Browsing by master's degree program "Kemian ja molekyylitieteiden maisteriohjelma"

The glymphatic system is a waste clearance system in the brain to prevent protein accumulation that negatively affects neural functions, leading to neurogenerative diseases such as Alzheimer's. Perivascular spaces surround the brain vasculature allowing cerebrospinal fluid (CSF) inflow into the brain parenchyma during NREM sleep. The CSF moves from perivascular spaces into the white matter through AQP4 water channels, and flows towards the efflux routes, from where the interstitial solutes are drained into the lymphatic system. The existence and functions of the glymphatic system raise controversies due to the lack of quantitative data. Imaging tools that do not negatively affect the flow are required to visualize the glymphatic system in health and disease. If the glymphatic flow could be intensified in a preventive or therapeutic manner or harness for CNS drug delivery, it would be revolutionary. Positron emission tomography (PET) is a rising imaging modality in glymphatic research. It provides an efficient and non-invasive method, even with nanomolar tracer concentrations, to follow the CSF within the entire animal, being also fully translatable for human studies. Albumin is the dominant protein the CSF and can act as a carrier for tracers extending the circulation time. More recently, truncated Evans Blue (EB) and 4-(p-iodophenyl)butyric acid (IP) have shown promising results in modulating the pharmacokinetics of radiopharmaceuticals through albumin-binding. This study aimed to develop and evaluate six new albumin- binding tracers ( three NODAGA conjugated and three DFO conjugated) for the PET imaging of the glymphatic fluid flow via binding of endogenous albumin in the CSF. The precursors were synthesized using coupling reactions and radiolabeled with [68Ga]GaCl3 (0.2M sodium acetate buffer, pH=4, 95°C or 25°C, 15 min). Radiochemical purities were determined by radio-TLC and radio-HPLC and LogD (octanol:PBS) with shake-flask method. The in vitro stability assays were done in rat serum, rat CSF, 2 mM EDTA, and 0.2 mM FeCl3 solutions at 37°C over 6 h of incubation. The in vitro albumin binding affinities were investigated at physiological rat CSF albumin concentration at 37°C over 1 h of incubation using radio-SEC- HPLC. The in vivo and ex vivo experiments of the three most stable tracers were done using healthy female Swiss mice, and the experiments included blood kinetic studies, ex vivo biodistribution studies, and analysis of urine, bile, and blood fractions (plasma, blood cells, proteins) for in vivo stability. The precursors were synthesized with high yields (69–96%) and radiolabeled with high radiochemical yield (64– 82%), radiochemical purity (97–99.5%), and molar activities sufficient for low-volume infusion into the CSF (9.2– 17.2MBq/nmol). The NODAGA conjugated tracers had higher radiochemical yield and molar activity than the DFO conjugated tracers. The radiolabeled DFO conjugated products were stable in rat serum and rat CSF, but stability assays with EDTA and FeCl3 showed major transchelation. The NODAGA conjugated tracers were stable in each medium, maintaining the percent of intact labeled compound above 97%. The albumin binding affinity studies showed full binding from the first time point of 5 min incubation until the last time point of 1 h. The blood kinetic studies of the three stable NODAGA conjugates showed decreasing %ID/g with varying biological half- lives depending on the tracers’ lipophilic properties. The ex vivo analysis of urine and bile showed eliminated free tracer or some metabolite of the free tracer with similar retention time with radio-HPLC, no free gallium was observed. The analysis of blood samples after fractionation showed that each tracer had the most significant %ID/g in the protein fraction, and no activity was observed in the plasma. The in vivo investigated tracers showed promising properties for possible future use as CSF tracer due to high radiolabel stability and rapid stable binding to albumin in vivo.

