Bio-based hybrid nanocomposites and ionic liquids-loaded hydrogels as new multifunctional sustainable solutions for stone conservation

(cc)2023 PABLO IRIZAR MERINO (cc by-nc-nd 4.0) Bio-Based Hybrid Nanocomposites and Ionic Liquids-Loaded Hydrogels as New Multifunctional Sustainable Solutions for Stone Conservation This Ph.D. Thesis has been developed in IBeA research group, in the Analytical Chemistry Department of the University of the Basque Country (UPV/EHU), as well as in the University of Messina in the Chemical, Biological, Pharmaceutical and Environmental Sciences Department, under the supervision of the Ph.D. directors Olivia Gómez Laserna and Irantzu Martinez Arkarazo. Pablo Irizar Merino September 2023 If you're going through hell, keep going..... Winston Churchill Acknowledgements A I am grateful to the Spanish Ministry of Economy and Competitiveness (MINECO) for my predoctoral fellowship (PRE2018-085888) as well as, to the Ikerkuntza eta Berrikuntza Analitikoa (IBeA) research group of the Analytical Chemistry Department for the technical and scientific support. Specifically, I would like to thank Prof. Marian Olazabal and Prof. Gorka Arana for making the funding of this PhD. thesis possible through the fulfillment of the aims involved in the following projects: ⇒ Synthesis, characterization and validation of multifunctional nano-reinforced sustainable hybrid products for the recovery and protection of stone surfaces (PHETRUM, ref. CTQ2017-82761-P) funded by MINECO. ⇒ Development of mortars resistant to environmental contamination and biodeterioration and innovative sustainable systems for cleaning and restoring Built Heritage (DEMORA, ref. PID2020-113391GB-I00) funded by the Spanish Ministry of Science and Innovation (MICINN). At this respect, the Research General Services of the UPV/EHU (SGIKER) is also acknowledged by the support of the Singular Coupled Multispectroscopy Laboratory (LASPEA), the Macroconduct, Mesostructure and Nanotechnology Unit as well as, the Analytical and High Resolution Microscopy in Biomedicine laboratory and the X-Ray Service of Rocks and Minerals Unit. After these, I would like to especially thank the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences of the University of Messina, specifically to Prof. Paola Cardiano, for welcoming me in their laboratories and cooperate to develop part of this research work. Finally, I would like to express my deepest gratitude to my thesis advisors, Dr. Olivia Gómez and Dr. Irantzu Martínez, whom I hold in very high regard for their unwavering support, and dedication, that made the development of this thesis work and my own skills possible. Lastly, I do not want to forget my friends, and my PhD. Colleagues for accompanying me in this challenge, and especially my family and couple that were always with me sharing the good and bad moments these last 4 years. I TABLE OF CONTENTS CHAPTER 1: Introduction __________________________________________________ 1 1.1. Decay of stone materials: Causes and degradation patterns _______________________ 2 1.2. Consolidating materials for stone recovery and protection ________________________ 7 Conventional products __________________________________________________________ 8 1.2.1.1. Silica-based materials _____________________________________________________ 8 1.2.1.2. Organic polymers: Epoxy resins ____________________________________________ 10 Advanced materials ___________________________________________________________ 12 1.2.2.1. Bio-based epoxy resins polymers ___________________________________________ 13 1.2.2.2. Bio-based epoxy-silica hybrid materials ______________________________________ 14 Multifunctional advanced solutions: Synthetic strategies _____________________________ 16 1.2.3.1. Ionic liquids ____________________________________________________________ 17 1.2.3.2. Plant extracts ___________________________________________________________ 19 1.2.3.3. Nanotechnology ________________________________________________________ 19 1.3. Cleaning technologies for stained stones _____________________________________ 20 Solid support and cleaning agents _______________________________________________ 21 1.4. References _____________________________________________________________ 23 CHAPTER 2: Objectives ___________________________________________________ 37 CHAPTER 3: Materials and methods ________________________________________ 39 3.1. Epoxy-silica hybrid material testing _________________________________________ 39 Microorganism growth tests ____________________________________________________ 39 Stone durability studies ________________________________________________________ 41 3.1.2.1. Stone materials _________________________________________________________ 41 3.1.2.2. Stone treatment ________________________________________________________ 42 3.1.2.3. Accelerated aging experiments ____________________________________________ 43 3.2. Ionic Liquids-loaded hydrogels testing _______________________________________ 43 Microorganism growth inhibition tests ____________________________________________ 43 Metal sequestering capability and cleaning tests ____________________________________ 44 3.2.2.1. Stone materials _________________________________________________________ 44 3.2.2.2. Artificial metallic stains production _________________________________________ 44 3.2.2.3. Natural metallic stains ___________________________________________________ 45 3.3. Instruments ____________________________________________________________ 45 Elemental Analysis ____________________________________________________________ 45 Chapter 1 2 economically viable, as it serves as a crucial pillar for maintaining and enhancing longterm profitability strategies for sustainable development. As a consequence, the development of new methods for monitoring and conserving stone building materials has become a major global business. Concerning this aspect, the International Charter for the Conservation and Restoration of Monuments and Sites emphasizes the importance of balancing conservation efforts with economic development and encourages sustainable approaches to manage it. Besides, these guidelines highlight that any conservation or restoration action must be carried out with the utmost respect, taking into account every (even predictable) aspect that has historical significance [5]. Hence, targeted prevention and remediation strategies must be suitably designed during the decision-making phase, avoiding relapsing into inappropriate protocols that do not act on the cause or do not take into account the stone material needs and evolution of its treatment along the time [6]. This challenge requires multidisciplinary knowledge, which has become increasingly crucial for rehabilitation and conservation endeavors in recent years. Among other fields, the integration of chemical science into this sector is instrumental in fostering the development of cutting-edge technologies. The main premise is to improve existing markets and conventional products while also addressing emerging needs. These efforts hold substantial significance in national development and contribute to the strategic framework of the global economy. The research and validation of tailored stone conservation treatments present an ambitious task for Research and Development (R+D). The advancements achieved in this field can be also extrapolated to promote urban regeneration and renewal, contributing to a sustainable reactivation of the construction sector, as well as the chemical industry. To make significant progress in this line, it is imperative to thoroughly know the risks to which lithic materials are subjected, and gain a comprehensive understanding of the physical, chemical, and/or biological underlying degradation processes. 1.1. Decay of stone materials: Causes and degradation patterns In this respect, as a summary extracted from the diagnosis studies carried out during the last decade, it has been demonstrated that the deterioration of stone materials is closely related to their inherent composition and their interaction with the environment [7–10]. Specifically, atmospheric pollution, water, and biodeterioration agents are generally Introduction 3 pointed out as the most critical factors in their state of conservation. Indeed, the pollutants exert a significant effect, particularly through the acidic attack of combustion gases (CO2, SO2, and NOx), whose degradative action is further amplified in the presence of water (humidity, rain, and infiltration) due to the synergistic effect produced. The water penetrates the capillary network of the stone, acting as a vehicle for contaminants, dissolving them, and activating other compounds inherent to the substrate composition. Both agents give rise to the presence of soluble salts, which are classified as one of the most dangerous pathologies for lithic materials by the International Council on Monuments and Sites (ICOMOS) [9]. An early stage of this pathology is often first identified by the presence of efflorescences. If left untreated, the chemical and physical processes of damage continue to progress, resulting in important losses of the cementitious matrix of the stone substrate. Therefore, it is manifested on a macro scale through the formation of internal fissures, fractures, and the occurrence of pathologies such as disaggregation, sanding, and/or scaling (Figure 1.1). These degradation patterns are a clear signal of important problems of structural integrity and micromechanics. Thus, if this pathology is overlooked or not addressed, it can potentially lead to the complete loss of the stone piece, or even in the most severe cases, the collapse of the architectural structure. Figure 1.1. Common deterioration patterns related to the soluble salt presence in stone materials. a) Efflorescence with white crust, b) alveolarization, c) scaling, d) sanding, and e) disaggregation. Chapter 1 4 In addition to the aforementioned factors, the presence of plants and microorganisms including bacteria, fungi, lichens, and algae, among others, also exert an important impact on the conservation of stone materials [11–16]. These substrates present minerals, environments with light, warm temperatures, and water, as well as rich nutrients from organic residues, creating ideal conditions for the growth of life. The general microorganisms, often associated with biofilms of various colors and textures ranging from green, brown, or black patches to slimy or crusty formations, can act individually or in combination with other environmental factors, thereby amplifying their capacity for degradation. Thus, their impact extends beyond mere aesthetic changes (see Figure 1.2). Several physical and chemical damages, commonly known as biopitting [17], include pore size enlargement and cracking, which can occur due to factors such as the opening of pathways for water circulation, penetration of hyphae, as well as the presence of soluble salts produced through the synergistic action of water, colonizing biota, and the organic and inorganic acids they secrete (such as those involved in the sulfur cycle or ammonia oxidation). Figure 1.2. Common examples of biodeterioration related to microorganisms presence in stone materials. a) Cyanobacteria, b) biopitting degradation, c) fungi, and d) lichens [17,18]. As a result, these processes pose a significant threat to the integrity and longevity of stone materials. Therefore, preventive actions are crucial, particularly during the early stages. This involves selecting suitable cleaning methods to remove these damaging agents and subsequently protecting the stone substrate through measures such as the application of biocidal products and coatings or consolidation treatments. Introduction 5 Furthermore, these various environmental factors can also be responsible for staining pathological manifestations, which are common issues encountered in the field of stone conservation. In addition to the formation of biofilms, the typical discolorations found can be directly attributed to environmental pollution, chemical reactions, and aging processes [19,20] (Figure 1.3). Figure 1.3. Common cases of stone deterioration related to environmental factors: a) Black crusts and dissolution of material caused by pollutants, b) chemical reactions, and c) aging processes. Specifically, metallic stains can be pointed out among the most frequent degradation patterns. These discoloration areas typically occur outdoors due to weathering processes affecting adjacent metallic materials, which are often used as decorative or functional items close to stone surfaces. The chemical attack on the metallic structure leads to the formation of corrosion products, and with the action of water, ions can migrate toward Chapter 1 6 the stone substrates [9–11]. Such leaching phenomenon not only impacts the aesthetic appearance of the lithic materials but also has the potential to compromise their longevity. Additionally, depending on the specific ions involved, it can even facilitate certain biological activities [21,22]. In detail, stains caused by iron and copper are of significant concern due to their widespread use in roofs, sculptures, and even buildings constructed with steel and bronze materials. It is known that leaching processes give rise to the formation of characteristic reddish-brown and greenish areas, which are composed of the cited oxides as well as various salts like chlorides, sulfates, carbonates, or nitrates [22–24]. These chromatic alterations can be developed in a short time, evidencing that they are not limited to ancient constructions but can also be observed in recent buildings where these metallic materials are extensively used (Figure 1.4). Figure 1.4. Examples of iron and copper metallic stains caused by the weathering of the metallic components of: a-b) building and c-e) statues The difficulty in removing these stains depends on several factors, including the underlying causes, the extent of their spread, their composition, and the depth of penetration into the stone substrate. As a result, this seemingly straightforward pathology can pose significant challenges for conventional cleaning methods, which may not always be suitable for removing complex stains without causing damage or altering the original appearance of the stone matrix [25,26]. Therefore, employing proper maintenance and conservation techniques is crucial to mitigate the effects of this pathology and ensure the long-term protection of stone materials along the time. Introduction 7 At this respect, although, the preservation of stone materials has become a significant multidisciplinary research field, there is still an increasing demand for more scientific and respectful approaches, that also prioritize environmental considerations [20,27]. Therefore, designing innovative conservation solutions is a complex and critical part that requires the collaborative work of many professional profiles (Conservators, archaeologist, chemists, architects and historians). Adding to this, the exploration of green alternatives to conventional treatments has not been widely implemented, as consolidating and coating products derived from harmful compounds are still widely used for treating large stone surfaces, posing risks to both human health and the environment [28]. Additionally, cleaning and preventive protocols often rely on the use of harmful biocides and aggressive chemical products that do not contribute to the principles of Sustainable Development Goals (SDGs) [29,30]. Hence, there is ever-growing social awareness that calls for innovative effective solutions that leave behind decisions based solely on cost-effectiveness and short-term outcomes and prioritize environmental considerations, establishing them as crucial factors that require special research divulgation in the professional sector and industrial attention. In accordance with this necessity, the following sections collect an overview of the products and methodologies conventionally employed highlighting their advantages and limitations for the consolidation, protection, and cleaning of stone materials. Moreover, the opportunities offered by ongoing chemical technologies to design advanced materials and methods to effectively address the current challenges in the field are also explored to focus a timely line of progress that directs the objectives of this Ph.D. Thesis, that specifically deals with treatments for silicate and carbonate stones. 