with little strength and are easily hydrolyzed by dilute acid or bases. Furtermore, while cellulose contains only anhydrous glucose as a monomer, hemicellulose can be build-up by a variety of monosugars. Xylan is a hemicellulose that can be found to different extends in hardwood and softwood. It has a typical weight content between 15 and 30 % in hardwood and is thereby the most abundant hemicellulose in hardwood (Sjostrom, 1981). In softwood, xylan tends to make up 7 to 15 % by weight, where it is the second most abundant hemicellulose after the glucomannans (Casey, 1980). Xylans from different sources differ in composition but some generalities can

9 2. Theory be constituted: Xylan consists of monomers of the pentose sugar xylose, which are linked by glycosidic bonds. The resulting 1,4-linked β-D-xylopyranose units form the backbone of xylan, illustrated in ﬁgure 2.4. When it comes to the degree of polymerization, for softwood xylan a DPn between 90-120 has been reported while hardwood xylan DPns vary between 100-220. The observed structure is essentially linear but some short chains of single monomers may exist.

Figure 2.4: Representative structural formula for the xylan backbone

An important characteristic of xylan are the side groups, attached to the xylopyranosyl residues. The frequency and composition of side groups are dependent on the source of xylan, and will be discussed with respect to softwood and hardwood. Present in both wood types is an uronic acid side group, namely 4-O- methylglucuronic acid (MeGlcA), which is α-1,2-linked to the xylan chain. In softwood, on average 1 MeGlcA residue can be found per 5-6 xylose units, while in hardwood it is 1 per 8-20. The greater occurrence of these groups is the reason why softwood xylans are typically more acidic. Furthermore, these side groups appear to be regularly distributed in softwood but irregularly in hardwood. The specific side group for softwood, is α-L-arabinofuranose - on average one per 8-9 xylan units. The characteristic xylan components in softwood are thus known as arabinoglucuronoxylan and are illustrated in figure 2.5. In contrast, hardwood xylan possesses acetyl ester groups, typically one per 10 xylopyranosyl residues. Most of the residues that carry a MeGlcA side group also contain an acetyl ester group, giving glucuronoxylan as the characteristic xylan component in hardwood (Teleman, 2008). The structure is illustrated in figure 2.6.

Figure 2.5: Representative structural formula for arabinoglucuronoxylan, encountered in softwood (Ebringerová et al. , 2005)

10 2. Theory

Figure 2.6: Representative structural formula for glucuronoxylan, encountered in hardwood (Ebringerová et al. , 2005)

Xylan during the Kraft and LignoBoost process The Kraft process employs alkaline conditions and high temperatures. The aim is to degrade and dissolve the lignin fraction in wood but it also has an effect on xylan structure and solubility. Several reactions can be made responsible for a change in xylan structure: • Hydrolysis reactions cleave the acetyl groups in hardwood xylan and are responsible for most of the acetate formed in kraft pulping. • Peeling reactions promote degradation of the polymer by splitting off the re- ducing end groups in the carbohydrate chains (Green et al. , 1977). Other carbohydrates are severely affected by peeling during kraft pulping but xylan only looses some of its MW as its substituents enable stopping reactions that form a stable metasaccharinic acid and set an end to peeling reactions. More detailes can be found elsewhere (e.g.(Gellerstedt, 2004)). • Elimination reactions of the methoxyl group within the MeGlcA group, resulting in methanol and the conversion of the MeGlcA group into a hexenuronic acid (HexA) group. A high solubility of xylan during Kraft cooking can be related to the presence of MeGlcA side groups, as they involve a carboxylic acid group with a pKa around 3.5-4. At neutral or alkaline conditions these groups are negatively charged and enable xylan to act as a weakly charged polyelectrolyte with an increased solubility. However, only part of the xylan ends up in the BL, as some of it redeposits onto the cellulose fiber surface (Yllner & Enström, 1956). The reason for this is still under investigation and not fully understood. What has been observed is that the ratio between xylan in pulp and BL depends very much on the wood type and on the exact process procedure. During the LignoBoost process, the dissolved xylan in the BL is exposed to acidic conditions. Under acidic conditions the hydrolysis of glycosidic linkages becomes an important degradation reaction, where the rate decreases in the following order: HexA > Xylan > MeGlcA (Teleman, 2004). The HexA linkage can already be selectively hydrolysed under mild acidic conditions without significant hydrolysis of xylan itself. Degradation of xylan requires more severe conditions. For example for birch xylan it has been shown that a temperature of 90 °C requires a pH below 2 and several hours of cooking for complete cleavage of xylan. The MeGlcA is a relatively acid resistant glycosidic linkage (Hilpmann et al. , 2014).

11 2. Theory

2.1.3 Ligno-Carbohydrate Complex In Kraft processing chemical linkages between lignin and carbohydrate components have been observed. These chemically linked complexes are often called “lignin- carbohydrate complexes” (LCC) (Du, 2013). Different bond types between lignin and carbohydrates have been reported: benzyl ether, benzyl ester, phenyl glycoside, acetal types (Watanabe, 2003). Tamminen et al. suggested that in the BL lignin was linked to xylan by an arabinose unit. However, the native form of LCCs is an on- going debate as the chemistry and MW changes with pulping processing condition. Under chemical pulping conditions, lower MW LCCs are expected due to cleavage of bonds in highly alkaline conditions (Sjöström, 1993), (Tenkanen et al. , 1999). The presence of LCC seems to cause difficulties in delignification procedure (Gierer & Wännström, 1986), (Iversen & Wännström, 1986), (Choi et al. , 2007) and in the LignoBoost Process. In the context of the LignoBoost process, Wallmo et al. (2009) indicated an increase in filtration resistance attributed to high MW carbohydrates (potentially LCCs). Another aspect found by Zhu et al. (2016) was that carbohydrate concentration (most likely in form of LCCs) in lignin precipitated from BL decreases with increasing precipitation yield (Zhu et al. , 2016).

2.2 Precipitation

2.2.1 Background Precipitation is the spontaneous formation of clusters in a colloidal suspension. In a colloidal suspension particles of 1-1000 nm in diameter are evenly distributed throughout the solution (Stepto, 2009). The particle formation dynamics are divided in nucleation (i.e. onset aggregation), particle growth and particle breakage. Nuclei are either present from the start or they form due to changing solution conditions. In this thesis, aggregation is specified as the pre-nucleation state, according to Nichols et al. (2002). The phase behavior of colloidal suspensions can be partially described by force balances using an extended version of the DLVO theory1. Aggregation and thus precipitation can occur when the attractive forces dominate the repulsive inter-particle forces within the colloidal suspension. Attractive forces include van-der Waals and hydrophobic forces. Repulsive forces are e.g. electrostatic forces and steric stabilization or electrosteric stabilization. Electrosteric stabilization combines electrostatic with configurational entropic repulsion between colloidal particles. The repulsive forces can be controlled by solution conditions such as solution pH, ionic strength and temperature (Norgren et al. , 2001). The aggregation of colloidal particles can be classified into two limiting regimes of kinetics: the rapid diffusion limited cluster (colloid) -cluster (colloid) aggregation (DLCA) and the reaction limited aggregation (RLCA). DLCA is when the Brow- nian motion of particles limits precipitation; meaning that the particle-particle in-

1The DLVO (Derjaguin-Landau-Verwey-Overbeek) theory is used to theoretically describe the aggregation of aqueous dispersions quantitatively by using a force balance. The theory considers two forces the van-der-Waals (attractive forces) and repulsive forces.

12 2. Theory teraction is dominated by attractive forces. RLCA is when multiple attempts are necessary to overcome an intermediate interaction potential and thus agglomeration can occur. DLCA forms loose and highly disordered aggregates while RLCA forms slightly denser aggregates (Norgren et al. , 2001). It has been shown that these processes are independent of the colloid nature, given that physical interactions are the same (Lin et al. , 1989). Therefore, it is suggested that RLCA, DLCA and their intermediate regime suﬃce to specify the kinetics of aggregation (Lin et al. , 1989), (Meakin et al. , 1990), (Lin et al. , 1990). Aggregation can switch from RLCA to DLCA at a certain concentration of ions, namely the critical coagulation concentration (CCC)(Norgren et al. , 2001).

2.2.2 Lignin Precipitation

KL macromolecules can be considered as polyelectrolytes in alkaline solution e.g. in BL. KL is negatively charged and soluble at high pH due to dissociated phenolic groups. The shape of lignin is often assumed to be a spherical amorphous macromolecule (Goring, 1962),(Lindström, 1980). KL macromolecules indicate a microgel structure having a dense core and a looser negatively charged surface (Rezanowich & Goring, 1960), (Lindström, 1980). It is assumed that the negative charges are evenly spread on the surface. In aqueous solution of high pH a double layer is formed where the negatively charged phenolic groups are surrounded by cations (mainly Na+ and K+), as shown in figure 2.7, left (Zhu & Theliander, 2011). Lignin precipitation depends on attractive and repulsive forces as qualitatively described by the extended DLVO theory (Norgren & Lindström, 2000). KL aggregation is favored if the attractive forces are predominant; if repulsive forces are predominent the KL stays in solution (Norgren et al. , 2001). Repulsive and attractive forces within the colloidal system are influenced by the KL chemistry and structure, (e.g. MW and functional groups). However, the solution conditions (KL concentration, pH, temperature, ionic strength) also influence the force balance (Rudatin et al. , 1989).

13 2. Theory

Figure 2.7: Model of a stable/ unstable KL molecule in BL (Zhu & Theliander, 2011)

KL precipitation from BL by acidification can be described in a simplified way when considering the pH as the key parameter for precipitation: In the alkaline BL, KL is in its ionized and stable state due to the dissociated phenolic groups. The negatively charged KL molecules electrostatically repel each other and create a stable colloidal system, see figure 2.7, left. This colloidal system is destabilized by acidification because the H+ ions protonate the negatively charged phenolic groups, see figure 2.7, right. The repulsive electrostatic forces are weakened by neutralization of the negative charges and attractive forces such as van der Waals and hydrophobic forces become dominant. As a result, KL becomes unstable, aggregates and eventually precipitates. However, the number of KL fragments that are susceptible to aggregation depends not solely on the solution condition such as the pH but also on the physiochemistry of KL fragments e.g its charge density (Norgren et al. , 2001). Nogren et al. suggest different modes of KL precipitation, reaching from macromolecular KL into colloidal KL particles (i.e. self-associated macromolecules) and finally as aggregation proceeds KL clusters are formed, as illustrated in figure 2.8 (Norgren et al. , 2002). Solution conditions that favor aggregation and precipitation of colloidal KL are according to Norgren: decreasing OH concentration, increasing ionic strength and/or increasing temperatures (Norgren & Lindström, 2000). As solution conditions change to favor precipitation, the first stage (i.e. the onset aggregation) is initiated. In the onset of aggregation, nuclei are formed by self- association of KL macromolecules and fractal KL aggregates are created. Then they grow into colloidal seed points (Evans & Fendler, 1994). High MW macromolecules

14 2. Theory serve as natural nuclei and may sorb low MW KL fragments before they aggregate (Leubner, 2000). Nogren et al. claim that the aggregate structure is determined by the aggregation rate - aggregates formed in the RLCA region are relatively dense. In the DLCA regime aggregates are formed faster, resulting in a looser aggregate structure. Norgren et al. suggest that this configurational difference between aggregates 2 formed in the DLCA and RLCA regime is reflected in their fractal dimensions (df ) being 1.8 for the DLCA and of 2.1 for the RLCA regime. (Norgren et al. , 2002)

Figure 2.8: Modes of KL aggregation starting from macromolecular KL and ﬁnally reaching KL clusters (Norgren et al. , 2001)

2.2.3 Inﬂuences on Lignin Precipitation

Studies on lignin precipitation have shown that there are multiple parameters besides the chemistry and stereochemistry (e.g. molecular weight, polydispersity of kraft lignin, functional groups) of the polyelectrolyte that influence the precipitation. A complex stereochemistry of the polyelectroylte can lead to stabilization of a colloidal suspension by high configurational entropic repulsive forces between the colloidal particles (Napper, 1983). External parameters are often related to the process conditions e.g. lignin concentration, pH, ionic strength and temperature of the solution as well as the agitation rate. It is reasonable to expect that the precipitation process can influence the filtration resistance of precipitated material in the filter cake. Thus, studying the precipitation conditions of KL may lead to find optimal precipitation conditions to reduce the filtration resistance.

