Comparative Study on the Effect of Chemical-Physical and UV- Mediated Crosslinking of Quaternized Chitosan/Poly(vinyl alcohol) Membranes

DIPLOMA THESIS

to achieve the university degree of Magistra pharmaciae (Mag.a pharm.)

submitted by Christina Spirk

submitted to University of Graz

Supervisor Ao.Univ.-Prof. Mag. Dr.rer.nat. Martin Schmid Institute of Pharmaceutical Sciences

Graz, April 2017 Acknowledgement

First, I would like to thank Ao.Univ.-Prof. Mag. Dr.rer.nat. Martin Schmid for supervising my diploma thesis and for passing on so much of his joy for the area of analytics and thus turning the characterization processes, approached in this study, into my favorite part of the work. Further gratitude I want to express to Ao.Univ.-Prof. Dr.phil. Volker Ribitisch for giving me the opportunity of working with his research group repeatedly since my first summer job there in 2012. The gained experiences and insights into research are indescribable valuable and I cannot be grateful enough for that. I would like to address my sincere appreciation to Ing.in Birgit Feketeföldi, for giving me the chance of almost two years of collaboration on the project e!Polycat and for enabling the six-month internship at JOANNEUM Research Forschungsgesellschaft mbH. Thank you so much for everything you have done for me. This thesis would have never been possible without your advice. I would like to address further thanks to Jakob Pusterhofer, for all his help with the syntheses, to Helga Reischl, for always contributing support in every organizational aspect and in the laboratory, and to Dipl.- Ing. Dr.techn. Martin Koller for revising my diploma thesis and the valuable advice regarding scientific phrasing. It is not possible to personally mention each and every one, who helped me or made my time at university brighter, funnier or more mind broadening, but I would like to especially thank Constanze Strasser and Margareta Nagele for all of that and for us sticking together since the very beginning. There will never be enough words to express my appreciation for my parents, not only for making my studying possible, but for all their love and support, for always reminding me of what really matters in life, and for building the foundation of everything I am today. Last but from the bottom of my heart, I would like to thank Sascha Winter, for the priceless kind of love, that makes us a team. The kind of love, that uplifts, strengthens and challenges me to grow into the best possible version of myself. Thank you for that and also for your understanding in moments when I am so far from my best version, that the light of “best version´s” sun would take approximately 2 years to reach me.

Motivation and Target

Polymeric membranes are extensively used in a variety of applications, such as wound dressing, drug delivery devices, biopolymer electrolytes in fuel cells or for separation techniques in waste water treatment. Chitosan and poly(vinyl alcohol) are popular compounds utilized for membrane preparation. Quaternization provides one possibility to improve the features of the native polymers, which has been in the focus of research due to its outstanding membrane forming traits as well as its good biological features. The properties of quaternized chitosan and poly(vinyl alcohol) products can be altered and even further enhanced by applying various crosslinking techniques. The aim of this work was the preparation of membranes encompassing quaternized chitosan and poly(vinyl alcohol) by two different crosslinking methods. The first approach is mediated by chemical- physical crosslinking with the crosslinking agents glutaraldehyde and ethylene glycol diglycidyl ether and the second by UV-induced crosslinking. Subsequent to the preparation process, the membranes should be characterized and compared, regarding their structural and optical features, flexibility, chemical, thermal and mechanical stability, ion exchange conductivity and ethanol permeability. This study aims to provide an overview on the effect of the applied crosslinking techniques on quaternized chitosan/poly(vinyl alcohol) membranes to assess future applications for the gained products.

Abstract

Membrane series of quaternized chitosan and poly(vinyl alcohol) have been successfully prepared by applying two different crosslinking methods- chemical-physical crosslinking with glutaraldehyde and ethylene glycol diglycidyl ether and UV-mediated crosslinking. The native polymers chitosan and poly(vinyl alcohol) were quaternized with (2,3-epoxypropyl) trimethylammonium chloride in the first modification step and utilized for chemical-physical membrane preparation with glutaraldehyde and ethylene glycol diglycidyl ether. In the following synthesis procedure, glycidyl methacrylate was grafted onto the afore quaternized products to make them approachable for crosslinking via UV light. The gained methacrylated polymers were blended to prepare membranes based on photopolymerization technique with assistance of the photoinitiator Irgacure 2959. The products were characterized and compared in terms of their structural and optical properties, chemical and thermal stability, alkaline swelling behavior and ion exchange capacity by following methods: alkaline uptake by mass change, back titration, ATF-Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis and ethanol permeability. Networks formed by chemical-physical crosslinking exhibited a darker, cloudier surface, while the UV- crosslinked hydrogels were transparent and more flexible. In both crosslinking variants, the properties of the end product could be influenced by applied amount of crosslinkers or photoinitiator. Membranes prepared with glutaraldehyde and ethylene glycol diglycidyl ether present higher chemical and thermal stability, withstanding 1 M KOH at 60°C for more than 20 days without disintegration and provide an onset degradation temperature above 280°C, while photo crosslinked products lasted a maximum of 5 days in alkaline medium and present a degradation temperature of 150°C. The swelling behavior of membranes gained from UV-crosslinking is higher than within chemical-physical crosslinked membranes and the ion exchange capacity reaches a maximum value of 1.300 meq/g for product-preparation by photopolymerization technique and a value up to 1.800 meq/g for crosslinking with glutaraldehyde and ethylene glycol diglycidyl ether. Due to the high swelling behavior of UV-crosslinked membranes, the ethanol crossover was only measured for chemical-physical products. The ethanol permeability was dependent on temperature and exhibited a value reduced to 3.30*10-7 cm2*s-1 at 60°C reliant on terms of crosslinker-concentration. This study shows that both crosslinking techniques can be applied on adequate modified derivatives of quaternized chitosan and poly(vinyl alcohol), forming promising membranes with different features, with high potential for applications in various fields, such as alkaline electrolytes in fuel cell preparation or hydrogels for medical usage.

Kurzfassung

Im Rahmen dieser Diplomarbeit ist es gelungen, Membranserien aus quaternisierten Chitosan und Polyvinylalkohol durch Anwendung zweier verschiedener Vernetzungsmethoden, chemisch-physikalisch bzw. UV-induziert, herzustellen. Die ursprünglichen Polymere Chitosan und Polyvinylalkohol wurden in einem ersten Modifikationsschritt unter Verwendung von (2,3-Epoxypropyl)trimethylammoniumchlorid quaternisiert und zur chemisch-physikalischen Membranherstellung verwendet. Um diese Produkte der UV-Vernetzung zugänglich zu machen, wurden sie in einem weiteren Syntheseverfahren mit Glycidylmethacrylat substituiert. Die erhaltenen methacrylierten Polymere wurden kombiniert, um mit Hilfe des Photoinitators Irgacure 2959 mittels Photopolymerisation Membranen herzustellen. Die erhaltenen Produkte wurden in Bezug auf ihre strukturellen und optischen Eigenschaften, sowie chemische und thermische Stabilität, alkalische Quellung und Ionenaustauschkapazität untersucht und verglichen. Dies erfolgte unter Anwendung folgender Analysenmethoden: Massenzunahme in alkalischem Medium, Rücktitration, ATR-FTIR Spektoskopie, Rasterelektronenmikroskopie, und Durchlässigkeit für Ethanol. Membranen, die durch chemisch-physikalische Vernetzung hergestellt wurden, hatten eine dunklere, trübere Oberfläche, wohingegen UV-vernetzte Hydrogele transparent waren und vergleichsweise bessere Flexibilität aufwiesen. Bei beiden Vernetzungsmethoden, konnten die Eigenschaften des Endproduktes durch die zugesetze Vernetzer-bzw. Photoinitatormenge beeinflusst werden. Membranen, die mit Hilfe von Glutaraldehyd und Ethylenglycoldiglycidylether hergestellt wurden, erreichen bessere chemische und thermische Stabilität, werden bis zu 20 Tage in 1 M KOH bei 60°C nicht zersetzt, und beginnen sich erst bei Temperaturen über 280°C abzubauen. UV-vernetzte Produkte waren bis zu 5 Tage in alkalischem Medium stabil und wiesen eine thermische Stabilität bis zu 150°C auf. Die alkalische Quellung dieser Membranen ist höher als die der chemisch-physikalisch hergestellten Produkte, und die Ionenaustauschkapazität liegt bei 1.300 meq/g, während die durch Glutaraldehyd und Ethylenglycoldiglycidylether vernetzten Membranen einen Maximalwert von 1.800 meq/g erreichen konnten. Aufgrund der hohen Quellung der UV-vernetzten Membranen wurde die Ethanoldurchlässigkeit nur für die chemisch-physikalisch vernetzen Produkte bestimmt. Die Ethanolpermeabilität ist temperaturabhängig und ließ sich in Abhängigkeit der Vernetzungsmittelkonzentration bis zu 3.30*10-7 cm2*s-1 bei 60°C reduzieren. Durch die vorliegende Arbeit wird gezeigt, dass es möglich ist, entsprechend modifizierte, quaternisierte Derivate von Chitosan und Poly(vinylalkohol) sowohl chemisch-physikalisch mit Vernetzungsmitteln als auch UV-induziert zu vernetzen, was zu vielversprechenden Membranen mit unterschiedlichen Eigenschaften führt. Diese neuen Membranen erscheinen sehr vielversprechend für potentielle Anwendungen in verschiedensten Gebieten, unter anderem als alkalische Elektrolyte in Brennstoffzellen oder als Hydrogele in medizinischen Anwendungen.

Contents

Contents ...... 1 Introduction ...... 3 Experimental ...... 21 Materials ...... 21 Modification of Polymers for Membrane Preparation ...... 21 Preparation of N-[(2-Hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) ...... 21 Preparation of Quaternized Poly(vinyl alcohol) (QPVA) ...... 22 Methacrylation of HTCC ...... 23 Methacrylation of QPVA ...... 24 Methods for Characterization of Synthesis Products ...... 25 1H-NMR ...... 25 Membrane Preparation via Chemical-Physical Crosslinking ...... 26 Membrane-Preparation via UV-Crosslinking ...... 26 Methods used for Membrane-Characterization ...... 27 Alkaline Swelling ...... 27 Ion Exchange Capacity (IEC) ...... 27 Chemical Stability ...... 28 ATR-FTIR ...... 28 Scanning Electron Microscopy (SEM) ...... 28 Thermogravimetric Analysis (TGA) ...... 29 Ethanol Permeability ...... 29 Results and Discussion ...... 30 Modification for Chemical-Physical Crosslinking via Crosslinking Agents ...... 30 Quaternization of Chitosan ...... 30 Quaternization of QPVA ...... 31 HTCC/QPVA-Membrane Preparation via Chemical-Physical Crosslinking ...... 32 Modification of HTCC for UV-Crosslinking ...... 41 Modification of QPVA for UV-Crosslinking ...... 43 HTCC-g-GMA/QPVA-g-GMA-Membrane Preparation via UV-Crosslinking ...... 45 Membrane Preparation in Dependence of the Degree of Methacrylation of Quaternized Chitosan ...... 45 Membrane Preparation with Altered Amounts of Irgacure 2959 ...... 47 ATR-FTIR Measurements...... 54 Scanning Electron Microscopy ...... 57

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Thermogravimetric Analysis ...... 58 Comparison of HTCC/QPVA and HTCC-g-GMA/QPVA-g-GMA Membranes ...... 60 Conclusion and Outlook ...... 62 References ...... 63

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Introduction

Different kinds of membranes find application in various tasks of our everyday life. They play important roles in water and waste water treatment, in various medicinal applications like wound treating, implants, drug delivery systems or separation techniques for several substances as for example petrochemicals.[1, 2] Another important application of membranes is their function as biopolymer electrolyte for fuel cells, such as direct methanol/ethanol fuel cells.[3] In general, there is a broad range of materials suitable for membrane preparations for all kinds of different applications, ranging from inorganic materials like metal oxides (γ-Al2O3, ZrO2, TiO2 or ZrTiO4) or glass to various polymers as used in the present work. Polymer membranes display the advantages of lower production cost and less complicated manufacturing procedures in comparison to most inorganic membranes.[1] The separation characteristics of membranes are in general classified in terms of permeability P and selectivity α. However, further requirements for different applications encompass mechanical stability, chemical resistance against substances the membranes encounter, as well as being thin but nonetheless stable. Those standards are tried to be fulfilled by using different improvement methods on the native membrane material, which are shown as an overview in Figure 1.[1] An often-used method to enhance membrane properties is blending at least two different polymers together to take advantage of the upgrading features of both compounds in the end product. Another possibility to improve especially the chemical and mechanical stability of the native materials are surface modifications, as for example plasma treatment[1], where functional groups, such as carboxyl or amino groups, are introduced by reaction gases, e.g., NH3, CO2, to considerably influence hydrophilicity. A further alternative of surface modification is grafting polymers carrying requested features on the native membrane surface by ozone oxidation or laser treatment.[4] By incorporating additives, it is possible to create mixed-matrix membranes. Using inorganic materials, for example zeolites, leads to changes in terms of permeability. The last modification method illustrated in Figure 1 is the possibility of crosslinking native polymers to influence features such as durability or selectivity of the end products; this will be further explained in details.[4] As mentioned above, crosslinking represents an important method to improve polymeric membrane materials. To achieve crosslinked membranes, there are various approaches, depending on the chosen native polymer and its available functional groups.[4] Since this works concerns different crosslinking methods of polymeric compounds, the concept of crosslinking will be further explained in the following paragraphs.

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Figure 1. Overview over different methods to improve the features of native membrane material adequate for the required applications.

