Browsing by discipline "Analyyttinen kemia"

Pollution is of concern for human health and the environment. Authorities have set concentration frames for many pollutants. Therefore, monitoring is required in industrial and farming activities, for example. Methods based on gas chromatography - tandem mass spectrometry are mainly utilized in the study of trace pollutants in the environmental samples. The main reason for that being versatility and robustness of the technique, which is capable of analysis of numerous compound groups covering all the main pollutants. This thesis focuses on recent gas chromatography - tandem mass spectrometry method development in environmental analysis. Samples of air particulate matter and from aqueous environment are covered. Optimization of tandem mass spectrometry parameters, such as excitation energies, the choice of precursor ions and product ions are shown. Tandem mass spectrometry techniques increase sensitivity and selectivity of the analysis by reducing baseline due to specific fragmentation. For the reason of comparison, conventional one dimensional mass spectrometric applications are shown and advantages and disadvantages of both approaches are discussed. Research papers published during past decade were reviewed. Studied material included research results of gas chromatography- tandem mass spectrometry applications for determination of organic compounds, such as endocrine disruptive compounds, organometals, illicit drugs, pharmaceuticals, pesticides, brominated and chlorinated organic compounds, some volatile organic compounds, organonitrogen compounds and polycyclic aromatic hydrocarbons, in environmental samples. Sample collection and preparation techniques utilized in applications are reviewed as well. In the experimental part of this study, gas chromatography - mass spectrometry method for the determination of carboxylic acids, phenols and sugars in aerosol particles was developed. The experimental part covers also the study of fatty acids, which are analyzed utilizing the same method. Fatty acids are qualitative trace markers for agriculture based pollution in air. Result of the practical application of fatty acids as trace marker is presented as well. Developed method for determining fatty acids was utilized in field campaign in Bologna, Italy. C18/C16 –ratio is trace marker of agricultural based emission. Campaign results of C18/C16 –ratio and is presented in this thesis. Day-of-week trend of fatty acid emission is covered as well. The utilization of tandem mass spectrometry will reduce common matrix effects of gas chromatography - mass spectrometry and in this way will make analysis more sensitive. Tandem mass spectrometry methods will complete to the requirements of environment monitoring and thus are highly recommended in environmental analysis.

The literature part of this thesis reviewed the process of obtaining affinity information with quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensors. Basic principles of these biosensors were also evaluated, along with the principles of data acquisition and finally the data processing. The raw data produced by QCM or SPR can be used to study biomolecular interactions qualitatively and quantitatively. These techniques are also powerful in obtaining kinetic and thermodynamic information of the biomolecular interactions. SPR and QCM can produce data easily, but data interpretation can be sometimes problematic. This is partly due to misconceptions on how the sensograms should be interpreted. Many of the interpretational problems can and should be avoided long before the modeling of the data takes place to obtain reliable affinity data. The literature part of this thesis also presents tools for developing good experimental design. Well-designed experimental set-up is the most important element for producing good biosensor data. One should also estimate from the sensogram shapes what kind of analysis is needed. This was explained in detail in the literature part, pointing out the key elements how sensograms with certain shape should be interpreted and further analyzed to obtain affinity constants. Data analysis part of the literature review provides also information how to use appropriate models (e.g. fitting equilibrium, kinetic or complex data) with extensive examples. Surface site distribution model will be also covered as the tool to analyze complex biomolecular interactions by QCM and SPR. In the experimental part, affinity of anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) towards different lipoproteins was studied with partially filling affinity capillary (PF-ACE) electrophoresis and QCM. PF-ACE with adsorption energy distribution (AED) calculations provided information on the heterogeneity of the interactions. For the first time, a modified surface site distribution model called Interaction map was utilized to model QCM data of lipoprotein interactions with anti-apoB-100 Mab. With the Interaction maps, it was possible to distinguish different kinetics of low-density lipoprotein (LDL) and anti-apoB-100 Mab interactions. Affinity constants obtained were used to evaluate thermodynamics of these interactions. Both methods were also used to evaluate interactions with other apoB-100 containing lipoproteins: intermediate-density lipoprotein (IDL) and very lowdensity lipoprotein (VLDL). It was found that the Interaction maps could distinguish two different kinetics from the mixture of IDL-VLDL with distinct affinity constants. Both methods agreed well with the affinity constants. It was found that the anti-apoB-100 Mab used in this study, had a high affinity towards apoB-100 containing lipoproteins. In the second part of the experimental, a convective interaction media (CIM) based LDL isolation platform was developed. In these studies, anti-apoB-100 Mab was immobilized on the CIM-disk and was used to isolate LDL from human plasma and serum samples. It was found that apolipoprotein based separation of LDL from plasma was possible, although not without difficulties, since apoB-100 is not only present in LDL, but also in VLDL and IDL. To circumvent this problem different antibodies (anti-apoE and anti-apoAI) were utilized to capture VLDL and IDL from the plasma before the interaction of LDL with the anti-apoB-100 CIM-disk. LDL was successfully isolated with this approach in a significantly reduced time compared to conventional ultracentrifugation method used for LDL isolation.

