Browsing by department "Department of Chemistry"

In the first part of this thesis the principles of capillary electrophoresis (CE) are presented from the aspects of steroid and sterol analysis focusing mainly on two separation techniques: micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC). Analytes are delineated in steroids, corticosteroids, phytosterols, and cholesterol. Conventional chromatographic methods: gas chromatography (GC) and high-pressure liquid chromatography (HPLC) are somewhat challenging for steroid and sterol analysis, since direct analysis of steroids/sterols and their conjugates is rarely feasible. Hence, alternative separation and analysis methods need to be approached. MEKC and CEC have provided intriguing new opportunities for steroids and sterols, respectively. The experimental part covers the study on finding out the steroid composition and concentrations of wastewater samples collected from wastewater treatment plants (WWTP) around Finland. In addition, the efficiencies of the WWTPs were resolved. There were two types of wastewater samples: influent and effluent. Influent is the unclean water and effluent is the cleaned water that has passed through the process steps. The sample pretreatment includes filtering (glass fiber and membrane filters), solid phase extraction (SPE) (with C18 (Strata-X) and quaternary amine (N+) sorbents), and liquid-liquid extraction (LLE) (with diethyl ether). SPE was effective in purifying and concentrating the water samples, with a concentration factor of 20,000. The analysis was performed with partial filling-micellar electrokinetic chromatography, utilizing UV detection. It was found that the method was suitable for both qualitative and quantitative analysis of endogenous steroids and their corresponding metabolites. Androstenedione, testosterone glucuronide, and progesterone were found from the samples. Some notable results are that biological treatment most likely increases the amount of androstenedione, whereas enzymatic processes remove efficiently progesterone. Overall, the lowest steroid concentrations were obtained from the samples of Espoo, Pori, and Uusikaupunki. On the contrary, highest concentrations were in Kajaani, Mikkeli, and Porvoo.

Lignocellulosic biomass has received widespread attention as environmentally benign feedstock for fuels. Biomass consists of cellulose, hemicellulose and lignin and has rather high oxygen content. Different techniques for the conversion of lignocellulose to liquid fuels have been suggested in the literature. In this thesis the emphasis is on the utilization of biomass-derived platform molecules. Platform molecules include eg. ketones, alcohols and carboxylic acids. In the literary section different deoxygenation and C-C coupling reactions for the conversion of biomass-derived platform molecules to larger hydrocarbons have been reviewed. Reaction routes for upgrading of the platform molecules 5-hydroxymethylfurfural, 2-furaldehyde, levulinic acid and some monofunctional compounds have been presented. These paths comprise mainly dehydration, hydrogenation, aldol condensation and ketonization. Heterogeneous catalysis, particularly bifunctional supported catalysts, dominates in this field. The selectivity that may be achieved with homogeneous catalysts is seen as highly desirable and served as the main incentive for the experimental work. Furthermore, the lack of publications in the area of homogeneously catalyzed C-C coupling of biomass-derived compounds also motivated for the work. Herein, 1st row transition metal acetates were utilized as catalysts for the ketonization of biomass-derived levulinic acid and other carboxylic acids. Some experiments were conducted with lignin as well. Reactions were performed under microwave heating or reflux conditions. Products were analyzed by GC-MS, GC-FID, NMR and FTIR. Combinatorial chemistry made it possible to conduct up to twelve reactions simultaneously. More than 160 reactions were performed in less than two months' time. The main product in many of the reactions was aromatic phthalic acid mono 2-ethylhexyl ester. Some other interesting products included hexadecanoic acid, 2,6-dimethyl-2,5-heptadien-4-one, diisooctyl and dibutyl phthalate. Despite small amounts of the products, their presence proves that various compounds may be produced from biomass by tailoring the catalyst and reaction conditions.

