DEGREE PROJECT IN CHEMICAL ENGINEERING AND TECHNOLOGY, FIRST LEVEL
STOCKHOLM, SWEDEN 2020
KTH ROYAL INSTITUTE OF TECHNOLOGY
KTH ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH
The impact of the pulping process on the properties of lignin nanoparticles
Kristoffer Guthenberg
DEGREE PROJECT
Bachelor of Science in
Chemical Engineering and Technology
Title: The impact of the pulping process on the properties
of lignin nanoparticles.
Swedish title: Massaframställningsprocessens påverkan på egenskaper hos nanopartiklar av lignin
Keywords: Lignin, Nano, Nanoparticles, Extraction, Kraft, Organosolv, Softwood, Properties
Workplace: Wood Chemistry and Pulp Technology:
School of engineering Sciences in Chemistry, Biotechnology and health.
Supervisor at
the workplace: Ievgen Pylypchuk, Olena Sevastyanova
Supervisor at
KTH: Ievgen Pylypchuk, Olena Sevastyanova
Student: Kristoffer Guthenberg
Date: 2020-05-27
Examiner: Prof. Mikael E. Lindström
i
List of abbreviations
NP: Nanoparticles
LNP: lignin nanoparticles
KL: Kraftlignin
OS: Organosolv lignin
PDI: Polydispersity index, a measurement on how the molecular weight of a polymer is distributed
ζ-potential: Zeta-potential
Mw: Weight average molecular weight
Mn: Number average molecular weight
SEM: Scanning electron microscope
TEM: Transmission electron microscopy
DLS: Dynamic light scattering
31P NMR: Nuclear magnetic resonance spectroscopy that studies phosphoric content.
Severity factor: An index coupled to the reaction conditions I regards to temperature, residence time and acid concentration.
ii
Abstract
Lignin valorization is a key component of the total utilization of biomass in the biorefinery industry. Lignin has seen some use in several different applications, but a breakthrough is still yet to happen, and there is still a need to find more areas where lignin can be used as an alternative feedstock or as the main component. Lignin nanoparticles (LNPs) could be an alternative route towards lignin valorization offering many areas of application. However, research around LNPs still has to overcome many challenges, primarily related to the complex structure of lignin, with composition and structure of lignin depending on its botanical origin and on the pulping process used to isolate the lignin from other components in biomass.
This study investigates how spruce lignin originating from Kraft and Organosolv pulping will affect the properties of lignin nanoparticles. Particles from organosolv spruce lignin were prepared using a solvent exchange method with acetone/water as solvent and water as antisolvent. This resulted in spherical LNPs with hollow centers, sizes ranging from 104.6- 270.3 depending on initial lignin concentration and average zeta-potential of -35mV.
Comparing Organosolv LPN’s with Kraft LNPs produced with the same experimental procedure, reviled that Organosolv LNPs were larger in and had lower absolute zeta potential, presumably due to the kraft lignin having higher phenolic-OH content. Furthermore, a larger comparison is made with LNPs from previous studies which indicated that LNP properties are further dependant Mw of lignin raw material, phenolic-OH content, and the method applied to produce the particles.
In conclusion, this study proves that the pulping process used to isolate lining will affect the properties of NPs. But to strengthen and generalize this conclusion beyond the limitations of this study, more experimental data are needed, to further investigate the relationship between LNP properties and the properties of lignin raw material
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Sammanfattning
En av utmaningarna för framtidens bioraffinaderier är att fullständigt utnyttja samtliga komponenter av råvaran. Historiskt sett har cellulosa varit den mest värdefulla komponenten av biomassan medan lignin har klassats som en biprodukt och har därför primärt bränts som bränsle vid framställning av pappersmassa. Även om lignin produceras på industriell skala saknas idag värdeskapande applikationsområden där lignin kan utnyttjas ur ett ekonomiskt hållbart perspektiv. Ett alternativ till valorisering av lignin är att använda det som råmaterial för framställande av nanopartiklar , vilket är ett relativ nytt område med stor potential framförallt inom biomedicin. Dock kvarstår en del utmaningar i forskningen runt lignin nanopartiklar. Framförallt relaterat till lignins komplexa och inhomogena struktur, som varierar beroende på botaniskt ursprung och vilken typ av massaframställningsprocess som används för att isolera ligninet från biomassan.
Den här studien undersöker hur granlignin från två olika massaframställningsprocesser, Organosolv- och Kraftprocessen, påverkar egenskaper hos NP av lignin. Under den experimentella delen av arbetet framställdes NP från Organosolv granlignin, vilket resulterade i sfäriska och ihåliga partiklar som varierade i storlek mellan 104.5–270.3 nm, beroende på den initiala lignin koncentrationen, samt en genomsnittlig zeta potential kring -35 mV.
Egenskaperna hos Organosolv nanopartiklarna som jämfördes med nanopartiklar av Kraflignin som producerats med samma metod. Slutsatsen drogs att organosolv partiklar var större och hade lägre absolut zeta-potential. Vilket troligtvis kan förklaras med den betydligt högre halten av fenoliska-OH enheter i Kraft ligninet. En bredare jämförelse med tidigare studier som producerat olika lignin nanopartiklar visar dessutom att molekylvikten, fenolisk-OH halt och produktionsmetoden, är bidragande faktorer till lignin nanopartiklars egenskaper.
Sammanfattnings visar den här studien att den massaframställningsprocess som används för att isolera lignin kommer påverka egenskaperna hos lignin nanopartiklar. Men för att kunna generalisera och stärka slutsatsen krävs dock utökad experimentella, för att vidare undersöka hur lignin nanopartiklars egenskaper beror på egenskaperna hos ligninet som använts för att producera partiklarna.