Extracellular vesicles (EVs) are bilayer nanoparticles ranging from 40 nm to 5 μm in size and are mainly categorized as exosomes, microparticles, and apoptotic bodies. Recent studies reveal their involvement in various metabolic mechanisms in the human body. In particular, certain types of platelet-derived EVs found in plasma and serum are related to blood coagulation diseases and strokes. Merely, the diversity and heterogeneity of EVs in cellular systems and the lack of their isolation and detection methods are known about EVs. The fact is that a wide range of nomenclatures and basic questions regarding their morphology, size, and phenotype has remained debatable. As a result, there is a need to discover and standardize a simple and robust protocol for the isolation and detection of EVs. The literature part of the thesis covers an overview of what is defined as EVs and their functions undertaken, followed by a summary of the conventional methods for isolation and detection of EVs. The literature part finally concludes with an insight into upcoming and novel isolation and detection methods. Ultracentrifugation is the “golden” method used in the isolation of EVs. The long extraction times and varying reproducibility makes ultracentrifugation less viable, resulting in the exploration of other methods including size exclusion chromatography and immunoaffinity methods. Currently, none of these techniques are able to effectively distinguish between subtypes of EVs and matrix contaminants such as proteins remaining in the isolates. Each isolation method has its own edging advantages and disadvantages. Nevertheless, immunoaffinity methods showed greater potential in EVs extraction due to their high specificity and selectivity to process purified EVs samples. Detection methods are hindered by the minute sizes of EVs and the presence of contaminants in the isolates. Most detection methods involve the use of antibodies to detect or microscopic imaging to verify their presence based on their morphology. The trend is, however, skewing towards more reliable detection methods such as the use of mass spectrometry and microfluidic devices. The aim of the experimental portion is the ability to isolate purified platelet-derived EVs from complex human plasma samples and enable fast isolation of EVs. The use of immunoaffinity chromatography using antibody immobilized carbonyldiimidazole (CDI) monolithic disk was explored. Studies using specific platelet EV biomarker, anti-human CD61 antibody had successfully isolated platelet-derived EVs from plasma samples. In these studies, diluted plasma in phosphate buffer saline (PBS) solution (1:20 v/v) was injected through the housing setup containing the anti-human CD61 antibody immobilized monolithic disk. The eluate was obtained by injection of carbonate-bicarbonate solution and neutralized with hydrochloric acid. Detection analyses were then performed with nanoparticle tracking analysis (NTA), protein assays, Western blot and transmission electron microscopy (TEM). The average particle counts obtained in plasma isolates were found to achieve 10,000 particles more than isolates obtained after ultra-centrifugation reported in the literature. Positive presence of cytosolic protein TSG101 and transmembrane protein CD9 in Western blots, and transmission electron microscopic images of circular bilayer particles confirmed the isolation of platelet-derived EVs. Optimization studies also showed the inverse relationship between the flow rate in isolation step and the yield of platelet-derived EVs obtained. Affinity chromatography using antibody immobilized monolithic disk proved its success in the quick isolation (≤45 minutes) of platelet-derived EVs from plasma samples. The fast, miniaturized, compact setup, and simple methodology make the method viable for automation such as high throughput screening and suitable for applications of microfluidic microchips and censoring techniques on-line coupled for non-invasive and portable diagnostics.

