Browsing by Subject "Kemian ja molekyylitieteiden maisteriohjelma"

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  • Sachan, Sinivuokko (Helsingin yliopisto, 2021)
    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.
  • Elliott, Todd (Helsingin yliopisto, 2019)
    An investigation into switchable polarity ionic liquids was carried out to find greener alternative substituents and still obtain a switchable polarity ionic liquid. First for fluorinated compounds (fluorinated alcohol and amine) with a non-fluorinated hydroxylamine to form a mixed carbamate, then replacing the superbase with a basic tertiary (or secondary) amine. The trigger molecule for switching polarity was CO2. It was found that O-hexylhydroxylamine was a suitable replacement for fluorinated ethanol and fluorinated ethylamine to work with DBU (superbase) to form a switchable polarity ionic liquid. The three amines of triethylamine (TEA), diisopropylethylamine (Hünigs base) and diisopropylamine (DIPA) were inconclusive or unsuccessful. Both TEA and DIPA require further alternative analysis for a conclusive result while Hünigs base was proven to be unsuccessful. These reaction products were characterised with 1H and 13C NMR and ReactIR spectral data. Synthesis of hydroxylamine was also approached for a greener improvement. A new synthesis method is demonstrated that is successful using water and methylamine in ethanol working on reaction equilibria. The new method proposed had a yield of 29.1%, while the patent literature method that used hydrazine monohydrate (which is highly toxic and unstable unless in solution) gave a yield of 54.3% of hydroxylamine. A secondary investigation was also undertaken in to basicity effects of caesium carbonate on the CO2 addition to aniline, with and without a superbase present. The superbase used was tertramethylguanidine (TMG). Aniline, p-nitroaniline and p-methoxyaniline were tested for CO2 addition by formation of an amide peak in ReactIR. There was formation of the amide peak with caesium carbonate, though not as much as with the already known TMG. A concentration series of caesium carbonate and TMG in aniline was also devised to observe the effect the added caesium carbonate had on the aniline-TMG system in absorbing CO2. This was also analysed using ReactIR spectra. It was seen generally that by increasing the concentration of both/either TMG/Cs2CO3 there is an increase in carbamate. However further concentration series data is required before a generalised rule can be defined.
  • Heiskanen, Ilmari (Helsingin yliopisto, 2021)
    Interest towards indoor air quality has increased for several decades from human health perspective. In order to evaluate the quality of indoor air in terms of volatile organic compound (VOC) levels, robust analytical procedures and techniques must be used for indoor air VOC measurements. Since indoor building materials are the greatest source of indoor VOC emissions, same kind of procedures must be used for analysis of emission rates from building materials and their surfaces. Theory part of this thesis reviews background of VOCs and human health, legislation and guideline values, common building materials with emissions and used sampling techniques/approaches for indoor air sampling and surface material emission rate sampling & analysis. Discussed sampling techniques include, for example, material emission test chambers, field and laboratory test emission cells, solid phase microextraction (SPME) fibre applications and Radiello passive samplers. Also new innovative approaches are discussed. Used common analysis instruments are Gas Chromatography (GC) with Mass Spectrometer (MS) or Flame Ionization Detector (FID) for VOCs and High-Performance Liquid Chromatography-Ultraviolet/Visible light detector (HPLC-UV/VIS) for carbonyl VOCs (e.g. formaldehyde) after suitable derivatization. Analytical procedures remain highly ISO 16000 standard series orientated even in recent studies. In addition, potential usage of new modern miniaturized sample collection devices SPME Arrow and In-tube extraction (ITEX) used in experimental part of this thesis are discussed as an addition to indoor air and VOC emission studies. The aim of the experimental part of this thesis was to develop calibrations for selected organic nitrogen compounds with SPME Arrow and ITEX sampling techniques and test the calibration with indoor and outdoor samples. A calibration was successfully carried out with SPME Arrow (MCM-41 sorbent), ITEX (MCM-TP sorbent) and ITEX (Polyacrylonitrile (PAN) 10 % sorbent) with permeation system combined with GC-MS for the following selected organic nitrogen compounds: triethylamine, pyridine, isobutyl amine, allylamine, trimethylamine, ethylenediamine, dipropyl amine, hexylamine, 1,3-diaminopropane, 1-methyl-imidazole, N, N-dimethylformamide, 1,2-diaminocyclohexane, 1-nitropropane and formamide. The overall quality of the calibration curves was evaluated, and the calibrations were compared in terms of linear range, relative standard deviation (RSD) % for accepted calibration levels and obtained Limits of Detection (LOD) values. Also, ways to improve the calibrations were discussed. The calibration curves were tested with real indoor and outdoor samples and quantitative, as well as semi-quantitative, results were obtained.
  • Kivinen, Anssi (Helsingin yliopisto, 2020)
    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.
  • Koivula, Juho (Helsingin yliopisto, 2021)
    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.
