Victor Garcia

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Keywords:

This thesis incorporates the application of several analytical methodologies to the speciation analysis of renewable feedstocks and petroleum heavy oil fractions. Separation techniques such as gel permeation chromatography (GPC) and supercritical fluid chromatography (SFC) have been combined with the elemental detection that inductively coupled plasma mass spectrometry (ICP-MS) provides to obtain a fingerprint of those species of interest, based on their hydrodynamic volume, present in samples with complex matrices. However, the combination of these separation techniques with ICP-MS was not an easy task.

With the purpose of providing to the analytical chemistry platform with novel approaches for the speciation analysis of complex samples, two novel tools have been devised during this three-year's project: (1) a cheap alternative to the current total consumption nebulisers, produced via 3D-printing and whose capillary has been built in PEEK, has been developed and validated for the analysis of trace elements in organic matrices via GPC-ICP-MS and (2) a novel interface has been designed and optimised for the trace element analysis via SFC-ICP-MS. Although very promising results have been obtained, further experiments must be carried out with these two methodologies to obtain a better understanding of the inorganic species present in renewable feedstocks and bio-oils.

The size distribution of sulfur, nickel and vanadium species in atmospheric residue fractions has been obtained via GPC-ICP-HRMS analyses to track and understand their evolution during two important industrial processes that are destined to convert these heavy oils into transportation fuels: hydrodemetallization and hydrodesulfurization.

Furthermore, the phosphorus-, sodium-, iron-, magnesium-and sulfur-containing species' size distributions in lignocellulosic fast pyrolysis oils and fatty acid feedstocks have been also obtained, demonstrating that high-molecular aggregates are formed within these complex samples when they are stored.

Alternative methodologies for the analysis of renewable feedstocks have been tested. The use of electrothermal vaporisation coupled to inductively coupled plasma optical emission spectroscopy (ETV-ICP-OES) has provided with highly promising results. A methodology

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -5-that does not require the use of freon-R12 gas has been optimised and validated for the total analysis of eight trace elements in complex samples. Besides, the distribution of the species containing trace elements such as P, Na, Mg and Al among others, based on their vaporisation temperatures, has been obtained.

RESUME

Ce manuscrit de thèse intègre l'application de plusieurs méthodologies analytiques à l'analyse de spéciation des matières premières renouvelables et des fractions de pétrole lourd. Des techniques de séparation telles que la chromatographie par perméation de gel (GPC) et la chromatographie en phase liquide supercritique (SFC) ont été combinées avec la détection élémentaire fournie par la spectrométrie de masse haute résolution à plasma à couplage inductif (ICP-MS), afin d'obtenir une empreinte digitale de ces espèces d'intérêt, basé sur leur volume hydrodynamique, présent dans les échantillons à matrices complexes.

La combinaison de ces techniques de séparation avec l'ICP-MS n'a pas été une tâche facile.

Cependant, afin de fournir à la plate-forme de chimie analytique de nouvelles solutions pour l'analyse de spéciation d'échantillons complexes (1), une alternative bon marché aux nébuliseurs à consommation totale actuelle, produite via l'impression 3D et dont le capillaire a été construit en PEEK, a été développé et validé pour l'analyse des éléments trace dans les matrices organiques via GPC-ICP-MS et (2) une nouvelle interface a été conçue et optimisée pour l'analyse des éléments trace via SFC-ICP-MS. Bien que des résultats très prometteurs aient été obtenus, de nouvelles expérimentations doivent être menées avec ces deux méthodologies afin de mieux comprendre les espèces inorganiques présentes dans les matières premières renouvelables et les bio-huiles.

La distribution en taille des espèces de soufre, de nickel et de vanadium dans les fractions de résidus atmosphériques a été obtenue via des analyses GPC-ICP-HRMS afin de suivre et de comprendre leur évolution au cours de deux processus industriels importants destinés à convertir ces fractions de pétrole lourd en carburants de transport: l'hydrodémétallisation et l'hydrodésulfuration. En outre, les distributions de tailles des espèces contenant du phosphore, du sodium, du fer, du magnésium et du soufre dans les huiles de pyrolyse lignocellulosique et les charges d'acide gras ont également été obtenues, démontrant que des agrégats de haut poids moléculaire se forment dans ces échantillons complexes lorsqu'ils sont stockés.

Des méthodologies alternatives pour l'analyse des matières premières renouvelables ont été testées. L'utilisation de la vaporisation électrothermique couplée à la spectroscopie

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -7-d'émission optique à plasma à couplage inductif (ETV-ICP-MS) a fourni des résultats très prometteurs. Une méthodologie qui ne nécessite pas l'utilisation de gaz fréon-R12 a été optimisée et validée pour l'analyse totale de huit éléments trace dans des échantillons complexes. De plus, la répartition des espèces contenant des éléments trace tels que P, Na, Mg et Al entre autres, en fonction de leurs températures de vaporisation, a été obtenue.

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -8-

ABSTRAKT

Denne PhD afhandling anvender forskellige analytiske metoder til specieringsanalyse af vedvarende råmaterialer og petroleumstunge oliefraktioner. I afhandlingen anvendes separationsteknikker som gel permeations kromatografi (GPC) og superkritisk vaeskekromatografi (SFC) i kombination med detektion af elementer somved induktiv koblet plasma-højopløsningsmassespektrometri (ICP-MS). Metoderne anvendes til at måle det kemiske fingeraftryk af specier i komplekse matricer , baseret på speciernes hydrodynamiske volumen. Kombinationen af kromatografiske separationsteknikker med ICP-MS var ikke en let opgave.

For at opnå en forbedre analyse af komplekse matricer blev der udviklet en raekke nye forbedrede metoder til specifikationsanalysen af komplekse prøver: (1) en billig alternativ til den nuvaerende "total consumption nebuliser" blev udviklet og valideret til sporstof anlayse i organiske matricer via GPC-ICP-MS. Den nye nebuliser blev produceret ved 3D print af PEEK materiale; (2) et nyt interface blev designet og optimeret til sporelementanalyse ved SFC-ICP-MS. Selvom der er opnået meget lovende resultater, skal yderligere eksperimenter udføres med disse to metoder for at opnå en bedre forståelse af specierne som er tilstede i vedvarende råmaterialer og bioolier.

Størrelsesfordelingen af svovl-, nikkel-og vanadiumspecier i atmosfaeriske restfraktioner er opnået via GPC-ICP-HRMS-analyser for at spore og forstå deres udvikling under to vigtige industrielle processer, der er designet til at omdanne ukonventionelle olier til transportbraendstof: hydrodemetallization og afsvovling.

Endvidere er størrelsesfordelingen af forfor-, natrium-, jern-, magnesium-og svovlholdige specier målt i lignocellulosiske pyrolyseolier og fedtsyreholdige-råmaterialer. Disse resultater viser, at der dannes højmolekylaere aggregater i disse komplekse prøver, når de opbevares ved normale forhold.

Alternative metoder til analyse af vedvarende råmaterialer er blevet testet. Der blev opnået lovende resultater ved anvendelsen af elektrotermisk fordampning koblet til induktivt koblet plasmaoptisk emissionsspektroskopi (ETV-ICP-OES). En ETV-ICP-OES metode som ikke kraever anvendelse af freon-12-gas, er blevet optimeret og valideret til analyse af summen af otte sporstoffer i komplekse prøver. Metoden blev anvendt til analyse af sammensaetningen af P, Na, Mg og Al specier baseret bl.a. på deres fordampningstemperaturer.

LIST OF TABLES

Table 1. Overview of the main reactions occurring during HDO. [START_REF] Dabros | Transportation Fuels from Biomass Fast Pyrolysis, Catalytic Hydrodeoxygenation, and Catalytic Fast Hydropyrolysis[END_REF] ...................................49 Table 5. ICP-OES parameters used for all the analysis carried out via ETV-ICP-OES. 117 Table 6. R 2 coefficient for each analysed wavelength. ...................................................... 125 Table 7. Concentrations in mg kg -1 , R 2 and detection limits in mg kg -

LIST OF FIGURES

INTRODUCTION

The production of renewable feedstocks and designed to obtain high-quality transportation fuels is growing considerably due to the upcoming shortage of fossil fuel products. Furthermore, the RED II legislation (Renewable Energy Directive for 2021-2030 in the European Union) has set a series of measures and obligations that will guarantee that at least 14% of the transportation fuel has been produced from renewable feedstocks by 2030.

The use of petroleum heavy oil fractions, such as atmospheric residues, and bio-oils, to produce high-quality transportation fuels, has increased rapidly during the last decade.

However, the presence of heteroatoms and other metals within these petroleum heavy oil fractions oils is causing several problems, such as the emission of poisonous compounds to the atmosphere and the catalyst poisoning during the hydrotreating processes among others. Thus, understanding the nature of these trace elements and their speciation is essential to improve the production yields and the quality of those energy sources produced from renewable feedstocks and petroleum heavy oil fractions. These elements, such as P, Fe, Mg, S and other trace elements present in renewable feedstocks; and V, Ni and S compounds, present at hundred ppm levels in crude oil and its heavy fractions, must be removed from the feedstocks in order to be processed and converted into transportation fuels.

Aims and objectives

Few analytical methodologies have been developed for the speciation analysis of those trace-element-containing species in crude oils and petroleum heavy oil fractions. However, in the present day, the development of such methodologies for the speciation analysis of bio-oils and renewable feedstocks have not been developed yet.

analytical tools for the speciation analysis of bio-oils and renewable feedstocks. This objective has been divided into three sub-objectives: o O1a: Development of a new total consumption nebuliser, produced through 3D-printing, that lowers the instrumental costs and allows the hyphenation of ICP-MS with LC. In addition, the hypothesis that suggest that this nebuliser would facilitate the speciation analysis of those species that might have interactions with the fused silica that was built in previous total consumption nebuliser versions has been tested.

Outline of the thesis

This PhD thesis has been divided into seven chapters. In the first two chapters a short literature review is presented, discussing (1) the production and composition of different bio-oils and renewable feedstocks and (2) the fundaments of the analytical methodologies that were used in this three-year project.

In the third chapter of this PhD thesis, the development and validation of a total consumption nebuliser that was made through 3D-printing technology is presented. This new nebuliser, which was built with a PEEK capillary, can connect either the outlet of a chromatographic column or a pump, allowing both chromatographic and total analyses of complex samples. The replacement of the fused silica capillary that was used in previous total consumption micronebulisers eliminates both the interactions and potential permanent retention that some elements might have with it and will allow the speciation analysis of other trace elements as well, like silicon in bio-oils and renewable feedstocks.

In the fourth chapter, a novel interface for the hyphenation of SFC and ICP-MS has been presented. Supercritical fluid chromatography can provide to the separation of complex matrices with a very high number of theoretical plates. However, hyphenating SFC with an elemental detection like ICP-MS had not been yet carried out for modern SFC instrumentation. Thus, an interface that allows the hyphenation between SFC, using CO2

and MeOH as the mobile phase, with ICP-MS has been developed. This chapter contains the preliminary results of the first use of this novel interface for both total and speciation analyses of renewable feedstocks and unconventional oils.

Chapter five discusses the development and application of an ETV-ICP-OES methodology that allows the speciation analysis of trace elements in complex matrices based on their vaporisation point. An optimisation of the gas flows, including the use of the alternative reaction gas SF6, has been carried out and the method has been validated for the total analysis of eight trace elements by analysing the NIST SRM 1085b. Finally, the distribution of several trace element species in fast pyrolysis oils, based on their vaporisation temperature, has been obtained. Monomodal, bimodal and multimodal distributions have been observed among the different heteroatom-containing species present within such complex samples.

In the sixth chapter a GPC-ICP-HRMS methodology has been applied to the speciation analysis of the vanadium, nickel and sulfur species that are present in the atmospheric residue that is hydrotreated to obtain transportation fuels. The results that are presented within this chapter describe how the high molecular weight species containing sulfur, vanadium and nickel have been removed differently than the rest of lower molecular weight compounds during the hydrodesulfurization and hydrodemetalation processes. In addition, the loss of activity of the catalyst showed how it is more noticed on the hydrotreating of the high molecular weight compounds.

CHAPTER 1: FROM BIOMASS TO BIO-OIL. PRODUCTION AND COMPOSITION

Definitions of Biomass and Bio-oil

Biomass is defined by the IUPAC as the material produced by the growth of microorganisms, plants of animals, 1 but it can also be considered as the biodegradable fraction of products, waste and residues from biological origin from agriculture (including vegetal and animal substances), as well as the biodegradable fraction of industrial and municipal waste among other products. 2 Biomass has been used as a source of energy all over the world for thousands of years until nowadays. 3 In 2017, the use of waste biomass and its derivates provided about 5% of the total primary energy use in the United States, becoming the most widely used renewable energy source at that time. 4 The use of natural biomass as energy resource possesses numerous advantages. It consists of a renewable source and it is also CO2 neutral 5,6 as all the CO2 produced during combustion will be equal to the CO2 naturally collected from the atmosphere during its formation by photosynthesis.

The use of industrial and municipal waste biomass cannot be considered a renewable source, but it still constitutes an important alternative to fossil fuels. It could also suppose an important improvement in general waste management strategies.

Bio-oils (also known as biofuels or agrofuels) are those solid, liquid or gaseous fuels that have been produced from biomass. They could also be defined as those fuels that are produced from organic matter rather than fossil fuels. With the application of different chemical processes, it is possible to produce bio-oil from biomass, which constitutes a potential alternative source of energy to fossil fuels.

Classification of bio-oils

Depending on the type of biomass feedstocks used to produce bio-oils, they can be classified into different groups:

➢ Primary bio-oils: Direct combustion of natural and unprocessed biomass such as wood, pellets, animal waste and crop residues among others. Normally used to supply heating or electricity production needs. [START_REF] Nigam | Production of Liquid Biofuels from Renewable Resources[END_REF] ➢ Secondary bio-oils: They are produced by processing biomass and consist of chemically modified primary bio-oils. Depending on the process and the material used, secondary bio-oils are classified into three subgroups:

• First-generation bio-oils: They are generally produced from edible crops such as sugars, grains or seeds. Bioethanol, which is produced by fermentation of the sugars and starch present in crops (wheat, corn, potato, etc.) and biodiesel, which is produced from different vegetable oils (rapeseed, soybeans, animal fats, etc.) by transesterification processes are clear examples of first-generation bio-oils.

Production of first-generation of bio-oils requires an outsize amount of land, initially used to produce food, which would be exclusively dedicated to feedstock.

Chisti et al. showed that even for palm crops, which have one of the most abundant yields, the 24% of the United States of America's total cropland would be required to meet only 50% of their transport fuel needs. 8

• Second-generation bio-oils: They are produced by processing the non-edible residues from lignocellulosic biomass (trees, grass, etc.) and municipal waste (tyres, plastics, etc.). Lignocellulosic biomass feedstocks have a high content of cellulose, hemicellulose and lignin, which could be considered as an important source of carbon. However, their high content in oxygen and other inorganic elements require hydrodeoxygenation processes and catalysis upgrading that will be explained in section 1.6 of this manuscript. These feedstocks have a lower cost than the first-generation ones, but the conversion process to transportation fuels will be quite expensive until numerous improvements are deployed and applied.

• Third-generation bio-oils: They derive from microalgae, which possess high lipid productivity. These lipids are produced in the form of triacylglycerols, which are ideal for the production of bio-oil. [START_REF] Shuba | Microalgae to Biofuels: 'Promising' Alternative and Renewable Energy[END_REF] Besides, microalgae biomass production can produce a large number of proteins and carbohydrates in short times under normal conditions by assimilating CO2 and sunlight and by consuming about 1.83 kg of CO2 per kilogram of dry algal biomass. [START_REF] Nigam | Production of Liquid Biofuels from Renewable Resources[END_REF][START_REF] Brennan | Biofuels from Microalgae-A Review of Technologies for Production, Processing, and Extractions of Biofuels and Co-Products[END_REF] One of the main advantages of third-generation bio-oils is explained by Chisti et al. [START_REF] Chisti | Biodiesel from Microalgae[END_REF] Their work illustrates the competence of microalgae yields by comparing them with the first-generation feedstock production: Only between 1 and 3% of the United States' total cropping area would be required to meet the 50% of their transport fuel needs if it were dedicated to producing microalgae biomass.

Renewable feedstocks

The major biomass feedstocks that are used to produce bio-oils and other renewable transportation fuels can be divided into groups, being these: Lignocellulosic biomass, sugars and starches, oils and fats, and industrial and municipal wastes. This review will focus on those whose samples will be analysed and studied during this work. • Hemicellulose: Consists of a group of polysaccharides that are present in most of the plants and vegetables. Their main role is to harden and strengthening the cell walls and their structure is formed by different monosaccharides linked through β(1→4)-glycosidic bonds that shape a long branched chain. Even though it might seem simple to break these glycosidic bonds to obtain glucose molecules, it is a difficult process. The difference between hemicellulose and cellulose is that the former normally consists of shorter chains, shaping a branched polymer with different sugar monomers.

• Lignin: Complex heteropolymer present in the cell walls in wood and bark that provides rigidity and strength to the plant. It possesses hydrophobic properties that make this polymer a key component in water transportation within the plants.

Knowing the actual molecular weight of this polymer is a challenge and numerous methods have been carried out to determine this value. [START_REF] Tolbert | Characterization and Analysis of the Molecular Weight of Lignin for Biorefining Studies. Biofuels, Bioprod[END_REF] It is characterized by its abundant aromatic groups and the numerous structures they can form. Its structure is formed by the dehydrogenate polymerization of three molecules classified as "monolignols". They are the p-coumaryl, p-coniferyl and synapyl alcohols. [START_REF] Sakagami | Distribution of Lignin-Carbohydrate Complex in Plant Kingdom and Its Functionality as Alternative Medicine[END_REF] The structure of a lignin-carbohydrate complex is shown in Figure 3. It is possible to observe the high presence of oxygen within the carbon-rich structure. This high carbon content could be employed as a source of energy.

These three major components are distributed in the cell walls shaping a complex structure where all they are connected through hydrogen bonds (mainly with cellulose) and covalent bonds. [START_REF] Wang | Lignocellulosic Biomass Pyrolysis Mechanism: A State-of-the-Art Review[END_REF] Hemicellulose structure, due to the presence of different monomers and its branched structure, follows a different degradation process: each monosaccharide is degraded to different products including char and gases. For instance, the xylan degradation can lead to the formation of various anhydrosugars and other sub-products such as acetic acid. [START_REF] Wang | Lignocellulosic Biomass Pyrolysis Mechanism: A State-of-the-Art Review[END_REF] Lignin structure, which is more complex, follows a completely different path. While the glycosidic bonds are degraded as for glucose and hemicellulose, the cleavage of ether bonds leads to the formation of new products. Re-polymerization and side-chain conversion reactions also take place, resulting in a wider range of products, including the formation of char, water and gas. Different varieties of crops are harvested to produce first-generation bio-oils. These crops contain materials such as sugar cane, wheat or fruits that are rich in sugars, and also rice, corn and potatoes, which are rich in starch.

Sugars, which consist of soluble carbohydrates, are easily converted into ethanol through a biological process called alcoholic fermentation, that also produces carbon dioxide as a by-product, whose simplified reaction scheme is shown in Figure 5. Starch, however, is a polymer formed by the glycosidic bond union of glucose monomers.

These glucose monomers can be extracted and, subsequently, through a fermentation process, they are transformed in bio-ethanol.