2A fractal dimension provides a statistical indication on the complexity of a self-similar structure. It is the ratio of self-similar pieces per magniﬁcation. In other words it shows the changes of details in a pattern with the scale at which it is measured.(Bandt et al. , 2015)

15 2. Theory

Lignin Concentration Nogren et al. state that for dilute lignin solutions, lignin concentration has relatively low inﬂuence on the precipitation mechanisms as indicated by turbidity measurements. They have found the same CCC for solutions having a lignin concentration varying between 0.26 and 3.3 g/l. (Norgren et al. , 2001) However, Öhman(2007) and Wallmo et al. (2009) indicated that lignin concentration inﬂuences the precipitation to some extend in the case for higher lignin concentrations. pH The pH of the solution is one of the most important factors in lignin precipitation. KL is has a very los solubilty in pure water but dissolves under alkaline conditions. The KL network comprises weakly acidic phenolic groups and thus its solubility is increased by increasing hydroxide ion concentrations, leading to dissociation of acidic groups. (Norgren & Lindström, 2000) On the other hand, below the pKa of the phenolic group the ionized phenolic groups become protonated which decreases lignin solubility. Softwood lignin mainly consists of coniferyl alcohol with a pKa of 10.2 at room temperature, but the apparent pKa of lignin is higher and depends on its MW (Norgren & Lindström, 2000). However, studies have shown that high MW KL with lower amount of phenolic groups possess higher pKa values for the phenolic groups (Norgren & Lindström, 2000), (Zhu & Theliander, 2011). Generally, self-association of KL may occur for pH below 9.5 in salt free solution at room temperature (Rudatin et al. , 1989), (Garver & Callaghan, 1991).

Temperature The inﬂuence of temperature on lignin precipitation is contradictory based on the available literature. On one hand, elevated temperatures increase the ionic dissociation constant of phenolic groups but the ionic dissociation constant of water is increased even more which compensates the dissociation of weakly acidic groups. Therefore, elevated temperatures reduce the degree of dissociation of the weakly acidic phenolic groups. As a result, electrostatic repulsion is reduced and aggregation mechanisms are favored. (Norgren & Lindström, 2000) Nogren reported that for increased temperatures aggregation of lignin can even occur at higher pH and lower ionic strength (Norgren et al. , 2001). However, in the studies of Zhu it was reported that increasing temperature increase the solubility of KL, although the underlying mechanism was not further investigated (Zhu & Theliander, 2011).

Ionic Strength It is generally known that precipitation of KL is stimulated by high concentrations of monovalent metal ion salts at neutral pH (salting out) (Norgren et al. , 2002). When increasing the ionic strength of the solution, charges on the KL are shielded and electrostatic repulsion is reduced. Lindström et al. reported that there is a clear CCC of added electrolytes for colloidal KL (Lindström, 1980). Norgren et al. showed that at high ionic strength (at and above 1.2 M NaCl) there is a formation of larger aggregates (hydrodynamic diameter of KL aggregates

16 2. Theory

> 1000 nm) at p0H of 3.5 and 70 °C. As a result, aggregation rate rises for larger hydrodynamic diameter. At high ionic strength the repulsive barrier should almost be removed (i.e. debye length3is short) and thus it is surprising that even at high ionic strength the aggregation rate still increases. This effect might be explained by the heterogenic nature of KL e.g. a large particle size distribution. At ionic strength above 1.3 M diffusion is the limiting factor.(Norgren et al. , 2001) The colloidal system can be destabilized (i.e. aggregation is initiated) by mono or polyvalent electrolytes. According to Norgren et al. polyvalent counterions have a larger effect on aggregation and shielding of the polyelectrolyte. Polyvalent counterions can serve as a bridge between charge groups of polyelectrolytes and create ionic complexes (Lindström, 1980). However, Norgren et al. indicated that increasing concentrations of certain ions and elevated temperatures can actually stabilize the colloidal suspension of KL (see (Napper, 1983)). They showed that the colloidal stability of a lignin suspension is drastically influenced by specific ions and it follows the Hofmeister series for anions, (Norgren & Lindström, 2000). The Hofmeister series classifies certain ions due to their ability to salt in or salt out proteins. The ions from the Hofmeister series have an effect on the secondary and − 2− tertiary structure of the proteins. It appears that anions (e.g. F > SO4 > HPO4 2− − − − − − − − > acetate > Cl » Br > NO3 > ClO3 > I > ClO4 > SCN ) may have a larger effect on the solubility of proteins than cations (Yang, 2009). However, the mechanism behind the Hofmeister series is still not completely understood. The lignin solubility follows the Hofmeister effect but only for the anion series, − − − showing that the aggregation ability of lignin is most favored by NO3 > Br > Cl . It is found that organic anions provide even more destabilization of the structure than inorganic anions; indicating a more pronounced effect of the destabilization ability of hemicellulose (e.g. xylan). It might be that xylan can destabilize the system even more (Norgren et al. , 2001). For cations (especially alkali metal ions) the aggregation increases with atomic number, meaning as the non-hydrated ion radius increases. Aggregation is most favored by Cs+ > Na+ > K+.

Molecular Weight and Polydispersity of Kraft Lignin

The MW of KL has an aﬀect on its precipitation behavior and solubility. High MW KL usually precipitates ﬁrst and can even serve as natural nuclei. Low MW KL macromolecules adsorb in the particle growth stage (Norgren et al. , 2001). Low MW KL has usually a higher solubility and stability than high MW KL. KL fragments of low MW are less compact and are less cross-linked. (Norgren et al. , 2001) Furthermore, there is a lower gain or loss in conformational chain entropy for high MW during precipitation (Flory, 1954). A high polydispersity of KL fragments are thus expected to change the solubility and the precipitation mechanism.

3Debye length (κ−1) is used to describe the screening eﬀect of monovalent ions and thus favoring aggregation. A short debye length corresponds to a larger screening eﬀect induced by high ionic strength and thus favors aggregation. The debye length can be calculated using the following −1 2 −1/2 equation:κ = ((0 r RT )/(2F I)) where is dielectric constants, R is the gas constant, T P 2 is the absolute temperature, F is the Farady constant, I is the ionic strength with (I = 0.5 (zj Cj) (where z and C are the ion valence and concentration, respectively).

17 2. Theory

2.3 Filtration

2.3.1 Background Filtration is one way of separating solid-liquid mixtures. A filter media allows the fluid to pass while retaining solid particles that are larger than the filter pore size. The driving force for fluid flow is a pressure difference across the filter media. Dead- end filtration is a mode of unit operation where the dominant direction of fluid flow is perpendicular to the filter. Retained particles are accumulated on the filter media over time and form a cake as illustrated in figure 2.9.

Figure 2.9: Dead-end ﬁltration, where a cake forms and growths over time due to a pressure gradient across a ﬁlter media Khean(2003)

The growing cake also acts as a filter medium as soon as a first layer of particles have accumulated. A growing filter cake impedes filtration since deposited particles constitute an obstacle for the fluid flow. The rate of flow progressively diminishes if the pressure stays constant. The degree to which the filtration performance is affected is expressed with the filter cake resistance Rc. Cake resistance increases with deposited material, making it useful to introduce the cake resistance per unit weight, known as the specific cake resistance α. The specific cake resistance is determined by the cake properties and can be calculated using equation 2.1.

1 α = (2.1) Kρsφ

Where K is the permeability of the cake, ρs is the solid density of the particles and φ is the solidosity of the cake.

Solidosity describes the solid density of the cake and is deﬁned as the volume based fraction of solids in the cake. Solidosity can thus be related to the porosity ε as seen in equation 2.2. V φ = solid = 1 − ε (2.2) V total

18 2. Theory

From a physical perspective porosity and permeability are key parameters for cake resistance. Cake porosity is a measure of the cake fraction that is available for fluid flow. Cake permeability is an indication of how easily the fluid can pass through the available cake pores under a pressure gradient. It means that permeability depends on cake porosity but also on physical and chemical specifications of the pores. Ac- counting for all the influencing parameters makes it virtually impossible to express permeability in a mathematically rigorous way and requires the use of simplified models. (Khean, 2003) A good first approximation for expressing permeability is based on the work of Kozeny (1927) and Carman (1938). It assumes that flow in a porous medium can be represented as flow through many parallel channels, which are formed by spherical particles. Therefore, permeability is determined by cake porosity and specific surface area of the particles. From a physical perspective, the flow resistance induced by the cake can be understood by an increase of drag force of the fluid on the particles. This is due to a higher solid-liquid contact area, which slow down fluid flow. A more elaborate model was developed by Endo and Alonso (2001), who considered particle size distribution (PSD) and particle shape in the description of permeability. It has been observed that a large PSD with a significant portions of fine particles forms a densely packed filter cake, which typically entails narrow channels (or capillaries). The capillary pressure increases with decreasing capillary size and is thus inhibiting the flow. Secondly, the particles are typically not spherical and hard but can appear in various shapes, resulting in different channel structures. Many more factors have been observed to influence permeability, such as tortuosity of capillaries, particle interactions or particle surface specifications, to name a few (Khean, 2003). Another concern about the specific cake resistance α is that it may vary with time and location in the filter cake. A uniform value for α is only obtained if one assumes an incompressible cake that does not deform over time. For an incompressible cake, the cake resistance Rc only increases due to additional deposited material. However, in reality deposited particles are often rearranged and /or deformed by the drag force of the liquid flow. In this case, it is a compressible cake, which forms a denser structure over time. Specific resistance α is therefore expressed as a local specific cake resistance αloc that varies along the flow direction and is highest close to the filter media. In an incompressible cake, pressure does not influence resistance and is thus directly proportional to flow rate. In case of a compressible cake, an increase of pressure does not necessarily result in an increased filtration performance as the filter cake gets more impermeable due to the compression.

2.3.2 Model for Dead-end Filtration

A model for the relation between flow rate and cake formation can be traced back to Darcy’s law (1856). Darcy found that the rate of filtration through a porous media is directly proportional to the driving force, where the factor of proportionality is the inverse resistance. Darcy’s law is thereby analogous to Ohm’s law in the field of electrical networks, or Fick’s law in diffusion theory for example. In the case

19 2. Theory of dead-end filtration a modified form of the Darcy’s law can be used, with the assumption of an incompressible filter cake, as in equation 2.3.

1 dV 4P u = = (2.3) A dt µRcRm

Where u is the flow rate of fluid [m/s], V is the filtrate volume [m3], t the filtration time [s], A the filtration area [m2], 4 P the pressure drop over the filter cake and −1 filter media [Pa], µ the viscosity [Pa s], Rc the filtration resistance of the cake [m ] −1 and Rm the resistance of the filter media [m ].

The cake resistance Rc can be expressed as specific cake resistance α times cake weight and therefore increases with the accumulation of filter cake. It is thus possible to relate Rc to the filtrate volume V , if one assumes a constant volume of cake deposited, per volume of filtrate. That means that applied pressure and slurry properties need to be constant. By rearranging equation 2.3 one can obtain a first order differential equation as seen in equation 2.4. A more elaborate derivation can be found elsewhere, e.g. (Holdich, 2002).

dt µαc µR = V + m (2.4) dV A24P A4P

Where c is the dry cake mass per ﬁltrate volume [kg/m3].

For an incompressible filter cake and at constant applied pressure, α is independent of time and the plotting of dt/dV against V describes a linear relationship during cake formation. The y-intercept of the linear region can be related to the resistance of the filter media and the slope of the line can be used to calculate α. Equation 2.4 works also well for the average specific cake resistance αav in weakly compressible filter cakes at constant applied filtration pressure. The premise is that αav is virtually independent of time, by being constant regardless of cake height at constant applied filtration pressure. That can easily be verified by observing that dt/dV versus V gives a straight line. Typically that condition is not met straight from the beginning of the filtration process as the first deposited particles on the medium are exposed to the full drag force of the liquid flow and are being particularly compressed.

2.3.3 The Inﬂuence of Xylan in the LignoBoost Process

Wallmo et al. (2009) investigated the filtration properties of BL after precipitation to pH 9.5. He observed a higher filtration resistance of BL, obtained from hardwood compared to softwood, which corresponds to previous findings of Öhman (2006). Wallmo found that this increase in filtration resistance could be correlated to higher hemicelluloses concentration, found in the hardwood BL’s. Furthermore, the same trend was observed when hemicelluloses concentration was substituted with xylose (i.e. xylan) content as can be seen in figure 2.10.

20 2. Theory

Figure 2.10: Specific filtration resistance of BL with different initial concentrations of hemicelluloses and xylose. Liquor A1 is a softwood BL and precipitated to pH 9.5 at 75 °C. Liquor C1 is a hardwood BL and precipitated to pH 9.5 at 60 °C. Liquor C2 is the same BL as C1 but was heat treated prior to precipitation.