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Table of Contents
1. An introduction to lignin ... 1
2. Lignin nanoparticles, towards valorization ... 2
3. The pulping and isolation processes of lignin ... 3
3.1. Kraft lignin ... 3
3.2. Organosolv lignin ... 5
4. The effect of the pulping process on LNP properties ... 7
4.1. Kraft lignin as starting material ... 7
4.2. Organosolv lignin as starting material ... 8
5. Summery of previous LNP studies... 9
6. Purpose of this project ... 10
7. Experimental ... 11
7.1. Materials ... 11
7.2. Method to produce spruce Organosolv LNPs... 11
7.3. Description of analytical methods ... 12
7.3.1. DLS ... 12
7.3.2. SEM ... 13
7.3.3. TEM ... 13
8. Results and Discussions ... 14
8.1. Description of Organosolv and Kraft spruce lignins ... 14
8.2. Experimental observations ... 15
8.3. Size, zeta potential and PDI from DLS analysis for spruce OS LNPs ... 16
8.4. SEM images, shape and surface morphology of spruce OS LNPs ... 19
8.5. TEM images, shape and morphology of spruce OS LNPs ... 20
8.6. Comparing properties of spruce OS and spruce Kraft LNPs ... 21
8.7. Comparing spruce Kraft and Organosolv with previous studies ... 23
9. Conclusion ... 25
References ... 26
Appendix I - Distribution graphs and zeta potential from DLS ... 29
Appendix II - Properties of Kraft spruce lignin NPs ... 33
Appendix III - Characterization of Kraft lignin ... 34
1
1. An introduction to lignin
Lignin is the second most abundant biopolymer in nature and one of the three main components in plant cells along with cellulose and hemicellulose. The main biological function of lignin is to provide structure, rigidity, and protection of the plant cells (Sjöström, 1981).
Lignin is an amorphous polymer with a complex and heterogeneous structure and consists of three types of phenolic units: p-hydroxyphenyl, guaiacyl, and syringyl, see figure 1. These units are covalently bonded together and form a polymer with condensed carbon-carbon bonds and carbon-oxygen-carbon-ether bonds (Laurichesse & Avérous, 2014).
Figure 1: Condensed molecular structure of phenolic units in lignin.
The chemical structure of native lignin depends on the plant it originates from, different plants have different ratios of the phenolic units which contribute to the heterogeneous structure of lignin. In general, softwood mainly contains guaiacyl units, hardwood guaiacyl and syringyl units, and grass lignin contain a mixture of all three types of units (Dence, 1992; Duval &
Lawoko, 2014). Besides the main phenolic units, lignin also contains several functional groups that determine its reactivity (Laurichesse & Avérous, 2014). These functional groups include methoxy, phenolic and aliphatic hydroxyls, and carboxyl groups.
Lignin is mainly produced as a by-product of pulping plants and biorefineries where lignin is chemically separated from the other lignocellulosic polymers in the biomass. The separation process greatly alters the chemical structure of the native lignin resulting in varying purity, functionality, and polydispersity (Calvo-Flores, Dobado, Isac-García, & Martín-Martínez, 2015;
Duval & Lawoko, 2014; Vishtal & Kraslawski, 2011). In comparison to cellulose which has a more homogeneous structure, lignin heterogeneity and lack of stereoregularity pose major limitations to produce products and materials based on lignin.
In the pulping and biorefinery industry the majority of lignin is used as boiler fuel providing energy to the processes and only an exceedingly small portion is used to produced high-value products.
2 Even though lignin is produced on a large industrial scale, the utilization of lignin in high-value applications and products has been limited. The development of processes that utilize lignin to produce a high-value product is, therefore, a major research area. The aim is to create a more economically sustainable pulping and biorefinery industry that unleashes lignins´
potential to replace fossil fuel-based products.
Lignin has long been regarded as a waste product of little value and as cited by (Graichen et al., 2017) “You can make anything you want from lignin, except money” has long been the general attitude toward the biopolymer. However, in recent times the concept of integrated biorefineries that utilizes every component in biomass has been a hot research topic.
Lignin should no longer be considered a waste product and the use of lignin solely as a biofuel has been disproven as economically viable in many cases, the value of lignin fuel is only 0.8$/kg whereas lignin used in chemical conversion processes has an estimated value of 1.08$/kg (Dessbesell, Paleologou, Leitch, Pulkki, & Xu, 2020; Vishtal & Kraslawski, 2011). But turning the concept of total biomass utilization requires the development of high-value products based on all components in the biomass, to create a sustainable biopolymer economy (Graichen et al., 2017).
2. Lignin nanoparticles, towards valorization
One of the promising routes to valorization is utilizing lignins as nanoparticles. Biodegradable nanomaterials produced from renewable sources have the potential to solve some of the challenges that face the nanomaterial field, such as concerns regarding environmental impact, biological accumulation and provide a less complex valorization route as there is less need for advanced depolymerization or upgrading process (Matsakas, Karnaouri, Cwirzen, Rova, &
Christakopoulos, 2018).
Nanoparticles are defined as solid colloidal particles with sizes ranging from 10-1000nm and can be prepared from several sources, synthetic or natural polymers, and inorganic compounds. Nanoparticles have unique properties compared to their parent material due to their large area to volume ratio, providing increased reactivity, stability and mechanical strength and can vary in size, shape depending on the application (Wurm & Weiss, 2014).
Polymeric nanoparticles have gained popularity in recent times due to their potential applications in the area of in-vivo drug delivery systems (Bettencourt & Almeida, 2012; Sahu, Solanki, & Mitra, 2018). The main advantage of using synthetic polymers for nanoparticle production is the ability to tune polymer functionality and surface properties to a specific application (Wurm & Weiss, 2014).
However, research has also shown that synthetic polymers nanoparticles, such as polystyrene, polyalkyl(meth)acrylats and polyurethanes have limitations in regards to their biocompatibility and biodegradability properties (Banik, Fattahi, & Brown, 2016)
3 Such limitations have shifted focus towards using natural polymers such as polysaccharides, polyesters, polypeptides, lignin. For example chitosan nanoparticles have been extensively studied as potential as a drug delivery system (Mohammed, Syeda, Wasan, & Wasan, 2017).
The big attraction of natural polymers is that they are sourced in abundance, obtained from natural sources, and offer a much more environmentally friendly route to nanoparticle production compared to fossil-fuel based synthetic polymers. Other major advantages of natural polymers are better biocompatibility, more economical, and often biodegradable depending on chemical modifications (Kulkarni Vishakha, Butte Kishor, & Rathod Sudha, 2012).