Oils and fats

Raw materials such as vegetable oils (palm oil, soybean oil, etc.) and several animal fat wastes such as tallow originating from pork, lamb, etc. can be used as a feedstock for biodiesel production. These materials contain a high concentration of triacylglycerides that after a transesterification process become fatty acid alkyl esters, which are the main component of biodiesel. [START_REF] Kordulis | Development of Nickel Based Catalysts for the Transformation of Natural Triglycerides and Related Compounds into Green Diesel: A Critical Review[END_REF] They can also be treated with H2 through a catalytic reaction to obtain a paraffin-based and completely deoxygenated fuel, also known as green diesel. generate bio-oils cause several ethical issues due to the lack of food in certain parts of the world, the use of non-edible materials such as wastes becomes a useful alternative. [START_REF] Doshi | Investigations into the Control of Odour SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS Victor GARCIA MONTOTO -21/09/2020 -196-and Viscosity of Biomass Oil Derived from Pyrolysis of Sewage Sludge[END_REF][START_REF] Mastral | Influence of Process Variables on Oils from Tire Pyrolysis and Hydropyrolysis in a Fixed Bed Reactor[END_REF] One of the most problematic waste materials in terms of storage and disposal, due to their long durability and non-biodegradability, is the scrap tyres. Thus, new methods for recycling scrap tyres have been developed during the last years. [START_REF] Mastral | Influence of Process Variables on Oils from Tire Pyrolysis and Hydropyrolysis in a Fixed Bed Reactor[END_REF] The main components of this feedstock are rubber, which consists mainly of organic polymers, and carbon black, a sub-product of petroleum. Thus, pyrolysis of tyres degrades the rubber polymers into smaller gaseous compounds that are condensed and used as biooils. Other sub-products such as char and gas that can be used in the energy industry are obtained by this method.

Bio-Oil from pyrolysis: Production and composition

Pyrolysis and fast pyrolysis

The rapid heating of biomass at temperatures between 400 and 800 °C in absence of oxygen results in a decomposition of its organic matter into three products whose proportion could vary depending on the process parameters and/or the biomass feedstock: char (also known as bio-char), [START_REF] Dhyani | A Comprehensive Review on the Pyrolysis of Lignocellulosic Biomass[END_REF] permanent gases and organic vapours that can be condensed, resulting in a liquid that is known as bio-oil. [START_REF] Bridgwater | Review of Fast Pyrolysis of Biomass and Product Upgrading[END_REF] In Figure 7 a diagram showing the different processes carried out to produce fast pyrolysis oils is detailed. While the use of high temperatures and long residence times during pyrolysis leads to gas formation, the use of low temperatures and long residence times leads to the formation of char. The optimum conditions for the production of bio-oils are medium temperatures with short residence times, which means high heating rates. [START_REF] Van De Velden | Kinetics and Endothermicity of the Biomass Pyrolysis Reaction[END_REF] These conditions are applied in the process called "Fast Pyrolysis", which consists of one of the most important processes in the production of bio-oils.

Fast pyrolysis tolerates the use of any form of biomass. Nearly 100 different biomass types (agricultural wastes, energy crops, solid wastes, etc.) have been tested by many laboratories. Bio-oil is the main product, with yields up to 75 wt. %, together with char and gas. [START_REF] Bridgwater | Renewable Fuels and Chemicals by Thermal Processing of Biomass[END_REF]

General composition of Pyrolysis Oil

Fast pyrolysis oil consists of a dense, sticky and dark brown coloured liquid. It possesses a strong smoky odour and normally, due to the feedstock moisture and the water produced during the whole process and storage, it presents two different phases (aqueous and organic).

GC-MS analyses of the aqueous phase were performed by Zhang et al. [START_REF] Zhang | Upgrading of Liquid Fuel from the Pyrolysis of Biomass[END_REF] This work shows its high concentration of water-soluble compounds such as acetic acid, hydroxyacetone and phenols among others. Therefore, this aqueous phase cannot be used as a fuel, but it is interesting as it could be a source of water-soluble chemicals.

Even though the major constituents of bio-oil can vary from each biomass feedstock, the organic phase generally possesses a high content in carbon and oxygen, mainly in the form of oxygenated hydrocarbons. These could be classified in these groups: acids, sugars, alcohols, ketones, aldehydes, phenols and derivates, furans and other mixed oxygenated compounds. [START_REF] Mohan | Pyrolysis of Wood/Biomass for Bio-Oil: A Critical Review[END_REF] Besides, it is possible to find some fractions of lignin that could reach the 5000 Dalton.

Main impurities present in Pyrolysis Oils

Char

Char is a carbon-rich solid residue that is produced after pyrolysis of biomass with high content in carbon and it is considered as one of the main sources of inorganic compounds, especially phosphorus, silicon, sodium, calcium and potassium containing species. 26 It acts as a vapour cracking catalyst, so it should be eliminated. To do so, cyclones and hot vapour filtration are the most common methods used. [START_REF] Bridgwater | Renewable Fuels and Chemicals by Thermal Processing of Biomass[END_REF] Collecting the char residue is important, as it can be used as a low-grade fuel due to its high content in carbon and as a soil additive.

Oxygenated compounds

The oxygen content of these bio-oils is usually 35 -40 wt. % which provides to the bio-oil with lower heating power, higher viscosity and corrosiveness: [START_REF] Bridgwater | Overview of Applications of Biomass Fast Pyrolysis Oil Overview of Applications of Biomass Fast Pyrolysis Oil[END_REF][START_REF] Vitolo | Catalytic Upgrading of Pyrolytic Oils to Fuel over Different Zeolites[END_REF] • Water is one of the main important oxygenated compounds (up to 25 wt % depending on the feedstock) present in raw pyrolysis oils and it cannot be separated. [START_REF] Mohan | Pyrolysis of Wood/Biomass for Bio-Oil: A Critical Review[END_REF] • The presence of carboxylic acids in bio-oils reduce the pH (2 -3) and make them more corrosive for the handling equipment and storage vessels. [START_REF] Dhyani | A Comprehensive Review on the Pyrolysis of Lignocellulosic Biomass[END_REF] • The presence of oxygenated compounds can promote etherification and esterification reactions whose main product is water, which reduces the bio-oil heating value. Besides, the condensation and polymerization reactions increase the average molecular weight of the bio-oil, which is directly linked with the increase of viscosity. [START_REF] Czernik | Stability of Wood Fast Pyrolysis Oil[END_REF] Figure 8 shows some of the typical oxygenated hydrocarbons found in pyrolysis oil: These compounds can be either monofunctional like phenol and benzaldehyde, or multifunctional such as 2-hydroxy-3-methoxybenzoic acid. [START_REF] Dhyani | A Comprehensive Review on the Pyrolysis of Lignocellulosic Biomass[END_REF] Thus, upgrading processes of pyrolysis oils must be carried out with the purpose of removing the high oxygen content and transforming this low-energetic renewable feedstock into high-quality transportation fuels.

Inorganic species

The presence of numerous inorganic elements causes numerous issues in engines, pipes and other equipment such as slagging, fouling and ash related problems. They also reduce the heating value of the pyrolysis oil and could cause environmental pollution due to the emission of some of these compounds.

One of the main sources of these inorganic compounds in pyrolysis oil due to their higher presence is char. Agblevor et al. 26 studied the alkali composition of pyrolysis oils and showed that about 100% of potassium in the initial feedstock is provided by char. Other elements, such as Calcium and Phosphorus could have different origins including char as well.

The main elemental inorganic constituents of pyrolysis oil are the following ones: 29

• Potassium: It is one of the most abundant metals present in plants and their nutrients as it is present in the cell-wall components and lignin. Nowakowski et al. [START_REF] Nowakowski | Uncatalysed and Potassium-Catalysed Pyrolysis of the Cell-Wall Constituents of Biomass and Their Model Compounds[END_REF] studied the effect of potassium on lignin pyrolysis and confirmed its high influence on the increase of char yield during pyrolysis.

• Calcium: Fifth most abundant element in earth crust, very important nutrient in plants and essential element in the cell walls, hence its presence in bio-oils such as lignocellulose and vegetable oils. It has been found that this element can form hard deposits in gas turbines airfoils, causing loss of performance and being difficult to be removed. 31 • Sulfur: Depending on the type of biomass that has been employed to produce biooils, sulfur can be present with different abundances. Pyrolysis oils from crops, which are rich in proteins, contains a high concentration of sulfur, as this element is present in amino acids such as methionine and cysteine. [START_REF] Knudsen | Sulfur Transformations during Thermal Conversion of Herbaceous Biomass[END_REF] Other types of biomass sources, such as tyres, could contain a much higher concentration of sulfur due to the vulcanisation process carried out to harden the rubber they are made from, which consists in heating this material with sulfur compounds. Among the different sulphur species in tyres pyrolysis oils, some sulphur PAHs have been already

identified. [START_REF] Williams | Sulfur-Polycyclic Tyre Pyrolysis Oil[END_REF] As one of the main impurities in crude oil, sulfur compounds are already well known since they cause corrosion in numerous alloys, catalyst poisoning and they are the cause of the emission of toxic gases to the atmosphere. Research into the speciation analysis of the different compounds containing sulfur in crude oil and its derivates have been performed thoroughly during the last years, focusing on achieving a better understanding of the characteristics of these species and thus adapt different desulfurization processes to obtain better results.

• Phosphorus: This element is present with a high abundance in biomass, as it consists of an essential nutrient for the growth of numerous organisms. This element can be found in fatty acids, phospholipids and sterols among other abundant biomolecules. Like potassium, it has negative effects on the pyrolysis oil stability since an increase of the pyrolysis oil viscosity caused by polymerisation reactions has been observed. In addition, its presence also increases the char yield during the pyrolysis process, which could cause future problems in engines and equipment. [START_REF] Banks | Impact of Potassium and Phosphorus in Biomass on the Properties of Fast Pyrolysis Bio-Oil[END_REF] Phosphorus speciation analyses in ash have been carried out in

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -40-different types of pyrolysis oils and it was found that it forms different phosphates with calcium, potassium and iron. [START_REF] Tan | Phosphorus Recovery from the Biomass Ash: A Review[END_REF] • Other trace elements, which are very abundant in the earth's crust, and, therefore, present in soils where the organic matter used as feedstock is grown, are amongst others, silicon, aluminium, magnesium and sodium. Biodiesel is far different from petroleum diesel, and some of its advantages are summarized below: [START_REF] Knothe | The Biodiesel Handbook[END_REF] • Derived from a renewable source.

Biodiesel and renewable diesel

• High biodegradability.

• Lower emission of toxic gases (except for NOX).

• Higher flashpoint.

• Excellent lubricity.

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -41-Nowadays, biodiesel is blended with petroleum diesel and used in diesel engines, due to its high viscosity. However, this blending is advantageous as the higher lubricity of the biodiesel improves the protection of the fuel injection systems in engines.

The industrial transesterification process takes place in a reactor where an alcohol, generally methanol, a catalyst that could be either acid or basic, and the fatty feedstocks are mixed under agitation for around 1h and at 60 °C. [START_REF] Knothe | The Biodiesel Handbook[END_REF] Once the reaction has been carried out, glycerol is removed from the products in a settling tank or a centrifuge and the methyl esters are neutralized and purified.

General composition of biodiesel

When the transesterification process is over, a mixture of different fatty acid methyl esters (FAME) is the main component of the products. These FAMEs are different for each feedstock that is used to produce them. Hoekman et al. [START_REF] Hoekman | Review of Biodiesel Composition, Properties, and Specifications[END_REF] studied the general composition of biodiesel produced from twelve different feedstocks.

Oleic, Palmitic and Stearic acid methyl esters are the principal species present in biodiesel produced from tallow, which is one of the feedstocks that will be analysed in this research project. FAMEs, whose carbon chains length vary from 6 up to 24 carbons and up to 4 instaurations, have been found for these types of feedstock.

However, Linoleic, Oleic and Palmitic were the principal FAMEs found in Soybean Oil, which is the other of the feedstocks analysed during this project.

Renewable diesel

Produced from feedstocks such as animal fats and vegetable oils, whose potential dieseltype molecules, independently of either the seed type or the animal, are the triglycerides.

These triglycerides can react under hydroprocessing conditions to form different n-alkanes whose number of carbons is within the diesel length depending on the pathway followed during the process.

This process is carried out in a reactor at conditions above 300°C and 45 bar and with a hydrogen-to-oil ratio of 500 nL/L and the reaction pathway carried out along the process is described in Figure 11, where the triglycerides present in the feedstocks react with the hydrogen in the presence of a catalyst saturating the double bonds within the triglycerides. 39 This first pathway is followed by a second reaction where the triglycerides are completely broken producing either H2O and propane (hydrogenation pathway) or CO2 and propane instead (decarboxylation pathway) as subproducts, added to the correspondent n-alkanes whose length is equal to each triglyceride length minus one.

One of the key factors in this process is the catalyst selection, since it must be able to handle the excess of CO produced within the reactor that inhibits the desulphurization process, among other factors that might reduce drastically the life of the catalyst and worsen the properties and composition of the process products.

Main impurities in Biodiesel, Renewable Diesel and their feedstocks

Not only can the type of FAMEs present in biodiesel or the n-alkanes within the green diesel vary depending on the feedstock. The nature of the impurities in these bio-oils can also have a high variation depending on the raw materials that have been used as a feedstock.

Phosphorus

As one of the main components of all the cell membranes, phospholipids are one of the main sources of phosphorus contamination. This contamination has been suggested by Van Gerpen et al. [START_REF] Gerpen | Biodiesel Processing and Production[END_REF] as a source of catalyst destruction during the transesterification process causing an important yield reduction: 50 ppm of phosphorus in a vegetable oil feedstock could reduce the yield up to a 5%.

Phospholipids, which are the major constituent of the lipid bilayer, are a class of lipids that are present in all the types of cell membranes. Their basic structure is constituted by a diacylglycerol backbone with a phosphate ester-linked to the third carbon of a glycerol molecule. Depending on the compound that is attached to the phosphate group of the phospholipid, different structures can be formed. The main compounds that are linked to this phosphate group are the choline, ethanolamine, serine and inositol. When they are part of the skeleton of the phospholipids, they modify their physical and chemical behaviour.

The structure of the most common phospholipids is detailed in Figure 12 and their chemical behaviour differences can be predicted from their structure, especially when paying attention to the different phosphate groups and the compound that is linked to them, which characterises the phospholipid. 42 From all the existing phospholipids, there are five of them that have been found as major components in nature: phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA) and phosphatidylethanolamine (PE).

These major components are divided into two groups concerning their interaction with water: hydratable (HPLs) and non-hydratable (NHPLs) phospholipids.

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -45-

The fatty acid chains that constitute the backbone of the phospholipids commonly have a length that varies from C4 to C26, with saturations that vary from one to six double bonds, located in different parts of the acyl group. Depending on the source of these phospholipids, such as vegetable or animal origins, the length and saturation rates of these compounds might vary.

Another issue that these compounds cause concerning the production of either biodiesel or renewable diesel is the formation of gums. The phospholipids possess a strong emulsifying action that can reduce the catalysts' life during the transesterification processes. Besides, other metallic species might be attached to these phosphatides leading to the formation of oxidant compounds such as aldehydes and ketones, amongst others, and ultimately degrade the biodiesel produced. 43,[START_REF] Sharma | Latest Advances in Degumming Feedstock Oils for Large-Scale Biodiesel Production. Biofuels, Bioprod[END_REF] Thus, a process known as "degumming" must be carried out to refine the vegetable oils and animal fats before they are used. This process consists of the removal of the maximum number of phospholipids via different phases.

Depending on the nature of the phospholipids present in a fatty acid feedstock, different degumming techniques can be used to obtain an optimal elimination of the phosphorus species within the sample in a faster way.

One of these methods is the water degumming, in which water is used to hydrate the HPLs.

These hydrates, which are insoluble in oils due to their aggregation rates, form oilinsoluble particles that can be eliminated by simple centrifugation. Regarding soybean oil, the HPLs fraction already means more than 50% of the phosphorus content. [START_REF] Sharma | Latest Advances in Degumming Feedstock Oils for Large-Scale Biodiesel Production. Biofuels, Bioprod[END_REF] Other chemical degumming methods have been reviewed by Sharma et al. [START_REF] Sharma | Latest Advances in Degumming Feedstock Oils for Large-Scale Biodiesel Production. Biofuels, Bioprod[END_REF] and Sengar et al. 43 who have studied the degumming process for soybean and rice bran oil samples respectively. A general overview is given below:

• Water degumming, which was already explained above, consists of the elimination of the hydratable fraction of the phospholipids present in fatty acid feedstocks. It also allows the recovery of lecithin, a mixture of phospholipids and other compounds, which can be used as an additive in the food industry.

• Chemical degumming groups all the different methods where the addition of a chemical reagent is carried out. The use of acids, such as citric acid and phosphoric acid help in the decomposition of numerous PL complexes with Na and Mg ions, amongst others, being ultimately eliminated by centrifugation. Other reagents, such as EDTA, a chelating reactive, or electrolyte solutions are also used for the removal of NHPLs.

• Enzymatic degumming. Considered as one of the best processes currently available to reduce the phosphorus content up to less than 5 ppm in rice bran oil, converts the NHPLs to HPLs, which are ultimately removed by centrifugation.

Phospholipase, in an acidic medium, is the most commonly used enzyme.

• Membrane degumming. The fundament of this process is based on both the molecular sizes of the phospholipid molecules, which are approximately 1.0 -1.5 nm and their molecular weights. Furthermore, these PLs can form micelles whose size can reach up to 200 nm of size and 20 kDa of weight. Thus, making these oil samples passing through a series of membranes with a determined pore size, depending on the sample, will allow the elimination of these undesirable species.

Amongst all the types of membranes, ceramic (Al2O3, TiO2, etc.) and polymeric (PVDF, PI, PES) membranes are currently in use. They have a high efficiency, but they are expensive and require larger amounts of time rather than other degumming methods.

1.5.4.2. Other inorganic species.

• Sulfur: This element is present in all type of living cells, hence its high abundance in both vegetable and animal feedstocks. Apart from the bad effect that sulfur emissions could have in the environment, these compounds have been found that can cause catalyst deactivation during the hydrodeoxygenation process of vegetable oils. [START_REF] Mohammad | Overview on the Production of Paraffin Based-Biofuels via Catalytic Hydrodeoxygenation[END_REF] • Silicon: Different species of this element are commonly present in biodiesel samples due to its presence as silicon oxide in feedstock crops. Their presence can cause damage to the engines, and the oxides formed during combustion can form slag deposits and corrosive compounds. [START_REF] De Oliveira | Determination of Silicon in Vegetable Oil and Biodiesel by High-Resolution Continuum Source Flame Atomic Absorption Spectrometry Using Sample Dilution with Xylene[END_REF][START_REF] Vakh | Determination of Silicon, Phosphorus, Iron and Aluminum in Biodiesel by Multicommutated Stepwise Injection Analysis with Classical Least Squares Method[END_REF] • Iron: Haemoglobin for animal feedstocks and poor storage conditions of vegetable feedstocks could be the main sources of iron contamination. Besides, iron species have been found to cause great damage in engines during their combustion. [START_REF] Vakh | Determination of Silicon, Phosphorus, Iron and Aluminum in Biodiesel by Multicommutated Stepwise Injection Analysis with Classical Least Squares Method[END_REF] • Magnesium, calcium, sodium and potassium: Their concentration in the biodiesel could vary depending on the type of feedstock employed. Together with K, the presence of Mg and Na could cause solid deposit in engines. [START_REF] Nowakowski | Uncatalysed and Potassium-Catalysed Pyrolysis of the Cell-Wall Constituents of Biomass and Their Model Compounds[END_REF]31 1.6. Bio Oils refining: Hydrodeoxygenation (HDO) Some renewable feedstocks, such as fast pyrolysis oil, can be burned directly in some static applications such as boilers, furnaces, engines and turbines. [START_REF] Bridgwater | Fast Pyrolysis Processes for Biomass[END_REF] However, as it has been mentioned before, the high content in water and oxygenated compounds reduces the pyrolysis oil's heating value. In addition, the low pH, instability and the presence of char and other impurities could cause solid deposits and corrosion on engines and pipes.