  • Martinmäki, Tatu (Helsingin yliopisto, 2020)
    Tiivistelmä – Referat – Abstract Molecular imaging is visualization, characterization and quantification of biological processes at molecular and cellular levels of living organisms, achieved by molecular imaging probes and techniques such as radiotracer imaging, magnetic resonance imaging and ultrasound imaging. Molecular imaging is an important part of patient care. It allows detection and localization of disease at early stages, and it is also an important tool in drug discovery and development. Positron emission tomography (PET) is a biomedical imaging technique considered as one of the most important advances in biomedical sciences. PET is used for a variety of biomedical applications: i.e. imaging of divergent metabolism, oncology and neurology. PET is based on incorporation of positron emitting radionuclides to drug molecules. As prominent radionuclides used in PET are of short or ultra-short half-lives, the radionuclide is most often incorporated to the precursor in the last step of the synthesis. This has proven to be a challenge with novel targeted radiotracers, as the demand for high specific activity leads to harsh reaction conditions, often with extreme pH and heat which could denature the targeting vector. Click chemistry is a synthetic approach based on modular building blocks. The concept was originally developed for purposes of drug discovery and development. It has been widely utilized in radiopharmaceutical development for conjugating prosthetic groups or functional groups to precursor molecules. Click chemistry reactions are highly selective and fast due to thermodynamic driving force and occur with high kinetics in mild reaction conditions, which makes the concept ideal for development and production of PET radiopharmaceuticals. Isotope exchange (IE) radiosynthesis with trifluoroborate moieties is an alternative labeling strategy for a reasonably high yield 18F labeling of targeted radiopharmaceuticals. As the labeling conditions in IE are milder than in commonly utilized nucleophilic fluorination, the scope of targeting vectors can be extended to labile biomolecules expressing highly specific binding to drug targets, resulting to higher contrast in PET imaging. A trifluoroborate functionalized prosthetic group 3 was synthetized utilizing click chemistry reactions, purified with SPE and characterized with HPLC-MS and NMR (1H , 11B-, 13C-, 19F-NMR). [18F]3 was successfully radiolabeled with RCY of 20.1 %, incorporation yield of 22.3 ± 11.4 % and RCP of >95 %. TCO-functionalized TOC-peptide precursor 6 was synthetized from a commercial octreotide precursor and a commercially available click chemistry building block via oxime bond formation. 6 was characterized with HPLC-MS and purified with semi preparative HPLC. Final product [18F]7 was produced in a two-step radiosynthesis via IEDDA conjugation of [18F]3 and 6. [18F]7 was produced with RCY 1.0 ± 1.0 %, RCP >95 % and estimated molar activity of 0.7 ± 0.8 GBq/µmol. A cell uptake study was conducted with [18F]7 in AR42J cell line. Internalization and specific binding to SSTR2 were observed in vitro.
  • Brasseur, Paul (Helsingin yliopisto, 2021)
    Plasmonic is an emerging field which has showed application for photocatlysis. Here we investigate a gold/platinum bimetallic catalytic system, and try to show how the catalytic properties of gold nanoparticles can be us to harvest visible light energy to increase the catalytic activity of platinum. Platinum being are rare and expensive metal, we also took the opportunity to find the optimal amount of catalyst to reduce platinum use. The catalyst is composed of a core spherical gold nanoparticles, of around 15 nm diameter. They were synthesized using an inversed Turkevich method, based on trisodium citrate, gold precursor salt and done in solution. Various amount of platinum was deposited on those nanoparticles using seeded growth method. The amount of platinum varied for single atoms to an atomic monolayer. This suspension of nanoparticles was deposited on ultrafine silica powder to be used for certain reaction and characterization. The material was characterized via several technics. UV-Visible and Diffuse Reflectance Spectroscopy were used to characterize its optical properties and showed a absorption peak around 524 nm characteristic of gold nanoparticles of this size. Imaging was done using electron microscopy (SEM and TEM) to study the morphology and showed monodisperse and spherical particles. The exact composition of the different catalyst were obtain using Atomic Emission Spectroscopy. The study was conducted by using reduction reaction as tests to investigate differences in conversion and selectivity under dark and monochromatic 525 nm and 427 nm light conditions. We chose to work on reduction of 4-nitrophenol, phenylacetylene and nitrobenzene, because they are widely used both in research and industry, and are easy to set up. Some catalyst showed good enhancement under 525 nm light, especially the one with the least amount of platinum. Different selectivity were also observed, indicating the presence of different reaction pathways under light conditions.
  • Pusfitasari, Eka Dian (Helsingin yliopisto, 2019)
    Urine can be used to determine human exposure to nerve agents through the analysis of specific biomarkers. Isopropyl methylphosphonic acid (IMPA) is an important marker of sarin nerve agent, a highly toxic chemical regulated under the Chemical Weapons Convention (CWC). A methodology for sensitive, reliable, and selective determination of IMPA in urine matrix was developed and validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample preparation method employs normal phase–solid phase extraction (NP-SPE) using silica based cartridge. Before conducting IMPA analysis, the instrument performance was controlled using a quality control sample. Three different ion sources, namely electrospray ionization (ESI), Unispray, and atmospheric pressure chemical ionization (APCI), were compared in order to define the best method for trace analysis of targeted IMPA. Parameters affecting the ionization process such as cone voltage, capillary voltage, impactor pin voltage, corona voltage, and mobile phase flow rate were optimized. Negative ion mode was selected as the best method for IMPA identification in all three ion sources, and multiple reactions monitoring (MRM) was employed to improve sensitivity and selectivity. The APCI source was shown to be the least sensitive and least efficient ionization technique for IMPA identification. In contrast, using ESI and Unispray resulted in satisfactory data with excellent limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The two latter ion sources share the same values of those parameters, i.e. 0.44 ng/mL, 1.46 ng/mL, < 4% precision bias, < 5% accuracy bias, for ESI; and 0.42 ng/mL, 1.38 ng/mL, < 4% precision bias, < 4% accuracy bias, for Unispray. Nonetheless, the Unispray shows better performance in comparison to ESI in producing higher signal intensity/peak area and has lower matrix effect.
