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O uso de DNA plasmidial (pDNA) visando a aplicações terapêuticas tem aumentado nos últimos anos. A cromatografia aparece como a técnica de purificação mais comum para obtenção de amostras de pDNA com o elevado grau de pureza exigido. Porém, as resinas cromatográficas disponíveis apresentam ainda uma série de desafios, nomeadamente no desenvolvimento de ligantes específicos e matrizes capazes de acomodar este tipo de molécula. Relativamente à apuração de novos ligantes, alguns estudos têm mostrado o potencial do aminoácido arginina para estabelecer interações específicas e preferenciais com o pDNA. Por outro lado, resinas monolíticas surgem como suportes interessantes devido às suas excelentes propriedades de transferência de massa e altas capacidades de adsorção. Neste estudo, diferentes ligantes baseados em arginina (arginina, di-arginina e tri-arginina) foram imobilizados em resinas de agarose previamente ativadas. Um primeiro estudo de adsorção em batelada foi realizado a fim de avaliar e compreender os mecanismos envolvidos no processo de adsorção dos ácidos nucleicos pDNA e RNA em resina com o aminoácido arginina. Na sequência, apresentamos uma proposta inovadora para o uso de ligantes de arginina em resinas de agarose, em um único passo de purificação em modo negativo a seguir ao passo de concentração por isopropanol. A capacidade da resina para o pDNA foi substancialmente maior do que a obtida para o mesmo tipo de resina no modo positivo, com notória vantagem de capacidade no uso de di-arginina face a arginina com rendimentos próximos de 100% do plasmídeo carregado. Os ligantes di-arginina e tri-arginina foram também imobilizados em resinas monolíticas. Em comparação com o aminoácido arginina, a imobilização dos homopeptídeos nas resinas monolíticas levou ao aumento da capacidade de adsorção (cerca de 2,5 vezes superior) e especicificidade de interações, mostrando-se como uma estratégia promissora para processos de purificação de pDNA. / The use of plasmid DNA (pDNA) for therapeutic applications has increased in recent years. Chromatography appears as the most common purification technique to obtain samples of pDNA with the high degree of purity required. However, the available chromatographic resins still present a series of challenges, namely in the development of specific ligands and matrices capable of accommodating this type of molecule. Regarding the determination of new ligands, several studies have shown the potential of the arginine amino acid to establish specific and preferential interactions with the pDNA. On the other hand, monolithic resins appear as interesting approaches due to their excellent mass transfer properties and high adsorption capacities. In this study, different arginine based ligands (arginine and di-arginine) were firstly immobilized on activated agarose resins. The first part of the work describes the adsorption equilibrium of plasmid DNA adsorption process, as well as the interaction with its main impurity (RNA) on arginine supports in a batch format, in order to compare and gather crucial information about adsorption mechanisms involved in this type of affinity system. Then, a new use for chromatographic bead matrixes based on arginine ligands was proposed, working as an adsorption matrix pDNA purification in negative mode after isopropanol concentration of the sample. The arginine based supports capacity for pDNA under negative mode for pDNA was substantially higher than that obtained with the same type of resin in the conventional positive mode, with a notable advantage of using di-arginine with recovery yields near 100%. The homopeptides (di-arginine and tri-arginine) were also immobilized on functionalized monolithic resins (BIA Separations, Slovenia). Effectively, the immobilization of the arginine homopeptides made the monolithic resins more functional compared to the (mono)arginine based resin, exhibiting greater binding capacities (around 2,5 times higher) and interaction intensities, proving to be a promising strategy for purification processes of pDNA.