Thus, transforming the crude bio-oil into a hydrocarbon fuel with high heating value, stability and low content of impurities requires an upgrading process.

Solvent addition, vacuum distillation and filtration are some of the physical upgrading processes that have been reviewed by Mostafazadeh et al. [START_REF] Khosravanipour Mostafazadeh | A Review of Recent Research and Developments in Fast Pyrolysis and Bio-Oil Upgrading. Biomass Convers[END_REF] However, the catalytical upgrading processes of bio-oils possess greater importance since the greatest improvements are achieved with these processes.

Catalytical processes designed for petroleum refining have been known and improved throughout the years. As it has been mentioned before, raw pyrolysis oils have a much higher oxygen content that must be lowered to transform these raw materials into stable products with high commercial value, efficient heating and low corrosion rates that will

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -48-allow them being used as liquid transportation fuels with a high content of hydrocarbons.

Besides, due to the much lower concentration of other elements such as S, it is clear that applying the same catalytical upgrading methods as those for fossil feeds (containing less than 0.1% oxygen) is not efficient.

Hydrodeoxygenation is one of the most used pathways to upgrade bio-oils to meet these criteria. Other catalytical methods, such as Hydrodemetallization and Hydrodesulfurization, will be reviewed briefly in the publication enclosed within the Chapter 4 of this manuscript on their application to the conversion of crude oil atmospheric residues into transportation fuels.

Reactions

In this catalytical process, among all reactions that may occur for a given catalyst at certain conditions, the oxygen present in the bio-oil is removed as the water product of a hydrogenolysis reaction in the presence of a catalyst. Table 1 shows an overview of the different reactions generally occurring during HDO and their most common used acronyms. [START_REF] Dabros | Transportation Fuels from Biomass Fast Pyrolysis, Catalytic Hydrodeoxygenation, and Catalytic Fast Hydropyrolysis[END_REF] These reactions occur depending on the choice of the catalyst and its selectivity towards the different reactions they are involved in. the sulphur present in these feeds. However, H2S must be added continuously to keep the catalyst within its sulphide form during HDO. A co-processing hydrodeoxygenation and hydrodesulfurization has been reported from Sepulveda et al. [START_REF] Sepúlveda | Hydrodeoxygenation and Hydrodesulfurization Co-Processing over ReS 2 Supported Catalysts[END_REF] In this research work, the H2S resulting from the HDS process acts as a sulfiding agent and extends the lifetime of the catalyst.

• Oxides, such as Mo, W, Ni and V oxides among others, require lower H2 pressure. [START_REF] Nolte | Ex Situ Hydrodeoxygenation in Biomass Pyrolysis Using Molybdenum Oxide and Low Pressure Hydrogen[END_REF] • Reduced transition metals, such as Pt, Pd, Ru and Rh, which are commonly used in organometallic chemistry have higher activity in reactions such as hydrogenation (HYD) and hydrodeoxygenation (HDO). However, in contrast to sulphides, they are not tolerant of sulphur, whose presence within the feed could cause catalyst poisoning. [START_REF] Bartholomew | Mechanisms of Catalyst Deactivation[END_REF] 1.6.

Catalyst deactivation

The catalyst's loss of activity over time is a problem which might cost large amounts of money to the industry. Although catalyst deactivation cannot be avoided over time, this degradation can be slowed down, and catalysts' lives can be extended.

Catalyst deactivation could be caused by different factors. Chemical deactivation is the most common, but other mechanisms such as fouling, consisting of the deposition of species on the catalyst surface and pores, or thermal degradation, causing thermally induced loss of activity/selectivity. [START_REF] Bartholomew | Mechanisms of Catalyst Deactivation[END_REF] • Water: In pyrolysis oil HDO, water is the main catalyst poison towards a broad group of catalysts. [START_REF] Dabros | Transportation Fuels from Biomass Fast Pyrolysis, Catalytic Hydrodeoxygenation, and Catalytic Fast Hydropyrolysis[END_REF] Since it is formed during fast pyrolysis of biomass and HDO, it can saturate the active sites of both oxide and sulphide catalysts and also decrease some of the transition metal catalyst's activity. For instance, Laurent et al. [START_REF] Laurent | Influence of Water in the Deactivation of a Sulfided NiMoγ-Al2O3 Catalyst during Hydrodeoxygenation[END_REF] concluded that under hydrotreating conditions, water causes the loss of twothirds of the initial activity of a NiMoS catalyst in less than 60 hours. Hence the importance of physical upgrading of pyrolysis oil and water removal before catalyst upgrading.

• Carbon deposition: Polymerisation condensation reactions could occur at the catalytic surface, leading to a deposition of carbon that blocks the active sites and causes lower activity and selectivity rates. Even though the carbon formation depends on the feed composition, it might be controlled by adjusting the process conditions, such as temperature and H2 pressure. [START_REF] Dabros | Transportation Fuels from Biomass Fast Pyrolysis, Catalytic Hydrodeoxygenation, and Catalytic Fast Hydropyrolysis[END_REF] • Inorganic species:

o Chlorine: Organically bound chlorine has been found that causes a considerable loss of activity in catalysts such as Ni-MoS2/ZrO2. [START_REF] Mortensen | Deactivation of Ni-MoS2 by Bio-Oil Impurities during Hydrodeoxygenation of Phenol and Octanol[END_REF] .

o Potassium: For the same type of catalyst ( Ni-MoS2/ZrO2), its impregnation with KNO3 caused a decrease of the degree of oxygenation up to 85%

showing that this loss of activity could be caused by the occupation of some the vacancy sites of the catalyst by potassium. [START_REF] Mortensen | Deactivation of Ni-MoS2 by Bio-Oil Impurities during Hydrodeoxygenation of Phenol and Octanol[END_REF] o Phosphorus: This element is the cause of several problems during the catalytical process. It has been reported that the presence of phosphorus compounds, normally present as phospholipids within these matrices, cause the deactivation of the catalyst: the phospholipids are decomposed into phosphoric acid, that acts as an oligomerization/polimerization catalyst, promoting the deposition of high-molecular-weight oligomers on the catalyst surface. In addition, the presence phosphorus increased the concentration of oxygenated compounds (fatty acids and fatty esters) at the end of the catalytical process. 56

CHAPTER 2: ANALYTICAL METHODS

In this chapter, a short review of the analytical techniques that have been used during this three-year PhD is given. With the purpose of organizing the structure of this chapter, the analytical techniques have been divided into two subgroups: those concerning the separation of the analytes and those concerning the detection.

Chromatographic techniques.

Complex matrices such as bio-oils and their feedstocks that have been analysed throughout this project require a high-efficiency separation of the species that form them.

With the purpose of achieving this high-efficiency separation, several techniques have been used throughout this three-year project.

Chromatography is a separation method used for the first time in the early 1900s by Mikhail Tswett, who used to separate vegetal pigments such as chlorophyll and xanthophyll by using a glass column filled with fine particles of CaCO3. Within the column coloured bands corresponding to the different components of these pigments appeared, hence the name of "chromatography".

Since then, the use of chromatography has grown exponentially until nowadays as it has become one of the standard methods for chemical separation. [START_REF] Skoog | Instrumental Analysis Principles[END_REF]

Principles of Chromatography

In Tswett's set up, the vegetal pigments were dissolved in a liquid that carried them through the column while they were interacting with the fine particles that defined the column filling. The carrying liquid is called "mobile phase" while the material within the column has been called "stationary phase". [START_REF] Skoog | Instrumental Analysis Principles[END_REF] The different interactions between the molecules of a mixture that has been dissolved in the mobile phase and the molecules of both mobile and stationary phases will cause a retention of certain molecules with higher affinity versus those molecules that have either less or no interactions at all. Thus, molecules that have been retained due to their higher affinity will be eluted later.

Since the separation is a result of the equilibrium between two immiscible phases, there exists a relation between the amount of solute in the stationary phase (s) and the amount of solute in the mobile phase (s). Thus, for a given solute, the retention factor (k) has been defined as the quantity of solute in the stationary phase divided by the quantity of solute in the mobile phase, and since the amount of solute in a solution is equal to its concentration (C) times the volume of solution (V), the retention factor can be expressed as:

𝑘 = 𝐶 𝑠 𝑉 𝑠 𝐶 𝑚 𝑉 𝑚 Equation 1
The retention time (tR) is then defined for a given solute as the time passed from the injection of the sample until the detection of this compound. By rearranging Equation 1, the retention factor (k) can be experimentally determined by the following equation:

𝑘 = 𝑡 𝑅 -𝑡 0 𝑡 0 Equation 2
being t0 defined as the column "dead-time" which consists of the retention time of the solvent peak which does not interact with the stationary phase. Equation 2 allows the experimental calculation of the retention factor for each peak in a chromatogram, being an important parameter that evaluates the performance of the separation of a certain solute.

Another important term that evaluates the separation of two chromatographic peaks is the chromatographic resolution (RS) which can be expressed as:

𝑅 𝑠 = √𝑁 4 (𝛼 -1) ( 𝑘 1 + 𝑘 ) Equation 3
Being 𝛼 a term expressing the selectivity of the column and N the column plate number, which is a concept that measures the efficiency of the column separation, consisting of the ability of a column to provide narrow peaks for a certain solute. The larger the value of N, the better is the separation and the narrower are the peaks obtained.

The value of N is dependent on the width of a peak (W) and its retention time (tR). However, the width of a peak is affected by multiple factors that will determine the quality of separation. By understanding these factors, it will be possible to select the optimal chromatographic conditions and columns to obtain the best results possible. [START_REF] Skoog | Instrumental Analysis Principles[END_REF][START_REF] Snyder | Introduction to Modern Liquid Chromatography[END_REF] • Eddy diffusion (A) is one of the factors that contribute to peak broadening. Since the stationary phase within a chromatographic column is not perfectly ordered, the molecules of the mobile phase will follow different paths before reaching the end of the column. • Longitudinal diffusion (B) of the analytes along the column is the second factor contributing to the peak broadening and it is inversely proportional to the mobile phase linear velocity. Since some of the molecules move faster than the others within the mobile phase, the effect will be higher the lower the flow is.

• Mobile phase mass transfer (C) is the last factor contributing to peak broadening.

During elution, analyte molecules are interacting with the stationary phase in an equilibrium. Since this process is not equal for all the analyte molecules as it is dependent on the particle size and porosity and viscosity and velocity of the flow among other factors, some analyses will spend longer time into a particle pore than others. During this time, other molecules will have moved further along the column and this fact will be reflected in the chromatogram as a broader peak.

If these three factors are put together and considering that the height equivalent to a theoretical plate (H) is the number of theoretical plates (N) divided between the length of the column (L), the following equation is obtained:

𝐻 = 𝐴 + 𝐵 𝑢 + 𝐶𝑢 Equation 4
where A, B and C are the three factors previously described and u the mobile phase velocity. [START_REF] Skoog | Instrumental Analysis Principles[END_REF][START_REF] Snyder | Introduction to Modern Liquid Chromatography[END_REF] Equation 4 is known as the "Van Deemter Equation" and if the height equivalent to a theoretical plate (H) is plotted versus the mobile phase velocity (u) the graph shown in Figure 14 is obtained: In the graph shown in Figure 14, the dotted line represents the variation of the height equivalent to a theoretical plate (H) versus the mobile phase velocity (u) including the three factors causing the peak broadening. It is possible to observe how for a certain mobile phase velocity there is a minimum of theoretical plate height. This velocity will be the optimal flow rate for a chromatographic separation. If the flow is lower, the longitudinal diffusion term (B/u) will cause an increase of H, leading to a worse separation efficacy and loss of resolution. The same would happen if the flow is higher: the mobile phase mass transfer (C) term will contribute to the peak broadening.

This relation between both terms H and u varies significantly among the different chromatographic methods. A general classification of these methods based on the physical state of the mobile phase is established:

• Gas Chromatography (GC) is a separation technique where the components of a vaporised sample carried by a gaseous mobile phase interact with the solid stationary phase of a column. However, in order to vaporise the analytes, they are heated in an oven at high temperatures. Excessive heating rates would make it unsuitable for molecules with a high molecular weight such as some of the samples analysed during this PhD.

• Liquid Chromatography (LC) is a separation technique where the components of a sample are carried out by a liquid mobile phase and interact with a liquid or solid stationary phase within a column or a plate. When column liquid chromatography separations are carried out, the columns are normally packed with quite small stationary phase particles (2 -10 µm), which increases the efficacy due to a decrease of the Eddy diffusion factor (A). However, the size of the stationary phase particles requires high pressures to achieve the elution of the mobile phase. This chromatography method of separation with high pressure is called as HPLC (High-Performance Liquid Chromatography).

Depending on the physical properties of the mobile phase in HPLC such as polarity and molecular weight, Liquid Chromatography can be further divided. The different types of HPLC are depicted in Figure 15. Applications of HPLC57. Gel Permeation Chromatography is one of the types of HPLC used in this project and its principles will be detailed in this chapter. As mentioned before, the A term (Eddy diffusion) is reduced due to the packing within the columns. Besides, the longitudinal diffusion term (B/u) is not significative in HPLC unless the mobile phase flow rates are extremely low. Thus, the optimal flow rate is slower than in other separations such as GC, and the van Deemter plot would resemble as it is depicted in Figure 16, achieving much higher efficiency separations with higher resolutions. [START_REF] Skoog | Instrumental Analysis Principles[END_REF][START_REF] Snyder | Introduction to Modern Liquid Chromatography[END_REF] Figure 16. Van Deemter plot for HPLC.

• Supercritical Fluid Chromatography (SFC) is a separation technique where the mobile phase is composed mainly of supercritical carbon dioxide and other coeluents such as methanol or ethanol among others. One of the advantages of this separation method is that it combines some of the advantages of both Gas Chromatography and Liquid Chromatography.

Gel Permeation Chromatography (GPC)

Size-exclusion chromatography (SEC), also called Gel Permeation Chromatography (GPC)

when an organic solvent is used as mobile phase, is one of the LC applications for the separation of molecules according to their hydrodynamic volumes. [START_REF] Striegel | Modern Size-Exclusion Liquid Chromatography[END_REF] Figure 17. GPC separation mechanism. [START_REF] Held | Tricks GPC/ SEC: What Are the Differences Between GPC, SEC, and GFC, and How Do You Get Started with the Technique?[END_REF] To achieve an effective separation, GPC column packings consist of small polymer particles containing pores (~ 5 μm) where the analyte molecules can be trapped and removed momentarily from the mobile phase, causing, therefore, retention. The residence time within the particles varies with the size of pore and the hydrodynamic volume of the molecule as it is described in Figure 17. Thus, molecules with larger diameters than the size of the pores are excluded and do not suffer retention and will be eluted together at the exclusion volume of the system whereas the molecules whose diameters are smaller than the pore size will penetrate within the pores. It is important to point out that neither chemical nor physical interactions take place between the analytes and the stationary phase. Hence, theoretically, there is an upper limit to retention time: no analytes are retained longer than the column total permeation time. Figure 18 explains visually the structure of a GPC chromatogram and it is possible to observe three different regions: [START_REF] Striegel | Modern Size-Exclusion Liquid Chromatography[END_REF] • The interstitial volume consists of the time that takes the mobile phase to reach the detector. If the mobile phase contained molecules whose size is much bigger than the pore diameter (exclusion limit), a peak would be observed at the end of this region.

• Pore volume consists of the region where the analytes reach the detector, corresponding the first peaks to the bigger size molecules and the opposite for the small peaks.

• Total permeation volume is the time when no more peaks are expected theoretically, as all the solute molecules have penetrated the pores completely, since no

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -62-interactions between the analytes and the mobile phase are expected. When the molecules with a significative smaller size in respect to the pore size are eluted, since they are very small, they elute all together in a single band labelled D.

With the purpose of understanding why the peaks are wider in this type of chromatography, it is necessary to pay attention to Equation 4 again. In gel permeation chromatography, the resolution of the peaks is strongly conditioned by the peak broadening, and this factor is controlled mainly by the mass transfer term (C), since the longitudinal effect (B) is generally insignificant: analyte molecules tend to have high hydrodynamic volumes and, therefore, very small diffusion coefficients. [START_REF] Striegel | Modern Size-Exclusion Liquid Chromatography[END_REF] However, when the analyte molecules have smaller hydrodynamic volume, a theory based on the coupling concept of both eddy and lateral diffusion terms has been formulated. This theory establishes that the molecules do not only follow their path within the column, but they can also travel between different paths through the column packing columns. This coupling results in a reduced band broadening respect to eddy diffusion term (A) by itself. [START_REF] Striegel | Modern Size-Exclusion Liquid Chromatography[END_REF] Other factors such as the column conditions, kinetic factors and experimental parameters such as temperature, solvent viscosity and sample concentration among others contribute as well to the peak broadening in gel permeation chromatography. [START_REF] Striegel | Modern Size-Exclusion Liquid Chromatography[END_REF] 2.1.3. Supercritical Fluid Chromatography (SFC) Supercritical fluid chromatography, as it has been introduced above, is a hybrid of liquid chromatography (LC) and gas chromatography (GC), since the mobile phase, in this case, is a fluid in supercritical conditions.

Carbon dioxide (CO2) is one of the most common substances used as a mobile phase in SFC since it consists of an abundant and inexpensive gas as well as non-toxic.

When both the temperature and pressure of a substance such as CO2 are above its critical point (Figure 19), this substance becomes a supercritical fluid. 57 This new physical state provides with new properties to the CO2. One of the main important ones is the behaviour as a gas, but with a density more likely to a liquid. This property is related to its ability to dissolve large non-volatile molecules such as PAHs and large alkanes among others. Besides, in order to recover these solutes, the only step required is the evaporation of the CO2 under laboratory conditions. [START_REF] Skoog | Instrumental Analysis Principles[END_REF] The state-of-the-art instrumentation used in SFC is quite similar to the instruments used for HPLC, being some of the most important differences the restrictors used to keep the pressure conditions within the chromatographic system and the oven where the columns are subjected to temperature control. These two elements are essential to keep the supercritical conditions.

Whereas CO2 is the main component of the mobile phase since it is an excellent solvent for a broad variety of nonpolar organic molecules, organic modifiers can be added to the mobile phase flow to improve the solubility of some molecules and avoid the precipitation of the analytes within the system. Modifiers such as methanol or ethanol among others are commonly used and their composition within the mobile phase can sometimes reach 100% of the flow, always depending on the stationary phases that are being used and the target analytes. 63 -64-chromatographic system and passes through the column/s that have been heated

previously within the column manager. Once the mobile phase exits the columns, it is sent to a detector, which may be a photodiode array, incorporated in numerous commercial instruments. Another detection method that is commonly used for SFC is the Mass Spectrometry. In order to avoid the precipitation of the analytes through the tubing towards the mass spectrometer, a make-up solvent can be pumped within the mobile phase before injection. In SFC there exists a huge variety of stationary phases, ranging from very polar (silica gel) to C18, being originally all of them designed for HPLC columns. The length and particle size of these columns may vary depending on the purposes of each analysis.