  • Sillanpää, Meri (Helsingin yliopisto, 2021)
    The literature study of this thesis focuses on the different analytical methods used to analyse amino acids in food and beverage samples. Amino acids are essential organic molecules and their concentrations in foods and beverages constitute, inter alia, the product’s nutritional value, quality, freshness, and flavour. Amino acid analysis of foodstuff has various applications, which exploit several analytical methods. These reviewed methods are founded on academic articles published during the past two decades. This literature review discusses the different sample matrixes, sample preparation methods, ways to derivate analytes, and different separation and detection methods utilized in the recent amino acid studies. The experimental part of this thesis was a modification of L-asparagine and L-aspartic acid test (L-Asp/L-AspAc) in Thermo Fisher Scientific Oy industrial R&D laboratory. An enzymatic photometric method is used to determine L-Asp/L-AspAc amino acids in food samples. The modification process entailed pre-testing of several candidate methods, from which the most suitable one was selected. The feasibility of the chosen test was affirmed before verification and validation of the modified test.
  • Tanskanen, Ville (Helsingin yliopisto, 2020)
    Microbial volatile organic compounds are emitted by diverse set of microbial organisms and they are known to cause health hazards when present in indoor air. Early detection of fungal contaminated buildings and species present is crucial to prevent health problems caused by fungal secondary metabolites. This thesis focuses on analysing emission profiles of different insulation materials and fungal cultures, which allows, in further studies, to develop efficient new ways to detect fungi from contaminated buildings. Studied insulation materials consisted of cellulose and glass wool, which were analysed in multiple different conditions. Humidity of atmosphere was varied between 0-10 microliters and temperature was varied between 30°C and 40°C. In fungal emission profile study 24 different cultures were analysed in two different atmospheres, ambient and micro- aerophilic, and in multiple different inoculums. Analysis for both insulation materials and fungal cultures was done using headspace solid phase microextraction Arrow -tool and headspace in tube extraction –tool together with gas chromatography – mass spectrometry. One goal for this thesis was also test suitability of these methods for detection of fungal secondary metabolites. Comprehensive fungal emission profiles were successfully formed and new information from behaviour of insulation materials in different settings was found. In addition, new information about analysis methods and fungal behaviour in different atmospheres was found. Headspace solid phase microextraction Arrow with gas chromatography – mass spectrometry was found to be efficient, sensitive and timesaving method for indoor air study purposes. There were also many potential fungal culture specific biomarker compounds found for further study purposes.
  • Rautsola, Iiro (Helsingin yliopisto, 2019)
    Multimodality imaging is an efficient, non-invasive method for investigation of molecular and cellular processes in vivo. However, the potential of multimodality imaging in plant studies is yet to be fully realized, largely due to the lack of research into suitable molecular tracers and instrumentation. Iodine has PET- and SPECT-compatible radioisotopes that have significant advantages over other radioisotopes applied in plant radioisotope imaging, and can be incorporated into small molecules via a variety of reactions. In this master’s thesis, a radioiodination method exploiting a novel, Dowex® H+-mediated addition of iodine for terminal alkynes was optimized and tested on two D-glucose analogues. The goal of the sugar analogue radioiodination was to develop a radioiodinated molecular tracer for plant carbohydrate metabolism studies. The parameters under optimization were activation Dowex® by HCl, reaction temperature, carrier amount, solvent, and evaporation of excess water. The most optimal results were achieved under the following conditions: Dowex® HCl-activated, reaction temperature 95 °C, amount of carrier 3.0 µmol of carrier, cyclohexanol as solvent, and excess water evaporated. The Dowex® approach was compared to electrophilic reactions with Chloramine T and Iodogen, and it was concluded that the Dowex® approach leads to superior radiochemical yields under the optimized conditions. The Dowex® method was successfully tested on the sugar analogues, resulting in a single main product at a satisfactory 50 – 56 % radiochemical yield. The main products were successfully characterized with NMR, and in addition the method was indicated to be regioselective. It is plausible that the developed method may be improved further in terms of radiochemical yield and molar activity, and that the method could prove to be a useful tool for developing novel radiodinated molecular tracers for plant studies.