1.2. Consolidating materials for stone recovery and protection Once weathering has begun, it is essential to treat the stones with products that can enhance their resistance to decay. They should ensure strong adhesion and intergranular cohesion within the stone matrix while protecting against the future detrimental effects of water, pollutants, and biodeterioration agents [31,32]. Moreover, the new generation of materials should adhere to current eco-sustainability criteria, and meet the mid-term treatment requirements to prevent issues that may arise from the use of untested materials, as seen with several products widely employed in past restoration activities. Therefore, the desired range of capabilities for a stone conservation product necessitates the specific design of advanced trifunctional materials with consolidating, hydrophobic Chapter 1 8 and biocidal properties that at the same time guarantees the use of minimal amounts of toxic compounds and includes studies of their behavior over time. Conventional products The products first employed for conservation purposes were selected according to their availability and the scientific advances at the time, being applied without the chemical knowledge of their behavior in the midand longterm. However, although the scientific community, and experience acquired from cases of studies, have already demonstrated their handicaps, the reason why are still currently used is often based on their extended application during the last decades instead of the long-term effectiveness and sustainability criteria. Some of them are highlighted by their low cost and short-term results rather than crucial features like chemical stability, polluting species or toxic side products formation upon UV aging, and so on. Thus, the majority of these conventional products, yet only partially matching the above requirements, in the case of stones of silicate and carbonate nature, mainly belong to two categories [33–36]. 1.2.1.1. Silica-based materials The inorganic consolidating materials [37,38] have been the most traditional products used from the 19th century by their compatibility with the lithic matrix and their ability to bind deteriorated stone particles by forming insoluble phases within the voids and pores through salt precipitation or chemical reactions with the stone substrate. Despite their historical use, alkali silicates and silicofluorides have been widely rejected by cause the formation of soluble salts or shallow crusts, and discoloration and exfoliation phenomena of the stone's surface. In the same way, alkaline earth hydroxides like the highly used calcium hydroxide have been also discouraged due to its consolidanting capability is limited to the surface and thus, not reach to treat the interior of the stone substrate and not provide long-term protection [38,39]. In contrast, from the century 20th, organosilica-based materials [40–42] obtained by solgel technology such as alkoxysilanes and alkylalkoxysilanes (i.e. tetraethoxysilane, triethoxymethylsilane, or poly(dimethylsiloxane) have been widely used, and in fact, they are currently preferred for consolidating siliceous sandstones and also by their feasibility to recover calcareous stones. These families of monomeric molecules can react with water by a polymerization process, which starts with the hydrolysis of the alkoxysilane and continues until all the alkoxy groups are liberated, resulting in the formation of either Introduction 9 alkylpolysiloxane or silica compounds intimately jointed to the stone substrate (Figure 1.5). The diluting alkoxysilanes with solvents helps to control their viscosity and reaction rate, facilitating a better penetration into the damage matrix. Figure 1.5. General polymerization reaction occurred during the sol-gel process and an example of an in situ polymerization process into a stone substrate of silicated nature. Thus, although they solve some of the above drawbacks, the influence of R groups (alkyl groups) and OR groups (alkoxy groups) on some fundamental properties of the sol-gel process is the main reason why the list of alkoxysilanes used for stone conservation is so limited. For a consolidant to fulfill its function, the material must have the ability to form a three-dimensional network, and therefore, it must have a minimum of three reactive groups. This requirement eliminates all bifunctional compounds, which only form linear polymers. On the other hand, some triand tetra-functional compounds, such as R-SiH3 and SiH4, are toxic, volatile, and generate hydrogen gas through hydrolysis, so they have also been discarded. Only triand tetra-functional alkoxysilanes are relatively non-toxic and non-volatile (lower vapor pressure), and the byproducts of their hydrolysis are Chapter 1 10 alcohols, which are not corrosive to stones. Additionally, they have low to moderate reactivity with water, which can be an advantage for a stone consolidant since an excessively fast reaction can limit the depth of penetration of the liquid into the stone material, causing it to gel before it can penetrate enough to fulfill its function [42]. However, the most used materials often display cracking upon drying, thus, reducing the consolidation efficiency and, due to their structural rigidity, show mechanical properties which are not compatible with the ones of weathered stones. Moreover, the low molecular weight starting compounds are also prone to evaporation before the polymerization process occurs inside the stone substrate [43,44]. 1.2.1.2. Organic polymers: Epoxy resins The use of synthetic organic polymers of thermoplastic and thermoset nature such as acrylic polymers (methylmethacrylate and butylmethacrylate), polyurethanes, and perfluoropolyethers have been implemented for stone consolidation from the early 1960s due to their potential to enhance the mechanical properties [37,44–47]. However, their application implies the use of harmful solvents (dichloromethane, dimethyl sulfoxide, and hexane among others) and, a part from the environmental issues, their characteristic rapid evaporation can draw the organic consolidants back to the stone's surface, leading to the formation of impervious hard surface crusts. In addition, the results at mid-term also showed negative effects due to their inherent susceptibility to degradation by oxygen and ultraviolet radiation, primarily affecting the chromatic features of the surface of the treated stone, as well as, with the passing of the time their capabilities to maintain the integrity of the stone matrix, typically causing problems. Among the conventionally used, epoxy resins are still widely accepted in consolidating tasks for limestone, marble, and sandstones, and even for mortars of integrity highly compromised [37]. These polymers are highlighted to exhibit excellent adhesive and hydrophobic properties, being also featured by suitable hardness and rigidity, with thermal and chemical resistances that surpass the durability of other polymers. In addition, these thermoset materials are capable to reach deep penetration, given that they are deposited in solution, and their in situ polymerization processes occur into the porous stone network. These advantages together with their customization options make them the traditional preferred option for stone consolidation among the products of organic nature [48,49]. Introduction 11 Epoxy resins stand out by their high versatility since, thanks to their epoxide rings, single or multiple, can be transformed into thermosetting polymers by a wide variety of curing reactions, which can convert the initially low molecular weight precursor into an infusible cross-linked three-dimensional polymer [50,51] with exceptional physicochemical properties. For this, compounds containing active hydrogens, such as polyamines, polyacids, polyphenols, polymercaptan, amines, and anhydrides, among others, are commonly used as hardening reactants. Through nucleophilic attack reactions, they are able to open the epoxy rings and lead to crosslinking by the formation of covalent bonds between the polymer chains to further develop the organic cross-linked network (Figure 1.6) [52,53]. Thus, the choice of the curing agent and the reaction conditions significantly impacts the properties of the final material, making them crucial factors in the thermosetting design for each specific application. Figure 1.6. The general cross-linking reaction of an epoxy resin polymer using a nucleophile compound as a curing agent. Given the capability of the oxirane ring to react through multiple pathways, together with their general excellent thermal, mechanical, and electrical properties, along with high adhesiveness and resistance to chemicals and moisture, make them extensively used in numerous sectors (aerospace, automotive, consumer goods, food packaging, electrical and electronic, building and civil engineering, and biomedicine) as general-purpose adhesives, fiber-reinforced materials, high-performance paints, and coatings or consolidants [51,54–57]. To meet the global demand, more than 75% of epoxy resin production still comes from Bisphenol A diglycidylether (DGEBA), which is based on Bisphenol-A (BPA) as parent product [58]. BPA is known as a controversial petrochemical-based compound that has been found to behave like a synthetic estrogen and to act as an antagonistic ligand to hormone nuclear receptors [59,60]. It has been shown to play an important role in the pathogenesis of various endocrine disorders, including female and male infertility, precocious puberty, hormone-dependent cancers, and several metabolic and neurological Chapter 1 18 Figure 1.9. Structural representation of commonly used constituents of ionic liquids, a) cations and b) anions [93]. In this respect, two types of conventional ILs that stand out because of their biocidal properties are quaternary ammonium (QASs) and phosphonium salts (QPSs) [106–109]. Regarding their structures, the long lipophilic substituent of the QASs and QPSs alongside the charged center allows it to become incorporated into the phospholipid bilayer. As a result, the cell loses its ability to maintain a stable internal environment, leading to the leakage of cellular contents and ultimately the death of the microorganism. This mechanism of action is non-specific, meaning that QASs and QPSs are active against a wide range of microorganisms, regardless of the species; that is, they are effective against bacteria, fungi, parasites, and even lipophilic viruses. This biocidal capability has been widely demonstrated in other industries in which, thanks to the broad-spectrum activity of QASs and QPSs, they have been employed for a variety of applications, including disinfection of surfaces, medical equipment, water treatment systems, topical antiseptics, and wound dressings and more recently, as antimicrobial material for air disinfection and sterilization. This way, covalently incorporating QAS/QPS moieties within an epoxy-silica hybrid network as multifunctional eco-substitutes for the toxic industrial biocides could achieve a broad-spectrum antimicrobial activity and an improved hydrophobic behavior given their aliphatic chain length. Introduction 19 1.2.3.2. Plant extracts An even greener alternative in this research line can be the incorporation of essential oils (EOs), such as eugenol, thymol, menthol, and so on, which have shown remarkable biocidal activity and suitable miscibility with consolidating formulations [110–112]. Their structure does not predict a significative chemical crosslinking with the epoxy-silica hybrid network, so they could remain dispersed into the polymeric matrix without modifying the main properties, or in change, altering the balance of the hybrid's base characteristics. However, their high volatility and degradation rate are handicaps to have into account in the material design phase [110,113]. 1.2.3.3. Nanotechnology Nanoparticles (NPs) are being currently exploited in the development of materials for maintaining stone substrates, offering new opportunities to design conservation products with innovative functionalities. Consequently, NPs are specifically employed for various purposes such as cleaning, consolidation, protection, and antimicrobial treatments i.e. metal NPs (Au, Ag, Pt), oxides and hydroxides NPs (TiO2, ZnO, SiO2, CaOH2, Mg(OH)2, Sr(OH)2) or even hydroxyapatite and carbonated derivates [89,114–116]. In fact, it is widely recognized that nanoscale materials often exhibit superior properties compared to traditional products, including enhanced mechanical and thermal characteristics, increased reactivity, and deeper penetration within stone matrices, among others. As a result, innovative studies have success in incorporating biocidal nanoparticles into hybrid consolidants and water repellent formulations to gain nanocomposite materials that achieve to mitigate the degradation processes of stones exposed to the environmental agents [31,32,117–119]. In particular, nano-TiO2 and lanthanide-doped TiO2 (in anatase form) have garnered significant attention due to their potent photocatalytic activity, making them highly effective catalysts for the degradation of various pollutants. Simultaneously, they impart strong self-cleaning and antimicrobial properties against bacteria and fungi to the materials in which they are incorporated. Furthermore, the advantages of nanotechnology have been also recently exploited to act as container capsules of EOs prior to the incorporation of biocidal formulations to the materials, for example, silica NPs or halloysite nanotubes [120,121]. These kinds of approaches could allow the incorporation of biocidal properties to a base material minimizing the handicaps directly related to the efficacy in the long term of EOs, as well as the possible negative effect on the final material features. Chapter 1 20 1.3. Cleaning technologies for stained stones Traditional physical and chemical methods such as mechanical abrasion, sandblasting, sand and water pressure, or acid washing have been widely used by their high effectiveness in removing stains from stone surfaces (Figure 1.10) [122]. However, they can be time-consuming, costly, and potentially hazardous to both the user and the environment. In addition, they are discouraged because they can also cause long-term damage to the stone substrate, particularly if used incorrectly or on a regular basis, causing excessive erosion, discoloration phenomena, and in some cases, even the deterioration of undamaged areas of the lithic material by acid attack or soluble salt processes, among others. As a consequence, conservators and researchers alike, ask for more advanced, respectful, and environmentally-friendly methods for selectively removing metallic stains from stone substrates, also