Tutkielman kirjallisessa osiossa perehdytään kevyiden hiilivetyjen kaasukromatografisen analyysin osa-alueisiin, joita ovat näytteenkäsittely- ja näytteensyöttömenetelmät, kolonnit, detektorit sekä kalibrointi. Kirjallisuuskatsauksen perusteella yleisimmin käytettyjä kolonneja ovat huokoisen seinämän avoputkikolonnit. Detektoreista eniten käytetty on liekki-ionisaatiodetektori. Näytteenkäsittelyssä korostetaan analyyttien haihtumista estävien menetelmien käytön tärkeyttä. Yleisimmin näyte syötetään kaasukromatografiin nestemäisen näytteen kaasufaasista. Kalibroinnissa puolestaan suositaan kaasukalibroinnin käyttöä. Tärkeitä sovellusalueita kevyille hiilivedyille ovat esimerkiksi vesistöjen saastumisen seuranta, ilmanlaadun tarkkailu sekä muuntajien vikadiagnostiikka. Pro gradu –työn kokeellisessa osassa kehitettiin kvantitatiivinen kaasukromatografinen vertailumenetelmä Vaisala Oyj:n OPT100-mittalaitteelle muuntaöljyyn liuenneiden kevyiden hiilivetyjen analysointia varten. Vertailumenetelmän tärkeimpiä kehityskohteita olivat näytteenkäsittely ja –syöttö, sillä haihtuvilla analyyteilla ne ovat todennäköisimpiä syitä analyysituloksien vaihteluihin. Tässä työssä kehitetty näytteenvalmistusmenetelmä saavutti tavoitteeksi asetetun alle 2 % (k=2) hajonnan. Näytteenvalmistuksen ja –syötön kehittämisen lisäksi kokeellisessa työssä tehtiin menetelmän validointi. Menetelmän validointi sisälsi epävarmuusanalyysin lisäksi toteamis- ja määritysrajan, spesifisyyden ja selektiivisyyden sekä lineaarisuuden ja mittausalueen määrittämisen.

Lipidomics or global lipid profiling is a branch of metabolomics that aims to comprehensive analysis of lipids in a biological system. Lipidomics has become an important research field, since the increased awareness of lipid functions in cell and their role in many common diseases. Also the development of analytical methods, especially mass spectrometry has driven the progress of lipid profiling methods. Lipid profiling allows detection and quantitation of hundreds of intact lipid species in parallel. Challenges in lipidomics are the diversity of different lipid structures and varying lipid concentrations in biological samples. Despite of the development of lipidomics there is not yet a single analytical method to screen all lipids in a biological system. Therefore targeted lipidomics methods are needed in addition to global lipidomics. The literature part of this study presents different analytical methods used in lipidomics studies. The most popular lipid profiling methods are liquid chromatography-mass spectrometry (LC-MS) and direct infusion mass spectrometry or so called shotgun analysis. LC-MS based methods usually utilize reversed phase columns including octadecylsilane stationary phases, but also normal phase chromatography has been applied. In shotgun lipidomics crude extracts are infused directly to mass spectrometer without prior separation. In lipidomics, both unit resolution mass spectrometers like triple quadrupole and ion trap as well as high mass accuracy mass spectrometers like Fourier transform ion cyclotron resonance, Orbitrap, and time of flight mass spectrometers have been used. Mass scanning methods have provided full scan acquisition with accurate mass as well as tandem mass spectrometry like precursor ion scan, neutral loss scan, and multiple reaction monitoring. From the atmospheric pressure ionization techniques, electrospray ionization has been the most applied ionization, but also other soft ionization techniques such as matrix-assisted laser desorption/ionization has been employed. Sample pretreatment in global lipid profiling relies on homogenization and extraction with chloroform-methanol. The most used extraction methods are Folch and Blight and Dyer extractions or modifications of these. In addition, other sample pretreatment methods like solid phase extraction, derivatization, and