Polystyrene behaviour in reversed phase high performance liquid chromatography was influenced mainly by the solvent system, but secondary affects were observed depending on the stationary phase. A variety of reversed phase columns were investigated using mobile phase combinations of dichlorom ethane-methanol, dichloromethane-acetonitrile, ethyl acetate-methanol and ethyl acetate-acetonitrile. Several different modes of behaviour were observed depending on the polymer solubility in the solvent system. In the dichloromethane-methanol solvent system, polymer-stationary phase interactions only occurred when the molecules had pore access. Retention of excluded polystyrene depended on the kinetics of precipitation and redissolution of the polymer. Peak splitting and band broadening occurred when the kinetics were slow and molecular weight separations were limited !o oligomers and polystyrenes lower than 5-10(4) dalton. Excellent molecular weight separations of polystyrenes were obtained using gradient elution reversed phase chromatography with a dichloromethane-acetonitrile mobile phase on C18 columns. The retention was based on polymer-stationary phase interactions regardless of the column pore size. Separations were obtained on large diameter pellicular adsorbents that were almost as good as those obtained on porous adsorbents, showing that pore access was not essential for the retention of high molecular weight polystyrenes. In the best example, the separation ranged from the monomer to 10(6) dalton in a single analysis. Very little adsorption of excluded polymers was observed on C8 or phenyl columns. Polystyrene molecular weight separations to 7-10(5) dalton were obtained in an ethyl acetate-acetonitrile solvent system on C18 columns. Adsorption was responsible for retention. When an ethyl acetate-methanol solvent system was used, no molecular weight separations were obtained because of complex peak splitting. Reversed phase chromatography was compared to size exclusion chromatography for the analysis of polydisperse polystyrenes. Similar results were obtained using both methods. However, the reversed phase method was less sensitive to concentration effects and gave better resolution.

The purpose of the studies included in this thesis is to develop an effective an efficient method to study the proteome using separation and detection of peptides, when only a limited amount of sample, 10 micrograms of total protein or less, is available. The analysis will be applied to multiple myeloma cancer cells using ultra high-performance liquid chromatography-mass spectrometry for expression proteomics to illustrate utility. To detect low abundance peptides in a complex proteome, we use different strategies, including basic pH reversed-phase liquid chromatography (bRPLC), mass-to-charge fractionation in the mass spectrometer, and various liquid chromatography gradients to increase peptide separation to improve opportunities for detection and quantification. The different methods are optimized and compared by the number of peptides detected. Step-wise elution of bRP spin columns proved to yield more than 36,000 peptides using only 10 μg of protein. Mass-to-charge (m/z) fractionation was tested in mass analyzer Q-Exactive Plus (Thermo Scientific). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of an unfractionated sample was analyzed 4 times at different mass ranges, each mass range width of 150 m/z, resulting from 4 spectra combined, 31,732 peptides representing 3,967 proteins. Showingcomparable results to those form high pH reversed phase fractionation spin columns 5 fractions. Establishing a benchmark where the LC-MS/MS analysis of 600 μg of 10plex TMT-labeled peptides fractionated with bRPLC into 24 fractions yielded over 74,000 peptides from 7,700 proteins, we compared those results with analysis of 10 μg of total TMT-labeled peptides fractionated by bRP spin columns into 5 fractions, which produced 14,019 peptides from 3,538 proteins. These experiments were used to relatively quantify protein expression in naïve and drug resistant multiple myeloma cells lines as an example application in cancer research.

The thesis consists of two parts targeting the characterization of chromatographic behavior of linear tryptic and cyclic peptides containing oxidized methionine (Met) in reversed-phased chromatography. The retention order of methionine-containing peptide analogues was observed to be the same in both studies: Met oxide < Met dioxide < Met. For linear tryptic peptides, the magnitude of the retention time shift may vary dramatically: from –9 % to +0.36 % acetonitrile. Particularly, large negative retention time shifts are found mostly associated with methionine being in the hydrophobic face of an amphipathic helix. Contrary to previously reported observations, I demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity, this occurs only when methionine is in the N3 position of the N-capping stabilization motif preceding an amphipathic helix. In the second study, the effect of peak splitting was observed for some Met oxide-containing cyclolinopeptides, which most likely appear due to diastereomerization.