claiming their testing at midand longterm. Figure 1.10. a) Examples of clean-up physical methods using a) high-pressure sandblasting, b) manual brushing, and c) laser. In this regard and specifically for metallic stains, various clean-up methods, as diverse as laser cleaning [123–126], biological [21,127–129], or based on complexing or chelating agents [130,131], are currently applied as alternative conservation solutions. Laser Introduction 21 cleaning (Figure 1.10) is minimally invasive and offers a controlled and selective removal action. It can be monitored in real-time and does not require solvents or mechanical and chemical contact with the stone matrix. However, the methodology requires high costs when large areas are treated, as well as extensive knowledge of the technique and substrate to avoid causing damage. Biocleaning could be a safe treatment for stained stones, as it allows highly versatile, effective, and selective application options. However, it also has some drawbacks; among them, the adherence to overly textured surfaces and the need to maintain optimal hydrothermal conditions for bioagents to remain viable are highlighted. Additionally, this method may not be eligible for application in the case of mixed stains with biological colonization, as it could potentially be detrimental by inducing their expansion. On the other hand, chemical methods based on ligands that (i.e. EDTA, cysteine, L-alanine etc.) form coordination complexes with the metallic ion contained in the stains are considered one of the most effective cleaning treatments. However, selecting sequestering agents is a complex task, as they should show a high specific affinity towards the ion of interest and a lower one towards the stone material composition to avoid damage. Besides, environmental and practical aspects such as non-toxicity, ease of handling and disposal, fast reaction, and low cost must also be considered. Accordingly, professionals in the field are continuously seeking innovative and gentle cleaning approaches to effectively remove the staining issues from stone surfaces. Solid support and cleaning agents In this regard, solid matrixes such as agar [132,133], polyvinyl-based materials [134–136], ionic exchange resins [137–139], or cellulose [130,140,141] have been recently used as supporting material of sequestering agents. They have demonstrated to be capable of acting as a suitable storage support and enabling a liquid-liquid exchange in which the metals diffuse. In addition, these malleable solids allow selective application treatments, and thanks to the slow release of the cleaning agent, the application can be controlled to preserve the treated substrate correctly. These advantages are crucial for the treatment of heterogeneous degradations that could require different cleaning needs. Among the mentioned supports, agar gel is a highly versatile and cost-effective threedimensional structure that is highlighted for being capable of holding significant amounts of water within its interstices. This characteristic allows a controlled water release with minimal risk on the substrate [133,142,143]. Moreover, its specific advantage over traditional aqueous methods lies in its ability to capture removed soiling within the gel Chapter 1 22 structure. Furthermore, several studies evidenced their capability to remove the typical metallic stains, thanks to the implementation of the chemical agents mentioned above, in which chelatants such as EDTA and citrates have been thoroughly tested [132,143–148]. Similarly, an inherently biocidal kind of starch called Kuzu has recently demonstrated structural characteristics and physicochemical properties that pose it as a potential candidate for solid support for similar purposes, in fact, it has been explored in the cleaning of old paper documents [149–151]. In the same manner, IL strategies can be also a promising alternative for classical chelating, they have proven to be suitable solvents to be used as effective substitutes for synthesis, catalysis, and extraction processes. In fact, several works have proved the significant ILs potential [94–99], either as direct solvent extraction or integrated into a solid matrix, to sequester metal ions in other application fields for example from solutions and even dissolve a variety of metallic oxides, typically the most prevalent species in metallic stains. Thus, it is highly interesting to investigate if agar and/or kuzu hydrogels are capable to retain ILs of long aliphatic chains while allowing them to act as ion sequesters in cleaning treatments. The information here collected demonstrates, there is a wide range of recent technological advancements that can be investigated along this Ph.D. thesis work to allow the development of more efficient and specific materials to respectfully address the most imperative conservation challenges associated with stone conservation. In pursuing this goal, sustainability must be also a fundamental aspect, requiring the search for environmentally friendly solutions that minimize negative impacts on human health and the natural surroundings. Indeed, the progress in the design of new consolidating and cleaning solutions will help us to preserve the stone heritage, ensuring its legacy for future generations, in the same way, that contributes to economic capacity, aligning with the general objectives of the global SDGs. Introduction 23 1.4. 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Prieto-Taboada, F.L. Hernando, M.D. Rodríguez Laso, J.M. Madariaga, A first evaluation of the usefulness of Kudzu starch in cultural heritage restoration, Sci Rep. 10 (2020) 15598. https://doi.org/10.1038/s41598-020-72643-x. [151] Y. Zhong, X. Song, Y. Li, Antimicrobial, physical and mechanical properties of kudzu starch–chitosan composite films as a function of acid solvent types, Carbohydrate Polymers. 84 (2011) 335–342. https://doi.org/10.1016/j.carbpol.2010.11.041. 36 37 CHAPTER 2: Objectives The aim of this PhD. thesis was to develop advanced bio-based materials as multifunctional sustainable alternatives to conventional treatment products used for the stone conservation. The overarching goal encompasses different conservation problems, on the one hand, developing tailor-made epoxy-silica hybrid materials, with consolidating, hydrophobic, and biocidal capabilities were developed. Moreover, Ionic Liquids-Loaded Hydrogels were also designed to clean metallic stains and to act as biocidal on stone substrates. According to the two groups of materials developed, the following operational objectives were established: ⇒ Bio-based epoxy-silica hybrids materials, with consolidating, hydrophobic, and biocidal properties, for advanced stone conservation.  To develop tailor-made BPA-free epoxy-silica hybrids that meets the specific multifunctional properties required for an advanced stone conservation treatment, as well as for the environmental policies of production and use, through a sequential building block design strategy composed of four steps. Accordingly, Step, named “Epoxy precursor”, consists of the selection and synthesis of a bio-based epoxy resin precursor. Subsequently, Step, named “Base thermoset”, focuses on the selection of the hardening agents to configure the thermo-mechanical and hydrophobic properties of the thermosetting Chapter 2 38 material, which acts as the base block of the design. Step, named as “Epoxy-Silica hybridation”, involves the functionalization of the previous thermoset material, with selected silica precursor agents, to gain an organic-inorganic network of thermoplastic nature and, to modulate the required thermo-mechanical and hydrophobic behaviour of its hybrid skeleton. Lastly, Step, defined as “Epoxysilica hybrid enrichment“, focuses on investigate various synthetic strategies, ionic liquids (ILs), essential oils (EOs) and nanoparticles (NPs), to finely adjust the main properties, and implement a biocidal capacity.  To characterize the materials developed and to conduct the decision making process in each step of the design through thermo-mechanical, water repellence and biocidal studies. To investigate the advantages addressed by the implementation of spectroscopic methodologies, along the different steps of the design process, complementarily to conventional techniques employed in the field of material development.  To determine the effectiveness of the most promising enriched epoxy-silica hybrid formulations as stone conservation treatments at mid-term, through their application on stone specimens, and a subsequent accelerated aging on an acid atmosphere. ⇒ Bio-based ionic liquid (ILs)-loaded hydrogels, with metal sequestering and biocidal properties, as respectful cleaning technology of stained stones.  To develop a bio-based hydrogels with loaded ILs for the respectfully removal of iron and copper-rich metallic stains from stone surfaces by a sequential design composed of two steps. Step, named “Solid support selection”, consists of selecting a bio-based hydrogel to allow the controlled encapsulation of specific ILs, and preserve the integrity of the stone surfaces. Subsequently, Step, named as “IL loading adjustment”, involves the selection of the ILs, loading level and way of application to obtain an effective and non-aggressive cleaning technology.  To evaluate the effectiveness of the developed hydrogels loaded with ILs for the described purpose, through laboratory simulated cleaning tests by using elemental and molecular imaging studies.  To test in situ the most promising materials to validate their efficience to clean naturally stained stones. Materials and methods 39 CHAPTER 3: Materials and methods Along this chapter, the general materials, methods and analytical techniques employed to assess the development of the materials, as well as to evaluate their effectiveness for preserving and cleaning stone substrates are provided. The related data are addressed in the following sections, according to the two groups of material developed. At this respect, it should be noted that, aiming to enhance the comprehension of the complex sequential synthetic approach followed, the detailed specifications of the synthesis, characterization and testing processes are provided in the respective dedicated chapters. In the same manner, the information regarding the chemicals that play a significant role is collected and categorized in Appendix A. 3.1. Epoxy-silica hybrid material testing Microorganism growth tests Disk-diffusion and live-dead staining methods were used to investigate the microorganism growth inhibition capability and biocidal power, respectively, of the most promising epoxy-silica hybrid materials developed. Specifically, disk-diffusion was employed in Chapter 6. For this purpose, the selected enriched hybrid film samples were cut into 10 mm diameter disks, dried at 60 °C in a vacuum oven (< 10−2 Torr) for 24 h, and subsequently sterilized by immersion in 70% ethanol for 10 min. Then, an strain of Chapter 3 40 Arthrobacter spp. acquired from the Spanish Type Culture Collection (CECT) (University of Valencia) was subcultivated under aseptic conditions [1–3]. Accordingly, tryptone soy agar (TSA) plates were inoculated with 100 μL of a freshly prepared bacteria suspension of approximately 1.5 × 108 CFU mL-1 and spread over the entire agar surface using a sterile cotton swab. After the disks were placed on agar surface, the plates were aerobically incubated at 25 °C for 48 h under visible irradiation. The antimicrobial halo (nwhalo) was calculated according to the following equation (3.1) where diz is the inhibition zone and d the disk diameter [2,3]. 𝑛𝑛𝑤𝑤ℎ𝑎𝑎𝑎𝑎𝑎𝑎 =�𝑑𝑑𝑖𝑖𝑖𝑖−𝑑𝑑 2� 𝑑𝑑 (Eq. 3.1) The live-dead staining method [4] was employed in Chapter 8. In this case, a strain of Micrococcus Luteus [5,6] acquired from the CECT was subcultivated in a TSA plate for 24h at room temperature. Then, a single colony was picked to the 40 mL LB (Condalab) at 37 °C and incubated for approximately 12 h, under shaking at 200 rpm, being finally adjusted to an appropriate optical density of ~0.1 by dilution. Then, LIVE/DEAD BacLight kit (Thermo Fisher Scientific Inc.) was used to stain the incubated bacteria. According to the kit protocol, 25 mL of the incubated solution was taken and, the culture media was removed by its centrifugation at 4000 x g for 10 min and the removal of the supernatant phase. Then, the resulting bacteria pellet was re-suspended on 2 mL of wash buffer and, 1 mL of this suspension was subsequently diluted on 20 mL of wash buffer. After, it was incubated for 50 min at room temperature and gently mixing every 15 min. The obtained suspension was again centrifuged, and the new pellet was re-suspended in 20 mL of clean wash buffer. Once the washing procedure was repeated twice, the bacteria are finally resuspended on 15 mL of wash buffer. When the growth medium was completely removed from the bacteria, equal volumes of SYTO 9 and propidium iodide compounds were combined in a microfuge tube. Then, 3 μL of the dye mixture were added per milliliter of the bacterial suspension, being the latter incubated at room temperature in darkness for 15 min. The stained bacteria were placed on the previously sterilized film hybrid materials for 10 min, and subsequently the bacteria on the surface of samples were observed by fluorescence microscopy. Materials and methods 41 Stone durability studies The most promising multifunctional epoxy-silica hybrids formulations were selected to test if the materials here designed meet the first premises of a mid-term stone conservation treatment. 