Regarding the performance characteristics of this type of chromatography, the advantages of using SFC have been studied by D. Gere who compared the Van Deemter plots of two separations carried out with a 5-µm octadecyl (C18) with both, SFC and LC elution. 65 [START_REF] Skoog | Instrumental Analysis Principles[END_REF] The plot of both curves can be observed in Figure 21 showing a significantly better performance for the analyses carried out with supercritical fluids: with SFC, at a mobile phase velocity of 0.6 cm/s (four times the HPLC's linear velocity) the column yields a plate height equivalent to three times less large than for HPLC. This fact can be reflected in a reduction of analyses while keeping the separation efficiency for SFC in respect to HPLC.

Detection

In the previous sections, an explanation about the methods used to perform the molecular separations which lead to the speciation analysis has been done. In this section, the description of different methods used for the obtention of the data will be carried out as well as their combination with the methods already introduced.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a method based on the detection of the isotopes present in a sample based on their mass/charge (m/z) and their

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -66-corresponding measured intensities. Before reaching the detector, the sample has been atomised and ionised within an argon plasma that achieves temperatures as high as 10,000 K.

Figure 22 presents the main components of an ICP-MS instrument and their role will be described below:

Sample introduction -Total consumption micronebulisers

The sample introduction is one of the most critical processes during ICP-MS analyses.

When the sample is a liquid, a nebuliser has to be used for these purposes. This nebuliser creates an aerosol where the sample is finely divided into small drops that are carried by a gas flow towards the plasma, where the sample is atomized and ionised.

The most common means of sample introduction in ICP techniques are the concentric glass nebulisers, whose fundamental parts are presented in Figure 23. At the tip of these nebulisers, a high-speed gas causes the aspiration of the liquid flow within the capillary that this gas surrounds by the Bernoulli effect. The aerosol is generated as the gas breaks up the liquid flow towards the plasma into fine droplets of different sizes. [START_REF] Skoog | Development of a Sheathless Interface between Reversed-Phase Capillary HPLC and ICPMS via a Microflow Total Consumption Nebulizer for Selenopeptide Mapping[END_REF] Generally, a spray chamber is used to filter the sample aerosol and to allow only those droplets with an appropriate size to reach the plasma. Since all the samples within this work are liquids that must be dissolved in organic solvents, they have been dissolved in THF. The introduction of organic solvents into ICP-MS is a challenging task since an excess of organic solvent can cause the extinction of the plasma, the formation of carbon deposits on the cone surfaces and an increase of the isotopic interferences during the ICP-MS analyses. Thus, the limitation of the amount of solvent that arrives to the plasma was achieved by using a total consumption microflow nebuliser that allows very low-flow ICP-MS sample injections.

Schaumloffel et al. 67 developed a sheathless interface equipped with a total consumption nebuliser that operated at flow rates in the range 0.5 -7.5 μL min -1 to hyphenate HPLC and ICP-MS. This nebuliser was designed from a previous micro-nebuliser that was designed for coupling capillary electrophoresis to ICP-MS. [START_REF] Pröfrock | Development and Characterisation of a New Interface for Coupling Capillary LC with Collision-Cell ICP-MS and Its Application for Phosphorylation Profiling of Tryptic Protein Digests[END_REF] The use of a spray chamber was no longer required and very low peak broadenings and low detection limits where achieved. With this total consumption nebuliser model, they managed to develop a method for the analysis of small amounts of uranium and plutonium in contaminated samples and another method for the analysis of selenium in protein tryptic digests. [START_REF] Schaumlöffel | Ultratrace Determination of Uranium and Plutonium by Nano-Volume Flow Injection Double-Focusing Sector Field Inductively Coupled Plasma Mass Spectrometry (NFI-ICP-SFMS)[END_REF][START_REF] Giusti | Interfacing Reversed-Phase NanoHPLC with ICP-MS and on-Line Isotope Dilution Analysis for the Accurate Quantification of Selenium-Containing Peptides in Protein Tryptic Digests[END_REF] of micro-and nano-LC with ICP-MS. [START_REF] Bustos | The Role of ICP-MS in Separation Science[END_REF] Giusti et al. [START_REF] Giusti | UFlow-Injection-ICP Collision Cell MS Determination of Molybdenum, Nickel and Vanadium in Petroleum Samples Using a Modified Total Consumption Micronebulizer[END_REF] succeeded in increasing the total consumption nebuliser capillary causing the elimination of clogging, pressure instability and memory effects that the previous version possessed. With this new version, an insight of the trace element composition in crude oil samples was accomplish in numerous works. [START_REF] Desprez | Study of the Size Distribution of Sulfur, Vanadium, and Nickel Compounds in Four Crude Oils and Their Distillation Cuts by Gel Permeation Chromatography Inductively Coupled Plasma High-Resolution Mass Spectrometry[END_REF][START_REF] Vargas | Development of a Chromatographic Methodology for the Separation and Quantification of V, Ni and S Compounds in Petroleum Products[END_REF][START_REF] Caumette | Fractionation and Speciation of Nickel and Vanadium in Crude Oils by Size Exclusion Chromatography-ICP MS and Normal Phase HPLC-ICP MS[END_REF][76] In the Chapter 3 of this PhD

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -68-thesis, the hyphenation of HPLC and ICP-MS with this total consumption nebuliser is discussed.

Total consumption nebulisers are currently used for numerous HPLC-ICP-MS applications such as the detection of As species in urine and Hg and Pb species in fish liver, among others. 77 In addition, new designs are still being carried out with respect to this type of nebulisers to improve the quality of the analysis of numerous samples via ICP-MS such as the development of a low-cost demountable nanoflow nebuliser to couple nano-HPLC with ICP-MS. 78 In this work, the development and optimisation of a 3D-printed total consumption nebuliser, based on the one that was developed by Giusti et al. [START_REF] Giusti | UFlow-Injection-ICP Collision Cell MS Determination of Molybdenum, Nickel and Vanadium in Petroleum Samples Using a Modified Total Consumption Micronebulizer[END_REF] , are described for the introduction of bio-oil samples dissolved in THF to reduce the interferences provided by the silica capillary commonly used in crude oil speciation analyses.

Another method of sample introduction for ICP-MS might be Electrothermal Vaporisation, which will be deeply described in Chapter 5.

Inductively Coupled Plasma (ICP)

A very intense and white plasma formed by ions and electrons is generated within a torch consisting of three concentric quartz tubes through which an argon gas flow streams to shape the plasma and cool down the quartz. When the sample as an aerosol reaches the plasma, its molecules are desolvated, atomised and ionised.

Although some elements can be ionized to higher charged states, the majority of the elements of the sample are ionized to singly charged positive ions with an ionization efficiency close to 100%. 79 Once the ionisation has been produced in the plasma, the ions pass through an interface based on two platinum cones (sample cone and skimmer cone) that separates the mass analyser, which is working in a very high vacuum, from the plasma, which works at atmospheric conditions. Within this interface, the positive ions are separated from electrons and molecular species by a negative voltage. Then, they are accelerated and focused by magnetic ion lenses onto the entrance of the mass analyser. [START_REF] Skoog | Development of a Sheathless Interface between Reversed-Phase Capillary HPLC and ICPMS via a Microflow Total Consumption Nebulizer for Selenopeptide Mapping[END_REF]

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Mass analysers in ICP-MS

The function of a mass analyser is to separate the ions produced in the plasma according to their mass-to-charge ratio (m/z). There are several types of mass analysers providing different properties, such as different mass ranges, analysis speed, transmission, mass accuracy and resolution.

Among the wide variety of mass analysers used in mass spectrometry, in this work two of them will be described: quadrupole and magnetic sector.

Quadrupole

The quadrupole is a compact, less expensive and rugged instrument that allows the transmission of ions with certain m/z values, acting as a mass filter. As it is shown in Figure 24 a quadrupole is formed by four parallel cylindrical rods acting as electrodes, being each opposite pair of rods connected electrically in such a way that one pair is connected to the positive pole of a variable dc source and the other pair to the negative one. At the same time, 180° out of phase ac voltages are applied to each pair of rods. This combination of ac and dc voltages will allow only those ions having a certain m/z value to pass through the quadrupole, whereas the rest of the ions will strike the rods and will be converted to neutral molecules.

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -70-A quadrupole can operate in two modes: Scan mode, in which the voltages increase simultaneously with time, and m/z ratios varying from 0 up to 4000 can be obtained within a few milliseconds; and Selected Ion Monitoring (SIM), in which only the ions with the m/z values within the specified range pass through the quadrupole.

One of the quadrupole properties is its high efficiency to focus the trajectory of ions.

However, the mass range is quite narrow, and ions with an m/z value higher than 4000 are poorly focused. Thus, based on the quadrupole principle, ion guides such as hexapoles and octapoles have been designed by adding one and two extra pair of rods respectively to a quadrupole, increasing, therefore, the mass range for simultaneous transmission of ions.

Double Focusing Magnetic Sector mass analysers

Whereas the quadrupole uses a combination of both alternating and direct potentials to separate the different ions originated in the ICP, the magnetic sector mass analyser applies a magnetic field that deflects the trajectories of the ions as they pass through.

At a given magnetic field intensity (B), two ions with the same mass (m), charge (q) and velocity will be deflected following a circular trajectory with a characteristic radius (r) defined by the following equation,

𝑟 = √2𝑚𝐸 𝑘 𝑞𝐵 Equation 5
being Ek their kinetic energy, which consists of the only factor affecting the dispersion of both ions. In order to avoid the dispersion of two ions with the same m/z value, an electrostatic field is applied employing an electric sector analyser, which focuses ions of the same mass onto the same point in space, which in this case it consists of a slit towards the detector. This double-focusing configuration, by coupling both magnetic and electrostatic fields and whose basis are schematically depicted in Figure 25, provides with a high mass resolution potential allowing to solve a wide variety of polyatomic interferences.

Detector

Once the ions have been selected as a function of their m/z values by either the quadrupole or the double magnetic sector analysers, they reach the detector where they are transformed into a signal.

There exist several types of detectors, but in this section, only two types will be described:

• Faraday cup: As it is shown in Figure 26, it consists of a cylinder with a small orifice and connected to the ground via a resistor. When the ions reach the inner part of the cylinder, they are neutralised by either donating or accepting electrons from the cylinder walls, becoming neutralised. This transfer of electrons generates a signal, that can be detected. However, despite their high precision and robustness, this type of detectors has very low sensitivity and slow response time.

• Electron multipliers: This device is composed of a series of dynodes that collect and convert positive ions into an electrical signal. Figure 27 shows a schematic diagram of an electron multiplier. When the ion beam strikes the first dynode, it generates a burst of electrons that are attracted to the next dynode, hitting it, being attracted to the following dynode and so forth, due to the decreasing negative potentials where these dynodes are held in. The cascade of electrons created along these dynodes will provide an electric current high enough that provides with a current gain of up to 10 7 . Compared to the Faraday Cup, this detector provides with faster response and higher sensitivity, and they are ideal to be placed behind the exit slit of a magnetic

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -73-sector mass spectrometer since the ions reaching the detector possess enough kinetic energy to eject electrons from the first dynode of the device. [START_REF] Skoog | Development of a Sheathless Interface between Reversed-Phase Capillary HPLC and ICPMS via a Microflow Total Consumption Nebulizer for Selenopeptide Mapping[END_REF] Some instruments, such as the Thermo Element XR used in this work, use the Electron Multiplier detector, which normally provides a linear response in a range over 10 9 , and then, by combining it with a Faraday Cup, the linear response is increased up to 10 12 .

Inductively coupled plasma -optical emission spectroscopy (ICP-OES).

Inductively coupled plasma -optical emission spectrometry is an analytical technique used for the detection of chemical elements based on their corresponding emission wavelengths generated within a plasma. [START_REF] Skoog | Development of a Sheathless Interface between Reversed-Phase Capillary HPLC and ICPMS via a Microflow Total Consumption Nebulizer for Selenopeptide Mapping[END_REF] The majority of the main components of an ICP-OES are similar to the components of an ICP-MS (Figure 28): The sample introduction and the excitation of the elements present in a sample consist of the same principle: Sample introduction (Section 0) and Inductively Coupled Plasma (Section 0). However, since this method is based on the detection of emission wavelengths and not ions, the mass analyser is replaced by an optical device that transmits a desired wavelength from the plasma, were several wavelengths are emitted from its core.

In ICP-OES, the wavelength originated in the plasma can be measured radially, perpendicular to its axis or axially (being this arrangement used in ICP-MS). In Figure 29, the main components of an ICP-OES with axial configuration are described.

The radiation generated in the plasma is focused through lenses towards a slit located before the monochromator, or, in some advanced instruments, polychromator, whose functions consist of separating the radiation into its different wavelengths in order to be able to detect and quantify the ones corresponding to the elements that are measured.

Separating these wavelengths can be carried out simultaneously with a polychromator whose main component is known as a Rowland circle whose grating surface (where the radiation wavelengths disperse) is located along a circumference in such a way that shapes a concave diffraction grating. 66 When the radiation hits this diffraction grating, numerous wavelengths will be directed to the detector, located on the opposite side of the circumference, where this radiation will be transformed into an electric signal.

The most common detector in optical emission spectrometry is the photomultiplier tube (PMT) whose fundament resembles the electron multipliers used in mass spectrometry, and whose components are showed in Figure 30. This plasma source spectroscopy provides with the lowest detection limits among all the emission spectroscopy techniques. Besides, the lack of mass interferences such as the ones occurring in ICP-MS that do not allow the detection of other ions with the same mass, make this detection method ideal for the speciation analysis of several elements.

CHAPTER 3: NOVEL ANALYTICAL TOOLS FOR THE SPECIATION ANALYSIS OF BIO-OILS: A 3D-PRINTED TOTAL CONSUMPTION NEBULISER

In this chapter, a novel alternative of a total consumption nebuliser, built through the use of the 3D-printing technology, is presented and validated in order to fulfil one of the main sub-objectives of this thesis (O1a). This alternative nebuliser, which does not have a builtin fused silica capillary that might cause interferences and permanent retention for the analysis of certain species (Si, K, etc.), can be produced in a very short time. The optimisation and validation steps of this new total consumption nebuliser are detailed in the Article 1, which has been attached to this chapter.

Methodology

The coupling of two of the techniques explained in Chapter 2, GPC and ICP-MS, which also allows carrying out a simultaneous analysis between GPC-ICP-MS and GPC-UV/Vis, is described in Figure 31 and consisted of one of the most used instrumental setups throughout this thesis project.

UV-VIS Detector

A constant flow of 1 mL min -1 of THF stabilised with BHT is delivered from the HPLC pump and it is used as mobile phase for the chromatographic separation, which is carried out using three Waters styrene-divinylbenzene GPC columns: an HR4 column (particle size of 5 μm with an exclusion limit of 600,000 Da of polystyrene equivalent), an HR2 column (particle size of 5 μm with an exclusion limit of 20,000 Da) and an HR0.5 column (particle size of 5 μm and an exclusion limit of 1,000 Da of polystyrene equivalent). Besides, to prevent any damages to these columns, a Styragel guard column (4.6 mm i.d. and 30 mm length) was also fitted between the columns and the pumping system.

Once the separation has been completed, a splitter is used to deliver a low amount of mobile phase towards the ICP, since an excess of THF might extinguish the plasma. With a split rate of approximately 20:1, most of the mobile phase is splitted towards a Dionex UV-Vis detector which can record up to six different wavelengths.

The splitted low flow is delivered towards the plasma within a fused-silica capillary belonging to a modified DS-5 microflow total consumption nebulizer (CETAC, Omaha, NE), described in Figure 32 and mounted with a laboratory-made single pass jacketed glass spray chamber heated up to 60 °C by a water/glycol mixture using an RTE-111 thermostat (Neslab, Thermo Fisher Scientific) to provide energy to the organic matrix with heat in order to increase the sensitivity of the plasma ionization process. [START_REF] Giusti | UFlow-Injection-ICP Collision Cell MS Determination of Molybdenum, Nickel and Vanadium in Petroleum Samples Using a Modified Total Consumption Micronebulizer[END_REF]76

3D-printed total consumption microflow nebuliser development for trace element analysis in organic matrices via

inductively coupled plasma mass spectrometry

Discussion and perspectives

In this work, the use of the 3D-printing has proven its usefulness to produce high-quality laboratory consumables and a new total consumption nebuliser has been validated

through the total analysis of several trace elements in a reference standard provided by NIST. Concerning the speciation analysis results, several differences can be observed and some suggestions for future analyses with this consumable would be useful.

In the Figure 6 of Article 1, two chromatograms showing the size distribution of an internal oil reference are showed. Each of the chromatograms was obtained by using a different total consumption nebuliser. A difference on the areas that correspond to the retention times between 24 and 30 minutes can be observed: a decrease in the area of these peaks with respect to the high-molecular weight area (20-24 min) is observed for the chromatogram obtained with the modified DS-5 nebuliser. Although these data agree with the hypothesis of having a permanent analyte retention within the DS-5 fused silica capillary, further experiments should be carried out in future analyses to contrast this hypothesis. Collecting the eluted fractions and measuring their total concentration of V and Ni could be one of the approaches. However, further experiments could be used, such as a washing-up of the silica capillary with acetonitrile in order to release any potential species that might be trapped within its structure and repeat the experiments afterwards to check whether the same results are obtained. By carrying out these experiments, the hypothesis that has been considered during this work could be contrasted and validated.

Other future analysis should also focus on the speciation analysis of Si. The high presence of Si-containing compounds in the previous total consumption nebulisers avoided the obtention of useful Si speciation data. However, with the PEEK tubing that links this new total consumption nebuliser with the HPLC column outlet, no interferences are expected and an insight on the speciation of silicon in complex samples such as bio-oils and renewable feedstocks would be obtained.

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CHAPTER 4: NOVEL SFC-ICP-MS HYPHENATION FOR THE SPECIATION ANALYSIS OF COMPLEX MATRICES

Previous SFC-ICP-MS interfaces

The coupling of Supercritical Fluid Chromatography with Inductively Coupled Plasma Mass Spectrometry possesses the potential of providing a huge number of theoretical plates to the separation of analytes from numerous families combined with the selectivity of ICP-MS detectors.

Coupling SFC and ICP-MS might be considered as an easy task, since the outlet of the SFC, once it is out of the restrictor, is in a gas state. However, when the mobile phase, which is held at high pressures, passes through the restrictor end, due to the free expansion of the gas that occurs, a significant decrease of temperature on this gas can be appreciated.

This effect is known as Thompson-Joule effect and can cause numerous issues when trying to couple the supercritical fluid chromatographs other detection techniques since the temperatures can reach very low values. 86 Hyphenation of SFC with other techniques has been widely researched, but in this case, as one of the main objectives of this thesis, this section will focus on the SFC-ICP-MS hyphenation.

In the 1990s, Carey et al. 87 developed an interface capable of coupling both instruments.

