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.

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.

This thesis describes bioanalytical methods for generating fingerprints of biological systems for extracting relevant information with (protein) drugs in focus. Similarities and differences between samples can reveal the hidden relevant information, which can be used to optimize the production and facilitate the quality control of such protein drugs during their development and manufacture. Metabolic fingerprinting and multivariate data analysis (MVDA) can also facilitate early diagnosis of diseases and the effects and toxicity of drugs. Currently, several protein drugs are available on the global market. Nevertheless, despite, the success of such biotherapeutics significant challenges remain to be overcome in maintaining their stability and efficacity throughout their production cycle and long-term storage. The native structure and functional activity of therapeutic proteins is affected by many variables from production to delivery, incl. variables assoc. with conditions in bioreactors, purification, storage and delivery. Thus, part of the work underlying this thesis focused on structural analysis of a protein drug using chemical labeling, peptide mapping, and evaluation of the charge state distributions of the whole protein generated by ESI. The other part focuses on non-targeted metabolomics with a view to optimizing the cell cultivation process and assessment of the drug’s toxicity. A combination of appropriate analytical methods and MVDA is needed to find markers that can facilitate optimization of the cultivation system and expression of the target proteins in early stages of process development. Rapid methods for characterizing the protein drugs in different stages of the process are also required for quality control. In order to obtain high quality fingerprints analytical separation techniques with high resolution (such as HPLC or UHPLC) and sensitive analytical detection techniques (such as ESI, quadrupole or TOF MS) have been used, singly or in combination. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.

In this thesis, the application of electrospray ionisation mass spectrometry (ESI-MS) to the analysis of intact proteins is demonstrated. In papers I and II, the use of ESI-MS for the analysis of noncovalent protein-ligand complexes were discussed. In addition, the interfacing of liquid chromatography (LC) with ESI-MS and the development of an LC-ESI-MS method were demonstrated in paper III for the quality control of recombinant proteins. Furthermore, this method was applied in paper IV for the analysis of covalent glycosyl-enzyme intermediates. The monitoring of noncovalent complexes by ESI-MS is well established. However, the varying characteristic of ESI-MS data, especially in the analysis of noncovalent complexes can make the quantification of such complexes troublesome. In paper I, it was demonstrated how the variation in the position of the ESI-emitter and the initial droplet size of the electrosprayed droplets, together with different partitioning of a protein and its ligand in these droplets, can be the cause of such varying characteristics. Furthermore, it was shown that the partitioning can be of electrostatic and/or hydrophobic/hydrophilic origin. Thus it was demonstrated that if the ligand is more hydrophobic and thereby more surface active relative to the protein, decreasing the droplet size or increasing the distance between the electrospray emitter and the sampling orifice will lead to more efficient sampling of the droplet bulk where the ligand concentration is low. This results in a favoured sampling of free protein relative to the protein ligand complex. The opposite was shown to occur if the ligand is more hydrophilic than the protein. In paper II, Ribonuclease A (RNAse) was used as a model for enzymes acting on polymeric substrates with different chain lengths. Nano-ESI-MS was applied to monitor the noncovalent interactions between RNAse and different target ligands. Among the single building blocks of RNA, including ribose, the bases adenine, guanine, cytosine and uracil, and phosphate, only phosphate was observed to interact at multiple RNAse sites at a higher cone voltage. Furthermore, monobasic singlestranded deoxycytidylic acid oligomers (dCx) of different lengths (X=6, 9 and 12), and RNAse were analysed with nano-ESI-MS. The deoxycytidylic acid with 12 nucleotides was observed with the highest complex to free protein ratio, hence indicating the strongest interaction. Finally, collision induced dissociation of the noncovalent RNAseA-dC6 complex resulted in dissociation of covalently bound cytosine from the nucleotide backbone rather than break up of the noncovalent complex, illustrating the cooperative effect of multiple noncovalent interactions. In paper III an LC-ESI-MS method was presented capable of analysing proteins 10-100 kDa in size, from salt-containing liquid samples. The proteins included human protein fragments for the largescale production of antibodies and human protein targets for structural determination, expressed in E. coli. Also, glycosylated proteins expressed in Pichia pastoris were analysed. The method provides fast chromatography, is robust and makes use of cheap desalting/trap columns. In addition it was used with optimised reduction and alkylation protocols in order to minimize protein aggregation of denatured and incorrectly folded proteins containing cysteins, which otherwise form adducts by disulfide bond formation. Furthermore, the method was used in paper IV for the quantification of covalent proteinligand intermediates formed enzymatically between PttXET16-34, a xyloglucan endo-transglycosylase (XET) from hybrid aspen, and the synthetic substrates GalGXXXGGG and GalXXXGXXXG designed in order to function as donor substrates only. Thus covalent GalG-enzyme and GalGXXXG-enzyme complexes were detected. Moreover, establishing of a pseudo equilibrium for the formation of the covalent GalGXXXG-enzyme complex enabled quantification of the saccharide and enzyme constituents of this equilibrium and determination of the free energy of formation (∆G0). The high mass resolution of the TOF-MS allowed unambiguous assessment of the covalent nature of the glycosyl-enzyme complexes. Morover, the formation of noncovalent complexes between excess substrate and protein, which can deteriorate MS-signal and increase spectrum complexity, was efficiently avoided by the chromatographic step, which separated the saccharide content from the protein content. / QC 20100913