At low concentrations, biogenic amines (BA) promote natural physiological activity, but at higher concentrations they can cause a wide variety of health hazards, especially for more sensitive individuals. The BA determination in wine is challenging due to the variation in physicochemical properties and the potential matrix effects of other compounds in the sample. It is important to develop efficient sample purification methods to minimize matrix interference. Derivatization is required for most biogenic amines due to the absence of chromophores. The conditions that promote the origin or formation of biogenic amines in wines are not yet fully understood, as many factors contribute to their formation. The main sources or stages of BA formation during wine-making should be identified in order to reduce BA levels by corrective measures. Currently, the analytical community is striving for more environmentally friendly methods. The literature review examines methods for determination of biogenic amines in wines from 2005 until 2020. The methods are high-performance liquid chromatography, ultra-high-performance liquid chromatography, high-temperature liquid chromatography, nano-liquid chromatography, micellar liquid chromatography, capillary electrophoresis, micromachined capillary electrophoresis, gas chromatography, immunoassay, sensor, colorimetric method, thin-layer chromatography and ion chromatography. The health disadvantages of biogenic amines and the problem areas associated with their determination from a complex wine matrix, such as matrix effect and derivatization, are also surveyed. In addition, changes in the BA profile during different stages of winemaking and storage, as well as the effect of the grape variety and lactic acid bacterial strain on the BA profile, are surveyed. Validation determines the suitability of a method for its intended use. In the methods for determining the literature review, measurement uncertainty - possibly the most important validation parameter - had not been determined in any of the validations. The aim of the research project was to obtain a functional and validated method for the determination of biogenic amines in wines for the Alcohol Control Laboratory at Alko Inc. In the method tested, histamine, tyramine, putrescine, cadaverine, phenylethylamine and isoamylamine derivatized with diethyl ethoxymethylene malonate were determined by high-performance liquid chromatography and diode array detector. The method was not sufficiently reliable, so a competitive enzyme-linked immunosorbent assay for the determination of histamine in wines was introduced, which provided a useful method for the Alcohol Control Laboratory. The validation determined specificity/selectivity, recovery, repeatability, systematic error, estimation of random error, measurement uncertainty, expanded measurement uncertainty, limit of detection and limit of quantification. The European Food Safety Authority has confirmed histamine and tyramine as the most toxic amines. The International Organization of Vine and Wine has not set legal limits for BA levels, but some European countries have had recommended maximum levels for histamine. Many wine importers in the European Union require a BA analysis even in the absence of regulations. Based on the literature review, high BA levels were found in the wines under study, including levels of histamine, tyramine, and phenylethylamine that exceeded the toxicity limits. Some wines had biogenic amines below the detection limit, so the production of low-amine wines is possible. In addition, certain strains of lactic acid bacteria were found to significantly reduce the BA levels in wine. High-performance liquid chromatography is the most widely used determination method. An increasing trend is to develop simpler methods such as the portable sensor-based method.

As nuclear reactors come to the end of their lifetime, they undergo decommissioning. All the construction materials and spend nuclear fuel must be characterized according to their activities before final disposal. For the analysis of difficult to measure radionuclides, such as beta emitters, sample decomposition methods are used. Acid digestion, alkaline fusion and thermal combustion are examples of sample decomposition methods. In this work, alkaline fusion methods were tested to gain knowledge on the suitability of the method for volatile and non-volatile radionuclides in decommissioning waste. The sample materials tested were concrete and two difficult to dissolve reference materials: soil and coal fly ash. Results obtained with alkaline fusion were compared with that of routinely used acid digestion method. In addition, activated graphite was studied to familiarize with the analysis of difficult to measure radionuclides. Non-volatile 63Ni and 55Fe and volatile 3H and 14C were studied. The results were compared with modelled activities. Alkaline fusions were performed with a borate flux in an automated fusion oven as well as with a carbonate flux in a muffle furnace. Acid digestion was performed in an open vessel with aqua regia. The dissolved samples were analysed with ICP-OES and ICP-MS for determination of elemental concentrations. Borate fusion seemed to be the most effective method, as it was suitable for all the sample materials and released even refractory elements. Some of the volatile elements were lost during the fusion methods (Cd and Zn), while some were not affected by the high temperatures used (Pb). Alkaline fusion methods appeared to be suitable for decommissioning studies, however further research is needed. In analysis of graphite, non-volatile elements were analysed by acid digestion. 55Fe and 63Ni were separated from the matrix by hydroxide precipitation and from each other by ion exchange in an AG-resin. Ni fraction was further purified by extraction chromatography with a Ni-resin. Volatile elements were analysed by thermal combustion in a pyrolyser. The activities of 55Fe, 63Ni, 3H and 14C were measured with liquid scintillation counting. The measured activity concentrations of 63Ni were partially in agreement with the modelled values, but some samples gave higher activities. No 55Fe activities were found in any samples, as was expected based on the modelled values and the short half-life of the radionuclide. Activities of the volatile 3H and 14C were accurate and close to the modelled values. In the future, the determined activity results will be used for improving scaling factors, for the assessment of radionuclide concentrations in graphite.