Lignin counts as one of the most abundant natural polymers and has many potential applications within the nanomaterial field, for example as antioxidants, as a component in nanocomposites, surface coating, and biocomposites (Figueiredo, Lintinen, Hirvonen, Kostiainen, & Santos, 2018). Lignin properties also make it a great potential candidate to be used as a nanoparticle targeted drug delivery system. Lignin itself is non-toxic and has good biocompatibility and stability, but the poor water solubility and dispersion properties have limited its application within the biomedicine field. However, utilizing lignin as nanoparticles overcomes some of these limitations making it a potential drug carrier. For example, LNPs can be produced as hollow spherical capsules, allowing the hollow centers to be loaded with a variety of substances and be used as a targeted drug delivery and release system (Figueiredo et al., 2018)
3. The pulping and isolation processes of lignin
To produce usable lignin from biomass it needs to be extracted and isolated from the other components in the biomass. Several industrial pulping processes that do this, and they produce so-called technical lignins, for example Kraft, sulfite, soda, and Organosolv. These processes not only extract lignin but also modifies the chemical structure by breaking bonds and introducing different impurities which makes the composition and structure of technical lignin’s different to native lignin (Vishtal & Kraslawski, 2011).
3.1. Kraft lignin
The most common industrial pulping process worldwide is the Kraft process that produces around 63x104 tons of lignin annually and stands for 85% of the world total lignin production (Vishtal & Kraslawski, 2011). The pulping procedure involves converting wood into wood pulp by treating it chemically with a solution of Na2S and NaOH at high temperatures, usually between 155-177 °C.
High pH leads to ionization of phenolic hydroxyl groups which extracts the lignin from the lignocellulosic biomass by solubilization (Ejaz Ahmad & Kamal K Pant, 2018). The treatment dissolves 90-95% of the lignin in the raw material which is accumulated in a lignin and cellulose rich liquid called black liquor (Vishtal & Kraslawski, 2011).
4 The harsh reaction conditions in the Kraft process fragments the native lignin into smaller polymer chains, leading to a wide range in molecular weight, from 600 to 180,000 g/mol and therefore a high degree of polydispersity (Duval, Vilaplana, Crestini, & Lawoko, 2016;
Sevastyanova et al., 2014; Vishtal & Kraslawski, 2011)
To produce usable technical lignin from the Kraft process it needs to be isolated from the black liquor and there are several different commercial methods used in the pulping industry. A common method for lignin isolation from black liquor is precipitation by acidification, using CO2 or mineral acids. MeadWestvaco (now called Ingevity) first produced commercially available pure Kraft lignin in late 1940, using acid precipitation. Lignin is produced in a batch process were the black liquor is acidified with sulphuric or hydrochloric acid (Fang & Smith Jr, 2016). MeadWestvaco (Ingevity) has been the predominant lignin supplier, ever since the late 1940’s, offering products like INDULIN AT a high purity, unsulfonated raw Kraft lignin.
However, in recent times several other actors started developing new isolation processes, refining the concept of acid precipitation.
STFI and Chalmers University of technology started developing a new patented isolation process in the late 1990’s called Lignoboost. The primary purpose of the Lignoboost process was to increase boiler capacity in existing Kraft pulping plants and create an alternative revenue stream beside the cellulose pulp (Nunes & Pardini, 2018). The Lignoboost process treats a part of the black liquor stream from the evaporation plant and precipitates the lignin by mixing in acid, usually CO2. The precipitate is then dissolved and undergoes another precipitation step before filtering out and washing the solid high purity lignin (Tomani, 2010).
FP Innovations and NORAM developed an isolation process similar to Lignoboost in that it utilizes acidic precipitation to isolate lignin. But before the addition of CO2 the black liquor is oxidized to reduce the amount of totally reduced sulfur in the liquor (TRS). This prevents the formation and release of volatile compounds such as hydrogen sulfide and mercaptan when the liquor is later acidified (Kouisni, Gagné, Maki, Holt-Hindle, & Paleologou, 2016). However, most of the lignin produced are burnt in recovery boilers to provide energy to recover the chemicals used in the pulping process.
Kraft lignin is characterized by containing large amounts of phenolic hydroxyl groups as a result of the cleaving β-aryl bonds, and the formation of condensed structures (Vishtal &
Kraslawski, 2011). Condensed structures relate to the number of carbon-carbon bonds in the lignin polymer. Kraft lignin also has a high ash content, which is removed by treating the lignin with sulphuric acid, reducing the ash content to 1-5%. (Gordobil, Moriana, Zhang, Labidi, &
Sevastyanova, 2016). Lignoboost and other lignin isolation processes provide high purity Kraft lignin with sulfur content around 1-3% (Kouisni et al., 2016; Tomani, 2010).
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3.2. Organosolv lignin
The Organosolv pulping process was originally developed as an eco-friendlier alternative to the more established pulping process, Kraft, soda, sulfite. The main principle of Organosolv is treating biomass with organic solvents at elevated temperatures, where the solvent acts as a delignifying agent and dissolves both lignin and hemicellulose (Figueiredo et al., 2018). To increase the delignification rate and lower reaction temperature an acidic catalyst is often used (Macfarlane, Mai, & Kadla, 2014). Common solvents are ethanol, methanol, acetic and formic acid (Figueiredo et al., 2018) while ethanol is the most commonly used as it’s very effective at dissolving lignin and easy to recover by distillation. (Rodríguez et al., 2018).
Two fractions are obtained from the treated biomass, cellulose, and liquor containing dissolved lignin and hemicellulose. Lignin can be isolated from the solvent liquor via precipitation, either by adding water and diluting the solvent or by evaporating the solvent.
This means the Organosolv process can produce three separate streams that can be used as high-value chemicals (Ejaz Ahmad & Kamal K. Pant, 2018), an important concept in utilizing every component in biomass.