Twenty different macrolide and ketolide antibiotics were analyzed by reversed phase high performance liquid chromatography on an ODS-2 cartridge column. Each of these compounds was uniquely separated and purified by varying the flow rate. Retention times of the individual drugs were proportional to the flow rate of the mobile phase. Recovery of antimicrobial activity for most of the drugs was greater than 90% based on a microbiological assay of material recovered from the column. Retention times were related to structural differences between these antimicrobial agents.

Highly cross-linked monoliths prepared from single cross-linking monomers were found to increase surface area and stability. Therefore, seven cross-linking monomers, i.e., 1,3-butanediol dimethacrylate (1,3-BDDMA), 1,4-butanediol dimethacrylate (1,4-BDDMA), neopentyl glycol dimethacrylate (NPGDMA), 1,5-pentanediol dimethacrylate (1,5-PDDMA), 1,6-hexanediol dimethacrylate (1,6-HDDMA), 1,10-decanediol dimethacrylate (1,10-DDDMA), and 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), were used to synthesize highly cross-linked monolithic columns in 75-µm i.d. capillaries by one-step UV-initiated polymerization using dodecanol and methanol as porogens for reversed-phase liquid chromatography (RPLC) of small molecules. Selection of porogen type and concentration was investigated in detail. Isocratic elution of alkylbenzenes at a flow rate of 300 nL/min was conducted for all of the monoliths. Gradient elution of alkylbenzenes and alkylparabens provided high resolution separations. Several of the monoliths demonstrated column efficiencies in excess of 50,000 plates/m. Monoliths with longer alkyl-bridging chains showed very little shrinking or swelling in solvents of different polarities. In addition, highly cross-linked monolithic capillary columns poly(1,6-HDDMA), poly(cyclohexanediol dimethacrylate) [poly(CHDDMA)] and poly(1,4-phenylene diacrylate) [poly(PHDA)], were synthesized and compared for RPLC of small molecules. Isocratic elution of alkylbenzenes was performed using 1,6-HDDMA and CHDDMA monolithic columns. Gradient elution of alkylbenzenes using all three monolithic columns showed good separations. Monolithic columns formed from 1,6-HDDMA, which had a linear alkyl-bridging chain structure, exhibited the highest column efficiencies (86,000 plates/m). Optimized columns showed high permeability and high run-to-run and column-to-column reproducibilities. Monoliths prepared from controlled/living polymerization was demonstrated exhibiting narrower molecular weight distribution and more homogeneous cross-linked structures due to the reversible character of this polymerization method. Thus, monolithic columns were developed from three cross-linking monomers, i.e., 1, 12-DoDDMA, trimethylolpropane trimethacrylate (TMPTMA) and pentaerythritol tetraacrylate (PETA) using organotellurium-mediated living radical polymerization (TERP) in 150-µm i.d. capillaries for RPLC of small molecules. Selection of the polymerization conditions for the 1,12-DoDDMA monolirh was investigated in detail. Isocratic elution of alkylbenzenes was achieved with good efficiency (47,700 to 64,200 plates/m for uracil) using all monolithic columns prepared using TERP.