The literature part of the thesis gives an overview of the occurrence, properties, biological activities, and analytical approaches used for the quantification of resin acids and their derivatives in tree resins, with the emphasis being on a balanced discussion of specific analytical merits and limitations. The quantification of resin acids is challenging due to their isomeric nature and limited stability, with these marker compounds being sensitive to light, oxygen, and strong acids. Methods employing reversed phase liquid chromatographic (LC) approaches in combination with UV/Vis, fluorescence, and mass spectrometric (MS) detection are covered. Gas chromatographic (GC) approaches, involving various derivatization schemes, are discussed, with detection being typically achieved by flame ionization (FI) and MS. Less frequently employed protocols, utilizing zone capillary electrophoresis with cyclodextrin additives and UV and/or fluorescence detection (FLD) are reviewed. Finally, emerging protocols employing supercritical fluid chromatography in combination with FI and MS detection are outlined. Baseline resolution of all major resin acids can only be readily achieved with GC and supercritical fluid chromatography (SFC) methods, while LC and CE protocols suffer from partial peak overlap. In terms of practical convenience, SFC may provide currently the most favorable analytical approach, obviating the need for derivatization and allowing for superior sample throughput. The objective of the experimental part of the thesis was to develop robust and expediate HPLC/UV methods for the detection and quantification of selected active marker compounds, i.e., vanillin, p-coumaric acid, pinoresinol and dehydroabietic acid, in Norwegian spruce resin extracts. Emphasis was placed on obtaining protocols characterized by operational simplicity and ready transferability to production settings. The developed methods involved continuous hot extraction of milled resin samples with 96% EtOH, followed by a simple filtration step and isocratic reversed phase HPLC/UV analysis. A preliminary validation of the developed reversed phase HPLC/UV methods was carried out to assess the limits of detection and quantification (LOD and LOQ); calibration linearity/range; precision and accuracy. LOD and LOQ values were lower than 3 µg/mL and 8.5 µg/mL for all quantified active marker compounds, respectively. To demonstrate the applicability of the developed methods, two batches of Norwegian spruce resin samples were analyzed in three replicate samples.

The analysis of volatile organics is growing by the year and there is a great interest in fast and simple sample preparation techniques. With solid phase micro-extraction, samples can be extracted non-destructively without a need for solvents. This is both cost effective and ecological, because even most eco-friendly solvents still cause strain on the environment. This thesis focused on studying the effect of extraction conditions on the extraction efficiency. The effect of different sorptive phase materials was tested as well. New single-step sample extraction and preparation method was developed for gas chromatographic mass spectrometric analysis. Three different sorptive phase materials were compared and the extraction conditions were optimized for each. The method developed was used to extract, analyze and determine unknown compounds from a butterfly specimen. Multiple extractions were performed from both headspace and with direct immersion. By progressively changing the extraction conditions, properties of the compounds such as volatility and polarity could be determined by their presence alone. Analysis was performed using with gas chromatography mass-spectrometer using electron ionization quadrupole mass detector in full scan mode.

Novichok nerve agents are persistent, highly toxic chemical weapons which were added to the Chemical Weapons Convention in 2020 after their use on civilians in England. The detection and characterization of Novichok nerve agents and the degradation products formed after their exposure to decontamination products can be accomplished through complementary instrumental analyses. Chromatographic methods such as LC-MS/MS can be utilized to qualitatively detect Novichok degradation products such as hydrolysates and LC-HRMS can provide information about their structure via the elemental composition and fragmentation pathways. By contrasting these data to spectroscopic techniques such as 1H and 31P NMR, structural elucidation of decontamination products is possible as well as the determination of the kinetics of the decontamination process itself. The literature review contains a summary of all published instrumental methods with which Novichok nerve agents, degradation products, biomarkers and adducts have been analyzed and the efficacy of those methods. In the experimental research, Novichok nerve agent A-234 was decontaminated via six different decontaminants and analyzed by LC-MS/MS to identify the mass spectra of the degradation products of each, followed by LC-HRMS analysis to determine the elemental composition and fragmentation patterns of the degradation products. The A-234 rate of hydrolysis kinetics were measured by 1H and 31P NMR spectroscopy in three of the decontaminants and when possible, two dimensional analysis was used to correlate the structural data from the chromatographic analysis. Lastly, the A-234 hydrolysate was derivatized via TMSDAM methylation for GC-MS/MS analysis after testing with two silylating and three methylating agents. Decontamination of A-234 was successful within 48 hours with three decontamination agents and complete hydrolysis was observed within 5 hours with an oxidizer-containing quaternary salt based decontamination agent.

