Dissertations / Theses on the topic 'Size exclusion chromatography (SEC)'

The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.

Le développement des outils analytiques pour l'analyse de la matière organique complexe en géochimie organique a connu de nombreuses avancées ces dernières années. Ce développement a permis de répondre à un grand nombre de questions quant à la composition de la matière organique. Cependant, beaucoup des points restent encore à élucider comme notamment la caractérisation des fractions de hauts poids moléculaires ainsi que le suivi de la réactivité de la matière organique. Ce travail de thèse a eu pour objectif (i) d'adapter les techniques de spectrométrie de masse déjà existantes pour l'analyse de la matière organique fossile (notamment par la sélection de la source d'ionisation atmosphérique la plus adaptée) mais également (ii) de développer un nouveau type de couplage entre la chromatographie d'exclusion stérique (SEC) et la spectrométrie de masse APPI-QTOF pour l'analyse des fractions peu polaires de hauts poids moléculaires. L'adaptation du l'APPI-QTOF a tout d'abord permis de mieux comprendre la réactivité de contaminants organiques polyaromatiques en présence de phases minérales. Le couplage SEC-APPI-QTOF a, quant à lui, permis d'améliorer les connaissances sur la structure des asphaltènes. Cependant, malgré la « simplification » rendue possible par la SEC, la très grande quantité d'informations reste difficile à interpréter et prend beaucoup de temps. Un modèle mathématique a donc été développé basé sur des analyses numériques et statistiques des spectres de masse, permettant de les comparer entre eux afin de distinguer l'origine des échantillons et de suivre l'impact de processus physico-chimiques (altérations naturelles - traitements de remédiation)
The development of analytical tools for organic geochemistry analysis has increased these past years. This development has allowed answering many questions about organic matter composition. However, many issues remain to be clarified including the characterization of high molecular weight fractions and monitoring the reactivity of organic matter. This thesis has focused on both (i) existing method improvements for fossil organic geochemistry analysis but also on (ii) developing a new type of coupling between the size exclusion chromatography (SEC) and the APPI-QTOF mass spectrometry for high molecular weight weakly polar fractions. Adjustments on APPI-QTOF mass spectrometry have allowed a better understanding of polyaromatic organic contaminant reactivity in presence of mineral matrices. The success of this coupling has allowed a better understanding of the structure of asphaltenes. However despite the "simplification" obtained by the SEC, the large amount of information remains difficult to interpret and time-consuming. A mathematical model has been developed based on numerical and statistical analysis of mass spectra, allowing direct comparison of mass spectra and being able to identify several types of information such as origins of samples, monitoring of physico-chemical processes and also the efficiency of soil recovery treatments as well as the identification of analytical protocols

Size exclusion PEGylation reaction chromatography was investigated using a model developed by Fee (2005). Column dispersion was neglected and the PEGylation reaction was modelled as second order. The model allowed up to four PEG groups to be attached to a protein and accounted for succinic acid hydrolysis from activated PEG. The model was adapted to simulate a-lactalbumin PEGylation and succinic acid hydrolysis from activated PEG in a batch stirred tank so rate parameters from stirred tank kinetic experiments could be obtained and the model verified. The model was solved using finite differences and simulations run in Matlab. The effect of reaction parameters such as timing, length and concentration of PEG and protein injection, reaction rates, and model resolution on model simulation results was explored. In the size exclusion PEGylation simulations it was found that increasing protein concentration increased MonoPEG concentrations and increased the ratio of MonoPEG to starting protein feed concentration. Increasing PEG pulse length and starting PEG concentration initially increased MonoPEG concentration and product ratio until all protein had been PEGylated at which point MonoPEG concentration the product ratio levelled out. Increasing PEG hydrolysis rates did not affect the amount of MonoPEG produced but reduced the activated PEG concentration and increased succinic acid concentration. Optimal conditions for producing MonoPEG were found to be equal concentrations of PEG and protein, with the PEG injection length twice as long as the protein injection, and the PEG injection done immediately after the protein injection.

Thesis (PhD) - Faculty of Life and Social Sciences, Swinburne University of Technology, 2008.
Presented for full assessment for the degree of Doctor of Philosophy, Faculty of Life and Social Sciences, Swinburne University of Technology - 2008. Typescript. Bibliography: p. 222-246.

Size exclusion chromatography (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance.

Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work.

The method developed was tested on cannabis, coffee and 'Spice' with good results.

Polymers capable of thermally controlled reversible bonding reactions are promising candidates for stimuli responsive materials, as required for self-healing or drug delivery materials. In order to investigate how the dynamic reactions can be controlled, effective analytical tools are demanded that are capable of analyzing not only the polymers but can also monitor the respective bonding reactions. Herein, we employ size exclusion chromatography in a newly developed temperature dependent mode (TD SEC) for the in situ characterization of polymers that undergo retro Diels-Alder (rDA) reaction at temperatures higher than 60 °C. Monitoring the evolution of the molar mass distribution of the polymers during the rDA reaction and evaluating the data quantitatively gives detailed information about the extent of the reaction and allows elucidating structural parameters that can be used for controlling the polymers debonding behavior. In contrast to spectroscopic techniques, TD SEC analyzes only the size of the polymers, hence the polymers do not need to fulfill any particular requirements (e.g. presence of detectable functional groups) but only need to be soluble in the TD SEC, which makes the method universally applicable. Side effects that might bias the results are minimized by using a high temperature chromatograph that allows performing the analysis in a broad temperature range (60 – 200 °C) and in different solvents. Thus, the analysis can be performed under the exact conditions that are required for the bonding reactions and an in situ image is provided.

The aim of this dissertation was to investigate and quantify the mass transfer processes occurring within a chromatography column by utilising Nuclear Magnetic Resonance (NMR). The dissertation describes the development of Magnetic Resonance Imaging techniques to enable the quantitative imaging of urea and lysozyme in one-, two- and three-dimensions, in situ and without chemical labelling. These images reveal the evolution of the distribution of both species simultaneously, and hence highlight that much of the distortion to the concentration distribution in the column arises from the inlet distributor. In addition to these novel imaging protocols, alternating pulsed gradient stimulated echo (APGSTE) NMR was used to obtain quantitative information on the microscopic motion of molecules over time periods from a few milliseconds up to two seconds. This enables quantitative measurement of eddy dispersion, mass transfer between mobile and stagnant liquid, and longitudinal diffusion. These effects can then be used to predict the microscopic (i.e. over displacements < 20 particle diameters) dispersion to within 25% over a range of flow rates. Furthermore, spatially resolved pulsed gradient stimulated echo NMR was used, in conjunction with the MRI techniques developed previously in this work, to isolate and quantify the effect of the inlet distributor and the packing heterogeneity on the macroscopic dispersion. Finally, a preliminary investigation of the influence of macroscopic flow heterogeneity on SEC protein refolding was performed. This confirmed an earlier hypothesis that sample distribution at the inlet of the column dominates the final refolding yield in SEC refolding reactors.

The chromatographic behaviour of a new column packing for high performance size exclusion chromatography based on crosslinked polyacrylamide particles was studied. Experimental retention data for poly(ethylene glycol)and poly(ethylene oxide) standards in water and water-methanol mixtures showed that the separation mechanism is size exclusion, since a universal calibration plot based on hydrodynamic volume was obtained. For polysaccharide standards in water and water-methanol (80/20), the universal calibration was valid for molecular weights above 4 * 10~. Below this value, secondary mechanisms appear to be taking part in the separation since water-methanol (80/20) is a poor solvent for polysaccharides. Crosslinked polyacrylamide packings showed an interactive behaviour with tetrahydrofuran and dimethylformamide as eluents with polystyrene and poly( ethylene glycol )/poly(ethylene oxide) standards and the universal calibration was not valid.

Branching in poly(methyl methacrylate) (PMMA) is produced by incorporating a pre-prepared polymeric chain transfer agent (PCTA) into a single stage radical polymerisation. Samples of PCTA having a range of transfer functionalities and molar masses were synthesised by modifying a methacrylate-based copolymer. Control of branching in PMMA has been studied as a function of transfer functionality and molar mass in the PCT A and a function of MMA and initiator concentrations in the MMA polymerisation. The branched samples of PMMA have been characterised by size exclusion chromatography (SEC) with multi-detectors to determine Mark–Houwink and other parameters to assess levels of branching. Some PCTA samples have been prepared with a UV chromophore to facilitate characterisation by SEC-UV.