From an environmental and sustainability point of view Organosolv provides several advantages compared to other pulping processes such as Kraft. There are less hazardous chemicals involved (this, of course, depend on the solvent used), and smaller energy requirements, especially if a catalyst is used (Nitsos, Rova, & Christakopoulos, 2018). Another advantage is that lignin can be isolated as the main product and not as a by-product compared to Kraft.
Several variations of Organosolv processes have been developed for commercial use, for example ALCELL, Organocell, ASAM, ASAE, and IDE. However, most of these processes are currently only in pilot-scale or used in small biorefineries with limited production capacity.
ALCELL process uses ethanol-water (50-50%) as the solvent and is the most common Organosolv process that utilizes alcohol without a catalyst. Figure 2 illustrates the ALCELL pulping process flowchart. Its best suited for hardwood but also works with other biomass sources.
Figure 2: Flowchart of the ALCELL pulping process (Z. Liu, Wang, & Hui, 2018).
6 Lignol Innovations Ltd uses the ALCELL process in a biorefinery pilot plant based in Burnaby Canada, using hard and softwood residue to produce ethanol and high purity lignin (Rabaçal, Ferreira, Silva, & Costa, 2017). In comparison to Kraft, ALCELL is more economical and produces higher yields of cellulose, hemicellulose, and lignin (Rodríguez, Rosal, & Jiménez, 2010). However, a major shortcoming for Organosolv pulping, in general, is the poor quality of usable cellulose pulp when using softwood as raw material. This limits the usability of the cellulose pulp to low-value products and makes it less economically viable. (Rodríguez et al., 2010).
Compared to lignin extracted via the Kraft process Organosolv lignin retains much of its native molecular structure. There is still cleavage of β-aryl bonds which leads to high phenolic functionality, and condensations reactions are quite limited leading to a less condensed structure compared to Kraft lignin. Other major differences include low molecular weight 500- 5000 g/mol, low PDI (1.5), and high purity, but this also varies depending on the lignin source.
(Gao & Fatehi, 2019; Matsakas et al., 2018).
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4. The effect of the pulping process on LNP properties
As stated earlier the structure and purity of technical lignin’s depend on the botanical origin of lignin as well as the pulp or refining process used to extract the lignin from biomass. This leads to difficulties when using lignin for nanoparticle synthesis. The molecular weight, polydispersity index, density of reactive functional groups all plays a major role in the size, shape and surface properties of LNP, not to mention the effect the many different synthesis methods have on the mentioned properties (Gao & Fatehi, 2019; Mishra & Ekielski, 2019).
An important factor directly associated with lignins ability to crosslink and form LNP is the amount of hydroxyl groups present. The number of crosslink-sites affect the tendency for noncovalent interactions, the main driving force for nucleation and particle growth in a lignin solution. Crosslinking opportunities varies depending on the lignin source and the pulping process used (Mishra & Ekielski, 2019).
For example, Richter et al. (2016) conducted a study comparing lignin nanoparticles using Organosolv lignin and Indulin Kraft lignin as starting material. The study provided lignin characterization data in regard to functional content, measured per 100 aromatic rings, see table 1.
Table 1:Functionl content of lignin starting material, adapted from (Richter et al., 2016).
Origin Mw (g/mol) Ph-OH content Aliphatic-OH content
Kraft Indulin - 73 68
Organosolv 969 34 51
Nanoparticles from Organosolv lignin had a uniform spherical shape, size 80nm and zeta- potential of -45 mV. LNP from Indulin lignin had an irregular shape and were larger in size 125nm and a zeta- potential of -35mV. However, an important note is that reported LNP size and zeta- potential was also proven to be affected by pH and synthesis parameters making it complicated to draw any certain general conclusions about Organosolv or Kraft derived LNP characteristics (Richter et al., 2016).
4.1. Kraft lignin as starting material
Zou et al. (2019) produced LNP from softwood Lignoboost Kraft lignin. An interesting note is that the lignin used was well characterized before LNP production, both molecular weight and total functionality was analysed. The LNPs were produced via solvent exchange method.
Different lignin concentrations were tested and with following results. 0.2 wt% lignin solution resulted in LNPs with a diameter of 97nm, PDI of 0.18 and ζ- potential of -27mv. 1-0.5 wt%
resulted in LNPs with a diameter 113nm, PDI of 0.19 and zeta- potential of -30mV (Zou, Sipponen, & Österberg, 2019).
8 Mattinen et al. (2018) studied the effect of lactase catalysed covalent cross-linking in LNP production. LNP were produced from softwood Kraft lignin, se table 2 for characterization, in a solution tetrahydrofuran. Several concentrations of lactase were investigated and compared to a control sample without lactase added to it. The control sample with no catalyst resulted in spherical LNP´s with an average size of 200nm and zeta- potential varying from -31 to -40mV (Mattinen et al., 2018).
Table 2: Characterization of softwood Kraft lignin used in (Mattinen et al., 2018), based on analysis made in (Lievonen et al., 2016).
Origin Mw
(g/mol)
Ph-OH content (mmol/g)
Aliphatic-OH content (mmol/g)
PDI
Softwood Kraft >4800 3.64 1.78 No data
4.2. Organosolv lignin as starting material
Z.-H. Liu et al. (2018) produced LNPs using Organosolv lignin from corn-stover and switchgrass.
The purpose of the study was to investigate how the severity factor of the Organosolv pre- treatment affected the properties of LNP. This resulted in uniform spherical particles with sizes ranging from 142-234nm, zeta- potential from -18.1mV to -50.8mV (Z.-H. Liu et al., 2018).
Matsakas et al. (2018) conducted a study on how two variations of Organosolv isolation of birch lignin affected the properties of LNP. Traditional Organosolv using ethanol and a hybrid method using Organosolv-steam explosion were tested. Resulting LNP were all spherical in shape but size, and charge proved to be highly affected by the isolation treatment with size varying from 200-4505nm, PDI 0.164-1 and zeta- potential -20.5mV – 47.1mV. The study provides a god insight in the unpredictable nature of LNP synthesis (Matsakas et al., 2018).