Wallmo et al. (2009) also showed that filtration resistance of BL decreases when hemicellulose concentration is reduced prior to precipitation, using ultrafiltration. Furthermore, he found that further reduction of hemicellulose via nanofiltration did not reduce the filtration resistance much more, indicating that not only the concentration but also the size of hemicellulose in the BL has an influence on filtration resistance. That also corresponds to his finding on the effect of heat treatment of BL. Wallmo observed that heat treatment at 170 °C prior to precipitation reduces filtration resistance. He concluded that this might be due to the reduction of high MW materials in the BL, which are constituted mainly of xylan and lignin. LCC’s would thus also fall under this definition. The effect of xylan on lignin filtration was also observed by Durruty by investigation of a model liquor. She prepared liquors where xylan was added additionally to softwood KL; i.e. there was no preexisting bond between the two compounds as in LCC’s. Durruty dissolved the compounds at highly alkaline conditions and precipitated at 80 to a precipitation pH between 2.5-3. When filtering the obtained slurries, she observed a substantial increase in filtration resistance for slurries where xylan had been added (LX), compared to slurries without additional xylan (L). In her experiments Durruty could furthermore observe an effect of salt addition on the influence of xylan. The increase of filtration resistance due to added xylan was significantly decreased at higher ionic strength, 2.11.

21 2. Theory

Figure 2.11: Filtration resistance for non-reprecipitated (mixing) and re- precipitated (via prior dissolution) slurries which were precipitated at 80°C to a precipitation pH between 2.5-3. Slurries diﬀer in xylan content and salt concentration.

22 3 Experiments

3.1 Materials

In this work softwood kraft lignin extracted from BL using the LignoBoost process was used. Its density was measured to be 1324 kg/m3 with a density meter (Mi- cromeritics AccuPyc II 1340). The xylan used in this study was highly puriﬁed xylan powder (Megazyme) from beechwood. Its molecular weight was measured to be 24030 g/mol with gel-permeations-chromatography and its density was measured to be 1522.7 kg/m3. The dye used in this study was Remazol Brilliant Blue R (RBBR) from Sigma Aldrich R-8001.

3.2 Preparation of Dyed Xylan

Synthesize of an ether bond between xylan and RBBR was performed, following the procedure of Biely et al. (1985). 10 g of xylan was suspended in 100 ml of distilled water at 50 °C. The solution was mixed with 100 ml of distilled water, in which 700 mg of RBBR had been dissolved. The mixture was kept under vigorous agitation and at a temperature of 50 °C for approximately 35 minutes. During this time interval, 20 g of Na2SO4 was added in small portions. Afterwards the pH was increased to around pH 12 by adding 3.2 g of Na3PO4. In the first synthesis (S1), the mixture was kept at 50 °C under agitation for 3 hours. In the second and third synthesis (S2 and S3) the mixture was kept at 50 °C under agitation for only 1 hour. After the synthesis was completed, dyed xylan was present in the mixture, alongside with unreacted RBBR and salts. In order to remove unreacted RBBR, dyed xylan was precipitated, allowing filtration and washing for purification. Two precipitation/ washing procedures were tested and two different washing liquids were applied to investigate the influence on xylan yield and on salt content in the final precipitate. The two precipitation/ washing procedures (i.e washing methods) dif- fered in temperature, precipitation time, time of pH adjustments and solid-liquid separation as illustrated in figure 3.1. The two procedures are in the following report referred to as washing method 1 and washing method 2. In washing method 1 the synthesis product was precipitated with EtOH and directly washed with an EtOH/ water mixture (2:1). Thereafter, the washed cake (i.e. synthesis product) was acidified to pH 3 with 0.1 M H2SO4 to ensure full pro- tonation of the dyed xylan. The synthesis batches using the washing method 1 were not filterable. This was due to the fact that when acid was added to the washed precipitate, the dyed xylan precipitate was finely dispersed in the liquid phase and

23 3. Experiments blocked the filter medium. Therefore, evaporation had to be used for solid-liquid separation. In washing method 2 the synthesis product was acidified first to pH 3 using 0.1 M H2SO4. Then, the acidified synthesis product was precipitated using EtOH (2:1) with respect to the acidified synthesis product. The suspension was precipitated over night. For solid-liquid separation, first decantation was applied. Then, it was washed with an EtOH/ water mixture (2:1) or ethanol only, depending on the synthesis batch. The solubility of RBBR in EtOH only was confirmed by an experiment. In the last solid-liquid separation step, filtration was applied.

(a) Washing/ Precipitation Method 1

(b) Washing/ Precipitation Method 2

Figure 3.1: Flow sheets of two diﬀerent precipitation/ washing procedures (a) washing/precipitation method 1 and (b) washing/ precipitation method 2.

For each washing procedure, washing was continued until a clear filtrate was obtained. The amount of washed out RBBR was measured using UV-vis. Xylan concentration in the final precipitate was measured using HPLC. Both results were used to calculate the substitution of RBBR on xylan as shown in the appendix equation A.1. Three syntheses of dyed xylan were performed (S1, S2, S3). S1 and S2 were split up in two batches (B1, B2) while S3 was not split. Overall 5 different precipitates were obtained, which are later on referred to as S1B1, S1B2, S2B1, S2B2 and S3. The washing/ precipitation method and respective washing liquids are summarized in table 3.1.

Table 3.1: Washing conditions for the synthesis of dyed Xylan

Synthesis Washing Method Washing Liquid

S1 B1 1 Ethanol/Water (2:1) S1 B2 1 Ethanol/Water (2:1) S2 B1 2 Ethanol/Water (2:1) S2 B2 2 Ethanol S3 2 Ethanol

24 3. Experiments

3.3 Precipitation Study

45 g of lignin (85 % dry content) was dissolved in 383g 1 M NaOH solution and stirred over night. Vacuum filtration was performed to remove remaining particles and reduce the background for FBRM measurements, using a 0.45 µm regenerated cellulose filter paper. 2.37 g of xylan was added to the solution after the pH had been lowered to pH 12 to avoid xylan cleavage. A xylan/ lignin ratio of 5 g/ 95 g was chosen, giving final concentrations of 121.4 g/l of lignin and 6.5 g/l of xylan in the solution. The solution was stirred at 250 rpm and kept at 45 °C. The FBRM probe was immersed into the solution to observe particle formation dynamics over the course of the experiment. The technique is explained more elaborately in chapter 3.5.5. The pH of the solution was lowered stepwise by adding 6 M H2SO4. For pH measurements, samples were withdrawn from the solution and cooled down to around 28 °C. A pH meter with automatic temperature correction for ideal solutions was used for pH measurements; afterward samples were returned to the solution. Titration was performed slowly and drop wise, to avoid localized precipitation as much as possible. The titration endpoint was determined by the precipitation onset, observable in the FBRM data. The slurry was stirred until no particle growth could be observed anymore in the FBRM data. Afterwards the slurry was filtered at 45 °C using a filter press and a filter media consisting of a 0.45 µm pore size regenerated cellulose membrane, see figure 3.2. Precipitation experiments were performed on lignin reference suspensions (L) (without xylan) as well as on a lignin-xylan suspension (L-X).

3.4 Filtration Study

Slurry Preparation Two slurry preparation procedures were used - the dissolution/ re-precipitation (DR) and the mixing (M) procedure, which are described below. For both procedures, the xylan/ lignin ratio was chosen to be 5 g/ 95 g, resulting in ﬁnal slurry concentrations of approximately 95 g/l of lignin and 5 g/l of xylan.

Dissolution/ Re-precipitation Experiments 2.205 g of dyed xylan was dissolved in 182 g 1.1 M NaOH solution. As the dyed xylan was present in a precipitate alongside with Na2SO4 and Na3PO4, also 6-7 g of those salts were simultaneously added to the solution (depending on the synthesis product that was used). Then, 41.63 g of lignin was added and the solution was heated to 80 °C and kept under stirring at 250 rpm for 1.5 hours to allow complete dissolution. The pH was measured to be around pH 12. Next 0.43 M of sulphuric acid (73 %) was added to the reaction mixture for precipitation at pH 3-4. The slurry was then kept for 1.5 hours under stirring (250 rpm) at 80 °C. The slurry was stirred over night at room temperature and ﬁltered the next morning. The described procedure holds for L-dX experiments. For L-X and L experiments the salt concentration

25 3. Experiments

was adjusted according to the L-dX experiment by adding Na2SO4 and Na3PO4 to the NaOH solution as a ﬁrst step. Furthermore, also a dX only experiment was performed, where no lignin was added to the solution to investigate the stability of bond between dye and xylan during DR experiment conditions. For one L-dX, one L-X and two L ﬁltration experiments a FBRM probe was immersed into the solution after the pH was lowered to roughtly pH 10.

Mixing Experiments

2.205 g of dyed xylan was suspended in 430 g of 0.25 M H2SO4 (73 %) solution. Like in the DR experiments, 6-7 g of Na2SO4 and Na3PO4 were added simultaneously. Then 41.63 g of dry lignin was added. After a well mixed suspension was obtained, the solution pH was adjusted to pH 4 by addition of 6 M of sulphuric acid (73 %). The solution was then heated to 80 °C and mixed under stirring (250 rpm) for 1.5 hours. The slurry was stirred over night at room temperature and ﬁltered the next morning. In a second experiment the slurry was not heated up but was kept at room temperature for the entire experiment. A third experiment was performed, where xylan was used instead of dyed-xylan. The salt concentration was adjusted according to the L-dX experiment by adding additional Na2SO4 and Na3PO4 as a ﬁrst step into the H2SO4 solution.

Filtration Experiment

All slurries were filtered using a filter unit designed for dead-end filtration as shown in figure 3.2. The slurries were filtered using 10 bars of applied filtration pressure and a regenerated cellulose membrane with 0.45 µm pore size, placed on a grade 5 muktell paper support. Piston position and mass of filtrate were measured and recorded every two seconds. The average specific filtration resistance was calculated using the classical filtration equation 2.4. The dry content of the filtrate was measured in order to calculate the precipitation yield of the dissolution/re-precipitation experiments (see appendix equation C.1- C.3).

26 3. Experiments

Figure 3.2: Schematic diagram of filtration equipment. 1) Piston press, 2) two sealing O-rings coated in grease to facilitate piston motion within the cell and prevent backflow of the slurry, 3) recipient to collect the filtrate, placed on a mass balance, 7) recording computer unit. (adapted from Durruty(2014))

3.5 Other Characterization Techniques

3.5.1 Confocal Fluorescent Microscopy

Confocal fluorescence microscopy (CFM) allows 3D optical resolution of fluorescent species (i.e. fluorophore) providing dimensional and spatial as well as quantitative information, which is correlated with its intensity (Waters, 2009). Fluorescence is the re-emission of light by a chemical species upon electromagnetic excitation. The fluorescent species absorbs light energy of a specific wavelength and re-emits light at a longer wavelength (i.e. lower energy). The difference in energy and wavelength between absorbed and emitted light is called the Stokes Shift and is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a high background of excitation photons. In a CF microscope, the specimen is illuminated using a laser. The light from the laser is directed through an excitation pinhole and is then reflected by a dichroic mirror. The reflected light is then focused by a microscope objective to a small spot in the specimen. The objective focuses the excitation light in the specimen and collects the fluorescent light. Ideally only emission reaches the detector, so that the resulting fluorescent structures are superimposed against a very dark background. The degree of brightness is often correlated with the concentration of the fluorophore. The 3D optical resolution is achieved by the design of the instrument by focused light illumination and a pinhole in front of the detector. The pinhole in front of the detector eliminates light coming from out-of focus planes but at the

27 3. Experiments same time allowing light to enter from in-focus plane. By raster scanning of the sample, a 3D picture can be obtained. The thickness of the focal plane depends on the light wavelength, the objective lens and the optical properties of the specimen. (Mueller, 1996) The detection efficiency of the microscope is limited technically but also by the fluorophore that possesses a limit in number and rate of emitted photons. Further- more, light is blocked by the pinhole, which increases resolution but comes with lower signal intensity. (Mueller, 1996) Another challenge is that the specimens are not uniform but contain spatial variations in refractive index that lead to significant aberrations in the signal (Cornea & Conn, 2014).

Procedure The CFM samples were grinded in a ball mill and the sample was placed between two glass plates before observation under the CFM. It was important to create a flat sample surface without air-bubbles to avoid misreading of the pictures. All samples were analyzed in cooperation with a specialist at SP Sveriges Tekniska Forskningsin- stitut (Gothenburg) using a Leica confocal microscope. The scanning layer depth was about 0.8 µm. Since lignin exhibits autofluorescence, the colocalization method was used for analysis. For this purpose, the emission spectra of RBBR and lignin were investigated in dX and L filter cakes, respectively by performing automated emission scans. In this way, one could identify wavelength ranges that would be specific for either RBBR or lignin due to a high emission intensity. Afterwards, a L-dX filer cake was analyzed, where lignin was identified by collecting the emitted light in the specific emission range of lignin, while dyed-xylan was identified by collecting the emitted light in the specific emission range of RBBR. Both collection wavelength ranges were attributed with a chosen colour; green for lignin and red for xylan. By superimposing both wavelengths ranges in one image, a colocalization image was created. If both ranges were present in one pixel, the pixel appeared yel- low, meaning that RBBR and lignin “colocalized”. If one of the ranges dominated, the pixel appeared either green or red. Red indicated in this case that dyed-xylan could be localized at this position.