  • Pakkanen, Noora (Helsingin yliopisto, 2021)
    In Finland, the final disposal of spent nuclear fuel will start in the 2020s where spent nuclear fuel will be disposed 400-450 meters deep into the crystalline bedrock. Disposal will follow Swedish KBS-3 principle where spent nuclear fuel canisters will be protected by multiple barriers, which have been planned to prevent radionuclides´ migration to the surrounding biosphere. With multiple barriers, failure of one barrier will not endanger the isolation of spent nuclear fuel. Insoluble spent nuclear fuel will be stored in ironcopper canisters and placed in vertical tunnels within bedrock. Iron-copper canisters are surrounded with bentonite buffer to protect them from groundwater and from movements of the bedrock. MX-80 bentonite has been proposed to be used as a bentonite buffer in Finnish spent nuclear fuel repository. In a case of canister failure, bentonite buffer is expected to absorb and retain radionuclides originating from the spent nuclear fuel. If salinity of Olkiluoto island´s groundwater would decrease, chemical erosion of bentonite buffer could result in a generation of small particles called colloids. Under suitable conditions, these colloids could act as potential carriers for immobile radionuclides and transport them outside of facility area to the surrounding biosphere. Object of this thesis work was to study the effect of MX-80 bentonite colloids on radionuclide migration within two granitic drill core columns (VGN and KGG) by using two different radionuclides 134Cs and 85Sr. Batch type sorption and desorption experiments were conducted to gain information of sorption mechanisms of two radionuclides as well as of sorption competition between MX-80 bentonite colloids and crushed VGN rock. Colloids were characterized with scanning electron microscopy (SEM) and particle concentrations were determined with dynamic light scattering (DLS). Allard water mixed with MX-80 bentonite powder was used to imitate groundwater conditions of low salinity and colloids. Strontium´s breakthrough from VGN drill core column was found to be successful, whereas caesium did not breakthrough from VGN nor KGG columns. Caesium´s sorption showed more irreversible nature than strontium and was thus retained strongly within both columns. With both radionuclides, presence of colloids did not seem to enhance radionuclide´s migration notably. Breakthrough from columns was affected by both radionuclide properties and colloid filtration within tubes, stagnant pools and fractures. Experiments could be further complemented by conducting batch type sorption experiments with crushed KGG and by introducing new factors to column experiments. The experimental work was carried out at the Department of Chemistry, Radiochemistry in the University of Helsinki.
  • Kilpeläinen, Wille Julius (Helsingin yliopisto, 2020)
    Inductively coupled mass spectrometry (ICP-MS) is a state-of-the-art technique for elemental analysis. The technique allows fast and simultaneous analysis of multiple elements with a wide dynamic range and low detection limits. However, multiple adjustable parameters and the complex nature ICP-MS instruments can make the development of new analysis methods a tedious process. Design of experiments (DOE) or experimental design is a statistical approach for conducting multi- variate experiments in a way that gives maximal amount of information from each experiment. By using DOE the number of experiments needed for analytical method optimization can be minimized and information about interrelations of di↵erent experimental variables can be obtained. The aim of this thesis is to address the utilization of DOE for ICP-MS method developement as a more e cient mean to optimize analytical methods. The first part of this two part thesis gives an overview on the basics of ICP-MS and DOE. Then a literature review on applying experimental design for ICP-MS method optimization is given and the current state of the research is discussed. In the second part, two new ICP-MS methods for simultaneous determination of 28 elements from six middle distillate fuels, diluted with xylene or kerosine, are presented. The method developement involved optimization of the integration times and optimization of test sample dilution ratios and viscosities using univariate techniques. In addition, experimental designs were succesfully utilized together with desirability approach in multivariate optimizations of the plasma conditions and sample matrix compositions to achieve the best possible analyte recoveries from various matrices.
  • Chou, Hao-Wei (Helsingin yliopisto, 2019)
    Diblock copolymers, poly(lactide)-block-poly(2-isopropyl-2-oxazoline) (PiPOx-b-PLA) and n-octadecyl poly(2-isopropyl-2-oxazoline) (PiPOx-C18), were characterized using the steady-state fluorescence with two fluorescence probes, pyrene and (1,6-diphenyl-l,3,4-hexatriene) (DPH), to investigate the core structure of the particles formed by the polymers. The two enantiomers of PiPOx-b-PLA, which are PiPOx-PLLA and PiPOx-PDLLA, reflected very different features in terms of polarity of the micellar core and the partitioning equilibrium constants of pyrene (Kv). This suggests that the core of the particles formed by PiPOx-PDLLA has higher polarity due to high water content in the range of the polymer concentrations from 0.03 g/L to 3 g/L, and more pyrene molecules are bound to the particles. Furthermore, the core of the particles formed by PiPOx-PDLLA reveals rigidity lower than that of PiPOx-PLLA, which is supported by the low anisotropy value (r) of DPH. r did not change upon heating from 15 ⁰C to 65 ⁰C for the PiPOx-PLLA particles. A slight decrease of r occurred above 50 ⁰C for the PiPOx-PDLLA particle. The PiPOx-C18 micelles exhibit higher critical micellar concentration (CMC), smaller Kv, and softer core in comparison to the PiPOx-b-PLA micelles. These differences owe to the nature of the alkyl chain end. Loading and release of a hydrophobic drug, curcumin, to/from the PiPOx-PLLA and PiPOx-PDLLA micelles were investigated by means of the steady-state fluorescence. The release test was conducted using the dialysis bag method. The PiPOx-PDLLA particles encapsulated more curcumin with encapsulation efficiency (EE) value being 98% while the curcumin-to-polymer ratio was 1:20. Curcumin intake of the PiPOx-PLLA particles was lower under the same condition (EE = 56%). The PiPOx-PDLLA particles show excellent ability of trapping curcumin. PiPOx-PDLLA bear 70% of curcumin inside the dialysis bag after 50 h, whereas 40% of curcumin was discharged from dispersions of PiPOx-PLLA. Precipitation of curcumin was observed in presence of PiPOx-PLLA in 4 days. No curcumin precipitating from the PiPOx-PDLLA dispersion occurred and a decrease in the intensity of curcumin owed to the degradation of curcumin. Finally, all the phenomena observed are well illustrated by the hypothesis of the morphology of the particles formed by PiPOx-PLLA and PiPOx-PDLLA. The particle formed by PiPOx-PDLLA consists of a loose hydrophobic core with hydrated tails extending to the aqueous phase, whereas the core of PiPOx-PLLA particle is dense.