This thesis deals with instrumental and methodological developments for speciation analysis of gaseous mercury (Hg(g)), based on isotope dilution analysis (IDA). The studied species include Hg0, (CH3)2Hg, CH3HgX and HgX2 (where X symbolises a negatively charged counter ion in the form of a halide or hydroxyl ion). Gas chromatography hyphenated with inductively coupled plasma mass spectrometry (GC-ICPMS) was used for separation and detection of Hg(g) species. Permeation tubes were used for the generation of gaseous isotopically enriched Hg standards (tracers). These tracers were continuously added to the sample gas stream during sampling of Hg(g). A mobile prototype apparatus incorporating both the permeation source and a sampling unit for collection of Hg(g) was developed and used for this purpose. Hg(g) species were pre-concentrated on Tenax TA and / or Carbotrap solid adsorbents. Au-Pt was used for pre-concentration of total Hg(g), either as the primary medium, or as backup. Collected species were eluted from these media and introduced to the instrument by thermal desorption. Various degrees of species transformations, as well as losses of analyte during pre-concentration and elution, were found to occur for both Tenax TA and Carbotrap. The performance characteristics of these media were shown to be species specific, as well as matrix dependent. The development of an on-line derivatisation procedure allowed for minimised species transformations, as well as reduced adsorption and memory effects of ionic Hg(g) species within the analytical system. In conclusion, IDA provides an important tool for identification, minimisation and correction of the above mentioned analytical problems. Furthermore, it offers significant advantages with respect to quality assurance, compared to conventional techniques, both when it comes to rational development of new methodology, as well as for continuous validation of existing procedures. The developed methodology for speciation analysis of Hg(g) has been tested in various applications, including the determination of Hg(g) species concentrations in ambient air (both in and outdoor) and in the head space of sediment microcosms. Hg(g) species were formed in the sediments as a result of naturally occurring redox and methylation processes, after addition of an isotope enriched aqueous Hg(II) precursor. The methodology has also been used for assessing the risk of occupational exposure to Hg(g) species during remediation of a Hg contaminated soil and for studying Hg0(g) transport and Au-Pt pre-concentration characteristics in natural gases. Hg0 was used as the model species in the latter experiments, since it is believed to be the dominating form of Hg(g) in natural gas. The results indicate that the occurrence of H2S can cause temperature dependent adsorption and memory effects of Hg0(g) in the presence of stainless steel, thereby providing a plausible explanation to the variability of results for sour gases occasionally observed in the field. Hg0(g) has demonstrated overall high recovery during collection on Au-Pt tubes for all gases tested in this thesis. Recent (unpublished) results however indicate that there are potential species specific and matrix dependent effects associated with the Au-Pt based pre-concentration of Hg(g) in natural gases.