Crosslinking describes a process, where two or more molecules are bound non-covalently or covalently, due to their availability of specific functional groups.[2] Adding crosslinks between polymer chains results in changes of properties, depending on the achieved degree of crosslinking. With this modification method product features, such as elasticity or viscosity, as well as strength and toughness, can be regulated. Furthermore, the solubility behavior of crosslinked polymers is altered. While crosslinked materials cannot be dissolved, they are able to absorb solvents, up to the degree of becoming a hydrogel. Another important effect induced by crosslinking is the change of thermoplasts to thermoset plastics, which means the polymers´ shape cannot be changed by reheating without destroying the formed product, as it was comprehensively reviewed.[5] The process of crosslinking can be achieved by applying different methods whereas a general overview is already given in Figure 1. Physically crosslinked polymers are networks, which are held together by attractive non-covalent forces, such as ionic interactions, hydrogen bonding or hydrophobic interactions, e.g., van der Waals interaction.[6] In physical crosslinking, ionic interactions can be achieved under mild conditions, very often at room temperature (RT) and physiological pH-value. Another important reason for the usage of these linking method is the possibility to waive crosslinking agents, such as epichlorohydrin, glyoxal or glutaraldehyde. These compounds are often toxic, affect the integrity of substances or must be removed laboriously prior to certain applications, such as hydrogels in the medical field or materials for food packaging.[7] Physically crosslinked hydrogels are also denoted as “reversible gels”, as they

4 | P a g e have a tendency of passing through a transition from a three-dimensional stable state to eventually disintegrating into a polymer solution.[5] Another important noteworthy fact is that blends and interpenetrating networks of two dissimilar polymers are also qualified to form interaction through noncovalent crosslinking. Collagen, hyaluronic acid, galantine or alginate are the most commonly known polymers for forming physical hydrogels. Other important examples regarding this linking method are represented by dextran, which forms a hydrogel in presence of potassium ions or by synthetic polymers such as the triblock copolymer poly(ethylene oxide)–poly(propylene oxide)– poly(ethylene oxide).[5-7] Polymer chains can be crosslinked through physical forces by specific environmental triggers, such as pH-value or temperature, or physicochemical interactions, whereby the most important to mention are hydrogen bonding, hydrophobic interactions, charge condensation, stereo complexation or, representing a more modern approach, supramolecular chemistry.[8] The listed forces for physical crosslinking will be explained further hereafter. Hydrogen bonding interactions form hydrogels for example by freeze-thawing processes, with poly(vinyl alcohol) (PVA) as a typical representative, as shown in Figure 2.

Figure 2. Crosslinking of poly(vinyl alcohol) (PVA) via hydrogen bonding actions (source: http://www.chemgapedia.de/ vsengine/vlu/vsc/de/ch/9/mac/netzwerke/vernetzung/physikalisch.vlu/Page/vsc/de/ch/9/mac/netzwerke/vernetzung/pva. vscml.html)

The viscoelastic features of polymers linked by hydrogen bonding are demonstrably more gel-like than the single compounds alone, but those networks can dilute easily in aqueous medium or can be disrupted by shear forces. Although hydrogels formed by hydrogen bonding are comparatively unstable, they are part of valuable biocompatible systems used in short acting drug release.[9] Hydrophobic interactions between polymers in aqueous environment can lead to crosslinking via reverse thermal gelation. This process is known under the term “sol-gel” chemistry.[8, 9] Sol-gel materials involve various inorganic and organic compositions, united by a certain preparation strategy. The so called “sol-gel processing” includes the preparation of colloid suspensions - in short “sols” - which are converted to gels, that usually exhibit high viscosity features and eventually become

5 | P a g e solid materials.[10] Gelation driven by hydrophobicity is reached by coupling a hydrophilic polymer with a hydrophobic segment (gelator), creating an amphiphilic compound[8, 10]; the gained product is soluble in water at low temperatures. However, by increasing the temperature, the hydrophobic parts of the polymer aggregate to minimize the hydrophobic surface contacting the bulk water. Due to this effect, the amount of water surrounding the hydrophobic groups is reduced and the solvent entropy is maximized.[8] Advantages of sol-gel synthesized materials are for example the low temperature used during the process, the mild chemical conditions regarding pH-value or expedient control over pore size or mechanical strength of the end product. Well-known compounds used for sol-gel preparations are triblock copolymers like poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide).[8] Products gained from thermal gelation find application as optical components, parts of drug delivery systems or for ceramic manufacturing.[8-10] Disadvantages of sol-gel-processing are very often time- consuming procedures, involving high costs.[10] Other physical forces being used for crosslinking are charge interactions, which may occur between two polymers or a polymer and a small molecule carrying opposite charges, as pictured in Figure 3. Examples for this crosslinking method between polymers are quaternized chitosan and glycerophosphate, which from ionically cross-linked networks at physiological temperature.[8] Small molecule cross-linking can be achieved within elastin-like polypeptides between lysine residues and organophosphorous groups.[8, 11] Charge interactions are also able to link micro- or nanoparticle gels resulting in three-dimensional particle assemblies. Dextran microspheres combined with anionic and cationic polymers can perform spontaneous gelation, which enables various applications as for example drug delivery systems.[8] Recent approaches for physical crosslinking represent stereocomplexation and supramolecular chemistry. Stereocomplexation sums up interactions that can occur between same chemical compositions that exhibit different stereochemistry. Polylactide blocks with L- and D-stereoisomers are an important example for forming hydrogels in this manner.[8, 12] The resulting products are highly biocompatible as no chemical crosslinkers or organic solvents are needed.[13] However, even small stoichiometric changes of the compounds can lead to significant weakening of the formed network.[8] The concept of supramolecular chemistry was for the first time addressed in 1978 with the definition of “chemistry beyond the molecule”, based on molecular assemblies and intermolecular bonds.[14] Supramolecular chemistry contains concepts of molecular recognition and self-organization, leading to programmed chemical systems, finding application in many scientific fields such as polymer chemistry, material science, nanoscience and nanotechnology. Other important scopes of application are solid-state chemistry, biological interactions and drug design, crystal engineering or sensor and diagnostic procedures.[14] Common types of network forming within this category are inclusion

6 | P a g e complexes consisting of poly(alkylene oxide) polymers and cyclodextrins, which have hydrophilic surfaces as well as hydrophobic pockets. Due to that structure features, they display geometrical compatibility with poly(alkylene oxide)-based polymers. Interactions between glycosamino glycans, like heparin, and polymer-grafted peptide sequences represent another example for this kind of crosslinking, resulting in rapidly forming networks.[8, 14, 15] Polypeptide-based systems are of great interest as they exhibit flexibility, good mechanical properties including the possibility of tuning the gelation time, to control further desired features needed for various applications as for example controlled drug release.[8, 16]

Figure 3. Charge interactions between a polymer carrying negative charges and a small molecule carrying positive charges shown schematically as an example for physical crosslinking. (Source: https://www.chemie.fu-berlin.de/chemistry /kunststoffe/quer.htm)

The second crosslinking method is referred to as chemical crosslinking, as already illustrated in Figure 1. Chemically crosslinked polymers are characterized by network forming due to covalent bonds. Such networks are usually more stable in comparison to physically crosslinked compounds, which results in chemically crosslinked hydrogels often being referred to as “permanent gels”.[6] On the one hand, chemical crosslinking represents a highly versatile method to improve mechanical properties, but, on the other hand, crosslinking agents are very often toxic and not environmentally friendly. There is a wide range of crosslinking compounds that are used for forming covalent polymer networks, e.g., glutaraldehyde, glycolaldehyde, formaldehyde or epoxy compounds, such as epichlorohydrin.[5] Their chemical structure is shown in Figure 4.

O O O O H O H H OH H H Cl

glutaraldehyde gylcolaldehyde formaldehyde epichlorohydrin Figure 4. Chemical structures of often used chemical crosslinking agents.

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The most important aspect of crosslinkers are their reactive groups as they establish the method and the mechanism of the chemical modification. The functional groups that are commonly targeted for conjugation reactions include primary amines, sulfhydryls, carbonyls, carbohydrates or carboxylic acids. The most common chemical crosslinking reactions are illustrated in Figure 5. The type and degree of crosslinking exhibit great influence on network properties, such as swelling behavior, elasticity or network permeability.[5, 8] Especially Michael additions between amines or thiols and a vinyl group are widely used for preparing membrane networks, because of the advantages of rapid reaction time, flexibility and biological inertness of the products.[8, 17] Further important factors to control the properties of the final product are the concentrations of the used product compounds, which are often limited by restricted solubility of particular polymers.[7]

2 O N R 1 R 2 1 H O + H2N R R + 2 H H aldehyde amine Schiff base

2 R O 1 H2N NH N NH R H O + 2 1 + 2 R R H H aldehyde hydrazine hydrazone

1 R O 1 O 2 2 R R O + H2N R O H2C acrlyate primary amine secondary amine

1 R O 1 O S 2 2 R R O + HS R O H2C acrlyate thiol sulfide

Figure 5. Overview of the most common chemical crosslinking reactions; the first reaction shows the formation of a Schiff base from aldehyde and amine; the second is the reaction of an aldehyde and hydrazine to hydrazone; the third and fourth are examples of a Michael reaction with acrylate and primary amine or thiol forming a secondary amine or a sulfide.

Chemically crosslinked hydrogels can be synthesized by different approaches, such as chain growth polymerization, addition and condensation polymerization or gamma beam, electron beam enclosing UV-polymerization. Chain-growth polymerization includes free radical polymerization, controlled free

8 | P a g e radical polymerization as well as anionic and cationic polymerization. It usually follows the three-step process of initiation, propagation and termination. Within this process free radical sites are generated, which link monomers up in a chain-like fashion.[5] In general membranes and hydrogels can be formed by cross-linking of various functional groups, such as hydroxyl, amine or hydrazide groups, within the polymer backbone by glutaraldehyde. Non-biodegradable synthetic hydrogels can be fabricated from copolymerization of various vinylated monomers or polymers, such as 2-hydroxyethyl methacrylate (HEMA), 2-hydroxypropyl methacrylate (HPMA), acrylamide (AAm), acrylic acid (AAc), poly(ethylene glycol) (PEG) or ethylene glycol diacrylate (EGDA) to mention some of the most common. Covalently crosslinked hydrogels can also be formed via enzymatic crosslinking, which is for example applied with gelatine.[5] One of the most common methods is crosslinking by aldehyde derivatives, such as glutaraldehyde. Due to its´ toxicity, alternatives have been developed, as for example gel preparation from crosslinking of gelatine with polyaldehydes from partial oxidation of dextran.[7] However, the disadvantage of the drastic conditions, such as low pH-value or high temperature needed for this crosslinking method remains, even without the usage of toxic crosslinking agents.[7] Examples of compounds for crosslinking via addition reaction are 1,6-diisocyanatohexane or 1,6-dibromohexane. Usually these reactions have to be carried out in organic solvents to prevent the agents from reacting with water [7], which leads to its limitation for water-insoluble substances. An efficient reagent for crosslinking by condensation reactions is N-ethyl carbodiimide, which can be used to crosslink alginate and PEG- diamines.[18] An important differentiation within chemical crosslinking has to be drawn between small-molecule crosslinking and polymer-polymer crosslinking.[8, 19] A recent example for small-molecule crosslinked hydrogels are products from dextrane tyramine and hyaluronic acid tyramide prepared with horseradish peroxidase and hydrogen peroxide as cross-linkers.[19] Human serum albumin crosslinked with an active ester of tartaric acid represents another example within this concept. The final product was described as highly tissue-adhesive hydrogel able for applications of controlling doxorubicin release.[20] A general disadvantage of small-molecule crosslinking methods is the occurrence of remaining unreacted molecules, which cause potential toxicity of the products.[8] Preparations using polymer-polymer crosslinking sometimes present the advantage of avoiding the use of potentially toxic small-molecule crosslinking agents. However, very often this method requires pre-modifications of the used polymers, which can include toxic compounds as well. The advantages of polymer-polymer linked products are rapid and usually stable crosslinking, as it was performed with hyaluronic acid crosslinked by hydrazone bonds.[8, 21] Another example of this crosslinking strategy were conducted using dextran, PVA, or poly(aldehyde guluronate).[8, 22, 23]

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One of the best-known chemical crosslinking processes is the vulcanization of rubber, which has been patented in 1844 and is shown in Figure 6. This crosslinking method greatly improves the durability of rubber, as it would start showing soft and sticky features at temperatures as low as 30°C.[24]

CH3 CH3 CH3 S S S S + S CH3 CH 3 CH3 S S S

CH 3 CH3 CH3

S S S S S S S S S S

H C H C 3 3 H3C Figure 6. Schematic representation of the vulcanization of rubber as one of the best known chemical crosslinking process.

As already shown in Figure 1, photochemical crosslinking represents a subsection of chemical crosslinking. In the field of photochemistry, chemical processes are caused by absorption of light, whereby different photochemical reactions can be initiated by infrared, visible or ultraviolet light.[25, 26] When an atom or molecule absorbs a quantum of light energy from a photon, the energy of the atom or molecule increases from its normal level to an excited or activated state.[26] The excited molecule can undergo various primary photochemical processes, as for example photoionization, photodissociation or luminescence. These processes are the consequence of the activated molecule returning to its initial state, by releasing excess energy or transfer that energy to another molecule by colliding. The term photoionization describes the process, when an electron escapes from the molecule due to high-energy absorption from the initial light. Photodissociation occurs when the excited molecule breaks into atomic and/or molecular fragments. This process can be followed by a rearrangement of the fragments leading to photoisomerization.[26] If the primary photochemical process leads to dissociation of a molecule into radicals (unstable fragments of molecules) the secondary step can involve a chain reaction, also called propagation, which describes a cyclic process where a radical attacks another molecule and produces another unstable radical , which continues the chain formation.[26] The photocrosslinking of compounds for membrane preparation, such as

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polymers, can be achieved using a photosensitizer and irradiation.[27] Most photosensitizers crosslink via formation of radicals following the pattern of initiation, propagation and termination, as shown in Figure 7.[26, 27]

Initiation

퐼푛𝑖푡𝑖푎푡표푟 + ℎ휈 → 푅 · 푅 · + 푀 → 푅푀 · Propagation

푅푀 · + 푀푛 → 푀푛+1 · Termination

푅푀푛 · + · 푀푚푅 → 푅푀푛푀푚푅

푅푀푛 · + · 푀푚푅 → 푅푀푛 + 푀푚푅

Figure 7. Overview of the scheme of photochemical reactions following the steps of initiation, propagation and finally termination.