  • Hirsso, Iida (Helsingin yliopisto, 2020)
    Kohdennettuun PET-kuvantamiseen sopivia spesifisiä radiolääkeaineita on kehitetty viimeisen kymmenen vuoden aikana aktiivisesti. Tiettyyn reseptorityyppiin sitoutuvien biomolekyylien käyttö kuvantamisessa sujuvoittaa syöpien diagnosointia ja nopeuttaa hoitosuunnitelmia. Tällaisia molekyylejä ovat muun muassa vasta-aineet, peptidit ja oligonukleotidit. Biologisesti aktiivisia molekyylejä on pitkään radioleimattu radiometalleilla, mutta tarve 18F-radioleimauksille on noussut radionuklidin positroniemissio-ominaisuuksien takia. Biomolekyylien radiolääkeainekehityksessä kulmakiveksi on noussut makromolekyylien herkkyys tyypillisille 18F-radio-leimausolosuhteille. Biomolekyylejä voidaan radioleimata joko suoraan biomolekyyliin tai epäsuoraan prosteettiseen ryhmän avulla. Kliinisesti käytössä jo pitkään olleita 18F-radiolääkeaineita, kuten [18F]fluori-deoksi-glukoosi ([18F]FDG) ja [18F]-N-sukkinimidyyli-fluori-bentsoaatti ([18F]SFB), on hyödynnetty PET-kuvantamisessa biokonjugoituna sopivaan peptidiin. Isotooppiseen 19F-18F vaihtoreaktioon (IEX, engl. isotopic exchange) soveltuvat prosteettiset ryhmät soveltuvat hyvin suoraan biomolekyylien radioleimauk-seen, kun ne on valmiiksi biokonjugoitu molekyylin klik-reaktiolla. Tällaisia prosteettisia ryhmiä ovat muun muassa Si-18F-, B-18F- ja Al-18F-sidoksia sisältävät molekyylit. [18F]Fluoriboraatit ovat osoittautuneet hyödyllisiksi työkaluiksi peptidien 18F-leimaukseen. Erityisesti alkyynirakenteista kahtaisionista [18F]alkyyliammonium-metyylitrifluoriboraattia ([18F]AMBF3 eli [18F]1) on radioleimattu biokonjugoituna useaan eri peptidiin hyvällä in vivo stabiilisuudella. [18F]1 biokonjugointi peptidiin perustuu kuparikatalysoituun atsidi-alkyyni-sykloadditioon. Tämän maisterin-tutkielmatyön tarkoituksena oli optimoida automatisoitu radiosynteesi [18F]1:lle. Lisäksi työssä määritettiin radioleimausolosuhteet kupa-rikatalysoidun atsidi-alkyyni sykloadditiossa muodostetulle trifluoriboraatin ja tetratsiinin johdannaiselle, [18F]AMBF3-PEG4-mTz:lle ([18F]F2). [18F]1 syntetisoitiin kaksivaiheisella synteesillä booripinakoliesterin substituutioreaktiolla, minkä jälkeen booripinakoliesteri fluorattiin trifluoriboraatiksi kaliumbifluoridilla, KHF2, 72 % saannolla. [18F]2 syntetisoitiin [18F]1:sta ja mTz-PEG4-atsidista kahden tunnin kuparikatalysoidulla klik-reaktiolla huoneenlämmössä 63 % saannolla. Molemmat prosteettiset ryhmät 18F-radioleimattiin IEX-reaktiolla pyridatsiini-HCl-puskuriliuoksessa (1.0 M, pH 2.0), johon radioaktiivinen [18F]fluoridi lisättiin µQMA-patruunasta konsentroituna 0,9 % NaCl-liuoksessa 10-20 µl:ssa. [18F]1 radioleimattiin toistuvasti automatisoidulla radiosynteesillä 47 % radiokemiallisella saannolla (RCY) 99,9 % puhtaudella radio-TLC-määrityksen perusteella (TLC, Thin Layer Chromatography = Ohutlevykerroskromatografia). [18F]2 radioleimattiin manuaalisella pipetoinnilla toistettavasti kohtuullisella RCY:llä 23-38 % puhtausluokassa 95-99,2 %. Molaariset aktiivisuudet (MA) olivat suoraan verrannollisia käytettyyn lähtöaktiivisuuteen: esimerkiksi yli 1 GBq lähtöaktiivisuudella [18F]1:n MA oli vastaavasti 1,12-1,22 GBq/µmol. Merkkiaineiden lipofiilisuusmääritysten perusteella LogD-arvoiksi saatiin: [18F]1:lle -1,28 ± 0,33 ja [18F]2:lle -0,43 ± 0,25. [18F]1 toimii hyvänä prosteettisena ryhmänä peptidien 18F-radioleimauksessa useisiin eri kuvantamiskäyttötarkoitukseen. Sen farmakologisia ja toksikologisia profiileja on tosin vielä tutkittava ennen kliinistä käyttöön ottoa. Tutkielmassa optimoitiin radiosynteesi, joka mahdollistaa myös muiden potentiaalisten trifluoriboraattien 18F-radioleimauksen säteilyturvallisesti. [18F]Fluoridin konsentrointi on radiosynteesin haastavin vaihe, joka voitaisiin korvata sopivilla vaihtoehtoisilla menetelmillä vielä tehokkaammaksi kuin µQMA-patruunalla.