The increased use of liquid biofuels has created a need for an accurate and a reliable technique for determining blend ratios of biofuel and fossil fuel due to technical reasons related to car engines and due to legislative reasons. The true portion of biological carbon in a fuel can be determined reliably only by radiocarbon measurement. Radiocarbon is created in upper atmosphere by cosmic radiation and is transferred to flora and fauna via photosynthesis. When an organism dies, the radiocarbon in its body starts do decay. Because the half-life of radiocarbon is very long and because biofuels are manufactured from relatively young feedstock materials, it is possible to calculate the biofraction of a fuel sample by determining its radiocarbon contents. The most popular techniques for determining this are, to date, accelerator mass spectrometry and liquid scintillation counting. Liquid scintillation counting is cheaper and easier to use, but in low concentrations the accuracy is not as good. In addition, the technique has the drawback of quenching effects. Accelerator mass spectrometry is the most accurate method, but the disadvantages are the price and size of the equipment and labor-intensive sample preparation process, which can take several days. In addition to the radioanalytical techniques, the biofractions of biofuels have been determined by infrared, Raman, nuclear magnetic resonance, X-ray and fluorescence spectroscopy and by gas and liquid chromatography, but these techniques have more limited applicability. In these techniques, the determination is usually based on direct or indirect detection of fatty acid methyl ester groups. However, the newer generation biofuels do not anymore contain these groups and their chemical composition is similar to fossil fuels. In addition, by using these techniques one cannot determine e.g. whether the ethanol in petrol blend is in fact manufactured from biological or fossil sources. In the experimental section of the thesis an elemental analyzer -based sample preparation method was developed, by which the time spent on sample preparation for accelerator mass spectrometer was decreased when compared to previous method, described by standard ASTM D6866-10. The biodiesel samples were combusted in the elemental analyzer and the carbon dioxide collected cryogenically. The carbon dioxide was reduced to graphite and their radiocarbon contents was measured by accelerator mass spectrometry. In addition, the results from elemental analyzer method were compared to previous results by closed-tube-combustion method. It was noticed that the elemental analyzer method was more accurate, faster and easier to use.

Many diseases are associated with oxidative stress caused by free radicals. Fungal endophytes are microbes that inhabit host plants without causing disease and have been recognized as potential sources of pharmaceutically valuable compounds. The extract of the endophytic fungi, Phialophora lignicola found in roots of Pinus sylvestris (Scots pine) seedlings has revealed protective bioactivity on human retinal pigment epithelial cells (hRPE) against oxidative stress which can lead to obtain compounds for the treatment or prevention of Age-related macular degeneration (AMD). The current research was directed towards finding the naturally-occurring antioxidant(s) in P. lignicola and to characterize and identify them by NMR spectroscopy and MS analysis. The bioactive aqueous extract of P. lignicola was fractionated by preparative HPLC and the antioxidant activity of each sample was evaluated by H2O2 scavenging test and bioassay with model cells of hRPE cells. The bioactive fractions were characterized by 500 Mhz NMR spectroscopy, performing both one- and two-dimensional NMR experiments, including 1H NMR, COSY, TOCSY, HSQC, ROESY, 31P-NMR and 31P-1H correlation. ESI-TOF MS in the positive mode was used for the determination of molecular weights found in the fractions. Additionally, LC-MSn was used for the separation of compounds and mass analysis to complement the information given by ESI-TOF MS. The present study provides evidence that two bioactive fractions of P. lignicola possessed the presence of antioxidative activity. The NMR experiments suggested arginine and polysaccharides. ESI-TOF MS results established the presence of arginine, a hexose-arginine conjugate and other compounds that based on NMR and mass spectrometry literature they could be chitin oligomers. Furthermore, LC-MSn identified fragments typical of L-arginine. For future research, some drawbacks of this study such as concentration of samples and chromatographic purification method should be improved to optimize the NMR and mass spectrometry analysis.

