<p>There is convincing research which shows that the enzyme alkaline phosphatase (ALP) has a central role in the mineralization of bone, more precisely that its catalytic activity is needed in the process. ALP is found on the surface of matrix vesicles where the mineral is formed. One theory about the function of the enzyme is that it binds to fibrous collagen in the bone and thereby incorporating the mineral into the bone. The purpose of this study is to establish whether ALP binds to collagen. If this is the case, more elaborate studies around this will be performed. The strength of the binding between collagen and the different types of ALP will be evaluated, as well as on which part of the collagen the binding occurs. The binding is going to be studied by constructing a method for the ÄKTApurifier system.</p><p> </p><p>Initially, the pureness of the different type of collagens was determined by using SDS-PAGE and the activity of the different types of ALP was established. These were also compared with a native PAGE. In SDS-PAGE, bovine type I collagen showed markings for a triple helix, a double helix and two single strains, α₁ and α₂. Bovine type II collagen showed markings for a double helix and α₁-strains. Human type I collagen showed markings for a triple helix, two double helixes, two α-strains and contaminations. Trials with collagen in Native PAGE did not provide any results. However, the trials with ALP revealed that the different types of ALP had different charge.</p><p> </p><p>Thereafter, blotting was performed. The results showed that all the different types of ALP, besides from E. coli, binds to bovine collagen type I and II and human collagen type I, however within various periods of time. In the trials with collagen coated plates the acquired results showed that some of the different types of ALP bind to collagen. ALP from liver binds the strongest to both collagen type I from rat and type IV from mouse. Intestinal ALP also binds to both types of collagen but not nearly as strong as liver ALP. Serum from rats did bind to collagen type I from rat but not to collagen type IV from mouse. ALP from kidney and human serum did not bind to either types of collagen. The trials concerning the ÄKTApurifier system were executed with ALP from liver alone because it had been proven to bind to bovine type I collagen through the previous methods. The results confirmed that ALP from liver binds to this type of collagen.</p><p> </p><p>The conclusions from this study are that ALP does indeed bind to collagen and does so to the triple helix and double helix form as well as the single strains of collagen. In other words the part of the structure in collagen that ALP binds to must exist in all three stages of collagen formation. Furthermore, it seems like some of the different types of ALP has a higher affinity for binding to collagen, as the time for binding to collagen varies for the different types of ALP. The results differed between methods concerning different types of ALP. Although, the method we consider to give the best result was blotting. However, the method using ÄKTApurifier can be complementary but needs further development.</p>

<p>The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%.</p><p>The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.</p>

<p>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.</p><p>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.</p><p>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: <em>Evaluating toxicity of asphalt leachates</em>.</p> / <p>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.</p><p>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.</p><p>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.</p><p>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: <em>Evaluating toxicity of asphalt leachates</em>. Delar av denna rapport är skrivna i samarbete med Gro Runeman.</p>

<p>Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum.</p><p>The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with <em>tert</em>-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible.</p>

<p>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.</p> / <p>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. </p>

<p>The magnitude of an electric field possible to apply in a capillary separation system is limited, because a high electric field causes a too high current through the capillary. Application of the electric field <i>in-line</i> will give an increased conductivity in the column, further increasing the risk of too high currents. The conductivity changes were found to result from an overall increase in ionic strength within the electric field. The increase in ionic strength is caused by the increase in mobile phase ions with electrophoretic velocity against the flow, together with OH^- or H₃O⁺ ions (depending on polarity) formed at the inlet electrode. Further it was found that the use of a pressurized reservoir or splitting of the flow at the inlet electrode could significantly limit the conductivity changes and thereby the maximum applicable electric field strengths could be increased.</p>

<p>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.</p>

<p>Gas chromatography (GC) has been utilized for the study of enantiomer resolution of the atropisomers of PCBs, o,p´-DDD and o,p´-DDT. Different substituents and concentrations of cyclodextrin, capillary dimensions and type of stationary phase films have been investigated to achieve the resolution of as many of the atropisomers on one column as possible. The results indicated that the butyl substitution of 6-hydroxyl and the methyl substitution of 2- and 3-hydroxyl were the most promising for the enantiomeric separation. Using Capillary Electrophoresis (CE), the trimers and monomers of PM-β-CDs were compared for enantiomeric resolution, as well as comparing the cationic PMMA-β-CD with the anionic HS-β-CD. In these studies the trimer did not show an improved resolution for mepivacaine, when compared to the equimolar concentration of the monomer. The cationic CD gave increased resolution values for ibuprofen when compared to the anionic CD. A scheme for reversing enantiomeric elution order of both the basic propranolol and acidic ibuprofen is also presented, with the aim of facilitating the detection of impurities in a high sample loading. The detection of 1% of each enantiomer of propranolol, and 1% of R(-)-ibuprofen, was demonstrated, with elution prior to the tailing peak of the corresponding enantiomer. Dimethylacrylamide-coated capillaries were used in this work, and the stability of this coating was demonstrated, giving a highly reduced electroosmotic flow for up to six months. Enantiomeric baseline separations of omeprazole and 5-hydroxyomeprazole have also been achieved with both CE and Open Tubular Capillary Electrochromatography (OT-CEC) methods. With CE-UV, both a non-aqueous method (using HDMS-β-CD) and an aqueous method (using HS-β-CD) were used for enantiomeric resolution of the two racemates. Resolution of omeprazole was also achieved using CE-Electrospray Ionization-Mass Spectrometry (ESI-MS). In OT-CEC, avidin was immobilized on the inside surface of a fused silica capillary and was employed as chiral selector for the enantiomeric baseline resolution of omeprazole and 5-hydroxyomeprazole.</p>

No description available.

This thesis covers sampling and analytical procedures for isocyanates (R-NCO) and amines (R-NH2), two kinds of chemicals frequently used in association with the polymeric material polyurethane (PUR). Exposure to isocyanates may result in respiratory disorders and dermal sensitisation, and they are one of the main causes of occupational asthma. Several of the aromatic diamines associated with PUR production are classified as suspected carcinogens. Hence, the presence of these chemicals in different exposure situations must be monitored. In the context of determining isocyanates in air, the methodologies included derivatisation with the reagent di-n-butylamine (DBA) upon collection and subsequent determination using liquid chromatography (LC) and mass spectrometric detection (MS). A user-friendly solvent-free sampler for collection of airborne isocyanates was developed as an alternative to a more cumbersome impinger-filter sampling technique. The combination of the DBA reagent together with MS detection techniques revealed several new exposure situations for isocyanates, such as isocyanic acid during thermal degradation of PUR and urea-based resins. Further, a method for characterising isocyanates in technical products used in the production of PUR was developed. This enabled determination of isocyanates in air for which pure analytical standards are missing. Tandem MS (MS/MS) determination of isocyanates in air below 10-6 of the threshold limit values was achieved. As for the determination of amines, the analytical methods included derivatisation into pentafluoropropionic amide or ethyl carbamate ester derivatives and subsequent MS analysis. Several amines in biological fluids, as markers of exposure for either the amines themselves or the corresponding isocyanates, were determined by LC-MS/MS at amol level. In aqueous extraction solutions of flexible PUR foam products, toluene diamine and related compounds were found. In conclusion, this thesis demonstrates the usefulness of well characterised analytical procedures and techniques for determination of hazardous compounds. Without reliable and robust methodologies there is a risk that exposure levels will be underestimated or, even worse, that relevant compounds will be completely missed.