  • Kurki, Satu (Helsingin yliopisto, 2021)
    Eturauhassyövän ja sen etäispesäkkeiden diagnostiikassa käytettävien radiolääkkeiden tärkeä kohdemolekyyli elimistössä on prostataspesifinen membraaniantigeeni (PSMA), jonka esiintyminen on eturauhassyövässä yliekspressoitunutta. Viimeisen kahden vuosikymmenen aikana on kehitetty lukuisia radioleimattuja PSMA-merkkiaineita, pääosin pienimolekylaarisia peptidomimeettejä, jotka inhiboivat PSMA:n toimintaa. Positroniemissiotomografiassa eli PET-kuvantamisessa käytettävistä fluori-18-leimautuista PSMA-inhibiittoreista merkittävimmät ovat [18F]DCFPyL ja [18F]F-PSMA-1007. Kyseisiä radiolääkkeitä kyetään tuottamaan teollisesti riittävän suurilla eräkohtaisilla aktiivisuuksilla ja synteesisaannoilla sekä hyödyntämään synteesissä suoria fluorinointireaktioita ja automatisoituja synteesiyksiköitä. Skaalattaessa tuotantoaktiivisuuksia tutkimusmittakaavasta teolliseen tuotantoon, haasteena on valmisteen radiolyysi eli hajoaminen joko suoraan säteilyn vaikutuksesta tai säteilyn muodostamien liuottimen radikaalien reagoidessa lääkeainemolekyylin kanssa. Radiolyysiä pyritään estämään lisäämällä formulaatioon radiolyysin estäjää tai vaikuttamalla radioaktiivisuustasoihin synteesiprosessin kriittisissä vaiheissa ja lopputuotteessa. Kokeellisessa osuudessa tutkittiin koesynteesien ja nestekromatografiamenetelmän avulla [18F]F-PSMA-1007-radiolääkkeen hajoamiseen vaikuttavia tekijöitä ja hajoamistuotteita tunnistettiin nestekromatografia-massaspektrometrisellä menetelmällä. Lisäksi vertailtiin kahden eri formulaatioliuoksen kykyä estää tuotteen radiolyysiä. Tutkimuksessa todettiin, että [18F]F-PSMA-1007:n hajoaminen tapahtuu suoraan beeta(+)-säteilyn ja radikaalireaktioiden kautta. Askorbaatti-ionit kykenevät tehokkaasti estämään tuotteen radiolyysiä. Radiolyysiä tapahtuu jo reaktioastiassa ja se kasvaa merkittävästi synteesin alkuaktiivisuuden noustessa yli 100 GBq. Hajoamistuotteita muodostuu lukuisia ja kokeellisessa osuudessa tunnistettiin muutama mahdollinen pilkkoutumiskohta tutkittavasta molekyylistä.