This project focused on developing a Size Exclusion Chromatography (SEC) methodwith Refractive Index (RI) detection for analysis of extraction samples from urinarycatheters to detect compounds that can be extracted from the catheter during use.Mobile phases, extraction fluids and sample concentrations were varied, as well aspore sizes of the columns, to investigate the applicability of this technique forcharacterization of the coating and potential leachables. Analyses of extractionsamples showed that this method can be used for analyses of polyvinylpyrrolidone(PVP), which is the main component in the coating, giving rise to the specialcharacteristics of the coating. No compounds could be detected from extraction ofuncoated catheters.Comparisons were made between different raw catheter materials, PVC and POBE,and differences in molecular weight distribution of the extracted PVP compoundswere seen, in spite of identical coating processes, indicating that the bonding of PVP inthe coating depends on raw catheter material. Furthermore, radiation effects alsodiffered, where a larger decrease in high-molecular weight fractions of PVP was seenwith increasing radiation dose from extracted PVC catheters, compared to thosefrom POBE. Analyses of radiated PVP powder showed opposite radiation effects thanthose from radiated catheters; that is, a steady increase in molecular weight withincreasing radiation dose, indicating cross-linking of PVP when radiated in powderform, and consequently chain-scission of high-molecular PVP fractions when radiatedbonded to the catheter coating.The SEC-RI method was concluded to be a useful tool for qualitative analyses of thedifferences in molecular weight distribution of PVP from the different extractionsamples, but showed low reproducibility in molecular weight calculations and therelative molecular weights calculated from these analyses differed significantly fromtrue molecular weights. This method could therefore not be used to give a goodestimation of true molecular weights.

Natural organic matter (NOM), ubiquitous in natural water sources, is generated by biogeochemical processes in both the water body and in the surrounding watershed, as well as from the contribution of organic compounds that enter the water as a result of human activity. NOM significantly affects the properties of the water source, including the ability to transport metals, influence the aggregation kinetics of colloidal particles, serve as a food source for microorganisms and act as a precursor in the formation of disinfection by-products (DBPs), as well as imparting a brown colour to the water. The reactivity of NOM is closely tied to its physicochemical properties, such as aromaticity, elemental composition, functional group content and molecular weight (MW) distribution. The MW distribution is an important consideration from a water treatment perspective for several reasons. For example, low MW NOM decreases the efficiency of treatment with activated carbon, and this fraction is thought to be the portion most difficult to remove using coagulation. The efficiency of membranes in the treatment of drinking water is also influenced by the MW distribution of NOM, while some studies have shown that the low MW fraction contributes disproportionately to the formation of bioavailable organic matter, therefore promoting the formation of biofilms in the distribution system. For these reasons, understanding the MW distribution of NOM is important for the treatment of natural waters for use as drinking waters. Optimisation of a high pressure size exclusion chromatography (HPSEC) method for analysis of the MW distribution of NOM in natural waters is described (Chapter 2). Several parameters influencing the performance of HPSEC are tested and an optimised set of conditions illustrated.
These parameters included eluent composition, ionic strength of the sample, flow rate and injection volume. Firstly, it was found that increasing the ionic strength of the HPSEC eluent resulted in less exclusion of NOM from the stationary phase. Stationary phases used in HPSEC contain a residual negative charge that can repel the negatively charged regions of NOM, effectively reducing the accessible pore volume. By increasing the ionic strength, interactions between the stationary phase and eluent enabled a larger effective pore size for the NOM analytes. However, increasing ionic strength of the eluent also resulted in a loss of peak resolution for the NOM portion able to access the pore volume of the stationary phase. Determining the ideal eluent composition required the balancing of these two outcomes. Matching of the ionic strength of the sample with the eluent was also an important consideration. Retention times were slightly lower when the sample ionic strength was not matched with the eluent, especially for the lowest MW material, although the effect on chromatography was minimal. Flow rate had no effect on the resolution of the HPSEC chromatogram for the portion of material able to permeate the pore space of the stationary phase. Changes in the volume of sample injected had a marked effect on the elution profile of the NOM sample. Besides the obvious limitation of detection limit, only minor changes in elution profile were obtained up to an injection volume of 100 µL. Volumes above this value, however, resulted in significant peak broadening issues, as well as an undesirable effect on the low MW portion of detected DOC.
In Chapter 3, high pressure size exclusion chromatography with UV254 [subscript] and on-line detection of organic carbon (HPSEC-UV254[subscript]-OCD) was used to compare the removal of different apparent MW fractions of DOC by two process streams operating in parallel at the local Wanneroo groundwater treatment plant (GWTP). One of these two process streams included alum coagulation (operating in an enhanced coagulation mode (EC) for increased DOC removal) and the other stream included a magnetic ion exchange (MIEX®) process followed by alum coagulation (MIEX®-C). The MIEX® process is based on a micro-sized, macroporous, strong base anion exchange resin with magnetic properties, which has been designed to remove NOM through ion exchange of the anionic sites in NOM. Water was sampled from five key locations within these process streams, and the DOC at each location was characterised in terms of its MW distribution. HPSEC was carried out using three different on-line detector systems, namely OCD, UV absorbance detection at 254 nm, and fluorescence detection (λex[subscript]= 282 nm; λim[subscript] = 353 nm). This approach provided significant information on the chemical nature of the DOC in the various MW fractions. The MIEX®-C process was found to outperform the EC process: these two processes removed similar amounts of high and low MW DOC, but the MIEX®-C process showed greater removal of DOC from the intermediate MW fractions. The two coagulation processes (EC and coagulation following MIEX®) showed good removal of the fractions of highest MW, while the MIEX® process alone was found to remove DOC across all MW fractions.
These results seem to indicate that anionic groups, particularly susceptible to removal with MIEX® treatment, are well distributed across all MW fractions of NOM. In agreement with previous studies, MIEX®-C outperformed EC in the overall removal of DOC (MIEX®-C removed 25 % more DOC than EC). However, 70% of the additional DOC removed by MIEX®-C was comprised of a surprisingly narrow range of medium-high MW fractions. The development of a novel online organic carbon detector (OCD) for use with HPSEC for determining the MW distribution of NOM is described in Chapter 4. With UV absorbance detection, the magnitude of the signal is based on the extinction coefficient of the chromophores in the analytes being investigated; whereas the signal from an OCD is proportional to the actual organic carbon concentrations, providing significantly more information. The development of an online OCD involved the separation of analytes using HPSEC, removal of inorganic carbon species which may interfere with organic carbon determination, oxidation of the organic carbon to carbon dioxide, separation of the produced carbon dioxide from the aqueous phase and subsequent detection of the gaseous carbon dioxide. In the new instrument, following separation of components by HPSEC, the sample stream was acidified with orthophosphoric acid to a concentration of 20 mmol L-1[superscript], resulting in a pH of ≤ 2, in order to convert inorganic carbon to carbon dioxide. This acid dose was found to remove greater than 99 % of inorganic carbon once the acidified sample was passed through a hydrophobic polytetrafluoroethylene (PTFE) membrane allowing the passage of dissolved gases (under negative pressure from a vacuum pump) but restricting the flow of the mobile phase.
Several factors influenced the oxidation of the organic carbon in the next step, including the dose of persulfate, the type and intensity of UV radiation and the composition of the capillary through which the sample stream passes. Through optimisation of this process, it was found that a persulfate dose of 0.84 mmol L-1[superscript] in the sample stream was required for optimum oxidation efficiency. A medium pressure UV lamp was compared to a vacuum UV lamp for its efficiency in oxidation of organic carbon to carbon dioxide. While the medium pressure lamp produced a far smaller percentage of its total radiation at the optimum wavelength for oxidation of organic compounds, the greater overall intensity of the medium pressure lamp was shown to be superior for this application. The composition of the capillary was shown to have a considerable effect on the oxidation efficiency. A quartz capillary, internal diameter 0.6 mm, was compared with a PTFE capillary, internal diameter 0.5 mm, for the oxidation of organic carbon by external UV treatment. While peak width, an important consideration in chromatographic resolution, was greater for the larger internal diameter quartz capillary, the lower UV transparency of PTFE combined with the shorter contact time, due to the reduced internal diameter of the capillary, resulted in a less efficient oxidation step using the PTFE capillary. The quartz capillary was therefore chosen for use in the UV/persulfate oxidation step for oxidation of organic carbon to carbon dioxide. Separation of the produced carbon dioxide from the sample stream was achieved by sparging with nitrogen and contacting the gas/liquid mixture with a hydrophobic PTFE membrane, restricting the passage of the liquid while allowing the nitrogen and carbon dioxide gases to pass to the detection system.
The only factor influencing this separation was the flow of the nitrogen sparge gas, with a flow of 2 mL min-1[superscript] found to be optimum. Detection of produced carbon dioxide was via a Fourier transform infrared (FTIR) spectrometer with a Iightpipe accessory. The Iightpipe accessory was designed for use as a detector for gas chromatography and the small size of the detector cell was ideal for use with this application. Using the new system described, concentrations of a single peak could be determined with a detection limit of 31 ng and a determination limit of 68 ng. The development of the new OCD allowed characterisation of NOM in terms of its MW distribution and the UV and fluorescence spectral properties of each MW fraction. Further characterisation of MW fractions of NOM from a local groundwater bore was carried out by separation of the fractions by preparative HPSEC, followed by off-line analysis. Preparative HPSEC involved the injection of a pre-concentrated groundwater sample multiple times, using a large scale HPSEC column, then collecting and combining material of identical MW. This allowed each MW fraction of the sample to be further characterised as described in Chapter 5. Preparative HPSEC has only previously been applied to a small number of samples for the concentration and fractionation of NOM, where the structural features of the various MW fractions were studied. In the current research, more extensive studies of not only the chemical characteristics, but also the disinfection behaviour, of the MW fractions were conducted. Separation of the sample was conducted on a large diameter silica-based HPSEC column, with fraction collection based on semi-resolved peaks of the HPSEC chromatogram. Nine MW fractions were collected by this method.
After concentration and dialysis to remove the buffer salts in the HPSEC mobile phase, each fraction was re-analysed by analytical HPSEC-UV254[subscript] and showed a single Gaussian shaped peak, indicating discrete MW fractions had successfully been collected. Analysis of the collected MW fractions indicated that 57 % of the organic carbon was in Fractions 3 and 4, with 41 % in Fractions 5-9, leaving only 2 % in Fractions 1 (highest MW) and 2. For each of the nine MW fractions, chorine demand and 7 day trihalomethane formation potential (THMFP) were measured on dilute solutions of the same DOC concentration, and solid state 13[superscript]C NMR spectra were recorded on some of the solid isolates obtained after Iyophilisation of the separate or combined dialysis retentates. The larger MW Fractions 3 and 4 were found to contain a greater proportion of aromatic and carbonyl carbon, and the lower MW Fractions 5 and 6 and Fractions 7-9 contained greater proportions of aliphatic and O-aliphatic carbon, by this technique. Chlorine demand experiments on each individual fraction with a normalised DOC concentration indicated that the largest MW fraction (Fraction 1) had the lowest chlorine demand. It was concluded that material in this fraction may be associated with inorganic colloids and unavailable for reaction with chlorine. Fraction 3 had the highest chlorine demand, just over two times more than the next highest chlorine demand (Fraction 4) and approximately three times the chlorine demand of Fraction 2. The organic material in Fraction 2 was postulated to contain a mixture of the reactive material present in Fraction 3 and the colloidal associated material present in Fraction 1.
NMR analysis indicated that the difference between Fraction 3 and Fraction 4 was a reduction in reactive aromatic carbon and hence the lower chlorine demand in the latter fraction. Fractions 5-8 had similar chlorine demands, lower than Fraction 4, while Fraction 9 had a very low chlorine demand similar to that of Fraction 1. For Fractions 5-9, the lower aromatic carbon content most likely resulted in the lower chlorine demand. The 7 day THMFP experiments showed some clear trends, with Fraction 1 and Fraction 2 producing the least amounts of THMs but having the greatest incorporation of bromine. Fractions 3 and 4 produced the greatest concentration of THMs with the lowest bromine incorporation, perhaps as they contained fast reacting THM precursors and the higher chlorine concentrations resulted in greater amounts of chlorinated THMs. Fraction 5 and Fraction 6 produced similar levels of THMs over 7 days to Fractions 7-9 (approximately 75% of the amount formed by Fractions 3 and 4), however, Fractions 7-9 formed these THMs more quickly than Fractions 5 and 6, with slightly greater amounts of bromine incorporation. It was thought that the increased speed of formation was due to the smaller MW of these fractions and a simpler reaction pathway from starting material to formation of THMs, as well as some structural differences. This research marks the first report of significantly resolved MW fractions being isolated and their behaviour in the presence of a disinfectant being determined. While the high MW fractions had the greatest chlorine demands and THMFPs, these fractions are also the easiest to remove during coagulation water treatment processes, as shown in Chapter 3. The lowest MW material formed significant amounts of THMs, and also formed THMs more quickly than other MW fractions.
This has important implications from a water treatment perspective, as the lowest MW material is also the most difficult to remove during conventional treatment processes. Solid samples of NOM were isolated from water samples taken from four points at the Wanneroo GWTP using ultrafiltration and subsequent Iyophilisation of the retained fractions, as described in Chapter 6. The sampling points were following aeration (Raw), following treatment by MIEX®, following treatment by MIEX®-C and following treatment by EC. Elemental analysis, FTIR spectroscopy, solid state 13[superscript]C NMR spectroscopy and HPSEC-UV254[subscript]-0CD analysis were used to compare the four isolates. Treatment with MIEX®-C was found to remove the greatest amount of NOM. Additionally, treatment with MIEX®-C was able to remove the largest MW range of NOM, with the remaining material being depleted in aromatic species and having a greater proportion of aliphatic and O-aliphatic carbon. EC treatment completely removed the NOM components above 5000 Da, but NOM below this was not well removed. NOM remaining after the EC train had a lower aromatic content and more aliphatic oxygenated organic matter than the RW. The remaining organic matter after MIEX® treatment contained less aromatic material compared to the RW, but had a greater aromatic content than either of the EC or MIEX®-C samples. HPSEC was a significant analytical technique used throughout this research. Initial optimisation of an HPSEC method was an important development which allowed improved resolution of various MW fractions. The application of this technique and comparison of three detection systems for the study of DOC removal showed, for the first time, the performance of MIEX® treatment at a full scale groundwater treatment facility.
The use of various HPSEC detection systems allowed significant characterisation of the MW fractions, more information than had previously been gathered from such a sample set. This work demonstrated the need for OCD when applying HPSEC to the study of NOM. As such, a system was constructed that built on previously developed systems, with the use of a small detector cell enabling detection limits capable of measuring even the most dilute natural and treated water samples. To study the individual MW fractions in detail, preparative HPSEC was applied and, for the first time, the disinfection behaviour of various MW fractions was examined. Interestingly, the lowest MW fractions, acknowledged to be the most recalcitrant to conventional water treatment processes, produced significant quantities of THMs. Also the formation kinetics of THMs from the low MW fractions indicated that THMs were formed as quickly as, or perhaps even at faster rates than from the larger MW fractions. Finally, structural characterisation of NOM at four stages of the Wanneroo GWTP indicated MIEX®-C treatment was superior to EC, of significant interest for the water industry.