3.5.2 UV-Vis Spectroscopy Ultraviolet-visible spectroscopy (UV-Vis) is used to determine concentration of analytes by measuring absorption, transmission and/ or reﬂectance spectra (190-1100 nm). 1 In the UV-Vis spectrometer, visible (halogen lamp) and UV light (deuterium lamp) is monochromatized in the spectrometer system. The monochromatic light is alternatingly directed (by mirrors) through the sample and through the blank (background solution) before it reaches the detector. Then, signals are ampliﬁed and send to the software which creates the spectra. The electromagnetic radiation causes electronic transition for molecules that absorb UV or visible light. In that

1The Beer-Lambert law shows the direct proportionality of the absorbance of a solution to the concentration of absorbing species and the path length. Thus, the concentration of the absorbing species can be related to the absorbance by using reference samples with known concentrations (i.e. calibration curve).(Harvey, 2016))

28 3. Experiments case, the light excites electrons from the ground state to an excited state, which is measured as absorption. The easier electrons in the molecules are excited, the longer is the wavelength of the light it can absorb. (Lampman et al. , 2001) In UV-vis, a relative error in the absorption spectra of 1-5 % in absorption can be reached. Problems causing inaccuracy include particulates in sample that scatter radiation or other species that react with analytes (Harvey, 2016). The equipment manual states a wavelength accuracy of ± 0.5 nm.

Procedure For UV-vis measurements, a double-beam spectrometer Specord 205 with a spec- tral bandwidth of 1.4 nm was used. The device was controlled and analyzed by WinASPECT software. The measurement equipment was always started one hour before sampling to let the power output stabilize. The blank and the sample cuvette containing 2.5 ml background and sample solution, respectively, were placed into the measurement equipment. RBBR concentration in the filtrates during dyed- xylan preparation was measured in plastic cuvettes at the local absorption maximum around 594 nm. The calibation curve for dyed xylan can be found in appendix fig- ureA.1. Lignin concentration in the filtrates of the filtration study experiments was measured in a quartz cuvette at the local absorption maximum around 288 nm. In this case, the same sample cuvette was used for all measurements to minimize the errors. The calibation curve of lignin can be found in appendix figure C.1.

3.5.3 High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC) is used to identify, and quantify different components in a mixture. In HPLC a pressurized liquid phase that contains the sample passes through a column filled with a solid adsorbent material (station- ary phase). The sample components are identified and separated because different components show different flow rates through the column. This is explained by each component showing different physical interactions with the adsorbent material (e.g. hydrophobic, dipole-dipole and ionic interactions). The sampler transfers the sampling mixture to the mobile stream and into the column while pumps generate the desired flow. The detector in turn generates a signal, which is proportional to the sample content. Most detectors are based on UV/Vis, photodiode array (PDA) or mass spectrometry.

Procedure For the HPLC samples a High-Performance Anion Exchange Chromatography with pulsed Amperometric Detection was used. Before conducting HPLC, 0.2 g of oven- dry samples were hydrolyzed according to the Klason method as presented by The- ander & Westerlund(1986). After hydrolysis, the sample was filtered using a 0.45 µm PVDF filter and the solid residue (i.e. Klason lignin) was measured gravimet- rically. The hydrolyzate was then diluted to 100 ml in a volumetric flask. Next, this solution was again 10 times or 30 times diluted for filter cake samples and for

29 3. Experiments dyed-xylan synthesis samples and 0.4 vol% of 200 mg/l frucose solution was added in each sample as an internal standard. This solution was analyzed with HPLC.

3.5.4 NMR Spectroscopy Nuclear magnetic resonance (NMR) spectroscopy is used to investigate molecular structures. It is based upon the magnetic spin of atomic nuclei with an uneven number of protons or neutrons such as 1H and 13C. If subjected to a magnetic filed, this quantum mechanical property gives a nuclei a characteristic resonance frequency. The resonance frequency is defined as the characteristic frequency where transition between nuclear energy levels takes place is the nuclei is exposed to electromagnetic radiation. Since the resonance frequency is characteristic for the atomic nuclei it allows NMR to be tuned to a selected isotope. Furthermore, the resonance frequency is also affected by the chemical environment of the atom, as the chemical environment may shield the nuclei from the externally applied magnetic field. Care- ful analysis of NMR spectra therefore allows the identification of functional groups within a molecule (Lampman et al. , 2001).

Procedure Two samples were investigated with NMR for detection of the ether bond between xylan and RBBR. The first sample was a dyed-xylan precipitate, obtained just after synthesis and washing. The second sample was a dyed-xylan precipitate after undergoing a DR experiment (in this case no lignin was present). As NMR relies on pure samples, salt was removed from both precipitates with the aid of dialysis tubing. The cut-off of the dialysis tubing was 10-12 µm. Distilled water was used as buffer solution and changed regularly over the course of a weak. Dialysis was considered complete when the conductivity of the buffer solution was approaching that of distilled water. The remaining content in the dialysis tubing was set to dry at 40 °C over several days. Deuterium oxide was used as the NMR solvent, where sodium deuteroxide (NaOD 40% w/w solution in D2O from Sigma) was added to enable sufficient dissolution of the samples. NMR was performed on 1H and 13C isotopes. The 13C and 1H NMR spectra of dyed xylan were analysed with respect to a NMR data set on beechwood xylan (Daus et al. , 2011), which is depicted in the appendix figure A.2. The 13C and 1H NMR spectra for RBBR were predicted by an automated software, see (Engineering for Institute of Chemical Science (Lausanne), 2016).

3.5.5 Focused Beam Reﬂectance Measurements Focused Beam Reﬂectance Measurement (FBRM) enables in-situ observation of particle formation at relatively high solid concentrations. The technique relies on a laser beam that is directed down a probe and focused in the medium to be analyzed, close to the probe tip. High speed optics allow rotation of the beam around an axis parallel to the probe so that the laser beam traces a circular path. Passing particles in front of the probe are thereby be scanned by the laser beam. The backscattered

30 3. Experiments light is detected by the probe, enabling the tracing of a chord along the particle silhouette. By noting the duration of the reflection the FBRM can deduce the chord length. FBRM provide a chord length distribution (CLD) when it is immersed in a flowing suspension of particles. The CLD is a reproducible characteristic of the particulate system and its behavior over time enables observation of process dy- namic changes, related to the particle formation process. Important to note is that CLD is different but related to the particle size distribution (PSD) as chords can be measured from every aspect of the particle. If a certain particle shape is assumed, the relationship between CLD and PSD can be evaluated with statistical methods but would require rather elaborate data processing and is not always successful (Li & Wilkinson, 2005)(Wynn, 2003).

Procedure Mettler-Toledo Focused Beam Measurement G400 probe was used for measurements with a detection range from 1 µm to 1000 µm in chord length. The probe was positioned at an angle around 60° with the vertical. Attention was payed to the exact positions of stirrer, buffles and probe to keep the set-up for the different experiments as constant as possible in order to achieve comparable FBRM results. For the precipitation study, measurements were performed in-situ over the entire course of the titration. For the filtration study, the probe was immersed into the solution after the pH had been lowered below pH 10 to avoid possible damage to the probe at employed elevated temperatures of 80 °C.

3.5.6 Laser Diffraction Analysis Laser diffraction analysis (LDA) is used for determination of particle size. LDA makes use of a laser beam, which is passed through a suspension, containing the particles to be analyzed. A detector on the opposite side measures the distribution of scattered light intensity. The data is processed to calculate the particle size distribution (PSD) of the solids in the solution, based on the Frauenhoer theory. The Frauenhofer diffraction theory relates the particle size to the angular variation in intensity of light scattered by the particle. As the Frauenhofer theory becomes invalid if the particle diameter approaches the wavelength of light, the detectable particle size by the measurement method is naturally limited around 0.007 µm . Furthermore, as the theory assumes spherical particles, LDA results decrease in accuracy the more the particle shape deviates from the spherical ideal. Finally, since the diffracted light needs to reach the detector, LDA requires highly diluted conditions. (McCave, 1986)

Procedure LDA was performed on a Malvern Instruments Mastersizer 2000 range for particles between 0.02 µm and 2000 µm in size. Obtained ﬁltrates and slurries were inserted directly, while ﬁlter cakes were crushed and suspended in background solution beforehand. The pH of the background solution was adjusted to match the respective sample in order to avoid dissolution and limit particle re-arrangement. Each sample

31 3. Experiments was measured once after insertion and again after the application of ultrasound for 1 minute at 75% of the maximum ultrasound intensity of the device.

3.5.7 BET Analysis BET measurements are used for determining the surface area of porous media. The measurement method relies on the phenomenon of physical adsorption of gases on solid surfaces. The speciﬁc surface area of the sample is then determined by relating it to the isotherm of the gas, i.e. the amount of gas adsorbed as a function of its pressure. The measurement method relies on the BET theory, which predicts adsorption isotherms for multi-layered gas adsorption (Brunauer et al. , 1938). However, that means that the analysis technique neglects micropores, since the small pore width (typically less than 20 Å) prevents proper layer adsorption due to ﬁlling of the pore. Therefore, BET measurements do not represent a real internal surface area, but a characteristic BET surface area.

Procedure Filter cakes were air-dried prior to the BET measurements. BET surface area measurements were performed on all samples using a Micrometrics Tristar 3000 with nitrogen as the adsorption gas.

32 4 Results and Discussion

4.1 Preparation of Dyed Xylan

4.1.1 Synthesis Yield

The dyed xylan synthesis yield was observed to be higher for washing method 2; however, also more salt was present in the synthesis product. Figure 4.1 shows the synthesis yield (%) of dyed xylan for different washing methods. It shows higher synthesis yields (70-93 %) for washing method 2 (dark grey columns) compared to washing method 1 (light grey columns). Most likely, the longer precipitation time in washing method 2 has lead to a higher precipitation yield. The precipitation time in washing method 2 was 1 night while in washing method 1 it was precipitated directly after the synthesis. Furthermore, the acidification step prior to washing (see washing method 2) simplifies the experimental procedure since it could be filtrated afterwards while evaporation had to be used for the first washing method. Another aspect shown in figure 4.1 is that the columns for S2B2 and S3 show around 90% synthesis yield while S2B1 shows a significantly lower synthesis yield around 70 %. In the former two cases (S2B2, S3) there was only ethanol (no water) used as the washing liquid. The dye makes xylan more hydrophilic and thus less dyed-xylan was lost when washing with ethanol only. However, washing with ethanol has the drawback of less salt being washed out resulting in less purity of the final precipitate. Therefore, varying salt concentrations were found within the different washed precipitates as depicted in table A.1 due to different washing methods and differing washing liquids used. The salt concentrations in the precipitates were estimated by a mass balance based on the xylan concentration from HPLC. The HPLC results were confirmed by the dilaysis tubing experiment1, in the course of NMR sample preparation. To summarize, pure ethanol might be prefered as a washing liquid in RBBR xylan washes since it leads to higher xylan yields even though more salt was present in the precipitate. Furthermore, longer precipitation time also increases the precipitation yield and acidification prior to washing is preferred for practical reasons.

1Salt was diffusing out of the tube whereas xylan was retained due to the cut-off of 10-12 µm. The resulting weight ratio of initial precipitate in the tubing (xylan + salt) vs. final material in the tubing (pure xylan) corresponded to the measured HPLC result.

33 4. Results and Discussion

Figure 4.1: Synthesis yield of dyed xylan (%) for the diﬀerent synthesis batches. The light grey displays synthesis batches precipitated/ washed with washing method 1 while the dark grey displays synthesis batches precipitated/ washed by washing method 2.

4.1.2 Substitution Degree

The results on the substitution degree (calculated as in Appendix equation refeq:sd) indicate that the substitution degree increases with reaction time. Figure 4.2 shows the substitution degree of dye molecules per xylan molecule. The substitution degree of S1 is slightly below 3 and for S2 and S3 it is slightly above 2. The substitution degree of S1 is most likely higher due to longer reaction time (3 hours) compared to S2 and S3 (1 hour). On average 2-3 dye molecules were attached to one xylan molecule, assuming that all unreacted dye was washed out.

Figure 4.2: Substitution degree of dye molecules per xylan molecule for the diﬀer- ent syntheses.