3.1.2.1. Stone materials A natural mediterranean calcarenite stone was used for testing the capability of protection (Chapter 6) and restoration (Chapter 8) provided for the developed hybrid materials due to its wide distribution across the Mediterranean Basin (Spain, Portugal, Italy, Greece, Tunisia, etc.) since the Bronze Age. Numerous quarries throughout Europe extract this type of stone, specifically the Albamiel variety used belong to the representative Rosales quarry (Albacete, Spain), and was purchased from Rosal Stones Company (Murcia, Spain). This lithotype is characteristic of Iberian culture and gained popularity during the Roman Empire through the Cartago Nova trade route, reaching the entire Iberian Peninsula and northern Europe and Africa. As a result, it was utilized by various cultures for a wide range of purposes, including fortifications, bridges, aqueducts, temples, tesserae production, mosaic floors and religious sculptures. Notable examples of its use can be seen in the Lady of Offering, Tolmo de Minateda, the archeological site of Taormina and the Alhambra. Even today, it is extensively employed in its region of origin for the construction of external facades and pavements, and also plays a crucial role in the restoration of heritage buildings [7–10]. According to the data provided by the supplier, the stone was characterized by an apparent density of 1.940 Kg.m-3, a porosity of 28.2% and a water absorption capacity of 14.53%. To conduct the material essays, the slab were cut to specimens of 2 x 2 x 1.5 cm size (Figure 3.1). Then, the standard stone samples were weighed, and colorimetrically studied. Chapter 3 48 eVISARCH [16] and eVISART [17] standard databases, the RRUFF online database [18] and a database self-developed for this purpose. Nuclear magnetic resonance spectrometry, 1H-NMR and 13C-NMR were used to carry out the characterization of the synthesized epoxy precursors (Chapters 5 and 7) by a Bruker AVANCE 500 equipment using CDCl3 as solvent. Data acquisition and treatment was carried out by Bruker Topspin 4.0.7 software. A potentiometric system Metrohm 720 titrino, equipped with an interchangeable 20 mL burette and a combined glass and Ag/AgCl(s) Ingold IL 9415 electrode, was employed for the determination of the polymer epoxy ratio (ER) by the back titration method (Chapter 7) [19]. Data acquisition was carried out with Metrohm Tiamo 2.5 software and then processed using the Gran’s method with MS Excel 2017 software. X-Ray Diffraction (XRD) analyses for the characterization of the NPs synthesized (Chapters 6 and 8) were carried out by means of a Bruker D2 Phaser desktop diffractometer, with a Cu tube (λ = 1.54056 Å), recording the data in the 2θ range of 20–90°, with an angular step size of 0.025°. The identification was made by using the database PDF2 [20]. A Shimadzu MultiSpec-1501 UV–Vis Diode Array spectrophotometer was used to measure the absorbance for the calculation of free amine groups in the funtionalizated NPs (Chapter 8) [21]. Thermomechanical and hydrophobic behaviour analysis The thermomechanical and hydrophobic properties for the design of the multifunctional epoxy-silica materials, from the thermoset base step to the enriched hybrid development (Chapters 5-8), were determined by a combination of the following instruments: Thermogravimetric analyses (TGA) to determine the suitability of the hardening agents and the silica forming additives for the hybrid development were carried out using a TA Instruments Q500 thermal analyzer, under nitrogen at 10 ml min-1, in the temperature range between 25 °C and 800 °C with a heating rate of 10 °C min-1. To complete the thermal data, differential scanning calorimetry (DSC) experiments were performed with a Mettler Toledo DSC3+, in the temperature range -60 to 220 °C, with a heating rate of 10 °C min-1, under a 20 ml min-1 nitrogen flow. Materials and methods 49 To investigate the mechanical properties and determine the plasticizing or load roles exerted by the forming silica compounds and enrichments used a dynamic mechanical thermal (DMA) analyser Epexor 100 N GABO Qualimeter was employed. For this purpose, rectangular hybrid films specimens of 9.0 mm x 13.0 mm x 2.9 mm were prepared and analysed in a temperature range from -70 to 150 °C, at a heating rate of 2 °C min-1 in tension mode. The tests were carried out at a strain rate of 0.5 % and 0.2 % for Static and Dynamic, respectively. DSC, TGA and DMA data were processed using Excel 2017 and Origin 2018 softwares. Finally, contact angle (CA) investigations were performed to determine the water repellence capability of the hybrid surfaces by means of two different contact angle instruments, Neurtek Instruments Dataphysics systems OCA 15EC and KRÜSS Drop Shape Analyzer - DSA25B. For this, Milli-Q water drops (2 μL/drop) were deposited onto the film surface and the average of 5 replicates was reported. The material behavior was classified in accordance to the established levels defined in Figure 3.5. [22]. Figure 3.5. Classification of the water repellence capability according to the contact angle values. Thus, surfaces that exhibit a CA greater than 150° were classified as superhydrophobic, indicating extremely high water repellency. Surfaces with contact angles between 90 and 150° were considered hydrophobic, meaning they have a moderate level of water repellency. Finally, surfaces with a CA less than 90° were classified as hydrophilic, and close to 0° super hydrophilic, indicating a tendency to attract and absorb water. Chapter 3 50 Microscopy image analysis A RoHS X4 digital microscope with a focus range of 15-40 mm was used for the optical microscopic visual inspection of the stones samples, treated and non-treated, during the durability stone tests to determine the effectiveness of the materials designed (Chapter 6 and 8). The images were obtained and processed using the HiView 1.4 software. Transmission Electronic Microscopy (TEM) analyses were performed to determine the average size of the synthesized NPs (Chapters 6 and 8) by a TECNAI G2 20 TWIN instrument, operated at 200 kV and equipped with LaB6 filament. The NPs samples were dispersed into methanol solvent and kept in suspension using an ultrasonic bath for 15 min. After, a drop of the suspension was spread onto a TEM copper grid (300 Mesh) covered by a carbon film followed by drying under vacuum. Finally, a Zeiss LSM 880 A1 fluorescence microscope was employed to determine the biocidal capability of the NPs and essential oils (Eos) epoxy-silica doped hybrid materials (Chapter 8) by staining assays, using a Fas Airy scan mode with excitation at 488 and 561 nm, and a sequential signal retrieval of 495-550 and 570-620 nm. The obtained images were processed using ImageJ 1.53c software. Colorimetric Analysis A PCE-CSM 5 colorimeter (PCE Instruments), with the 8 mm measuring aperture and CIE 10°observation angle and equipped with a silicon photoelectric diode sensor, was employed to monitor the chromatic alteration (color difference, ∆E) (n=5) [15] caused by the stone aging experiments and the application of the different hybrid materials before and after the treatment (Chapters 6 and 8). Besides, this type of analysis was also used to assess the cleaning effectiveness of the ILs-loaded hydrogels treatment (Chapter 9). For this purpose, the instrument was calibrated on a standard white cap reference before each measurement and the L*a*b* color space average values were collected following the ASTM D-1925 and CIE 2000 requirements [23]. Stone aging experiments The stone aging experiment were carried out (Chapters 6 and 8) using an Autonics Kesternich chamber (CCI, Spain). The camera was equipped with a TK4S High Accuracy PID Materials and methods 51 control module with a fixable temperature range, up to 50 °C, and humidity up to saturation. For these studies, the CO2 was used as the acid atmosphere precursor. 3.4. References [1] O. Gómez-Laserna, G. Lando, L. Kortazar, I. Martinez-Arkarazo, I. Monterrubio, E. Sevillano, P. Cardiano, M.Á. Olazabal, Eco-friendly nanocomposite products based on BPA-free epoxy–silica hybrid materials for stone conservation, Archaeol Anthropol Sci. 11 (2019) 5799–5812. https://doi.org/10.1007/s12520-019-00904-6. [2] A.W. Bauer, W.M.M. Kirby, J.C. Sherris, M. Turck, Antibiotic Susceptibility Testing by a Standardized Single Disk Method, American Journal of Clinical Pathology. 45 (1966) 493–496. https://doi.org/10.1093/ajcp/45.4_ts.493. [3] M. Martí, B. Frígols, A. Serrano-Aroca, Antimicrobial Characterization of Advanced Materials for Bioengineering Applications, JoVE. (2018) 57710. https://doi.org/10.3791/57710. [4] X. Xu, S. Ma, S. Wang, J. Wu, Q. Li, N. Lu, Y. Liu, J. Yang, J. Feng, J. Zhu, Dihydrazonebased dynamic covalent epoxy networks with high creep resistance, controlled degradability, and intrinsic antibacterial properties from bioresources, J. Mater. Chem. A. 8 (2020) 11261–11274. https://doi.org/10.1039/D0TA01419B. [5] P.J.A. Skipper, L.K. Skipper, R.A. Dixon, A metagenomic analysis of the bacterial microbiome of limestone, and the role of associated biofilms in the biodeterioration of heritage stone surfaces, Sci Rep. 12 (2022) 4877. https://doi.org/10.1038/s41598022-08851-4. [6] P. Descheemaeker, J. Swings, The application of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present in decaying Ledestone of the St. Bavo Cathedral in Ghent, Science of The Total Environment. 167 (1995) 241–247. https://doi.org/10.1016/0048-9697(95)04585-O. [7] L.M. Gil-Martín, M.A. Fernández-Ruiz, E. Hernández-Montes, Mechanical Characterization and Creep Behavior of a Stone Heritage Material Used in Granada (Spain): Santa Pudia Calcarenite, Rock Mech Rock Eng. 55 (2022) 5659–5669. https://doi.org/10.1007/s00603-022-02946-0. [8] A. Zornoza-Indart, P. Lopez-Arce, N. Leal, J. Simão, K. Zoghlami, Consolidation of a Tunisian bioclastic calcarenite: From conventional ethyl silicate products to nanostructured and nanoparticle based consolidants, Construction and Building Materials. 116 (2016) 188–202. https://doi.org/10.1016/j.conbuildmat.2016.04.114. [9] A. Calia, A.M. Mecchi, D. Colangiuli, L. Scudeler Baccelle, Conservation issues with calcarenites used as historical building materials in Syracuse (Southern Italy), QJEGH. 46 (2013) 485–492. https://doi.org/10.1144/qjegh2012-050. Chapter 3 52 [10] C. Jimenez-Lopez, F. Jroundi, C. Pascolini, C. Rodriguez-Navarro, G. Piñar-Larrubia, M. Rodriguez-Gallego, M.T. González-Muñoz, Consolidation of quarry calcarenite by calcium carbonate precipitation induced by bacteria activated among the microbiota inhabiting the stone, International Biodeterioration & Biodegradation. 62 (2008) 352–363. https://doi.org/10.1016/j.ibiod.2008.03.002. [11] M. Torabi-Kaveh, M. Moshrefyfar, S. Shirzaei, S.M.A. Moosavizadeh, B. Ménendez, S. Maleki, Application of resin-TiO2 nanoparticle hybrid coatings on travertine stones to investigate their durability under artificial aging tests, Construction and Building Materials. 322 (2022) 126511. https://doi.org/10.1016/j.conbuildmat.2022.126511. [12] R.J. Flatt, F. Caruso, A.M.A. Sanchez, G.W. Scherer, Chemo-mechanics of salt damage in stone, Nat Commun. 5 (2014) 4823. https://doi.org/10.1038/ncomms5823. [13] M. Drdácký, J. Lesák, S. Rescic, Z. Slížková, P. Tiano, J. Valach, Standardization of peeling tests for assessing the cohesion and consolidation characteristics of historic stone surfaces, Mater Struct. 45 (2012) 505–520. https://doi.org/10.1617/s11527011-9778-x. [14] L. Gianni, G. Gigante, M. Cavallini, A. Adriaens, Corrosion of Bronzes by Extended Wetting with Single versus Mixed Acidic Pollutants, Materials. 7 (2014) 3353–3370. https://doi.org/10.3390/ma7053353. [15] K. Bieske, C. Vandahl, A Study about Colour Difference Thresholds, in: Ilmenau, Deutschland, 2007. [16] M. Perez-Alonso, K. Castro, J. Madariaga, Vibrational Spectroscopic Techniques for the Analysis of Artefacts with Historical, Artistic and Archaeological Value, CAC. 2 (2006) 89–100. https://doi.org/10.2174/157341106775197385. [17] K. Castro, M. Pérez-Alonso, M.D. Rodríguez-Laso, L.A. Fernández, J.M. Madariaga, On-line FT-Raman and dispersive Raman spectra database of artists’ materials (eVISART database), Anal Bioanal Chem. 382 (2005) 248–258. https://doi.org/10.1007/s00216-005-3072-0. [18] D.R. T, The RRUFF project : an integrated study of the chemistry, crystallography, Raman and infrared spectroscopy of minerals, Program and Abstracts of the 19th General Meeting of the International Mineralogical Association in Kobe, Japan, 2006. (2006). https://cir.nii.ac.jp/crid/1571417124253896704 (accessed November 11, 2022). [19] H. Panda, Epoxy Resins Technology Handbook (Manufacturing Process, Synthesis, Epoxy Resin Adhesives and Epoxy Coatings), 2nd Revise, ASIA PACIFIC BUSINESS PRESS, 2019. [20] S. Gates-Rector, T. Blanton, The Powder Diffraction File: a quality materials characterization database, Powder Diffr. 34 (2019) 352–360. https://doi.org/10.1017/S0885715619000812. [21] L. de Oliveira, K. Bouchmella, A. Picco, L. Capeletti, K. Gonçalves, J.H. dos Santos, J. Kobarg, M. Cardoso, Tailored Silica Nanoparticles Surface to Increase Drug Load and Materials and methods 53 Enhance Bactericidal Response, J. Braz. Chem. Soc. (2017). https://doi.org/10.21577/0103-5053.20170017. [22] A.K. Kota, G. Kwon, A. Tuteja, The design and applications of superomniphobic surfaces, NPG Asia Mater. 6 (2014) e109–e109. https://doi.org/10.1038/am.2014.34. [23] NORMAL Protocol 43/93 (NORMAL 1993), (n.d.). 54 Bio-based epoxy thermosetting polymers 55 CHAPTER 4: Potential of bio-based epoxy thermosetting polymers for the advanced design of stone conservation materials This chapter was focused on a comprehensive research review on epoxy thermosetting polymers of natural origin to guide Step and Step of the hybrid material design. Thus, bio-based epoxy resins potentially suitable for stone conservation and the influence of curing agents on the materials properties are collected and discussed here. In addition, aspects such as resources, synthetic routes, and thermo-mechanical behavior were also addressed to gain valuable information and determine the future modulation possibilities to be designed in the following chapters for the restoration of silica-based stones. Renewable resources of bio-based epoxy resins In recent years, several works have focused on synthesizing and modifying bio-based polymers to cover a wide range of industrial uses [1–6]. Among them, compounds such as cardanol, vanillin, soy oil, gallic or itaconic acids, and sugar derivatives, or even industrial and cooking wastes have recently gained attention (Figure 4.1). Their increasing importance as starting materials is linked to their availability, low price, and unique Chapter 4 56 reactive chemical structure, which can easily lead to epoxy resins with low toxicity and intrinsic biodegradability [7]. In addition to be employed as green alternatives to BPAbased epoxy resins, they could be also very attractive for the field of stone conservation if they are subjected to further modification processes. Figure 4.1. Renewable natural resources of BPA-free epoxy resins with potential application in the stone conservation field. In this sense, the selection of specific cross-linking agents could allow to obtain thermosetting polymers that generally fulfill certain well-established criteria to be directly tested as consolidating stone treatments, or to be the basis of material designs for its modulation with similar purposes. In fact, as was commented in the Introduction section, different synthetic approaches could achieve the adjustment of crucial features, including thermal-mechanical behavior, UV resistance, or even compatibility with the stone substrate, among others. In these customization processes, a part of their ability to restore the integrity of the lithic matrix, it is also preferable that they prevent water penetration, meanwhile maintaining the aesthetic of the stone surface unaltered [8–12]. Bio-based epoxy thermosetting polymers 57 Consequently, in the following sections the most promising resins are classified according to their origin and discussed paying special attention to the different properties observed with respect to their DGEBA (diglycidylether of bisphenol A) counterpart. In addition, all the molecular structures and material characterization data reported are collected as a summary table and figures to facilitate their consultation (see Appendix B). Cardanol Cardanol is an oil obtained from cashew nutshell liquid (CNSL), a non-edible agricultural byproduct extracted from the shell of the cashew nut from the Anacardium Occidentale tree. CNSL is a dark brown viscous liquid composed of a mixture of phenolic compounds, precisely, anacardic acid (74-77%), cardanol (1-9%), and cardol (15-20%). Given that, it has been recently highlighted as an appealing renewable alternative to petroleum-derived phenols [13–15]. Specifically, cardanol is a long aliphatic side chain with mono-, diand tri-unsaturated bonds, and although there are possible ways of extraction, a higher yield is obtained if the CNSL is directly heated and later subject to a distillation process. The obtained product reaches an industrial-grade purity of 90 %, with small quantities of cardol and methylcardol [13,16–18]. Given its nature and chemical structure, several studies have been conducted on cardanol epoxidation and hardening reactions to gain thermoset materials [19–21]. Rocha da Silva et al. [19] exploited a direct CNSL valorization to prepare anticorrosive coatings for steel pieces. The epoxidation of the double bonds of the cardanol and cardol aliphatic chains, in acidic solvent-free