Crosslinked poly-4-vinylpyridine (XPVP) has not previously been used as a size exclusion chromatographic packing material. This material appears attractive for the analysis of nitrogen containing polymers because of its basic nitrogen functionality, and desirable physical properties. Its use as such is investigated with probe compounds in several different mobile phases. Various methods of separation of the XPVP material by particle size are employed. In addition, four column packing techniques were examined.
Master of Science

In the present thesis two analytical methods forcharacterisation of underivatised pulp and lignin samplesobtained from kraft pulping of hardwood and softwood areevaluated. The first method is the use of lithiumchloride/N,N-dimethylacetamide (LiCl/DMAc) for dissolution andsize-exclusion chromatography (SEC) of pulps. The applicabilityof LiCl/DMAc-SEC is demonstrated for birch wood kraft pulpswith different relations between zero-span tensile strength andviscosity. The second method concerns the applicability ofcapillary zone electrophoresis (CZE) for characterising blackliquor and isolated lignin samples with respect to mobility,i.echarge density. A method for determining the averagemobility (µav) of the mobility distributions to easecomparison between samples is also presented.

The solubility in LiCl/DMAc and the elution behaviour in SECdiffer between hardwood and softwood kraft pulps. Hardwoodkraft pulps are completely dissolved in LiCl/DMAc, whereas highamounts of lignin and presence of glucomannan restrict thesolubility of softwood kraft pulps. The undissolved fraction ofsoftwood kraft pulps consists of larger amounts of mannose andlignin but has a diminished xylose content compared with theinitial pulp. Xylan and cellulose of hardwood kraft pulps arefairly well separated by LiCl/DMAc-SEC. In contrast, themolecular weight distributions (MWD) of softwood kraft pulpsare more complex. It was found that the hemicelluloses ofsoftwood pulps elute over the entire molecular weight range,indicating various degrees of association with cellulose. Theneutral monosaccharide composition implies that associationsbetween galactoglucomannan and cellulose increase withdecreasing amount of galactose. The elution behaviour ofsoftwood kraft pulp xylan suggests that this xylan consist ofsubstructures with varying propensity for associating withcellulose and/or mannan. In absence of associations betweencellulose and hemicellulose, cellulosic solutions of LiCl/DMAcconsist of cellulose aggregates, which is seen as a shoulder onthe high molecular weight end of the MWD of cellulose.According to the profiles of the MWD and light scatteringmeasurements, it is possible to break these aggregates bymechanical treatment of the solutions, without causing severecleavage of the glycosidic bonds. The relation between MWD andzero-span tensile strength was studied on hardwood kraft pulpsdegraded by gamma irradiation, oxygen/alkali or alkali. For alltreatments, the MWD of cellulose is shifted to a lowermolecular weight range as degradation proceeds. In thechemically treated pulps, a shoulder on the lowmolecular-weight end of the cellulose distribution graduallydevelops, which is not seen for the gamma treated pulps. Theobserved decrease in shape factor/fibre strength of thechemically treated-pulps is proposed to be due to a combinationof heterogeneous degradation and removal of hemicellulosewhereas the decrease in Mw of cellulose is of minorimportance.