The efficiency of photochemical processes is measured by the quantum yield, which provides insight in the amount of energy absorbed from each photon. For the primary process the quantum yield ranges from 0 to 1. However, for chain reactions the value can be as high as one million, as for every photon absorbed many products are received.[26] The process of UV-crosslinking has the advantage of being executable at RT and physiological pH-value without the usage of toxic crosslinking agents, which allows it to be classified as “green” process and contributes to its popularity as crosslinking method.[27] Other important benefits of photopolymerizable systems include powerful spatial and temporal control of the reactions kinetics and minimal heat production.[28] Due to these reasons, as well as to the fact that it does not require expensive devices, photocrosslinking systems have come to use for biomedical applications, drug/cell delivery systems, coatings for biosensors or cell transplantations.[29] There are compounds, which are naturally photosensitive, like coumarin or natural acids, while others require preparatory modifications to be able to react under UV irradiation.[28] One frequently used method to achieve this ability is to insert photocrosslinkable functional groups, like (meth)acrylate-derivatives. A collection of examples often applied for the modification of various polysaccharides like cellulose, hyaluronic acid or chitosan are shown in Figure 8.[28]

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O CH2 CH2 H C H2C O O 2 O O O O CH3 CH3 CH3 Cl

glycidyl methacrylate methacrylic anhydride acryloyl chloride

O O O OH O H2C NH2 O NH H2C

CH3 H2C CH3 N C O

aminoethyl methacrylate N-methylol acrylamide 2-isocyanatoethyl methacrylate

Figure 8. Typical compounds that are used to insert photocrosslinkable functional groups to polymers. The photosensitive groups of each chemical structure are highlighted in red.

Methacrylates have excellent features for photopolymerization because of their high photocuring rate and - according to literature - suitable mechanical properties of the resulting products.[28] From the examples given in Figure 8, glycidyl methacrylate (GMA) and methacrylic anhydride (MA) are the most often used compounds to modify polysaccharides. A favored approach is the reaction of glycidyl methacrylate with the hydroxyl groups of various polysaccharides such as dextran, heparin or hyaluronic acid.[28, 29] The process of grafting GMA onto polysaccharides is usually performed in aqueous media or buffer media, such as phosphate buffer solution.[28-30] Glycidyl methacrylate is only slightly soluble in aqueous media often chosen to dissolve the polysaccharides, but it can be degraded in water. The degradation takes place via hydrolysis of the epoxy group. The esterification of the hydroxyl groups of the polysaccharide compound is achievable in aqueous medium as well.[28] As already mentioned before, photopolymerization reactions are driven by free radicals, produced by certain chemicals when exposed to specific wavelengths of light. Usually a photoinitiator is used to induce the formation of free radicals. Thereby a photon from the UV light source excites the utilized photoinitiator and leads to its dissociation. The gained radical then induces the following polymerization. There is a variety of photoinitiators with different absorption spectra available and research continues to develop further examples. 2-hydroxy-1-[4-(hydroxyethoxy) phenyl]-2-methyl-1- propanone (Irgacure 2959), 1-hydroxycyclohexyl phenyl ketone (Irgacure 184) and 2,2-dimethoxy-2- phenylacetophenone (Irgacure 651) represent often applied examples. However, it is known that high- energy radicals cause damage to cell membranes, nucleic acids and proteins, usually causing cell death.[31]

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Most chemical initiators have toxic properties and are difficult to remove from the final product. However, it was shown that the photoinitiator Irgacure 2959 (see Figure 9) caused minimal toxicity over a broad range of mammalian cell types and species.[31] One possible approach to renounce the usage of a photoinitator is to choose monomers like acrylic acid, which form free radicals directly through adsorption of UV radiation.[32] As it was shown, the water soluble photoinitator Irgacure 2959 is well tolerated over a wide range of cells types, hence it was chosen for the UV crosslinking experiments within this work.[31]

O OH

CH3 H3C HO O

Irgacure 2959

Figure 9. Chemical structure of the photoinitiator 2-hydroxy-1-[4-(hydroxyethoxy) phenyl]-2-methyl-1-propanone, which was used for UV mediated crosslinking within this work.

UV-mediated crosslinked polysaccharides usually form hydrogels, but it is possible to alter their properties by blending different polysaccharides or polysaccharides with other polymers. Applying for crosslinking with chemical agents as well as UV-mediated crosslinking, the resulting materials are termed as hybrid polymer networks (HPN), interpenetrating polymer networks (IPN) or semi interpenetrating polymer networks (SIPN).[28] These specific terms will be explained later. Hydrogels in general represent crosslinked hydrophilic polymeric structures capable of incorporating large amounts of water or biological fluids. This feature entitles them to numerous biomedical and pharmaceutical applications, such as protein delivery systems, production of contact lenses, implants or scaffolds. The soft and rubbery nature of hydrogels ensures low risk of issue irritation, and cells and proteins show a low tendency to adhere to the hydrogel surface. Furthermore, hydrogels are applied in fiber and metallic cable sealing, thickening agents for cosmetics, feminine hygiene products and water technologies, among many others.[2, 5] The formation of HPN, IPN or SIPNS can be initiated by combining synthetic or natural polymers.[28, 33] In the most common formed network, the so-called hybrid polymer network (HPN), blended polymers are modified with the same crosslinkable functional groups. Interpenetrating networks (IPN) however are defined as a combination of two or more network polymers, without a link between the different polymers. To sum up the IUPAC definition, an IPN is a polymer comprising two or more networks, which are at least partially interlaced but not covalently bound to each other, however the compounds

13 | P a g e can only be separated if chemical bonds are broken. This entanglement leads to a “forced miscibility” between usually incompatible polymer blends and results in the manufactured product exhibiting dimensional stability. This defines the main difference to the term “blend”, which represents a mixture of two or more preformed polymers. Semi-interpenetrating polymer networks (SIPN) are composed of one linear or branched polymer entrapped within the network of another polymer.[28] They differ from IPNs, as the linear or branched polymers can be separated from the constituent network without chemical bonds being broken.[28, 34] As already explained in detail, the fabrication of crosslinked networks can be approached using different methods, including irradiation, thermal annealing or the use of chemical crosslinking agents.[5] The chosen methods for approaching crosslinking of the main polymers within this work - chitosan and PVA - will be discussed in a separate point, following a general introduction of the deployed polymers. Characteristics of chitosan and PVA were reviewed before.[2] Chitosan has been chosen as a polymer for membrane preparation within this work, because it is proven to possess very good film forming capabilities as well as biocompatibility and biodegradability. Further appreciated features of chitosan are its good muco-adhesive and antibacterial properties. The hydrophilicity of chitosan, resulting from the presence of amino and hydroxyl functional groups within its repeating structural unit, is responsible for the solubility of chitosan in water. This feature also induces the formation of a rubbery hydrogel in aqueous solutions. The properties of chitosan can be adjusted by chemical modification or by blending with other polymers, such as poly(vinyl alcohol), which represents the second utilized polymer in this study.[2] PVA is an inexpensive hydrophilic semi crystalline polymer, which - just as chitosan - shows excellent film forming properties. It is reported to produce effective electrolytes after doping with potassium hydroxide (KOH) solution, resulting in PVA matrices being outstanding ionic conductors.[2] These two polymers have been successfully used alone or combined for a variety of applications, and were therefore implemented in the membrane preparations within this work.[2, 36] In the following sub-points the origin, characteristics and modifications of chitosan and poly(vinyl alcohol) are examined in more detail. Starting with chitosan, it is an N-deacetylated derivative of chitin, which represents one of the most abundant natural polysaccharides, second only to cellulose.[2, 38] Chitin can be found as an important component of exoskeletons of arthropods, such as crustaceans like crabs or shrimps, or various insects. Furthermore, it represents a constituent of the beaks and internal shells of cephalopods, including octopuses and squids and the radulae of mollusks.[2] Another

14 | P a g e important natural deposit of chitin are cell walls of various fungi, where it fulfils tasks such as maintaining the overall strength of the cell wall or enabling protective modifications under cell wall stress conditions.[39] Beholding Chitin (poly(1-4)-N-acetyl-D-glucosamine) from chemical aspects, it is a biopolymer consisting of ß-(14)-linked D-acetylglucosamin units. Although the glucosamine units are arranged in the same linkage as cellulose, the replacement of the hydroxyl groups of cellulose by acetamido groups leads to the forming of stronger hydrogen ponds, resulting in the mechanically more stable features of chitin compared to cellulose. Naturally occurring chitin represents a mixture of statistic copolymers, containing D-glucosamine and N-acetyl-D-glucosamine. The resulting degree of acetylation determines the characteristic of chitin. The transition from chitin to the N-deacetylated derivate chitosan is defined by an extent of acetylation of less than 50%, as it is illustrated in Figure 10.[2, 37, 40, 41]

OH OH OH

HO O O O O O HO HO HO OH R R n R

R : 1 NH2

R2: NH O H C 3

Figure 10. Structure of chitin and chitosan. If R is more than 50% R1 (-NH2) it is defined as chitosan; if R is more than 50% R2 (-NHCOCH3) it is defined as chitin.

Besides the degree of acetylation, the characteristics of chitosan are influenced by its molecular weight, which can vary between 10000 - 1 million Da approximately.[2, 40] While chitin is virtually insoluble, chitosan is soluble in dilute organic acids, e.g., in acetic, formic or lactic acids. At a neutral pH-value, chitosan is not water soluble, however solubility can be achieved at pH-values lower than 6.0.[40] Chitosan and its derivatives have been in the focus of research due to its biological features and broad areas of application, such as in medical, food, industrial or agricultural fields. Chitosan presents itself

15 | P a g e as a very attractive material concerning its good biocompatibility with the human body, its hydrogen- bonding capability as well as its biodegradability properties, to mention but a few examples. Furthermore, chitosan is the only pseudonatural cationic polymer. This feature expands its application possibilities to for example flocculants for protein recovery or industrial depollution tasks.[2, 37] The versatile properties of chitosan make it a basic material for the preparation of various hydrogels, films, fibers or sponges.[37, 40] The most promising chitosan products find application in pharmaceutical and biological areas and within cosmetics, especially for hair care in relation to electrostatic interactions.[37] Compositions including chitosan include drug delivery systems of oral, nasal, parenteral or transdermal application.[37] The mucoadhesivity of chitosan enhances the adsorption of drugs especially at neutral pH-value.[42] Chitosan gels or layer-by-layer polyelectrolyte capsules are popular systems for controlled release of drugs or proteins.[37] Another important feature of chitosan is its antiviral and antiphagal activity as well as its ability to inhibit the growth of bacteria and therefore to prevent bacterial infections.[43] Products including chitosan or chitosan derivatives are also important elements of wound dressing or bone tissue engineering.[44, 45] To improve the potential of chitosan even further and to develop it into an advanced functional material, it has been subject to several various chemical modifications, for example N-alkylation, N- acrylation or N-carboxyalkylation.[46] By inserting specific side chains, new or increased material properties, such as better solubility behavior in water by adding hydrophilic substituents, are established.[2] Chitosan displays three different functional groups - hydroxyl, primary amine and ether groups - per unit, whereas the -OH und -NH2 groups enable different chemical modifications, such as saccharization, alkylation, acylation, quaternization and metallization, making derivatized chitosan available for several specific applications.[41, 47] Important examples for chitosan derivatives are O- and N-carboxymethyl chitosan, which are amphoteric polymers and exhibit a solubility in dependence of pH-value[37] or chitosan 6-O-sulfate, which represents an anticoagulant.[48] Other interesting modification products are N-methylene phosphonic chitosan, which can form complex ions with metals such as cupper or zinc and therefore provides corrosion protection for metal surfaces[49] or carbohydrate branched chitosan, which achieves excellent water solubility.[50] One of the most explored derivatives of chitosan is poly(ethylene glycol)-grafted chitosan, which is soluble in water as well.[37] In this work, chitosan was derivatized by introducing quaternized groups via (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC), achieving, amongst others, better solubility. After that modification, glycidyl methacrylate was grafted onto the gained quaternized product, to make it accessible for UV-crosslinking. On that account, these two modifications will be explained in detail.

16 | P a g e

The first applied modification - quaternization - is one of the main chemical modifications performed with chitosan. It can be carried out in aqueous solutions under relatively mild conditions to obtain randomly distributed substituents along the polymer chain of chitosan with controllable degree of substitution. This chemical modification is applied to discard the limitations of usage for chitosan due to its poor solubility and to introduce positively charged functional groups leading to an improvement in ionic conductivity in the final membrane preparation.[3] Quaternized polymers hold a high degree of hydrophilicity, which results in moderate mechanical stability, whereby trimethylammonium groups ensure better chemical stability than other functional groups, such as pyridinium, sulfonium of imidazolium.[3, 51] Common reagents for quaternizing chitosan are methyl iodide or glycidyl trimethylammonium chloride, whereas the second was used for preparing [(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) in this work. The chemical equation regarding this process will be explained in detail in the experimental part of this work. In order to make the afore prepared HTCC accessible for the application of UV-crosslinking, which represents the second linkage method processed in this work, it is necessary to modify the product with certain functional groups capable of crosslinking via photopolymerization.[52] Through polymer grafting, a significant alteration of physical and mechanical features of the modified product can be achieved.[53] Based on previous works glycidyl methacrylate (GMA) has been chosen as grafting reagent, because of its two reactive functional groups, firstly a highly reactive epoxy group and secondly an methacrylic group.[52] Furthermore, it is reported that GMA favorably reacts with chitosan by minimizing steric hindrance along the hydroxyl groups of the chitosan backbone.[52] In this work two different synthesis prescriptions were utilized for grafting HTCC with glycidyl methacrylate, based on previously existing literature[52, 55], although both describe the modification processes for chitosan. However, the general reaction processes were still adaptable for HTCC, as the derivatization with GMA takes place on the accessible hydroxyl group, instead of the afore occupied amino group of chitosan.[54] The performed modification procedures will be shown in detail in the experimental part of this work. The second polymer PVA - is a linear synthetic polymer, which is prepared by partial or full hydrolysis of poly(vinyl acetate), resulting in removing acetate groups, as shown in Figure 11.