thin layer chromatography have been used, especially with targeted lipidomics methods. Lipidomics platforms have been applied to several studies of different diseases like diabetes, schizophrenia, and cancer. Many bioactive signaling lipids cannot be detected with lipid profiling methods, since they exist at low concentration and have polar structure. In the experimental part a targeted method was developed for analysis of signaling lipids by ultrahigh-performance liquid chromatography-mass spectrometry. Selected compounds were lysophosphatidic acid, sphingosine-1-phosphate, and phosphoinositides. Method development was mainly done with triple quadrupole mass spectrometer, but also Orbitrap was applied. Several different columns, eluent systems, and pretreatment methods were tested, as well as direct infusion. The best chromatographic separation to signaling lipids was achieved with a reversed phase column at alkaline conditions. However, with this method also several drawbacks were encountered. Peaks were shifting and broadening, there were carryover effects, and problems with repeatability and sensitivity. Direct infusion on the contrary turned out to be problematic because of the unstable electrospray and formation of lysophosphatidic acid from lysophosphocholine in the ionization chamber. Development of a single analytical method for these signaling lipids turned out to be a challenging task and a complete working method for all the studied compounds was not attained.

Synthetic polymer matrices are found in many materials being part of people's everyday life. Metals are utilized in the polymerization process, and added to plastic polymers as colorants and additives. Toxic metals, such as Cd, As and Pb should not exceed certain limits within polymeric materials. Various analytical techniques may be utilized for quantification of metal concentrations in polymer samples. Many analysis methods require liquid samples with low carbon concentration. Therefore, efficient sample preparation methods are needed. Different sample digestion techniques and analytical instruments employed for these tasks are reviewed in the literature part of this thesis. Because polymers are a wide group of material with very different properties, there is no single sample preparation method applicable to all of them. Even within one sample digestion technique, there are multiple optimization parameters to be considered; generally these parameters need to be studied for every polymer type separately. In addition to the variable properties of polymer classes, also the specific properties of each metal and its chemical forms affect the choice of the analytical approach. The most frequently employed sample preparation technique is closed-vessel microwave digestion, attractive because of its efficiency and its ability to retain volatile elements. Other techniques discussed in this thesis include dry and wet ashing, fusion, and microwave induced combustion (MIC). Most commonly used instrumentation for metal analysis are inductively coupled plasma optical emission and mass spectroscopy (ICP-OES and ICP-MS), atomic absorption spectroscopy (AAS) and x-ray fluorescence XRF. In the experimental part, a microwave digestion method for quantification of As, Cd, Cr, Cu, Hg and Pb in selected polymers and monomers and using ICP-MS analysis was developed. For every studied sample, suitable digestion conditions using nitric acid as reagent were identified. Water insoluble polymers were found to be more challenging to digest as compared to water soluble and solid polyethylene samples. In addition, a full evaluation of method uncertainty was performed for the method addressing the water soluble polymer. Final expanded method uncertainty was found to be approx. 15 % at 95 % confidence level at a measurement level of 2 μg l-1 for every studied metal.

