Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 N·s·m-2 and elastic shear moduli of ~ 105 N·m-2. Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli. Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn ≥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn ≥ 0.7). These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces. / Ph. D.

Polysaccharides are one of the most abundant and diverse families of natural polymers, and have an incredibly wide range of natural functions including structural reinforcement, energy storage, aqueous rheology modification, and communication and identity. Application of native polysaccharides like cellulose as sustainable materials is limited by some inherent drawbacks such as insolubility in common solvents including water, and poor dimensional stability. To increase their functionality and utility, researchers have sought to tailor the chemical and physical properties of cellulose and other polysaccharides using a variety of chemical modification techniques, resulting in a number of important, useful commercial derivatives. Because of their greater biocompatibility and biodegradability, and low immunogenicity, naturally derived cationic polymers including cationic polysaccharide derivatives are very attractive candidates for biomedical applications, due to the fact that they are capable of binding with anionic biomolecules, such as nucleic acids and certain proteins, via electrostatic interactions. However, there are relatively few practical synthetic methods reported for their preparation. We demonstrated a useful and efficient strategy for cationic polysaccharide salt preparation by reaction of 6-bromo-6-deoxypolysaccharides such as 6-bromo-6-deoxycellulose esters with pyridine or 1-methylimidazole exclusively at the C-6 position, resulting in high degrees of substitution (DSs). These permanently cationic polysaccharide derivatives have been demonstrated to dissolve readily in water, and bind strongly with a hydrophilic and anionic surface. Availability of these cationic polysaccharides will facilitate structure-property relationship studies for biomedical uses including drug delivery and bioelectronics applications. We also extended the chemistry, reacting 6-imidazolo-6-deoxycellulose with propane sultone, leading to a new synthetic pathway to zwitterionic cellulose derivatives. In addition to cationic and zwitterionic derivatives, we found a simple, efficient route to carboxyl-containing polysaccharide derivatives from curdlan esters via regioselective ring-opening reactions catalyzed by triphenylphosphine (Ph3P) under mild conditions. Curdlan, a polysaccharide used by the food industry and in biomedical applications, was employed as starting material for preparing these carboxyl-containing derivatives by a reaction sequence of bromination, azide displacement and ring-opening reaction with cyclic anhydrides, affording high conversions. These modification techniques have been demonstrated to display essentially complete regio- and chemo-selectivity at C-6. These novel polysaccharide-based materials starting from abundant and inexpensive curdlan are promising for some applications such as amorphous solid dispersion (ASD) oral drug delivery. / Ph. D. / Polysaccharides are chains of natural sugars. They constitute one of the most abundant and diverse families of natural polymers (polymers are chains of small molecules, and polysaccharides are a class of polymers), and in nature polysaccharides play an incredibly wide range of functions such as structural reinforcement, energy storage, changing the viscosity of solutions of things in water, and communication. Cellulose, a polymer comprising long chains of linked glucose molecules, may be the most abundant natural polysaccharide on earth. Application of native cellulose as a sustainable material is limited by its inability to dissolve in water or commonly used organic solvents, poor dimensional stability, inability to melt and flow when heated, and the fact that it degrades when exposed to the environment. In order to increase its functionality and utility, a number of research groups have tried to tailor the chemical and physical properties of things made from cellulose (cellulose “derivatives”) using various chemical modification techniques, resulting in some important, useful commercial cellulose derivatives. The Edgar group, in the recent years has developed a series of new techniques to synthesize various cellulose derivatives for effective oral drug delivery. We have demonstrated that these cellulose derivatives are capable of preventing drugs from forming insoluble crystals, meanwhile protecting the drugs from the harsh environment of the stomach. As a result, these formulations based on cellulose derivatives enhance the solubility of drugs in the digestive tract, and the ability of the drug to permeate to the blood stream, thereby enhance distribution to the parts of the body where it is needed, is enhanced as well. Cellulose- and other polysaccharide-based polyelectrolytes are very attractive candidates for biomedical and therapeutical applications. However, currently, the set of commercially available cellulose derivatives is limited in number and diversity, and contains no positively charged derivatives. This dissertation focuses on the development of new ways to make charged polysaccharide derivatives using chemical modification of cellulose, cellulose esters, and other polysaccharides. Unlike conventional methods which require harsh reaction conditions or metal catalysts, the new approaches in this dissertation offer simple and efficient ways to make a wide variety of charged derivatives of cellulose or other polysaccharides under mild conditions. Availability of these polysaccharide-based charged polymers will help us design more useful, economical materials for biomedical, pharmaceutical, and other applications including gene or drug delivery, oral delivery of potent and selective protein drugs, agricultural applications, and coatings.