17 | P a g e

CH3 CH3

O O O O OH OH Hydrolysis

n n Figure 11. Preparation scheme of PVA via hydrolysis of poly(vinyl acetate).

The degree of hydroxylation determines mechanical, chemical and physical properties of PVA, whereby levels of hydrolysis are typically ranging from 80-99%. The resulting polymer is soluble in water but insoluble in most organic solvents. PVA has a broad field of possible applications and has been used as a pharmaceutic aid, ophthalmic lubricant, synthetic alternative for native cartilage replacement in medicine, cosmetics as well as in fields of surface coatings and modifications for the past 30 years, as comprehensively reviewed.[56] One of the reasons explaining the wide ranged applications for PVA is the fact that its manufacturing process can be diverted in different directions to generate diverse biomechanical properties. Adjustments in the thawing and freezing protocol, saline addition or crosslinking agents modulate the characteristics of the end product.[56] PVA exhibits the characteristic of high hydrophilicity, low toxicity, as well as biocompatibility. Therefore, it is commonly used in the textile industry, food packaging industry, paper products manufacturing as well as in the medical field. In medical devices, PVA represents a popular compound because it is nontoxic, noncarcinogenic and bioadhesive. Membranes prepared with PVA are furthermore reported to show high stability in acidic or alkaline environments.[56] Another advantage supporting the broad usage of PVA is that the wear PVA particles are less harmful than wear particles of comparably polymers, like Ultra-high-molecular-weight poly(ethylene) (UHMWPE) or metals.[57] PVA also represents expedient hydrogel and membrane forming properties, which enable applications such as contact lenses, artificial pancreases, hemodialysis or implantable medicinal materials to name just a few.[56, 58, 59] In addition, PVA is a low cost hydrophilic polymer and has a high selectivity of water compared to alcohol, which leads to low methanol as well as ethanol permeability and makes PVA-based membranes deployable for anion exchange fuel cells. Products prepared with PVA also show high tensile strength and elongation at break[56], which adds up to the reasons why PVA was chosen for combination with quaternized chitosan for various membrane preparations in the present work. PVA provides one functional group per unit accessible for chemical modification reactions. The most common modifications of the hydroxy groups are esterification, etherification or acetalization.[61] Other important approaches are the incorporation of tosyl, azide and amine groups leading to an

18 | P a g e improvement of the reactivity of PVA.[61] In this work, PVA was modified via quaternization with (2,3- epoxypropyl) trimethylammonium chloride. PVA is reported to produce effective electrolytes after doping with a KOH solution. The chemical interactions, such as dipole-dipole interaction and hydrogen bonding, can be achieved between PVA and KOH during alkali doping, which increases the ionic conductivity of PVA.[60] It has been reported that the introduction of quaternary ammonium groups enhances the hydrophilicity and water selectivity of prepared membranes. Due to the positively charged functional groups, the ion-exchange capacity of quaternized PVA (QPVA) is higher than that of native PVA.[36] Based on literature, (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC) was grafted onto the accessible hydroxyl groups using KOH, with the exact description and reaction mechanism explained in the experimental part of this work. The introduced positively charged amino group acts as charge carrier in the PVA matrix.[60]

H3C

HO CH O 2

O

OH O

O O O O O HO O n HO n NH NH

OH OH

+ + H C N CH H3C N CH3 3 3 - CH - CH3 Cl 3 Cl

HTCC HTCC-g-GMA

O

HO CH3 O - Cl CH + 3 CH2 OH OH O N OH O OH CH3 OH CH3 H C CH 3 3 H3C CH3 n n

QPVA QPVA-g-GMA

Figure 12. Chemical structures of the modified polymers in this work; quaternized chitosan prepared with (2,3-epoxypropyl) trimethylammonium chloride; glycidyl methacrylate grafted onto HTCC; quaternized poly(vinyl alcohol) (QPVA) prepared with (2,3-epoxypropyl) trimethylammonium chloride; glycidyl methacrylate grafted onto QPVA.

19 | P a g e

To make the afore prepared QPVA approachable for crosslinking via UV light, it has to be chemically modified by introducing a photocrosslinkable functional group. Therefore, the same reagent and a similar procedure that were used for gaining chitosan modified with GMA (HTCC-g-GMA) was applied.[52, 53] The reaction conditions and chemical structures of the process will be shown in detail in the experimental chapter of this work. Both quaternized chitosan and PVA are capable of being crosslinked by various dialdehydes such as glyoxal or glutaraldehyde[3, 62] as well as by epoxide compounds such as epichlorohydrin or ethylene glycol diglycidyl ether.[3, 63, 64] Based on its free hydroxyl groups, QPVA is also enabled to crosslink via hydrogen bonding, as already shown in Figure 2.[3, 5] Covalently crosslinked quaternized chitosan is capable of crosslinking with itself as well as with QPVA forming an interpenetrating network. In addition to the covalent bonds, secondary interactions, such as hydrogen bridges or hydrophobic interactions, occur within chitosan networks. For the physical-chemical crosslinked membrane series within this work, the crosslinking is formed as dialdehydes react with the hydroxyl groups of QPVA and the amine groups of chitosan in order to create stable acetal and imine bonds, respectively. The epoxide compounds contributed by ethylene glycol diglycidyl ether are forming networks with hydroxide and amine groups by opening the epoxide ring. Additionally, the crosslinking process can be supported by thermal treatment with the aim of improving thermal and chemical properties of the composite membrane.[3] The crosslinking procedure will be described in detail in the experimental part of this work and the formed interpenetrating networks will be shown within the results. By grafting glycidyl methacrylate onto quaternized chitosan as well as QPVA, both polymers became capable to be crosslinked by photopolymerization technique. Based on this concept, a sol-state can be transformed to a hydrogel by free radical polymerization[32, 53] as it is demonstrated schematically in Figure 7. The forming of interpenetrating networks between the modified polymers was initiated with Irgacure 2959.[31] The schematic of the radical formation of Irgacure 2959 will be shown in the experimental part followed by descriptions of the formed hydrogels within the results. The prepared products of both crosslinking concepts are characterized in terms of their structural properties, flexibility and chemical, thermal and alkaline stability and their ion transport capabilities and ionic properties are investigated within this work. The results of their features will be prepared leading to a comparative study on the effect of chemical-physical and UV-mediated crosslinking of quaternized chitosan/PVA membranes.

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Experimental

Materials

Chitosan (low molecular weight), poly(vinyl alcohol) (Mw 89,000-98,000, >99% hydrolyzed), (2,3- epoxypropyl) trimethylammonium chloride (EPTMAC), ethylene glycol diglycidyl ether (EGDGE), glutaraldehyde (25 wt.% content in distilled water), 2-hydroxy-4´-(2-hydroxyethoxy)-2- methylpropiophenone (Irgacure 2959), poly(ethylene glycol)diacrylate (average Mn 250), deuterium oxide (99.9 atom-% D) and acetic acid-d4 (99,5 atom-%) were purchased from Sigma-Aldrich HandelsgmbH Austria and N,N-dimethylformamide (MERCK, 99.9% p.a.) was provided by VWR International GmbH, Vienna, Austria. Acetic acid (ROTIPURAN, 100% p.a.), acetone (ROTIPURAN, 99.8% p.a.), ethanol (ROTH, >99%) and tetrahydrofuran (THF) (ROTH, >99%) was obtained from LACTAN VertriebsgmbH & Co KG, Graz, Austria and were used as described in the following procedures without further purification.

Modification of Polymers for Membrane Preparation

Preparation of N-[(2-Hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC)

OH O H3C O + CH3 O + N HO O CH H3C 3 CH3 NH2 n Chitosan EPTMAC

60°C 4h

OH H C 3 O O HO O CH3 NH n

OH

CH3 + N CH3

H3C

HTCC Figure 13. The quaternization of chitosan with glycidyl trimethylammonium chloride to gain HTCC.

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6.0 g Chitosan were dispersed in 60.0 g deionized water and heated to 80°C. After 10 minutes 17.7 g (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC) were added and the mixture was continuously stirring at 80°C under reflux for a maximum of 4 hours. After the reaction period, the product was precipitated in cooled acetone and filtered off before being dispersed in ethanol for a minimum of 10 minutes. The washing step was followed by another filtration before repeating the precipitation step in acetone once more. Afterwards the yellow-white product was gathered and dried overnight at RT in the vacuum oven. The characterization of the product was ensured by 1H-NMR, whereas the degree of quaternization was calculated using Equation 1.

Preparation of Quaternized Poly(vinyl alcohol) (QPVA)

5.0 g PVA were dissolved in 100.0 g deionized water over night and filtered using a 5 µm PFTE filter. 3.6 g 2 M KOH, which was prepared separately, together with 10.0 g EPTMAC were added to the solution and followed by a reaction time of 4 hours at 65°C under constant stirring. Subsequently the product was precipitated in cooled acetone. After about 10 minutes, when the formation of a white spongy solid was observable, it was transferred into ethanol and kept boiling for 20 minutes repetitively. The obtained product was dried overnight at RT in a vacuum oven. For characterizing the prepared solid, a 1H-NMR spectrum was recorded and interpreted by using Eq. 2.

OH OH OH CH 3 - + N Cl + CH H3C CH3 3 n O H3C PVA EPTMAC

KOH 65°C, 4h

- Cl + CH3 OH O N CH3 OH CH3 H C CH 3 n 3 QPVA

Figure 14. The quaternization of poly(vinyl alcohol) (PVA) with glycidyl trimethylammonium chloride (EPTMAC) in alkaline medium to gain quaternized poly(vinyl alcohol) (QPVA).

22 | P a g e

Methacrylation of HTCC

Synthesis Prescription 1

1.0 g of the afore prepared N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) was dissolved in 60 mL 0.4 M acetic acid while stirring at 60°C. When the polymer was completely dissolved, the solution was cooled down to RT before various amounts of GMA were added dropwise through a septum using a syringe whilst stirring was continued. After adjusting the pH-value to 3.8 using 0.4 M KOH solution, the reaction was allowed to proceed for 3 hours at 60°C under nitrogen atmosphere. At the end of the reaction time, the solution was cooled down in a refrigerator (4°C) for a minimum of 30 minutes to stop any further reactions before the solution was poured dropwise into acetonitrile under constant stirring to precipitate the product. The obtained white, fiber-like substance was washed with THF and dried at least overnight at RT using the vacuum oven. After drying, the gained synthesis product was characterized via 1H-NMR and the degree of methacrylation was calculated using Equation 3.

Synthesis Prescription 2

1.0 g quaternized HTCC was dissolved in 60.0 g deionized water whilst stirring at 60°C and afterwards cooled to RT. 5.0 ml GMA were added dropwise in the same way as in synthesis prescription 1. The pH-value was adjusted to 3.8 using 0.4 M acetic acid before adding 60 mL N,N-dimethylformamide . Afterwards the reaction was stirred at RT for various periods of time (5-14 days). After the given reaction time, the product was precipitated in acetone while stirring and then washed repetitively in THF. The obtained yellow-white product was dried at RT overnight and characterized following the same procedure as described in Synthesis prescription 1.

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OH O CH3 O H2C O O O HO O + H3C R 2 n CH3 HTCC GMA

R1: OH KOH O pH 3.8 CH3 O O

OH CH2

R1 NH R2: 2 CH3 O CH3 O HO O + H3C N CH3 R2 n CH3 NH OH HTCC-g-GMA

Figure 15. The chemical reaction of grafting glycidyl methacrylate onto N-[(2-Hydroxy-3-trimethylammoinium) propyl] chitosan chloride (HTCC) in alkaline medium at a Ph-value adjusted to 3.8; the possible conditions for R1 on the end product are either a non-substituted hydroxyl group or substituted with glycidyl methacrylate; R2 on the start- as well as end product can either exist as a non-substituted amino group or quaternized with glycidyl trimethylammonium chloride from the previous polymer modification step;

Methacrylation of QPVA

1.0 g QPVA was dissolved in 60.0 g deionized water under stirring at 90°C for at least 1 hour. 40.0 g 3 M KOH solution was added and the mixture was stirred at 60°C for 1 hour. Afterwards the solution was slowly cooled to RT before 5.0 mL GMA were added dropwise through a septum using a syringe whilst stirring. The composite was left to react overnight at a temperature of 60°C under reflux. The following day the product was precipitated in ethanol, whereas a white, flaky substance was obtained. The mixture was centrifuged (12 min, 10,000 rpm) and separated ethanol was discarded while the remaining solid was dried at RT in vacuum overnight. Subsequently the product was characterized via 1H-NMR and the degree of substitution was calculated using Equation 4.

