Oligonucleotides may become a new class of therapies with the potential of curing many today untreatable diseases. Oligonucleotides becomes increasingly more difficult to separate with an increase in length since the relative difference in retention of these very similar compounds becomes increasingly smaller. Therefore, coelution of impurities formed during synthesis may result in insufficient purity, which is necessary for therapeutic treatments. Oligonucleotides are also relatively large biomolecules, possibly consisting of hundreds of nucleotides. As a result, oligonucleotides may have limited diffusion through the stationary phase pores which affects separation performance. Surprisingly few studies have be published in this research area and a wider knowledge in how this affects separation is needed. In this master thesis, separation of deoxythymidine oligonucleotides with 5-30 mers in length were separated with 60, 100, 200 and 300 Å pore size reversed phase C4 columns. It was concluded that pore size resulted in more restricted diffusion if insufficient pore size was used. Poor peak performance was also observed with too large pore sizes which lead to less efficient separations.

Seizures containing large numbers of units of narcotics, goods dangerous to health and doping are often sent to the Swedish National Forensic Centre (NFC). Only a fraction of these capsules or tablets can be analyzed, therefore the samples need to represent the whole seizure. If the samples show content variations, Near Infrared (NIR) spectroscopy in combination with hyperspectral imaging has been shown to be a promising tool to gauge the homogeneity in the seizures based on chemical content. The objective of this thesis was to further develop and then validate a method for the separation of pregabalin and gabapentin capsules using NIR hyperspectral imaging and Principal Component Analysis (PCA). Capsules containing different amounts of pregabalin and gabapentin were prepared and analyzed. Additionally, authentic seizures were analyzed to confirm that the method fulfilled its purpose. The result of this study showed that use of hyperspectral data in the wavelength range 1650-1750 nm gave the best differentiation between pregabalin and gabapentin capsules. Capsules containing the ratio 70-30 % gabapentin and pregabalin could be separated distinctively from capsules containing pure gabapentin. Multiple authentic seizures could be separated into groups correctly depending on the capsules or tablets content.

Cereals are rich in phenolic acids, a group of secondary plant metabolites that are associated with reduced risk of chronic diseases. The objective was to develop and internally validate a method for extraction and quantification of phenolic acids in cereals using HPLC-UV and to apply this method for quantification of the content of phenolic acids in several species of Swedish cereals. Different procedures for extraction of phenolic acids from cereal grains using acid or base hydrolysis with and without subsequent enzymatic treatment were tested. Both the extraction procedure and the chromatographic conditions for quantification with HPLC-UV were optimized. Phenolic acids from 14 cereal samples, representing different cultivars of rye, wheat, barley, and oat, were extracted and analyzed under optimized conditions. Using the optimized method, 15 phenolic acids could be quantified with limits of detection and quantification ranging from 0.4 to 11.4 µg/g and from 1.3 to 38.0 µg/g, respectively. The hydrolysis procedure and further sample treatment showed a substantial effect on the yield of phenolic acids from cereals. The highest yield was achieved by 90‑minute base hydrolysis at room temperature using sodium hydroxide solution containing ascorbic acid and EDTA. Mean recoveries ranged from 88 to 108%. The following phenolic acids were found in the analyzed cereal grains with ferulic acid being the most abundant one: p‑hydroxybenzoic acid, vanillic acid, vanillin, caffeic acid, syringic acid, ferulic acid, sinapic acid, and 3,4‑dihydroxybenzaldehyde. A further compound was p‑coumaric acid or the co‑eluting syringaldehyde or a mixture of both. The content of phenolic acids in Swedish cereals ranged from 6 µmol/g DM in rye to 3 µmol/g DM in oat and a barley cultivar. In conclusion, a simple and accurate method for extraction and quantification of phenolic acids in cereals was developed and successfully applied. / Getreide ist reich an Phenolsäuren, einer Gruppe pflanzlicher Sekundärmetabolite, die mit einem verringerten Risiko für chronische Erkrankungen in Verbindung gebracht wird. Ziel war es, eine Methode zur Phenolsäure-Extraktion aus Getreide und Quantifizierung mittels HPLC-UV zu entwickeln, intern zu validieren und diese im Anschluss anzuwenden, um den Phenolsäure-Gehalt in mehreren schwedischen Getreidearten zu quantifizieren. Verschiedene Verfahren zur Phenolsäure-Extraktion aus Getreide unter Verwendung von Säure- oder Basenhydrolyse mit oder ohne nachfolgender enzymatischer Hydrolyse wurden getestet. Es wurden sowohl das Extraktionsverfahren als auch die chromatographischen Bedingungen zur Quantifizierung mittels HPLC-UV optimiert. Phenolsäuren von 14 Getreideproben, darunter Kultivare von Roggen, Weizen, Gerste und Hafer, wurden unter optimierten Bedingungen extrahiert und analysiert. Mit der optimierten Methode konnten 15 Phenolsäuren mit Nachweisgrenzen von 0,4 bis 11,4 µg/g und Bestimmungsgrenzen von 1,3 bis 38,0 µg/g quantifiziert werden. Hydrolyseverfahren und weitere Probenbehandlung haben die Phenolsäure-Ausbeute von Getreide wesentlich beeinflusst. Die höchste Ausbeute wurde durch eine 90‑minütige Basenhydrolyse bei Raumtemperatur unter Verwendung von Natronlauge mit Ascorbinsäure und EDTA erzielt. Die mittlere Wiederfindung betrug 88 bis 108%. In den untersuchten Getreideproben wurden folgende Phenolsäuren gefunden mit Ferulasäure als häufigster Verbindung: p-Hydroxybenzoesäure, Vanillinsäure, Vanillin, Kaffeesäure, Syringasäure, Ferulasäure, Sinapinsäure und 3,4‑Dihydroxybenzaldehyd. Eine weitere Verbindung war p‑Cumarsäure oder der co‑eluierende Syringaldehyd oder eine Mischung aus beiden. Der Phenolsäure-Gehalt reichte von 6 µmol/g DM in Roggen bis 3 µmol/g DM in Hafer und einem Gerstenkultivar. Zusammenfassend wurde eine einfache und genaue Methode zur Phenolsäure-Extraktion und Quantifizierung in Getreide entwickelt und erfolgreich angewendet.