The composition of the non-starch polysaccharides in sweet potato was determined and their flatulence-inducing potential evaluated. A preparative adaptation of the AOAC enzymatic method was used to isolate the insoluble and soluble non-starch polysaccharides. The insoluble non-starch fraction was mainly cellulose, but contained a substantial amount of hemicellulosic glucose, suggesting the presence of an independent glucan fraction. Relatively small amounts of xylose, galactose, arabinose, mannose, rhamnose and fucose were present. Galactose was the predominant sugar in the soluble non-starch polysaccharide fraction, although substantial quantities of arabinose and mannose were also present. Xylose, rhamnose, glucose and fucose were also present in small quantities. Uronic acids also made up a substantial portion of the soluble non-starch polysaccharides. Appreciable protein and ash were present in both the insoluble and soluble NSP fractions. The in vitro test for gas production using Clostridium perfringens as a test organism showed that both the insoluble and soluble non-starch polysaccharides are potential flatulence-inducers in sweet potato. However, the presence of the hemicellulosic glucans in the insoluble fraction make the latter a more likely candidate for flatus-inducer. / Master of Science

The relationship between substituent chemistry and melt behavior for xylan derivatives was investigated by differential scanning calorimetry and parallel-plate dynamic viscometric measurements. Xylan esters exhibit characteristic Tg values which decrease with increasing size of substituent. However, these materials do not flow at temperatures well above Tg. The ether derivative hydroxypropyl xylan was found to flow at substitution levels higher than 0.5 degree of substitution, with melt viscosity decreasing as the degree of substitution increased. The influence of viscosity ratio and composition on the texture of melt-blends of hydroxypropyl cellulose and polystyrene was studied for viscosity ratios of 0.08 to 0.55. Blends were examined by SEM and TEM. It was found that composition was the determining factor in texture, and not viscosity ratio over the range tested. Extrusion problems limited the range of compositions tested to 40% biopolymer or less. Phase inversion was not observed, although inversion was expected for some samples based on predictive models in the literature. / Master of Science

Calcified skeletons are produced within complex assemblages of proteins and polysaccharides whose roles in mineralization are not well understood. Researchers have long postulated that living organisms utilize the macromolecules of organic matrices to actively guide the formation of crystal structures. The timing and placement of the subsequent minerals that form are most easily controlled during nucleation; however, a physical and chemical picture of how organic functional group chemistry influences the initial stages of nucleation is not yet established. These processes are further complicated by the realization that carbonate biominerals can form by an amorphous to crystalline transformation process, which has prompted the question of how chemical signatures are recorded during mineralization. Investigations of mineralization processes such as the kinetics of nucleation and the transformation of amorphous calcium carbonate (ACC) to crystalline products are critical to building a better understanding of biomineral formation. Only from that fundamental basis can one begin to decipher changes in climate and seawater chemistry over geologic time and by recent anthropogenic effects. This dissertation presents the findings from experimental studies of the thermodynamics and kinetics of multiple mineral formation processes, including nucleation and transformation from an amorphous phase. The kinetics of calcite nucleation onto a suite of high-purity polysaccharide (PS) substrates were quantified under controlled conditions. Nucleation rates were measured as a function of 1) supersaturation extending above and below ACC solubility and 2) ionic strength extending to seawater salinity. These conditions decipher the chemical interactions between the PS substrate, calcite crystal, and solution. These investigations show the energy barrier to calcite formation is regulated by competing interfacial energies between the substrate, crystal, and liquid. The energy barriers to nucleation are PS-specific by a systematic relationship to PS charge density and substrate structure that is rooted in minimization of the competing substrate-crystal and substrate-liquid interfacial energies. The data also suggest ionic strength regulates nucleation barriers through substrate-liquid and crystal-liquid interfacial energetics. In a second experimental study, stable isotope labeling was used to directly probe the transformation pathway. Four processes were considered: dissolution-reprecipitation, solid-state, or combinations of these end member processes. Isotope measurements of calcite crystals that transform from ACC have signatures that are best explained by dissolution-reprecipitation. The extent of isotopic mixing correlates with the amount of ACC transferred and the time to transformation, suggesting the calcite crystals are recording the changing local solution environment during the transformation. These investigations into different mineralization mechanisms build a framework for how functional group chemistries of organic molecules regulate mineralization and the resulting isotopic and elemental signatures in the calcite. This may provide useful insights to interpreting chemical signatures of carbonate biominerals in fossil record and understanding ocean chemistry changes throughout geologic time. / Ph. D.