  • Liang, Zehong (Helsingin yliopisto, 2020)
    Tämä tutkimus on osa DNA-oligonukleotidien liuossynteesin menetelmien kehittämishanketta. Kemiallinen oligonukleotidisynteesi tehdään tyypillisesti kiinteän kantajan päällä. Suurempien ainemäärien valmistamiseksi liuosfaasissa toimiva menetelmä olisi kuitenkin toivottava. Vaiheittain etenevässä synteesimenetelmässä on keskeistä löytää yhteensopiva sarja suojaryhmiä. Kiintokantaja- ja liuosfaasisynteeseissä suojaryhmiltä vaaditaan erilaisia ominaisuuksia, koska reaktiot suoritetaan eri tavalla. Liuosfaasisynteesissä reaktioiden tasapaino pitää asettua vahvasti tuotteiden puolelle, koska toisin kuin kiintokantajamenetelmässä sivutuotteita ei voi huuhtoa pois ja reagenssien määrää ei voida pitää niin vakaana. Olemme kehittäneet sarjan suojaryhmäreagensseja nukleosidien 5’-hydroksyylisuojaryhmän suojaamiseksi asetaalina. Asetaalisuoja voidaan liuossynteesissä poistaa kokonaan ja suojaryhmällä on tarkoitus korvata samaan ortogonaaliseen ryhmään kuuluva trityylisuojaus, joka ei soveltunut liuosfaasisynteesin. Nyt esiteltävässä työssä paneudutaan menetelmään soveltuvien 2’-deoksinukleosidi 3’-O-vetyfosfonaattiestereiden valmistamiseen. Fosfori(V)-yhdisteiden reaktiivisuus ei useinkaan ole riittävä fosfaattiryhmän liittämiseen kohdemolekyylissä olevaan hydroksyyliryhmään, joten fosforyloinnissa käytetään usein fosfori(III)-yhdisteitä, kuten fosfiitteja, fosforamidiitteja ja vetyfosfonaatteja. Liitettävä fosfaattiryhmä, kuten monet muut reaktiiviset funktionaaliset ryhmät, suojataan usein suojaryhmillä. Suojaryhmillä voidaan niiden päätarkoituksen lisäksi muokata molekyylin rakennetta ja fysikokemiallisia ominaisuuksia. Reaktiotuotteen liukoisuutta voidaan muokata suojaryhmillä sen eristyksen tai jatkoreaktioiden helpottamiseksi. Tutkielman kirjallisuusosassa tarkastellaan suojaryhmiä, joita on käytetty fosfaateille ja vetyfosfonaateille. Fosfaattisuojaryhmällä voidaan mahdollisesti suojata vetyfosfonaattimonoesteri diesteriksi, jolloin negatiivinen varaus saadaan naamioitua ja tuotteesta tulisi lipofiilisempi. Kokeellisen tutkimuksen tavoite oli valmistaa 5’-asetaalisuojattuja tymidiinin vetyfosfonaatteja difenyylivetyfosfonaattireagenssilla. Saanto jäi hyvin pieneksi, koska tuote osoittautui liian vesiliukoiseksi pienikokoisen ja heikosti lipofiilisen asetaalisuojan kanssa. Ratkaisuksi kehitettiin menetelmä, jossa muodostettiin ensin tymidiinin vetyfosfonaattidiesteri syanoetanolin kanssa. Vetyfosfonaattidiesteri on todennäköisesti varauksettomana tuotteena ja lisätyn alkyyliryhmän kanssa poolittomampi. Suojaryhmän avulla tymidiinin vetyfosfonaatti voitaisiin eristää, jonka jälkeen ryhmä poistetaan emäksellä β-eliminaatioreaktiossa. Vetyfosfonaattidiesterin eristäminen ja suojaryhmän poistaminen vedettömissä olosuhteissa helpottaa vetyfosfonaattimonoesterin talteenottoa. Vaihtoehtoisesti tymidiinin liukoisuusominaisuuksia muokattiin suojaamalla tymiinin 4-O β-eliminoituvalla poolittomalla suojaryhmällä. Fosfitylointimenetelmä ei tarvitsisi muokkausta jos, emäsmuokattu tymidiini osoittautuu veteen liukenemattomaksi.
  • Teittinen, Henry (Helsingin yliopisto, 2020)
    Ligniini on maailman yleisin biopolymeeri selluloosan jälkeen. Kuitenkin puiden biomassan rakenteen vuoksi ligniinin eristäminen muusta biomassasta ei ole yksinkertaista vaan vaatii erityisesti sitä varten kehitettyjä menetelmiä. Lisäksi ligniinin heterogeenisen rakenteen vuoksi sen hyödyntäminen kemiallisesti ei ole helppoa, vaan biomassan jalostusprosesseissa ligniini päätyy sivuvirtojen mukana poltettavaksi. Ligniinin yleisyyden ja sen uusiutuvuuden vuoksi ligniinin käytöstä ollaan kiinnostuneita ja ligniinistä yritetäänkin saada sitä muokkaamalla käyttökelpoista materiaalia teollisuuden eri tarpeisiin. Japanilainen professori Masamitsu Funaoka on kehittänyt menetelmän, joka pyrkii samanaikaisesti eristämään ligniinin muusta biomassasta sekä muokkaamaan ligniinin rakennetta niin, että sitä voitaisiin hyödyntää erilaisissa sovelluksissa tekemällä siitä homogeenisempaa. Funaokan menetelmässä ligniinin bentsyyliseen α-asemaan liitetään selektiivisesti haluttu fenolinen yhdiste, joka lisää ligniinissä olevien aromaattisten hydroksyyliryhmien määrää ja katkoo ligniiniyksikköjen välisiä sidoksia alentaen ligniinin moolipainoa. Tämän tutkielman kirjallisessa osassa perehdytään Funaoka-menetelmään lignoselluloosamassan fraktionnissa. Lisäksi tarkastellaan saatujen lignofenolien rakenteellista muokkaamista, potentiaalisia käyttökohteita ja niistä jatkokäsiteltyjä materiaaleja. Kokeellisessa osassa puolestaan analysoidaan eräitä kotimaisia biomassan prosessoinnissa syntyneiden sivuvirtojen biomassatähteitä, erityisesti kiinnittäen huomiota biomassanäytteiden ligniinipitoisuuksiin. Biomassanäytteiden kemiallisen karakterisoinnin lisäksi kokeellisessa osassa testattiin ligniinipitoisten biomassanäytteiden fraktiointia Funaoka-menetelmällä ja tämä toistettiin myös näytteiden lähtöaineille eli havupuuhakkeelle.