The interface that they developed consisted of a tee union where heated argon was introduced as the carrier gas of the plasma. However, the end of the restrictor was located near the end of the torch's injector and the gas-free expansion was produced a few centimetres from the plasma source. Thus, the role of the heated argon was to compensate this decrease of temperatures, so that the outlet of the restrictor would not have such low temperatures, and the plasma energy can be employed on ionising the components of the mobile phase. Other interface prototypes were also used for achieving this hyphenation, following the same principle of adding heat to the outlet of the restrictor to assure ionisation efficiency and avoiding the plasma switching off. 88 However, all these interfaces were designed for capillary SFC, which is carried out in gas chromatography ovens and it is not adapted to the modern instrumentation, detailed in the Chapter 2.

Modern SFC, also known as convergence chromatography, does not only use one single carrier gas. The combination of different solvents and gases allows setting gradient programs that improve the chromatographic separation and increases the number of theoretical plates. However, adding an extra solvent when using these types of interface would cause the immediate extinction of the plasma, since an aerosol would probably be formed at the outlet of the restrictor and the droplets, of any size, would be injected directly into the plasma.

Thus, as the main objective of this thesis (O1b), an interface that allows the coupling of SFC and ICP-MS has been developed. The optimisation procedure and the results obtained are presented in the following article (Article 3), published in the RSC Journal of Analytical Atomic Spectrometry in October 2020.

Hyphenating supercritical fluid chromatographyinductively coupled plasma mass spectrometry: Interface development and analysis of unconventional oils CHAPTER 5. ETV-ICP-OES FOR THE ANALYSIS OF BIO-

OILS

In this chapter, the development of a methodology that allows both the quantitative and speciation analysis in complex samples is detailed. Unfortunately, the research work on this topic could not be finished in time and some hypotheses have not been tested. However, the continuation of this work will be carried out in the near future within the scope of other projects and further analysis will be done by following the methodology explained within this chapter, providing with a better understanding of the behaviour and the composition of some of trace element species present in complex samples, such as the feedstocks that were analysed in this three-year project. Nevertheless, in this work, the basis of a novel methodology that hyphenates ETV and ICP-OES have been stablished, being able to fulfil one of the main sub-objectives of this PhD thesis (O1c).

Introduction

As it was introduced in Chapter 1, the common presence of trace elements in renewable feedstocks becomes an undesirable factor that can affect negatively to the yield of their conversion into hydrocarbon fuels and their quality and environmental properties.

Numerous techniques can determine the presence of these undesirable trace elements in complex samples such as crude oil and biomass products. One of these techniques, ICP-MS, has been used throughout this PhD providing high-quality results. However, other methodologies might be considered to obtain a fingerprint of these trace elements species, like Electrothermal Vaporisation -Inductively Coupled Plasma -Optical Emission Spectroscopy.

With this methodology, quantifying and determining the fingerprint of trace elements based on their pyrolysis or boiling temperatures was possible by using the hyphenation of an Electrothermal Vaporiser Spectral Systems 4000-c with an ICP-OES Spectro Arcos.

An electrothermal vaporiser is an evaporator that can be used as a sample introduction technique for a plasma source spectrometry. As it is shown in Figure 35,Figure 35.

Experimental setup of an electrothermal vaporiser (Adapted from Asfaw et al. 89 ). the vaporiser consists of an oven where a graphite boat containing the sample, which can be a liquid or a solid, is placed. The oven is electrically heated to the desired temperatures, normally by following a heating program, and the evaporated sample is carried by an inert gas such as argon that flows towards a detector such as ICP-OES, which is the detection method used in this work. A reaction gas is used to avoid the formation of carbides from elements such as Cr, Ti and V among others. 90 One of the most common ETV reaction gases is CCl2F2, also known as Freon R12, which improves the volatilization efficiency of the inorganic elements by reacting and forming volatile halides. However, due to its damaging effect on the ozone layer, its manufacture and commercialisation have been banned from most of the developed countries, including the US and Europe. 91,[START_REF]Refrigerant Transition & Environmental Impacts[END_REF] Hence the need to test and to find a new reaction gas that allows the volatilisation of all the inorganic species in samples such as bio-oils. In this work, SF6 gas has been used alternatively, since it is cheaper and more environmentally friendly.

One of the main advantages of using this technique is the nonrequirement of sample preparation steps and the low amount of sample required to carry out the analysis, which normally is less than 1 mg. Besides, it consists of a less time-consuming technique with shorter times of analysis (less than 2 minutes) with no dilution needed, which improves significantly the sensitivity, are some of the other advantages that make this technique ideal for the aim this project.

The mode of operation of this instrument can be summarized in four steps:

I.

Deposition of the sample:

A liquid or solid sample is deposited in a graphite boat, which is introduced within the ETV graphite furnace.

II.

Heating: A temperature programme is designed with temperatures that allow the first step of desolvation of the sample, followed by the ashing of the matrix and finally the vaporisation of the analytes.

III.

Transport towards the detector: Via a transfer tube which normally is made of Teflon, vaporised analytes are transferred towards a detector, which normally consists of a plasma source.

IV. Recording of the transient signal in function of time.

The electrothermal vaporised that has been used in this work consists of a state-of-the-art model. Spectral Systems, which is the provider of this instrument, has been specialised in ETV instrumentation for more than 16 years. [START_REF]Spectral Systems. ETV[END_REF] The use of ETV-ICP-OES has been mainly

focused on the determination of several trace elements in numerous types of samples and rarely used for speciation analysis purposes: The most recent articles that have been published on ETV-ICP-OES analyses show the high efficiency of this methodology for fast analysis in solid samples: Vogt et al. [START_REF] Vogt | Quantitative Multi-Element Analysis of Argonne Premium Coal Samples by ETV-ICP OES -A Highly Efficient Direct Analytical Technique for Inorganics in Coal[END_REF] quantified Al, Ba, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, S, Si, Ti, V and Zn by ETV-ICP-OES in Argonne Premium Coal samples; Santos et al. [START_REF] Santos | Multielement Determination in Medicinal Plants Using Electrothermal Vaporization Coupled to ICP OES[END_REF] determined As, Cd, Cr, Cu, Mn, Ni, Pb and Zn in medicinal plants and Silva et al. [START_REF] Silva | Feasibility of Rare Earth Element Determination in Low Concentration in Crude Oil: Direct Sampling Electrothermal Vaporization-Inductively Coupled Plasma Mass Spectrometry[END_REF] determined the concentration of 16 rare elements in crude oil. Bauer et al. [START_REF] Bauer | Direct Determination of Sulfur Species in Coals from the Argonne Premium Sample Program by Solid Sampling Electrothermal Vaporization Inductively Coupled Plasma Optical Emission Spectrometry[END_REF] developed for the first time an ETV-ICP-OES methodology for the speciation of sulfur in coals. This methodology did not require any sample preparation and was validated by analysing a standard reference material.

These recent experiments were carried out by using the same instrument (ETV 4000c purchased from Spectral Systems). However, all these methodologies have used Freon-R12 as reaction gas during the analyses, which, as it has been mentioned before, its use in the European Union has been forbidden.

Materials and methods

Instrumentation

All the measurements have been carried out in an ETV Spectral Systems 4000-c. During the electrothermal vaporisation, both carrier gas and bypass flows were optimised, and these optimum values were 0.05 and 0.15 L min -1 respectively. An SF6 flow of 0.35 mL min - 1 was used during the analyses to improve the volatilization efficiency of the compounds that were analysed. The graphite boats and graphite furnaces used during this work have been purchased from Spectral Systems (Germany). A Sea-spray nebuliser was used for the continuous addition of an internal solution containing 5 ppm of Rh. Figure 36 shows a picture containing the typical appearance of this ETV-ICP-OES setup, and Table 2 resumes the main parameters of the ICP-OES. For both method optimisation and quantification purposes, a Conostan S21 containing 50 ppm of Al, B, Ba, Ca, Cd, Cr, Cu, Fe, Mg, Mn, Mo, Na, Ni, P, Pb, Si, Sn, Ti, V and Zn, purchased from SCP science, was employed.

The standard reference material, SRM 1085b, purchased from NIST (National Institute for Standards and Technology, Gaithersburg, MD, USA) containing 300 ppm wear metals in lubricant oils (Al, As, Ba, B, Cd, Ca, Cl, Cr, Cu, Fe, Pb, Mg, Mn, Mo, Ni, P, Si, Ag, Na, Sn, V and Zn) was used for method validation.

Six different fast pyrolysis oil samples have been analysed via ETV-ICP-OES. Two of them (Samples P and T) consist of commercial pyrolysis oils that were obtained from Haldor Topsoe (Lyngby, Denmark) and the other four, whose production will be discussed in Chapter 7, were provided by RTI. The feedstocks employed to produce the fast pyrolysis oils and their respective names in the figures are detailed below:

• Sample A: Pyrolysis oil produced from a 50:50 mixture of milorganite and loblolly pine wood.

• Sample B: Pyrolysis oil produced from a 50:50 mixture of milorganite and red oak wood.

• Sample C: Pyrolysis oil produced from milorganite fertilizer.

• Sample D: Pyrolysis oil produced from red oak wood.

• Sample P: This sample consists of a pyrolysis oil produced from pine wood.

• Sample T: Pyrolysis oil produced from used tyres.

Procedure

A dilution (DF = 10) was carried out to obtain a 5 ppm solution which was used for the optimisation of the gas flows. For each run, 10 µL of this solution were deposited in the graphite boat, after having waited for one minute to let the THF evaporate and then starting the analysis. Dilutions of the S21 and K standards were carried out in order to obtain solutions containing 0, 0.001, 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10 and 20 ppm of the corresponding elements, to perform an external calibration.

The SRM 1085b was diluted 10,000 times in THF. The Conostan multielement solution was diluted in THF and added to aliquots of the diluted SRM 1085 b in order to have five different solutions containing each analyte plus an added concentration of 0, 0.5,1, 2 and 4 ppm. 10 µL of each standard was introduced in the graphite boat and ran by triplicate.

Each of the pyrolysis oils analysed in this work was diluted 10 times with THF to avoid the saturation that a high concentration of some elements might cause in the detector. 20

µL of each diluted sample was injected by triplicate.

The transient signal obtained via the methods used in this work has been corrected with the intensities obtained for the internal standard via the division of their correspondent transients (Sanalyte / SIS) followed by an integration of the intensities that conform the corresponding peaks observed.

Matlab 2018b and Microsoft Excel were used for the data treatment and graphic design of the results obtained.

Results and discussion

ETV-ICP-OES Optimisation

To obtain a good sensitivity is a critical step in the acquisition of ETV-ICP-OES data since an inaccurate combination of the three different gas flows that are used in the electrothermal vaporised could drop significantly the sensitivity rates.

To perform this optimisation, a temperature ramp with a very fast increase of the graphite furnace temperature was chosen to obtain the highest transient signal in the shortest possible time for one sample.

Figure 37 shows the temperature ramp that was chosen for these optimisation experiments. It starts with a smooth increase of the temperatures (20 °C s -1 ) which is held for 5 minutes up to 20 s of analysis. Then, a very fast gradient has been programmed in order to reach a temperature of 2200 °C which will vaporise and send towards the ICP-OES the target analytes. This temperature is held for 20 seconds and then, a second gradient of temperature is programmed to completely pyrolyze and vaporise those potential compounds that might have been retained in the graphite boat. Thus, temperature raises for 10 seconds (40 °C s -1 ) and finally held at 2600 °C for the last 9 seconds. The fist flow to be optimised was the SF6 reaction gas. To do so, ETV gas flow rates were set up using the same values that Silva et al. [START_REF] Silva | Feasibility of Rare Earth Element Determination in Low Concentration in Crude Oil: Direct Sampling Electrothermal Vaporization-Inductively Coupled Plasma Mass Spectrometry[END_REF] used for determining rare earth elements in crude oil samples. Whereas both the carrier and the bypass flows were set up to 0.4 L min -1 , the transient signal generated by the analysis of the same standard solution was fully integrated and corrected with the Rh internal standard transient values.

Figure 38 shows the intensity variations of different wavelengths with the different gas flow values. It is interesting to mention that, for all the elements, with a few exceptions, there are common patterns: (1) the absence of reaction gas reduces the sensitivity of the detection and (2) an excess of reaction gas also reduces the sensitivity of the detection. In order to choose accurately the optimum reaction gas flow value, the relative standard deviations of each analyte were studied. Figure 39 shows the relative standard deviation of the integrated signals for each measured wavelength. There is a region, between 3 and 5 mL min -1 where higher precision is obtained. Thus, considering the sensitivity values and the precision obtained, a reaction gas flow of 3.5 mL min -1 was elected.

The carrier gas flow was optimised once the reaction gas rates were optimal. To perform this optimisation, the gas reaction flow was held at 0.35 mL min -1 and the bypass flow was held at 0.5 L min -1 . Figure 40 shows the intensity average values obtained for the different wavelengths analysed. Except for 4 different wavelengths (on the right side of the figure), generally, the sensitivity of the transient signals decreases with higher carrier gas flows.

Besides, as it can be observed in Figure 41, the precision increases at low gas flow rates.

Taking into account this information and considering that a very low carrier gas flow might not be able to transport all the analytes to the plasma, a compromise was taken and a flow of 0.05 L min -1 was chosen. Finally, once both reaction and carrier gas flows were set up, the same procedure has been followed to evaluate the sensitivity concerning the different bypass gas rates. Figure 41, Figure 42 and Figure 43 show respectively the different sensitivities and relative standard deviations obtained for different bypass flow rates. There is a flow rate that provides high intensities with a low relative standard deviation. Since a compromise had to be taken, the gas flow that has been chosen for the argon bypass was 0.15 L min -1 . Once the three gas flows that control the electrothermal vaporisation process and the transportation of the analytes to the plasma were optimized, a calibration was carried out

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020

-124-to check the linearity of the method and its limit of detection for the different elements that have been analysed. Figure 44 shows the regression curve of three different elements analysed simultaneously in this experiment. In Table 3, the coefficients of determination for the different transient wavelength are depicted. In general, there is a great correlation between the concentration of each element and the intensities detected. However, certain elements like Fe do not have good linearity, probably due to the high presence of this element within the instrument joints, since the graphite furnace is placed inside of a steel compartment.

Method validation.

The quantitative analysis of a reference sample, SRM 1085b, has been carried out with this configuration with the purpose of validating the ETV-ICP-OES methodology that has been optimised.

The results, detailed in Table 4, show that the methodology that has been employed to carry out the analyses do not possess a very high precision. This fact might be associated with the use of a less-reactive reaction gas due to legislation issues.

However, the accuracy of the method is acceptable for the simultaneous quantification of up to 8 different elements. Considering that this method does not require any sample preparation but the dissolution of the sample in a solvent (in this case THF) and carrying out the standard additions, the results prove that this methodology could be established in the industry since it provides with cheaper and faster alternatives to other methodologies that might be more costly and slower. Other elements, however, did not provide with good results. That is the case for phosphorus, aluminium, calcium, iron and zinc. The only reason why the analysis of these elements has not provided with a good result might be related to the quality of SF6 as a reaction gas. The standard additions were performed using the Conostan S21 standard solution, which contains 100 ppm of 21 elements. Since these elements are dissolved in the standard matrix as salts, SF6 may not be reactive enough to achieve the quantitative vaporisation of all the different element salts.

Speciation analysis of fast pyrolysis oils via ETV-ICP-OES

In order to obtain the elemental distribution of some of the trace elements present within different fast pyrolysis oils based on their evaporation/pyrolysis temperatures, the length of the analyses has been increased up to 250 seconds using a different temperature gradient. Figure 45 shows the temperature program that was designed for this experiment. In this method, the temperature was initially increased up to 300 °C during the first 10 seconds of sequence. This temperature was then increased up to 400 °C during the following 50 seconds to eliminate all those volatile compounds that do not provide with useful information to the analysis (solvent, water, etc.). Then, a heating ramp of 10 °C s -1 was applied for 160 seconds until it reached 2000 °C. Finally, a last increase of the temperature up to 2500 °C was applied for 10 seconds and held for another 10 seconds to pyrolyze all those potential residual compounds that might have not been removed from the graphite boat, to be able to be reused.

All the samples were diluted (with a dilution factor of 10) in THF and 20 µL of each solution were deposited in the graphite boat by triplicate.

One of the trace elements, Phosphorous, and whose characterisation via GPC-ICP-HRMS was achieved for 4 of the following samples (Samples A, B, C and D), presents the ETV-ICP-OES distribution showed in Figure 46. Concerning the analysis of potassium, a high abundance of this element can be observed in the sample C (milorganite pyrolysis oil). This high abundance was expected since milorganite possesses a high concentration of K2O, which constitutes an essential nutrient for plants. 100 The analysis of potassium oxide via ETV-ICP-OES would allow the confirmation of this hypothesis. However, since this compound is not expected to be found at high concentrations in other natural materials, such as red oak wood (120D), the presence of a peak within the transient signal for its corresponding pyrolysis oil might indicate a presence of potassium salts in the sample. Magnesium, however, presents peaks that can be observed when very high temperatures are applied. Magnesium is expected to be found in this type of samples since Mg 2+ is present within the chemical structure of chlorophyll.

The aluminium ETV-ICP-OES distribution is one of the most interesting. At least four different compounds containing these elements are expected from the distribution of the transient. Temperatures above 500 °C suggest the presence of aluminium as salts, although a bad elimination of ashes from the fast pyrolysis oil would be another cause.

Aluminium is a very abundant element in the earth's crust, its higher presence within the lignocellulosic pyrolysis oil than within the tyres pyrolysis oil agrees with the data obtained with this methodology.

Finally, concerning zinc, the data suggests that at least two different species are present within the samples that have been analysed (bimodal distributions). As one of the essential micronutrients within the cell plants, its presence within these samples was expected. However, a monomodal distribution with a high abundance has been found for the tyres fast pyrolysis oil: zinc is highly present within the tyres' pyrolysis ashes.

However, with this methodology, it is not possible to confirm the hypothesis of the presence of ashes within the samples without the analysis of a reference ash containing zinc.

Conclusion

The development of a methodology that allows not only the speciation analysis of trace elements via ETV-ICP-OES but as well the quantitative analysis of certain trace elements in complex samples has been achieved and validated for certain elements. This methodology, which uses SF6 as a reaction gas, has shown a lower efficiency than previous methodologies that used gases with a higher reactivity and whose commercialisation has been forbidden by the public institutions.

By using ETV-ICP-OES, the industry could be benefitted from a very fast methodology that does not require very expensive instrumentation. While the presence of ICP-OES instruments is quite common in this sector, coupling it to an electrothermal vaporiser should be an easy task.

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -133-However, further experiments must be carried out to fully determine the speciation of these trace elements present within this set of samples: while the analysis of standards, such as phospholipids and salts, would help with the identification of the peaks that have been obtained with this methodology.

CHAPTER 6: IMPORTANCE OF GPC-ICP-HRMS ON THE SPECIATION ANALYSIS OF COMPLEX MATRICES

This chapter focuses on the great advantages of coupling gel permeation chromatography and inductively coupled plasma mass spectrometry to carry out speciation analysis in complex matrices.