In the first part of this thesis literature about the determination amines in food samples of the past decade (2007 – 2017) has been reviewed. The sample preparation methods and chromatographic determination methods have been reviewed. The review is focused on biogenic amines (BA) since BAs are the most relevant in food samples. Monitoring the concentration levels of BAs in foods is important because elevated levels of amine concentrations in food products can indicate spoilage which can lead to food poisoning. Food samples are complex matrices therefore sample preparation is required prior to analysis. Amine extraction methods are reviewed in more detail, including conventional solid-liquid extraction (SLE) and solid phase extraction (SPE), and novel and miniaturized methods: solid phase micro extraction (SPME), liquid phase micro extraction (LPME) and dispersive liquid-liquid micro extraction (DLLME). The derivatization methods of Bas have also been reviewed including derivatization with o - phthaldialdehyde (OPA), dansyl chloride, benzoyl chloride and diethyl ethoxymethylenemalonate (DEEMM). Chromatographic methods are well researched tools in determination of amines in food samples. In the past decade only few application were found of thin layer chromatography (TLC). The gas chromatography (GC) has been used more often, especially in the analyses of beverages. However, the high performance liquid chromatography (HPLC) is the main method of choice in determination of amines in food samples as demonstrated by the large numbers of research articles. Recently also the ultra-high performance liquid chromatography (UHPLC) has been gaining popularity. In this master’s thesis experimental part several sets of experiments were performed. Adsorbing materials were synthesized using suspension polymerization and silica gel functionalization. Compositions of materials were estimated by FTIR. The materials were characterized in terms of their suitability for amine adsorption. Ion exchange capacity was determined by titration. Static and dynamic binding capacity was determined by HPLC-UV. Derivatization studies of atmospheric amines by 9-Fluorenylmethoxycarbonyl chloride (Fmoc-Cl) were carried out by HPLC-UV. Potential imine formation was investigated by HPLC-UV. The most promising adsorbing material was a hydrolyzed copolymer of divinylbenzene and methacrylic anhydride (DVB-(MAA)2O). Its ion exchange capacity was 4.8 meq/g, static binding capacity was 0.95 mmol/g of tertiary amine and dynamic binding capacity was 2.0 mmol/g for primary amine and 0.8 mmol/g for tertiary amine.

Porphyrins and porphyrin derivatives are naturally occurring molecules, whereas carbaporphyrinoids are synthesized porphyrin derivatives. They have received much attention in recent years by the scientific community due to their diverse potential applications in technological developments such as molecular electronic devices and conversion of solar energy. However, the full utilization of this class of compounds can not be realized without an in-depth understanding of their chemical and physical properties. Two of such properties are aromaticity and optical properties. In this thesis, the aromatic properties and the light absorption spectra in the ultraviolet and visible (UV/Vis) range have been studied computationally for some recently synthesized carbaporphyrins and carbachlorins using first-principle computational approaches. In the first part of the thesis, the background of carbaporphyrinoids and some examples of naturally occurring porphyrins and porphyrin derivatives are delineated. The second and third part review theoretical and computational methods that are employed in studies of the molecular aromaticity and electronic excitation spectra of molecules. The computational studies of magnetically induced current densities and electronic excitation energies are discussed in the fourth chapter. The obtained results are also presented in chapter four and the main conclusions are summarized in the last chapter. The study shows that all the carbaporphyrinoids studied sustain a magnetically induced ring current in the porphyrin macro ring. This indicates that they are aromatic according to the ring-current criterion. However, the calculated ring-current pathways differ from those predicted from the nucleus independent chemical shift (NICS) calculations and the current pathways deduced from H NMR spectroscopy studies. The vertical excitation energies which is akin to the ultraviolet-visible spectrum obtained experimentally for some of the selected carbaporphyrinoids also showed deviations from those of the experimental values. These deviations can be ascribed to solvent effects as in the calculation of the vertical excitation energies, solvent effects were not accounted for.

The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.