Two chemical methods, multi-reducing end modification and bleach oxidation, were used to prepare polysaccharide aldehydes and ketones. Their derived hydrogels and microgels were made for potential drug-delivery applications. Polysaccharide aldehydes and ketones are reactive intermediates for adding other functional moieties through chemo selective reactions such as Schiff-base formation or reductive amination. The most widely used method to prepare polysaccharide aldehydes is periodate oxidation. However, this method impacts higher-order polysaccharide structure, decreases degree of polymerization (DP), and increases polysaccharide instability, leading to degraded mechanical properties. Developing a new method to prepare polysaccharide aldehydes while preserving DP, stability, and desirable physical properties is challenging. Inspired by the reactive reducing ends of polysaccharides, which are the anomeric carbons (at the chain end), one per natural polysaccharide molecule, that (for aldose-based polysaccharides) is in equilibrium between a ring-closed hemiacetal and an open-chain aldehyde form, we developed a novel method to prepare polysaccharide aldehydes by attaching monosaccharides to polysaccharide chains. Herein, we describe the approach of attachment through amination between amine group at the C2 position of the monosaccharide and carboxylic acid groups on polysaccharides. In this way, more reducing ends (C1 of the monosaccharide) can be introduced to the polysaccharides. We have chosen to call this new family of polysaccharides "multi-reducing end polysaccharides (MREPs)". We call this method "multi-reducing end modification". We then fabricated injectable, self-healing, fast gelling Schiff base hydrogels based on MREPs. Previous methods to fabricate Schiff base polysaccharide hydrogels usually required periodate oxidation which leads to degraded mechanical properties, with gelation time typically from minutes to hours. We employed acetic acid to induce fast gelation of our MREPs hydrogels within seconds. The Schiff base MREP hydrogels exhibited self-healing and injectable behavior with limited cytotoxicity, which is promising for future biomedical applications such as targeted drug delivery or tissue engineering. Microgels are dispersible but undissolvable colloids of three-dimensional polymer networks with numerous applications. We synthesized all-polysaccharide microgels (herein, we use the general term "microgels" to describe small gel particles of nanometer to micron diameters) using oxidized hydroxypropyl cellulose (Ox-HPC), carboxymethyl chitosan (CMCS), and calcium chloride. By tuning the calcium concentration, uniform microgels can be obtained with gel size in the hundreds of nanometers. Model amine-containing drugs such as picloram or p-aminobenzoic acid (pABA) can be chemically attached to Ox-HPC through Schiff base chemistry, creating imine bonds that are reversible in water, thereby permitting slow release. This class of all-polysaccharide microgels showed promising applications in agriculture, such as controlled release of agrochemicals. We anticipated that these strategies would benefit future polysaccharide chemistry research and permit synthesis of novel hydrogel or microgel systems with potential drug-delivery applications. / Doctor of Philosophy / Polysaccharides are long chains composed of sugar units ("sugar polymers"). Many natural-derived polysaccharides are sustainable, biodegradable and have low toxicity. Hydrogels are composed of porous solids and water, similar to the structure of human tissues. "Microgels" are used herein to describe small gels of nanometer to micron diameters. Fabrication of polysaccharides into hydrogels or microgels can be advantageous for drug-delivery applications. Chemical modification of polysaccharides is usually required before making polysaccharide-based hydrogels or microgels. However, previously described methods usually destroy the chemical structure of polysaccharides and cause degradation. To overcome this challenge, we developed a non-destructive chemical modification method to prepare hydrogels without these disadvantages. This method also introduced a new concept in polysaccharide science. Following our novel chemical modification method, polysaccharide-based hydrogels were made. Compared to the previous polysaccharide hydrogels which usually required long gelation times, our polysaccharide hydrogels gel within seconds with addition of tiny amounts of vinegar. Besides, our polysaccharide-based hydrogels are injectable and spontaneously repair themselves with low toxicity to cells. These properties make our hydrogels promising for cancer-targeted drug delivery. Food is the first necessity of human beings. Pesticides are often used in excessive amounts and in broad distribution, to guarantee high crop productivity. Excess use and/or distribution of pesticides can pollute to the environment and pose threats to human health. To solve this problem, we made all polysaccharide microgels, dispersed in benign water, that can permit slow release of pesticides, applied in a form that can promote great precision. Overall, we developed new ways to modify polysaccharides to create effective and harmless hydrogels or microgels. We aim to push the boundaries of science and benefit human society through our research.