24 | P a g e

OH R1 OH CH2 + O O H3C CH3 H3C n O QPVA GMA

OH R1, R2: KOH

CH3 + CH3 O N OH R2 OH OH CH3

H3C CH3 O n R2: CH3 QPVA-g-GMA O O

OH CH2 Figure 16. Reaction of grafting glycidyl methacrylate (GMA) onto quaternized poly(vinyl alcohol) (QPVA) in alkaline medium; R1 of the start product can either exist as a non-substituted hydroxyl group or quaternized with glycidyl trimethylammonium chloride from the previous modification step. As there is only one functional group per polymer unit, the possible conditions for R2 are either a non-substituted hydroxyl group, quaternization via glycidyl trimethylammonium chloride or grafted with glycidyl methacrylate.

Methods for Characterization of Synthesis Products

1H-NMR

1H-NMR measurements were carried out on a nuclear magnetic resonance spectrometer Bruker AVANCEIII spectrometer with UltraShield magnets at 300 MHz. As prearrangement for making the solid synthesis products accessible for 1H-NMR measurement, HTCC as well as HTCC-g-GMA was dissolved in 0.1-0.3 vol.-% CD3COOD/D2O, whilst QPVA and QPVA-g-GMA samples were dissolved in

0.1-0.3 vol.-% D20. For interpreting the gained spectra following equations were used:

퐼 ( 3.1) 9 퐷푆 = ∗ 100 퐼 Equation 1 3.1 ∗ 5 퐼 − ( 9 ) 2.5−4.5 9

25 | P a g e

퐼 ( 3.1) 9 Equation 2 퐷푆 = ∗ 100 퐼 ( 1.4−1.6) 4

퐼5.8 퐷푆 = ∗ 100 Equation 3 퐼 ( 3.0−4.5) 21

퐼 + 퐼 ( 5.2 5.5) 2 Equation 4 퐷푆 = ∗ 100 퐻 ( 1.8) 2

Membrane Preparation via Chemical-Physical Crosslinking

0.250 g HTCC, which was modified as described before, was dissolved in 5.0 g 1 wt.% acetic acid and a differing amount of previously prepared 10 wt.% (ethylene glycol) diglycidyl ether solution (EGDGE) in ethanol was added. The mixture was allowed to stir at 60°C for 1 hour before being filtered through a 5 µm PTFE filter. 0.250 g previously prepared QPVA was dissolved in deionized water for at least 1 hour at 90°C in order to gain a 5 wt.% solution. After the solution was filtered using a 5 µm PTFE filter and cooled to RT, the pH-value was adjusted to 5.0 using 0.1 N HCl. A prescribed amount - differing within the various membrane series - of 10 wt.% glutaraldehyde (GA) in 0.01 N HCl was added. The two separately prepared mixtures were poured in one glass vial and stirred together at 60°C for another hour. Afterwards the solution was coated onto the surface of a petri dish and dried at 60°C overnight, followed by a temperature program, whereby the obtained membrane was heated to 130°C for the duration of 2 minutes.

Membrane-Preparation via UV-Crosslinking

Variation 1

0.250 g HTCC-g-GMA was dissolved in 5.0 g deionized water at a temperature of 60°C. 0.250 g QPVA- g-GMA was dissolved separately in 5.0 g deionized water at 90°C. After cooling the QPVA-g-GMA down to 60°C, it was mixed together with the HTCC-g-GMA solution. Both compounds were stirred together for a few minutes before the mixture was filtered with a 5 µm PTFE filter. Afterwards various amounts of Irgacure 2959 were dissolved before the mixture was degassed by using sonication and poured into

26 | P a g e a petri dish. The time of crosslinking under UV-irradiation (365 nm) in nitrogen atmosphere was varied for the different membrane series. The gained hydrogels were dried at RT and heated from 20 to 100°C to remove the remaining water.

Variation 2

In order to facilitate the dissolution process, especially of HTCC-g-GMA which displays higher degrees of methacrylation, ethanol was added as a solvent. For this procedure, to an amount of 0.250 g HTCC- g-GMA, 4.0 g deionized water as well as 1.0 g ethanol were added and stirred at 60°C. 0.250 g QPVA- g-GMA was dissolved in 5.0 g water at 90°C separately before being mixed with the HTCC-g-GMA solution. The combined compounds were stirred for 30 minutes before being filtrated using a 0.45 µm PTFE filter. Afterwards 2.5 mg of Irgacure 2959 were added and the UV-crosslinking was carried out under the same conditions as in Variation 1.

Methods used for Membrane-Characterization

Alkaline Swelling

To define the swelling percentage of the individual produced membranes, the mass change was examined by weighing the membranes before and after treating them in 1 M KOH solution for 24 hours at RT. The swelling percentage AW of the membranes was calculated by applying Eq. 5 to the obtained mass values. The mass [g] of the wet membrane is symbolized by mwet while the mass [g] of the dry membrane before the storage in 1 M KOH-solution is defined with mdry.

푚푤푒푡 − 푚푑푟푦 Equation 5 퐴푊 = ∗ 100 푚푑푟푦

Ion Exchange Capacity (IEC)

For computing the ion exchange capacity (IEC) of the individual membranes prepared under specific conditions, the method of back titration was deployed. For this technique, a defined mass of membrane [g] was soaked in 1 M KOH solution for 24 hours and was subsequently washed by using deionized water for another 24 hours. Following this scheme, the membrane was suspended into an exact amount of 40 mL 0.01 M HCl standard solution for 24 hours before undergoing a potentiometric

27 | P a g e titration with 0.01 M NaOH standard solution. To determine a blank value, an additional assay was usually carried out without added membrane pieces. The IEC was calculated by applying Equation 6.

(푉푏푙푎푛푘 − 푉푚푒푚푏푟푎푛푒) ∗ 푐퐻퐶푙 Equation 6 퐼퐸퐶 = ∗ 1000 푚푚푒푚푏푟푎푛푒

Vblank and Vmembrane represent the respective volume [mL] of 0.01 M NaOH solution consumed while performing the back titration with and without added membrane. The term cHCl symbolizes the concentration of the used HCl solution [M], while mmembrane shows the mass [g] of the membrane sample before the IEC determination procedure.

Chemical Stability

For evaluating the long-term durability in alkaline medium the membrane was immersed in 1 M KOH solution at 60°C for the achievable amount of time before the compactness, which was verified visually, decreased and structural flaws were observed.

ATR-FTIR

For getting insight into different structure features due to differently mediated crosslinking methods and times as well as the variety of membrane compositions, Fourier transform infrared spectroscopy was applied. The measurements were carried out on a Bruker ALPHA infrared spectrometer within a range from 4100 to 400 cm-1 using a wavenumber resolution of 4 cm-1.

Scanning Electron Microscopy (SEM)

The different morphology, resulting from various crosslinking and preparation processes, was examined using a JOEL JSM 5600 scanning electron microscope at 5 kV. In advance, the samples were fractured in liquid nitrogen and sputtered with gold, before being investigated at 2,000-fold magnification.

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Thermogravimetric Analysis (TGA)

Thermogravimetric assay was carried out applying a TGA/DSC system (NETZSCH STA 449). For the determination, the membranes were heated in the range of 60°C to 600°C at a heating rate of 10°C/min using nitrogen as inert gas with a flow rate of 20 mL/min.

Ethanol Permeability

Before the measurement, the membrane was soaked in 1 M KOH for 24 hours, followed by a washing step in deionized water for another 24 hours. The ethanol permeability measurement was carried out in a diffusion cell with the membrane fixated between the two compartments. The first compartment was filled with 1 M ethanol solution and the other with deionized water. The solutions were agitated with magnetic stirrers. The concentration of permeated ethanol was determined at a temperature of 20°C, 40°C and 60°C by refractive index (Knauer Smartline RI Detector 2300).[3] The ethanol permeability was calculated via the slope of the concentration increase as a function of the elapsed time multiplied by the source reservoir volume and the membrane thickness, ensued by a division of the effective membrane area and the primary ethanol concentration in the reservoir, as shown in Equation 7.

푃 ∗ 퐴 ∗ 퐶퐴 Equation 7 퐶퐵(푡) = 푉 ∗ 퐿 ∗ (푡 − 푡0)

CB(t) is the ethanol concentration in the receptor cell at time t, whereas t0 defines the time lag. CA depicts the primary ethanol concentration of the donor cell, while V is the volume of each half cell. A and L represent the area and thickness of the membrane available for permeation.[71]

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Results and Discussion

Modification for Chemical-Physical Crosslinking via Crosslinking Agents

Quaternization of Chitosan

The reaction products obtained from the synthesis and purification process were white powdered solids, as shown in Figure 19. As HTCC was used for membrane preparation as well as starting product for the grafting with glycidyl methacrylate, its preparation was performed repeatedly and controlled via 1H-NMR. An overview of the most important functional groups within the 1H-NMR spectra is illustrated in Figure 17. The band at 3.1 ppm represents the hydrogen groups of the quaternized ammonium group. The hydrogens of the backbone of chitosan as well as hydrogen 2b are summed up between 3.2 to 4.5 ppm and the bands of hydrogen 2a and 2c are located between 2.5 and 3 ppm; R1 represents either a non-substituted amino group or the quaternized functional group gained through the modification with (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC). The quaternization process of low molecular weight chitosan with EPTMAC resulted in a relatively high degree of quaternization of up to over 90%, as it was published previously.[3]

+ N (CH3)3 OH

CH3 4 6 O O 5 HO 2 O H3C 3 1 n R1

R1:

1) NH2

CH3 2c + N CH3 2a 2) 2b CH3 NH OH

H1,2(b), 3, 4, 5, 6 H2(a,c)

Figure 17. Overview of the 1H-NMR Interpretation method to evaluate the quaternization process of chitosan.

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Quaternization of QPVA

The product gained after the modification and purification process of PVA is white and shows a sponge-like structure after drying, as shown in Figure 19. With the performed quaternization concept, the degree of quaternization was relatively low, fluctuating between 9% and 15%.[3] As this product, as well as HTCC, serves as basis for further processing, the modification was repeatedly performed and controlled via 1H-NMR. An example of an 1H-NMR spectrum, with focus on the quaternization process, is shown in Figure 18. The band at 3.1 ppm represents the hydrogen atoms of the quaternized ammonium group. The resulting bands for H2 are located at 1.5 ppm and H1 as well as H1(a, b, c) are found between 3.1 and 4 ppm. The possible conditions for R2 are either a non-substituted hydroxy group or the quaternized functional group gained through the modification with (2,3-epoxypropyl) trimethylammonium chloride.

+ H1(a, b, c), N (CH3)3 H2

OH R OH 1 H C CH 3 2 n 3

R: 1) OH

CH3 2) 1c + N CH3 1a 1b CH H 3 1 O OH

Figure 18. Overview of the H-NMR Interpretation method to evaluate the quaternization of QPVA.

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Figure 19. Final products after the quaternization, purification and drying process of chitosan and poly(vinyl alcohol); HTCC (left) and QPVA (right).

HTCC/QPVA-Membrane Preparation via Chemical-Physical Crosslinking

The first membrane series in the scope of this study was prepared in dependence of the polymer ratio of HTCC versus QPVA. Therefore, the ration between the 5% polymer solutions was varied between 1:1 to 9:1. For membrane series 1, the crosslinker concentration was kept constant, whereas for membrane series 2, the concentration of the crosslinker glutaraldehyde (GA) was adjusted. The influence of the QPVA/HTCC ratio as well as the varied GA concentration was examined regarding alkaline swelling and ion exchange capacity as well as optical features of the membrane and alkaline durability.

Membrane-series 1

Constant Parameters: Mass of HTCC in the polymer solution (5%) 0.25 g

Mass of QPVA in the polymer solution (5%) 0.25 g Amount of GA/EGDGE each 0.3 mmol*mmol-1 blend membrane Crosslinking time 60 min Crosslinking temperature 60°C Temperature treatment 60°C over night, heated up to 130°C for 2 minutes

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Table 1. Results of alkaline swelling and IEC measurements of HTCC/QPVA membranes in dependence of the polymer solution ratio of HTCC and QPVA with constant amounts of crosslinking agents. Sample number 5% HTCC/5% QPVA solution Alkaline swelling [%] IEC [meq/g] 1 5 g/ 5g (1:1) 154 1.323 2 6 g/4 g (1.5:1) 155 1.464 3 7 g/3 g (2.3:1) 158 1.588 4 8 g/2 g (4:1) 156 1.802 5 9 g/1 g (9:1) 201 1.749

Membrane-series 2

Constant Parameters: Mass of HTCC in the polymer solution (5%) 0.25 g

Mass of QPVA in the polymer solution (5%) 0.25 g Amount of EGDGE 0.3 mmol*mmol-1 blend membrane Crosslinking time 90 min Crosslinking temperature 60°C Temperature treatment 60°C over night, heated up to 130°C for 2 minutes

Table 2. Results of alkaline swelling and IEC of HTCC/QPVA membranes in dependence of the polymer solution ratio of HTCC and QPVA with varied glutaraldehyde concentration. 5% HTCC/5% QPVA Alkaline Sample number GA [mmol*mmol-1] IEC [meq/g] solution swelling [%] 6 5 g/5 g 0.30 143 1.373 7 6 g/4 g 0.24 182 1.522 8 7 g/3 g 0.18 204 1.603 9 8 g/2 g 0.12 191 1.639 10 9 g/1 g 0.06 225 1.878

With increasing amount of HTCC solution and decreasing amount of GA, the membrane surface became cloudier. As demonstrated in Figure 20, alkaline swelling, as well as ion exchange capacity, increase with higher HTCC ratio. However, the alkaline stability decreased from 22 days (5 g HTCC- solution/5 g QPVA-solution and 0.3 mmol/0.3 mmol GA/EGDGE) to 15 days (9 g HTCC-solution/1 g QPVA-solution and 0.06 mmol/0.3 mmol). Regarding alkaline durability, the ratio of 1:1 HTCC/QPVA with 0.3 mmol/0.3 mmol GA/EGDGE showed the best result of 22 days and was applied to all further experiments, as increasing alkaline stability represents an important point for membranes prepared with chemical crosslinking agents. It is observed that the swelling behavior and IEC are interrelated as a result of the formation of transferring channels due to higher water uptake capacity, which leads to better transport of hydroxide ions.[3]

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Figure 20. Demonstration of the alkaline swelling and ion exchange capacity of membrane samples with varied ratios of HTCC/QPVA polymer solution (1:1, 1.5:1, 2.3:1, 4:1, 9:1) with constant amounts of added crosslinking agents GA and EGDGE.