The mobility distributions obtained by CZE differ betweenblack liquor, isolated dissolved lignin and isolated residuallignin. The µav measured at pH 12 reveals that theresidual lignin isolated from pine wood kraft pulp has asignificantly lower charge density than the lignin removed fromthe pulp throughout the cook. At the end of the kraft cook ofbirch, the µav of the isolated residual lignin is aboutthe same as that of the isolated dissolved lignin, whichsuggests that the solubility is sufficient for the pulp ligninto be dissolved. Comparisons between the µav at pH 12 andpH 10 indicate that the isolated dissolved lignin samplesobtained in the middle of the cook have a lower acidity thanthe other samples. The observed difference in µav betweenblack liquor and isolated dissolved lignin may be due toassociations between lignin fragments and carbohydrate polymersin the black liquor.

Keywords:Kraft pulps, Black liquor, Cellulose,Hemicellulose, Lignin, LiCl/DMAc, Dimethylacetamide,Size-exclusion chromatography, Capillary zoneelectrophoresis

Rat hepatocytes are isolated via a modified collagenase portal vein perfusion method and encapsulated within alginate-polylysine microcapsules to be used as a biohybrid artificial liver support system. The microcapsule membrane must allow the diffusion of small metabolites including peptides and small proteins and the exclusion of cells and immunoglobulins of molecular weight >150 kDa. The success of a complete hybrid liver depends on the preservation of hepatocyte multiple functions including the removal of ammonia amongst other toxic substances, urea synthesis, gluconeogenesis and secretion of proteins in the molecular weight range of 5--150 kDa while allowing the transport of lower MV biomolecules. Five types of alginate-polylysine microcapsules are examined (i) standard single membrane microcapsules; (ii) microcapsules prepared with a two step procedure where special attention is taken to prevent the entrapment of cells within the membrane matrix; (iii) double membrane microcapsules where intracapsular alginate is minimized for improved intracapsular diffusion and cell contact; (iv) new microcapsules where microcapsules are encapsulated within larger microcapsules, macrocapsules, with distinct and non interacting membranes separated by a liquid intracapsular space; and (v) microcapsules with polyanion/polycation multilayering. The effect of process variables is considered in the design of the microcapsules with a size exclusion range limit as close to 150 kDa as possible. A method is devised to simultaneously analyze the exclusion limit and permeability to macromolecules diffusing into microcapsules. A polydisperse dextran mixture, proteins as single solutes and in mixtures are used as model diffusants in a batch mode high performance size exclusion chromatography analysis. High performance size exclusion chromatography is complimented with image analysis to evaluate microcapsule designs for biohybrid artificial liver in different biological fluids; plasma, s

Purification of Biosynthetic Human Insulin (BHI or Humulin), involves multimodal chromatographies as well as intermediate crystallizations. Size exclusion chromatography (SEC) is the final chromatographic step in BHI purification, which is conducted in acetic acid at high protein concentrations. The purpose of the SEC step is to remove the higher-molecular weight species proinsulin (molecular weight (MW) ≈ 10 kDa) and covalently linked insulin dimer (MW ≈ 11.6 kDa) from native insulin monomer (MW 5808 Da). Because covalent insulin dimers are recognised as a significant factor in immunological responses seen in diabetic patients (Darrington & Anderson 1995), limiting the amount of covalent in the final active pharmaceutical ingredient (API) is a key requirement for the development of the purification process. The presence of covalent dimers in insulin process streams results from chemical degradation of BHI either through intermolecular disulphide bond formation or from transamidation. Complete characterisation of the covalent dimeric species present in commercial SEC feed streams has not been performed. However, because specific purification technology is employed to remove disulphide bonded polymeric impurities upstream of the SEC step, dimeric impurities are thought to be predominantly transamidated species. Further, recent laboratory-scale SEC studies have indicated that there is a covalent dimer form present in the SEC feed streams that is difficult to separate from the native BHI molecule by SEC. The objectives of this study were to determine the types of insulin covalent dimer entering the BHI SEC step, understand the impact of the covalent insulin dimer type on SEC separation of dimer from the native insulin and to learn more about the SEC step itself i.e., investigations into the effect the SEC resin (G-50 Sephadex), may have had on the BHI structure. In order to achieve this, various techniques were explored to analyse BHI conformation, purity and degradation trends. Such techniques included Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE, High Performance Liquid Chromatography (Reverse Phase and Size Exclusion Chromatography), Circular Dichorism and Surface Enhanced Laser Desorption Ionisation (SELDI). Breakdown studies on BHI were carried out using reducing agents DTT and GdnHCl and the effects of environmental conditions (pH, temperature and protein concentration), on native BHI as well as the reduced protein were investigated. Further to this, studies carried out at Eli Lilly and Company explored the occurrence of the fronting observed after SEC purification. Here the human proinsulin (HPI), high molecular weight polymer (HMWP) and BHI were thought to exist together and were separated from the main BHI peak, and then recycled in order to separate and recapture BHI in order to increase yield. Studies were conducted to understand the occurrence of the fronting better, as well as to see if factors such as presence of Zinc could alter the fronting and whether this could ultimately lead to steps being taken to modify the manufacturing process to make it more efficient and economically sound. Finally, the folding reaction where human proinsulin s-sulfonate (HPSS) is converted to HPI during BHI manufacture was investigated. During this step, the HPI purity and behaviour was assessed in order to determine whether changes could be made here in order to make the manufacturing process more efficient. Overall it was found that the species present during BHI analysis were BHI monomer and transamidated dimer. Upon reduction by denaturing agents such as DTT with GdnHCl, the BHI monomer was found to become reduced to form its constituent A and B chains. More of a dimer peak was observed also, which was a combination of transamidated dimer, but also BHI monomer being eluted sooner during HPLC due to its reversible formation of self-associated dimer as it flowed through the column. An additional small peak was also observed which was suspected to be an AA-AA dimer, but further experimentation is required to confirm its identity. Addition of Sephadex caused BHI monomer to be more stable, thus more difficult to reduce using denaturing agents such as DTT and GdnHCl to its constituent A and B Chains. The sephadex induced a shift in equilibrium between BHI and Chains A and B such that the BHI protein remained in a more folded state, thus making it harder for the DTT to access and reduce the disulphide bonds. With regards to environmental conditions, it was found that an increase of temperature lead to an increase of degradation/reduction of BHI monomer, resulting in an increase of all the other peaks. The general trend observed with regards to pH was that BHI monomer was most stable at pH 3 at all the temperatures tested, and pH 4 was the most unstable except at 40°C where an increase of pH appeared to encourage misfolding and avoid aggregation. Complete unfolding of BHI was only found to occur at pH 9 (where all the pH’s examined were 1.5, 2, 3, 3.5, 4, and 9). Concentration changes resulted in an expected increase in peak areas in SEC HPLC, and it did show that more BHI dimer was likely to form as a result of increased concentration. A number of theories were tested to understand the occurrence of fronting in the BHI elution peak from SEC. It was found to result from the BHI forming a self-associated dimer on the column, thus eluting earlier but then returning to its original monomeric state. This explained why BHI monomer was found in the fronting area along with HMWP and HPI, and therefore had to be recycled and repurified. It was also found that chelating zinc ions from the BHI sample did not have an effect on the equilibrium between monomer and dimer, or on the fronting observed on the large scale SEC HPLC. It was seen however that when the G-50 SEC column was saturated with ZnCl2 the BHI monomer peak shifted towards being eluted at a higher MW (towards the left), thus promotion of zinc induced BHI dimerisation. Although this resulted in increased fronting, this is more likely now to be due to the harmless self-associated dimers that are reversibly formed as opposed to non-reversible covalently formed dimers, so the need for the cut and recycle step to remove the covalent dimer, would no longer be needed. However it was also found that the HPI content was significantly increased in the Main Stream (MS) step (as illustrated in Figure 7.1) which decreased the final BHI purity, and so overall the saturation with zinc did not allow the recycle step to be avoided during manufacturing. Thus it was better to keep the initial fronting and avoid Zn induced dimerisation. Finally with regards to HPI purity during the manufacturing step where HPSS was converted to HPI, it was found that disulphide shuffling occurred with a corresponding increase of HMWP, but only for the first 40-50 hours, after which the polymer fraction decreased and HPI fraction increased until a plateau was reached. A variation in the time to reach the plateau is likely to be due to slight variations in added cysteine concentration. It is recommended that the cysteine concentration should be investigated in further experimentation in order to speed the reaction up so that the plateau could be reached faster. This decreased manufacturing time would result in a more time and cost efficient process.