In another study Matsakas et al. 2020 tried to develop a production method for that allowed size control of LNPs. In this study both spruce and birch OS (ethanol) lignins were used for particle synthesis. The result showed that a two-step method involving solvent dilution followed up by particle isolation, centrifugation or freeze drying, produced well defined spherical particles. The shape and size of LNPs proved to correlate to the ratio of dilution.
Good results were observed when diluting a lignin ethanol solution with water at a ratio of 1:6. This produced well defined LNP´s of both spruce and birch with sizes varying between 35- 70nm an zeta- potential of -31.6mV for birch and -24.8 for spruce. The study also reports many more nanoparticles with varying characteristics depending on production parameters (Matsakas, Gerber, Yu, Rova, & Christakopoulos, 2020).
Beisl et al. (2020) conducted a study producing ethanol Organosolv LNP´s from wheat straw using a continuous direct precipitation method. The effects of process parameters such as flowrate, antisolvent ratio and pH on LNPs properties studied were investigated. The particles sizes recorded varied between 97.3 to 219.3nm and zeta- potential between -30mV to -45mV depending on process parameters. No characterization of lignin starting material was provided in this study (Beisl, Adamcyk, & Friedl, 2020).
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5. Summery of previous LNP studies
To summarize, the difference in characteristics of LNP from Kraft or Organosolv lignin depends on a variety of factors. It´s difficult for example to say that Organosolv lignin produces LNP with a distinct property regarding size and shape compared to Kraft lignin. One thing worth highlighting is that Organosolv LNPs seem to have slightly lower zeta- potential. High zeta- potential is related to the hydroxyl and carboxyl content in the lignin (Lievonen et al., 2016), which in turn greatly depends on the lignin source and pulping process.
Many previous studies, see table 3, show that solvent exchange is a common method to produce LNP´s. However, the process conditions are often quite different regarding solvent used, dilution factors, the concentration of lignin and pH. Witch probably also has an effect on LNPs properties. Thus, making comparisons between studies difficult. Another major issue is the lack of information about the characteristics of the lignin starting material. If the functionality of lignin in terms of hydroxyl and carboxyl content play a major role in the self- assembly process of LNP, there is a need to characterize lignins functional content before any well motivated conclusion can be drawn as to how lignin extraction affects LNP properties.
Table 3: Summary of lignin NP´s studies.
Type of lignin Source Synthesis Shape Size ζ-potential Source
OS Corn Stover
Antisolvent
precipitation Uniform nano spheres
142-234 nm -18 to -50.8mV (Z.-H. Liu et al., 2018)
OS Poplar
Dialysis
Solvent shift Spheres 197 nm -35.8mV (Tian et al.,
2017)
KL Unspecified
Solvent
exchange Spheres 320-360 nm -60mV (Lievonen et al.,
2016)
OS Spruce
Solvent
Exchange Spheres 35-70 nm Spruce: -24.8mV
Birch: -31.6mV
(Matsakas et al., 2020)
OS Wheat straw
Continuous process using solvent exchange.
Undefined particles
97.3-219.3 nm -30 to -45mV (Beisl et al., 2020)
KL Softwood
Solvent
exchange Spheres 97-113nm -27 to -30mV (Zou et al.,
2019)
KL Softwood
Evaporation and redispersion
Spheres 200nm -30 to -41mV (Mattinen et al., 2018)
OS Corn stover
Solvent exchange dialysis bag
- 132nm -56.1mV (Z.-H. Liu et al.,
2019)
OS Unspecified
Solvent
exchange Spheres 160nm -37mV (Capecchi et al.,
2018)
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6. Purpose of this project
The aim of this project is to investigate how pulping methods affect the properties of lignin NP´s. To do this, two different types of technical lignins, one originating from Kraft and one from Organosolv pulping processes will be used to produce NP´s with the same experimental procedure. Differences in particle properties such as size, zeta potential and PDI will be determined with DLS and differences in morphology determined by SEM and TEM. The working hypothesis is that the properties and structure of starting material as a result of the pulping process, will affect the morphology, size, PDI and surface charge of LNPs.
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7. Experimental
This chapter comprises materials, the lignin NP production method and analytical methods used to determine particle properties. Due to external circumstances only LNPs from Organosolv spruce lignin were produced and characterized in this study. LNPs from Kraft spruce lignin was instead produced in a yet to be published study by Pylypchuk et al. (2020)
7.1. Materials
Organosolv spruce lignin was provided by the Department of Fibre and Polymer Technology Department, KTH Royal Institute of Technology. The lignin was isolated and characterized in a collaborative study by Department of Fibre and Polymer Technology Department, KTH Royal Institute of Technology and Department of Chemical and Environmental Engineering, University of the Basque Country and is described in Gordobil et al. (2016). Acetone was provided by VWR chemicals (VWR chemicals, lot #19F064007). Deionized water used was Ultrapure water with specific resistance of 18.2 MΩ/cm.
7.2. Method to produce spruce Organosolv LNPs
The method applied to produced LNPs follows the same protocol described in Pylypchuk et al.
(2020). 60 mg of dry Organosolv spruce lignin powder was weighed and transferred to an empty vial. The lignin sample was then dissolved with 10ml 4:1 (v/v) acetone-water solution.
Sonication of the sample was performed to completely dissolve the lignin. This sample is referred to as the stock solution. From the stock solution several diluted samples were prepared in empty microcentrifuge tubes, according to the following dilation scheme seen in table 4.
Table 4: Dilution scheme for lignin samples.
Sample Stock solution (ml) Acetone-water (4:1) (ml) Final Lignin concentration (mg/ml)
1 0.1 0.9 0.6
2 0.2 0.8 1.2
3 0.4 0.6 2.4
4 0.6 0.4 3.6
Each of the samples in table 4 was transferred into separate vials, together with magnetic stirrers. The test tubes were placed on a magnetic plate and 4ml of deionized water was slowly added to each sample. The samples were left on the magnetic stirrer for three hours to allow the complete precipitation of nanoparticles.
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7.3. Description of analytical methods
Several analytical methods were used in order to characterize produced lignin NPs in regard to their size, zeta-potential and shape.