<p>An intrinsic mathematical model for simulation of fixed bed chromatography was demonstrated and compared to more simplified models. The former model was shown to describe variations in the physical, kinetic, and operating parameters better than the latter ones. This resulted in a more reliable prediction of the chromatography process as well as a better understanding of the underlying mechanisms responsible for the separation. A procedure based on frontal liquid chromatography and a detailed mathematical model was developed to determine effective diffusion coefficients of proteins in chromatographic gels. The procedure was applied to lysozyme, bovine serum albumin, and immunoglobulin γ in Sepharose™ CL-4B. The effective diffusion coefficients were comparable to those determined by other methods.</p><p>Molecularly imprinted polymers (MIPs) are traditionally prepared as irregular particles by grinding monoliths. In this thesis, a suspension polymerization providing spherical MIP beads is presented. Droplets of pre-polymerization solution were formed in mineral oil with no need of stabilizers by vigorous stirring. The droplets were transformed into solid spherical beads by free-radical polymerization. The method is fast and the performance of the beads comparable to that of irregular particles. Optimizing a MIP formulation requires a large number of experiments since the possible combinations of the components are huge. To facilitate the optimization, chemometrics was applied. The amounts of monomer, cross-linker, and porogen were chosen as the factors in the model. Multivariate data analysis indicated the influence of the factors on the binding and an optimized MIP composition was identified. The combined use of the suspension polymerization method to produce spherical beads with the application of chemometrics was shown in this thesis to drastically reduce the number of experiments and the time needed to design and optimize a new MIP.</p>

<p>This thesis describes the use of nuclear magnetic resonance (NMR) spectrometry as an analytical tool. The theory of NMR spectroscopy in general and quantitative NMR spectrometry (qNMR) in particular is described and the instrumental properties and parameter setups for qNMR measurements are discussed. Examples of qNMR are presented by impurity determination of pharmaceutical compounds and analysis of urine samples from rats fed with either water or a drug (metabolic profiling). The instrumental parameter setup of qNMR and traditional data pre-treatments are examined. Spectral smoothing by convolution with a triangular function, which is an unusual application in this context, was shown to be successful regarding the sensitivity and robustness of the method in paper II. In addition, papers III and IV comprise the field of peak alignment, especially designed for ¹H-NMR spectra of urine samples. This is an important preprocessing tool when multivariate analysis is to be applied. A novel peak alignment method was developed and compared to the traditional bucketing approach and a conceptually different alignment method.</p><p>Univariate, multivariate, linear and nonlinear data analyses were applied to qNMR data. In papers I–II, calibration models were created to examine the potential of qNMR for these applications. The data analysis in papers III–VI was mainly explorative. The potential of data fusion and data correlation was examined in order to increase the possibilities of analysing the highly complex samples from metabolic profiling (papers V–VI). Data from LC/MS analysis of the same samples were used with the ¹H-NMR data in different ways. Correlation analyses between the¹H-NMR data and the drug metabolites identified from the LC/MS data were also performed. In this process, data fusion proved to be a valuable tool.</p>