In this thesis, the main concern has been to improve the reliability of different parts of the analytical workflow (Paper I, II, IV &V). Additionally, one of the resulting optimized methods was used in a real application (Paper III). Paper I-II concerned the extraction of acrylamide (AA) from foods. In Paper I different parameters such as sample particle size, extraction solvent, extraction time and extraction temperature were optimized, leading to a method that showed good agreement with the assigned AA levels of several proficiency test samples. Later, after the publication of the paper, the method showed good performance in a collaborative trial validation, in terms of trueness, repeatabil­ity and reproducibility figures. It was labeled “undoubtedly fit for the purpose”. In Paper II, it was shown that the ‘extra’ amounts of AA obtained during extraction of foods with an alkaline aqueous solution was not due to improved extractability of AA from the food matrix. Strongly alkaline conditions seemed to rather induce net formation of AA from water-soluble precursors formed during thermolysis. This phenomenon should therefore be regarded as an extraction artifact. Paper III was an application of the optimized method from Paper I, where it was used to study the reduction of AA in potato chips (crisps) by using pre-treatments and frying at reduced pressure. There were significant reductions in AA, down to below the limit of quantification (5 µg/kg) for the method. Paper IV-V concerned analysis of anthocyanins (AC) in red onion. In Paper IV, a new separation method using capillary electrophoresis was developed, and its rapidness combined with an acidic background electrolyte helped in preventing AC degradation. Furthermore, its alternative separation mechanism is a complement to that of the more commonly used liquid chromatography technique. In Paper V, simultaneous extraction and degradation of anthocyanins from red onion was studied in a static batch reactor at 110ºC. The extraction and degradation kinetics were successfully separated, and an ideal theoretical extraction curve was constructed by compensating mathematically for degradation effects, showing that more anthocyanins, 21 to 36% depending on different species, could be extracted if no degradation occurred. The results give important information about the different kinetics competing during an extraction procedure, and also show that quantitative extraction is not to recommend in the batch system used in the study.

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. 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. 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. 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. 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. This method is still in its cradle and needs further development.

The aim of this study was to develop methods for sample preparation for 8-OHdG in blood plasma samples with different solid phase extraction techniques using HPLC with an elec- trochemical detector. The solid phase extraction cartridges used were Chromabond® C18, Oasis® MAX, and three types of SupelMIPTM cartridges for chloramphenicol, riboflavin, and nitroimidazoles. The SupelMIPTM cartridges are based on molecularly imprinted polymers- technique. The separation of 8-OHdG in samples extracted from blood plasma was carried out with a Thermo Quest Hypersil Division ODS column (250 mm × 4 mm, 3μm I.D.) and methanol:buffer (10:90, v/v) as mobile phase. Recovery and selectivity was studied for the different solid phase extraction methods. The highest recovery was obtained using the Chromabond C18 cartridge with a recovery of 92%, and CV coefficient 9.5% (n = 4). 8-OHdG could not be extracted on MIP-cartridges for chloramphenicol or riboflavin, but was retained on MIP columns for nitroimidazoles, and the highest recovery was 49%.

In the biopharmaceutical industry, identification of glycans in a glycoprotein is a regulatory requirement and is a part of the characterization of the protein. Glycans are constructed of several monosaccharides linked together. N-linked glycans, which have been studied in this project, are attached to the nitrogen atom in asparagine. A method for separating N-linked glycans by high performance anion exchange chromatography had already been developed at the department. To develop a method for identification of the N-glycans by mass spectrometry, a desalting method on porous graphitic carbon (PGC) columns was used and optimized resulting in the eluents A (0,05% TFA in ACN:water 5:95 v/v) and B (0,05% TFA in ACN:water 50:50 v/v). Also the sample introduction on the mass spectrometer was optimized and resulted in a sensitive on-line liquid chromatography mass spectrometry (LC-MS) approach which gave mass spectrometric peaks with high signal to noise ratios and with high mass accuracy. The developed procedure was then successfully used on glycans cleaved from a glycoprotein separated by high performance anion exchange chromatography with pulsed amperometric detector.