34 4. Results and Discussion

4.1.3 Bond between Xylan and Dye Several analysis methods indicated that a bond has been established between RBBR and xylan. Firstly, the synthesis product showed a clear blue color after the washing was completed (i.e. no RBBR was detected by UV-vis in the filtrate). Secondly, the dialysis tubing experiments also indicated that a bond was established between RBBR and xylan. The cut-off of the dialysis tubing would have allowed the leaching of single RBBR molecules into the buffer solution while dyed- xylan molecules were retained due to their size. A bond between xylan and RBBR was indicated since the liquid inside the dialysis tube stayed blue while the buffer solution stayed colorless over the entire salt leaching period. NMR was conducted for an ultimate proof of bond between xylan and dye. However, carbon as well as hydrogen spectra could not give an ultimate proof. This is possibly a matter of detection limitations of NMR since low concentrations of dye on xylan might lead to an absence of peaks characteristic for RBBR. The 13C and 1H NMR spectra for dX, dX after DR as well as for xylan and RBBR are discussed in appendix A. The bond stability between the dye and xylan seems to suffer under the applied DR experiment conditions of the filtration study, as indicated by the dialysis tubing experiments. In the dialysis tubing experiment of dX after the synthesis, no dye was leached out. Contrarily, for the dialysis tubing experiment of dX after DR, a substantial amount of dye was leached out while a lot of xylan was retained. The substitution degree of RBBR on one xylan molecule for the dX after DR was recalculated using dialysis tubing masses and UV-vis data (see A.1). The recalculated substitution degree of the dX after the DR experiment was decreased by a factor of 5.2 with respect to the substitution degree after synthesis. Since the difference in substitution degree is quite substantial, dX indicates poor bond stability of the ether bond in dyed-xylan under the harsh conditions of DR. However, in the real lignin-dyed xylan experiment the lignin might buffer the effect of the harsh reaction conditions and “protect” the dyed xylan. Therefore, higher bond stability might be expected in the experiments where dX is dissolved and re-precipititated together with lignin. In the highly diluted suspension (used in experiment) dX becomes much more vulnerable to the possible degradation reactions compared to the real L-dX case with much more concentrated suspensions.

35 4. Results and Discussion

4.2 Precipitation Study

The precipitation experiments are speciﬁed and summarized in table 4.1, showing investigated substances, onset pH, ﬁnal pH, salt content, temperature as well as the analysis techniques applied. The L experiment was performed twice to investigate reproducibility of results.

Table 4.1: Speciﬁcations of precipitation experiments in the alkaline regime.

Substance Onset pH Final pH Salt Content Temperature Analysis Technique wt% [°C] L-X 9.17 8.87 5.9 45 FBRM L 9.20 8.50 5.8 45 FBRM L 9.09 8.59 5.8 45 FBRM

4.2.1 Particle Formation Dynamics

The FBRM study of L and L-X precipitation experiments indicates similar particle formation dynamics in the alkaline regime. Figure 4.3 and figure 4.4 show the FBRM results of L and L-X, respectively. The figures show the recorded counts as a function of time for different chord length as well as the titration data as a function of time. The FBRM data of the L replicate and its titration data can be found in appendix B, figure B.1. The replicate showed the same behaviors as the other L experiment, which strengthen the experimental validity of the results. When comparing figures 4.3 and 4.4, it is observed that L and L-X have a similar aggregation onset pH and both experiments display the same particle formation dynamics in the alkaline regime. After the onset aggregation, a CLD equilibrium is reached relatively fast. The measured onset pH of L-X at pH 9.17 lies within the onset pH of the two L experiments and thus the onset pH is assumed to be the same for L and L-X. The average onset pH of L, L replicate and L-X experiments was calculated to be around pH 9.15. The observed similarities between L and L-X particle formation dynamics might result from xylan being still soluble at such high pHs. This would imply that the observed aggregation onset pH and particle formation dynamics reflect only lignin aggregation/ precipitation mechanisms and thus would be similar between L and L-X experiments. This hypothesis could be further investigated with FBRM by monitoring step-by-step titration into a strongly acidic pH regime for which the precipitation of xylan can not be questioned.

36 4. Results and Discussion

Figure 4.3: FBRM results of the L-X experiment depicting counts as a function of time for diﬀerent chord length (primary axis). The secondary axis shows the titration data of the experiment (amount of acid added or pH).

Figure 4.4: FRBM results of the L experiment depicting counts as a function of time for diﬀerent chord length (primary axis). The secondary axis shows the titration data of the experiment (amount of acid added or pH).

Figure 4.5 shows similar titration data of L and L-X experiments. However, in the L-X experiment slightly more acid was consumed. This can be explained by higher concentration in buﬀering substance due to the addition of xylan in the L-X experiment.

37 4. Results and Discussion

Figure 4.5: Titration data of L, L replicate and L-X precipitation experiments depicting pH as a function of H2SO4 added

The FBRM study in the alkaline precipitation regime indicates similar equilibrium CLDs for both the L-X and the L experiments. Figure 4.6 shows the normalized CLD at the apparent steady state (100 min after onset), depicting recorded counts as a function of chord length for L-X, L and L replicate precipitation study experiments. It can be seen that the L and L replicate experiment follow the same CLD while L-X shows a similar CLD. All precipitation experiments have maximum counts of cord length in the range of 5-10 µm. Chord length higher than 50 µm were not detected. The absolute CLD at the apparent steady state for L, L replicate and L-X is found in figure 4.7. It also shows the same trend in the final CLD but lower number of recorded counts for the L-X experiment. The smaller number of recorded counts for L-X could be explained by a difference in final pH. The final pH was higher in the L-X compared to L and L replicate experiment. A higher final pH suggests less precipitation, which leads to a lower concentration of agglomerates and thus a lower amount of counts measured by FBRM. The replicate of L showed the same CLD as the L experiment, which strengthens the reproducibility of the results.

38 4. Results and Discussion

Figure 4.6: Normalized chord length distribution (at 100 min after onset precipitation) depicting counts as a function of chord length (log-scale) for L and L-X precipitation experiments

Figure 4.7: Absolute chord length distribution (at 100 min after onset precipitation) depicting counts as a function of chord length (log-scale) for L and L-X precipitation experiments

In general, the precipitation study in the alkaline regime indicated that the L-X and L behaves relatively similar in terms of particle formation dynamics, onset pH and size distributions at least under these speciﬁc experimental conditions (low salt concentration, 45 °C). Conclusively, the results suggest that there is no reason to believe that presence of xylan in small amounts (5 wt% of total xylan and lignin weight) inﬂuences precipitation mechanisms of KL in the alkaline regime. In order to strengthen this conclusion, precipitation in the alkaline regime could be executed for lignin-xylan and lignin only experiments at 80 °C as temperature might change the results.

4.2.2 Filtration Properties The filtration properties of the precipitation experiments could not be determined due to the inability to filter any of the precipitation experiments. Both, filter media

39 4. Results and Discussion of 0.45 µm cut off and 2.5 µm cut off filter were plugged immediately when starting filtration. This indicates that smaller particles or partially dissolved matter, that was not detected by the FBRM, have plugged the filter medium and the filter cake.

4.3 Filtration Study

Information on the filtration experiments is provided in table 4.2, showing the investigated substances, experimental conditions and analysis techniques applied. The calculated precipitation yield for the DR experiments varied between 98-106 %. A correlation between the salt concentration and the yield could be observed, where the highest salt concentrations corresponds to the highest yield. However, this correlation is a result of the assumption that all salt is washed out during filtration. All results on precipitation yield and salt concentration of filtration study experiments can be found in the appendix, table C.1.

40 4. Results and Discussion

Table 4.2: Speciﬁcations of ﬁltration experiments precipitated in the acidic regime.

Substance Precipitation pH Salt Content Temperature Analysis Technique wt% [°C] Mixing (M) Experiments

L-dX 3.92 1.5 80 Filtration, HPLC, UV-Vis, LD L-dX 3.71 1.5 25 -

L-X 3.88 1.5 80 Filtration

Dissolution/ Re-precipitation (DR) Experiments

L-dX 4.04 4.4 80 Filtration, HPLC, UV-Vis, LD L-dX 4.31 4.7 80 Filtration, UV-Vis, LD, FBRM, BET L-dX 2.89 4.3 80 Filtration, HPLC, UV-Vis, LD L-X 4.25 5.1 80 Filtration, UV-Vis, LD, FBRM, BET L-X 2.97 4.2 80 Filtration, HPLC, UV-Vis, LD, BET L 6.15 4.4 80 Filtration, UV-Vis, LD L 4.19 4.3 80 Filtration, UV-Vis, LD, BET L 5.95 4.9 80 Filtration, UV-Vis, LD, FBRM L 4.07 5.6 80 Filtration, UV-Vis, LD, FBRM, BET dX 2.85 3.1 80 Filtration, UV-Vis, LD

4.3.1 Interaction between Lignin and Xylan The DR in comparison with the M experiments showed that the slurries diﬀer substantially for the two procedures. Figure 4.8 shows pictures of the slurries of DR and M experiments for L-dX experiments. For the DR experiments a brownish supernatant above the solid brown phase in the bottom can be seen, while for the M experiments a blue supernatant is visible above the solid brown phase in the bottom. The blue colour of the supernatant originates from dyed xylan being in the aqueous phase. The eﬀect is possibly enhanced by the use of dyed xylan compared to xylan without dye, as the dye makes xylan more hydrophilic. A possible explanation

41 4. Results and Discussion for this is that xylan did not fully interact with lignin in the M experiment, since the compounds are simply mixed together. On the contrary, the complete brown colour of the supernatant in the DR experiment, suggests an increased interaction between xylan and lignin during joint re-precipitation so that xylan is incorporated in lignin particles. This tentative explanation is discussed further using HPLC data see chapter 4.3.3.1.

(a) DR experiment (b) M Experiment

Figure 4.8: Slurries of ﬁltration experiments: (a) DR experiment of L-dX, pH 4.04, 80 °C; (b) M experiment of L-dX, pH 3.92, 80 °C. Note that the proportion between solid and supernatant is not representative.

The visual observation of the filtrates shows that decreasing pH leads to more lignin precipitation which is also supported by literature (see chapter 2.1.1). Figure 4.9 shows pictures of the filtrates of the DR experiments with L-dX and L slurries. It is observed that the brownish color of the filtrates gets weaker as the pH decreases. Lignin is mainly responsible for this brown-yellowish color and thus the lighter the filtrate, the lower the lignin content. This last observation is supported by the determination of the lignin concentrations in filtrates using UV-vis, as shown in figure 4.10. The associate calibration curve for lignin concentration versus absorption can be found in the appendix, figure C.1.

42 4. Results and Discussion

(a) L, pH 6.3 (b) L, pH 4.19

Figure 4.9: Filtrates of DR experiments at diﬀerent precipitation pH’s

Figure 4.10: Lignin concentration in ﬁltrates for DR experiments, determined by UV-vis as function of precipitation pH.

43 4. Results and Discussion

4.3.2 Filtration Properties

The filtration properties of low ionic strength slurries show that increasing salt concentration in the slurry, result in a decrease in filtration resistance as well as an increase in solidosity. The trend becomes observable when looking at the filtration resistances of slurries as well as cake solidosities for experiments with similar precipitation pH, as shown in figure 4.11 (a) and (b), respectively. The results on the influence of ionic strength are in agreement with previous findings of Durruty (not published yet). Furthermore, figure 4.11 (a) and (b) shows a correlation between a low filtration resistance and a high solidosity. This relationship is a common phenomenon, because even though a high solidosity leaves less volume of the cake fraction available for fluid flow, it also involves less solid surface area which decreases fluid flow due to friction at the solid-liquid interface. Furthermore, it was found that the L slurry gave significantly lower filtration resistance and higher solidosity compared to filtration of L-dX slurries, as shown in figure 4.11. The same phenomenon was observed by Durruty (not published yet) for L, compared to L-X filter cakes. She proposed that xylan is sorbed at the surface of lignin particles, increasing the contact area between solid and liquid and making the particles structure more porous. A similar effect has been observed by others for different materials (Lee et al. , 2005), who attributed the increase of filtration resistance to an increase in fractal dimensions. Thus, this is worth of further investigation as it might provide the answer to the increased filtration resistance due to xylan addition. However, it has to be noted that the L slurry that features the lowest filtration resistance also involves the highest salt concentration. Therefore, the distinct influence of xylan on filtration properties is not provided by these data. Since the effect of ionic strength was underestimated when designing the experiments, more data points would be necessary, ideally with L and L-X slurries at the same pH and with equal ionic strengths.

44 4. Results and Discussion

(a) Filtration resistance of slurries with increasing salt content.

(b) Cake solidosity of slurries with increasing salt content.

Figure 4.11: Filtration properties for dissolution/ re-precipitation slurries at similar acidic precipitation pH.

Filtration of slurries from M experiments (M-L-dX 80 °C, M-L-dX 25 °C and M-L-X 80 °C) could not be completed due to plugging. L and L-X slurries, obtained by mixing below pH 3 were found to be easily ﬁlterable in a previous study (Durruty, not published). Therefore, it is suspected that mixing around pH 4, without prior dissolution results in insuﬃcient interaction between lignin and xylan. One possible

45 4. Results and Discussion explanation is, thus, that the fine xylan particles in the slurry cause plugging of the filter cake or filter medium. This conclusion is also in accordance with the visible observation of the mixed slurry (M-L-dX, 80 °C) as discussed in chapter 4.3.1.

4.3.3 Location of Xylan in Filter Cake

4.3.3.1 High Pressure Liquid Chromatography The distribution of xylan was found to be uniform throughout the filter cake of DR experiments, as shown by the HPLC results in figure 4.12. Two different filter cakes were analyzed, where samples were taken from different vertical positions in the filter cake. Small deviations were found to be within the experimental error range.