Ce projet visait à mesurer l’effet de l’enrichissement en polysaccharides d’une matrice laitière sur son comportement rhéologique en digestion in vitro. Le comportement rhéologique de solutions de protéines laitières (3,9%), de gomme xanthane (0,7%), de gomme de caroube (0,7%) et de leurs mélanges, acidifiés par HCl sous agitation, a été suivi durant une digestion in vitro. Le comportement de solutions de poudre de lait écrémé (3,5 et 4% de protéines) enrichies des mêmes polysaccharides (0,7%) et fermentées a aussi été suivi en digestion in vitro. Le mode d’acidification affectait le comportement rhéologique des solutions contenant des protéines. La viscosité des solutions a diminué en cours de digestion, à l’exception des solutions contenant protéines et gomme xanthane dont la viscosité augmentait de l’étape gastrique à l’étape intestinale. L’ajout de polysaccharides à une matrice laitière permettrait donc de moduler sa viscosité lors de la digestion et pourrait ainsi procurer des bénéfices physiologiques. / This project aims to measure the effect of enrichment in polysaccharides of a dairy matrix on its rheological behaviour when subjected to in vitro digestion. The rheological behaviour of solutions containing milk protein (3,9%), xanthan gum (0,7%), carob gum (0,7%) and their binary or ternary mixtures, acidified by HCl with stirring, was followed during in vitro digestion. The rheological behavior of solutions of skimmed milk powder (3,5% and 4% of protein) enriched or not with same polysaccharides (0,7%) and fermented has also been followed during in vitro digestion. The mode of acidification affected the rheological behavior of solutions containing proteins. The viscosity of the acidified and fermented solutions decreased throughout digestion, except for the protein solutions containing xanthan gum for which viscosity increased at the transition from gastric to intestinal stage. Integration of polysaccharides to a dairy matrix would, therefore, modulate its viscosity during digestion and could provide physiological benefits.

Les nutriments des matrices alimentaires doivent être bioaccessibles puis biodisponibles pour atteindre les organes vitaux et être métabolisés. Les protéines laitières (caséines et protéines de lactosérum) ont une valeur nutritionnelle importante par leur composition en acides aminés (AA) essentiels et en acides aminés branchés qui ont un impact sur plusieurs réponses métaboliques postprandiales. Ces protéines ont aussi un rôle technologique essentiel dans le processus de fabrication des yogourts. Des polysaccharides (PS) peuvent être ajoutés dans les yogourts pour les stabiliser. Des études précédentes ont montré que des amidons, des pectines (PS communs utilisés par les industriels laitiers) et des β-glucans (stabilisants d’intérêt comme fibre soluble) pouvaient moduler la digestion des protéines (in vitro et in vivo) et certaines réponses métaboliques postprandiales. Un modèle de digestion gastro-intestinale (GI) in vitro a été mis au point pour analyser la digestion des protéines laitières de yogourts standardisés variant par leur composition en PS (sans PS, 0.75 % d’amidon, 0.4 % de β-glucan, 0.2 % de pectine). Les formulations ont montré des viscosités différentes dans des conditions de cisaillement contrôlées représentant les conditions GI. La bioaccessibilité des AA a été différente selon le PS ajouté et il n’y avait pas de corrélation avec la viscosité du yogourt. Une étude in vivo chez l’animal réalisée avec les mêmes yogourts a aussi montré un impact de l’ajout de PS de nature différente sur la biodisponibilité de certains AA et sur certaines réponses métaboliques. En particulier, l’ajout d’amidon, par rapport au yogourt sans PS, a significativement diminué la vidange gastrique et la concentration plasmatique de Phe, un AA associé à l’incidence du diabète de type 2. Quinze minutes après l’ingestion du yogourt avec β-glucan, la glycémie a significativement diminué et la clairance à l’insuline a augmenté par rapport aux autres yogourts. Ces résultats ont