Although the topic of this thesis consists of the speciation analysis of bio-oils, familiarisation with the instrumentation that was going to be used through this PhD project had to be done. Thus, through collaborations with Haldor Topsoe, a Danish company specialised in the production of heterogeneous catalysts and the design of catalytic processes, different samples were received to get a better understanding of their elemental composition through two very important catalytical processes designed to improve the quality of crude oil with the elimination of the different compounds containing V, Ni and S within its matrix: Hydrodemetallization (HDM) and Hydrodesulfurization (HDS). In addition, one of the sub-objectives of this thesis (O2a) was fulfilled, confirming the hypothesis that suggested that a different reaction pathway might occur for the different species present in this type of samples.

Performing inorganic speciation analysis of such complex matrices via GPC-ICP-HRMS was a success. Fingerprints of the V, Ni and S aggregates present in atmospheric residue samples, based on their molecular size, have been obtained at different stages of the respective catalytical processes, providing insights into the catalyst behaviour concerning the different hetero-compounds present within such complex samples.

Future analyses should be carried out in order to fully understand the composition of these aggregates. One of the techniques that might provide with this information is the FT-ICR-MS since its high-resolution power will allow the full identification of each of the aggregates that were detected via GPC-ICP-MS.

Crude oil composition and its derivates

Crude oil, also known as petroleum, is a black liquid that consists of a naturally occurring mixture of hydrocarbons and other complex organic compounds that can be found in several geological formations (also known as petroleum reservoirs) which are located beneath the earth's surface. Crude oil has been the most used source of primary energy in terms of heating, transportation and production of energy. During 2019, the production of crude oil reached an average of 2.2 millions of barrels per day. 101 As it has been already mentioned, hydrocarbons such as alkanes, cycloalkanes and aromatic hydrocarbons are the main components of crude oil: carbon and hydrogen can represent between 83-87% and 11-14% of its mass composition. 102 These hydrocarbons can be separated through a distillation process that is carried out in the petroleum refineries.

Figure 49 shows the typical distillation cuts that are obtained from crude oil. [START_REF] Desprez | Study of the Size Distribution of Sulfur, Vanadium, and Nickel Compounds in Four Crude Oils and Their Distillation Cuts by Gel Permeation Chromatography Inductively Coupled Plasma High-Resolution Mass Spectrometry[END_REF] Apart from their main composition based on hydrocarbons, crude oils also contain low percentages of hetero-elements such as sulfur (0.04-8%), oxygen, nitrogen and other metals, such as vanadium and nickel, with concentrations that are at the ppm levels. Their presence consists of an issue in terms of corrosion and reduction of the efficiency of the catalysts during the refining process. Besides, their presence can also contribute to the emission of environmental pollutants. 102 The upcoming shortage of crude oil reserves is accelerating the research on alternative sources of hydrocarbon fuels. One of these alternatives consists of the conversion of the heavy distillation fractions of crude oil into transportation fuels.

Atmospheric residue, which is one of the heavy fractions of crude oil, consists of a mixture of hydrocarbons that have carbon numbers greater than C11 and boiling temperatures above 200 °C. 103 These crude oil cuts have a higher content of hetero-elements, which are pre-concentrated during the distillation process and remain within this residue. Hence the need for carrying out two very important processes that will guarantee that none of these hetero-compounds is present in the products: Hydrodemetallization and Hydrodesulfurization. Both processes will be shortly explained in the publication enclosed in this chapter.

In this chapter, the results on the speciation analysis of V, Ni and S in atmospheric residue is reported through the article (Article 2) published in "Fuel Processing Technology" in December 2019: "Understanding the removal of V, Ni and S in crude oil atmospheric residue hydrodemetallization and hydrodesulfurization.

This study has provided with very valuable information related to the size distributions of the S, Ni and V species present in atmospheric residue feedstocks and how their size distributions change during the HDM and HDS processes. One detail that has not been mentioned in the following article is that, in Figures 2 and4, a little shift can be observed from the feed peak with respect to the product peaks. Further investigations should be carried out in order to clarify whether any type of aggregation process has occurred. The analysis of both feed and products via FT-ICR-MS could provide with useful information about this topic.

OILS

One of the aims of this fifth chapter, together with the sub-aim O2b that was presented in the Introduction of this thesis, consists of providing to the reader with a short review of the current methods of analysis of inorganic compounds (trace elements) within the biomass feedstocks, pyrolysis products and other bio-oils that were introduced in Chapter 1, together with a brief overview of the composition of these samples. As it was mentioned, numerous compounds can be found within these matrices. For example, considering the pyrolysis oils as one of the most complex samples analysed in this work, up to 300 different oxygenated compounds have been identified within only one sample.

The second, and most important aim of this chapter, consists of reporting the results that were obtained by studying the speciation of phosphorus in different types of fast pyrolysis oil and fatty acid feedstocks. As it was explained before, phosphorus is one of those elements present within most of the bio-oils feedstocks that would cause serious issues during the catalyst process that transforms these bio-oils into high-quality transportation fuels. Thus, both the phosphorus content and its speciation in three different lignocellulosic fast pyrolysis oils and three fatty acid feedstocks, are under discussion in this chapter. Even so, ICP-MS is not the only method that has been used for the analysis of this type of feedstocks. Nerin et al. 110 analysed the metal content of different waste industrial oils by graphite-furnace atomic absorption spectrometry (GF-AAS), after an acid digestion sample treatment.

Respect to biodiesel samples, since the content of certain trace elements is regulated and standardised, numerous methods for biodiesel total analysis have been developed. 111 Sanchez et al. 112 reviewed the main techniques employed for the determination of metals and metalloids in biodiesel. They agree on the challenging of working with these complex samples since the target elements are present in very low concentrations and the calibration with organic complex matrices reduces the accuracy of these determinations.

In their review, they describe numerous procedures for sample preparation, including emulsification, micro emulsification, dilution with ethanol to work in aqueous mode and acid digestion amongst others, followed by either ICP-MS or ICP-OES detection.

Regarding the total analysis carried out for the trace elements in bioethanol, there exists numerous publications discussing the topic, but it seems that microwave acid assisted digestion using HNO3 and posterior analysis by ICP-MS is the optimal method to obtain the highest, as it was reported by Sanchez et al. 112,113 , obtaining acceptable LODs and both high short and long term precision within their method.

However, other techniques such as MP-ICP-OES that have been tested for the analysis of Si in complex samples such as diesel and biodiesel might be very useful for the determination of other trace elements in other fatty feedstocks and biodiesel. 114,115

Pyrolysis Oils

The total analysis of trace elements in all types of pyrolysis oils is mainly carried out by ICP techniques once they have been acid-digested. Among all the methods that have been designed for these purposes, different variations on the acid composition or the detection may occur. For example, while acid digestion using H2O2 and HNO3 and ICP-OES detection was the method chosen by Al Chami et al. 116 , Chiang et al. 117 performed a more aggressive digestion using a mixture of HNO3, HCLO4 and HF. Posteriorly, and after a preconcentration, two different methods were chosen to carry out the analyses: an ICP-OES for the analysis of Al, Ca, Fe, K, Mg, Na, S, Co and Zn, and an ICP-MS for the analysis of As, Ba, Cd, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sr, Ag and V concentrations, achieving in both cases detection limits at the ppb level.

However, other techniques have been explored for the quantification of trace elements in these bio-oils, such as the TGA method for the determination of Na, Ca, Mg and K in bioslurry fuels (mixtures of biochar and bio-oil) developed by Zhang et al. 118 .

Speciation Analysis

Strictly speaking, "speciation" is considered by the IUPAC as the "distribution of an element amongst defined chemical species in a system". 119 However, due to the lack of literature related to the speciation of metalloids and trace elements in some of the samples that are referred in this work, other methods that help to characterize and elucidate species containing certain elements will be discussed in this section.

7.1.2.1. Biodiesel, bioethanol and their feedstocks

As it has been discussed in the previous section, both biodiesel and bioethanol samples possess already established methods of quantification of their trace elements and possible future applications of these methods that might be used for speciation analysis. However, extra information needs to be obtained to understand the presence of these trace elements within the matrices they belong to.

One technique that has been mentioned in this thesis and that was used for the determination of certain trace elements in ethanol fuel, ETV-ICP-MS obtained good calibration figures and LODs by using an isotopic dilution calibration method. 120 The optimised coupling of these two techniques might be used, with a different temperature ramp, for the speciation of these trace elements and obtaining their fingerprint concerning their boiling/pyrolysis temperature.

One interesting method, that helps to understand the behaviour of some trace elements species throughout the production process for bioethanol, was developed by Sanchez et.

Al. 121 They quantified and compared several trace elements from samples taken at different phases of the industrial process and, as it is shown in Figure 51, by ICP-MS analysis.

FT-IR

Fundamental vibrations

• Functional groups.

• Ageing mechanisms.

GC-MS

Ions (m/z) • Quantification of PAHs, organic acids, ketones, alcohols, etc.

FT-ICR-MS

Ions (m/z) • Hydrocarbon and small organic molecules characterisation.

NMR

Magnetic properties • Functional groups, quantification.

• Degradation mechanisms.

HPLC-UV HPLC-RI

Absorbance

• Polar/apolar species identification.

• Size distribution fingerprinting.

Another speciation method for the analysis of heavy metals (Cu, Cr, Pb, Zn, Cd, and Ni) in sewage sludge pyrolysis oil was carried out by Yuan et al. 124 . They carried out an extraction procedure where four fractions were extracted. These four fractions, consisting of (1) exchangeable metals and carbonate-associated fractions, (2) Fe and Mn oxides fraction (3) organic-mater-bonded fraction and (4) the residuals. Then, the total concentration of these elements was obtained by ICP-OES. They found out that these trace elements are present within all the four fractions, being Cu and Cr the most abundant ones within the organic-mater-bonded fraction, demonstrating that these metals can be present as free ions but also bonded to organic compounds within the pyrolysis oil matrices.

These results are shown in Figure 52 being F1, F2, F3 and F4 the four fractions described before.

Figure 52. Speciation of heavy metals in pyrolysis oil from sewage sludge

The speciation analysis of P and the transformations that its species have during the hydrothermal treatments of animal manures has been reported in the literature. Huang et al. 125 performed a sequential extraction to consider all the P species by using different solvents, acids and bases among others. They carried out the analyses with three different techniques: X-ray powder diffraction analysis, liquid 31 P NMR spectroscopy and X-ray absorption near-edge structure (XANES) analysis and obtained information about the abundance of P species along the hydrotreating process of beef, chicken and dairy manure samples.

However, apart from a few publications where speciation analyses are performed targeting the trace elements present within pyrolysis oils, the rest of the publications focus on the speciation analysis of organic compounds, such as sugars, aldehydes, PAHs, etc, making the work carried out within this project quite novel and contributing a new point of view to the research in pyrolysis oil characterisation. Nonetheless, the following reviews cover these speciation and characterization methods for the main components of pyrolysis oil samples. [126][127][128][129][130] • Sample VO: Unprocessed soybean oil.

• Sample TRIG.X: Unprocessed pork fat.

Except for the Sample P, all the other pyrolysis oils were produced at the RTI facilities in the United States. A fast pyrolysis process was carried out in a pilot-scale catalytic fast pyrolysis unit with γ-Al2O3 being the catalyst, with a catalyst-to-biomass ratio average equal to 5.

The concentration of the trace elements obtained for some of the samples whose speciation is studied in this chapter is detailed in Table 6. The reader might have noticed that the details concerning three of the samples described above are not reflected in Table 6. These samples are the commercial pyrolysis oil sample, the tallow fat and the soybean oil.

In order to determine the concentration of trace elements within these three samples, Haldor Topsoe carried out the round-robin study, described in Section 5.1.1., for the quantification of several trace elements, including P, S, Na, Fe and Mg. 104,131 The results of this round-robin study concerning the trace elements studied in this chapter are detailed in Figure 53.

While the analyses carried out for the quantification of trace elements in the vegetable oil sample are quite precise, the animal fat and pyrolysis oil results indicate how challenging the quantitative analysis becomes by presenting very high deviations among laboratories that followed the same methodology. For all the analyses carried out within this section via GPC-ICP-HRMS, the same instrumentation and solvents that are described in the article displayed within the Section 4.3 of this manuscript were used. Each of the samples was diluted with a factor of 5 in THF. Injections of 20 µL were carried out by triplicate.

Methodology and calibration of the columns

With the purpose of having an estimation of the molecular size of the eluting compounds, the chromatographic profile of nine commercial polystyrene standards was obtained via GPC-UV/VIS. ICP-MS detection was not possible for these compounds, since they are composed of carbon and hydrogen, and detecting these elements via this technique is not possible.

These polystyrene standards were dissolved in three different solutions with THF and injected by triplicate. Figure 54 shows the profile obtained for the three different solutions.

Solution 1 contains PSs with a MW of 3 152 000, 1 044 000 and 466 300 Da respectively; A common peak for the three PS solutions can be observed after approximately 32 min of analysis. This peak, which corresponds to the solvent peak, determines the total permeation volume of the GPC columns. Theoretically, if there are no other interactions between the analytes and the stationary phase (meaning that the separation is based only on the hydrodynamic volume of the analytes), the elution of compounds beyond this point will not occur.

A calibration curve was obtained by plotting the logarithm of the molecular weight of each PS standard versus their corresponding retention times and it is shown in Figure 55. 7. Four classes have been stablished, resembling to those ones that were defined for crude oil analysis in previous works. [START_REF] Vargas | Development of a Chromatographic Methodology for the Separation and Quantification of V, Ni and S Compounds in Petroleum Products[END_REF]132 However, in this case, the retention time and, therefore, the size of each group of species varies with respect to these previous works.

High molecular weight (HMW), medium molecular weight (MMW), low molecular weight (LMW) and tailing fractions have been defined for this classification purposes. Although the same distribution can be observed and the retention time of each of the peaks is common to all the feedstocks used to produce these pyrolysis oils, some differences can be observed:

• Milorganite fast pyrolysis oils show higher intensities than the rest of the samples.

This higher presence of sulfur-containing species might be linked with the numerous salts containing sulfur that are present in fertilizers, such as ammonium sulfates or potash sulfates, amongst others, intended to provide to the plants either with very essential nutrients for their growth or with protection against pests and insects.

• The commercial pyrolysis oil sample shows a high intensity for the peak eluting after 32 minutes of analysis. The molecular size of this species might suggest a high concentration of sulfur-containing salts that do not interact with any other compounds within the pyrolysis oil matrix. These salts might have been caused

through the rupture of bigger aggregates during the production of this commercial unit. One methodology that might help to confirm this hypothesis could be the injection of a sulfur-containing salt in a low concentration. Besides, the presence of compounds within the MMW and LMW fractions, indicates that either aggregation, complexation or polymerisation reactions have taken place within all the samples. By performing a fractionation via preparative GPC of these samples, further analysis might be carried out to determine the structure of these compounds, such as the use of FT-ICR-MS.

The contribution of each fraction with respect to the total area of the chromatogram was calculated and the results can be observed in Figure 58. A high presence of MMW and LMW compounds is common within all the pyrolysis samples, no matter their feedstocks.

However, regarding the commercial pyrolysis oil produced from pine, a change can be noticed, since an increase of the tailing compounds (probably salts) and a reduction of the MMW compounds has taken place.

Sodium species

The GPC-ICP-HRMS chromatograms corresponding to the distribution of Na-containing species in the same set of samples are detailed in Figure 59 and Figure 60.

results have been plotted in Figure 61. It can be observed how the commercial pyrolysis oil produced from pinewood lacks HMW sodium species within its matrix, whereas, for the rest, the abundance of all the size fractions is observed in decrescent order, from lower to high molecular weight compound.

Iron species

The fingerprints obtained for the Fe-containing species based on their size are shown in Figure 62 and Figure 63 for the same samples, where a common peak can be observed.

This peak, which corresponds to a Fe-containing compound with a molecular weight of approximately 21,000 Da is present amongst all the lignocellulosic fast pyrolysis oils that have been analysed.

As it has been mentioned in Chapter 1, iron is one of the most common elements in the earth crust and, therefore, it is present within both vegetable and animal tissues. It is very usual to see natural complex and other organometallic compounds with iron in nature. 133 Besides, the data suggests that bigger complexes have been formed during the fast pyrolysis process, as the elution of iron species takes place from after 15 minutes of analysis except for the commercial fast pyrolysis oil produced from pinewood.

A very small peak can also be observed after minute 30 of analysis, near the limit of permeation of the columns, which might be caused by the presence of Fe 2+ and Fe 3+ ions that are not complexed.

Thus, the data obtained via GPC-ICP-HRMS suggests that a high molecular weight ironcontaining complex is formed during the fast pyrolysis of lignocellulosic feedstocks. In Figure 64, the distribution of the different fractions based on their contribution to the total chromatogram area is observed, where the absence of high molecular weight iron species for the commercial pyrolysis oil can be observed clearly. However, the medium molecular weight species are the more abundant ones for all the lignocellulosic fast pyrolysis oils. The fractionation of this samples and posterior analysis with other techniques such as FT-ICR-MS would allow the identification of these species.

Magnesium species

Magnesium-containing species, which are expected in lignocellulosic feedstock due to the presence of chlorophylls in all the vegetable tissues. The weight distribution of these compounds obtained via GPC-ICP-HRMS analyses is detailed in Figure 65 and Figure 66. An eluting peak with approximately the same size as the most abundant peak found for the analysis of iron in the same set of samples can be observed. This peak, which corresponds to several compounds with an approximate mass of 21 KDa, is present among all the fast pyrolysis oils. Furthermore, bigger compounds, which start eluting after 15 minutes of analysis can be observed, suggesting that aggregation of Mg-containing compounds occurs during the fast pyrolysis process.

Since a high presence of different chlorophyll high-molecular-weight aggregates in complex matrices was found under different matrices and temperature conditions, [134][135][136] the data obtained via GPC-ICP-HRMS analyses suggest that aggregation reactions have occurred either during the fast pyrolysis process or the storage of the sample.

A GPC-ICP-HRMS analysis of chlorophyll should be carried out in the near future to study the size distribution of Mg. Although chlorophyll is an expensive reagent, with a TLC plate and a similar methodology to the one that Tswett carried out with vegetal pigments (mentioned at the beginning of the Chapter 2 of this PhD thesis), the extraction of chlorophyll from spinach could be achieved. The TLC spot where the chlorophyll is eluted could be extracted and collected in order to be injected via GPC-ICP-HRMS.

Figure 67 shows the bar plot corresponding to the different size fractions based on their relative area with respect to the total chromatogram area. Same abundances are observed for all the samples, with exception of the 50:50 red oak wood and milorganite fast pyrolysis oil, where the low molecular weight compounds are the most abundant ones. In addition, as it happens for all the trace elements concerning the commercial pyrolysis oil sample, a lower number of high-molecular-weight compounds has been found as well in this sample. The results that concern the analysis of the phosphorus-containing compounds in fast pyrolysis oils and fatty acid feedstocks have been summarised in the Manuscript 1 that is attached below. This manuscript is not in its final version. Further changes will be considered before its submission in the RSC Green Chemistry journal.

CONCLUDING REMARKS AND PERSPECTIVES

The main objectives of this thesis project were to provide with novel alternatives for the speciation analysis in bio-oils and other petroleum heavy oil fractions and to achieve a better knowledge and understanding on the behaviour of the hetero-atomic species present in these samples. Through combining different modern separation techniques with the elemental detection that the ICP provides, different solutions have been proposed in order to understand the behaviour of some of the trace elements present in such complex samples.

The first main objective of this thesis, which consisted of introducing, developing and validating novel analytical tools for the speciation analysis of bio-oils and renewable feedstocks, has been partially fulfilled.