7.3.1. DLS
Measurements of zeta-potential, average size and size distribution of each LNP sample were carried out using a Zetasizer Nano ZS instrument (Malvern-Pananalytical, Malvern, UK, figure 3). Zeta potential relates to how LNPs are electrically stabilized in solution under given conditions, regarding pH and ionic strength.
High absolute zeta potential indicates good stability and low aggregation of particles. Low absolute zeta potential indicates poor stability and a tendency for particles to aggregate (Matsakas et al., 2018).
To conduct the DLS analysis 1ml of each LNP sample was injected with a syringe into a folded capillary Zeta cell making sure no air bubbles were trapped inside, see figure 3. The particle concentration for samples 3 and 4 was suspected to be too high due to their darker color and was diluted with deionized water, see table 5, to keep the attenuator setting for the DLS between 4-9.
Table 5: Sample preparation for DLS analysis.
Sample Volume of sample (ml) Volume of deionized water (ml) Att factor
1 1 0 6
2 1 0 5
3 0.5 0.5 5
4 0.5 0.5 5
Figure 3: Folded capillary zeta cell loaded with lignin (right). Zetasizer Nano ZS instrument (left).
13 7.3.2. SEM
A S-4800 microscope was used for experiments. EDS was performed on TEM grid on Hitachi field-emission scanning microscope with EDS detector (S-4800 Hitachi, Japan, figure 4), at accelerating voltage 15 kV and emission current 10.5 µA. Sample preparation: 5 mkl of nanoparticles suspension (approx. 0.1 mg/ml) was deposited on 200 mesh copper grid (Ted Pella Inc., USA; prod No 01800-F; see figure 5) and air dried for 30 min.
7.3.3. TEM
TEM analysis was performed on a Hitachi HT7700 series instrument, figure 5, (Hitachi, Japan), at accelerating voltage 100.0 kV and emission current 8.0 µA. Sample preparation: 5 mkl of nanoparticles suspension (approx. 0.1 mg/ml) was deposited on a holey TEM grid (Ted Pella Inc., USA; prod No 01800-F; see figure 5) and air dried for 30 min.
Figure 4: LNP sample on a 200-mesh copper grid (right).
Hitachi HT7700 Cryo-TEM (left)
Figure 5: LNP sample on a holey TEM grid for TEM analysis (right).
Hitachi S-4800 (left)
14
8. Results and Discussions
This chapter describes and compares the characteristics of nanoparticles produced from Organosolv (OS) spruce lignin prepared in this study, with Kraft spruce lignin nanoparticles as described by Pylypchuck et al. (2020). A broader comparison is also made with different studies listed in table 3 to generalize and make conclusions about how the lignin pulping process affect NP properties.
8.1. Description of Organosolv and Kraft spruce lignins
The spruce Organosolv lignin powder (figure 6) used to produce LNPs was characterized in a previous study by Gordobil et al. (2016). The Organosolv treatment used to prepare the lignin powder was as follows: Spruce fibers was treated at 180 °C for 60 min with a mixture of ethanol (50/50 w/w) together with 𝐻2𝑆𝑂4 (1.2 % w/w) as a catalyst to dissolve lignin from the fibers. Dissolved lignin was precipitated using cold water and filtered, washed, and dried.
Lignin properties such as molecular weight and PDI were determined using gel-permeation chromatography (GPC). Phenolic and aliphatic functionality content was determined by 31P NMR.
Figure 6: Sample of spruce OS lignin powder.
The spruce Kraft lignin fraction used in Pylypchuck et al., (2020) was produced and characterized as described by Tagami et al. (2019). In this study the impact of solvent fractionation on properties of Lignoboost spruce lignin was investigated. The molecular weight and PDI were determined using size-exclusion chromatography (SEC) and lignin functionality determined by 31P NMR. Molecular weight and functionality of both Organosolv and Kraft spruce lignin are shown in table 6. The choice of original lignin materials was based on a similar molecular weight.
Table 6: Functionality and properties of spruce OS lignin (Gordobil et al., 2016) and spruce Kraft lignin (Tagami et al., 2019).
Fraction Mw
(g/mol)
Ph-OH content (mmol/g)
Aliphatic-OH content (mmol/g)
PDI
KraftMeOH 3080 4.2 2.0 2
OSEtOH 3081 2.99 0.75 2.89
15 As table 6 shows, both the OS and Kraft lignin have the same Mw. However, the kraft lignin has lower PDI compared to Organosolv, which is surprising since Kraft lignin is usually associated with high inhomogeneity and therefore high PDI. This low PDI value is probably a result of the solvent fractionation described in Tagami et al., (2019), meaning that it might not represent kraft lignins in general. The Kraft lignin also has higher phenolic-OH and aliphatic- OH content compared to the OS lignin.
8.2. Experimental observations
When deionized water was added to the lignin solution samples a change in opacity was observed, primarily in the samples with higher lignin concentration. This is a good indication of the formation of nanoparticles. The white foam observed in figure 7 is probably caused by the lignin acting as a surfactant.
Figure 7: Samples with precipitated lignin NPs
16
8.3. Size, zeta potential and PDI from DLS analysis for spruce OS LNPs
Particle size, zeta- potential and PDI of lignin NPs was obtained from the DLS analysis. Raw data in form of distribution graphs can be seen in appendix I. The average size for each sample can be seen in figure 8.
Figure 8: The Average size of lignin nanoparticles for each sample, based on DLS analysis, complete set of data can be seen in appendix. Trendline (blue dotted line) and equation are also displayed. Error bars are based on the standard deviation provided by the DLS analysis.
Sample 2 (1.2 mg/ml) produced particles with the smallest size. Increasing lignin concentration leads to increasingly larger particles. Sample 1 (0.6 mg/ml), with the lowest concentration also produced larger particles compared to sample 2.
To represent the average size, distribution by number measurements were chosen.
Measurements of distribution by volume are sensitive to larger particle sizes as shown in figure 9, meaning that the average size can be misrepresentative if there are any larger particles or agglomerates in solution. The number average size is ,in this case, considered more representative.