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Figure 21. Results of alkaline swelling and IEC of membrane samples with varied ratios of HTCC/QPVA polymer solution (1:1, 1.5:1, 2.3:1, 4:1, 9:1) and varied amounts of added glutaraldehyde (0.3 mmol, 0.24 mmol, 0.12 mmol, 0.06 mmol) but constant amount of ethylene glycol diglycidyl ether (0.3 mmol).

Membrane-series 3

For this membrane series, the glutaraldehyde concentration was altered from 0.3 mmol to 1.3 mmol. The influence of the concentration of GA was examined regarding the alkaline swelling and IEC of the prepared membranes, as well as its optical and mechanical features.

Constant Parameters: Mass of HTCC in the polymer solution (5%) 0.25 g

Mass of QPVA in the polymer solution (5%) 0.25 g Amount of EGDGE 0.3 mmol*mmol-1 blend membrane Crosslinking-time 90 min Crosslinking temperature 60°C Temperature treatment 60°C overnight, heated up to 130°C for 2 minutes

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Table 3. Summarized results of alkaline swelling and IEC measurements of HTCC/QPVA membranes in dependence of glutaraldehyde concentration within the range of 0.3 to 1.3 mmol. Sample number GA [mmol*mmol-1] Alkaline swelling [%] IEC [meq/g] 11 0.3 151 1.478 12 0.6 118 1.308 13 0.9 94 1.145 14 1.1 112 1.515 15 1.3 104 1.118

The resulting membranes became less flexible and more brittle with increasing amount of glutaraldehyde added to the membrane preparations. The alkaline swelling as well as the ion exchange capacity decreased within the range of 0.3 to 0.9 mmol and increased again within a small range from 0.9 to 1.3 mmol, as demonstrated in Figure 22. Due to the high brittleness of those membranes prepared with high amount of glutaraldehyde, another test series was carried out with varied amounts of glutaraldehyde within a lower concentration range between 0.2 to 0.5 mmol.

Membrane-series 4

Constant Parameters: Mass of HTCC in the polymer solution (5%) 0.25 g

Mass of QPVA in the polymer solution (5%) 0.25 g Amount of EGDGE 0.3 mmol*mmol-1 blend membrane Crosslinking time 90 min Crosslinking temperature 60°C Temperature treatment 60°C overnight, heated up to 130°C for 2 minutes

Table 4. The results of alkaline swelling and IEC measurements of HTCC/QPVA membranes in dependence of glutaraldehyde concentration within the range of 0.2 to 0.5 mmol. Sample number GA [mmol*mmol-1] Alkaline swelling [%] IEC [meq/g] 16 0.2 182 1.092 17 0.3 155 1.266 18 0.4 139 1.191 19 0.5 118 1.265

The membranes gained from this experimental series feature by far better flexibility properties than the membranes prepared with higher glutaraldehyde concentrations. However, they still exhibited an alkaline stability of more than 20 days. The decrease of the alkaline swelling in dependence of the added amount of glutaraldehyde solution is illustrated in Figure 22. The IEC varies between 1.092 to

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1.515 meq/g, whereby the membranes with GA amount of 0.3 mmol and 1.1 mmol showed the highest almost equivalent values and therefore the lower GA amount was chosen as standard concentration of glutaraldehyde for the following membrane experiments.

Figure 22. Demonstration of alkaline swelling and ion exchange capacity of membrane series 3 and 4 with varied amounts of GA (0.2 mmol, 0.3 mmol, 0.4 mmol, 0.5 mmol, 0.6 mmol, 0.9 mmol, 1.1 mmol, 1.3 mmol) and constant amount of EGDGE (0.3 mmol*mmol-1).

In the last experimental series with crosslinking agents, the added amount of the second crosslinker EGDGE was varied between 0.3 to 0.9mmol. Again, the resulting changes regarding alkaline swelling, ion exchange capacity, alkaline durability and ethanol permeability of the prepared membranes, as well as its optical and mechanical features, were examined.

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Membrane-series 5

Constant Parameters: Mass of HTCC in the polymer solution (5%) 0.25 g

Mass of QPVA in the polymer solution (5%) 0.25 g GA concentration 0.3 mmol*mmol-1 blend membrane Crosslinking time 90 min Crosslinking temperature 60°C Temperature treatment 60°C overnight, heated up to 130°C for 2 minutes

Table 5. Results of alkaline swelling and IEC measurements of HTCC/QPVA membranes in dependence of EGDGE in the range of 0.3 to 0.9 mmol. Sample number EDGDE [mmol*mmol-1] Alkaline swelling [%] IEC [meq/g] 20 0.3 148 1.598 21 0.43 157 1.581 22 0.6 103 1.289 23 0.75 108 1.289 24 0.9 78 1.170

Figure 23. Demonstration of alkaline swelling and ion exchange capacity of membrane samples with varied amounts of ethylene glycol diglycidyl ether (0.3 mmol, 0.43 mmol, 0.6 mmol, 0.75 mmol, 0.9 mmol) and a constant amount of glutaraldehyde (0.3 mmol).

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With increasing amount of ethylene glycol diglycidyl ether, the membranes lost their flexibility and became brittle up to a degree already challenging their handling, especially for the products containing a concentration of 0.75 and 0.9 mmol EGDGE. On the contrary, the membranes with crosslinker concentrations of 0.3 and 0.43 mmol showed excellent elastic features, as shown in Figure 25. The alkaline swelling and IEC decrease with higher crosslinker addition, as it can be seen in Figure 23. The highest ion exchange capacity was obtained with 0.3 mmol EGDGE as well as 0.3 mmol GA. The membrane prepared through chemical-physical crosslinking lasted up to 25 days in alkaline medium before decomposition. The average ethanol permeability was 3.17*10-8 cm2/s at 20°C, 1.99*10-7 cm2/s at 40°C and 5.21*10-7 cm2/s at 60°C, as already published.[3] A demonstration of the ethanol permeability in dependence of the temperature for sample 18 (HTCC/QPVA 0.4 mmol GA/0.3 mmol EGDGE) is shown in Figure 24.

Figure 24. Ethanol permeability of a HTCC/QPVA (1:1) membrane containing 0.4 mmol GA and 0.3 mmol EGDGE at 20°C, 40°C, 60°C.

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Figure 25. HTTC/QPVA membrane prepared through chemical-physical crosslinking containing 0.3 mmol EGDGE and 0.3 mmol GA and demonstration of its flexibility.

A schematic demonstration of the interpenetrating network (IPN) formed within the illustrated membranes, prepared with the crosslinking agents GA and EGDGE, is shown in Figure 26. Here, it is illustrated that the membrane is formed through reaction of dialdehyde with the non-substituted hydroxyl groups of QPVA and the amino groups of chitosan in order to create stable acetal and imine bonds, respectively. The epoxide compounds contributed by EGDGE are forming networks with hydroxide and amine groups and, additionally, the hydroxyl groups of both HTCC and QPVA interact by forming hydrogen bonds.[3]

Figure 26. Schematic visualization of the networks formed between HTCC and QPVA with I) ethylene glycol diglycidyl ether and II) glutaraldehyde due to the chemical-physical- crosslinking process.[3]

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Modification of HTCC for UV-Crosslinking

For making HTCC accessible for UV-crosslinking, GMA was grafted onto HTCC using two different synthesis methods (synthesis prescription 1 and 2), each processed with various amounts of added GMA and varying reaction times. The yellow, gel-like products obtained by afore mentioned methods showed poor solubility in water and therefore they were washed several times with THF until white fibers of water soluble HTCC-g-GMA were received.

Figure 27. Comparison of HTCC-g-GMA gained from synthesis prescription 1 before (left) and after (right) the optimized purification steps.

To verify the substitution of HTCC with GMA, the synthesis products were characterized via 1H-NMR and the degree of methacrylation was determined. The interpretation concept for the gained spectra is shown in Figure 28. The peaks between 5 and 6 ppm represent the marked hydrogens of the

methylene group summarized under the term H7. Due to the possibility that the peak closer to the solvent peak at 5 ppm could falsify the result, only the posterior peak at 5.8 ppm was consulted for the determination of the degree of methacrylation. The other highlighted hydrogens remain the same,

as already explained in the spectra of HTCC. The functional groups presumably represented by R1 are

either a non-substituted hydroxy group or a methacrylic group. The possible conditions for R2 can either be a non-substituted amino group or the quaternized functional group gained through the modification with (2,3-epoxypropyl) trimethylammonium chloride.

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+ H1, 2 (a, b, c), 3, 4, 5, 6 (a, b, c), CH3, (CH3)3N

R1 CH3 4 6 O O 5 HO 2 O H3C 3 1 n R2

R1:

1) OH

O 6a 6c 2) 6b CH3 O O

OH CH2 7 R2:

1) NH2

CH3 2c + N CH3 2a 2b CH3 H 2) 7 NH OH

Figure 28. Demonstration of the 1H-NMR interpretation scheme to determine the degree of methacrylation through grafting GMA onto HTCC.

As shown in Table 6, starting with amounts of 2 mL/g GMA and a reaction time of 2 hours under nitrogen atmosphere, it was possible to detect the added methylene groups to HTCC via 1H-NMR, resulting in a DS of 0.6%, while lower amounts of GMA did not result in detecting any degree of methacrylation. By extending the reaction time by one hour, the degree of substitution could be increased to 1.5%. It was further observed that, by redoubling the amount of GMA from 2 mL/g to 4 mL/g, a DS increased by 6% was achieved. However, further increase of reaction time did not deliver any notable advantage in the degree of substitution, thus resulting in appointing synthesis prescription 2, in order to gain higher degrees of methacrylation, which are shown in Table 7.

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Table 6. Overview of the gained DS values from synthesis prescription 1 for grafting glycidyl methacrylate onto HTCC in dependence of added GMA and reaction time. GMA [mL/g] Reaction time [h] DS [%] 0.5 2 0 0.5 3 0 2 2 0.6 2 3 1.5 4 3 6.0 4 5 6.2

By applying the afore-remarked methacrylation method, it was possible to obtain higher degrees of substitution varying between 10 to 53% in dependence on the reaction time. The water solubility HTCC-g-GMA declined with increasing degree of substitution. However, it was possible to dissolve the products up to a DS of 45%, while products of even higher degree of methacrylation could only be dissolved in a water-ethanol mixture. Though the dissolution of HTCC-g-GMA could be improved by the addition of ethanol to the reaction mixture, the amount of ethanol used was limited due to the other compound – QPVA-g-GMA – precipitating at higher ethanol concentrations.

Table 7. Results of the gained DS for the products of synthesis prescription 2 for grafting glycidyl methacrylate onto HTCC in dependence of reaction time for two parallel reaction mixtures. Reaction time [d] DS [%] Reaction mixture I DS [%] Reaction mixture II 6 10 19 7 26 29 9 36 40 13 53 45

Modification of QPVA for UV-Crosslinking

In order to improve the membrane properties regarding compatibility of the compounds and mechanical features, HTCC was blended with QPVA for the afore prepared membranes; this concept was maintained for the UV-crosslinking preparations.[3] Therefore, it was necessary to graft photosensitive functional groups onto QPVA. As already mentioned before, GMA was used for this modification process.[66] The white, spongy solids gained from the modification and purification process were analysed via 1H-NMR. The concept for the interpretation of the gained spectra with focus on the functional groups providing information on the degree of methacrylation, is shown in Figure 29. The peaks between 5 and 6 ppm represent the marked hydrogens of the methylene group summarized under the term H3. The average of the two intensities was calculated and consulted for the determination of the degree of methacrylation. The other highlighted hydrogens remain the same as already explained in the spectra of QPVA; R can either represent a non-substituted hydroxy group,

43 | P a g e a methacrylic group gained from modification with glycidyl methacrylate, or the quaternized group gained from modifying with (2,3-epoxypropyl) trimethylammonium chloride.

+ H1 (a, b, c, d, e, f), CH3, (CH3)3N H2

OH R OH 1 H C CH 3 2 n 3

R: 1) OH O 1a 1c 2) 1b CH2 O O 3

OH CH3

CH3 1f + N CH3 1d 1e CH3 3) O OH

H3

Figure 29. Demonstration of the 1H-NMR interpretation scheme to determine the degree of methacrylation after grafting GMA onto QPVA.

The DS gained for the performed QPVA-g-GMA synthesis is in the range of 48 to 61%, resulting in an average degree of methacrylation of 55%. The minor variation in reaction implementation as well as the inaccuracy of the evaluation method of a few percent in both directions is most likely the reason for variation of the DS regarding the different reaction mixtures.

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Table 8. Overview of the calculated DS values received from the conducted synthesis processes of grafting glycidyl methacrylate onto QPVA. Reaction mixture DS [%] 1 61 2 48 3 66 4 52 5 49 6 55

HTCC-g-GMA/QPVA-g-GMA-Membrane Preparation via UV-Crosslinking

Membrane Preparation in Dependence of the Degree of Methacrylation of Quaternized Chitosan

In membrane series 6, HTCC-g-GMA gained from synthesis method 2 with various degrees of methacrylation was used. As shown in Table 7 the DS from two different reaction mixtures varied between 10% and 53%. For better dissolution of the HTCC-g-GMA products with a DS higher than 40%, ethanol at a ratio of one to ten in respect to water was added to all membrane solutions. A higher amount of ethanol would result in precipitation of QPVA-g-GMA, the second membrane component. However, the membrane solution with HTCC-g-GMA with a degree of methacrylation of 53% showed unwanted foaming that could not be removed by ultrasonic treatment. The influence of the degree of methacrylation was examined regarding the alkaline swelling and ion exchange capacity of the prepared membranes, as well as its optical and mechanical features.