Figure 4.12: Xylan content in the filter cake along the filter cake height. Filter cakes from two DR experiments were analyzed. Samples were taken along 8 positions in the cake, where number 1 corresponds to the top layer, number 8 to the bottom layer. Sample number 8 of experiment L-X, pH 2.97 was analyzed three times by HPLC to obtain a confidence interval of 0.19 wt% for the involved measurement error.

The average xylan content in the filter cake was found to vary substantially between DR and M experiments, as shown by the HPLC results in figure 4.13. Although the M experiment was not completely filterable due to plugging, a thin filter cake for HPLC analysis was obtained. For the M experiment, a lower xylan concentration in the filter cake was observed, which indicates less interaction between lignin and xylan compare to the case where the solids are dissolved and re-precipitated together. This result matches with the visible observation of the mixed slurry, as discussed in chapter 4.3.1. Figure 4.13 also shows that the DR experiments feature a similar xylan concentration. A slightly higher xylan concentration can be observed for experiments precipitated at a lower pH. This may be explained by the chemical structure of xylan, as the MeGlcA side groups are known to have a pKa around 3.5-4. Exper- iments that were precipitated to a pH around 3 are therefore expected to involve fully protonated MeGlcA groups, which is enhancing precipitation of xylan.

46 4. Results and Discussion

Figure 4.13: Average xylan content in ﬁlter cake for dissolution/ re-precipitation experiments at diﬀerent acidic precipitation pHs and a mixing experiment at 80 °C.

4.3.3.2 Confocal Fluorescent Microscopy

CFM was performed on pure L and pure dX filter cakes to identify the emission spectra of lignin and RBBR as the respective fluorophores. The normalized spectra can be seen in figure 4.14 and were used to define the collection ranges, which would enable a differentiation between lignin and RBBR. However, the spectra show that lignin and RBBR have rather broad emission ranges and therefore also fluoresce in the collection range of the respective other (although with a weaker intensity). Furthermore, the spectra show a very low emission intensity for both compounds, making it necessary for the CF microscope to operate at its lowest detection limit. The absolute emission spectra can be found in appendix figure C.2. The low emission intensity of dyed-xylan was unexpected as RBBR was chosen based on its strong fluorescent properties.

47 4. Results and Discussion

Figure 4.14: Normalized emission spectra for pure lignin and pure dyed xylan where 488 nm laser light was used for excitation. The respective collection ranges for the identiﬁcation of lignin and RBBR are highlighted in green and red, respectively.

The tracking of dX on lignin particles was attempted via colocalization analysis of an L-dX filter cake. The filter cake was probed for several hours but only two spots could be detected, where the collection range for RBBR was found to be predominant. One of these cases is shown in figure 4.15 where a small red dot can be seen in the middle of the colocalized image (figure 4.15 (b)).

48 4. Results and Discussion

(a) Collected at emission (b) Colocalization of all (c) Collected at emission range from 500-540 nm collected emission ranges. range from 620-722 nm

Figure 4.15: CEM images of the ﬁlter cake from experiment L-dX, pH 4.04. Im- ages were “pseudo-coloured” for better visualization of the diﬀerent collection wavelengths.

The red dot might be due to the presence of dX. However, HPLC results indicate that dX is evenly distributed through the filter cake, which implies that colocalization analysis should detect spots with predominant wavelengths in the collection range for RBBR through the entire filter cake. The measurement is therefore considered to be unsatisfying in tracking of dX on lignin particles, where possible reasons have been identified: • Overlaping emission spectra of lignin with low intensity of RBBR. • Low concentration of RBBR in the filter cake, since the concentration of dX is below 5 wt% in the filter cake. • Questionable bond stability between RBBR and xylan as discussed in chapter 4.1.3, which might result in an even lower RBBR concentration in the filter cake than assumed for the original dX starting material.

Based on the identified problems some recommendations for a refinement of the method can be given: • More favourable emission spectra by using another dye. • A higher RBBR concentration by increasing dX concentration (although that might eventually result in a different precipitation and filtration behavior). • Preserve bond stability between RBBR and xylan by performing DR experiments at milder conditions, for example at lower temperatures.

4.3.4 Particle Sizes and Shapes 4.3.4.1 Confocal Fluorescence Microscopy While CFM images were unsatisfying regarding the location of xylan in the filter cake, they could be used for analysis of particle sizes and shapes. Figure 4.16 shows more CFM pictures for comparing sizes and shapes of filtration experiments re- precipitated at acidic pHs. As can be seen in figure 4.16 the preparation methods of the samples have a large influence on appearance under the microscope. The

49 4. Results and Discussion expressed cake in figure (d) involves the highest dry content and does not allow the distinction of particle outlines. A wet filter cake (figure (a) and (b)) or re-suspension in acidic water of a matching pH (figure (c)) are therefore considered more suitable preparation methods for the observation of particle size and shapes. The observed particles in the different filter cakes seem to feature predominant particles between 1-10 µm in size with a few exceptions of larger particles. Furthermore, sample L, pH 6.15 in figure (b) appears to have smaller particles overall, most likely arising due to a higher precipitation pH, which results in less precipitated material. Furthermore, the particles appear more homogeneous in size and spherical in structure. In this experiment, less acid had been added (resulting in the higher pH), meaning that the driving force for precipitation was smaller. A possible explanation is that precipitation mechanisms took place over a longer time, allowing for homogenization of the suspension, compared to faster precipitation mechanisms that are expected to take place at lower pH’s.

50 4. Results and Discussion

(a) L, pH 4.19, non-expressed cake (b) L, pH 6.15, non-expressed cake

Figure 4.16: CEM images of ﬁlter cakes with L, pH 4.19 (a); L, pH 6.15 (b); and L-dX, pH 4.04 (c)(d). Samples underwent diﬀerent treatments beforehand. Images were obtained by collecting wavelengths in the range of 500-540 nm and 620-722 nm. Resolution corresponds to 10 µm, indicated by the white mark.

4.3.4.2 Focused Beam Reﬂectance Measurement

In the following, it is assumed that the particular L-X (pH 4.25) and L (pH 4.07) experiments are comparable as they show similar pHs and salt concentrations (i.e. L-X, 5.1 wt% and L, 5.6 wt%). Additional FBRM graphs for experiments L-dX and L at pH 5.95 are shown in the appendix ﬁgure C.3 and C.4. They show similar behavior to the L-X and L experiments represented here, respectively. The FBRM data shows similarities in the particle formation dynamics of L-X

51 4. Results and Discussion and L experiments, as well as a strong effect of temperature. Figure 4.17 shows FBRM data for L-X (a) and L (b) experiments. In the L-X (pH 4.25) (a) and L (pH 4.07) (b) experiments, the heating was switched off at 210 minutes and 110 minutes, respectively. A general observation for all experiments is that as soon as the temperature is switched off, the counts of smaller chord length (1-10 µm) increase while the larger chord length (10-100 µm) start to decrease (see figure 4.17). This trend might indicate that larger particles are less stable at lower temperatures and tend to decompose. About these results, literature is contradictory. However, Nogren et al. (2001) reported that increased temperatures favor aggregation of lignin as discussed in chapter 2.2.3 which is more in line with the results compared to the results of Zhu et al. (2015).

52 4. Results and Discussion

(a) L-X

(b) L

Figure 4.17: FBRM results depicting diﬀerent chord length as function of time for L-X, precipitation pH 4.25 (a) and L, precipitation pH 4.07 (b) for the ﬁltration study experiments. The heat was removed at 210 minutes for the L-X experiment (a) and at 110 min for the L experiment (b). 53 4. Results and Discussion

Furthermore, FBRM data showes differences in the CLDs between L-X and L experiments. Figure 4.18 shows a higher CLD for L-X (pH 4.25) compared to L (pH 4.07) experiments for both temperatures at 80 °C (a) and 25 °C(b). This indicates that at low pH the presence of xylan and lignin in the DR experiment tends to form larger particles compared to the DR experiment of lignin only. For this statement it is assumed that chord length can be associated with particle sizes in a relative comparisons. Nogren et al (2001) already suggested a destabilization effect in a colloidal suspension by inorganic anions and that resulting aggregation/precipitation, might be even more pronounced for organic anions (e.g. xylan). In addition, FBRM data indicates different responses to a change in temperature between L-X and L experiments, which can be seen in figure 4.18. At 25 °C the CLDs of L-X and L become more similar compared to the CLD at 80 °C; meaning that the particle sizes become more similar at lower temperatures. It might be hypothesized from this that L-X precipitates are less stable at lower temperatures compared to precipitates, consisting only of lignin.

54 4. Results and Discussion

(a) CLD at 80 °C

(b) CLD at 25 °C

Figure 4.18: CLD at 80 °C (before removing the heat) (a) and 25 °C (100 minutes after removing heat) (b) showing counts as function of chord length for L-dX at pH 4.31, L-X at pH 4.25 and L at pH 4.07 and L at pH 5.95 for the ﬁltration study experiments.

4.3.4.3 Laser Diffraction LDA of slurries that were precipitated to around pH 4, show a difference in number based PSD between L and L-dX, as can be seen in figure 4.19. The PSD was found to be roughly between 1-10 µm, which thereby corresponds with the observed particle size range of CFM images in figure 4.16. The most abundant particle size in LDA was found to be around a diameter of 1.66 µm for L-dX slurries, 1.6 µm for the L-X slurry and around 1.9 µm for the L slurry. The PSD of the L slurry appears to be shifted towards larger particles. However, pH and ionic strength influence precipitation and the discussed L slurry shows a relatively low pH and a high salt content compared to the other slurries. Therefore, from these results it cannot be certain that the PSD is influenced by the presence of xylan in the slurry or only by

55 4. Results and Discussion precipitation conditions. As already mentioned, it is not possible to directly compare PSD obtained with LDA and CLD obtained with FBRM as they are based on different measuring principles, with different detection limits and at different conditions. Nevertheless, it was unexpected that all slurries show a larger CLD (1-100 µ) compared to PSD (1-10 µ). This might be explained by the measurement conditions of LDA, which measures at room temperature and at highly diluted concentrations. Furthermore, it is observed that the L slurry (pH 4.07), showing the largest particles according to PSD exhibits the smallest chord lengths, compared to L-(d)X slurries. This might be explained by the hypothesized difference in thermal stability between L and L- (d)X particles. Following this hypothesis, L-(d)X particles would decompose more strongly compared to L particles since LDA was performed under highly diluted conditions and a few days after slurry preparation (thus L-(d)X particles had time to decompose/ rearrange at 25 °C).

Figure 4.19: Number based PSD for slurries of DR experiments. The measured duplicate is congruent with the ﬁrst measurement.

4.3.4.4 BET Analysis

BET measurements indicate an observable trend of a decrease in BET surface area with increasing ionic strength, as shown in ﬁgure 4.20.

56 4. Results and Discussion

Figure 4.20: BET surface area measurements on ﬁlter cake samples as a function of ionic strength in the slurry. Measurements were performed on air-dryed samples, using nitrogen as the adsorption gas.

. BET findings thereby also correspond with LDA as the sample with the largest particle sizes (L, pH 4.07, salt content = 5-6 wt%) was found to yield the lowest BET surface area. Furthermore, the results also correspond with results on filtration properties as they show a decrease in filtration resistance for increasing salt concentration that can be related to a decrease in surface area. BET measurements indicate no trend in surface area due to the presence of xylan. However, differences between L and L-X might be reduced by the preparation method of BET samples, which relied on air-dried samples. Air drying is expected to induce a collapse of organic porous agglomerates due to an increase of water capillary forces during evaporation. The measured BET surface area is therefore expected to be an underestimation compared to actual surface area of particles in suspension. BET measurement on freeze-dryed samples might therefore give more accurate results (Durruty, 2014) and be more representative for a potential influence of xylan. Regarding the sensitivity of BET analysis one should also bear in mind that micropores are neglected. An increase in ruggedness of particles due to xylan addition might therefore not be detectable with BET measurements.

