This project was aimed to study the degradation of polystyrene-butadiene-styrene block copolymer (SBS). SBS is used to modify bitumen, the binder in asphalt. From earlier studies it is known that SBS degrades in bitumen, but the degradation products have not been identified. To be able to determine the long term effects of SBS in the environment, degradation products need to be identified. Polystyrene forms rigid blocks with polybutadiene as a rubbery matrix between the ridgid blocks. When blended in bitumen, the polystyrene blocks are crosslinked to form a three dimensional network. SBS is an elastomer and has the ability to regain its shape after being a subject of mechanical force. According to the pre references the polymers had to be degraded in similar ways as they would in bitumen. Since bitumen is a very complex matrix with high boiling point and viscosity, there would be difficulties separating polymers from the bitumen. We made an assumption that it would be plausible for the degradation products formed in bitumen to form even with no bitumen present. Four different SBS polymers were used. One of the polymers was branched. Another had a high content of 1-2 polybutadiene. The third was a diblock copolymer (SB) with low styrene content compared to the others. The last was a linear SBS. The polymers were degraded in pieces of apparatus used for study ageing characteristics of bitumen as well as they were aged by refluxing in hexane. The degradation products were extracted by Solid Phase Extraction (SPE) and identified by GC-MS. Due to instrumental limits there were not many degradation products identified. The main products detected included saturated hydrocarbons in the range of 16-31 carbon atoms as well as squalene. / I det här projektet studerades nedbrytning av styren-butadien-styren block kopolymerer (SBS). SBS används för att modifiera bitumen vars användningsområde är som bindemedel i asfalt. Tidigare studier har visat att SBS bryts ned i bitumen, men nedbrytningsprodukterna har inte identfierats. För att kunna avgöra vad SBS har för långsiktiga effekter på miljön måste nedbrytnignsprodukterna identifieras. I SBS bildar polystyren styva block medan polybutadien fungerar som flexibla bryggor mellan polystyrenblocken. När SBS blandas med bitumen korslänkas polystyrenblocken så att ett tredimensionellt nätverk bildas. Eftersom SBS är en elastomer har den förmågan att återfå sin ursprungliga form efter att ha blivit utsatt för mekanisk stress. Enligt de givna förutsättningarna för studien skulle polymererna brytas ned på samma sätt som om de skulle ha varit lösta i bitumen. Eftersom bitumen är en komplex matris med hög kokpunkt och viskositet skulle det varit svårt att separera polymererna och deras nedbrytningsprodukter från bitumenet. Vi gjorde ett antagande om att det förmodligen bildas samma nedbrytningsprodukter som om bitumen varit närvarande även om nedbrytningen sker utan bitumen närvarande. Fyra olika SBS polymerer användes. En av polymererna var grenad. En annan hade en stor andel 1,2-polybutadien. Den tredje var en diblock kopolymer (SB) med lågt styren innehåll jämfört med de andra. Den sista polymeren var linjär. Polymererna åldrades i apparatur framtagen för att studera åldring av bitumen. Polymererna åldrades även genom att återloppskokas i hexan. Nedbrytningsprodukterna extraherades via fastfasextraktion (SPE) och identifierades med GC-MS. Genom instrumentella begränsningar kunde bara ett fåtal nedbrytningsprodukter identifieras. Bland de produkter som identifierades fanns alkaner och alkener med mellan 16 och 31 kolatomer, men även skvalen identifierades som nedbrytningsprodukt. Lågmolekylära ämnen kan ha ventilerats bort för att vi inte hade tillgång till apparatur för adsorbtion av flyktiga ämnen för injektion i GC-MS.