Size exclusion chromatography is the method of choice for characterizing molecular weights and molecular weight distributions of polymers. An important advancement in SEC is multidetection SEC which includes multi-angle laser light scattering, viscometry, refractive index and UV spectroscopy to analyze block and graft copolymers as well as polymers with oligomeric molecular weights. Oligomeric molecular weights present special challenges since the light scattering and viscosity detectors are more sensitive to higher molecular weights and both detectors have low molecular weight threshold values. The molecular weights and distributions of poly(2-ethyl-2-oxazoline) oligomers and block copolymers as well as poly(2-ethyl-2-oxazoline) were investigated by SEC using multiple detectors. Both a universal calibration method and light scattering were used to determine molecular weights and molecular weight distributions. The solvent was N-methylpyrrolidone that contained 0.05M LiBr used to minimize interactions among the polymers and solvent. SEC was used to establish that the diblock copolymers had heterogeneous compositional distributions. The low molecular weights of the diblock and homopolymer made it necessary to use the universal calibration method with combined refractive index and viscometry detectors to determine absolute molecular weights.
Master of Science

Ammonium ionenes are macromolecules with quaternized nitrogen groups in the main chain. Ionenes are commonly referred to as x,y-ionene, where x and y represent the number of methylene groups between quaternized nitrogens. Synthesis of aliphatic ammonium ionenes has been studied since the early twentieth century; however, absolute molecular weight characterization has only been performed using extensive light scattering and viscosity experiments. Performing aqueous size exclusion chromatography (SEC) on ammonium ionenes provides absolute molecular weight determinations while eliminating the need for separate viscosity and light scattering experiments. We developed a mobile phase composition that provides reliable separation of aliphatic ammonium ionenes using aqueous SEC. For the first time, we report absolute molecular weights of aliphatic ammonium ionenes using this technique. We investigated the influence of charge density and structural symmetry on thermal and mechanical properties of ammonium 6,6-, 12,6- and 12,12-ionenes. Thermal properties were measured using differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), and mechanical properties were measured using dynamic mechanical analysis (DMA) and an Instron. Incorporating low molecular weight polymer segments into the main chain of the ionene allows tailoring of polymer characteristics. Poly (tetramethylene oxide) segments decrease hydrophilicity and increase elastomeric character. Linear PTMO based ionenes have been synthesized previously, and we were interested in how branching affected thermal and mechanical properties. We synthesized bis(dimethylamino) poly(tetramethylene oxide) segments, and subsequently, synthesized linear and branched ionenes to study the effects of topology on thermal and mechanical properties. Polymers were analyzed using DMA, DSC, TGA, SAXS, and an Instron.
Master of Science

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking: Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein). My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions. Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

This dissertation focuses on the synthesis and characterization of functional poly(2-ethyl-2-oxazoline) (PEtOx) containing homo- and block copolymers that are potential materials for membrane-based water purification and gas separation, drug delivery, magnetic resonance imaging and tissue engineering applications. The polymerization of 2-ethyl-2-oxazoline (EtOx) was investigated with regard to the effects of initiator structures and reaction parameters such as polymerization time and temperature on molecular weight control and molecular weight distribution, endgroup functionality, living characteristics, and mechanism and kinetics. The structure of initiators was shown to significantly affect the molecular weight control and molecular weight distribution of PEtOx oligomers. Methyl triflate initiated polymerizations were found to result in oligomers with low polydispersity (PDI) values around 1.10-1.15 and symmetrical chromatograms were obtained via size exclusion chromatography (SEC) studies with the use of refractive index, light scattering and viscosity detectors. However, EtOx polymerizations initiated by halide containing initiators such as benzyl chloride, dibromo- and diiodo-p-xylene, and vinylsilylpropyl iodides yielded PEtOx oligomers with higher PDI values ~ 1.30-1.40. Higher molecular weight distributions can be attributed to the presence of covalent species during polymerization and slower initiation rate as evidenced by kinetic studies when compared to PEtOx prepared from methyl triflate initiators. In all cases, termination reactions with aliphatic cyclic amines were quantitative. Mono- and diamine functional PEtOx oligomers with controlled molecular weight and excellent end-group functionality may be used as prepolymers for incorporation into multiblock and graft copolymer and crosslinked structures for a variety of applications such as membranes and hydrogels for tissue engineering matrices. Poly(2-ethyl-2-oxazoline) containing block copolymers were prepared using the macroinitiator method. First, amphiphilic triblock copolymers with hydrophobic poly(arylene ether sulfone) (PSF) central block and hydrophilic PEtOx side blocks were synthesized via polymerization of EtOx sequences from tosylate functional telechelic PSF macroinitiators. PSFs are well-known engineering thermoplastics with excellent resistance to hydrolysis and oxidation, as well as displaying good mechanical properties, thermal stability and toughness. Phenol functional PSFs were prepared via step-growth polymerization of dichlorodiphenylsulfone and bisphenol-A (slight excess) monomers. Phenolic chain ends were then converted to aliphatic hydroxyethyl endgroups by reaction with ethylene carbonate. Upon treatment with p-toluenesulfonyl chloride, tosylate functional PSF macroinitiators were prepared. PEtOx-b-PSF-b-PEtOx triblock copolymers (pendent acyl groups of PEtOx side blocks) were partially hydrolyzed in an acidic medium to introduce random charged poly(ethylene imine) units to prepare ionomer structures that may show good salt rejection, water flux and antibacterial properties for membrane-based water purification applications. Phosphonic acid modified poly(ethylene oxide)-b-poly(2-ethyl-2-oxazoline) (PEO-b-PEtOx) diblock copolymers were prepared via cationic ring opening polymerization of EtOx monomers from tosylate functional PEO macroinitiators and subsequent functionalization reactions on the polyoxazoline block. Post-modification reactions included controlled partial pendent acyl group hydrolysis under an acidic medium to form the random block copolymers of PEtOx and poly(ethyleneimine) (PEI), Michael addition of diethylvinyl phosphonate groups to PEI units and hydrolysis of the ethyl groups on the phosphonates to yield pendent phosphonic acid groups on the polyoxazoline block. After each step of functionalization reactions, structures and compositions were confirmed utilizing 1H NMR and the degree of phosphorylation was found to be > 95%. Both PEO and PEtOx are biocompatible polymers and the anionic quality of the phosphonic acid has the potential to be pH controllable and provide an environment where cationic drugs and contrast agents can be attached. Thus, these polymers have potential as drug carriers and contrast enhancement agents for magnetic resonance imaging applications.
Ph. D.

In this thesis, size-exclusion chromatography (SEC) of woodpolymers dissolved in lithium chloride/N,N-dimethylacetamide(LiCl/DMAc) has been used to characterize the molar massdistributions (MMD) of wood polymers in pulp fibers afterchemical degradation.

Characterization of birch kraft pulps subjected to ozonedegradation and acid hydrolysis, respectively, rendereddifferent changes in the MMD. Ozone degradation resulted inlarge redistributions of the original MMD, observed as thedevelopment of a distinct fraction of cellulose withintermediate molar mass. Acid hydrolysis resulted in minorchanges of the original MMD compared to ozonation. Fiberssubjected to acid hydrolysis were considerably weaker thanozonated fibers. These results indicated that there aredifferences in how the two chemicals degrade the fiber.

The solubility of softwood kraft pulp fibers was enhanced byderivatization of the fiber polymers with ethyl-isocyanateduring simultaneous dissolution in LiCl/DMAc. Thederivatization made it possible to achieve reliable estimationsof the MMD, and hence molar masses, of softwood kraft pulps.The derivatization procedure made it possible to dissolve 90 %of softwood kraft pulps with kappa numbers over 50.

Severe alkaline degradation of birch and Norway spruce woodchips was studied both by varying the pulping time and byvarying the initial alkali concentration. Differences werefound in the MMD of the two fiber types, and the alkalinedegradation was found to affect polymers in the entire MMD.

Multi-angular laser light scattering (MALLS) was used as adetection technique with SEC on cellulosic samples. The MMD andaverage molar masses obtained through directstandardcalibration with commercial standards were compared with MMDand molar masses as obtained by MALLS-detection. Largediscrepancies were found, and two methods of correcting forthese discrepancies were developed.

Theoretical simulations of polymer degradation wereperformed. Random, or homogeneous degradation was used as amodel for alkaline cellulose chain scission, and a resemblancewith experimental data was observed. End-wise depolymerizationof cellulose was also simulated and the results are discussedin the light of experimentally observed MMD.