4.4 Comparison between Precipitation and Fil- tration Study

The CLDs of the precipitation (P) and filtration (F) study experiments can be compared for L and L-X using the respective FBRM data as shown in figure 4.21. However, the comparison can only be taken as an indication since experimental conditions differ between the P and F study, as shown in table 4.3. As can observed in this table, the salt concentrations of the P experiments are relatively similar but slightly higher compared to F experiments. What differs clearly is the temperature of

57 4. Results and Discussion

45 °C for P experiments and 80 °C for F experiments as well as the final precipitation pH, which is in the alkaline regime (pH 8.5-8.87) for P experiments and in the acidic regime (pH 4.07-5.59) for F experiments. The FBRM data for F experiments showed a clear temperature effect on the particle sizes, where higher temperatures favor formation of larger particles. Thus, for comparing the CLDs of P and F, the temperature difference between the experiments was minimized by using F CLDs at 25 °C and P CLDs at 45 °C. Table 4.3: Specifications of precipitation (alkaline regime) and filtration (acidic regime) study experiments experiments

Substance Precipitation pH Salt Content Temperature wt% [°C] Precipitation Experiments L-X 8.87 5.9 45 L 8.50 5.8 45 L 8.59 5.8 45 Filtration Experiments L-dX 4.31 4.7 80 L-X 4.25 5.1 80 L 5.95 4.9 80 L 4.07 5.6 80

Figure 4.21 compares the CLDs of filtration experiments at 25 °C with CLDs of P experiments at 45 °C, where (a) combines L-X experiments and (b) combines L experiments of both studies. It can be seen that all CLD curves of F experiments are shifted to higher chord length compared to P experiments, which can be related to an increase in particles size for lower precipitation pH. The increase in particle size for F experiments can be explained by the fact that low precipitation pH favors precipitation. Another feature shown in figure 4.21 is that the width of the CLD curve for the P experiments (alkaline regime) is more narrow compared to the CLD curve of the F experiments. The shape of the CLD curve is closely related to PSD and particle shape. Therefore, it might be concluded that particle shapes and PSD are more homogenous in the P experiments compared to particles formed in the F study. This would be reasonable because the acid addition in the precipitation study was executed over hours, which provides time for the system to equlibriate and form particles more homogeneously. In contrast, in the F study it was precipitated in the acidic regime and acid was added at once which leads to a stronger driving force for precipitation and faster formation of particles. The lignin CLD shows an unexpected CLD for the F study experiment precipitated at pH 4.07 (see figure 4.21 (b)). The CLD of the F study experiment at pH 4.07 shows more similarities in counts and width of the CLD curve with the P experiments, compared with the CLD of other F experiments. Contrary to the expectations, the pH 5.95 experiment also shows higher maximum chord lengths. In order to be conclusive about these results, the experiment should be repeated to

58 4. Results and Discussion exclude experimental and measurement errors. However, a possible explanation for the unexpected CLD of the F experiment at pH 4.07 might be that this particular experiment stayed for approx. 20 minutes just below the onset pH (around pH 8) and was then acidiﬁed down to pH 4.07. It might be that nuclei have already formed that are below the FBRM detection limit. Further, acidiﬁcation from pH 8 to pH 4.07 therefore led to the formation of more particles (higher counts), leaving less material for the formation of big particles (lower cord lengths), compared to other F experiments. Since P experiments were also kept around the onset pH for hours, they had time to homogenize and form many nuclei, yielding a CLD similar to the discussed F experiment, pH 4.07.

(a) L-X experiments for ﬁltration (F) and precipitation (P) study

(b) L experiments for ﬁltration (F) and precipitation (P) study

Figure 4.21: CLD depicting counts as a function of chord length (log-scale) of L-X (a) and L (b) experiments for the ﬁltration study (F) at 25 °C (100 min. after removing the heat) and the precipitation study (P) at 45 °C (100 min. after onset precipitation).

In short, higher chord lengths were observed for F compared to P study experiments, which is attributed to lower pHs in F experiments. It might also be concluded that formation of particles and PSD is more homogeneous when precipitating in the alkaline regime (P study) compared to precipitation in the acidic regime (F study), which is supported by the discussion of CFM pictures see chapter 4.3.4.1.

59 4. Results and Discussion

60 5 Conclusion

Preparation of dyed Xylan

• The preparation method of dyed xylan was successful and a bond between Remazol Brilliant Blue R (i.e. dye) and xylan could be established, as supported by ﬁltration and dialysis tubing experiments. The substitution degree of dye on xylan was calculated to be 2-3 dye molecules per xylan molecule, based on HPLC results. However, the bond stability between dye and xylan is suspected to suﬀer under experimental conditions (precipitation from pH 12 to around pH 4, at 80 °C) as indicated by dialysis tubing experiment. • In the washing step, after the synthesis of dyed xylan, washing with pure ethanol showed highest precipitation yields up to 93% but also led to less salt being washed out, compared to washing with an ethanol/ water solution.

Precipitation Study

• The FBRM results indicate an onset pH for lignin-xylan and lignin experiments around pH 9.15 for low ionic strength solutions, precipitated at 45 °C. • The FBRM suggest that there is no reason to believe that presence of xylan in small amounts (5 wt% of total xylan and lignin weight) inﬂuences precipitation mechanisms of KL in the alkaline regime.

Filtration Study

• At the low ionic strengths of the slurries (4.2-5.6 wt%), deviations in salt concentrations showed a strong influence on filtration properties and particle size. It was observed that a higher salt concentration results in a lower filtration resistance, a lower solidosity, a lower BET surface area, as well as larger particles when measured with laser diffraction. • Lignin-(dyed)xylan experiments show higher filtration resistance but lower solidosity compared to lignin only experiments. However, the degree of xylan influence on filtration properties could not be studied in detail as ionic strength varied between different experiments. • Xylan showed a stronger interaction with lignin particles when both solids were dissolved and re-precipitated under acidic conditions, compared to the case where the solids were mixed without prior alkaline dissolution.

61 5. Conclusion

• HPLC measurements showed that xylan was evenly distributed through the entire filter cake when xylan and lignin were dissolved and re-precipitated in the acidic regime before filtration. • Confocal microscope pictures and laser diffraction measurements indicate a particle size distribution between 1-10 µm sized particles. • FBRM results indicated a clear influence of temperature on the particle chord length distribution in the acidic precipitation regime. A temperature change from 80 °C to 25 °C favored simultaneous formation of smaller particles (1- 10 µm) and decomposition of bigger particles (10-100 µm) for lignin as well as lignin-(dyed)xylan experiments. It was observed that lignin-(dyed)xylan particle responded more strongly to a change in temperature, leading to the hypothesis of a lower thermal stability of particles, when xylan was added to lignin. • Dyed xylan could not be tracked within the lignin particles, using a confocal fluorescence microscope. The main reasons for this was found in the autoflu- oresence of lignin, low emission intensity of dyed xylan, as well as in the low concentration of xylan within the filter cake.

62 6 Recommendations

• The particle size distributions, shapes and sizes of lignin-xylan compared to lignin should be further investigated as they might explain the filtration resistance increase due to xylan addition. The precipitation pH and salt concentration should be kept constant to be conclusive about difference in sizes, shapes, and particle size distribution. • The filter cakes obtained after filtration of lignin and lignin-xylan slurries could be analyzed using scanning electron microscopy. Thereby, a potential difference in surface properties of lignin-xylan and lignin filter cakes could be investigated. • The BET surface area could be investigated on freeze-dried samples since air- dried samples are expected to show a lower surface area compared to particles in suspension due to collapsing of structures. Measurements on freeze-dryed samples might thus be more representative and also more sensitive to a potential influence of xylan. • The confocal fluorescence microscopy measurements for tracking of xylan within the lignin particles might be worth repeating. However, a much higher emission intensity for dyed xylan should be reached, e.g. by using a different dye. • The influence of temperature on chord length distribution of lignin compared to lignin-xylan could be investigated over a longer time period to test the hypothesis of a lower thermal stability for lignin-xylan particles.

63 6. Recommendations

64 Bibliography

Amarasekara, Ananda S. 2013. Separation and Uses of Lignin. Pages 357–380 of: Handb. Cellul. Ethanol. John Wiley & Sons, Inc.

Bandt, Christoph, Falconer, Kenneth, & Zähle, Martina. 2015. Fractal Geometry and Stochastics. Progress in Probability, vol. 70. Cham: Springer International Publishing.

Brännvall, Elisabeth. 2004. Overview of pulp and paper processes. Chap. 18 of: Wood Chem. Stockholm: Fiber and Polymer Technology, KTH.

Brewster, J. 2007. Economic impact of modern kraft recovery boilers. In: Int. Chem. Recover. Conf.

Brunauer, Stephen, Emmett, P. H., & Teller, Edward. 1938. Adsorption of Gases in Multimolecular Layers. J. Am. Chem. Soc., 60(2), 309–319.

Casey, J. P. 1980. Pulp and Paper: Chemistry and Chemical Technology. New York: John Wiley and Sons.

Choi, Joon Weon, Choi, Don-Ha, & Faix, Oskar. 2007. Characterization of lignin- carbohydrate linkages in the residual lignins isolated from chemical pulps of spruce (Picea abies) and beech wood (Fagus sylvatica). J. Wood Sci., 53(4), 309–313.

Cornea, Anda, & Conn, P Michael. 2014. Fluorescence Microscopy: Super- Resolution and other Novel Techniques. Elsevier.

Daus, Stephan, Petzold-Welcke, Katrin, Kötteritzsch, Manuela, Baumgaertel, Anja, Schubert, Ulrich S., & Heinze, Thomas. 2011. Homogeneous sulfation of xylan from diﬀerent sources. Macromol. Mater. Eng., 296(6), 551–561.

Deng, Weiping, Zhang, Hongxi, Xue, Laiqi, Zhang, Qinghong, & Wang, Ye. 2015. Selective activation of the C-O bonds in lignocellulosic biomass for the eﬃcient production of chemicals. Cuihua Xuebao/Chinese J. Catal., 36(9), 1440–1460.

Du, Xueyu. 2013. Deepening the insights of lignin structure : Lignin-carbohydrate complex (LCC) fractionation and characterization and Kraft lignin amination. KTH, Träkemi och massateknologi.

Durruty, Julie. 2014. Studies on LignoBoost lignin and the inﬂuence of ionic strength.

65 Bibliography

Ebringerová, A., Hromádková, Z., & Heinze, Th. 2005. Hemicellulose. Adv. Sci, Polym., 128(1-68).

Ek, Monica, Gellerstedt, Göran, & Henriksson, Gunnar. 2009. Pulp and Paper Chemistry and Technology Volume 2. Pulping Chemistry and Technology. Vol. 2.

Engineering for Institute of Chemical Science (Lausanne). 2016. Tools for NMR Spectroscopists.

Evans, Fennell, & Fendler, Janos H. 1994. The colloidal domain: Where physics, chemistry, biology, and technology meet.

Flory, Paul J. 1954. Principles of Polymer Chemistry. New York: Cornell Univ. Press.

Garver, T. M., & Callaghan, P. T. 1991. Hydrodynamics of kraft lignins. Macro- molecules, 24(2), 420–430.

Gellerstedt, Göran. 2004. Chemistry of pulping. Chap. 20 of: Cellul. Technol. Stockholm: Fiber and Polymer Technology, KTH.

Gierer, Josef, & Wännström, Sune. 1986. Formation of Ether Bonds between Lignins and Carbohydrates during Alkaline Pulping Processes. Holzforschung, 40(6), 347– 352.

Goring, D. A. I. 1962. The physical chemistry of lignin. Pure Appl. Chem., 5(1-2).

Green, J., Pearl, I., Hardacker, K., Andrews, B., & Haigh, F. 1977. The peeling reaction in alkaline pulping. Tappi 60, 10(120 - 125).

Harvey, D. 2016. UV/Vis and IR Spectroscopy.

Hilpmann, G., Becher, N., Pahner, F. A., Kusema, B., M??ki-Arvela, P., Lange, R., Murzin, D. Yu, & Salmi, T. 2014. Acid hydrolysis of xylan. Catal. Today, 259, 376–380.

Höglund, E., & Otterbeck, H. 2004. Biobränslet Lignin. Deﬁnition och analys av potentiell marknad. Ph.D. thesis, Royal Institute of Technology.

Holdich, Richard G. 2002. 04: Filtration of Liquids. Fundam. Part. Technol., 29–44.

Iversen, Tommy, & Wännström, Sune. 1986. Lignin-Carbohydrate Bonds in a Resid- ual Lignin Isolated from Pine Kraft Pulp. Holzforschung, 40(1), 19–22.

Jönsson, Ann Soﬁ, & Wallberg, Ola. 2009. Cost estimates of kraft lignin recovery by ultraﬁltration. Desalination, 237(1-3), 254–267.

Kadla, John F, Kubo, Satoshi, Gilbert, Richard D, & Venditti, Richard A. 2002. Chemical Modiﬁcation, Properties, and Usage of Lignin. Boston, MA: Springer US.

66 Bibliography

Kalogiannis, Konstantinos G., Stefanidis, Stylianos D., Michailof, Chrysoula M., Lappas, Angelos A., & Sjöholm, Elisabeth. 2015. Pyrolysis of lignin with 2DGC quantiﬁcation of lignin oil: Eﬀect of lignin type, process temperature and ZSM-5 in situ upgrading. J. Anal. Appl. Pyrolysis, 115(August), 410–418.

Kamm, Birgit, & Gruber, Patrick R. 2006. Handbook of Fuels Beyond Oil and Gas : The Methanol Economy Bailey ’ s Industrial Oil and Fat Products Oil Reﬁneries in the 21st Century. Vol. 1.

Khean, T. S. 2003. Studies in Filter Cake Characterisation and Modelling. Environ. Eng.