Capillary electrophoresis (CE) has a wide range of applications in the field of analytical chemistry. In general the most expensive part in a CE system is the detector due to the fact that the detector must have a high sensitivity for small detection volumes and low concentrations. Building portable instruments is one way to make the instruments cheaper and has the advantage that they can be used virtually everywhere. However, downscaling of CE instruments puts some extra demands on the detector. This report describes the design and building of two homemade light-emitting diode (LED) based detectors; a LEDabsorbance photometric detector (LED-AP) and a LED-induced fluorescence (LED-IF) detector. The main goal was to install them inside a portable CE and make a simple separation. The performance of the two detectors had to be evaluated before the main goal could be achieved. p-Nitrophenol was used to create a sensitivity graph for the LED-AP detector, calculating the upper linearity to 5.6 mM when the sensitivity had dropped 10 % caused by non-linearity. The sensitivity graph also showed that the detector had an effective pathlength of 74.2 µm and a stray light of 4.5 % for a 75 µm i.d fused-silica capillary. The LED-IF detector was evaluated by determining the limit of detection (LOD) for fluorescein, at a signal to noise ratio of 3. The LOD was 0.72 µM ± 0.01 µM when immersion oil was used to limit the light scattering from the optic fibres in to the capillary and 0.58 µM ±0.02 µM when silicone oil was used. Without doing any improvements only the LED-AP detector could be used in the portable CE. As a common application area for portable CE instruments is environmental analysis, indirect detection using p-nitrophenol as a probe for separating anions was done to test the system. All analytes were eluted in less than 4 minutes.

In this thesis interactions in complex fluids, such as serum and meat juice, were analysed with optical biosensor techniques. Panels of lectins immobilised on gold surfaces were used for investigation of differences in protein glycosylation pattern in sera and meat juices between various species. The present panel was also used for investigation of global glycosylation changes of serum proteins in type 1 diabetes patients. Biorecognition was evaluated with null ellipsometry and scanning ellipsometry combined with multivariate data analysis techniques (MVDA). Principal component analysis (PCA) showed that the lectin panel enabled discrimination between sera from the different species as well as for the different meat juices. The results also indicate that there is a measurable global alteration in glycosylation pattern of serum proteins in type 1 diabetic patients compared to healthy subjects. Using an artificial neuronal net (ANN), it was also possible to correctly categorise unknown serum samples into their respective class or group. The analytical potential of combining information from lectin panels with multivariate data analysis was thereby demonstrated. Also, a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D) has been developed. When measuring trace molecules, such as autoantibodies, in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labelled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (from 4 days to 50 min), while sensitivity is maintained at a level comparable to that offered by RIA. Finally, the assay was used in a screening study of newly diagnosed type 1 diabetes patients and non-diabetic subjects.

Self-assembly has emerged as a promising technique for fabrication of novel hybrid materials and nanostructures. The work presented in this thesis has been focused on developing nanoscale materials based on synthetic de novo designed polypeptides. The polypeptides have been utilized for the assembly of gold nanoparticles, fibrous nanostructures, and for sensing applications. The 42-residue polypeptides are designed to fold into helix-loop-helix motifs and dimerize to form four-helix bundles. Folding is primarily driven by the formation of a hydrophobic core made up by the hydrophobic faces of the amphiphilic helices. The peptides have either a negative or positive net charge at neutral pH, depending on the relative abundance of Glu and Lys. Charge repulsion thus prevents homodimerization at pH 7 while promoting hetero-dimerization through the formation of stabilising salt bridges. A Cys incorporated in position 22, located in the loop region, allowed for directed, thiol-dependent, immobilization on planar gold surfaces and gold nanoparticles. The negatively charged (Glu-rich) peptide formed homodimers and folded in solution at pH < 6 or in the presence of certain metal ions, such as Zn2+. The folding properties of this peptide were retained when immobilized directly on gold, which enabled reversible assembly of gold nanoparticles resulting