Keywords:cellulose, kraft pulp, birch, spruce,ozonation, acid hydrolysis, degradation, MMD, sizeexclusionchromatography, light scattering, molar mass, chainscission

O Brasil é atualmente o maior produtor e exportador mundial de açúcar e a aguardente de cana aqui produzida é a terceira bebida destilada mais consumida no mundo. Contudo, estes produtos ainda têm a sua qualidade afetada pela presença de Dextranas. Devido à sua origem (produto secundário do metabolismo bacteriano) e por conta das suas propriedades físico-químicas, as dextranas constituem um importante meio para a avaliação da qualidade do açúcar. No âmbito industrial, as dextranas podem causar diversos problemas ao setor alimentício que utiliza o açúcar como matéria-prima, além de estarem diretamente relacionadas à formação de precipitados na aguardente de cana adoçada. Este trabalho apresenta o primeiro perfil da distribuição de massa molar das dextranas presentes no açúcar brasileiro em função dos seus valores de Mn, Mw, Mz e polidispersividade, os quais exercem influência significativa sobre suas características físico-químicas. Estes parâmetros foram determinados via cromatografia líquida de exclusão por tamanho. O perfil de distribuição de massa molar traçado exibiu, majoritariamente, a presença de dois grupos de dextranas com valores de Mw médios de 5,0 x 106 e 4,8 x 104 Da, e, ocasionalmente, a presença de dextranas que foram classificadas num terceiro grupo com massas molares da ordem de 105 Da. Através deste perfil, foram avaliadas, em sistemas modelo aguardente-dextrana, as influências dos fatores temperatura, acidez, presença dos íons metálicos CuII, FeIII, CaII e MgII e incidência de luz sobre a velocidade de formação dos depósitos de dextranas na aguardente de cana. Demonstrou-se que a acidez e a temperatura foram os fatores que mais influenciam na velocidade de precipitação das dextranas em aguardentes de cana adoçadas.
Brazil is the largest producer and exporter of sugar in the world, and the cachaça is the third most produced distilled beverage in the world. However, these products have their quality affected by the presence of dextrans. Because of its origin (a secondary compound of bacterial metabolism) and due to its physical-chemical properties, the dextrans are an important indicator to assess the sugar quality. Dextrans can lead to problems in processed food production, and it has a strong relation with insoluble deposits in sugared sugar cane spirits. The dextran molecular mass distribution profile in terms of Mn, Mw Mz and polydispersity in Brazilian sugars are reported for the first time. The analyses were accomplished by Size-exclusion chromatography, using a refraction index detector. In most of the sugar samples, it was possible to identify two major groups of dextrans with Mw averages of 5.0 x 106 e 4.8 x 104 Da. A third group of dextrans with Mw of 105 was occasionally observed. With this data, model systems were built to assess the influence of the temperature, acidity, presence metalic ions CuII, FeIII, CaII, and MgII, light incidence in relation of dextran precipitation velocities. It is demonstrated that factors temperature and acidity are the most influential in the dextran precipitation velocity in sugar cane spirits.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Human interferon-α2a (hIFN-α2a) is a natural protein produced by the cells of the immune system with antiviral, antiproliferative and immunomodulatory properties. A size exclusion liquid chromatography (SE-LC) method was validated for the determination of recombinant interferon-α2a (rhIFN-α2a) in pharmaceutical formulations without human serum albumin. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.) maintained at 25°C. The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M sodium phosphate dibasic and 0.2 M sodium chloride buffer, pH 7.4, run at a gradiente flow rate and using photodiode array (PDA) detection at 214 nm. The chromatographic separation was achieved with retention time of 17.2 min, and was linear over the concentration range of 0.5-50 MUI/mL (r2 = 0.9996). The accuracy was 101.39% with bias lower than 1.67%. The limits of detection and quantitation were 0.22 and 0.5 MIU/mL, respectively, and the method validation demonstrated acceptable results for precision and robustness. The proposed method was applied for the analysis of rhIFN-α2a in pharmaceutical dosage forms, and the content/potencies correlated to the previously validated reversed-phase (RP-LC), and the in vitro bioassay. The pharmaceutical samples were analyzed by the chromatographic methods and compared to the bioassay, showing mean differences between the estimated potency of 1.50% higher for SE-LC, and 2.45% lower for the RP-LC. The alternative methods studies contribute to improve the quality control, assuring the therapeutic efficacy of the biological medicine. Moreover, the pharmacokinetic parameters of the formulations A and B were evaluated by subcutaneous injection in rats, showing comparable profiles with Cmax of 7924.60 and 8698.68 pg/mL, respectively, and Tmax= 60 min.
O interferon-α2a humano (hIFN-α2a) é uma proteína produzida pelas células do sistema imune com propriedades imunomoduladora, antiviral e antiproliferativa. No presente trabalho, foi validado método por cromatografia líquida por exclusão molecular (CL-EM), para determinação de interferon-α2a recombinante (rhIFN-α2a) em formulações farmacêuticas sem albumina. O método CL-EM foi validado empregando coluna BioSep-SEC-2000 S (300 mm x 7,8 mm d.i.) mantida a temperatura de 25°C. A fase móvel foi composta de tampão fosfato de potássio monobásico 0,001 M, fosfato de sódio dibásico 0,008 M e cloreto de sódio 0,2 M, pH 7,4, eluída em gradiente de fluxo e utilizando arranjo de diodos (DAD) com detecção em 214 nm. A separação cromatográfica foi alcançada com o tempo de retenção de 17,2 min e o método foi linear no intervalo de concentração de 0,5-50 MUI/mL (r2 = 0,9996). A exatidão média foi de 101,39%, com erro calculado inferior a 1,67%. Os limites de detecção e quantificação foram de 0,22 e 0,5 MUI/mL, respectivamente e a validação demonstrou parâmetros aceitáveis de precisão e robustez. O método proposto foi aplicado para análise de formulação farmacêutica de rhINF-α2a e os teores/potências correlacionados com aqueles fornecidos pelos métodos previamente validados por CL-FR e bioensaio in vitro. Estabeleceu-se correlação entre os métodos, demonstrando que os biofármacos comerciais apresentaram diferenças entre as médias 1,5% maiores para o CL-EM, e 2,45% menores para CL-FR, em relação ao bioensaio. Assim, contribuiu-se para estabelecer alternativas que aprimoram o controle de qualidade, assegurando a eficácia terapêutica do produto biológico. Além disso, avaliaram-se os parâmetros farmacocinéticos das formulações A e B após injeção subcutânea em ratos, demonstrando perfis comparáveis com Cmax de 7924,60 e 8698,68 pg/mL, respectivamente, e Tmax= 60 min.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19025号
農博第2103号
新制||農||1030(附属図書館)
学位論文||H27||N4907(農学部図書室)
31976
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 加納 健司, 教授 植田 和光, 教授 植田 充美
学位規則第4条第1項該当