<p>Computer simulations can be useful aids for both developing new analytical methods and enhancing the performance of existing techniques. This thesis is based on studies in which computer simulations were key elements in the development of several new tools for use in gas chromatography and dielectrophoresis. In gas chromatography, gaseous analytes are separated by exploiting differences in their partitioning between different phases, and after their partitioning parameters have been determined the separations can be computationally predicted, and optimized, for a wide range of operating conditions. Similarly, in dielectrophoresis, particles with differing polarizability or size can be separated, and since particle trajectories within a separation device can be predicted using computations, the suitability of new designs, applications of forces and combinations of operational parameters can be assessed without necessarily making or empirically testing all of the variants.</p><p>Using two existing numerical methods combined with semi-empirical determinations of retention behavior, temperature-programmed gas chromatograms were predicted with less than one percent deviations from experimental data, and a new method for improving the capacity of a gas-trapping device was predicted and experimentally verified. In addition, two new concepts with potential capacity to enhance dielectrophoretic separations were developed and tested in simulations. The first provides a promising way to improve the trapping of bacteria in media with elevated conductivity by using super-positioned electric fields, and the second a way to increase selectivity in the separation of bio-particles by using multiple dielectrophoretic cycles. The studies also introduced a more accurate method for determining the conductivity of suspensions of bacteria, and a new computational method for determining the dielectrophoretic behavior of particles in concentrated suspensions.</p><p>The scientific studies are summarized and discussed in the main text of this thesis, and presented in detail in seven appended papers.</p>

<p>Liquid chromatography is an important separation technique for a vast number of analytes. This thesis mainly focuses on the development of stationary phases for liquid chromatography based on zwitterionic sulfobetaine polymers.</p><p>In the thesis, various ways to prepare zwitterionic polymers in an aqueous environment using reversible addition fragmentation chain transfer (RAFT) polymerization are described. Both telomers, i.e. short soluble polymer chains containing a functional terminal group, as well as graft polymers on various supports have been synthesized. The RAFT polymerization technique provides an increased degree of control of the final polymers, which may aid in the preparation of more specifically tailored separation materials.</p><p>Sulfobetaine polymers carry both a positive and a negative charge within a single entity, which results in interesting solution properties as well as highly biocompatible features. These unique features make them especially suited for separation of highly polar and/or charged compounds. An example of the successful separation of short peptides using a stationary phase synthesized with the RAFT technique is given.</p><p>The unusual properties of sulfobetaine-type polymers are believed to be associated with the structure of water close to the polymer. A study of water structure in some silica based stationary phase grafted with zwitterionic sulfobetaine polymers was conducted. The impact of water structure on retention characteristics was investigated.</p>

<p>A new method for analysis of acidic herbicides, mostly phenoxy acids and their esters, in cereals with liquid chromatography-tandem quadrupole mass spectrometry (LS-MS/MS) has been developed. Samples were hydrolyzed with sodium hydroxide in order to release covalently bound compounds followed by neutralization and finally extraction with acidified ethyl acetate. The extraction efficiency for both ester formulations and acids were studied. Acceptable results (70-120 %) were obtained for 2,4-D, dichlorprop, MCPA and mecoprop for both esters and acids. However, low recoveries were observed for ester formulations of dicamba, fluroxypyr, fluazifop and haloxyfop, possibly due to the complex structure of the compounds in combination with the matrix and/or incomplete hydrolysis step. The limit of quantification (LOQ) for targeted pesticides was 0.01 mg/kg. The method has been tested in the EU Proficiency Test for cereals with good results.</p>