in aggregates with well-defined interparticle separations. Particle aggregation was found to induce folding of the immobilized peptides but folding could also be utilized to induce aggregation of the particles by exploiting the highly specific interactions involved in both homodimerization and hetero-association. The possibility to control the assembly of polypeptide-functionalized gold nanoparticles was utilized in a colorimetric protein assay. Analyte binding to immobilized ligands prevented the formation of dense particle aggregates when subjecting the particles to conditions normally causing extensive aggregation. Analyte binding could hence easily be distinguished by the naked eye. Moreover, the peptides were utilized to assemble gold nanoparticles on planar gold and silica substrates. Fibrous nanostructures were realized by linking monomers through a disulphide-bridge. The disulphide-linked peptides were found to spontaneously assemble into long and extremely thin peptide fibres as a result of a propagating association mediated by folding into four-helix bundles. / Ingenjörer och vetenskapsmän har ofta inspirerats av naturen i sökandet efter lösningar på tekniska problem. Allt ifrån byggnadskonstruktioner, flygplansvingar, kompositmaterial till kardborrebandet har skapats med utgångspunkt från förebilder i naturen. Många av de material och konstruktioner som återfinns i naturen har åtråvärda egenskaper som är svåra att erhålla i syntetiska matrial med traditionell teknik. Även om vi i flera fall kan härma sammansättningen och formen blir resultatet inte nödvändigtvis det samma. Den största skillnaden mellan syntetiska material och material producerade av levande organismer är hur deras komponenter sinsemellan är organiserade och sammansatta. I syntetiska material är komponenterna ofta inbördes mer eller mindre slumpvis ordnade medan de i biologiska material är organiserade med en oerhörd precision som sträcker sig ända ned på molekyl- och atomnivå. Naturens byggstenar har genom evolutionens gång förfinats för att spontant kunna organisera sig och bilda komplexa material  och strukturer. Denna process, som styrs genom att många svaga krafter inom och mellan byggstenarna samverkar, kallas ofta för självorganisering och är en förutsättning för allt liv. Självorganisering har också blivit en allt viktigare metod inom nanotekniken för att konstruera material och strukturer med nanometerprecision. I den här avhandlingen beskrivs en typ av självorganiserande material där byggstenarna utgörs av nanometerstora guldpartiklar och syntetiska proteiner. De syntetiska proteinerna är designade för att efterlikna naturliga biomolekyler och antar en välbestämd tredimensionell struktur när två av dem interagerar med varandra. Denna interaktion är mycket specifik men kan styras genom att variera kemiska parametrar som surhet och jonstyrka vilket ger en möjlighet att påverka och kontrollera proteinernas struktur. Proteinerna har vidare modifierats för att spontant organisera sig till fibrer som är flera mikrometer långa men endast några nanometer tjocka. Proteinfibrer utgör en mycket viktig typ av strukturer i biologiska system och finns i alltifrån spindelväv till muskler. Syntetiska proteinfibrer är därför både ett intressant modellsystem och ett material med många potentiellt intressanta användningsområden. Genom att fästa de syntetiska proteinerna på ytan av guldnanopartiklar går interaktionerna mellan partiklarna att kontrollera på samma sätt som interaktionerna mellan proteinerna. Krafterna mellan proteinerna och interaktionerna involverade i proteinernas veckning har använts för att reversibelt aggregera och organisera nanopartiklarna. Ett antal olika byggstenar har studerats och utvecklats till något som liknar ett mycket enkelt nano-Lego, som på en given signal spontant bygger ihop sig eller trillar isär. Guldnanopartiklar är intressanta eftersom de är stabila och lätta att modifiera kemiskt men också på grund av deras optiska egenskaper som ger dem en ovanligt vacker vinröd färg. Färgen uppstår på grund av partiklarnas ringa storlek och varierar naturligt med egenskaperna hos den omgivande miljön. Detta gör det enkelt att studera hur partiklarna interagerar eftersom de byter färg när de närmar sig varandra, men gör dem också intressanta för sensortillämpningar. En enkel och robust sensor beskrivs i avhandlingen där syntetiska proteiner, speciellt utformade för att upptäcka och binda andra molekyler, har fästs på nanopartiklarna. Med partiklarnas hjälp går det att med blotta ögat detektera ett mänskligt protein i koncentrationer under ett tusendels gram per liter. En tidig diagnos av sjukdomstillstånd kan i de flesta fall avsevärt underlätta behandlingen och behovet av enkla sensorer för att bestämma närvaro och koncentration av medicinskt intressanta molekyler är därför mycket stort.

Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA polymerase α (Pol α) synthesize short primers required for DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) to carry out the bulk synthesis. The role of Pol δ and Pol ε at the replication fork has been unclear. The aim of this thesis was to examine what role Pol ε has at the replication fork, compare the biochemical properties of Pol δ and Pol ε, and to study the function of the second largest and essential subunit of Pol ε, Dpb2. To identify where Pol ε replicates DNA in vivo, a strategy was taken where the active site of Pol ε was altered to create a mutator polymerase leaving a unique error-signature. A series of mutant pol ε proteins were purified and analyzed for enzyme activity and fidelity of DNA synthesis. Two mutants, M644F and M644G, exhibited an increased mutation rate and close to normal polymerase activity. One of these, the M644G gave rise to a specific increase of mismatch mutations resulting from T-dTMP mis-pairing during DNA synthesis in vitro. The M644G mutant was introduced in yeast strains carrying a reporter gene, URA3, on either side of an origin in different orientations. Mutations which inactivated the URA3 gene in the M644G mutant strains were analyzed. A strand specific signature was found demonstrating that Pol ε participates in the synthesis of the leading strand. Pol δ and Pol ε are both stimulated by the processivity clamp, PCNA, in in vitro replication assays. To clarify any differences they were challenged side by side in biochemical assays. Pol ε was found to require that single-stranded template (ssDNA) was entirely coated with RPA, whereas Pol δ was much less sensitive to uncoated ssDNA. The processivity of Pol δ was stimulated to a much higher degree by PCNA than of Pol ε. In presence of PCNA the processivity of Pol δ and Pol ε was comparable. In contrast, Pol ε was approximately four times slower than Pol δ when replicating a single-primed circular template in the presence of all accessory proteins and an excess of polymerase. The biochemical characterization of the system suggests that Pol ε and Pol δ are loaded onto the PCNA-primer-ternary complex by separate mechanisms. A model is proposed where the loading of Pol ε onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand. The essential gene DPB2 encodes for the second largest subunit of Pol ε. We carried out a genetic screen in S.cerevisiae and isolated a lethal mutant allele of dpb2 (dpb2-200). When over-expressed together with the remaining three subunits of Polε, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify. The biochemical property of Pol2/Dpb3/Dpb4 complex was compared with wild-type four-subunit Pol ε (Pol2/Dpb2/Dpb3/Dpb4) and a Pol2/Dpb2 complex in replication assays. The absence of Dpb2 in the complex did not significantly affect the specific activity or the processivity, but gave a slightly reduced efficiency in holoenzyme assays when compared to wild-type four-subunit Pol ε. We propose that Dpb2 is not essential for the enzyme activity of Pol ε.

Apoptosis is a special form of cell death that if non-functional may lead to diseases such as cancer. A reduction of the intracellular potassium ion (K+) content is necessary for activating enzymes important for the execution of apoptosis. Pharmacological modulation of K+ fluxes to reduce intracellular K+ in cancer cells might therefore force the cells into apoptosis and decrease tumour cell mass. Human malignant pleural mesothelioma (MPM) is a form of cancer often caused by asbestos exposure. Although asbestos has been banned in the Western World, the incidence of MPM is expected to increase. Cisplatin is the first-line chemotherapy for MPM, but acquired resistance to the drug is a clinical problem. This thesis is mainly based on work with the human malignant pleural mesothelioma cell line (P31 wt) and a cisplatin-resistant sub-line (P31 res). The aim was to first characterize K+ fluxes in P31 wt and P31 res cells, and then manipulate them in order to reduce intracellular K+ and induce apoptosis with K+ manipulation alone or in combination with cisplatin. Characterization of K+ fluxes in P31 wt cells showed that: 1) ouabain, a digitalis-like drug, and specific blocker of the Na+, K+, ATPase pump, effectively inhibited K+ uptake, 2) bumetanide, a diuretic, and an inhibitor of the Na+, K+, 2Cl-¬-cotransporter, had a transient effect on K+ uptake, and 3) the antifungal drug amphotericin B stimulated K+ efflux. In order to determine intracellular K+ content, the potassium-binding fluorescent probe PBFI-AM was used in a 96-well plate assay. After a 3-h incubation with ouabain, with or without bumetanide, combined with amphotericin B, the intracellular K+ content was reduced in P31 wt cells but not in P31 res cells. Ouabain induced apoptosis in both P31 wt and P31 res cells. P31 res cells were sensitized to cisplatin by ouabain, since 10 mg/L cisplatin in combination with ouabain induced about the same percentage of apoptotic cells as 40 mg/L cisplatin. Apoptosis was executed via caspase-3 activation in both P31 wt and P31 res cells. Amphotericin B enhanced ouabain-induced apoptosis in P31 wt cells via caspase-9 activation, with increased caspase-3 activation and DNA fragmentation as consequences. Ouabain-induced apoptosis in P31 res cells was executed via increased expression of pro-apoptotic Bak. The combination of cisplatin with ouabain and amphotericin B was stressful to both P31 wt and P31 res cells, since SAPK/JNK a known factor in stress-induced apoptosis was activated. In conclusion, K+ flux manipulation with clinical used drugs can induce apoptosis per se and also enhance cisplatin-induced apoptosis in P31 wt and P31 res cells.