The main purpose of the project was to identify potential environmentally harmful substances which can be leached from rubber asphalt. A method for analysing asphalt was developed and three rubber asphalt materials were analysed after being cryogrinded. One of the materials was also tested in a road machine made for testing of asphalt paving. The particles created in the machine were analysed in the same manner as the cryogrinded asphalt materials. The asphalt materials were leached by water during 24 hours. The leachates were extracted with dichloromethane, dried with sodium sulphate and concentrated to a small volume. The extracts were analysed with gas chromatography-mass spectrometry (GC-MS). Due to low concentrations of substances the GC-MS was operated in SIM-mode (Selected Ion Monitoring). Thirteen substances were chosen for the analysis. The substances were aniline, benzothiazole, butyl benzyl phthalate, bisphenol-A, decanoic acid, dibutyl phthalate, diethylhexyl phthalat, phenanthrene, chrysene, naphthalene, 4-n-nonyl phenol and 4-tert-octyl phenol. Benzothiazole and 4-tert-octyl phenol were detected and quantified in the leachates. In addition to the analysis of organic substances, pH was measured too. The leachates produced in this project were also sent to an analysis company for several analyses, for example analysis of metals and sulphur. Toxicity tests were performed on the same leachates within an exam work made by Gro Runeman at Lund University. The results of the metal analysis, sulphur analysis, and toxicity tests are not covered within the scope of this report. For the results of these tests and analyses, see Gro Runeman’s report: Evaluating toxicity of asphalt leachates. / Detta examensarbete är utfört vid Statens Geotekniska Institut (SGI). Huvudsyftet med arbetet är att identifiera potentiellt miljöfarliga ämnen som kan laka ut från gummiasfalt. En metod för analys av asfalt har tagits fram och tre olika gummiasfalter har analyserats efter att ha kryomalts. En av dessa asfalter har också använts i en provvägmaskin där det damm som bildades har samlats upp och analyserats. Inom ramen för examensarbetet har också en litteraturstudie gjorts för att bland annat ta reda på vad som är gjort inom området sedan tidigare. För att ta reda på vad asfalten innehåller för föreningar gjordes först en fastfasextraktion av krossad (ej kryomald) asfalt där diklormetan användes som lösningsmedel. Från början var tanken att en GC/MS-screening skulle göras för att på så sätt få en överblick av samtliga organiska ämnen som finns i asfalten men på grund av de väldigt låga halterna av i gaskromatografen analyserbara föreningar var det nödvändigt att begränsa analysen till några få föreningar. De föreningar som analysen inriktade sig mot var anilin, bensotiazol, bensylbutylftalat, bisfenol-A, dekansyra, dibutylftalat, di(etylhexyl)ftalat, fenantren, krysen, naftalen, 4-n-nonylfenol, pentaklortiofenol och 4-tert-oktylfenol. Laktester utfördes genom att de kryomalda asfaltmaterialen lakades med vatten under 24 timmar. Efter extraktion, torkning och koncentrering analyserades lakextrakten med avseende på de föreningar som hittats vid fastfasextraktionen. I lakvattnen hittades bensotiazol samt 4-tert-oktylfenol. Dessa föreningar kvantifierades genom att en enpunktskalibrering gjordes. Utöver analyserna ovan mättes även pH på lakvattnen. Lakvattnen skickades också på metall- och svavelanalys samt turbiditets- och fenolindexmätning till ett större analysföretag. Toxicitetstester har utförts på samma lakvatten av Gro Runeman inom ramen för ett examensarbete vid Lunds Universitet. För resultaten från toxicitetstester, metallanalyser samt turbiditets- och fenolindexmätningar hänvisas till Gro Runemans rapport: Evaluating toxicity of asphalt leachates. Delar av denna rapport är skrivna i samarbete med Gro Runeman.