For the first time, in order to fulfil the sub-objective O1a, a 3D-printed total consumption nebuliser has been developed and optimised for speciation analysis purposes. 3D-printing technology has proven its usefulness in the analytical chemistry field, being able to provide with cheap and high-quality consumables. Furthermore, the use of PEEK tubing within this 3D-printed total consumption nebuliser will avoid the potential interferences that other species might have with the former silica capillary present in previous total consumption nebulisers.

Further analysis will be carried out in the near future with this consumable for the speciation analysis of both crude oil products and renewable feedstocks. In addition, the development of a complemental heating device, designed to increase the temperature of the spray chamber that links the nebuliser with the ICP torch, has already started.

Through the combination of these two novel consumables, a higher sensitivity on the ICP-MS analysis will reduce the DL of this technique and improve the quality of the GPC-ICP-HRMS chromatograms, specially of those elements that require a higher resolution, such as K and Si.

Besides, with the purpose of fulfilling the objectives O1b and O1c, the use of two Concerning the second main objective of this thesis, which consisted of obtaining a better understanding of the trace element composition of petroleum heavy oil fractions and renewable feedstocks, significative advances have been achieved during this three-year project.

In Chapter 6, alongside a familiarisation with the state-of-the-art GPC-ICP-HRMS technique that allows the study of the trace elements in petroleum compounds, a better understanding of how V, Ni and S compounds during the hydrodemetallization and hydrodesulfurization process of crude oil atmospheric residue has been achieved, together with the fulfilment of the objective O2a. In addition, this study has provided with insights into the preference that the catalysts possess towards some of the compounds and nanoaggregates that are present in crude oil atmospheric residues.

The results obtained in the Chapter 6 of this thesis might be very useful for further analyses carried out with other analytical techniques, since the elucidation of the structure of such aggregates would be useful for the industry, as better catalysts could be designed to avoid this undesirable reduction of their activity during the hydrothermal processes. For this purpose, FT-ICR-MS would be one of the best candidates to achieve this goal in future projects. By knowing the structure of these compounds, a novel generation of longer-life catalysts might be designed, providing to the petroleum industry with important economic savings and a faster production process of high-quality transportation fuels.

This familiarisation with the GPC-ICP-MS technique has eased the optimisation of a method, which allowed the analysis of other trace elements in fast pyrolysis oils and fatty acid feedstocks, and which has been described in the Chapter 7 of this PhD thesis, fulfilling the sub-objective O2b. A better understanding of the behaviour of the phosphorus compounds present in this type of samples has been achieved through the obtention of their corresponding GPC profiles: phosphorus compounds form high molecular weight compounds, potentially micelles, liposomes or long polymers, when they are present in these feedstocks. Furthermore, an aggregation/polymerisation phenomenon of these compounds has been confirmed by analysing the same samples over a period of three months. These resulting big aggregates might have a great contribution to the catalyst poisoning and the pressure drops that occur during the processes that transform these feedstocks into high-quality transportation fuels.

By performing a fractionation of these different compounds based on their molecular size, added to the use of complementary techniques such as FT-ICR-MS it will be possible to achieve a new insight into the structure and reactivity of these compounds, which would allow the synthesis of novel and more refractory catalysts towards these compounds.

Furthermore, for the first time, a fingerprint based on the molecular size of the sulfur-, sodium-, iron-and magnesium-containing compounds in pyrolysis oil feedstocks has been obtained via GPC-ICP-HRMS analysis. The early retention times that have been obtained

for the different pyrolysis oil feedstocks that have been analysed, concerning these four elements, suggest that they are present as complexes or high molecular weight compounds within the pyrolysis oil matrices. In addition, the different distributions that have been observed in the GPC-ICP-HRMS chromatograms suggest that each of the detected elements form different species on each of the analysed feedstocks. As mentioned before, the size fractionation of these samples and their posterior analysis with a technique such as FT-ICR-MS might help for the elucidation of the structures of these species. The hypothesis that suggests that the presence of chlorophyll derivates and iron and sodium complexes would be confirmed.

This technique has showed a great performance on the obtention of molecular fingerprints.

By using the novel 3D-printed nebuliser that was designed and developed during this work, the speciation analysis of more elements such as Si and K could be carried out. In addition, a sampling approach, similar to the one that is detailed in Article 2, of hydrotreating pyrolysis oil products would provide with insights on the behaviour of such hetero-atomcontaining species during the hydrodeoxygenation process, through their analysis via GPC-ICP-HRMS.

6. 2 .

 2 Understanding the removal of V, Ni and S in crude oil atmospheric residue hydrodemetallization and hydrodesulfurization .......................................................... 137 Chapter 7: GPC-ICP-MS speciation analysis in bio-oils ....................................... 145 7.1. Current trends on inorganic speciation and total analysis of bio-oils ................... 145 7.1.1. Total analysis ...................................................................................................... 145 7.1.1.1. Biodiesel, bioethanol and their feedstocks ................................................... 146 7.1.2. Speciation Analysis ............................................................................................. 148 7.1.2.1. Biodiesel, bioethanol and their feedstocks ................................................... 149 7.1.2.2. Pyrolysis Oils................................................................................................. 150 7.2. Speciation analysis of trace elements in renewable feedstocks via GPC-ICP-HR MS ....................................................................................................................................... 153 7.2.1. Samples, methodology and instrumentation ................................................... 153 7.2.2. Methodology and calibration of the columns ................................................... 156 7.2.3. Analysis of S, Na, Fe and Mg in fast pyrolysis oils ......................................... 158 7.2.4. Phosphorus speciation analysis of fatty-acid based feedstocks and fast pyrolysis biocrudes via gel permeation chromatography inductively coupled plasma high resolution mass spectrometry. ................................................................................... 170 Concluding remarks and perspectives .................................................................... 190 Bibliography .................................................................................................................. 194 Annexes .......................................................................................................................... 213 Poster presented at the 48 th International Symposium on High-Performance Liquid Phase Separations and Related Techniques ................................................................. 213 Simplification of heavy matrices by liquid-liquid extraction: Part I -How to separate LMW, MMW and HMW compounds in maltene fractions of V, Ni and S compounds.215 Simplification of heavy matrices by liquid-solid extraction: Part II -How to separate LMW, MMW and HMW compounds in asphaltene fractions for V, Ni and S compounds. ....................................................................................................................................... 222 Spatial and temporal trends of Polycyclic Aromatic Hydrocarbon (PAH) contamination in sediments from different locations in western Greenland. ...................................... 231

  1 determined for the SRM 1085b via ETV-ICP-OES. ........................................................................................ 126 Table 2. Main techniques used for the speciation analysis of pyrolysis oils. 123 ............... 151 Table 3. Sample name, type of feedstock and total concentration in ppm of P, S, Na, Fe and Mg............................................................................................................................... 154 Table 4. Definition of the molecular weight interval for the different fractions obtained via GPC-ICP-MS. .................................................................................................................... 158

Figure 1 . 25 Figure 2 . 31 Figure 3 . 32 Figure 4 . 33 Figure 5 . 33 Figure 6 . 34 Figure 7 . 35 Figure 8 . 38 Figure 9 . 40 Figure 10 . 57 Figure 15 . 60 Figure 18 . 65 Figure 22 . 72 Figure 31 . 74 Figure 33 . 76 Figure 36 .Figure 42 .Figure 66 .• Article 1 :• Article 2 :• 3 :• 5 :• Manuscript 1 :• Manuscript 2 :

 125231332433533634735838940105715601865227231743376364266123512 Figure 1. Outline of the objectives covered in this thesis. .................................................25 Figure 2. Chemical structure of cellulose. ..........................................................................31 Figure 3. Schematic structure of Cellulose, Hemicellulose and Lignin fibres in the cell wall. 14 ..................................................................................................................................32 Figure 4. Schematic representation of some lignocellulosic fast pyrolysis products. 14 .....33 Figure 5. Alcoholic fermentation diagram..........................................................................33 Figure 6. Main routes for the production of bio-oils from triglycerides. 16 .........................34 Figure 7. General fast pyrolysis of biomass diagram. 21 .....................................................35 Figure 8. Typical oxygenated hydrocarbons in pyrolysis oils. 19 .........................................38 Figure 9. Transesterification process for the production of biodiesel. 36 ............................40 Figure 10. Schematic diagram of an industrial biodiesel production process. 37 ...............41 Figure 11. Reaction pathway for the production of n-alkanes from triglycerides 40 . .........42 Figure 12. Structure of the most common phospholipids in animal fats and vegetable oils. 42 .............................................................................................................................................44 Figure 13. Representation of the different paths of various molecules in a packed chromatographic column. 59 .................................................................................................55 Figure 14. General variation of H versus mobile phase velocity. (Van Deemter Equation). .............................................................................................................................................57 Figure 15. Applications of HPLC 57 . ....................................................................................58 Figure 16. Van Deemter plot for HPLC. ............................................................................59 Figure 17. GPC separation mechanism. 61 ..........................................................................60 Figure 18. Example of a GPC chromatogram with RI detection for a mixture of four different polystyrenes. 61 ......................................................................................................61 Figure 19. CO2 P-T diagram 62 . ............................................................................................63 Figure 20. Block diagram of a SFC ACQUITY UPC 2 System. 64 ........................................64 Figure 21. Van Deemter plots for SFC and HPLC separations in a C18 column. 57 ...........65 Figure 22. Main components of an ICP-MS instrument. ...................................................66 Figure 23. Schematic diagram of a concentric glass nebuliser. 66 ......................................67 Figure 24. Quadrupole mass filter. 80 ..................................................................................69 Figure 25. Double Focusing Magnetic Sector mass analyser diagram. 81 ..........................71 Figure 26. Faraday Cup Diagram. 81 ...................................................................................72 Figure 27. Schematic diagram of an Electron Multiplier detector. ...................................72 Figure 31. Main components of an ICP-OES. ....................................................................73 Figure 32. ICP OES and Rowland circle components. 66 ....................................................74 Figure 33. Photomultiplier tube components diagram. .....................................................75 Figure 35. GPC-ICP-MS / UV-Vis diagram of the set up used during this work. 82 .........76 Figure 36. Diagram of the modified DS-5 total consumption nebulizer. 72 ........................77 Figure 37. SFC-ICP-MS interface proposed by Carey et al. ..............................................88 Figure 38. SFC ICP MS chromatogram at m/z = 52 for a mixture of two Cr compounds. 88 Figure 39. Experimental setup of an electrothermal vaporiser (Adapted from Asfaw et al. 89 ). .................................................................................................................................. 114 Figure 40. ETV-ICP-OES set up. ...................................................................................... 116 Figure 41. Temperature ramp design for the optimisation of the ETV-ICP-OES parameters. ....................................................................................................................... 120

oo

  O1b: Development and validation of an SFC-ICP-MS interface that will contribute to the improvement of the chromatographic resolution of the inorganic speciation analysis in complex samples. o O1c: To develop and validate a Freon-12-free methodology based on the coupling of ETV and ICP-OES for the inorganic speciation analysis of complex matrices based on their vaporisation temperatures. • O2 (instrumental applications): To obtain a better understanding of the composition and chemical structure of the hetero-atomic composition of bio-oils, renewable feedstocks and petroleum heavy oil fractions. This objective has been divided into two sub-objectives: o O2a: To adapt an existing GPC-ICP-MS methodology for the speciation O2b: To adapt an existing GPC-ICP-MS methodology for the speciation analysis of several hetero-atomic containing compounds in renewable feedstocks. SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS Victor GARCIA MONTOTO -21/09/2020 -25-Objectives O1a, O1b and O1c are covered in Chapters 3, 4 and 5, respectively, of this thesis; and the objectives O2a and O2b are covered in Chapters 6 and 7, respectively. This distribution is illustrated in Figure 1.

Figure 1 .

 1 Figure 1. Outline of the objectives covered in this thesis.

•

  Cellulose: Consists of an organic polymer with the formula (C6H10O5)n and it is the most abundant compound in the cell wall of the plant cells. Its structure consists of a straight-chain formed by the condensation of D-glucose monomers through β(1→4)-glycosidic bonds, which are detailed in Figure 2.

Figure 2 .

 2 Figure 2. Chemical structure of cellulose.

  microfibers, while lignin and hemicellulose polymers are inserted in the inner space, as it can be observed from Figure3. Schematic structure of Cellulose, Hemicellulose and Lignin fibres in the cell wall.[START_REF] Wang | Lignocellulosic Biomass Pyrolysis Mechanism: A State-of-the-Art Review[END_REF]

Figure 3 .

 3 Figure 3. Schematic structure of Cellulose, Hemicellulose and Lignin fibres in the cell wall.[START_REF] Wang | Lignocellulosic Biomass Pyrolysis Mechanism: A State-of-the-Art Review[END_REF]

Figure 4 . 14 1. 3 . 2 .

 41432 Figure 4. Schematic representation of some lignocellulosic fast pyrolysis products.[START_REF] Wang | Lignocellulosic Biomass Pyrolysis Mechanism: A State-of-the-Art Review[END_REF]

15

 15

Figure 5 .

 5 Figure 5. Alcoholic fermentation diagram.

Figure 6 .

 6 Figure 6. Main routes for the production of bio-oils from triglycerides.[START_REF] Kordulis | Development of Nickel Based Catalysts for the Transformation of Natural Triglycerides and Related Compounds into Green Diesel: A Critical Review[END_REF]

Figure 7 .

 7 Figure 7. General fast pyrolysis of biomass diagram.[START_REF] Bridgwater | Overview of Applications of Biomass Fast Pyrolysis Oil Overview of Applications of Biomass Fast Pyrolysis Oil[END_REF]

Figure 8 .

 8 Figure 8. Typical oxygenated hydrocarbons in pyrolysis oils.[START_REF] Dhyani | A Comprehensive Review on the Pyrolysis of Lignocellulosic Biomass[END_REF]

1. 5 . 1 .

 51 Figure 9:

Figure 9 .

 9 Figure 9. Transesterification process for the production of biodiesel.[START_REF] Naik | Production of First and Second Generation Biofuels: A Comprehensive Review[END_REF]

Figure 10

 10 Figure 10 depicts a schematic diagram of the industrial process for the production of biodiesel in feedstocks like soybean oil.

Figure 10 .

 10 Figure 10. Schematic diagram of an industrial biodiesel production process.[START_REF] Knothe | The Biodiesel Handbook[END_REF]

Figure 11 .

 11 Figure 11. Reaction pathway for the production of n-alkanes from triglycerides 40 .

Figure 12 .

 12 Figure 12. Structure of the most common phospholipids in animal fats and vegetable oils.[START_REF] Caballero | Encyclopedia of Food Sciences and Nutrition[END_REF]

  The main groups of HDO catalysts can be summarized in sulphides, oxides, transition metals. The type of catalyst, added to other operating conditions such as temperature, residence time and hydrogen pressure, is crucial to obtain a high-value fuel.• Sulphides, such as Co-/NiMoS and Re2S among others, have been widely used in bio-oil (HDO) and crude oil upgrading. One of their advantages is the tolerance to SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS Victor GARCIA MONTOTO -21/09/2020 -50-

Figure 13 .

 13 Figure 13. Representation of the different paths of various molecules in a packed chromatographic column.[START_REF]Multipath) Broadening in Chromatography -Chemistry LibreTexts[END_REF]

Figure 14 .

 14 Figure 14. General variation of H versus mobile phase velocity. (Van Deemter Equation).

Figure 15 .

 15 Figure 15. Applications of HPLC 57 .

Figure 18 .

 18 Figure 18. Example of a GPC chromatogram with RI detection for a mixture of four different polystyrenes.[START_REF] Held | Tricks GPC/ SEC: What Are the Differences Between GPC, SEC, and GFC, and How Do You Get Started with the Technique?[END_REF]

Figure 19 .

 19 Figure 19. CO2 P-T diagram 62 .

Figure 20 explains

 20 Figure 20 explains schematically the main components of a SFC instrument: Two pumps compress the mobile phase fluids up to critical conditions and it is then that they are mixed and sent towards the column through the injector, where the sample joins the

Figure 20 .

 20 Figure 20. Block diagram of a SFC ACQUITY UPC 2 System. 64

Figure 21 .

 21 Figure 21. Van Deemter plots for SFC and HPLC separations in a C18 column.[START_REF] Skoog | Instrumental Analysis Principles[END_REF]

Figure 22 .

 22 Figure 22. Main components of an ICP-MS instrument.

Figure 23 .

 23 Figure 23. Schematic diagram of a concentric glass nebuliser.[START_REF] Skoog | Development of a Sheathless Interface between Reversed-Phase Capillary HPLC and ICPMS via a Microflow Total Consumption Nebulizer for Selenopeptide Mapping[END_REF]

Figure 24 .

 24 Figure 24. Quadrupole mass filter. 80

Figure 25 .

 25 Figure 25. Double Focusing Magnetic Sector mass analyser diagram. 81

Figure 26 .

 26 Figure 26. Faraday Cup Diagram. 81

Figure 27 .

 27 Figure 27. Schematic diagram of an Electron Multiplier detector.

Figure 28 .

 28 Figure 28. Main components of an ICP-OES.

Figure 29 .

 29 Figure 29. ICP OES and Rowland circle components.[START_REF] Skoog | Development of a Sheathless Interface between Reversed-Phase Capillary HPLC and ICPMS via a Microflow Total Consumption Nebulizer for Selenopeptide Mapping[END_REF]

Figure 30 .

 30 Figure 30. Photomultiplier tube components diagram.

Figure 31 .

 31 Figure 31. GPC-ICP-MS / UV-Vis diagram of the set up used during this work. 82

Figure 32 .

 32 Figure 32. Diagram of the modified DS-5 total consumption nebulizer.[START_REF] Giusti | UFlow-Injection-ICP Collision Cell MS Determination of Molybdenum, Nickel and Vanadium in Petroleum Samples Using a Modified Total Consumption Micronebulizer[END_REF]

Figure 33 .

 33 Figure 33. SFC-ICP-MS interface proposed by Carey et al.

Figure 34

 34 Figure 34 shows one of the SFC-ICP-MS chromatograms obtained by Carey et al. with this interface. A 10 ng mixture of two chromium organometallic compounds was injected while using a nitrous oxide mobile phase. They achieved detection limits at the picogram level and high reproducibility levels, which can explain the high intensities of both peaks and their high separation's resolution.

Figure 34 .

 34 Figure 34. SFC ICP MS chromatogram at m/z = 52 for a mixture of two Cr compounds.

Figure 35 .

 35 Figure 35. Experimental setup of an electrothermal vaporiser (Adapted from Asfaw et al. 89 ).

Figure 36 .

 36 Figure 36. ETV-ICP-OES set up.

Figure 37 .

 37 Figure 37. Temperature ramp design for the optimisation of the ETV-ICP-OES parameters.

Figure 38 .

 38 Figure 38. Corrected intensities for each wavelength and normalised vs. reaction gas flow.

Figure 39 .

 39 Figure 39. Relative standard deviation (n = 3) of each wavelength vs. reaction gas flow.

Figure 40 .

 40 Figure 40. Corrected intensities for each wavelength and normalised vs. carrier gas flow.

Figure 41 .

 41 Figure 41. Relative standard deviation (n = 3) of each wavelength vs. carrier gas flow.

Figure 42 .

 42 Figure 42. Corrected intensities for each wavelength and normalised vs. bypass gas flow.

Figure 43 .