conditions, resulted in an oligomeric mixture obtained as a reddish-brown viscous liquid (e-CNSL) (yield of 90 % and epoxide equivalent weight, EEW, of 469 g·eq-1) (Figure 4.2). Chapter 4 64 Xu et al. [29] exploited the aldehyde group on the vanillin performing a Wolf-Kishner reaction with hydrazine to obtain the 4,4'-(-hydrazine 1,2diylidenebis(methanylylidene))bis(2-methoxyphenol) (HBP). Then, it was epoxidized via ECH, using tetrabutylammonium bromide (TBAB) as transfer catalyst, and the diepoxydihydrazone (HBE) was obtained as a white solid product with a yield of 84.8%. Then, the HBE was cross-linked using IPDA and poly(propylene glycol) bis(2-aminopropyl ether) (D400), and the results were compared with DGEBA (DER331) as reference. The HBE/IPDA and HBE/D400 mixtures showed Tg of 146 and 76 °C, respectively, which are 23 °C higher than the DGEBA-based thermoset. This fact suggests that the high cross-linking of HBEbased thermosets could be due to the rigidity of the hydrazone group conjugated structure. The same explanation could also justify the higher Young's modulus values of HBE/D400 and HBE/IPDA and tensile strength data with respect to the ones observed for the DER331/D400 thermoset. On this basis, the high potential of HBE to be employed as epoxy resin for the preparation of high-performance thermosets is highlighted. However, static contact angle measurements evidenced a slightly hydrophilic behavior that should be properly tuned to meet the first requirements requested for stone conservation. Besides, the HBE-based thermosets have an extra advantage over DGEBA-based materials since the study also revealed a bactericidal activity against E. coli, thanks to the hydrazone group, showing killing rate values of 96 %. Therefore, it could address multifunctional properties that are very compelling for the conservation of lithic substrates. Gallic acid Gallic (GA) acid is the common name for 3,4,5-trihydroxybenzoic acid, a naturally occurring antioxidant found in several plants, vegetables, red fruits, nuts, and coffee. It is known to exhibit significant biological activity, and in addition to various pharmacological uses, GA is widely employed in the food industry as a preservative and flavor additive [30– 34]. Besides, the presence of three phenolic -OH groups and the carboxylic moiety has led to several epoxidation attempts for different purposes. Tomita et al. [35] reported a patent, in which GA-based epoxy resins were obtained with various grades of functionalization, i.e., from mono to tetra epoxy derivatives. Subsequently, for thermosets development, Patil et al. [36] started from the preparation of tetradiglycidylether of gallic acid (TGEGA) synthetic route (Figure 4.8). Bio-based epoxy thermosetting polymers 65 Figure 4.8. Route for the synthesis of the tetradiglycidylether of gallic acid (TGEGA) via epichlorohydrin (ECH). The tetra epoxy compounds obtained were cured with different ratios of polyamide, and the resulting materials were compared to DGEBA. Then, DSC investigations provided Tg of 54, 56, and 63 °C for the TGEGA:Polyamide ratios of 1:8, 1:12, and 1:16, respectively. This fact evidenced a clear upward trend in the cross-linking density. In addition, these biobased materials displayed Tg between 23-42% higher than the one observed for the DGEBA material. TGA tests also revealed that all the examined thermosets had a similar thermal resistance to the DGEBA-based polymer. Therefore, data indicates that the biomaterials displayed, at least, thermal properties that could match the ones requested for an effective stone conservation treatment. So, further investigations are needed to assess whether these polymers could maintain their features, for instance, if they are prepared with green solvents, or to check their mechanical and hydrophobic behavior. Tarzia et al. [37] reported on the glycidylation of GA to gain triand tetra-glycidyl ethers of gallic acid (TGEGA) through a two-step synthesis. The first one involves the alkalineassisted allylation of hydroxylic groups (both phenolic and non phenolic), and then it was followed by the epoxidation of the resulting tetra-allylated GA (AGA) (Figure 4.9), obtained as a yellow oil mixture with a yield of 89%. Chapter 4 66 Figure 4.9. Route for the synthesis of the tetradiglycidylether of gallic acid (TGEGA) via allyl bromide using alkene oxidation. TGEGA curing was then carried out at room temperature using IPDA or Jeffamine D230 (DPG) in a stoichiometric ratio, whereas the tertiary amine N,N-dimethylbenzylamine (BDMA) was also used as an initiator. DSC scannings showed Tg of 158, 136, and 98 °C for TGEGA/IPDA, TGEGA/BDMA, and TGEGA/DPG formulations, respectively. These values indicate the formation of well-developed cross-linked networks, generally also associated with high stiffness and low flexibility, which are not suitable for materials to be used in stone conservation. Nevertheless, TGA investigations indicated excellent thermal properties, achieving the highest resistance upon heating for TGEGA/BDMA homopolymerized thermoset (mass residue 29%). As expected, mechanical analyses show that the thermoset based on the more flexible amine, DPG, provided the highest elongation at break and the greatest strength. In addition, the elastic moduli were relatively high and similar for all the investigated systems, showing brittle fracture. Itaconic acid Itaconic acid is a di-carbonic unsaturated weak acid with antimicrobial properties obtained from the biological activity of several fungi, mainly Aspergillus terreus, that is already used as a bio-based substitute for acrylic acid [38–40]. Bio-based epoxy thermosetting polymers 67 Kumar et al. [41] carried out the allylation of itaconic acid, followed by oxidation with mchloroperoxybenzoic acid (mCPBA) to form the tri epoxy itaconic acid (TEIA) (yield 75%, EEW 1.02 mol/100 g) (Figure 4.10). Figure 4.10. Synthetic procedure to obtain the tri epoxy itaconic acid (TEIA), via allyl bromide route, by double bond oxidation. The TEIA cured with MHHPA showed a Tg of 133 °C, only 11 °C lower than the one displayed by DGEBA/MHHPA used as reference. In addition, the TEIA thermoset featured high thermal stability, quite similar to that of DGEBA/MHHPA. Both thermosets showed similar tensile strength, although TEIA/MHHPA exhibited higher flexibility in elongation tests. These properties indicate that TEIA/MHHPA has great potential for replacing petroleum-based epoxy thermosets in a wide range of high-performance applications. Unfortunately, its dark yellow color represents a limit for its potential use in conserving lithic materials. Ma et al. [42] followed the same route shown in Figure 4.10, and obtained a TEIA monomer with a yield of 60 % and an EEW of 1.16 mol/100 g. In order to compare the properties exhibited by the bio-based thermosets with the ones shown by the conventional ones, both TEIA and DGEBA were cured using MHHPA and DPG. The TEIA/MHPPA thermoset displayed thermal and mechanical properties comparable to those described by Kumar et al. [41] for an analogous material. Furthermore, TEIA/D230 was featured by a Tg of 61 °C, significantly lower than the other materials investigated, both bioand petroleum-based. This fact can be explained in terms of a higher mobility of the chain segments in TEIA/D230, whereas the rigid MHHPA hindered the movement of the chain segments of the epoxy network, resulting in higher Tg values. On the other hand, Chapter 4 68 DGEBA-based thermosets featured higher thermal stability than bio-based materials. The mechanical investigations evidenced that the flexural strength of TEIA/D230 and DGEBA/D230 was similar, whereas TEIA/D230 flexural modulus and strain at break were much higher than the ones exhibited by DGEBA/D230. Moreover, the flexural strength, flexural modulus and strain at break were all higher for TEIA/MHHPA mixtures than for the DGEBA/MHHPA one. From all the above, it can be affirmed that TEIA systems had better processability than the DGEBA ones, and that, it could be considered as a viable alternative for high performance materials synthesis, evidencing once again the high performances that can be obtained from the bio-based materials. Isobutyric acid Isobutyric acid, also known as 2-methylpropanoic acid, is a short-chain saturated carboxylic acid found in Coffea Arabica [43], strawberries [44], and in the root of Arnica montana [45], and even as a synthetic metabolic pathway from E. coli that enables the biosynthesis of this compound from glucose [46]. This colorless liquid is highly soluble in water and organic solvents and easily reacts to form amide, ester, anhydride, and chloride derivatives [47]. For this reason, it is commonly used as an intermediate in the cosmetic industry and also can be found in a wide variety of foods to impart different flavors. Another interesting point of this natural compound is that its pyrolysis can be used to gain a dimethylketene, which spontaneously dimerizes to a cyclic diketone. Then, it can be easily hydrogenated to obtain a natural cyclodiol, 2,2,4,4-tetramethyl-1,3cyclobutanediol (CBDO) [48]. This cyclical structure and the arrangement of both OH groups make this unexplored compound a very attractive building block for a wide variety of applications. A simple synthesis route was patented by Daniel Schmidt [48], who exploited the traditional epoxidation approach with ECH, in an alkaline medium, to obtain a pure cis/trans mixture of 2,2,4,4-tetramethyl-1,3-cyclobutane diglycidylether (CBDO-DGE) with a yield of 74% and an EEW of 128 g·eq-1. The study obtained thermoset materials employing TETA amine and different curing temperatures, comparing their thermal properties to those displayed by DGEBA/TETA. Depending on the thermal curing process, CBDO-DGE/TETA showed Tg occurring between 68 and 82 ᵒC versus values of 100 and 120 ᵒC for DGEBA/TETA. TGA studies revealed higher thermal stability of DGEBA/TETA, with respect to CBDO-DGE/TETA (Tonset of 326 °C), whereas Tmax reached 381 ᵒC in the case of the bio-based thermoset and 364 ᵒC for DGEBA/TETA. The reported data suggests that Bio-based epoxy thermosetting polymers 69 cross-linking and stiffness of CBDO-DGE/TETA were milder than those gained for DGEBA/TETA. Although there was no more data available, the results obtained with this aliphatic amine, and the epoxy solubility in sustainable solvents, together with the low temperature used to obtain a well cross-linked thermosetting material, pointed to the CBDO-DGE as a promising candidate for being further studied. In fact, the structural resemblance between CBDO-DGE and petroleum-derived epoxy precursors, such as 1,4cyclohexanedimethanol diglycidyl ether (CHDM-DGE), which have already been previously investigated by our research group [49] as a promising building block for the well-controlled development of consolidating products. In this work, a thermoset material derived from CHDM-DGE and diaminooctane (DAO) was combined with 3glycidoxypropylmethyldiethoxysilane (GPTMS) as a coupling agent and silica forming additive to gain epoxy-silica hybrid material with excellent thermal and hydrophobic responses (Tg of 53°C, Tonset of 375.3 ᵒC, and CA of 105ᵒ). Soybean oil Soybean oil is one of the cheapest renewable sources available and widely used in fabricating several bio-based products [50,51] as well as in the cooking, animal feed, biodiesel production, and cosmetics industries. This versatility is due to the lipidic fraction presented, mainly composed of triglycerides with primarily unsaturated fatty acids that could be easily epoxidized by an oxidation reaction [51–53]. Given that, epoxidized soybean oil (ESO) also has the advantage of being commercially available. Kumar et al. [41] reported the comparison between commercial ESO (EEW 222-230 g mol1) and DGEBA cured with MHHPA. The bio-based thermoset showed Tg of 64 °C, which corresponds to a compatible range for designing a coating for stone conservation purposes. In addition, ESO/MHHPA materials evidenced quite good thermal stability with T5% and T50% of 237 and 359 °C, respectively. Nevertheless, the DGEBA-based thermoset displayed higher thermal resistance, 100 °C more for T5% and 45 °C more for T50%. Another significant finding to determine the ESO suitability for stone applications is that rheological investigations highlighted a lower viscosity of ESO with respect to DGEBA one, which could facilitate its penetration in the lithic substrate, minimizing a common drawback associated with conventional epoxy-based treatments. Moreover, ESO/MHHPA mechanical properties were featured by high elongation and impact strength, with significant flexibility attributed to the long aliphatic chains into the network, which may Chapter 4 70 be of great interest for the design of advanced materials for stone conservation. However, the curing temperatures needed to obtain such successful results were impossible to apply in outdoor in situ treatment. Zhao et al. [54] cured a commercial ESO resin with dihydroxydiphenylmethane (DHM) and a vanillin-derived Schiff base (VSB), a hardener obtained from the reaction of the vanillin with 4,4-diaminodiphenyl methane (DAM). The curing reactions were performed employing different ESO/VSB ratios and a fixed ESO/DHM ratio of 0.7 for comparison purposes, using 1,2-dimethylimidazole (DMI) as a catalyst. The ESO/VSB Tg values were gradually increased, ranging from 30 to 66 °C for 0.5 to 1 ratios. Conversely, the crosslinking density increased up to 0.7 ratio, and then it started to drop. Besides, the maximum storage modulus was found below the Tg for 0.9 ratio and above the Tg for 0.7 ratio. In addition, ESO/DHM thermoset showed Tg of 19 °C and lower E’ values with respect to the other investigated samples, due to a lower network stiffness imparted by the lower content of aromatic rings, which is one-half of the VSB one. Besides, all the thermosets displayed excellent thermal stability with T5% at ca. 400 °C, and much higher char residues at 700° than the ESO/DHM ones. These results are attributed to the conjugated aromatic rings and the cross-linking of the imine at high temperatures. The ESO/DHM thermoset displayed poor mechanical properties with respect to the ESO/VSB ones, which showed elongation break decrease and tensile strength, and Young’s modulus increased with the ratio increasing. From all the collected data, it appeared that the studied thermosets displayed a variety of tuneable characteristics, being the ESO/VSB materials soft and with no yielding stress curves at 0.5–0.6 ratios, whereas ratios between 0.7-0.8 produce the ductile-brittle transition and for ratios higher than 0.8 a brittle fracture occurred. Thus, ESO/VSB thermosets could present thermo-mechanical properties very appealing and highly modulable to develop a range of versatile materials by simply tuning the ratio reactions, and therefore great attention should be paid to their potential applications. However, their dark yellow-orange color is complex to solve for the field of stone conservation. Eugenol 4-Allyl-2-methoxyphenol, also known as eugenol (Eu), is a natural phenolic compound that can be found in essential oils of clove, basil, and pepper, among others. Due to its inherent biocidal activity, Eu has already been investigated to assess its suitability against cultural heritage biocolonization [55–58]. Bio-based epoxy thermosetting polymers 71 Respect its possibilities as an epoxy product, Wan et al. [59] developed a diepoxy resin by a Williamson etherification reaction between α,α’-dichloro-p-xylene, and Eu, to obtain a product bearing two allyl groups, which was then epoxidized with mCPBA