As a consequence of industrialization, metal concentrations in nature are big enough to have harmful effects on humans, animals and the environment. On that grounds, EU legislates maximum metal concentrations in several matrices. More sensitive and more selective analytical techniques are required in making sure these norms are being followed. To assess the biological activity and toxicity, along with the total metal concentration, the knowledge on the oxidation state of the metal and speciation is to be signified. In the literature part of the thesis the most used sample pretreatment and analysis techniques in metal determination in the years 2007–2017 are covered. Atomic absorption spectrometry and inductively coupled plasma based techniques are compared in determination of total metal concentration. Liquid chromatography and capillary electrophoresis coupled with mass spectrometry detection are presented as techniques used in speciation analysis. Also, sample pretreatment techniques are presented – especially solid-phase extraction – as well as parameters influencing extraction efficiency and selectivity. New sorbent materials, their synthesis and characterization, and applications are discussed in the last section of the literature part. A few studies regarding metal speciation analysis are also presented. Some future aspects of solid-phase extraction are evaluated in the end of literature part. In the experimental part of the thesis, metals were determined with inductively coupled mass spectrometer (ICP-MS) from boric acid containing waters in Loviisa Nuclear Power Plant. Determination of metals relates directly to the safe operation of the plant. However, the large concentration of boric acid in the process water samples prevents their analysis with ICP-MS. In the experimental part, boric acid concentration was diluted with two sample pretreatment methods: esterification or on-line aerosol dilution. Aerosol dilution was a better approach in process water analysis than esterification. Thus, three analytical methods using aerosol dilution in different boric acid and metal concentrations were optimized. Validation of two of these methods are presented in the end of the experimental part.

Rapid detection of bioactive compounds plays an important role in the phytochemical investigation of natural plant extracts.The hyphenated techniques, which couple on-line chromatographic separation and biochemical detection, are called high-resolution screening methods. In this system, high-performance liquid chromatography separates complex mixtures, and a post-column biochemical assay determines the activity of the individual compounds present in the mixtures. At the same time, parallel chemical detection techniques (e.g., diode-array detection, mass spectrometry, and nuclear magnetic resonance) identify and quantify the active compounds. In recent years, bioassays for radical scavenging (antioxidant) activity and immunoassays for antibodies have particularly been developed and applied. Assays for enzymes and receptors are limited. In the literature section of this thesis the development of on-line, post-column biochemical detection systems for the screening of bioactive compounds from complex mixtures was investigated. The interaction of drugs with proteins has gained significant importance in various areas of analytical chemistry. It can also be expected that more drugs will certainly be discovered with the development of biotechnology in the future. In the experimental section of this thesis comprehensive two-dimensional gas chromatography- time-of-flight mass spectrometry was used for screening for chemical composition of birch bark (Betula pendula). The exploitation of the mass spectra and retention index information allowed the identification of more than 600 organic compounds. Altogether, 59 phenolic compounds were identified in the inner layer of birch bark. To the best of our knowledge some of these compounds (e.g., raspberry ketone and tyrosol) have not been reported as extractives of Betula species before. The results achieved by gas chromatography mass spectrometry showed that several phenols with biological activity were present at relatively high concentrations in the sample. It was noticed that content of the compounds were dependent on the solvents with different polarities and volatilities. Phenols were extracted from birch bark using an environmental friendly pressurized hot water extraction technique. It provided good extraction efficiencies for phenolic compounds compared to those achieved with Soxhlet extraction. With pressurized hot water extraction the amount of extractable phenolic compounds approached for up to 23% of the dry weight whereas the amount was 2-5% (w/w) by Soxhlet extraction. Typical extraction time varied from 20 to 40 minutes. Most of the phenolic compounds were extracted at 180 °C for 40 minutes. Increase in the extraction temperature from 150 to 180°C resulted in an increase in the number of phenols extracted. However, enhanced temperature can accelerate hydrolysis and oxidation, so that unwanted or thermo-labile compounds can compose. Pressurized hot water extraction using water as a solvent proved to be a very promising extraction technique, and it surely has a great potential for the extraction of phenols from birch bark in the future.