This thesis reports on interactions between microgels and oppositely charged proteins. Two types of negatively charged microgels are investigated: poly(acrylic acid) microgels of 60-80 µm in diameter, and colloidal poly(NIPAM-co-acrylic acid) microgels of around 1 µm in diameter. The proteins used are lysozyme and cytochrome c, which both have positive net charge. The experimental techniques used in the studies of the larger microgels are mainly micromanipulator-assisted microscopy and confocal microscopy, while the smaller microgels are studied mainly with dynamic light scattering. It is observed that large amounts of protein are absorbed by the microgels, and that the uptake involves a substantial deswelling of the microgel. The uptake generally decreases as the ionic strength is increased, which is characteristic of electrostatic interactions. An ionic strength optimum is however observed in the case of lysozyme and poly(acrylic acid) microgels, where the highest uptake (10 gram lysozyme / gram microgel) is observed at ionic strength 40 mM. Cytochrome c uptake in poly(acrylic acid) microgels results in homogenous cytochrome c distribution throughout the microgel, whereas lysozyme uptake results in core-shell formation; the lysozyme concentration becomes much higher in the shell (outer part of the microgel) than in the core (inner part of the microgel). The shell constitutes a stress-bearing network which is sufficiently porous to allow protein diffusion through the shell. The different protein distributions are associated with different protein-protein interactions; strong protein-protein attraction promotes shell formation. In the case of colloidal microgels, lysozyme uptake decreases the electrophoretic mobility and the colloidal stability of the microgels. The microgels flocculate as the uptake reaches charge ratio 0.6-0.7 (positive lysozyme charges/negative microgel charges), largely independent of ionic strength. Initial experiments on the combination of cytochrome c and colloidal microgels show that colloidal stability is maintained at a range of conditions (ionic strength, protein concentration) where flocculation occurred in the case of lysozyme.

The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.

Vattenhaltsbestämning är av stor vikt i många sammanhang. T.ex. kan vattenhalten påverka utbytet av en kemisk syntes, eller ha negativ inverkan på hållbarheten av läkemedel och livsmedel. Standardmetoden för vattenhaltsbestämning är Karl Fischer-titrering, baserad på antingen volymetri eller coulometri. I den här avhandlingen presenteras nya infallsvinklar för bestämning av mycket låga halter vatten i komplexa provmatriser, som t.ex. tekniska oljor och substanser som interfererar med alkoholbaserade Karl Fischer-reagens. Vattnet avskiljs ofta från oljematrisen före titrering genom förångning. I samband med framtagningen av nya referensmaterial för vatten i olja ifrågasattes förångningsteknikernas effektivitet av National Institute of Standards and Technology (NIST). NIST menade att en fraktion av vattnet bands hårt i oljefasen och att det inte kunde frigöras och detekteras annat än med en modifierad volymetrisk metod där reagenset innehöll minst 65% kloroform. I den här avhandlingen presenteras en alternativ metod som uppfyller det ställda kravet för en fullständig upplösning av oljefasen. Med denna metod visas att det inte finns någon anledning att ifrågasätta förångningsteknikernas effektivitet och att den modifierade metoden som NIST använder ger systematiskt för höga resultat. Fördelar som enklare handhavande, kortare konditioneringstider och att endast ett reagens behövs har gjort att diafragmafri coulometri har blivit allt mer populär. Spårhaltsbestämning med denna teknik ställer dock speciellt höga krav på reagensen eftersom strömtätheten vid katoden är låg. Med anledning av detta testades olika typer av kommersiella reagensblandningar för bestämning av små vattenmängder och kritiska parametrar identifierades. Dekanol visade sig ha en gynnsam effekt på katodreaktionen i reagens modifierade med xylen enligt standardmetodbeskrivningen för bestämning av vatten i oljor. För provtyper som inte går att analysera med alkoholbaserade reagenser presenteras en ny typ baserad på N-metylformamid. Med ett sådant reagens bestämdes vattenhalten i ett reaktivt salt som används i litiumjonbatterier. Liknande alkoholfria reagens undersöktes mer utförligt i en djupare studie som även inkluderade formamid och dimetylformamid. För- och nackdelar med dessa alternativa lösningsmedel diskuteras och möjliga reaktionsförlopp föreslås. Det visade sig att läget på jämvikten mellan svaveldioxid och vätesulfit är en avgörande faktor för att förklara den stora skillnaden i reaktionshastighet i dessa lösningsmedel. / Moisture determination is of great importance in the production and use of many substances. For example, the moisture content can affect the efficiency of a chemical reaction or determine the shelf life of pharmaceuticals or foods. The standard method for moisture determination is Karl Fischer (KF) titration, based on either volumetry or coulometry. This thesis concerns new approaches to trace