to gain a dieugenol-based diepoxide (DEu-EP) (Figure 4.11). Figure 4.11. Synthetic procedure to obtain the dieugenol epoxy (DEU-EP) using α,α’-dichloro-pxylene as a binder and alkene oxidation. DEu-EP (yield 43%, EWW 0.380 mol/100 g,) obtained as yellowish needle crystals, was cured with 4,4´-diaminodiphenyl methane (DDM) and compared with DGEBA. DMA studies indicated that DGEBA/DDM material was featured by a higher Tg than DEu-EP, 153.6 and 114.4 ᵒC, respectively, probably given the mobility imparted by the xylene fragment to the network. This effect was also confirmed by the E’ value, which was 17% higher than the one detected for the DGEBA-based thermoset. Thermogravimetric scannings evidenced similar thermal stability up to 270 ᵒC, although the bio-based material exhibited both lower T5% and Tmax than DGEBA/DDM (i.e., 341 and 379 °C; 377 and 393 ᵒC, respectively), being the latter also featured by a faster decomposition rate. Furthermore, the char yield at 700 °C of DEu-EP/DDM was 38%, more than double that found for the petroleum-based thermoset, demonstrating the capacity presented by the bio-based epoxy to overpass the petroleum based thermoset. However, they characteristic made it an interesting candidate for other kinds of applications, for example, materials for flame retardant use. Chapter 4 72 In a work by Qin et al. [60], a Eu derived diepoxy resin (Eu-EP) was obtained by exploiting a synthetic strategy involving mCPBA and ECH as epoxidation reactants (Figure 4.12). First, the phenolic group was protected via acetylation to safely oxidize the double bond with mCPBA. Then, deacetylation and glycidylation was performed in a single synthetic step using ECH in alkaline conditions to obtain Eu-EP as a white powder (yield 51.62%, EEW 129 g mol-1). Figure 4.12. Synthetic procedure to obtain the eugenol epoxy (Eu-EP) via alkene oxidation with hydroxyl group protection and via epichlorohydrin (ECH). Then, Eu-EP was cured using hexahydrophthalic anhydride (HHPA) and abietic acid (MPA) in the presence of 1-ethyl-4-methylimidazole (EMID) as a mandatory catalyst to ensure the complete curing kinetics. The data showed excellent thermo-oxidative stability for both thermosets, with a T5% value above 300 °C. In addition, DSC scannings evidenced Tg of 155.3 °C and 114.2 °C, whereas DMA provided very similar storage modulus (i.e., 2.9 and 2.8 GPa) for Eu-EP/MPA and Eu-EP/HHPA, respectively. These data demonstrated once again the possibilities of Eu-derived materials in other fields. Furans 5-hydroxymethylfurfural (HMF) can be obtained from different saccharides, such as glucose and fructose, and biomass [61,62]. HMF is a highly versatile precursor due to its functional groups, an aldehyde, and alcohol, as it can be converted into various added value products [63]. HMF hydrogenation easily leads to 2,5-bis(hydroxymethyl)furan (BHMF), a highly versatile building blocks to gain a variety of functional materials [64]. Hu et al. [65] obtained the monomeric 2,5-bis[(2-oxiranylmethoxy)methyl]-furan (BOF) by the epoxidation of BHMF, with the ECH route, as a yellow liquid with a 99% of purity and a yield of 60% (Figure 4.13a). Bio-based epoxy thermosetting polymers 73 Figure 4.13. The synthetic procedure of the a) Bis[(2-oxiranylmethoxy)methyl]-furan (BOF) via epichlorohydrin (ECH), and b) the Diels-Alder reaction with N-Hexyl Maleimide (HMI) to form the Bis[(2-oxiranylmethoxy)methyl]-furan (DA-BOF). BOF was then cured with 4,4'-Diaminodicyclohexylmethane (PACM) and diethyl toluene diamine (Epikure W), and the results obtained were compared to DGEBA ones. The Tg values reached 80 and 94 °C for BOF/PACM and BOF/EpikureW mixtures, respectively, significantly lower than the ones gained for the DGEBA analogous thermosets. These data, together with the high storage modulus of BOF-based thermosets make it an interesting candidate to be further studied for its potential exploitation in the field of conservation. Meng et al. [66] worked with the aforementioned bio-epoxy resin for the development of flame retardant thermosets, using 3,3′-diamino diphenyl-sulfone (33DDS) and 4,4′- diamino diphenyl-sulfone (44DDS) as hardeners. 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In parallel to this work, innovative analytical methodologies were also developed to define the advantages addressed by the implementation of spectroscopic techniques, together with the classical ones used in the field, to assess the progress of the developing processes. To achieve this, the sequential material development previously indicated was here specifically followed until Step- Epoxy-Silica hybridation, and the corresponding enrichment of the product obtained in this chapter was approached in Chapter 6 to achieve the final multifunctional epoxy-silica hybrid target material. Concretely, Step- Epoxy precursor was faced according to the review research carried out. Thus, the cycloaliphatic diol 2,2,4,4-tetramethyl-1,3-cyclobutanediol (CBDO) was fixed as starting point to synthesize the bio-based epoxy precursor 2,2,4,4-tetramethyl- Chapter 5 88 1,3-cyclobutanediol diglycidylether (CBDO-DGE). During the course of its synthesis and clean-up process, a method based on a combination of FT-IR and Raman analysis was applied to systematically monitor the structure and purity of the product obtained during the different phases. In this manner, the advantages of exploiting spectroscopic analytical strategies, over the conventional material characterization techniques such as in this case 1H-NMR and 13C-NMR, were here studied. Afterwards, Step- Base thermoset was undertaken to obtain an adequate material foundation that make easier the implementation of the thermal-mechanical and hydrophobic properties required by adding of silica-forming additives. Thus, knowing that the selection of the curing agent plays a key role to set up the performances of the epoxy thermoset that will act as base layer for the hybrid material development, according to the review, the amines were chosen as hardener agents. Bearing in mind the data compiled and, with the intent to avoid any cracking phenomena upon cross-linking or chromatic alterations of the epoxy resins, as well as, to enhance hydrophobic and ductility thermoset properties, two kind of amines (linear and cycloaliphatic), namely 1,8diaminooctane (DAO) [1,2], triethylenetetramine (TETA) [3,4], and 5-amino-1,3,3trimethylcyclohexanemethylamine (IPDA) [5,6] were tested to evaluate the effect imparted in the organic network. Furthermore, as the hardener ratio needed to open the epoxy rings does not correspond to an exact stoichiometric value, and its excess or defect has a strong influence in the final material performance, the addition process of the amine was first followed, and later delimited, by a method based on ATRFT-IR and Raman analysis, whereas their suitability as organic counterpart of the hybrids was finally established by their thermal characterization data. Lastly, Step- Epoxy-Silica hybridation was performed to develop the thermoplastic materials according to the CBDO-DGE thermoset polymer characterization study. Then, multiple epoxy-silica hybrids were obtained exploiting the sol-gel technology through the CBDO-DGE epoxy hardening reactions in presence of different silica-forming precursors. Specifically, (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and octyltriethoxysilane (OcTES) were selected as not hazardous inorganic additives to finely tune both the flexibility and hydrophobic properties of the resulting thermoplastic materials and, also to increase the product compatibility with the lithic substrate [1,7–9]. In parallel to this, a combination of SEM-EDS, ATR FT-IR and Raman analysis was employed to establish a systematic method to evaluate the distribution and interpenetration of silica domains into the organic matrix formed with each formulation. Lastly, the material properties imparted Design of bifunctional epoxy-silica hybrids derived from arnica root 89 by the different mixtures were investigated by thermo-mechanical analysis (DSC, TGA and DMA) and contact angle measurements. In this way, the suitability of each blend to be fixed as base formulation to fulfil the main requirements for an advance stone conservation material was determined. In this manner, the work carried out and the main results obtained, together with their corresponding discussion, are detailed along the following sections according to the three steps of development described. 5.1. STEP: Epoxy Precursor Synthesis of CBDO-DGE CBDO-DGE was synthesized according to the patent (Figure 5.1) [10]. Briefly, in a two-neck round bottom flask, CBDO (2.8 g, 0.019 mol) and epichlorohydrin (ECH) (15.5 mL, 0.19 mol) were added and vigorously stirred. Then, the phase transfer catalyst tetra-nbutylammonium bisulfate (TBAB) (0.65 g, 0.0019 mol), sodium hydroxide (NaOH) (7.5 g, 0.19 mol) and few drops of Milli-Q water were added. Figure 5.1. Synthesis reaction of the epoxy precursor CBDO-DGE, via epichlorohydrin (ECH) in basic medium, from the natural diol CBDO. The yellow reaction mixture, left under stirring at 40°C for 2 hours, turned to reddishorange. Once it reached room temperature, dichloromethane was added and the formed suspension was filtered (Ø 45 µm). The organic phase was washed with Mili-Q water until the aqueous phase obtained was clear, adding a final wash with an acetic acid solution (pH ≈ 4.5). Then, the organic phase was recovered and treated with anhydrous sodium Chapter 5 96 although, CT was featured by higher elasticity and tenacity than CI, which was instead quite fragile. Conversely, CD sample was flexible but slightly sticky, featured by visible surface roughness, with small yellowish spots. The FT-IR spectra taken to verify the extent of cross-linking [13] showed for all the samples the increase of the band at 3600-3200 cm-1 (due to the –OH groups forming as result of the epoxy rings cleavage) together with the disappearing of the oxirane signal at 849 cm1. At the same time, new peaks corresponding to primary amines appeared at 1640 and 1650 cm-1, mainly, in samples CD and CI, suggesting the presence of significant unreacted amounts of DAO and IPDA, respectively. Raman analysis showed as a common feature that the secondary oxirane signals at 781 cm-1 completely disappeared, whereas a weak intensity for the main peak at 1260 cm-1 was observed as result of the curing reaction progress. Hence, the opening of the epoxy groups was double spectroscopically confirmed. Moreover, unreacted primary amines were again detected by the bands at around 1447 cm-1, both in CD and CI spectra [1,14]. Since often the amount of the amine does not correspond to the required theoretical stoichiometric ratio, and keeping in mind that an excess of the amine in the bulk of the resin may cause unwanted darkening due to its easy oxidation, the proper amine amount for both DAO and IPDA hardening agents were followed studied. Thermoset material: Optimization by spectroscopic techniques Additional synthetic trials were carried out using epoxy/amine ratios of 2:0.75 and 2:0.5 for both amines. Herein, CBDO-DGE/DAO (CD-1 and CD-2) and CBDO-DGE/IPDA (CI-1 and CI-2) samples were prepared (Table 5.2) following the previous synthetic process. Unfortunately, samples CD-2 and CI-2 were discarded of the next studies after the curing process due to their semi-solid and sticky nature which is unappropriated for the defined material purpose. Design of bifunctional epoxy-silica hybrids derived from arnica root 97 Table 5.2. Relative amounts of the selected hardeners employed at different ratios for the syntheses of the CBDO-DGE thermoset. Sample CBDO-DGE DAO IPDA CD-1 0.30 g 1.17 mmol 0.06 g 0.43 mmol CD-2 0.30 g 1.17 mmol 0.04 g 0.30 mmol CI-1 0.30 g 1.17 mmol 0.07 g 0.44 mmol CI-2 0.30 g 1.17 mmol 0.05 g 0.29 mmol Then, the valid samples were subjected to the spectroscopic study to determinate the effect in the cross-linking exerted by the modification of the epoxy/amine ratio used. The normalized absorbance intensity data of the ATR-FTIR signals due to –OH and to epoxy rings, with respect to the ether group at 1098 cm-1, is reported in Figure 5.6 for their comparison. Figure 5.6. Comparison between the ATR FT-IR absorbances at 3500 (-OH), 1650 and 1640 (N-H) cm-1 bands of CBDO-DGE cured samples, using 1,8-diaminooctane (DAO) and 5-amino-1,3,3trimethylcyclohexanemethylamine (IPDA) at different epoxy:amine ratios, 2:1 for CD and CI, and 2:0.75 for CD-1 and CI-1. The results obtained clearly indicate that the maximum extent of the ring opening together with lower amount of primary amine was accomplished for samples CD-1 and CI, with an epoxy/amine ratio of 2:0.75 and 2:1, respectively. Moreover, given the Chapter 5 98 importance of the thermal transitions (in terms of Tg) in the final purpose of these materials, the characterization of the thermal behavior was also determined to finally verify if they present a proper suitability. Thermoset material: Thermal study DSC investigations evidenced that all the CBDO-DGE/DAO samples display a small exothermic peak at approximately 175 °C during the first scanning and, in addition, the Tg detected during the second scan was higher, indicating a post-curing effect due to the first heating and/or suggesting that the crosslinking was not total. More in detail, Tg of 25.9 and 16.9 °C were found for CD-1 and CD, respectively, suggesting that CD-1 matrix is more cross-linked (i.e. epoxy/amine ratio of 2:0.75), in agreement with the findings coming from FT-IR study. Similarly, as the bibliographic data indicated, the CT reached 26.8 °C [10]. Yet, samples CI and CI-1 showed Tg values of 52.0 and 49.4 °C, respectively, indicating that, the decrease of epoxy/amine ratio did not exert any significant variation on the IPDA thermoset networks. Moreover, the thermal stability of the most promising thermoset (i.e. CT, CD-1, and CI) was investigated by TG-DTA. The main degradation temperatures and the obtained % of mass residues from the thermograms curves (Figure 5.7) are summarized in Table 5.3. Figure 5.7. Thermogravimetric traces of cross-linked networks based on CBDO-DGE and DAO, TETA IPDA amines. Design of bifunctional epoxy-silica hybrids derived from arnica root 99 Table 5.3. Thermal properties of CBDO-DGE cross-linked with DAO, TETA and IPDA: CD-1, CT and CI samples, respectively. Samples CD-1 CT CI First event T onset [◦C] 158 154 128 T max [◦C] 321 356 335 % mass loss 59.9 95.9 93.9 Second event T onset [◦C] 428 T max [◦C] 451 % mass loss 33.3 Residual mass % * 3.3 3.9 3.4 T g [◦C] 25.9 26.8 50.0 *at 800 ◦C The results indicated that the degradation occurs in two steps for CD-1 and showed Tonset values of 158 and 428°C, with Tmax at 321 and 451 °C and a % mass loss of 60 and 33%, respectively, and displaying a residual mass of 3.3%. However, samples CT and CI displayed a single peak featured by Tonset values of 154 and 128 °C, with maximum degradation rates at 356 and 335 °C, detecting carbonaceous residues at 800 °C of about 4%. Therefore, on the basis on the results reported, in terms of macroscopic texture and thermal features, CI and CT samples were the most suitable and easily tunable for conservation purposes, so that further investigations were carried out to check which silica-forming additives could generate a hybrid network with the specific properties required. 