Ionic liquids (ILs), also known as molten salts or fused salts, are a unique class of non-volatile and low-melting solvents which are composed entirely of cations and anions. These salts are liquid at room-temperature or below 100°C, thus the term 'room-temperature ionic liquids' (RTIL). Ionic liquids are known to possess some very intriguing properties such as negligible vapour pressure, remarkable thermal and electrochemical stability, non-flammability and high ionic conductivity. They have notable feature of tunability, due to which they are commonly known as 'designer solvents'. Interest in the different aspects and application of ILs in diverse fields has increased rapidly in the past few years which is pretty evident from the increasing number of research publications. The main aim of this thesis is to study the applicability of phosphonium-based ionic liquids in the field of analytical chemistry, focusing on extraction techniques. In this work, novel phosphonium-based ILs are synthesized by maintaining a common phosphonium-based cation and varying the counter-anion with carboxylate ones. Furthermore, these prepared ILs were tested for their critical micelle concentrations by using capillary electrophoresis as the mode of determination. In this work, different prospects of the use of phosphonium ILs in traditional as well as modern extraction techniques have been studied. This sub-set of ionic liquids has been the favourite choice of researchers in liquid-liquid extraction as a co-solvent as replacements of traditional volatile organic solvents. Similarly, in various microextraction techniques, phosphonium ionic liquids have been favored as a co-solvent or an ion-pair reagent while in the membrane extraction processes, they have been used as carrier in the transport of metal ions. Moreover, different parameters affecting the extraction process, for example, the effect of alkyl chain length of the ionic liquid cation and anion, branching, or even the effect of temperature have been studied. Phosphonium based ionic liquids not only find their place in the extraction of metal ions, phenolic compounds and radiochemical elements. ILs with desirable properties can be obtained by playing with the design of cation and anion of a particular IL. Taking advantage of this unique feature, many researchers have synthesized ILs to test their applicability in the extraction methods with remarkable extraction efficiencies. In the experimental section of this work, the critical micelle concentration of the synthesized phosphonium based ILs has been determined by using CE. Also, the electrosmotic flow in the fused silica capillaries are measured with the synthesized ILs and the influence of the alkyl chain length on the same is determined. Such ILs have great potential as novel capillary coatings for CE.

The aim of this thesis was to present some non-antibody-based analytical techniques and methods for protein analysis and separation. The first part of the thesis focused on the possibilities of aptamers, chemically synthesized oligonucleotide affinity tools, in protein analysis. Aptamer-based affinity chromatography, affinity capillary electrophoresis, and biosensor applications in protein analysis published in the past ten years (2007−2017) were studied. Also, microfluidic applications are discussed shortly. The literature review revealed the outstanding prospects of aptamer-based techniques in clinical diagnostics and biomedical research. Especially aptamer-based biosensors are expected to become an important technique in diagnostics assays of disease-related biomarkers. The detection limits achieved with aptasensors were in the picomolar to attomolar range in most of the reviewed studies, thereby, being comparable (or even superior) to many of the detection limits achieved with traditional antibody-based methods. However, challenges, such as the special expertise needed when working with aptamers and the low availability of appropriate aptamers mean that a commercial breakthrough of these applications is still being waited to happen. The second, the experimental, part of the thesis studied the possibility to separate lipoproteins (HDL2, LDL, VLDL, and IDL) by capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) using UV-VIS detection. Different parameters, such as separation voltage, injection style, and capillary dimensions were investigated. The capillaries were coated with poly(N-methyl-2-vinylpyridinium iodide-block-ethylene oxide (P2QVP-b-PEO) in order to prevent the adsorption of lipoproteins onto the capillary wall. It was found that CZE under physiological conditions (phosphate buffer at pH 7.4) resulted in broad low-intensity lipoprotein peaks. Furthermore, a lipoprotein mixture of all three fractions (HDL2, LDL, and VLDL/IDL) migrated as two peaks. CGE with linear polyacrylamide, on the other hand, resulted in sharp high-intensity peaks for the lipoproteins. The separation of lipoprotein mixture, however, did not succeed. The gel used in CGE studies consisted of 4% m/v acrylamide and 0.1% sodium dodecyl sulfate in Tris-aspartic acid buffer at pH 8.0. For further studies, a more systematic research plan needs to be constructed.