Membrane-series 6

Constant Parameters: Mass of HTCC-g-GMA in the polymer solution (5%) 0.25 g

Mass of QPVA-g-GMA in the polymer solution (5%) 0.25 g Irgacure 2959 2.5 mg Crosslinking conditions UV light of 365 nm under nitrogen atmosphere Crosslinking time 15 min Crosslinking temperature RT

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Drying conditions RT overnight, heated from 20° to 100°C to remove remaining water to gain a fully dried product

The obtained membranes, prepared from HTCC-g-GMA with DS values between 10 and 36%, exhibit a clear, bubble-free surface. The product gained from methacrylated HTCC with a DS of 53% showed a comparatively inhomogeneous, uneven surface. It was observed that the flexibility of the membranes decreases with higher degree of methacrylation.

Figure 30. HTCC-g-GMA/QPVA-g-GMA membrane prepared with HTCC-g-GMA with a DS of 53%; the surface of the product exhibits inhomogeneous and uneven features.

The analytical determinations of the membranes included water stability, alkaline stability, alkaline swelling and IEC determination. The determination of water stability showed a durability of 10 days with all the samples from this series before their decomposition. Values for alkaline swelling and IEC were only obtainable from the membrane prepared from HTCC-g-GMA with a DS of 53% (shown in Table 9). The other samples were decomposed during the analyzing step in 1 M KOH within 24 hours and therefore, were not accessable to further measurement. The second test series was prepared exactly the same way as the previous except for the crosslinking time, which was increased from 15 minutes to 60 minutes.

Membrane-series 7

Constant Parameters: Mass of HTCC-g-GMA in the polymer solution (5%) 0.25 g

Mass of QPVA-g-GMA in the polymer solution (5%) 0.25 g Irgacure 2959 2.5 mg

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Crosslinking conditions UV light of 365 nm under nitrogen atmosphere Crosslinking time 60 min Crosslinking temperature RT Drying conditions RT overnight, heated from 20° to 100°C to remove remaining water to gain a fully dried product

Once again it was observed that the solubility of the HTCC-g-GMA product with a DS of 45% was poorer than of those products with lower methacrylation degrees. This effect is caused by the increasing hydrophobicity due to the indroduced methacylate groups. The flexibility tended to be slightly lower compared to the membrane series exposed to 15 minutes of crosslinking. Extended times of UV- exposure cause higher degrees of crosslinking, reducing the flexibility of the products. The water durability was increased from 10 to 12 days, while alkaline durability was still not achieved. The only analyzable value for both alkaline swelling and IEC was recived from the membrane encompassing HTCC-g-GMA with a DS of 45%. The gained values did not differ significantly, which seems to indicate that the chemical stability cannot be increased by simply extending crosslinking time.

Table 9. Results of water durability, alkaline durability and alkaline swelling of UV-crosslinked HTCC-g-GMA-QPVA-g-GMA membranes containing HTCC-g-GMA with highest DS after crosslinking times of 15 and 60 minutes; DS of HTCC-g-GMA 53%, DS of QPVA-g-GMA 49%. Sample Crosslinking Water Alkaline IEC Alkaline swelling [%] number time [min] durability durability [meq/g] 25 15 10 days 0 days 714 1.236 26 60 12 days 0 days 728 1.301

Membrane Preparation with Altered Amounts of Irgacure 2959

For the following series of experiments HTCC-g-GMA with a DS of 6%, prepared with synthesis prescription 1 and HTCC-g-GMA with a DS of 40%, gained from synthesis prescription 2, were utilized. The degree of methacrylation of 40% was chosen, because it was barely manageable to dissolve in water without addition of ethanol. The concentration of Irgacure 2959 was altered from 0.5 to 5 wt.%. The membranes were characterized regarding their water stability, alkaline stability, alkaline swelling and ionic exchange capacity.

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Membrane-series 8

Constant Parameters: Mass of HTCC-g-GMA in the polymer solution (5%) 0.25 g

DSHTCC-g-GMA 40%

Mass of QPVA-g-GMA in the polymer solution (5%) 0.25 g

DSQPVA-g—GMA 49% Crosslinking conditions UV light of 365 nm under nitrogen atmosphere Crosslinking time 60 min Crosslinking temperature RT Drying conditions RT overnight, heated from 20° to 100°C to remove remaining water to gain a fully dried product

The flexibility and elastic features decreased with increasing amount of Irgacure, but even the membrane containing the highest photoinitiator concentration of this series was not brittle enough to break. However, the surfaces of the membranes with amounts higher than 5 wt.% were extremely rough and inhomogeneous, containing white crystals all over. The afore dissolved photoinitiator precipitated during the crosslinking process (see Figure 31) and seemed to disrupt the formation of links between the photosensitive groups, as the membranes showed very instable features when getting in contact with liquid. Due to this, only the membranes containing an amount of Irgacure 2959 between 0.5 wt.% and 5 wt.% were analyzed. The membranes where tested regarding their water as well as alkaline durability and alkaline swelling. It was observed that with higher amount of photoinitiator, the membrane became more stable in water and alkaline medium. A decrease in swelling behavior was also measurable. With an amount of 5 wt.% of Irgacure 2529 the stability in water increased up to 20 days and the alkaline stability up to 5 days; in comparison, the membranes prepared with 0.5 wt.% of Irgacure completely decomposed within a few hours when exposed to 1 M KOH.

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Table 10. The results of water durability, alkaline durability and alkaline swelling of UV-crosslinked HTCC-g-GMA/QPVA-g- GMA membranes (DS of HTCC-g-GMA 40%, DS of QPVA-g-GMA 49%, crosslinking-time 60min) in dependence of Irgacure concentration; * measure was not possible due to decomposition. Sample number Irgacure [wt.%] Water durability Alkaline durability Alkaline swelling [%] 27 0.5 12 days 0 days -* 28 1.5 12 days 0 days 897 29 2.5 14 days 3 days 536 30 5 18 days 5 days 386

Figure 31. HTCC-g-GMA/QPVA-g-GMA Membrane with Irgacure 2959 amount of 10 wt.% after the crosslinking process; the afore dissolved photoinitator participated and formed white crystals on the membrane surface.

Depending on the added amounts of Irgacure 2959, the alkaline swelling can be altered between 897%, which represents the first measurable value within this membrane series, to a value of 386%. The reduced swelling behavior enables the membranes to withstand up to 5 days in 1 M KOH without decomposing, as well as longer endurance in deionized water up to 6 days longer than the membrane containing only one tenth of the amount of Irgacure. In this test series, it was explicitly demonstrated that the amount of photoinitator used for membrane preparation has significant influence on stability features and alkaline swelling. For comparison between membranes that contain HTCC-g-GMA with a DS of 40% gained from synthesis method 2 and HTCC-g-GMA with low DS preserved from synthesis prescription 1, the experiment was repeated with HTCC-g-GMA with a DS of 6% under the same conditions and preparation. The added Irgacure 2959 concentrations altered from 0.5 wt.% to 5 wt.%.

Membrane-series 9

Constant Parameters: Mass of HTCC-g-GMA in the polymer solution (5%) 0.25 g

DSHTCC-g-GMA 6%

Mass of QPVA-g-GMA in the polymer solution (5%) 0.25 g

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DSQPVA-g—GMA 49% Crosslinking conditions UV light of 365 nm under nitrogen atmosphere Crosslinking-time 60 min Crosslinking temperature RT Drying conditions RT overnight, heated from 20° to 100°C to remove remaining water to gain a fully dried product

The resulting membranes showed more flexible and elastic features than the membranes prepared with higher DS. The flexible features of the samples in membrane series 9 remained unchanged, even with the highest added amount of Irgacure 2959, which presents an advantage in comparison to the membranes from series 8. The products prepared with HTCC-g-GMA with DS of 6% are also less brittle and can therefore be handled more easily. The performed analytical tests showed similar values regarding the increase of stability with higher amount of photoinitator. In comparison to membrane series 8, the water durability slightly differs, but the alkaline durability stays the same. In this membrane series, the sample prepared with the lowest amount of Irgacure (0.5 wt.%) was not accessible for determination of alkaline swelling due to disintegration as already shown. However, the percentage of alkaline swelling of the products with 2.5 wt.% and 5 wt.% of Irgacure 2959 exhibited slightly lower values than the membranes prepared from HTCC-g-GMA with higher DS%, while the results of the products containing 1.5 wt.% of photoinitator remain almost the same. An overview of the measurements´ outcome is provided in Table 11, whereas a comparison of the influence of photoinitiator concentration within membrane series 8 and 9 is visualized in Figure 32.

Table 11. Results of water durability, alkaline durability and alkaline swelling of UV-crosslinked HTCC-g-GMA-QPVA-g-GMA membranes in dependence of Irgacure 2959 concentration after crosslinking time of 60 minutes; DS of HTCC-g-GMA 6%, DS of QPVA-g-GMA 52%; * measurement was not possible due to decomposition. Sample number Irgacure [wt.%] Water durability Alkaline durability Alkaline swelling [%] 31 0.5 11 days 0 days -* 32 1.5 12 days 0 days 912 33 2.5 20 days 3 days 448 34 5.0 23 days 5 days 320

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Figure 32. Comparison of alkaline swelling of HTCC-g-GMA/QPVA-g-GMA membranes containing HTCC-g-GMA of low (6%) and higher (40%) degree of methacrylation in dependence of photoinitator concentration

The IEC of the membranes containing 0.5 wt.% of Irgacure were determined and showed similar results with 1.255 meq/g (membrane preparation containing HTCC-g-GMA DS 40%) and 1.055 meq/g (membrane prepared with HTCC-g-GMA DS 6%), respectively. The slightly lower IEC of the membrane encompassing HTCC-g-GMA DS 6% is matching the observed decreased swelling behavior. As already explained before, the ion transport is enhanced when the network is less tight due to higher alkaline swelling.[3] Within the following membrane series, the time of UV irradiation at a wavelength of 365 nm under nitrogenic atmosphere was reduced from 60 minutes to 10, 5, and 2 minutes, respectively, in order to test the according crosslinking effects.

Membrane-series 10

Constant Parameters: Mass of HTCC-g-GMA in the polymer solution (5%) 0.25 g

DSHTCC-g-GMA 6% and 40%

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Mass of QPVA-g-GMA in the polymer solution (5%) 0.25 g Irgacure 2959 25 mg

DSQPVA-g—GMA 55% Crosslinking conditions UV light of 365 nm under nitrogen atmosphere Drying conditions RT overnight, heated from 20° to 100°C to remove remaining water to gain a fully dried product

Once again, the experiments were performed using HTCC-g-GMA with high (40%) and low (6%) DS. Due to the previous results regarding alkaline stability, 5 wt.% Irgacure was added to each membrane. The compounds resulted in a clear, non-foaming membrane solution during the preparation. The obtained membranes exhibited clear and homogeneous surfaces and showed superb flexibility and elastic features as demonstrated in Figure 34. The products were analyzed regarding their alkaline swelling behavior as well as selected samples were compared by ATR-FTIR measurements and their surface structure was analyzed via scanning electron microscope (SEM).

Table 12. Results of alkaline swelling of membranes prepared with HTCC-g-GMA of low (6%) and high (40%) degree of methacrylation in dependence of crosslinking time with constant Irgacure 2959 concentration of 0.5 wt.%; *measure was not possible due to decomposition. Sample number HTCC-g-GMA-DS [%] Crosslinking time [min] Alkaline swelling [%] 35 6 2 min 532 36 6 5 min -* 37 6 10 min 486 38 6 60 min 386 39 40 2 min 730 40 40 5 min 767 41 40 10 min 505 42 40 60 min 320

Due to the missing value of alkaline swelling for the 5 minutes crosslinking measurement, only the values of 2, 10 and 60 minutes were comparable. A clear decrease in alkaline swelling in dependence on the duration of the crosslinking process can be observed for the membranes prepared from HTCC- g-GMA with low as well as high DS. While the alkaline swelling of the membranes with HTCC-g-GMA DS 6% showed a value of 532% after 2 minutes of crosslinking, the membrane prepared from HTCC-g- GMA with a DS of 40% presented with 730% a much higher swelling behavior. However, the alkaline swelling after 10 minutes as well as 60 minutes of crosslinking exhibited almost the same value for the different methacrylation degrees, as demonstrated in Figure 33.

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Figure 33. The decrease of alkaline swelling of HTCC-g-GMA/QPVA-g-GMA membranes prepared with HTCC-g-GMA with low (6%) and high degree (40%) of methacrylation in dependence of crosslinking time with constant amount of Irgacure 2959 of 5 wt.%.

Figure 34. HTCC-g-GMA/QPVA-g-GMA membrane (HTCC-g-GMA DS 6%, QPVA-g-GMA DS 49%) after 60 minutes of crosslinking with 5 wt.% of Irgacure 2959; the products exhibit clear, homogenous surfaces, as well as great flexibility.

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A schematic demonstration of the interpenetrating network formed within the membranes is shown in Figure 35. Irgacure 2959 is excited by UV-light leading to the formation of radicals[67], which attack the methylene group of glycidyl methacrylate grafted onto HTCC as well as QPVA leading to further radical formation.[68] This process results in a chain reaction[68], linking the polymers via their introduced methacrylated functional groups.