Figure 9: Distribution by volume and number from DLS analysis of sample 2 (1.2 mg/ml). Distribution by number is not affected by large agglomerates compared to distribution by volume.
139.7nm
104.6 nm
219 nm 270.3 nm
y = 52,26x + 81,493 0
50 100 150 200 250 300 350 400 450
0 0,5 1 1,5 2 2,5 3 3,5 4
nm
Concentration (mg/ml)
Average size (nm)
17 As figure 10 shows, the zeta- potential only varied very slightly with lignin concentration. The size and zeta-potential could be said to correlate as in that larger particles have higher absolute zeta-potential. This may be explained by larger particles having larger surface areas and therefore more functional groups to interact with the solution. However, the differences in zeta-potential are still minuscule.
Figure 10: zeta-potential of lignin nanoparticles for each sample, based on DLS analysis, complete set of data can be seen in appendix. Trendline (blue dotted line) and equation are also displayed. Error bars are based on the standard deviation provided from the DLS analysis.
PDI measurement, displayed in figure 11, show large variations between samples. Sample 2 and 4 have fairly high polydispersity, which can be also be observed by looking at the volume distribution graph for the two samples, see appendix I. Both samples have a significant amount of larger volume particles or agglomerates, which in turn increases PDI.
Figure 11: PDI of lignin nanoparticles for each sample, based on DLS analysis, complete set of data can be seen in appendix.
-39 mV
-34,4 mV
-36,8 mV
-38,7 mV
y = -0,3701x - 36,503 -60
-50
-40
-30
-20
0 0,5 1 1,5 2 2,5 3 3,5 4
mV
Concentration (mg/ml)
Zeta potential (mV)
0,259
0,457
0,15
0,342
0 0,1 0,2 0,3 0,4 0,5
0 0,5 1 1,5 2 2,5 3 3,5 4
PDI
Concentration (mg/ml)
PDI
18 Sample 2 has the highest PDI (0.457) and sample 3 the lowest PDI (0.15), a surprisingly large difference. This is probably due to sample 2 having significantly larger agglomerates present, which can be seen when comparing the distribution by volume graphs from DLS, figure 12.
Figure 12: Distribution by volume for sample 2 (top) and sample 3 (bottom). Sample 2 has much more agglomerates present .
19
8.4. SEM images, shape and surface morphology of spruce OS LNPs
The SEM analysis provided information about the surface structure and shape of produced lignin NPs. Only sample 2 (1.2 mg/ml) was analysed and image is displayed in figure 13.
Figure 13: SEM image from sample 2 (1.2mg/ml).
As the images in figure 13 shows, some particles are well separated with a spherical shape, ranging from 68.8-103nm in size. This correlates well with previous studies were spherical particles are the prominent shape reported, see table 2. Some agglomerated structures can also be observed where particles have stuck together and formed large clusters. The size distribution from SEM also correlates well with the size distribution from DLS as shown in figure 14. The majority of particles are between 50-90 nm in size.
Figure 14: Size distribution by number for sample 2 (1.2 mg/ml) from DLS analysis.
20
8.5. TEM images, shape and morphology of spruce OS LNPs
TEM analysis provided further information about particles shape and structure. Only sample 3 (2.4mg/ml) was analysed and images are displayed in figure 15.
Figure 15: TEM images from sample 3 (2.4mg/ml).
The TEM images confirm that the spherical shape seen in the SEM for sample 2 (1.2mg/ml), is also observed at a higher lignin concentration (2.4 mg/ml). Some particles in figure 15 are also hollow or solvent filled, which is indicated by the slightly lighter color in the center of some particles. The reason for selecting sample 3 for TEM analysis and not sample 2 (used in the SEM analysis) was to find an explanation for the large PDI difference between the two samples. But even though sample 3 produced particles with the lowest PDI, there are still some larger agglomerates present and quite a bit of unreacted lignin polymer. To further clarify as to why the PDI varies between different samples, SEM, and TEM images for all 4 samples are needed.
21
8.6. Comparing properties of spruce OS and spruce Kraft LNPs
Comparing spruce Organosolv LNPs produced in the current study with spruce Kraft LNPs produced in Pylypchuck et al. (2020) see appendix II, reveals that Organosolv particles have a lower absolute zeta potential and slightly larger size throughout the entire concentration range, see figure 16 and 17.
The effect of lignin concentration on particle size i.e. an increase of concentration produces larger particles, this was observed for both the Organosolv and Kraft particles as shown in figure 16. Organosolv LNPs also increase more in size with higher initial concentration compared to raft lignin according to the trendline equations in figure 16. Another interesting observation is that concentration under 1mg/ml also produced larger particles, indicating that there is a small concentration range optimal for producing small particles.
Figure 16: Comparison of size between Kraft (black) and Organosolv (orange) lignin NP´s.
The Kraft lignin NPs were produced using the same method as the Organosolv particles, experimental parameters should therefore not be a large contributing factor to particle properties. However, the difference in properties may be explained by looking into the functionality of the lignins themselves. In this case the Kraft lignin had a much higher phenolic content and lower PDI, whereas the Mw´s were identical between the two lignin, see table 6.
Lignin with higher phenolic content has more crosslinking sites within the polymer strands which might lead to better self-assembly capability and therefore smaller particle sizes. To summarize, even though particle size and zeta potential differ the general trends are fairly similar.
y = 52,26x + 81,493
y = 25,692x + 95,527
0 50 100 150 200 250 300
0 1 2 3 4 5 6 7
Size (nm)
Concentration (mg/ml)
Size of Organosolv and Kraft lingin NP's
OS spruce size Kraft spruce size (Pylypchuck et al., 2020)
22 Higher phenolic-OH and aliphatic-OH content might also explain why the Kraft LNPs resulted in higher absolute zeta potential, as seen in figure 17. The zeta potential of OS LNPs seems to increase with higher initial lignin concentration, compared to the zeta potential of Kraft LNPs which seems to be fairly stable at higher initial lignin concentration.
Figure 17: Comparison of zeta potential between Kraft (black) and Organosolv (organosolv) lignin NP´s.