Several novel polymer monoliths for the analysis of peptides and proteins were synthesized using polyethylene glycol diacrylate (PEGDA) as crosslinker. Photo-initiated copolymerization of polyethylene glycol methyl ether acrylate and PEGDA yielded an inert monolith that could be used for size exclusion liquid chromatography of peptides and proteins. This macroscopically uniform monolith did not shrink or swell in either water or tetrahydrofuran. More importantly, it was found to resist adsorption of both acidic and basic proteins in aqueous buffer without any organic solvent additives. A strong cation-exchange polymer monolith was synthesized by copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and PEGDA. A ternary porogen (water, methanol and ethyl ether) was found suitable to prepare a flow-through monolith with moderate pressure drop in aqueous buffer. The resulting monolith showed excellent ion exchange capillary liquid chromatography of peptides using a simple salt gradient. Extremely narrow peaks were obtained for the analysis of synthetic peptides, natural peptides and a protein digest. A peak capacity of 179 was achieved. Although the poly(AMPS) monolith demonstrated extraordinary performance, one main drawback of this monolith was its relatively strong hydrophobicity. A decrease in hydrophobicity was achieved by using more hydrophilic monomers (e.g., sulfoethyl methacrylate or vinyl sulfonic acid). The most hydrophilic poly(vinyl sulfonic acid) monolith provided high resolution cation-exchange liquid chromatography of protein standards and lipoproteins. Use of the new PEGDA biocompatible crosslinker over the conventional ethylene glycol dimethacrylate crosslinker for the preparation of polymer monoliths was found to be advantageous for the analysis of biological compounds in several chromatography modes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The interferon is a cytokine with antiviral, antiproliferative, and immunomodulatory properties. It is a protein synthesized by cells in response to viral infection, producing successive biochemistry alterations. The chromatographic methods for evaluation of recombinant interferon-alfa 2a (rhIFN-α2a) in pharmaceutical products were validated in the present work. The reversed-phase liquid chromatography method (RP-LC) was developed and validated using a Jupiter C4 column (250 mm x 4.6 mm), maintained at ambient temperature (25°C). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of 0.1% TFA in acetonitrile, run in gradient: 0.01 1 min, 38% of B; 1 5 min, 38 43% of B; 5.01 20 min, 43 45% of B; 20.01 30 min, 45 48% of B; 30.01 40 min linear back to 38% of B and 40 42 min, 38% of B. The flow rate used was 1 mL/min with detection at 214 nm. The chromatographic separation was obtained within 42 min and it was linear in the concentration range of 0.5 50 MIU/mL (r2=0.9999). The size exclusion method was developed and validated using a BioSep-SEC-S 2000 (300 mm x 7.8 mm), maintained at ambient temperature (25°C). The mobile phase consisted of 1mM potassium phosphate monobasic, 8mM sodium phosphate dibasic and 200mM sodium chloride buffer, pH 7.4, run at a gradient flow rate: 0.01 20 min, 0.5 mL/min; 20 25 min, 0.5 1.7 mL/min; 25 35 min, 1.7 mL/min; 35 38 min, 1.7 0.5 mL/min; 38 40 min, 0.5 mL/min. The method was linear in the concentration range of 0.5 - 50 MIU/mL (r2=0.9996). The procedures were validated by the parameters of specificity, linearity, precision, accuracy, robustness, limit of quantitation and limit of detection. The methods were applied for the evaluation of the rhIFN-α2a in pharmaceutical products, contributing for the establishment of alternatives which improve the quality control, assuring the safety and therapeutic efficacy of the biological product.
O interferon é uma citocina que possui ação antiviral, imunomoduladora e antiproliferativa. É uma proteína sintetizada pelas células em resposta a infecção viral, gerando sucessivas alterações bioquímicas. No presente trabalho foram validados métodos cromatográficos para a avaliação de interferon-alfa 2a (rhIFN-α2a) em produtos farmacêuticos. O método por cromatografia líquida em fase reversa (CL-FR) foi desenvolvido e validado empregando coluna Júpiter C4 (250 mm x 4,6 mm), mantida a temperatura ambiente (25°C). A fase móvel A foi composta de ácido trifluoracético 0,1% e a fase móvel B de ácido trifluoracético 0,1% em acetonitrila, eluídas no gradiente: 0,01 1 min, 38% de B; 1 5 min, 38 43% de B; 5,01 20 min, 43 45% de B; 20,01 30 min, 45 48% de B; 30,01 40 min, 48 38% de B, mantendo-se nesta proporção até 42 min. Utilizou-se vazão de 1 mL/min e detecção no ultravioleta a 214 nm. A separação cromatográfica foi obtida no tempo de 42 min, sendo linear na faixa de concentração de 0,5 - 50 MUI/mL (r2=0,9999). Paralelamente, desenvolveu-se e validou-se método cromatográfico por exclusão molecular (CL-EM) empregando coluna BioSep-SECS 2000 (300 mm x 7,8 mm), mantida a temperatura ambiente (25°C). A fase móvel foi composta de tampão fosfato de potássio monobásico 1mM, fosfato de sódio dibásico 8mM e cloreto de sódio 200mM, pH 7,4, eluída no gradiente de fluxo: 0,01 20 min, 0,5 mL/min; 20 25 min, 0,5 1,7 mL/min; 25 35 min, 1,7 mL/min; 35 38 min, 1,7 0,5 mL/min; 38 40 min, 0,5 mL/min. O método foi linear na faixa de concentração de 0,5 - 50 MUI/mL (r2=0,9996). Ambos os procedimentos foram validados com base nos parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limite de quantificação e detecção. Os métodos foram aplicados para avaliação de rhIFN-α2a em produtos farmacêuticos, contribuindo para o estabelecimento de alternativas que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biológico.

La cellulose est un biopolymère naturel et très abondant. Selon son origine et son mode d’extraction, elle présente des propriétés de cristallinité et de longueur de chaînes variables. Les interactions hydrogène entre les chaînes de cellulose sont en grande partie responsables de son organisation et forment un réseau très dense qui limite sa solubilité. Sa dissolution, complète et non-dégradante, est donc compliquée et dépend de la capacité du solvant à rompre ces liaisonshydrogène intermoléculaires. L’analyse de la cellulose par chromatographie d’exclusion stérique (SEC) pour suivre l’évolution des distributions de masses molaires (DMM) au cours des procédés de transformation est donc toujours problématique. Le système de solvant le plus commun estactuellement le chlorure de lithium/N,N-diméthylacétamide (LiCl/DMAc) qui est extrêmement toxique. L’objectif de ce travail est le développement d’une méthode d’analyse des DMM de la cellulose dans des conditions moins toxiques et adaptée à tous les types de cellulose. Parmi les nombreux systèmes de solvant décrits dans la littérature, 3 systèmes, plus verts et nondérivatisants, ont été sélectionnés afin d’identifier un système de solvant permettant la dissolution,sans dégradation, d‘échantillons de cellulose de cristallinité et de masse molaire moyenne variables afin de remplacer LiCl/DMAc. Les analyses thermogravimétriques et les mesures viscosimétriques ont permis d’évaluer et de comparer les modifications de 4 échantillons de cellulose dissous dans ces 3 systèmes et dans le LiCl/DMAc. LiCl/DMAc dégrade les celluloses de plus haute masse molaire (-cellulose et Vitacel), réduisant de 50 % les longueurs de chaîne après dissolution. Le système tétrabutylammonium/diméthylsulfoxide (TBAF/DMSO) permet une dissolution rapide des 4 échantillons de cellulose sans dégradation ou modification majeure. Le système TBAF/DMSO a, par la suite, été privilégié pour la caractérisation des échantillons en SEC. Du fait d’interactions possibles entre les groupements aromatiques composant la phase stationnaire et les molécules de TBAF, le système complet n’a pas pu être utilisé comme éluant. L’éluant choisi a donc été le DMSO mais qui, seul, ne solubilise pas les échantillons cellulosiques. Les molécules de TBAF sont cependant indispensables au mécanisme de dissolution et laconcentration en TBAF dans le DMSO a dû être adaptée en fonction des échantillons. Pour les échantillons cellulosiques de faible masse molaire, une concentration en TBAF de l’ordre de 1 %(m/v) est suffisante et permet de réaliser une analyse des masses molaires satisfaisantes. Pour les échantillons de plus haut poids moléculaire, cette concentration n’est plus suffisante pour les dissoudre convenablement. Or, avec des concentrations plus élevées, des phénomènesd’agrégation provoque l’élution d’une partie importante des macromolécules dans le volume mort, comme observé avec l’analyse de standards de pullulane
Cellulose is a very abundant natural biopolymer. According to its origin and to its extraction mode, it presents various cristallinity rate and molar mass. Its organization relies mainly on intermolecular hydrogen bonds that form a strong network and thus limitating cellulose solubility. Complete andnon-degradative dissolution is then complicated and depends on the solvent ability to disrupt these hydrogen bonds. Analysis of the molar mass distribution (MMD) of cellulose by size exclusion chromatography (SEC) of cellulose is consequently problematic while the study of the evolution of cellulose molar mass during transformations could be extremely useful in many processes. The most common solvent used in SEC is lithium chloride/N,N-dimethylacetamide (LiCl/DMAc), which is extremely toxic. The aim of this PhD study is then to develop a new analytical method to characterize cellulose MMD using safer solvents and adapted to all kinds of cellulosic sample. Among the numerous non-derivatizing solvent systems described in the literature, three of themhave been selected. Greener and less toxic than LiCl/DMAc, their ability to dissolve cellulosic sample of various cristallinity and average molar mass without degradation was then tested. Thermogravimetric analyses and viscosimetric studies allowed to evaluate and to compare the modifications involved by the dissolution for four different cellulosic samples. LiCl/DMAc degraded the samples of higher molar mass (-cellulose and Vitacel) decreasing their degree ofpolymerization by 50 % after dissolution. Tetrabutylammonium/dimethylsulfoxide (TBAF/DMSO) system allows rapid dissolution of the 4 cellulose samples, without major degradation or modification. TBAF/DMSO system was then studied as solvent for SEC analysis of these cellulose samples. Due to the interactions between the aromatic groups composing the stationary phase with TBAF molecules, the complete system could not be used as eluant. Chosen eluant was then DMSO alone. However, as the TBAF molecules are mandatory for the dissolution of cellulose, TBAF concentration was adapted according to the cellulose nature for the preparation of the samples. For low molar mass cellulose samples, a TBAF concentration of 1 %(w/v) was sufficient and allowed to performed correctly the chromatographic analysis. For the samples of higher molar mass, this concentration was not high enough to complete the dissolution. Using, higher concentrations caused aggregation phenomena resulting in the elution of a large amount of the macromolecules in the dead volume, as observed with the analysis of pullulan standards