  • Gasques Rocha Pinheiro, Beatriz (Helsingin yliopisto, 2020)
    Geometric isomers are of extreme importance due to the different properties of E and Z compounds. The interconversion between these forms allow to explore a vast amount of applications since their use in perfume and food industry until the development of photoactive drugs and advanced polymers. Included in this scenario are the E/Z isomers of pepper alkaloids, whose broad range of desirable pharmacological activities, such as anti-inflammatory, antioxidant and anti-cancer effects, makes them a focus in multidisciplinary research. Black pepper contains several of these alkaloids, among which piperine is the most abundant. Its properties have been studied for many years, with highlight for its pungency and flavour, in addition to medicinal applications that date from the development of ancient Indian and Chinese medicine. Piperine and structure-related compounds undergo rapid double bond isomerization in the presence of light, equilibrating to a mixture of four geometrical isomers, due to the two conjugated double bonds present in their structures. The biological activity of these isomers differs from those of the natural abundant E/E molecules. Thus, emphasizing the importance of having reliable analytical assays for their separation, detection and quantification. The current project pursued the development of a robust HPLC assays for isomers separation for piperine and some analogues. The effort included the extraction of piperine from black pepper and its use for the synthesis of highly pure piperylin and piperlonguminine standards. Piperine extraction kinetics was also studied to optimize the extraction procedure. The standards of alkaloids were isomerized using sunlight and then HPLC separation methods on chiral stationary phases were successfully established to resolve their E/Z isomers. Isocratic runs were also developed for piperine, piperylin and piperlonguminine, with the goal of adapting these methods to LC/MS application in the future. These last separations could be accomplished within 25 minutes with critical resolutions values larger than 1.8.
  • Juvonen, Mari (Helsingin yliopisto, 2020)
    Viime vuosikymmeninä on kasvanut huoli ympäristöön joutuvien kemikaalien kasvavasta määrästä. Vesistöön kulkeutuvien sukupuolihormonien, erityisesti estrogeenien, on havaittu vaikuttavan haitallisesti kalojen ja muiden vesieliöiden kehitykseen, häiritsevän kalojen lisääntymiskykyä ja hormonitoimintaa. Jätevedenpuhdistuslaitokselle tulevat steroidihormonit ovat peräisin yhdyskuntavesistä, maataloudesta, lääketeollisuudesta ja sairaaloista. Vedenpuhdistusprosessi ei poista kaikkia steroidihormoneja ja osa hormonijäämistä kulkeutuu ympäristöön kontaminoiden pohja- ja pintavesiä sekä maaperää. Tässä työssä tarkastellaan steroidien määritystä jätevesistä nestekromatografialla ja kapillaarielektroforeesilla vuosina 2010-2020. Steroideja on tutkittu jätevesistä pääasiassa GC-MS (kaasukromatografia-massaspektrometri) ja LC-MS tai LC-MS/MS (nestekromatografia-massaspektrometri tai nestekromatografia-tandem-massaspektrometri) -menetelmillä. Kaasukromatografia soveltuu vain haihtuville ja termisesti stabiileille yhdisteille ja vaatii usein paljon näytteiden esikäsittelyä. Tästä syystä nestekromatografia on tällä hetkellä yleisin menetelmä steroidien määrittämiseen. Kapillaarielektroforeesilla (CE) tutkimuksia on tehty vielä aika vähän, mutta se on osoittautunut lupaavaksi analysointitekniikaksi steroidien tutkimisessa. CE:n etuna on korkea erotustehokkuus rakenteellisesti samankaltaisillakin yhdisteillä, kuten steroidit ja niiden metaboliitit. CE-tekniikat jaetaan alalajeihin eri erotusperiaatteiden perusteella. Misellinen sähkökineettinen kapillaarikromatografia (MEKC) perustuu pinta-aktiivisen aineen käyttöön puskuriliuoksessa. Kun pinta-aktiivisen aineen pitoisuus ylittää niin sanotun kriittisen misellikonsentraation, liuokseen syntyy misellejä. Erottuminen perustuu näiden misellien ja analyyttien vuorovaikutukseen. Osittaistäyttöisessä misellisessä sähkökineettisessä kromatografiassa (PF-MEKC) vain pieni osa kapillaarista on täytetty miselliliuoksella. MEKC -tekniikka soveltuu sekä neutraalien että varautuneiden yhdisteiden erottamiseen. Koska steroidit esiintyvät jätevesissä hyvin pieninä pitoisuuksina (ng/l), näytteet on esikonsentroitava analyysia varten. Tähän käytetään useimmiten kiinteäfaasi-uuttoa (SPE). Uusia kiinteäfaasi-uuttotekniikoita on myös otettu käyttöön. Nämä ovat usein niin sanottuja mikrouuttotekniikoita, jotka kuluttavat vähemmän liuottimia ja näytteitä.