Lampman, G. M., Pavia, D. L., Kriz, G. S., & Vyvyan, J. R. 2001. Spectroscopy. Mary Finch.

Lee, S A, Fane, A G, & Waite, T D. 2005. Impact of natural organic matter on floc size and structure effects in membrane filtration. Env. Sci Technol, 39(17), 6477–6486.

Leubner, Ingo H. 2000. Particle nucleation and growth models. Curr. Opin. Colloid Interface Sci., 5(1-2), 151–159.

Li, Mingzhong, & Wilkinson, Derek. 2005. Determination of non-spherical particle size distribution from chord length measurements. Part 1: Theoretical analysis. Chem. Eng. Sci., 60(12), 3251–3265.

Lin, M. Y., Lindsay, H. M., Weitz, D. A., Ball, R. C., Klein, R., & Meakin, P. 1989. Universality in colloid aggregation. Nature, 339(6223), 360–362.

Lin, M Y, Lindsay, H M, Weitz, D A, Ball, R C, Klein, R, & Meakin, P. 1990. Universal reaction-limited colloid aggregation. Phys. Rev. A, 41(4), 2005–2020.

Lindström, T. 1980. The colloidal behaviour of kraft lignin. Colloid Polym. Sci., 258(2), 168–173.

Meakin, M Y Lin, Lindsay, H M, Weitz, D A, Klein, R, Ball, R C, & P. 1990. Universal diﬀusion-limited colloid aggregation. J. Phys. Condens. Matter, 2(13), 3093.

Mueller, Michiel. 1996. Confocal Fluorescence Microscopy. Spie Press B., 2(1), 1–8.

Napper, Donald H. 1983. Polymeric stabilization of colloidal dispersions. Vol. 36. Akademie Verlag GmbH.

Nichols, Gary, Byard, Stephen, Bloxham, Mark J, Botterill, Joanne, Dawson, Neil J, Dennis, Andrew, Diart, Valerie, North, Nigel C, & Sherwood, John D. 2002. A review of the terms agglomerate and aggregate with a recommendation for nomen- clature used in powder and particle characterization. J. Pharm. Sci., 91(10), 2103–2109.

67 Bibliography

Norgren, Magnus, & Edlund, Håkan. 2014. Lignin: Recent advances and emerging applications. Curr. Opin. Colloid Interface Sci., 19(5), 409–416.

Norgren, Magnus, & Lindström, Birger. 2000. Dissociation of Phenolic Groups in Kraft Lignin at Elevated Temperatures. Holzforschung, 54(5).

Norgren, Magnus, Edlund, Håkan, Wågberg, Lars, Lindström, Birger, & Annergren, Göran. 2001. Aggregation of kraft lignin derivatives under conditions relevant to the process, part I: phase behaviour. Colloids Surfaces A Physicochem. Eng. Asp., 194(1-3), 85–96.

Norgren, Magnus, Edlund, Håkan, & Wågberg, Lars. 2002. Aggregation of Lignin Derivatives under Alkaline Conditions. Kinetics and Aggregate Structure. Lang- muir, 18(7), 2859–2865.

Öhman, F. 2006. Precipitation and separation of lignin from kraft black liquor. Ph.D. thesis, Chalmers Technical University.

Öhman, F. 2007. Precipitation and ﬁltration of lignin from black liquor of diﬀerent origin. Nord. Pulp Pap. Res. J., 22, 188–193.

Rezanowich, A, & Goring, D.A.I. 1960. Polyelectrolyte expansion of a lignin sulfonate microgel. J. Colloid Sci., 15(5), 452–471.

Rudatin, Sri, Sen, Yasar L., & Woerner, Douglas L. 1989. Association of Kraft Lignin in Aqueous Solution.

Sjostrom, E. 1981. No TitleWood Chemistry: Fundamentals and Applications. Lon- don: Academic Press, Inc.

Sjöström, Eero. 1993. Chapter 4 - LIGNIN BT - Wood Chemistry (Second Edition). San Diego: Academic Press.

Stepto, Robert F. T. 2009. Dispersity in polymer science (IUPAC Recommendations 2009). Pure Appl. Chem., 81(2).

Teleman, Anita. 2004. Hemicelluloses and pectines. Chap. 5, pages 1–10 of: Wood Chem. Stockholm: Fiber and Polymer Technology, KTH.

Teleman, Anita. 2008. Hemicelluloses and pectines. Chap. 5, pages 7–13 of: Wood Chem. Fibre and Polymer Technology, KTH.

Tenkanen, M., Tamminen, T., & Hortling, B. 1999. Investigation of lignin- carbohydrate complexes in kraft pulps by selective enzymatic treatments. Appl. Microbiol. Biotechnol., 51(2), 241–248.

Theander, Olof, & Westerlund, Eric A. 1986. Studies on dietary ﬁber. 3. Improved procedures for analysis of dietary ﬁber. J. Agric. Food Chem., 34(2), 330–336.

Uloth, V. C., & Wearing, J. T. 1989. Kraft lignin recovery: acid versus ultraﬁltra- tion. Pulp Pap. Canada, 90(9), 67–71.

68 Bibliography

Wallmo, Henrik. 2008. Lignin Extraction from Black Liquor - Precipitation, ﬁltration and washing. Ph.D. thesis, Göteborg.

Wallmo, Henrik, Theliander, Hans, Jönsson, Ann-Soﬁe, Wallberg, O, & Lindgren, Karin. 2009. The inﬂuence of hemicelluloses during the precipitation of lignin in kraft black liquor. Nord. Pulp &Amp, 24(2), 165–171.

Watanabe, T. 2003. Analysis of Native Bonds Between Lignin and Carbohydrate by Speciﬁc Chemical Reactions.

Waters, Jennifer C. 2009. Accuracy and precision in quantitative ﬂuorescence microscopy. J. Cell Biol., 185(7), 1135–1148.

Wynn, E.J.W. 2003. Relationship between particle-size and chord-length distributions in focused beam reﬂectance measurement: stability of direct inversion and weighting. Powder Technol., 133(1-3), 125–133.

Yang, Zhen. 2009. Hofmeister eﬀects: an explanation for the impact of ionic liquids on biocatalysis. J. Biotechnol., 144(1), 12–22.

Yllner, S., & Enström, B. 1956. Studies of the absorption o f xylan on cellulose ﬁbers during the sulphate cook. Part 1. Sven. Papperstidning 59, 6(229).

Zhu, W., Westman, G., & Theliander, H. 2016. Lignin separation from kraft black liquor by combined ultraﬁltration and precipitation: a study of solubility of lignin with diﬀerent molecular properties. Nord. Pulp Pap. Res. J., 31(02), 270–278.

Zhu, Weizhen, & Theliander, Hans. 2011. Equilibrium of lignin precipitation. Proc. 16th ISWFPC.

69 Bibliography

70 A Appendix - Preparation of dyed Xylan

A.1 Calculations

UV-vis Calibration Curve of Remazol Brilliant Blue R

Figure A.1: UV-Vis Calibration Curve of RBBR with absorption as a function of concentration

Substitution Degree of dye on Xylan The substitution degree of dye mol per xylan mol was calculated using equation A.1. The amount of dye washed out (md,f ) was determined by UV-vis and the calibration curve for RBBR concentration versus absorption is shown in ﬁgure A.1. The amount of dye in the precipitate (mX,p) was determined using HPLC. The calculation is based on the assumptions that all dye which was not washed out is bonded to xylan and that the substitution of dye on xylan is evenly distributed over all xylan molecules. md,i−md,f nd,p MWd = mX,p (A.1) nX,p MWX

Where nd,p is the amount of dye in the precipitate [mol], nX,p is the amount of xylan in the precipitate [mol] md,i is the amount of dye initially added in the synthesis [g]

I A. Appendix - Preparation of dyed Xylan

md,f is the amount of dye washed out (in the ﬁltrate) [g] and MW is the molecular weight of dye (index d) and xylan (index x) [g/mol], respectively.

A.2 Results

Salt Concentration for diﬀerent Synthesis Batches

Table A.1: Synthesis of dyed Xylan with salt concentrations for diﬀerent synthesis batches.

Synthesis Salt Concentration wt% S1 B1 70 S1 B2 70 S2 B1 70 S2 B2 73 S3 B3 74

Bond Stability- NMR Spectra

Figure A.3 shows the 13C (a) and 1H (b) NMR spectra for dX after synthesis, dX after dissolution and re-precipitation experiment and xylan without the dye. The 1H (b) NMR spectra also include a reference spectrum of RBBR. In the 13C NMR spectra it is visible that the xylan structure reappears in both the dX and dX after reactiontion spectrum. This holds true for both the C and H NMR spectra. The C NMR spectrum of dX indicates a peak at 110 ppm which does not appear in neither, the C NMR spectrum of dX after reaction and xylan. However, this peak was predicted to appear for RBBR C NMR spectra. This is an indication that dye is present in dX but not an ultimate proof for the bond due to other carbon peaks predicted for RBBR that are missing. Furthermore, the signal intensitiy was relatively low due to medium concentration of polymer in the NMR sample. The loading could not be further increased because high viscosities should be avoided. In addition, the H NMR spectra do not provide an indication for the chemical linkage between RBBR and xylan. Figure A.3 (b) shows no peaks at 7.65-4.3 ppm in the dX and dX after experiment spectra. The peaks at 7.65-4.3 ppm are attributed to aromatic groups in RBBR. These peaks cannot be detected in neither the dX after synthesis nor for the dX after dissolution experiment, which indicates that no dye was detected in the HNMR spectra of dX and dX after experiment. The absence of the peaks in the H NMR spectra of dX and dX after experiment might be due to low concentrations of dye in the NMR sample compared to xylan.

II A. Appendix - Preparation of dyed Xylan

Figure A.2: 13C-NMR data of beechwood xylan (reference) Daus et al. (2011)

III A. Appendix - Preparation of dyed Xylan

(a) 13C NMR spectra

(b) 1H NMR spectra

Figure A.3: 13C (a) and 1H (b) NMR spectra for X, dX and dX after experiment. The 1H NMR spectrum of RBBR is also included in ﬁgure (b).

IV B Appendix - Precipitation Study

B.1 Results

Particle Growth of L Replicate

Figure B.1: FRBM results of L replicate depicting counts as a function of time for diﬀerent chord length. The secondary axis shows the titration data of the experiment (amount of acid added or pH).

V B. Appendix - Precipitation Study

VI C Appendix - Filtration Study

C.1 Calculations

UV-vis Calibration Curve of Lignin

Figure C.1: UV-Vis Calibration Curve of Lignin with absorption as a function of concentration

Precipitation Yield The precipitation yield is calculated by using equations C.1- C.3. The calculations are based on the assumption that all salt ends up in the filtrate, so only the remaining solid material in the filtrate is attributed to lignin and xylan. The Uv-vis measurement on the filtrate show that lignin concentration is very low and lies below 0.1 g/l.

DCfiltrate ∗ H2Oadded Sfiltrate = (C.1) 1 − DCfiltrate

LXdissolved = Sﬁltrate − Saltadded (C.2) LX − LX yield = added dissolved ∗ 100 (C.3) LXadded

Where Sfiltrate is the solid material in the filtrate [g], DCfiltrate is the dry content of the filtrate [g/g], H 2Oadded is the total amount of water added [g], Saltadded is the

VII C. Appendix - Filtration Study

total amount of salt added [g], LXadded is the total amount of lignin and xylan added [g] and LXdissolved is the amount of lignin and xylan that ends up in the ﬁltrate [g].

C.2 Results

Precipitation Yields of Filtration Experiments

Table C.1: Speciﬁcations of ﬁltration experiments including precipitation pH, salt concentration and precipitation yield for the dissolution experiments.

Date Substance Precipitation pH Salt Concentration Precipitation Yield

[wt%] [wt%] Dissolution Experiments 16/03/09 L-dX 4.04 4.37 102.48 16/04/14 L-dX 4.31 4.71 102.72 16/03/20 L-dX 2.89 4.32 100.29 16/04/20 L-X 4.25 5.13 16/05/03 L-X 2.97 4.22 98.78 16/03/17 L 6.15 16/03/20 L 4.19 4.30 16/04/19 L 5.95 16/05/23 L 4.07 5.57 109.23 16/03/09 dX 2.85

VIII C. Appendix - Filtration Study

Confocal Fluorescence Microscopy

(a) Absolut emission spectra of lignin (b) Absolut emission spectra of dyed xylan

Figure C.2: Absolute emssion spectra for lignin and dyed xylan excited with laser light at 488nm..

IX C. Appendix - Filtration Study

Focused Beam Reﬂectometry Measurement

Figure C.3: FRBM results of the L-dX (pH 4.31) experiment depicting counts as a function of time for diﬀerent chord length. The heating was removed at 100 min.

Figure C.4: FRBM results of the L (pH 5.95) experiment depicting counts as a function of time for diﬀerent chord length. The heating was removed at 100 min.