Biomacromolecules are large particles found in biological fluids. The upregulations and downregulation of some biomacromolecules, such as extracellular vesicles (EVs) have been linked to cancer and infectious diseases. The study of these biological particles can help us in understanding the progression of those conditions better. Furthermore, studying naturally occurring biological molecules, e.g., immunoglobulin G (IgG), DNA, nucleic acids and glycoproteins can help us to gain more insight to important biological processes in the human body. The first part of this thesis is a literature review of monolithic columns in the separation of large biological molecules in liquid chromatographic and capillary electrochromatographic applications. Columns, including novel monolithic stationary phases, also known as monoliths, have been developed to counter some of the problems associated with the traditionally used packed beds in separation science. Monoliths have a unique structure of interconnecting porous channels, which allows faster separation with better resolution, reproducibility and mass transfer characteristics compared to packed beds. Organic-based polymer monoliths are the most widely used monolithic materials in biological applications, but the use of inorganic-based silica monoliths and hybrid monoliths have grown in the last couple of decades. Monolithic columns are versatile and they can be utilized in several chromatographic techniques, such as reversed-phase chromatography, affinity chromatography, ion-exchange chromatography, capillary electrochromatography and mixed-mode chromatography. Due to the growing interest, miniaturized monoliths e.g. in microfluidic devices, small capillaries and microarrays have been exploited to allow faster separation using sample volumes even as low as a few femtolitres. For higher sample throughput, monoliths in the format of 96-well plates, tips, sheets and disks have been introduced, especially for sample pre-treatment purposes. In the experimental part, affinity monolithic chromatography was employed for the isolation of lipoproteins and EVs in both exomere and exosome size range. The main function of EVs is transporting signal molecules from cell-to-cell to maintain homeostasis of the body. Low-density lipoprotein (LDL), very-low-density lipoprotein and chylomicrons are lipoproteins that transport different lipids in the human blood stream. The study of these particles is important because lipoproteins and especially LDL have been associated with atherosclerotic cardiovascular diseases. The experimental part of this thesis is focused on studying the feasibility of Convective Interaction Media (CIM) monoliths in disk (1.3 µm pores, 0.34 ml) and 96-well plate (2.1 µm pores, 0.1 ml) formats in purifying nanosized biomacromolecules from human plasma. The preparation of the affinity monoliths and the isolation of particles in the disk format was conducted following existing protocols and methods, which were modified for the monolithic 96-well plate. Six different monoclonal antibodies (mAbs), anti-CD9, anti-CD34, anti-CD61, anti-CD63, anti-CD81 and anti-CD82 were immobilized on the monolithic supports to target EVs. Anti-apoB100 mAb was used in targeting apolipoprotein B100 present on the surface of apoB100-containing lipoproteins. The isolation in the disk format was done using an on-line immunoaffinity chromatography – asymmetric flow field-flow fractionation method connected to ultraviolet, dynamic light scattering and diode array detectors. To compare the two different formats with different pore sizes in lipoprotein and EV isolation, the immobilization protocol and isolation conditions were optimized for the monolithic well plate. The isolation on the monolithic 96-well plate was done within 20 minutes, and the operation consumed three times less sample and buffer than in the disk format. Both monolithic formats were suitable for LDL isolation and the disks could also be used in EV isolation and separation. However, due to the larger pore size, EVs were found to be unstable in the monolithic wells.

Kirjallisuuskatsauksessa käydään läpi erilaisia menetelmiä C3-substituoitujen indolien synteeseihin 2-alkenyylianiliinityyppisistä lähtöaineista, joiden bentsyylinen asema oli substituoitu. Erityistä huomiota kiinnitetään menetelmiin, joiden reaktiomekanismeiksi ehdotettiin radikaalimekanismeja. Myös näitä ehdotettuja radikaalimekanismeja esitellään tutkielmassa. Kokeellisessa työssä tutkittiin C3-subsituoitujen indolien hapettavaa synteesiä hiilikatalyytin avulla. Lähtöaineina käytettiin bentsyylisesti aryylisubstituoituja 2-alkenyylianiliinijohdannaisia. Joidenkin lähtöaineiden typpeen oli kiinnitetty metoksipyridiini, jonka kiinnitystä varten kehitettiin Buchwald-katalyysi. Hiilikatalyysit tuottivat hyviä saantoja. Korkea elektronitiheys, etenkin aniliinin bentseenirenkaan ja/tai typen aromaattisen substituentin korkea elektronitiheys, oli eduksi. Reaktion mekanismin ehdotetaan alkavan hapettumisella radikaalikationiksi, ja näitä hapetuspotentiaaleja laskettiin aiemmin raportoidun menetelmän mukaisesti. Mikäli indolin 5-renkaan substituutiot (N1, C2, C3) olivat tarpeeksi samankaltaisia, korkea elektronitiheys, matala hapetuspotentiaali ja hyvä saanto korreloivat. Indolin 5-renkaan substituutio on kuitenkin merkittävämpi tekijä kuin hapetuspotentiaali ja/tai korkea elektronitiheys. Pyridiini typen suojaryhmänä toimi katalyysissä ja se onnistuttiin poistamaan helposti. Metoksipyridiini toimi katalyysissä hyvin, mutta sen kvantitatiivinen poistaminen ei onnistunut.