permis de mettre en évidence l’intérêt d’étudier l’impact de la composition de la matrice alimentaire sur son intégrité et sur la cinétique de libération des nutriments azotés dans l’appareil GI, et sur des réponses métaboliques associées pouvant ici être bénéfiques aux diabétiques. / Nutrients of food matrixes have to be bioaccessible and bioavailable to reach vital organs and be metabolized. Dairy proteins (caseins and whey proteins) possess an important nutritional value because of their high content in essential and branched amino acids (AA) affecting postprandial metabolic responses. These proteins have an essential technical function in yogurt process. Polysaccharides (PS) can be added in yogurts as stabilizers. Previous studies showed that starches, pectins (common PS used by the dairy industry), and β-glucans (stabilizers of interest as soluble fiber) modulated protein digestion (in vitro and in vivo) and postprandial metabolic responses. An in vitro gastrointestinal (GI) model was developed to analyze protein digestion in standardized dairy products with different PS composition (without PS, starch 0.75%, β-glucan 0.4%, pectin 0.2%). The formulations showed different viscosities under controlled shear representative to GI conditions. AA bioaccessibility was different depending on the added PS but was not related to the yogurt viscosity. The in vivo study, realised with animal model, using the same yogurts showed different impact of added PS on AA bioavailability and on metabolic responses. In particular, in comparison to the control yogurt the starch supplementation decreased significantly the gastric emptying and the plasmatic Phe concentration, an AA associated with incidence of type 2 diabetes. Fifteen minutes after yogurt with β-glucan consumption, glycemia decreased significantly and insulin clairance increased in comparison to other yogurts. These results show the interest to study the impact of food matrix composition on matrix integrity and on release kinetics of nitrogenous nutrients in GI tractus, and on associated metabolic responses here beneficial for diabetic subjects.

Les fucanes, ou fucoïdanes, sont des polysaccharides caractéristiques des algues brunes comme Saccharina longicruris. Leur hydrolyse par des enzymes bactériennes amplifie leurs activités biologiques. Cependant, peu d'enzymes capables d'hydrolyser les fucoïdanes ont été découvertes et leurs actions sont spécifiques à la structure de chaque fucoïdane. Le but de ce projet est d'identifier des bactéries marines capables d'hydrolyser le fucoïdane. Des bactéries fucosidase positives ont d'abord été isolées par des préenrichissements à partir d'échantillons d'algues et d'eau de mer prélevés dans le fleuve St-Laurent (Ste-Luce, mai 2007) et sélectionnées par une réaction chromogénique. Le séquençage d'une section de la petite sous-unité du gène d'ADNr 16S a révélé que les bactéries sélectionnées appartenaient à Pseudoalteromonas spp. La cinétique de croissance d'une souche de Pseudoalteromonas dans un milieu à base d'eau de mer (Marine Broth) avec ou sans les différents sucres composant le fucoïdane (glucose, fucose ou galactose) n'a pas révélé de différence entre les milieux testés. Le dosage des activités enzymatiques de type fucosidase et fucoïdane hydrolase des extraits bruts de biomasse a été déterminé par la mesure de la libération du nitrophénol lors de l'hydrolyse du PNP-oc-L-fucoside et par le dosage des sucres réducteurs, respectivement. L'activité spécifique de la fucosidase, optimisée à 20°C, est d'environ 0,014 U/mg et elle est constante dans le temps. Celle de la fucoïdane hydrolase, atteint sa valeur maximale de 0,67 U/mg après une heure d'incubation et est optimale à 50°C. Ce travail a permis l'isolement d'une bactérie productrice de deux enzymes capables de dégrader les fucoïdanes, soit la fucosidase et la fucoïdane hydrolase. Les gènes codant pour ces enzymes pourront ultérieurement être identifiés et servir au développement d'un procédé commercial de fractionnement du fucoïdane par bioconversion.