<p>Traditionally, rolling mills use emulsions based on a mixture of oil and water for lubrication. Since two years ago SAPA has been using (instead of oil) a synthetic lubricant so called conditional emulsion for hot-rolling of aluminum. This lubricant is water based and homogenous at ambient temperature, but switches to a two-phase system at heating above the cloud point.</p><p>This project aims to validate and if necessary modify an existing HPLC method for quantifying two out of three key components (A, B and C) in the conditional emulsion. Attempts to develop a method to quantify the pH adjusting components, X and Y were also made. These two methods are required to optimize the lubricant.</p><p>Due to the complexity of the components, it has been difficult to present a method for quantification, and HPLC with ELS detection was chosen after a long series of trials. Due to a few uncontrollable parameters the proposed analysis method has tendencies to be unstable. The column used is sensitive to changes in equilibrium and ELSD is also less sensitive and less reproducible than the commonly used UV-detector.</p><p>While the proposed assay method shows somewhat large relative standard deviations the method has been shown to produce sufficiently precise and accurate data for the intended purpose.</p><p>Development of a method for the pH-adjusting components X and Y was more difficult than expected. For some reason their difference in chemical properties does not show satisfying impact in the chromatograms.</p><p>This method is still in its cradle and needs further development.</p><p> </p>

<p>Platelets are important for the healing of damaged blood vessels since they have an importantpart to play in the coagulation process. At the same time, the blood must be kept fluid and notcoagulate at the wrong time. Therefore there are factors that effect the aggregation of plateletsin a positive or a negative way.</p><p>Previous investigations have shown that platelets during stirring conditions produce reactiveoxygen species (ROS) that weaken the inhibiting effect of nitric oxides (NO) on platelets andthat the drug Dexamethasone (Dex) can reduce the ROS-production.</p><p>The aim of this project was to investigate if glucocorticoids, in this case Dexamethasone,could restore the inhibiting effect of NO on platelets and if there was any decrease in ROS-production.</p><p>The result of the ROS-measurements showed a great variance and it was difficult to draw anyconclusions from them, but a clear decrease in ROS, as previous reported, was not shown. In the aggregation experiments the inhibiting effect of NO was observed through the drug S-nitroso-N-acetylpenicillamine (SNAP), a NO-donator.</p><p>From the aggregation experiments, the result seemed to be that SNAP during longerincubation time lost its inhibiting effect, probably because the cells become desensitized.With superoxid dismutase (SOD), the effect of SNAP increased, both in the experiment withlonger and shorter incubation times. Dex seemed to reinforce the aggregation in relation toboth SOD and SNAP. To understand this relation further, more investigations must be done.Another interesting experiment would be to do combinations of experiments monitoring bothaggregation and ROS-production at the same time.</p> / <p>Trombocyterna, blodplättarna i blodet, är livsviktiga för att människor inte ska förblöda vid enskada. Samtidigt måste blodet hållas flytande och inte koagulera i onödan och därför finns deti kroppen en mängd faktorer som verkar pro- eller antiaggregerande.</p><p>Tidigare undersökningar har visat på att trombocyter har en omrörningsberoendesyreradikalproduktion (ROS) som försvagar kväveoxids (NO) antiaggregerande effekt och attläkemedlet Dexametason (Dex) kan minska denna produktion.</p><p>Detta projekt syftade till att ytterligare studera om glukokortikoider, i detta fall Dexametason,kunde återställa NO:s effekt på trombocyterna och om de i någon grad minskaderadikalproduktionen.</p><p>Resultatet av ROS-mätningarna blev väldigt varierande och svårtolkade och några säkraslutsatser kunde inte dras, men en tydlig minskning i produktionen som tidigare observeratskunde inte upptäckas. I aggregationsförsöken observerades NO:s inhibitoriska verkan genomS-nitroso-N-acetylpenicillamine (SNAP), en NO-donator.Resultaten tyder på att SNAP under en längre inkuberingstid tappar sin inhiberande förmågapå trombocyterna, vilket förmodligen beror på att cellerna desensibiliseras.Superoxiddismutas (SOD) verkar ha en förstärkande effekt på SNAP oavsett ominkuberingstiden innan dosresponstillsats av trombin är lång eller kort, medan Dex tenderaratt förstärka aggregeringen både i förhållande till SNAP och SOD. För att få mer klarhet omdessa resultat är korrekta måste fler upprepningar göras och dessutom borde man genomförakombinationsförsök där man samtidigt övervakar ROS-produktion och aggregering.</p>