Worth mentioning is that the Kraft lignin used to produce the NP´s in this comparison had been solvent fractionated, thereby lowering the PDI. Which means it does not represent the inhomogeneity that Kraft lignin is usually associated with.
-70 -60
-50
-40
-30 -20
-10
0
0 1 2 3 4 5 6 7
Zeta potential (mV)
Concentration (mg/ml)
Zeta potential of Organosolv and Kraft lingin NP's
OS spruce zeta Kraft spruce zeta (Pylypchuck et al., 2020)
23
8.7. Comparing spruce Kraft and Organosolv with previous studies
To compare the particles produced in this report with results from previous studies, the initial lignin concentration used in particle production has to be the same, otherwise, properties such as size might be affected by initial lignin concentration, and comparison would be irrelevant. In many studies initial lignin is not reported, or it is reported in (w%), in that case a unit conversion has been made from (w%) to (mg/ml) to estimate lignin concentration.
The initial lignin concentration used in figure 18 and 19 is 2 mg/ml. No particle sample of OS spruce made in this study has this specific concentration. Therefore, an estimation of size and zeta potential is calculated using the trendline equations in figure 8 and 10, for a theoretical 2 mg/ml sample. This is made under the assumption that particle size and zeta potential is directly proportional to initial lignin concentration.
Figure 18: Comparrisson with previous studies. Relating size and zeta potential to Mw of raw lignin from different pulping processes.
When comparing Organosolv and Kraft LNPs with several previous studies based on lignin Mw, some trends can be observed. In general, higher Mw leads to larger particles. When looking at the same type of technical lignin, Kraft or Organosolv, the size of the lignin NP´s increase with higher Mw, see figure 18. However, no obvious correlation can be seen between Mw and zeta potential.
969 g/mol OS (Richter et al.,
(2016))
3080 g/mol Kraft (Pylypchuk et
al., (2020))
3081 g/mol OS (Current study)
>4800 g/mol Kraft (Leivonen et
al., 2016)
Size (nm) 50 155,8 186 320
Zeta (mV) -45 -47 -37,2 -55
-60
-50
-40
-30
-20
-10
0 0
50 100 150 200 250 300 350
Zeta potential (mV)
Size (nm)
Size and zeta potential based on Mw(g/mol)
(2 mg/ml initial lignin concentration)
24 Figure 19 looks at how the phenolic-OH content affects size and zeta potential for different Kraft and Organosolv LNPs. Very high phenolic content might relate to smaller particle size, but no clear trend is observed as to how phenolic-OH content affects particle size. Since particle size also corresponds to the molecular weight, it is difficult to make any specific statements that phenolic content itself is a substantial factor for particle size, only that it is a contributing factor.
Figure 19: Comparrisson with previous studies. Relating size and zeta potential to phenolic content of raw lignin from different pulping processes.
Furthermore, high phenolic content and small particles size is also associated with high absolute zeta-potential. Probably due to more functional groups being present on the surface of the particles. However, its difficult to see an obvious correlation between phenolic-OH contentand zeta potential in figure 19.
An important side note is that the studies cited in the comparisons have not all used the same method to produce the LNPs, and the botanical origin of the lignin is not the same either. This further complicates a comparison and ignores two large contributing factors to particle properties. Therefore, the comparisons made in figure 18 and 19 are better used as an indication that there is a need for more informative and precise research data to accurately compare particle properties.
2. 87 mmol/g OS (Capecchi et al., 2018)
2. 99 mmol/g OS (Current
study)
3.37 mmol/g OS (Tian et al.,
(2016))
3.64 mmol/g Kraft (Leivonen et
al., 2016)
4.2 mmol/g Kraft (Pylypchuk et
al., 2020)
Size (nm) 160 186 197 320 155,8
Zeta (mV) -37 -37,2 -35,8 -55 -47
-60
-50
-40
-30
-20
-10
0 0
50 100 150 200 250 300 350
Zeta potential (mV)
Size (nm)
Size and zeta potential based phenolic-OH content (mmol/g)
(2 mg/ml initial lignin concentration)
25
9. Conclusion
• Spherical and hollow LNPs from Organosolv spruce lignin were produced, ranging from 104,6-270,3 nm in size, with average zeta-potential of -35mV.
• The pulping process used to obtain the lignin raw material is proven to affect the properties of lignin NPs. Comparing Kraft and Organosolv spruce lignin nanoparticles produced with the same experimental procedure, revealed that Kraft lignin resulted in smaller particles with higher absolute zeta potential compared to Organosolv spruce lignin
• Initial lignin concentration has a notable effect on particle size. Concentration under 1 mg/ml or over 2mg/ml produces significantly larger particles compared to concentrations around 1 mg/ml.
• The Mw of lignin used to make NPs seem to affect particle size in that higher Mw leads to larger particles.
• The phenolic-OH content of lignin also affects the properties of LNP. However, it is difficult to observe any clear trends, therefore difficult to make any certain conclusion since the synergy between phenolic content and Mw has to be further investigated where these parameters are predetermined and the same LNP production procedure is used.
A significant limitation when comparing the result is the lack of information included in previous studies. To make any certain claims about how the pulping process affects the properties of lignin NPs, the lignin raw material needs to be characterized regarding functionality, molecular weight, and PDI. This is necessary if a conclusion is to be made, that a certain set of properties connected to a certain pulping process would lead to specific particle properties. One more thing worth mentioning, is that to compare particle properties it is also necessary to produce the particles using the same procedure and the same initial lignin concentration. Otherwise differences in properties might relate to the production method or initial lignin concentration, instead of the properties of the lignin itself.
To conclude, this study has proven that the lignin extraction method will affect the properties of NPs. Using Kraft spruce lignin will lead to smaller particles with higher zeta potential compared to Organosolv spruce lignin. But in order to strengthen and generalize this conclusion, beyond the limitations of this study, more LNP experimental data is needed. A good way to do this would be to use several types of lignin from different pulping processes.
Determine functionality, Mw and PDI of the lignin raw material, and apply the same LNP production procedure for all lignin types. This would make it possible to do a broader and more accurate comparison of particles properties and eliminate the effects of experimental parameters.
26
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