determination in complex sample matrices and is focused on oils and substances that interfere with alcoholic KF reagents. Moisture is frequently separated from oil matrices before titration by means of evaporation techniques. In connection with the preparation of new reference materials for moisture in oil, the National Institute of Standards and Technology (NIST) questioned the efficiency of such evaporation techniques. NIST claimed that some of the moisture was sequestered in the oil phase and that it could only be released and detected by using a modified volumetric KF method with a reagent containing at least 65% chloroform. In this thesis, an alternative KF method that meets the proposed requirement for a complete dissolution of the oil sample is presented. With this method it is shown that there is no reason to question the efficiency of the evaporation techniques and that the criticized volumetric method used by NIST is biased high. Ever since its introduction diaphragm-free coulometry has gained popularity due to its ease of use, with a single reagent and short conditioning times. Trace determination with this technique sets great demands on the reagent due to the resulting low current densities at the generator cathode. The performance of several commercial reagents is evaluated under such unfavorable conditions and critical titration parameters are identified. It is also shown that decanol has a favorable effect on the cathode process when using reagents modified with xylene according to standard methods for moisture determination in oils. For samples that are incompatible with the alcohol component in ordinary KF reagent a new reagent based on N-methylformamide is presented. It is shown that is works well for determinations of moisture in a conductive salt used in lithium-ion batteries. The concept of alcohol-free KF reagents is taken a step further in a systematic investigation, also including formamide and dimethylformamide. Advantages and disadvantages with these solvents are discussed and possible reaction paths are surveyed. It is shown that the position of the sulfur dioxide/hydrogen sulfite equilibrium is the main explanation for the large differences in the KF reaction rates in these solvents.

Electron paramagnetic resonance (EPR) dosimetry using the well established dosimeter material alanine is a generally accepted dosimetric method for measurements of high absorbed doses. Alanine EPR dosimetry is however not sensitive enough for high precision measurements of low (< 5 Gy) absorbed doses using reasonably measurement times and small dosimeters. It has therefore not been possible to fully exploit the benefits of EPR dosimetry for applications in radiation therapy. The aim of this thesis was to show that sensitive EPR dosimetry is a competitive method for applications in radiation therapy fulfilling the requirements of measurement precision. Our strategy for reaching this goal was to search for new, more sensitive, EPR dosimeter materials fulfilling the criteria of being tissue equivalent, having a high radical yield and having a narrow EPR spectrum suitable for dosimetry. The best materials were found among formates and dithionates. Doping with small amounts of metal ions and recrystallisation in D2O were tested to further increase the sensitivity. Four promising candidate materials were tested regarding radical stability and dose response and among them lithium formate was chosen for dosimetry in radiation therapy applications. A high precision EPR dosimetry method was developed using lithium formate. The method included the development of a production method for EPR dosimeters with very homogenous shape, mass and composition. A read-out process was developed with maximal measurement precision for reasonably short measurement times. The method also included a dosimeter quality control before actual dose measurements. Measurement accuracy was controlled for every new dosimeter batch. This high precision lithium formate EPR dosimetry method was evaluated for pretreatment verifications of intensity modulated radiation therapy (IMRT) treatment plans. The precision and accuracy was shown to be sufficient (< 5 %) for measurements of doses above 1.5 Gy using one single dosimeter and a measurement time of 15 minutes. The described evaluation is therefore a demonstration of the improved precision at low dose determinations that is available with our sensitive EPR dosimeter materials. While the EPR signal intensity is proportional to absorbed dose, the signal shape is in some cases dependent on the radiation quality. A new method is presented for simultaneous measurements of beam LET (linear energy transfer) and absorbed dose in heavy charged particle beams using potassium dithionate EPR dosimetry. The study shows that when irradiating a dosimeter with 35 MeV carbon ions, the ratio of the signal amplitudes from two radicals in potassium dithionate vary along the track indicating a dependence on linear energy transfer, LET. Potassium dithionate may therefore be a promising EPR dosimeter material for simultaneous measurements of absorbed dose and LET in heavy charged particle radiation fields.