Chan Pui Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 98-104). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.i / LIST OF FIGURES --- p.iv / LIST OF TABLES --- p.vii / ABBREVIATIONS --- p.viii / Chapter Chapter one --- Research Background / Chapter 1.1 --- Carbohydrates --- p.2 / Chapter 1.2 --- Impact of molecular weight of polysaccharides --- p.5 / Chapter 1.3 --- Molecular Weight Determination of polysaccharides --- p.6 / Chapter 1.3.1 --- Laser Scattering --- p.6 / Chapter 1.3.2 --- Gel Permeation Chromatography --- p.7 / Chapter 1.3.3 --- Mass spectrometry --- p.9 / Chapter 1.4 --- Matrix assisted laser desorption/ ionization (MALDI) --- p.10 / Chapter 1.4.1 --- Laser desorption --- p.10 / Chapter 1.4.2 --- Matrix-assisted laser desorption / ionization (MALDI) --- p.11 / Chapter 1.5 --- MALDI-TOFMS analysis of polymers --- p.14 / Chapter 1.6 --- Outline of the present work --- p.16 / Chapter Chapter two --- Experimental and Instrumentation / Chapter 2.1 --- Matrix-assisted laser desorption/ ionization Time of flight Mass Spectrometry (MALDI-TOFMS) --- p.18 / Chapter 2.2 --- Delayed extraction --- p.20 / Chapter 2.3 --- Time of flight mass spectrometry (TOFMS) --- p.20 / Chapter 2.3.1 --- Linear time-of-flight mass spectrometry --- p.20 / Chapter 2.3.2 --- Reflectron --- p.21 / Chapter 2.4 --- Instrumentation --- p.23 / Chapter 2.4.1 --- Laser system --- p.24 / Chapter 2.4.2 --- Ion source --- p.26 / Chapter 2.4.3 --- Ion deflection --- p.26 / Chapter 2.4.4 --- Detection --- p.27 / Chapter 2.4.5 --- Reflector --- p.27 / Chapter 2.4.6 --- Data acquisition --- p.29 / Chapter 2.5 --- Experimental --- p.29 / Chapter 2.5.1 --- Sample preparation --- p.29 / Chapter 2.5.2 --- Calibration --- p.33 / Chapter 2.6 --- Data analysis --- p.33 / Chapter Chapter three --- Use of ammonium fluoride as co-matrix / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Results and discussion --- p.37 / Chapter 3.2.1 --- Effect of co-matrix --- p.45 / Chapter 3.2.2 --- Effect of sample preparation --- p.49 / Chapter 3.2.3 --- Analysis of dispersed dextran --- p.52 / Chapter 3.3 --- Conclusion --- p.55 / Chapter Chapter four --- Effect of sample preparation / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Experimental --- p.57 / Chapter 4.2.1 --- Sample preparation --- p.57 / Chapter 4.3 --- Results and discussion --- p.59 / Chapter 4.4 --- Conclusion --- p.71 / Chapter Chapter five --- Development of liquid matrix systems / Chapter 5.1 --- Introduction --- p.73 / Chapter 5.2 --- Experimental --- p.75 / Chapter 5.2.1 --- Sample preparation --- p.75 / Chapter 5.3 --- Results and discussion --- p.76 / Chapter 5.3.1 --- Formulation of matrix solutions --- p.76 / Chapter 5.3.2 --- Use of liquid matrix system --- p.87 / Chapter 5.3.3 --- Analysis of dispersed dextran --- p.90 / Chapter 5.4 --- Conclusion --- p.93 / Chapter Chapter six --- Conclusion / Chapter 6.1 --- Conclusion --- p.95 / References --- p.98 / Appendix / Appendix 1 Chemical structure of matrices / Appendix 2 Chemical structure of solubilizing agents / Appendix 3 Chemical structure of liquid supports / Appendix 4 Chemical structure of additives