O O OH C OH h + CH C H C 3 3 H3C CH3

O O CH C O 3 O

H2C C + O O

CH3 OH HO O +

CH3 O O

O H2C H3C O C CH OH HO 3 O O HO

Chain reaction

Figure 35. Schematic visualization of the network-formation reactions of HTCC-g-GMA/QPVA-g-GMA induced by the photoinitator Irgacure 2959 leading to crosslinking over the methacrylated functional groups grafted onto HTCC an QPVA.

ATR-FTIR Measurements

To observe the crosslinking process during UV exposure, ATR-IR spectra were recorded after 0, 5, 10, 15 and 60 minutes of crosslinking under nitrogenic atmosphere, as illustrated in Figure 36. A summary of the assigned bands is shown in Table 13.

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Table 13. Overview over the identified bands of the obtained ATR-IR spectra Frequency [cm-1] Interpretation 3594-3390 OH stretching 3550-3350 NH-valence 2935-2915 Methylene C-H stretch 1740-1725 C=O -valence 1715-1680 C=O -valence 1680-1620 Alkenyl C=C stretch, Aromatic ring stretch C=C-

1400-1470 CH3 and CH2-bending 1350-1000 C-C stretch 1100 C-O stretch

At 3300 cm-1, the characteristic band for OH-stretching vibration from -CH-OH and remaining water, most probably overlaping with amino stretching vibration, is observed. The typical C-H stretching vibrations at 2935-2915 cm-1 are only visible after 60 minutes of crosslinking time, which can be an indication for increasing formation of -CH-groups in consequence of photopolymerization. Another evidence for the crosslinking process could be the increasing bands between 1350 and 1000 cm-1 that can be assigned to C-C stretching. It can be observed that after 0 and 5 minutes of crosslinking, those bands are unaffected, but start getting more intense after 10 minutes. The band is clearly increased after 60 minutes of crosslinking, which seems to be an indicator for the proceeding crosslinking process.[3, 66-70] For the IR-spectrum shown in Figure 37, the membranes containing different amounts of Irgacure 2959 were analyzed. The first membrane was prepared with 7.5 mg of photoinitator and the second membrane encompassed 25 mg of Irgacure 2959. At 3300 cm-1 the characteristic OH-stretching vibrations are observed. The typical C-H stretching vibrations located at 3300-3010 cm-1 seem to be overlapped by the broad OH-band. However, the bands at 2926 cm-1 to 2852 cm-1 represent the methylene C-H-stretching bands. The C=O stretching vibration at 1740-1680 cm-1 belongs to the according functional groups of Irgacure 2959 and GMA. The bands at 1640-1620 cm-1 most likely belong to the alkenyl stretching bands of GMA. Between 1400

-1 -1 and 1470 cm , the bands for CH2-bending can be observed, and the band at 1100 cm can be assigned to the C-O groups.[3, 66-70]

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Figure 36. ATR-IR spectrum obtained after UV-beam exposure of HTCC-g-GMA/QPVA-g-GMA membrane solution with 5 wt.% of Irgacure after 0, 5, 10, 15, and 60 minutes of crosslinking.

Figure 37. ATR-IR spectrum obtained from HTCC-g-GMA/QPVA-g-GMA membranes prepared with 1.5 wt.% (7.5 mg) and 5 wt.% (25 mg) of Irgacure after 60 minutes of crosslinking.

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When comparing the two spectra it can be observed that the hydroxy group band becomes more intense, as well as the bands at 1740 to 1680 cm-1 representing the C=O stretching vibration. This is likely to be caused by the higher Irgacure 2959 amounts used for the membrane preparation. The bands at 1640-1620 cm-1 belong to the alkenyl stretch bands of GMA. The C=C stretching bands show decreasing intensity from 7.5 to 25 mg Irgacure 2959 and could be evidence that the methylene groups of GMA form C-C- bonds via the photopolymerization process. The area between 1350 and 1000 cm-1 contains the C-C stretch bands. The increase of the intensity of those bands could be an indicator for the -C-C- bonds formed during the crosslinking process mediated by photopolymerization.

Scanning Electron Microscopy

Selected samples from membrane series 9 and 10 were analyzed via scanning electron microscopy (SEM) to get an insight on the surface properties of membranes prepared with various amounts of Irgacure 2959 as well as different crosslinking times.

Figure 38. Membrane containing 7.5 mg Irgacure 2959 (crosslinking time 60 min); membrane containing 12.5 mg Irgacure 2959 (crosslinking time 60min); membrane containing 25 mg Irgacure 2959 (crosslinking time 60 min); membrane containing 25 mg Irgacure (crosslinking time 10 minutes); upper left to lower right.

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The SEM images showed that low Irgacure 2959 concentrations lead to inhomogeneous distribution of the detected crystals on the surface. With an Irgacure 2959 concentration of 7.5 mg (1.5 wt.%) the interstices between the particles were the largest, what could be an indicator, that the crosslinking process does not have fully proceeded. With increasing amount of Irgacure 2959, the distance between the observed crosslinked polymer matrix became smaller, leading to a denser membrane surface. By comparing the highest applied Irgacure 2959 concentration of 25 mg (5 wt.%) after a crosslinking time of 10 minutes to 60 minutes, it is clearly observed that after 10 minutes there are still interspaces visible, while after 60 minutes of crosslinking time the membrane surface is densely covered with crystalline structure as well as fibrous particles. After 60 minutes of crosslinking time with 5 wt.% of Irgacure 2959, the surface seems to be a comprehensive network.

Thermogravimetric Analysis

The thermal degradation behavior of UV-crosslinked HTCC-g-GMA/QPVA-g-GMA membranes in dependence of the added Irgacure 2959 was determined and compared to HTCC/QPVA composite membrane crosslinked with GA and EGDGE. The chemically-physically crosslinked HTCC/QPVA membranes exhibit three stages of thermal degradation. At the first stage at between 70°C and 100°C bonded water evaporated, while the onset degradation temperature (Td) observed for the second stage is about 280°C and for the third stage approximately around 320°C. Similary, for the UV- crosslinked membranes, the thermal degradation also proceeds in three stages. The first stage occurs between 70°C and 100°C analogous to HTCC/QPVA membranes. However, it is observed that the weight loss of membranes crosslinked via UV-radiation is higher and that the onset degradation temperature is lower than that of membranes crosslinked via GA and EGDGE. The second stage exhibits an onset degradation temperature of about 150°C and the second at 240°C for the sample encompassing the highest amount of Irgacure 2959 (25 mg). The membranes prepared with lower photoinitator amounts (12.5 mg and 7.5 mg) showed even lower onset degradation temperatures of approximately 130°C for the first stage and around 220 °C for the second stage. An overview of the weight loss of the membranes from stage 1 to 3 is provided in Table 14.

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Figure 39. Thermogravimetric analysis of HTCC/QPVA crosslinked with GA and EGDGE in comparison with UV-crosslinked HTCC-g-GMA/QPVA-g-GMA membranes in dependence of Irgacure 2959 amount; 1. HTCC/QPVA membrane containing 0.3 mmol*mmol-1 GA and 0.3 mmol*mmol-1 EGDGE; 2. HTCC-g-GMA/ QPVA-g-GMA membrane with 25 mg Irgacure 2959; 3. HTCC-g-GMA/QPVA-g-GMA containing 12.5 mg Irgacure 2959; 4. HTCC-g-GMA/QPVA-g-GMA with 7.5 mg Irgacure 2959.

Table 14. Overview of the weight loss of the membranes analyzed via TG measurement from Fig.41. First stage Second stage Third stage Number Weight Weight Weight Membrane Td [°C] Td [°C] Td [°C] in graph loss [%] loss [%] loss [%] HTCC/QPVA 1 70 5 260 35 320 20 HTCC-g-GMA/ QPVA-g-GMA (25 2 70 12 150 28 240 20 mg Irgacure 2959) HTCC-g-GMA/ QPVA-g-GMA (12.5 3 70 16 130 29 220 20 mg Irgacure 2959) HTCC-g-GMA/ QPVA-g-GMA (7.5 4 70 16 130 28 220 25 mg Irgacure 2959)

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Comparison of HTCC/QPVA and HTCC-g-GMA/QPVA-g-GMA Membranes

Comparing the optical features of HTCC/QPVA and HTCC-g-GMA/QPVA-g-GMA membranes, it is observed that the networks formed by crosslinking agents show a darker and cloudier surface, while the UV-crosslinked products were more transparent. Both membrane types showed flexible features, but the HTCC-g-GMA/QPVA-g-GMA membranes prepared through UV-crosslinking technique exhibit even more flexibility. The properties of the end product of both crosslinking methods highly depend on the amount of added crosslinker or photoinitiator. HTCC/QPVA membranes with lower GA and EGDGE concentration as chosen in this work (less than 0.2 mmol for GA and less than 0.3 mmol of EGDGE) exhibited poor mechanical properties or leaded to unexaminable membranes, as published previously.[3] Higher amounts of crosslinkers, as used in this study (1.3 mmol for GA and 0.9 mmol for EGDGE), caused crumbling or breaking of the prepared membranes.[3] The mechanical features of HTCC-g-GMA/QPVA-g-GMA membranes developed in great dependence on added photoinitator Irgacure 2959. Membranes containing 0.5 wt.% of Irgacure 2529 were less stable and showed higher alkaline swelling than products with 5 wt.% photoinitator. Higher amounts of photoinitiator caused a rough and inhomogeneous membrane surface, containing white crystals all over, resulting in dissatisfying membrane features. The membranes gained from chemical-physical crosslinking with crosslinking agents exhibited inferior swelling behavior than the UV-crosslinked membranes, which corresponds to the stability results described before. For HTCC-g-GMA/QPVA-g-GMA products, there is no need for toxic crosslinking agents, but the modification step for grafting a photosensitive group onto the utilized polymers was performed by using toxic GMA, but can be substituted by less toxic compounds for future approaches, such as polyethylene glycol diacrylate.[72] However, hydrogels can be formed after only a few minutes of being exposed to UV-light, while the crosslinking agents GA and EGDGE need more time for the network-forming process.[3] Furthermore, the HTCC/QPVA membranes had to be crosslinked under stirring at 60°C as well as being dried over night at 60°C, followed by undergoing a temperature program up to 130°C, while HTCC-g-GMA/QPVA-g-GMA membranes can be prepared and dried at RT. Regarding the thermal degradation behavior of the membranes, products prepared with GA and EGDGE provided an onset degradation temperature above 280°C, while photocrosslinked products with the highest amount of Irgacure present an onset degradation temperature of 150°C. In addition to superior thermal stability, HTCC/QPVA membranes crosslinked with GA and EGDGE provided a much higher alkaline stability by lasting up to 22 days in 1 M KOH at 60°C, while UV-crosslinked membranes endured 5 days under the same conditions. The results of alkaline swelling show suitable results, which are inversely proportional to the observed stability. UV- crosslinked membranes exhibit alkaline swelling of 320% with highest Irgacure concentrations and

60 | P a g e crosslinking time, while networks formed with GA and EGDGE showed values between 90 and 225% in dependence of the amount of crosslinker. The ionic exchange capacity was similar for both membrane types, up to 1.300 meq/g for membranes prepared via UV-crosslinking technique and up to 1.878 meq/g for products gained from chemical-physical crosslinking with GA and EGDGE. The ethanol permeability could only be determined of HTCC/QPVA membranes, as the UV-crosslinked membranes did not exhibit enough stability to be installed in the diffusion cell after the preparatory alkaline treatment. The measured ethanol permeability was temperature-dependent, with an average value at 60°C of 5.21*10-7 cm2*s-1.

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Conclusion and Outlook

In this work two crosslinking concepts, chemical-physical crosslinking via crosslinking agents GA and EGDGE and UV-mediated crosslinking have been successfully applied on quaternized chitosan and PVA membranes. At first, the native polymers have been quaternized with EPTMAC for physical-chemical crosslinking with GA and EGDGE. In order to introduce the photosensitive groups necessary for UV- mediated crosslinking, GMA was successfully grafted onto HTCC as well as onto QPVA. The achieved degree of methacrylation for HTCC-g-GMA could be altered between 0.6-53%, while QPVA-g-GMA exhibited an average DS of 55%. For chemical-physical crosslinking, the utilization of the toxic agents glutaraldehyde and ethylene glycol diglycidyl ether turned out to be necessary, while the photoinitiator (Irgacure 2959) used for UV-mediated crosslinking exhibit very low toxic features. The time necessary for forming networks was longer for chemical-physical crosslinking than for UV-mediated crosslinking, whereby hydrogel networks could be received after only a few minutes. The required temperature for crosslinking with GA and EGDGE was 60°C including a temperature treatment of up to 130°C, while UV-mediated crosslinking enables network formation at RT. Membrane series of both crosslinking types have been prepared in dependence of various factors and analyzed regarding their alkaline swelling behavior, ion exchange capacity, chemical, mechanical and thermal stability. The membranes prepared via crosslinking agents show lower swelling behavior and therefore higher chemical stability. This feature, as well as the average IEC of about 1.400 meq/g and low ethanol crossover, reduced to 3.30*10-7 cm2*s-1 at 60°C, qualify the prepared HTCC/QPVA membranes as a promising candidate for applications as anion exchange membranes in alkaline fuel cells such as direct ethanol fuel cells.[3] The publication of these outcomes in a dedicated scientific journal, as well as further investigation, in particular on increasing the IEC of the membranes, are planned. The membranes prepared via UV-crosslinking result in hydrogels, which swelling behavior can be controlled up to a certain degree. Chitosan-based hydrogels are promising materials for applications in the biomedical field. Further research can pave the way for grafting other photosensitive functional groups onto HTCC and QPVA, such as poly(ethylene glycol) diacrylate, as it has already been demonstrated for native chitosan[54]; this will enable avoiding the use of the toxic compound GMA for the modification process. To summarize, UV-crosslinking can be interpreted as a “green process” that has the potential to replace fossil based chemicals, which qualifies it as a promising method for future chemical industry in many fields.

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