Volvo Cars är ett välkänt företag som säljer bilar över hela världen. Idag skickar Volvo Cars sin kylvätska till leverantörer för analys. Processen är komplicerad och tar mycket tid. Det finns ett intresse hos Volvo Cars att själva kunna analysera kylvätskan. För att kunna göra detta krävs det att en metod som de kan använda för att analysera kylvätskan utvecklas. Examensarbetet syftar till att utveckla en metod som gör det möjligt att analysera koncentrationen av organiska syror i Volvos kylvätska. Analysen ska göras med hjälp av en High Performance Liquid Chromatography - HPLC. De organiska syrorna är inhibitorer som skyddar material i motorn från korrosion när det kommer i kontakt med kylvätskan. Metoden utvecklas i flera steg, det är viktigt att veta hur olika organiska syror kan analyseras med en HPLC. HPLC-analyser är mycket beroende av de intrumentella inställningarna. Inställningarna som ändrar när metoden utvecklas är pH, typ av kolonn, våglängd på detektorn, flödeshastighet i kolonnen, injektionsvolymen, sammansättning av eluent (lösningsmedel) och analystid. Kalibreringskurvor konstrueras för att sedan kunna användas i fortsatta studier. Tre av fem organiska syror som skulle analyseras var möjliga att analysera med två olika metoder. Den enda skillnaden mellan metoderna var våglängden på detektorn. En av de organiska syrorna hade en mer linjär kalibreringskurva vid en högre våglängd. / Volvo Cars is a well-known company and they are selling their cars all over the world. Today, Volvo Cars send their engine coolant to the supplier of engine coolant for analysis. This process is complicated and takes a lot of time. It is in the interest of Volvo Cars to be able to analyze the engine coolant themselves. In order to do that, a method where they can analyze the engine coolant must be developed. This exam work aim to develop a method where it is possible to analyze the concentration of organic acids in Volvo´s engine coolant with a High Performance Liquid Chromatography - HPLC. The organic acids are inhibitors that protect the materials in the engine from corrosion when in contact with the engine coolant. The method is developed in several steps. First it is important to know how different organic acids could be analyzed with an HPLC. HPLC-analysis is very dependent of the instrumental parameters. The parameters you change when developing a method are pH, kind of column, wavelength of the detector, kind of detector, flow rate in the column, injections volume, composition of the eluent (solvent) and, analysis time. Calibration curves are created and could be used as a reference in further analysis. Three of the five organic acids that were supposed to be analyzed were possible to analyze. Three organic acids were analyzed with one method and the two remaining organic acids with another method. The only difference between the methods is the wavelengths of the detector.

The thesis is focused on application of ion chromatography in pharmaceutical analyses of organic ions. Ion chromatography is increasingly used in the field of pharmaceutical analysis. This includes the analysis of impurities and metabolites. In the first part of this thesis, ion chromatography is compared with common separation techniques used in pharmacy, such as gas chromatography and high performance liquid chromatography. In the second part development and validation of methods of ion chromatography for purity evaluation and quality control of active pharmaceutical substances Rivastigmine hemitartrate and Pramipexole hydrochloride were carried out. Key words: ion chromatography, reversed-phase chromatography, ion-pair chromatography, ion-exclusion chromatography, ion-exchange chromatography, GC, HPLC

Capsaicinoids are a class of compounds responsible for the "heat" of hot peppers. Capsaicin and dihydrocapsaicin have the highest burning effect. The aim of this work is to separate and quantify the two major capsaicinoids in fruits harvested at different stages of development and at different seasons. Simple and rapid HPLC method involves 73:27% methanol water mobile phase with C18 stationary phase and UV-Vis detector set at 210 nm. The method showed good reproducibility with 1.74% - 4.72% relative standard deviations, a linear response within 0.65–45.5 and 0.25-17.5 μg/mL for capsaicin and dihydrocapsaicin, respectively. The method achieved average recovery of 106% for capsaicin and 102% dihydrocapsaicin. Determination of capsaicinoids in four naturally grown chili and commercial source habanero were analyzed. The amount in the sample ranged from 1184-8156 μg/g for capsaicin and 430-3299 μg/g for dihydrocapsaicin.

Cholesterol is a fat-like molecule found among lipids in animal (including human) tissues. It is needed for maintaining good health. However, health issues have been raised because of the strong correlation between high levels of cholesterol in the body and cardiovascular disease. An HPLC method for quantitative determination of cholesterol in foods is presented. This involves a C-18 stationary phase using a 70:30 methanol: 2-propanol mobile phase with an UV detector set at 212 nm. The method showed linearity in the range 5.0 to 100.0 μg/mL and also good reproducibility with relative standard deviation of 4.22%, 2.71%, 4.8%, and 3.7% for the different samples analyzed. The mean recovery of the butter sample was 106.5%. The samples under investigation were common food items such as butter, lard, and two different types of cheese.

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