5.3. STEP: Epoxy-Silica hybridation Synthesis of CBDO-DGE epoxy-silica hybrids In order to test the capability to generate proper organic-inorganic networks of tailored properties, 10 different hybrid formulations were prepared using sol-gel technology. For this, methanol was selected as solvent of slow down to promote the formation of more Chapter 5 100 homogeneous materials, taking the advantage of its capability to provide low reactivity to alkylalkoxysilanes, ensuring a controlled sol-gel reactions [15–17]. Then, each formulation started with a 5% w/w epoxy-containing methanol solution and included one or two epoxy compounds. Figure 5.8 and Table 5.4 shows the structures and specific amounts of reactants and additives selected for each sample. Figure 5.8. Chemical structures of the alkylalkoxysilanes studied as coupling agents and silica forming additives, (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and octyltriethoxysilane (OcTES), respectively. In detail, samples 1T and 1IP were obtained by dissolving 0.5 g of CBDO-DGE in 7 mL of methanol and adding the hardener (TETA or IPDA, respectively), previously dissolved in 3 mL of MeOH. The solutions were stirred for 60 min, and OcTES was added at 10% w/w with respect to CBDO-DGE, then left to stir, for 90 min. To calculate the stoichiometric amine required, GPTMS and OcTES were prepared as well. Samples 2T and 4T were prepared using 0.3 g and 0.25 g of CDBO-DGE, respectively, with two different amounts of GPTMS (0.2 g and 0.25 g) dissolved in 2 mL of MeOH. After stirring for 60 min, TETA amine dissolved in 2 mL of MeOH was added and left under stirring for 90 min. Samples 3T and 5T were prepared similarly, but with the addition of 0.025 g of prehydrolyzed OcTES. Samples 2IP and 4IP were prepared using IPDA as the hardener for the two different epoxy mixtures, and samples 3IP and 5IP were obtained by adding 0.025 g of OcTES. All the samples were left in teflon Petri dishes capped with parafilm for 4 days, dried at room temperature, and thermally cured at 60 °C for 24 hours, reached at a heating rate of 5 °C min-1. Design of bifunctional epoxy-silica hybrids derived from arnica root 101 Table 5.4. Relative amounts of the CBDO-DGE, hardeners and silica precursors employed for the syntheses of epoxy-silica resins. Sample CBDO-DGE GPTMS OcTES TETA IPDA 1T 0.50 g 0.05 g 0.095 g 1.95 mmol 0.18 mmol 0.65 mmol 2T 0.30 g 0.20 g 0.071g 1.17 mmol 0.85 mmol 0.49 mmol 3T 0.30 g 0.20 g 0.025 g 0.071g 1.17 mmol 0.85 mmol 0.09 mmol 0.49 mmol 4T 0.25 g 0.25 g 0.067 g 0.98 mmol 1.06 mmol 0.46 mmol 5T 0.25 g 0.25 g 0.025 g 0.067 g 0.98 mmol 1.06 mmol 0.09 mmol 0.46 mmol 1IP 0.50 g 0.05 g 0.17 g 1.95 mmol 0.18 mmol 1.00 mmol 2IP 0.35 g 0.15 g 0.13 g 1.37 mmol 0.63 mmol 0.76 mmol 3IP 0.35 g 0.15 g 0.025 g 0.13 g 1.37 mmol 0.63 mmol 0.09 mmol 0.76 mmol 4IP 0.30 g 0.20 g 0.12 g 1.17 mmol 0.85 mmol 0.70 mmol 5IP 0.30 g 0.20 g 0.025 g 0.12 g 1.17 mmol 0.85 mmol 0.09 mmol 0.70 mmol The, transparent pale yellow solid films were obtained as macroscopically homogenous, flat and crack-free samples; roughly, their elasticity and tenacity seemed to increase with the GPTMS content, being their hardness maintained. Formulations based on OcTES as the only silica additive, lead to films featuring higher coloration (which turned to brown over time), opacity and flexibility, but slightly sticky textures. On the contrary, samples based on the combination of both alkylalkoxysilanes seemed to display a good balance for stone conservation purposes. Chapter 5 102 Characterization of CBDO-DGE epoxy-silica hybrids 5.3.2.1. Spectroscopic assessment To investigate the organic and inorganic domains distributions, secondary electron images (SE) and elemental mappings were studied (Figure 5.9). Figure 5.9. SEM-EDS images of 1T-5T and 1IP-5IP hybrid samples, where silicon distribution mappings are overlayed in red color on the SE images acquired at a magnification of 1000X. Design of bifunctional epoxy-silica hybrids derived from arnica root 103 The results showed a homogeneous morphology for all samples, except 2IP, 4IP and 5IP, where surface empty areas suggested slight phase separation phenomena. Their silicon mappings confirmed a significant local non-uniformity, whereas the homologous samples synthesized with TETA (2T and 4T) suggested that GPTMS was able to promote the formation of a hybrid matrix, featured by different domains intimately jointed to provide a homogenous network [17,18]. However, according to the GPMTS ratio employed, networks of different micro-morphologies were observed. On the contrary, epoxy-silica formulations with OcTES as the only silica forming additive, 1T and 1IP, showed a slightly heterogeneous surface morphology with detectable aggregates no bigger than 15 and 65 µm, respectively. This fact suggests that OcTES may be prone to self-condensation thus forming small hybrid clusters; that is, acts as a filler rather than introducing joint points between organic and inorganic domains [19,20]. Both samples cured with TETA or IPDA amines showed comparable results. However, this kind of behavior in the curing with diamines was not surprising since the implementation of prefunctionalized polysiloxanes bearing epoxy groups commonly produces the formation of single-phase systems featured by a segregation of siloxane domains in the epoxy matrix [21,22]. The results obtained for samples 3T and 3IP indicated that the combination of both alkylalkoxysilanes, regardless of the amine used, facilitated the interpenetration between domains, and thus, a lower tendency towards the micro-phase separation. However, by increasing GPTMS ratio, significant morphologic and distribution changes were observed. In detail, 5T and 3T hybrids, which only differed in the relative amount of the two different epoxy precursors, i.e. CBDO-DGE and GPTMS, were both featured by a homogeneous surface, yet displaying different morphologies (Figure 5.9). In addition, 5T and 2T surface distributions were comparable, as well as 5IP and 4IP even though 2T and 4IP were only based on GPTMS as silica forming additive. In addition, higher GPTMS ratios in the alkylalkoxysilanes mixtures seem to hinder the interaction with OcTES, suggesting that the two silica forming additives give rise to distinct concurring reactions in competition. Furthermore, to gain a deeper insight into the organic-inorganic cross-linking extent, ATR FT-IR and Raman spectra were studied. Infrared spectra of samples containing GPTMS (all except for 1T and 1IP) showed the disappearing of the epoxy peaks at 849 cm-1, as a result of the opening by reactive sites of amines, although weak signals at 1650 cm-1 evidenced the presence of free primary amines in the case of IPDA containing samples. Chapter 5 104 Consequently, a further amine amount adjustment for the synthesis is not required; this evidence also suggested that the Si-OH formed during the sol-gel reactions is not significantly involved in the epoxy ring opening. On the contrary, hybrid samples based on OcTES only showed the presence of an excess of the curing agent that can, very likely, explain the film darkening observed. It should be highlighted that the detection of partially reacted and/or unreacted amine in the final hybrid composition is crucial for the potential feasibility of the products on historical lithic substrates [23]. As far as the degree of cross-linking is concerned, only TETA hybrids showed significant signals at 1038 and 947 cm-1 due to the Si-O-Si and Si-OH bonds respectively, formed during the hydrolysis and condensation steps. In addition, the cited signals were not clearly observed for IPDA hybrids since their weak intensity did not allow a straightforward distinction with the CBDO-DGE ones, i.e. bands at 1098 and 930 cm-1. Only for 1IP and 5IP samples a little shoulder attributed to Si-OH signal was identified. Consequently, in order to assess which formulations has provided the most developed silica network, the hydrolysis of alkoxysilane moieties in TETA cured samples was studied by means of polycondensation ratios (PR) (see Figure 5.10). Figure 5.10. ATR-FTIR spectra of the samples 3T and 4T comparing their polycondensation ratio and showcasing the polycondensation ratio equation. Design of bifunctional epoxy-silica hybrids derived from arnica root 105 According to the average intensity ratio between Si-O-Si and Si-OH bonds [24], 4T and 2T samples showed the highest values, 2.6 and 2.1 respectively, explaining the difference observed by SEM-EDS and highlighting the positive effect of GPTMS as coupling agent. PR values of around 1.8 were obtained for 3T and 5T hybrids, indicating that the combination of both silica additives promoted the presence of partially hydrolyzed -Si(OR)3 groups or uncondensed silanoles. As expected, due to the presence of the OcTES ethoxy groups, sample 1T showed the lowest PR ratio. Raman spectroscopy confirmed the previous results and, although weak signals of epoxy rings were identified at 1260 cm-1 for all the samples, the secondary characteristic peak at 781 cm-1 was not detected in any case. In addition, although Raman (parameters: 785 nm laser, 180s laser bleaching, power of 10 %, exposure time of 10 s and 20 accumulations) signals attributed to partially hydrolyzed -Si(OR)3 groups at 666 cm-1 were overlapped, the development of inorganic network was observed only for TETA hybrid samples by the identification of weak Si-O-Si bond signals at 466 cm-1 as shown in Figure 5.11. Figure 5.11. Raman spectrum of the sample 4T indicating the developed Si-O-Si bond. Chapter 5 112 amount of GTPMS as the unique compatibilizer between organic and inorganic counterparts (2IP and 4IP hybrids) did not seem to favor hydrophobicity. 5.4. General remarks Along this chapter, the successful synthesis reaction and purification of the CBDO-DGE epoxy resin derived from the arnica root was successfully achieved. During this process, a multianalytical approach that implements a variety of spectroscopic analysis for the systematic monitoring of these different phases was also here developed and successfully applied to control the quality of the bio-epoxy production process. In this manner, the advantages addressed by exploiting the spectroscopic techniques in the material field evidenced a reduction of the resources and time consumed for the process of quality assessment, in addition to the minimization of using potentially harmful solvents. The potentially of these techniques in material design processes were further demonstrated in relation to the selection of the curing agent, and addition ratio, for the optimization of the thermosetting material performance, since it has the role of building up the foundation of the hybrid formulation and making easier the design based on the building blocks system. In pursue of developing epoxy-silica hybrids materials, specifically designed for being applied on the field of stone conservation, this work demonstrated the suitability of TETA as crosslinking agent for the organic-inorganic network formation. Moreover, the combination of the organic and inorganic domains employing GPTMS as a coupling agent led to different cross-linked systems that allow the homogenous dispersion of OcTES, resulting in a properties compromise between rigidity and elasticity, while providing the highest hydrophobic properties, thus, ensuring a balance for the bifunctional requirements of an advanced stone conservation material. For all the mentioned, two mixtures, namely 3T (CBDO-DGE/TETA/GPTMS) and 4T (CBDODGE/TETA/GPTMS/OcTES), were established as base formulations that meeting the recommended consolidating and hydrophobic capabilities. 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Luz, Use of DMA-material pocket to determine the glass transition temperature of nitrocellulose blends in film form, Carbohydrate Polymers. 226 (2019) 115288. https://doi.org/10.1016/j.carbpol.2019.115288. [29] C.A. Gracia-Fernández, S. Gómez-Barreiro, J. López-Beceiro, J. Tarrío Saavedra, S. Naya, R. Artiaga, Comparative study of the dynamic glass transition temperature by DMA and TMDSC, Polymer Testing. 29 (2010) 1002–1006. https://doi.org/10.1016/j.polymertesting.2010.09.005. 116 117 CHAPTER 6: Multifunctional epoxysilica hybrid materials derived from arnica root for advanced stone conservation This chapter was aimed to develop a specific tailor-made bio-based stone conservation material that exhibits a biocidal capacity to achieve the desired trifunctional role, maintaining a suitable balance of properties such as elasticity, adhesion, thermal and chemical stability, and hydrophobicity. Accordingly, the sequential building block addition was completed through the development of Step- Epoxy-silica hybrid enrichment, using the most promising formulations accomplished in Chapter 4 as the design basis. To finely adjust them and achieve microorganisms growth inhibition without compromising the features displayed by the base hybrids, various synthetic strategies based on ionic liquids (ILs), essential oils (EOs), and nanoparticles (NPs) were here investigated. Furthermore, the potential of the multifunctional materials designed as advanced conservation treatment was determined at mid-term. In accordance with the characterization study of the CBDO-DGE-based hybrid materials, 3-glycidyloxypropyl)trimethoxysilane (GPTMS) containing formulation, namely CBDODGE/GPTMS, was pointed out as a highly cross-linked and slightly rigid network with high potential to be modulated by a plasticizing effect exerted by the incorporation Chapter 6 118 of long aliphatic ILs chains or EOs. Thus, dimethyloctadecyl[3(trimethoxysilyl)propyl] ammonium chloride (QAS), trihexyl(tetradecyl)phosphonium chloride (TP), and thymol were here investigated for the first time with two possible functions: to act as network modulating dopings and to introduce biocidal capabilities [1–3]. As for NPs addition, the opposite effect could be theoretically predicted, expecting to act as a reinforced load of the epoxy-silica network [4], so the formulation based on GPTMS and octyltriethoxysilane (OcTES), namely CBDO-DGE/GPTMS/OcTES, that showed higher flexibility was selected for testing this doping strategy. Specifically, cerium-doped TiO2 NPs were tested as nanofiller as they have proven capabilities [5–7] to improve coating hydrophobicity, and impart significant self-cleaning and biocidal activity to epoxy-silica hybrids similar to the ones here developed. In addition, knowing the volatility handicap of EOs and the possible detriment of the hybrid network properties (caused by the direct addition of EOs), as well as the loss of biocidal property, an alternative approach based on the encapsulation of thymol into the mesoporous of NPs was also explored in parallel. In fact, SiO2 NPs were selected not only by their encapsulation advantages but also by the potential load and hydrophobic effects provided that could result in an advantageous finding of a balance of material properties [8–11]. A combination of ATR-FTIR, XRD, TEM, and µ-XRF analysis first checked the synthesized NPs suitability. Once the enrichment step was carried out, distribution studies were performed by SEM-EDS analysis. The suitability of each formulation to match the main requirements for a stone conservation material was evaluated in terms of thermostability, hydrophobicity, and inhibition of the microbiological growth by a combination of thermomechanical studies, contact angle and disk-diffusion against bacteria measurements. The formulations with the most promising results were further tested on stone specimens by artificial aging experiments under an acidic atmosphere. Colorimetric investigations, mass loss, and Scotch tape tests evaluated the potential shown by the treatment. Finally, microscopic imaging analysis of treated stone surfaces was performed for visual comparison. Accordingly, the work carried out and the main results