 43 Figure 43. Relative standard deviation (n = 3) of each wavelength vs. bypass gas flow.

Figure 44 .

 44 Figure 44. Regression curves obtained for three elements: Ag 338.289 nm (blue), Al 167.078 nm (orange) and K 766.491 (grey).

Figure 45 .

 45 Figure 45. Temperature program for the speciation analysis of trace elements in fast pyrolysis oils via ETV-ICP-OES.

Figure 47 .

 47 Figure 47. ETV-ICP-OES transient signal for Na, Cr, Mn and Ca species in different fast pyrolysis oil samples.

Figure 48 .

 48 Figure 48. ETV-ICP-OES transient signal K, Mg, Al and Zn species in different fast pyrolysis oil samples.

Figure 49 .

 49 Figure 49. Schematic representation of the crude oil distillation cuts.[START_REF] Desprez | Study of the Size Distribution of Sulfur, Vanadium, and Nickel Compounds in Four Crude Oils and Their Distillation Cuts by Gel Permeation Chromatography Inductively Coupled Plasma High-Resolution Mass Spectrometry[END_REF]

6. 2 .

 2 Understanding the removal of V, Ni and S in crude

7. 1 . 7 . 1 . 1 .

 1711 Current trends on inorganic speciation and total analysis of bio-oils Total analysisAs an example of how challenging quantifying inorganic compounds in biomass feedstocks and bio-oils, in 2018, Haldor Topsoe carried out a Round Robin analysis for the determination of different elements in three type of samples which are included in this work. The results of this study agree on the difficulties of achieving good reproducibility among the 12 laboratories that took part in this study. Out of these three samples, one, consisting in a vegetable oil, provided with reproducible results and a very good

Figure 50 .

 50 Figure 50. (A) Determination of Co, Ni and V in a certified reference material by three different methods. (B) Determination of Cu, Fe and Mn in four different samples by GF AAS and ETV-ICP-MS 109 .

Figure 53 .

 53 Figure 53. Results of the analyses of P, S, Na, Mg and Fe (in ppm) for the different laboratories that participated in the round-robin study directed by Haldor Topsoe.104,131

Solution 2 contains

 2 PSs with a MW of 206 000, 67 600 and 27 060 respectively and the Solution 3 contains PSs with a MW of 12 980, 6 660, 1 140 and 162 Da. The peaks are eluted from the highest to the lowest molecular size, which in this case, agrees with their molecular weight.

Figure 54 .

 54 Figure 54. GPC calibration using 10 polystyrene standards dissolved in three different solutions and detected by a UV-VIS detector.

Figure 55 .

 55 Figure 55. GPC calibration curve.

Figure 56 and

 56 Figure56and Figure57show the GPC-ICP-HR MS distribution of sulfur species in six different fast pyrolysis oils. Sulfur's distributions in fast pyrolysis oils are very different from the distributions found in petroleum products. In these renewable feedstocks, the sulfur species possess a slightly lower size (below 30 KDa) and all of them show trimodal profiles of those S-containing species.

Figure 56 .

 56 Figure 56. Sulfur ( 32 S) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from red oak, milorganite and a 50:50 blend of both.

Figure 57 .

 57 Figure 57. Sulfur ( 32 S) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from milorganite, a blend of milorganite and loblolly pine wood and a commercial fast pyrolysis oil sample.

Figure 58 .

 58 Figure 58. Distribution of the S-containing species for the five samples analysed via GPC-ICP-HRMS.

Figure 60 .

 60 Figure 60. Sodium ( 23 Na) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from milorganite, a blend of milorganite and loblolly pine wood and a commercial fast pyrolysis oil sample.

Figure 61 .

 61 Figure 61. Distribution of the Na-containing species for the five samples analysed via GPC-ICP-HRMS.

Figure 62 .

 62 Figure 62. Iron ( 56 Fe) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from red oak, milorganite and a 50:50 blend of both.

Figure 63 .

 63 Figure 63. Iron ( 56 Fe) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from milorganite, a blend of milorganite and loblolly pine wood and a commercial fast pyrolysis oil sample.

Figure 64 .

 64 Figure 64. Distribution of the Fe-containing species for the five samples analysed via GPC-ICP-HRMS.

Figure 65 .

 65 Figure 65. Magnesium ( 24 Mg) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from red oak, milorganite and a 50:50 blend of both.

Figure 66 .

 66 Figure 66. Magnesium ( 24 Mg) size distribution via GPC-ICP-HRMS for pyrolysis oils produced from milorganite, a blend of milorganite and loblolly pine wood and a commercial fast pyrolysis oil sample.

Figure 67 . 7 . 2 . 4 .

 67724 Figure 67. Distribution of the Fe-containing species for the five samples analysed via GPC-ICP-HRMS.

  alternative techniques has been studied and a methodology has been established for the analysis of unconventional oils for each of them: SFC-ICP-MS and ETV-ICP-OES. Further experiments must be carried out in order to improve the quality of the information that these methodologies can provide.Although the results obtained with the use of 2-Picolylamine as stationary phase in the hyphenation of SFC and ICP-MS did not provide with broad information about the speciation of the trace elements studied in the correspondent section of this thesis, the objective O1b has been partially achieved, since a proof-of-concept that comprises a novel interface that allows the hyphenation of modern supercritical fluid chromatographs with ICP-MS has been validated, allowing the use of two co-eluents (CO2 and MeOH) during the separation process. Further experiments, considering the evaluation of different stationary phases should be carried out.With regard to the objective O1c, the development of a new speciation technique has been achieved. ETV-ICP-OES analyses of fast pyrolysis oil samples have been carried out with an alternative reaction gas, less reactive but more environmentally friendly than the former gases that were commercialised (Freon-R12). However, further experiments need to be carried out to understand the speciation of these trace elements in renewable feedstocks. The analysis of standards that resemble those analytes of interest might provide with useful information about the vaporisation temperatures of different chemical structures and families.

  At the same time, cellulose polymers are gathering to form

	SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS
	Victor GARCIA MONTOTO -21/09/2020
	-32-

Table 1 . Overview of the main reactions occurring during HDO. 50

 1

Table 2 . ICP-OES parameters used for all the analysis carried out via ETV-ICP-OES. ICP/AES (SPECTRO ARCOS)

 2

	Observation plasma	Radial
	Power	1650W
	Nebuliser	Sea-spray
	Plasma Ar flow rate	15L/min
	Auxiliary Ar flow rate	1.8L/min
	Integration time	10 ms
	5.2.2. Samples and reagents

THF, Multisolvent GPC-grade, ACS-grade, and stabilised with 250 ppm of butylated hydroxytoluene (BHT) to prevent the formation of peroxides, purchased from Sharlab (Spain) was used to prepare the corresponding dilution of the samples analysed in this work.

Table 3 . R 2 coefficient for each analysed wavelength.

 3

	Element		Element
	(Wavelength in nm)	R 2	(Wavelength in nm)	R 2
	Ag (338.289)	0.990	Mn (260.569)	0.860
	Al (167.078)	0.990	Na (589.502)	0.944
	Ca (422.673)	0.994	Ni (221.648)	0.999
	Cr (267.716)	0.997	Ni (341.476)	0.999
	Cr (283.563)	0.992	P (178.287)	0.997
	Cu (219.226)	0.998	Pb (261.418)	0.999
	Cu (219.958)	0.998	Pb (283.305)	0.996
	Cu (224.700)	0.998	Si (288.158)	0.493
	Fe (259.941)	0.797	Sn (242.949)	0.999
	Fe (373.486)	0.850	V (309.311)	0.897
	K (766.491)	0.997	V (439.523)	0.969
	Mg (279.553)	0.971	Zn (213.856)	0.992

Table 4 . Concentrations in mg kg -1 , R 2 and detection limits in mg kg -1 determined for the SRM 1085b via ETV-ICP-OES.

 4

	Element	Certified Value	Measured Concentration	R 2	DL
	Cr	302.9 ± 2.1	291 ± 55	0.992	0.14
	Cu	295.6 ± 8.5	300 ± 111	0.968	0.27
	Mg	297.3 ± 4.1	345 ± 34	0.997	0.08
	Mn	289	334 ± 87	0.993	0.13
	Ni	295.9 ± 7.4	287 ± 69	0.987	0.17
	Pb	297.7 ± 6.8	247 ± 53	0.991	0.14
	Sn	294	310 ± 85	0.987	0.23
	V	297.8 ± 6.8	290 ± 26	0.991	0.14

Table 5 . Main techniques used for the speciation analysis of pyrolysis oils. 123

 5

	Technique	Properties measured Highlights
	TGA	Loss of mass	• Content of water, carbon, ash,
			cellulose, lignin, etc.

Table 6 . Sample name, type of feedstock and total concentration in ppm of P, S, Na, Fe and Mg.

 6

	Sample	Feedstock	P	S	Na	Fe	Mg
	name
	A	50:50 blend of pinewood and milorganite.	150 n.m. 23	220	42
	B	50:50 blend of red oak wood and milorganite 34	n.m. 19	690	24
	C	Milorganite	260 n.m. 150	320	230
	D	Red oak wood	7.3 n.m. 65	1210 27
	P	Pinewood (commercial sample)
	TRIG.X	Pork fat	105 20.1 7.87 14.3 15.8
	n.m. stands for "not measured".

Table 7 . Definition of the molecular weight interval for the different fractions obtained via GPC- ICP-MS.

 7

	Fraction / Class	Molecular Weight
	HMW	≥ 3152 -91 KDa
	MMW	91 -21 KDa
	LMW	21 KDa -318 Da
	Tailing	≤ 318 Da
	7.2.3.1. Sulfur species.

-27-The appendix of this thesis contains one poster and other manuscripts that were either published or redacted during this three-year project. In two of these manuscripts, which were published in the journal ACS Energy & Fuels, the application of the GPC-ICP-HRMS method presented in this thesis is carried out to obtain a better understanding of the crude oil hetero-atomic species composition. The last manuscript, which is slightly out of the scope of this thesis, was redacted at the University of Copenhagen after having carried out several GC-MS experiments in an arctic station in Greenland, with the purpose of understanding the PAH pollution trends in the sediments of such pristine environment.

Thus, in this thesis, the development and validation of a set of novel tools and methodologies designed for the speciation analysis of bio-oils and renewable feedstocks are investigated. The work that is discussed in this thesis can be broken down into several objectives (O): SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS Victor GARCIA MONTOTO -21/09/2020 -24-• O1 (instrumental development): To introduce, develop and validate novel

RTI

-Research Triangle Institute (U.S.

An O2 gas flow of 0.08 mL/min was delivered into the plasma to avoid carbon deposition on the cones' surfaces due to the introduction of organic matrices in the plasma. Both the instrument and mass calibration settings were optimized daily at resolutions of 300 (low resolution) and 4000. These optimizations were carried out using a tuning solution containing 1.0 ng g-1 of Ag, Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, In, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Sc, Si, Sn, Ti, V, Zn and Y in THF. A mass offset was also applied to compensate for the mass drift coming from the sector field magnet.

Numerous works have been carried out using this modified DS-5 nebulizer for the inorganic speciation in petroleum derivates. [START_REF] Caumette | Fractionation and Speciation of Nickel and Vanadium in Crude Oils by Size Exclusion Chromatography-ICP MS and Normal Phase HPLC-ICP MS[END_REF]76,[83][84][85] However, to carry out speciation analysis in bio-oils, the size distribution of different elements, such as Si, K and Na amongst others, will be studied. The presence of Si within the fused-silica capillary carrying the mobile phase towards the nebulizer might interfere with the future target analytes. Hence, in this chapter, this hypothesis is tested and a new interface is introduced after having been optimised and validated for its use on the speciation analysis of complex samples via GPC-ICP-MS. From this distribution of the phosphorus species based on their evaporation temperature, it is possible to conclude that there exist at least two different species with a very different chemical nature. An early peak is observed for all the samples when the temperature has reached 300 °C, which is the temperature where the pyrolysis process takes place. This fact suggests that the phosphorus species could be covalently bonded to an organic compound. However, this peak is much broader in the tyres pyrolysis oil and it is observed at higher temperatures than for the rest of the samples. While the presence of phospholipids is evident in the feedstocks that were employed to produce the corresponding lignocellulosic fast pyrolysis oil, their presence in tyres might be justified since the tyre's thread has been fabricated from rubber and phosphorus compounds, such as orthophosphates, sugar phosphates and phospholipids that have been found in rubber analysis. [START_REF] Loadman | Inorganic Fillers and Trace Metal Analysis[END_REF] In order to confirm this hypothesis, a standard containing either phospholipids or other phosphate salts should be analysed with this current method: the temperature when the corresponding peaks are detected will determine whether any similarities are found with respect to the phosphorus peaks found in this sample set.

Concerning the peaks observed for the samples C and A (milorganite and the blend of milorganite and loblolly pine wood fast pyrolysis oils respectively) at a temperature of approximately 600 -700 °C, they might be linked to the high presence of phosphorus

SPECIATION ANALYSIS IN RENEWABLE FEEDSTOCKS AND PETROLEUM HEAVY OIL FRACTIONS

Victor GARCIA MONTOTO -21/09/2020 -129-pentoxide in milorganite. 100 However, although the presence of this compound is expected in the milorganite:red oak blend pyrolysis oil, no peak is observed. Further analysis should be carried out, considering the analysis of P2O5 via ETV-ICP-OES to either confirm or reject this hypothesis, since another potential cause of the presence of phosphorus in pyrolysis oils would be inefficient removal of ashes within the sample matrix during the fast pyrolysis process. [START_REF] Tan | Phosphorus Recovery from the Biomass Ash: A Review[END_REF] In Figure 47, the transient signals obtained via ETV-ICP-OES for Na, Cr, Mn and Ca are shown for the same set of samples.

These four elements present almost the same distribution: only one peak with different widths presented for each sample, which agrees with the hypothesis that these species share the same chemical structure or reactivity with the reaction gas and suggests that the same compound is present on all the samples. The presence of Na and Ca, which are very important nutrients in plants and essential element in the cell walls, is therefore justified, whereas the presence of Mn and Cr is assumed to be originated from the presence of these elements in soils as salts.

Furthermore, these trace elements should be linked to non-volatile compounds: these peaks are detected when the graphite furnace was working at high temperatures (above 1000 °C), which agrees with the hypothesis that suggests that the presence of these trace elements in the pyrolysis oil samples is originated from the salts that are present in the soils.

performance concerning the quantification of inorganic contaminants (Al, Ca, Fe, K, Na, Mg, P, Si and Zn). However, concerning the other two samples, a pyrolysis oil produced from pine wood and an animal fat (pork tallow), the results show clearly how the large deviations obtained among the different laboratories make the analysis of these samples a highly challenging task. 104 Some of these results have been reported in the Section 5.2.1. of this thesis.

Biodiesel, bioethanol and their feedstocks

Acid digestion before ICP-MS analysis is one of the most used analytical techniques for the quantification of metals and other elements in the whole spectrum of bio-oils and their feedstocks. From vegetable oils and animal fats, up to biodiesel samples follow the same guidelines for their total analysis.

In vegetable oil feedstocks, several ICP-MS quantification methods have already been developed. Like many other environmental samples, acid digestion is a good choice of sample preparation before ICP-MS analysis. For example, two research groups, Llorent et al. 105 and Benicasa et al. 106 investigated the detection of trace elements in vegetable oils and developed methods where the samples were microwave digested by using nitric acid prior to ICP-MS analysis. However, this methodology is not the only one for the total analysis of trace elements in vegetable oils. Different authors developed and optimised different sample preparation procedures, such as Jimenez et al. 107 and Castillo et al. 108 , who developed a sample preparation procedure where the vegetable oil samples were mixed with Triton-X emulsified and then, directly introduced into the plasma.

The sample introduction method can change to perform total analysis. This is the case of the study that Chaves et al. 109 have carried out by using ETV-ICP-MS for the determination of Co, Cu, Fe, Mn, Ni and V in diesel and biodiesel samples, using a fast gradient for the quantification of the analytes and having performed a sample emulsion preparation before the analyses. Good linear correlations and low LODs and LOQs (at the order of ng g -1 ) were obtained, suggesting that ETV as a method for sample introduction is trustworthy for the analysis of samples with complex matrices. Figure 50 describes in bar plots the accuracy regarding the concentrations found for different elements by using different techniques, which is practically similar to the figures obtained for GF-AAS. The information obtained from both the increase and decrease of Na, K, Ca and Mg respectively within the bioethanol production process can facilitate the determination of the source of these emerging contaminations and study their degradation and elimination.

A similar method has been reported for sorghum biomass samples and their metal distribution along the bio-oil process. 122 However, further information might be obtained when coupling HPLC-ICP-MS to each one of the collected samples, in term of obtaining a fingerprint based on their polarity, size, etc., depending on the stationary phase and the solvents used to this end.

Pyrolysis Oils

Even though numerous analytical methods for the characterisation of pyrolysis oil samples have been developed, there are not a high abundance of speciation methods focusing on the trace elements present within these samples. A review of some of these methods have been written by Kanaujia et al. 123 , and some of the main techniques used for the general speciation analysis are resumed in Table 5.

Speciation analysis of trace elements in renewable feedstocks via GPC-ICP-HR MS

Samples, methodology and instrumentation

As it was demonstrated in Chapter 4, GPC-ICP-HRMS is a powerful technique that can provide with very useful information about the size distribution of numerous trace elements. Thus, in this chapter, the application of the already developed methodology is carried out in a set of renewable feedstock samples.

Speciation analyses based on their molecular size are carried out for phosphorus, sulfur, sodium, iron and magnesium in five lignocellulosic fast pyrolysis oil samples and three fatty acid feedstocks.

Five lignocellulosic fast pyrolysis oils were provided by Haldor Topsoe (Denmark) and RTI (United States). To elaborate these fast pyrolysis bio-oils, woods from red oak, loblolly pine and pine (commercial sample) were used. Besides, three fatty acid samples, consisting of two types of unprocessed animal fat (pork and tallow) and a soybean oil were also provided by Haldor Topsoe.

The origin of the lignocellulosic fast pyrolysis oils is described below:

• Sample A: Pyrolysis oil produced from a 50:50 mixture of milorganite and loblolly pine wood.

• Sample B: Pyrolysis oil produced from a 50:50 mixture of milorganite and red oak wood.

• Sample C: Pyrolysis oil produced from milorganite fertilizer.

• Sample D: Pyrolysis oil produced from red oak wood.

• Sample P: This sample consists of a commercial pyrolysis oil sample produced from pine wood.

The origin of the fatty acid feedstocks used in this chapter is described below:

• Sample AF: Unprocessed tallow fat.

A trimodal distribution is observed as well for all the pyrolysis oils except for red oak pyrolysis oil, which presents a multimodal distribution with, at least, five different nonresolved peaks.

Red oak pyrolysis oil presents some compounds eluting at very early retention times and suggesting that Na-containing compounds with a molecular weight higher than 91 KDa are present within this sample. These compounds, however, do not seem to be present when milorganite is blended to with red oak wood to produce a fast pyrolysis oil.

Another difference that can be observed between the Na-containing and the S-containing compounds is the absence of an intense peak near the permeation volume of the column, where the solvent peak was detected for the PS standards during the mass calibration of the columns. The absence of this peak could suggest that the sodium present within these samples might be strongly linked to other compounds via complexation. The contribution of each fraction with respect to the total area of the chromatogram was also calculated for the Na-containing species that were eluted from the GPC columns. The