: L’une des méthodes qui consiste à optimiser la production d’hydrocarbure est basée sur l’injection d’eau, viscosifiée par addition de polymères de type polyacrylamides, afin d’améliorer l’extraction de pétrole (RAH). Or, les propriétés viscosifiantes des polymères dépendent de leurs masses molaires. L’enjeu de ce travail de thèse a donc été de mettre en place de nouvelles méthodes pour la détermination de larges distributions en masses molaires et de grande dispersité, caractéristiques propres aux échantillons d’intérêt industriel dans le domaine pétrolier. La stratégie d’analyse retenue repose sur la chromatographie d’exclusion stérique (CES) et le fractionnement par couplage flux force (A4F) couplés à la diffusion de lumière et à la réfractométrie. Des polymères modèles ont été synthétisés par un procédé de polymérisation par transfert de chaîne réversible par addition fragmentation (RAFT/MADIX). Cela a permis d’obtenir des polymères dans une gamme de masses molaires comprises entre 103 et 107 g/mol et avec une dispersité inférieure à 1,4. Via l’utilisation de ces polymères, les performances et limites des deux méthodes séparatives investies ont été évaluées. Les conditions opératoires en A4F ont été déterminées et cette méthode s’est avérée être adaptée à l’analyse de polymères industriels distribués sur 3 décades de masses molaires. Au delà de l’analyse dimensionnelle, le couplage avec l’A4F a également permis une analyse conformationnelle
One method for optimizing the production of hydrocarbon is based on the injection of water, viscosified by the addition of polymers such as polyacrylamides, in order to enhance the oil extraction (EOR). The viscosifying properties of polymers depend on their molecular masses. The aim of this thesis was therefore to develop new methods for determining wide molecular mass distributions and high dispersity, which are specific characteristics of samples of industrial interest in the oil sector. The analytical strategy used is based on size exclusion chromatography (SEC) and flow field flow fractionation (A4F), coupled to light scattering and refractometer. Model polymers were synthesized by a polymerization process by reversible addition fragmentation chain transfer (RAFT/MADIX). This allowed to obtain polymers in a range of molar masses between 103 and 107 g / mol and with a dispersity lower than 1.4. Through the use of these polymers, the capabilities and limitations of the two separation methods invested were evaluated. The A4F operating conditions were determined and this method has proved to be adapted to the analysis of industrial polymers distributed over 3 decades of molecular masses. Beyond the dimensional analysis, A4F-based coupling also allowed conformational analysis

Submitted by Milena Rubi (milenarubi@ufscar.br) on 2016-11-09T14:11:31Z No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5)
Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2016-11-09T14:11:44Z (GMT) No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5)
Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2016-11-09T14:11:50Z (GMT) No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5)
Made available in DSpace on 2016-11-09T14:11:57Z (GMT). No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5) Previous issue date: 2016-03-28
Não recebi financiamento
The need to obtain biomaterials in order to reduce environmental impacts has been the focus of research groups in recent years, and cellulose, a dominant component at most forms of plants is a promising resource because of its abundance. In order to improve the ability processing, the chemical modification of cellulose has been widely studied. Among the most important reactions of cellulose are: etherification, esterification, acetylation and oxidation; being cellulose acetate, viscose, nitrocellulose and cellulose ethers, the main cellulose derivatives. The chemical modification with isocyanates presents some unique properties, such as absence of by-products and chemical stability of the urethane group. In this work we were synthesized gels obtained by modified cellulose acetate (CA) with a degree of substitution (DS) 2,5 by crosslinking, with 4,4' - Diphenylmethane diisocyanate (MDI) in stoichiometry of 1:1, in homogeneous by varying the humidity and the homogenization time. For characterization were used the following techniques and tests: vibrational infrared absorption spectroscopy (Fourier Transform Spectrometer - FTIR), size exclusion Chromatography (SEC), molecular absorption spectrophotometry UV-VIS, density determining of the gels by pycnometry, determination of the coefficient swelling, determination of cross-links by Flory-Rehner theory, thermogravimetry (TG) and scanning electron microscopy (SEM). Crosslink density results showed that the gel synthesized in the absence of moisture suffered greater crosslinking with an average number of repeat units between the crosslinking points of about 1000 times lower. The potential applications of these gels were tested, by using than as stationary phase in size exclusion chromatography, having been assessed its efficiency in the fractionation and separation of natural and synthetic polymers. Results showed the effectiveness of the gel as stationary phase on separation of polymers, opening up a range of opportunities, taking into consideration the simplicity of the process and lower costs attributed to it.
A necessidade de se obter biomateriais na tentativa de reduzir impactos ambientais tem sido o foco de grupos de pesquisa nos últimos anos e, a celulose, um componente dominante na maioria das formas de plantas, é um recurso promissor devido à sua abundância. A fim de melhorar a capacidade de processamento, a modificação química da celulose tem sido amplamente estudada. Dentre as reações mais importantes da celulose estão: eterificação, esterificação, acetilação e oxidação; sendo o acetato de celulose, viscose, nitrocelulose e éteres de celulose, os principais derivados da celulose. A modificação química com isocianatos apresenta algumas propriedades únicas, como ausência de produtos secundários e estabilidade química do grupo uretano. Nesse trabalho foram sintetizados géis obtidos por meio da modificação de Acetato de Celulose (AC) com grau de substituição (GS) 2,5 através da reticulação com 4,4' - Difenilmetano Diisocianato (MDI), na estequiometria 1:1, em meio homogêneo, variando a umidade e o tempo de homogenização. Para caracterização foram utilizadas as seguintes técnicas e ensaios: espectroscopia vibracional de absorção no infravermelho por Transformada de Fourier (FTIR), cromatografia de exclusão por tamanho (SEC), espectrofotometria de absorção molecular UV-VIS, determinação de densidade dos géis por picnometria, determinação do coeficiente de intumescimento, determinação de ligações cruzadas pela teoria de Flory-Rehner, termogravimetria (TG) e microscopia eletrônica de varredura (MEV). Resultados da densidade de ligações cruzadas mostraram que o gel sintetizado na ausência de umidade sofreu uma maior reticulação, com um número médio de unidades de repetição entre os pontos de reticulação cerca de 1000 vezes menor. As aplicações potenciais desses géis foram testadas como fase estacionária em cromatografia de exclusão por tamanho, tendo sido avaliada sua eficiência no fracionamento e separação de polímeros naturais e sintéticos. Resultados mostraram a eficácia do gel como fase estacionária na separação de polímeros, abrindo uma gama de oportunidades, levando-se em consideração a simplicidade do processo e os baixos custos a ele atribuídos.

Les oligonucléotides antisens (ASO) ont la capacité d’inhiber ou de moduler l’expression d’un gène cible par différents mécanismes. La bioconjugation de l’ASO avec un lipide est une approche très prometteuse qui a montré une amélioration de la délivrance de la séquence antisens et par conséquent, de l’efficacité thérapeutique de l’oligonucléotide. Ces nouveaux agents thérapeutiques, les oligonucléotides lipidiques (LON), sont des molécules amphiphiles capables de s’auto-assembler sous forme d’objets supramoléculaires. Leur développement pharmaceutique requiert des méthodes analytiques adaptées pour évaluer la pureté des LON synthétisés et pouvoir les quantifier dans les formulations mais aussi pour caractériser les assemblages supramoléculaires formés.Dans ce travail, différentes méthodes ont été évaluées en chromatographie liquide (HPLC) à polarité de phase inversée par appariement d’ions et à interactions hydrophiles, en électrophorèse capillaire (CE) et en chromatographie d’exclusion stérique (SEC) pour l’analyse de LON de structures chimiques variées. Malgré les nombreux paramètres étudiés, l’asymétrie des pics obtenus en HPLC, limite son utilisation à des fins de dosage. En revanche, une méthode quantitative a été développée et validée en CE en présence de cyclodextrines (CD). La constante de complexation entre le LON libre et les CD ainsi que la mobilité électrophorétique du complexe ont été déterminées. Enfin, le potentiel de la SEC et de la CE pour la caractérisation des objets supramoléculaires de LON a été évalué
Antisense oligonucleotides (ASO) have the ability to inhibit or modulate the expression of a target gene by various mechanisms. Bioconjugation of ASO with a lipid is a very promising approach which has shown an improvement in the delivery of the antisense sequence and therefore, in the therapeutic efficacy of the oligonucleotide. These new therapeutic agents, lipid oligonucleotides (LON), are amphiphilic molecules and are able to self-assemble to form supramolecular objects. Their pharmaceutical development requires suitable analytical methods to study the purity of the LONs synthesized and to be able to quantify them in the formulations but also to characterize the supramolecular assemblies formed.In this work, different methods were investigated in ion-pairing reversed-phase HPLC and hydrophilic interaction chromatography, capillary electrophoresis (CE) and size exclusion chromatography (SEC) for LON analysis with various chemical structures. Despite the different parameters studied, the asymmetry of the peaks obtained by HPLC limits its use for assays. On the other hand, a quantitative method has been developed and validated in CE in the presence of cyclodextrins (CD). The complexation constant between free LON and CDs as well as the electrophoretic mobility of the complex were determined. Finally, the potential of SEC and CE for the characterization of supramolecular objects of LON was assessed