and discussion, were detailed along the following sections. Multifunctional epoxy-silica hybrids derived from arnica root 119 6.1. STEP: Epoxy-silica hybrid enrichment Synthesis and characterization of NPs: Thymol-SiO2 The synthesis of SiO2 mesoporous NPs loaded with thymol was carried out by mixing tetraethyl orthosilicate (TEOS) (380 μL), ethanol (12 mL), an aqueous solution of thymol (60 μL, 10% m/v) and ammonium hydroxide (1 mL, 26% m/v). The reaction mixture was stirred for 48 h at room temperature. The resulting NPs were then separated by centrifugation (30 min at 10000 rpm), washed with ethanol, and dried at room temperature for one day [8]. The expected product was obtained as a fine white powder. The suitability of the NPs obtained was confirmed by XRD diffractograms displaying its characteristic signal at 23.2° 2θ, and further corroborated by the presence of the bands at 1097, 957, and 799 cm-1 in the infrared spectrum (Figure 6.1), which were attributed to the Si-O-Si asymmetric stretching, Si-OH stretching, and Si-O-Si symmetric stretching vibrations, respectively. Figure 6.1. FTIR spectrum of the synthesized thymol-SiO2 mesoporous NPs. The absence of the peak at 1634 cm-1, commonly attributed to O-H bending vibration mode of physisorbed water molecules inside the pores, suggested that the pores, very likely, being water-free, were filled with thymol. This assumption was further ascertained Chapter 6 120 by means of biocidal tests (see section 6.1.4.4). Besides, the –OH groups of the Si-OH moieties on SiO2 NPs surface were assigned to the broad band at 3445 cm-1 [12]. TEM analysis showed an uniform average NPs size of 100 nm (Figure 6.2), without NP aggregates formation. Figure 6.2. Transmission Electronic Microscope (TEM) images of thymol-loaded SiO2 NPs. Synthesis and characterization of NPs: Ce-doped TiO2 Titania nanoparticles were synthesized by mixing titanium isopropoxide (TTIP, 0.5 g) with isopropanol at a ratio of 1:30. Once the colloidal suspension was formed, cerium nitrate hexahydrate (0.023 g) was added (0.03:1 ratio, with respect to TTIP) and stirred for 1 h. Then, the NPs were washed and centrifuged three times using isopropanol, ultrapure water, and ethanol, respectively [5]. The solid was left at 80 °C for 48 h and then thermally cured at 450 °C for 3 h, with a heating rate of 2 °C min−1, to obtain specifically the anatase polymorph form. This way a pale-yellow solid was obtained. The successful synthesis of the Ce-doped TiO2 NPs in anatase form was confirmed by the Raman signals detected at 145, 398, 515, and 639 cm-1. The XRD diffractograms revealed peaks at 25.6, 38.2, 48.3, 54.2, 55.4, 62.8, 69.1, 70.3, and 75.3° 2θ (Figure 6.3), evidencing again the occurrence of anatase as the major phase (> 98%), i.e., the TiO2 polymorph with Multifunctional epoxy-silica hybrids derived from arnica root 121 biocidal properties[13]. The presence of cerium was roughly confirmed by the quite low spectral signal/noise ratio obtained in the diffractogram, as already observed [5,14]. However, the Ce3+ inclusion was finally corroborated by XRF data that showed its characteristic elemental signals at 34,719.7 eV (Ce Kα). Figure 6.3. XRD diffractogram of the synthesized Ce-doped TiO2 NPs. TEM analysis showed that the titania NPs average size achieved was around 200 nm, a double value compared to the silica ones (Fig. 6.4). Chapter 6 128 Table 6.2. Thermal and mechanical data of the enriched hybrid samples 1-5 and the base hybrids CBDODGE/GPTMS (G) and CBDO-DGE/GPTMS/OcTES (GO). Sample 1 2 3 4 5 G GO First step T onset [°C] 270 259 305 253 276 273 290 T max [°C] 333 335 336 340 331 317 345 % mass loss 67.6 58.0 95.1 57.2 54.2 63.2 67.0 Second step T onset [°C] 407 442 410 396 394 403 T max [°C] 452 463 431 435 437 410 % mass loss 17.8 20.8 21.3 25.9 14.6 14.9 % residual mass* 14.6 21.2 4.9 21.5 19.9 22.5 18.8 T g [°C] 24.4 19.9 20.2 36.6 31.0 48.4 39.1 T α [°C] 36.3 39.7 39.1 44.0 54.0 55.4 55.8 *at 800 °C The DSC thermograms showed glass transition temperatures (Tg) values (Table 6.2) reduced by more than a half for samples 1, 2, and 3 of 24.4, 19.9, and 20.2 °C, respectively, with respect to the 48.4 °C achieved by the non-doped hybrid. These findings clearly indicated that the GPTMS capability to act as a coupling agent was strongly affected by the presence of ILs and EO, so a significant decrease in the cross-linking degree in the resulting hybrids may occur [16]. Although, in principle, SiOMe groups of QAS, after hydrolysis, could actively concur to the condensation of silanols coming from GPTMS, thus providing additional anchor points for the hybrid development, the rather low Tg obtained for sample 1 suggests that the plasticizing effect due to the alkyl chains of QAS actually prevailed over the cross-linking with GPTMS so that the efficient incorporation of QAS into the hybrid network seemed unlikely. By contrast, for samples 4 and 5, the detected Tg values were slightly lower (i.e. values of 36.6 and 31.0 °C, respectively) than the one (39.1 °C) observed of the non-doped sample, evidencing that the use of thymol-containing NPs, rather than neat thymol, could be a successful strategy to provide biocidal properties of the EO to the products without a substantial negative impact on the hybrid development. The DMA investigations (Table 6.2 and Figure. 6.8) displayed for sample 1 a Tα value of 36.3°C, close to the Tg, thus suggesting that QAS may actively concur to the development Multifunctional epoxy-silica hybrids derived from arnica root 129 of the organic-inorganic network, although not to a great extent. Again, it can be noticed that the use of TP and thymol as additives in samples 2 and 3 resulted in a clear plasticizing effect, as the difference between Tg and Tα values was in both cases around 20 °C, with Tα of 39.7, and 39.1 °C, respectively. In contrast, the NPs loadings, as for samples 4 and 5, provided Tα of 44 and 54 °C, respectively, thus confirming that their incorporation preserved the cross-linking degree. This was even more evident for sample 5, suggesting that the efficient incorporation of NPs in the hybrid matrix occurred very likely due to the co-condensation of silica -OH groups with the silanols coming from the alkylalkoxysilanes, without compromising the intrinsic flexibility of the non-doped original formulation. Figure 6.8. Log storage modulus (E′) and damping (tan δ) of hybrid samples 1-5 as a function of temperature. Chapter 6 130 6.1.4.3. Water repellence studies The hydrophobic behavior of samples 1-5 was assessed according to the contact angle average data (n=5) displayed in Figure. 6.9, where two groups can be clearly distinguished. Figure 6.9. Contact angle average values CA (M) and standard deviations obtained for the enriched hybrid samples 1-5. Samples 1, 2 and 3 showed values lower than 90°, thus indicating that the hydrophobic capability of the original hybrid was not improved by the incorporation of ILs or neat thymol. On the contrary, the nano-enrichment with thymol-SiO2 NPs managed to preserve the original hydrophobic capability, with a CA value of 100°. However, the most significant result was obtained for sample 4, where the enrichment of the formulation with Ce-doped TiO2 NPs imparted an increase of 12° in the hydrophobicity with respect to the non-doped hybrid [17]. From the whole of the collected data, it clearly appears that nano-enriched hybrid samples 4 and 5 are the ones that displayed thermo-mechanical and hydrophobic properties suitable for stone conservation purposes. Accordingly, further studies were carried out to determine the biocidal capability and validate their multifunctional properties on stone specimens. Multifunctional epoxy-silica hybrids derived from arnica root 131 6.1.4.4. Anti-microbial capacity The anti-microbial test against the bacteria Arthrobacter spp. demonstrated for nanoenriched formulations 4 and 5 a bacterial inhibition effect in both cases (Figure 6.10). This confirmed that the NPs embedded into the hybrid matrix preserved their biocidal capability and that they were also able to provide additional properties to CBDO-based hybrids. Figure 6.10. The anti-microbial capacity of the hybrids samples 4 and 5 enriched with Ce-TiO2 and thymol-SiO2 NPs, respectively, against Arthrobacter spp. after 48 h of incubation under visible light. The nwhalo average values for samples 4 and 5 were 0.25 and 0.31 mm, respectively. However, although cerium doping was employed to enhance self-cleaning and antimicrobial activity in the visible light range, the Petri dishes used did not allow the UV light fraction to pass, and thus, the highest biocidal effect of Ce-doped TiO2 NPs of sample 4 is expected to be obtained in outdoor conditions where UV radiation enables a complete photocatalytic effect [5,6,18]. 6.2. Stone durability study Colorimetric analysis Colorimetric measurements on the lithic samples revealed that the treatments based on the selected nano-enriched hybrid formulations, namely 4 and 5, did not result in significant color changes. Concretely, color difference values (ΔE) of 3.27 and 2.23 were Chapter 6 132 obtained, respectively, for the Ce-TiO2 and thymol-SiO2 NPs (GPTMS/OCTES base formulations), corresponding both to the just noticeable chromatic alteration level, according to the CIE*Lab Colour-Difference Thresholds classification [19]. Furthermore, by comparing colorimetric data obtained for non-treated stone samples with those collected on treated, after aging experiments, it resulted that ΔE values were below 3.8, i.e. chromatic alterations below the distinctively perceptible level, thus confirming that exposure to an acidic atmosphere did not cause significant chromatic changes on the developed nano-enriched materials. The values obtained are also below the chromatic changes attributed to conservation treatments, which allow ΔE values of 5 [20]. Microscopic image analysis Subsequently, in order to evaluate the consolidating effect provided by the hybrid products on the stone samples, images by the magnification glass and SEM analysis were studied. As it is shown in Figure 6.11, it is evident that the aging test under the acidic CO2 atmosphere modified the porous network of the non-treated stone, enlarging the pore size and suggesting an alveolarization phenomena, which is a very typical degradation pattern suffered by these kind of stone. However, although both treated samples also appeared to be lightly affected by the aging, the affection degree was clearly inferior. Upon a closer examination of the magnified images, it can be inferred that the proposed Treatment 5, based on thymol-loaded SiO2 NPs enrichment, seemed to provide greater protection against the degradative action of the carbonate substrate by the environmental agent attack. Accordingly, these findings were supported by SEM investigations, which clearly showed a smooth and uniform surface for non-aged sample and an evident undergoing a degradation process (see Figure 6.12). At this scale, the protection provided by the treatments were evidenced without showing significant differences between their protection capacities to maintain an internal integrity similar to that of the original stone matrix. In fact, although a micro alveolarization areas were visible in the stone surfaces before the consolidating essays, the results collected after the application of the treatments demonstrated that the holes and superficial imperfections presented a minor depthless and thus, the matrix was less exposed the adverse effects of the environmental agents and achieved a prevention of further decay. Multifunctional epoxy-silica hybrids derived from arnica root 133 Figure 6.11. Magnification photographs of the stone samples, untreated and treated with the nano-enriched hybrid formulations 4 and 5 (obtained from GPTMS and OCTES as silica precursors with Ce-TiO2 and thymol-SiO2 NPs, respectively) for the visual examination of their ability as coating. Chapter 6 134 Figure 6.12. SE images of the stone samples, untreated and treated, with the nano-enriched hybrid formulations 4 and 5 (obtained from GPTMS and OCTES as silica precursors with Ce-TiO2 and thymol-SiO2 NPs, respectively) for the visual examination of their protection capability. Mass loss analysis These results was finally confirmed by the mass loss average values collected after aging experiments, as non-treated samples displayed a weight mass loss of 1.1 %, whereas stone Multifunctional epoxy-silica hybrids derived from arnica root 135 samples treated with 4 and 5 formulations showed values between 0.4 and 0.3 %, respectively. The Scotch tape test revealed the same trend, with 87% and 92% less disaggregated material observed on the stone surfaces. From the whole of the discussed results, it is reasonable to conclude that the epoxy-silica hybrid materials enriched with NPs exhibited a significant capability to mitigate the midterm decay of the tested stone specimens, being the SiO2 doped product the one that displayed the most significant multifunctionality overall. 6.3. General remarks Throughout this chapter, a sustainable multifunctional bio-based epoxy-silica hybrid nanocomposite specifically for stone conservation purposes was successfully developed using a building block design approach by the combination of CBDO-DGE/GPTMS/OcTES with thymol-loaded SiO2 NPs. The resulting material exhibited potential outstanding properties, including excellent consolidation, flexibility, hydrophobicity, and remarkable anti-microbial inhibition capacity. This multifunctional balance was possible thanks to the implementation of nanotechnology as a synthetic strategy. The opportunities presented by the hydroxyl surface of the silica NPs are highlighted for the enrichment of epoxy-silica hybrid materials via the sol-gel process, given that they facilitated an intimate joint between the additive doping and the organic-inorganic hybrid network. In fact, 3% content in silica NPs not only acts like a nano-filler capable of finely modulating the final material properties but also demonstrated to be a valuable support to add biocidal properties without compromising consolidating and hydrophobic features already displayed by the hybrid base used as a building block. Moreover, this work evidences the advantages presented by the EOs adsorption on silica NPs surface versus its direct addition to hybrid systems, achieving a clear minimization of the handicaps in the in final features, opening an innovative synthetic approach in the field of the building block design with vast advantages for developing advanced materials for different application purposes. However, in contrast to NPs strategy, the direct addition of ILs demonstrated not being suitable for building block designs of epoxy-silica hybrids materials since they induced substantial changes in the base network and produced an excessive plasticizer effect, while lowered the water repellence of the final products below the hydrophobicity Chapter 6 136 threshold. Nevertheless, the ILs potential in the field should not be underrated since other synthetic strategies can be studied in future lines of work. Finally, the stone specimen treatment proved that the product can be easily applied thanks to its low density, due to the use of methanol as solvent, ensure a homogenous and deep penetration into the stone substrate. Besides, the polymerization reaction takes place directly within the stone (in situ) and allows its suitable integration into the stone matrix while decreasing the incompatibility handicaps presented by conventional organicbased products. 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