This paper features two parts; a literature review discussing the recent development in using electrochemical gas sensors for pollutant detection and the use of sensor nodes in real-life locations, and an experimental section focusing on the kinetic study of nitrogen containing compounds utilizing in-tube extraction device. Growing interest towards personal safety have led to development of low-cost electrochemical sensors for personal safety, indoor air quality and leak detection applications. Heterojunctions and light illumination have emerged as an effective way to improve sensor performance, but the selectivity of electrochemical sensors remains relatively poor. Multiple sensors can be combined to create ‘E-noses’ which significantly improve the selectivity and compound identification. These E-noses have been deployed in some indoor locations, either being stationary in sensor networks or moved around by a robot or drone. All approaches have benefits and caveats associated to them, with the differences between individual sensors limiting sensor network use, and slow response and recovery times limiting the use of moving sensors. A novel micropump system was constructed to be used in the active air sampling together with in tube extraction (ITEX) and thermal desorption gas-chromatography (TD-GC-MS). The repeatability of this method was tested in a kinetic study of 10 selected nitrogen containing compounds in a custom-built permeation chamber. The breakthrough times and volumes of the compounds were investigated. Kinetic modelling was successful for 9 out of the 10 compounds with 1 compound behaving significantly different from the rest. The breakthrough times were always over 20 minutes and breakthrough volumes were around the 1000 ml region. Reproducibility was tested with multiple ITEX’s and samples were taken from five indoor locations. Three of the tested compounds were found in some of the samples.

As part of the SAFER2028, ABCRad (Alternative Buffer/Backfill Characterization and Radionuclide Interactions) research project, two alternative bentonite materials supplied by Posiva Oy were investigated in this thesis. The aim of the thesis was to investigate and determine the sorption behavior of these two buffer material candidates, with a deliberate reference to a well- known Na-Wyoming type bentonite serving as a benchmark. In order to closely imitate conditions relevant to repository settings, a synthetic reference water was prepared, and the experiments were conducted within a glove box in N2 atmosphere excluding CO2 and O2. This thesis provides valuable perspectives on the behavior and attributes of the alternative bentonite materials, which is crucial for guiding decisions in the design of repositories for radioactive waste and strategies for managing spent nuclear fuel. More specifically, this thesis provides thermodynamic sorption models (TMS) for two risk-driving radionuclides, uranium (U) and cesium (Cs). Batch sorption isotherms were made using a 1:20 solid-to-liquid ratio including 0.5 g of bentonite in 10 cm3 of reference water. Gamma spectroscopy and Liquid Scintillation Counting (LSC) were employed for the analysis of reaction supernatants. Complementary to these techniques, additional bentonite properties, including Cation Exchange Capacity (CEC) and Exchangeable Cations (EC), were determined. Pre-characterization was done for the bentonites using Fourier Transform Infrared Spectroscopy (FTIR) and the Specific Surface Areas (SSA) were determined. These analyses provide a comprehensive characterization of the alternative backfill materials under investigation. This thesis focuses on combining quantitative sorption data (e.g., distribution coefficient, Kd) with mechanistic understanding (e.g., FTIR spectroscopy). This contributes to an improved understanding of radionuclide sorption mechanisms, thereby bolstering safety considerations. The CEC determined for the bentonites, Laviosa, LMS, and Na-Wyoming were 87 (±0,048) meq/100 g, 95 (±0,34) meq/100 g, and 91 (± 1,23) meq/100 g, respectively. The distribution coefficient (Kd) values of uranium ranged from 130–135 cm3/g with Laviosa and 78–110 cm3/g with LMS, while those of cesium ranged from 130–280 cm3/g with Laviosa and from 150–425 cm3/g with LMS. Cesium demonstrated sorption of 95% within the 10-10 to 10-6 M range, decreasing slightly to 85–95% at concentrations up to 10-2 M. Uranium showed sorption in the range 80–100% across both clays, peaking at lower concentrations and declining at higher concentrations. These data align with those of the reference buffer material, indicating that these bentonites could potentially serve as feasible alternatives if they exhibit additional favorable sorption capacity with other risk-driving radionuclides (e.g., Eu, Ni, Th).