Accurate and sensitive analysis of mono-, di-, and oligosaccharides is desired in several different scientific areas due to the wide appearance of saccharides. This work focuses on the detection of mono-, di-, and oligosaccharides utilizing capillary electrophoresis (CE). Saccharide analysis with CE is challenging due to the lack of UV-absorbing chromophores in the molecular structure. CE also requires that the analytes are in their charged form, which is demanding in the case of mono-, di-, and oligosaccharides due to their high pKa-values. The first part of this work presents several detection methods and procedures to succeed in saccharide analysis with CE. A selection of the scientific work published in this area is presented to highlight the different detection possibilities. Derivatization of the analytes is commonly used to transform the saccharides into UV absorbing species. Special compositions of the background electrolyte, e.g. borate buffers and copper(II) containing buffers can be exploited to form charged complexes with the saccharides, which enhance the separation. Indirect UV detection is not as sensitive as direct UV detection of saccharide derivatives, but it is fast and useful in applications where high sensitivity is not required. Electrochemical detection (pulsed amperometric detection and contactless conductivity detection) is especially useful in miniaturized and portable systems. An advantage of electrochemical detection is also that no sample pretreatment or special reagents are required. Mass spectrometry (MS) detection is a powerful tool when detailed information about oligosaccharide structures is required and when the sample amounts are small. MS detection is therefore especially suitable in biochemical applications. In the second part of this work, CE was utilized for the separation and quantification of five novel ionic liquids and the quantification of acetate and xylose in ionic liquid matrices. The internal standard method was used in the quantitative work. The novel ionic liquids were detected with direct UV detection and the limit of detection ranged from 2-5 µg/mL. Resolution and number of effective plates were calculated from the separation studies. In the quantitative work, calibration curves were obtained for four of the novel ionic liquids. CE with indirect UV detection was used for the quantification of acetate, which is a typical counter ion in ionic liquids. A calibration curve for acetate was obtained and the linearity ranged from 0.0025 to 0.2 mg/mL. The method was successfully applied to the determination of the concentration of acetate in a standard sample containing the ionic liquid [MTBDH][OAc]. In the last part of the work, solid phase extraction was utilized to extract ionic liquids from industrial samples. CE with direct and indirect detection was used to check if the extraction was complete and if saccharides were present in the extracts. A calibration curve for xylose was constructed and the linear range for xylose was 0.05 to 3 mg/mL. It was found that the developed method for xylose detection was not sensitive enough to detect possible saccharide residues in the extracts and the analytical procedure requires further development.

Sugar and Sugar alcohol are indicative compounds in the environmental aerosol which make them really important. The concentration of sugar and sugar alcohol reveal biogenic and anthropogenic information such as climate, air quality, wood consumption, the activity of plantation and pollution. The conventional analysis methods of sugar and sugar alcohol are reverse phase High Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS/MS), and Gas Chromatography-Mass Spectrometry (GC-MS/MS). However, both of them have some limitations due to the sugar and sugar alcohol aerosol sample which are not easy to analyze. For reverse phase HPLC-MS/MS, the separation of analytes is not satisfied. For the GC-MS/MS, the derivatization process requires extra work and the derivatization compound is not stable. Besides, the matrix effect from the aerosol sample is a significant challenge which needs to be solved. Hence, the hydrophilic interaction chromatography (HILIC) and the Solid Phase Extraction (SPE) are introduced. The retention factors of HILIC column are the hydrophilic partition, the hydrogen bonding, and the electrostatic interactions. Polar stationary phase is used in HILIC mode, and the highly organic solvent is employed in mobile phase. Hence, a stagnant aqueous-rich layer is generated in HILIC mode, which can separate sugars and sugar alcohol efficiently. Furthermore, the interference and the matrix effect are solved by SPE. The development and the optimization of SPE-HILIC-MS/MS method for sugars were done in the experimental part. Eventually, the real environmental aerosol was analyzed by the optimized parameters and methods. The sugars and sugar alcohols were analyzed successfully from atmospheric aerosol samples.