Microfluidics is a vibrant and expanding field that has the potential for solving many analytical challenges. Microfluidics shows promise to provide rapid, inexpensive, efficient, and portable diagnostic solutions that can be used in resource-limited settings. Microfluidic devices have gained immense interest as diagnostic tools for various diseases through biomarker analysis. My dissertation work focuses on developing electrokinetically operated integrated microfluidic devices for the analysis of biomarkers indicative of preterm birth risk. Preterm birth (PTB), a birth prior to 37 weeks of gestation, is the most common complication of pregnancy and the leading cause of neonatal deaths and newborn illnesses. In this dissertation, I have designed, fabricated and developed several microfluidic devices that integrate various sample preparation processes like immunoaffinity extraction, preconcentration, fluorescent labeling, and electrophoretic separation of biomarkers indicative of PTB risk. I developed microchip electrophoresis devices for separation of selected PTB biomarkers. I further optimized multiple reversed-phase porous polymer monoliths UV-polymerized in microfluidic device channels for selective retention and elution of fluorescent dyes and PTB biomarkers to facilitate on-chip labeling. Successful on-chip fluorescent labeling of multiple PTB biomarkers was reported using these microfluidic devices. These devices were further developed using a pH-mediated approach for solid-phase extraction, resulting in a ~50 fold enrichment of a PTB biomarker. Additionally, this approach was integrated with microchip electrophoresis to develop a combined enrichment and separation device that yielded 15-fold preconcentration for a PTB peptide. I also developed an immunoaffinity extraction device for analyzing PTB biomarkers directly from a human serum matrix. A glycidyl methacrylate monolith was characterized within microfluidic channels for immobilization of antibodies to PTB biomarkers. Antibody immobilization and captured analyte elution protocols were optimized for these monoliths, and two PTB biomarker proteins were successfully extracted using these devices. This approach was also integrated with microchip electrophoresis for combined extraction and separation of two PTB biomarkers in spiked human serum in <30 min. In the future, these optimized microfluidic components can be integrated into a single platform for automated immunoaffinity extraction, preconcentration, fluorescent labeling, and separation of PTB biomarkers. This integrated microfluidic platform could significantly improve human health by providing early diagnosis of PTBs.

Du fait du coût élevé de certains matériaux adsorbants d’intérêt pour le traitement de la micropollution organique, l’étude a porté sur la régénération de matériaux adsorbants de type zéolithes hydrophobes et monolithe de carbone dans le cas de l’adsorption du bisphénol A et du diclofénac comme micropolluants réfractaires. Des procédés d’oxydation avancée impliquant des espèces radicalaires HO• (réaction de Fenton, électro-Fenton) et SO₄• – (activation de persulfate par voie thermique) ont été utilisés pour assurer la régénération des matériaux par désorption et dégradation oxydative des polluants fixés. La production de radicaux HO• au sein de la phase aqueuse circulant au niveau de l’adsorbant n’est pas suffisamment efficace pour sa régénération. Il a donc été envisagé de générer les radicaux au plus près des molécules adsorbées. Au cours de ce travail, une méthode sensible d’analyse par polarographie de H₂O₂ a été développée et validée pour le suivi des expériences avec les procédés mettant en jeu la réaction de Fenton. Pour différentes zéolithes, le catalyseur de la réaction de Fenton à base de fer a été incorporé préalablement dans la zéolithe. Pour le monolithe de carbone, les propriétés de conduction du matériau ont été mises à profit en l’utilisant comme cathode pour l’application du procédé électro-Fenton permettant de produire les radicaux HO• directement au sein du matériau. Cela a conduit à améliorer les performances de la régénération avec toutefois une diminution de son efficacité au cours de cycles successifs adsorption/régénération. / The elimination of organic micropollutants often requires the use of adsorption processes among the water treatments. The aim of our study is to regenerate two expensive materials (hydrophobic zeolites and carbon monoliths) to increase their life expectancy and decrease their investing cost. Two organic contaminants were targeted : diclofenac and bisphenol A, which are refractory pollutants. Advanced oxidation processes involve radical species, HO• (Fenton and electro-Fenton reactions) and SO₄• – (thermal activation of persulfate ion). These oxidants were used to decompose the adsorbed pollutants and thus regenerate the adsorbents. The HO• production, within the core of aqueous phase, did not reach satisfactory regeneration, and a loss of adsorption capacity was observed. Furthermore, during this study, a sensitive polarographic analytical method was developed and validated for the quantification of H₂O₂ in the aqueous phase. This method was used to follow in situ the Fenton reaction. The location of the catalyst in a closer vicinity of the adsorbed species was then optimized and the iron catalyst was impregnated in the host, prior to the adsorption, on different types of hydrophobic zeolites. Concerning carbon monolith, the electro-Fenton process was carried out using the material as the cathode thanks to its electrical conductivity. Consequently, HO• are produced in the porosity of monolith. This latter property enhanced the degradation of adsorbed solutes. The overall performances were increased compared to the homogeneous Fenton process. Nonetheless, a decrease of the adsorption capacities with adsorption-regeneration cycles was observed.

Sample preparation is often a bottleneck in systems for chemical analysis. The aim of this work was to investigate and develop new techniques to address some of the shortcomings of current sample preparation methods. The goal has been to provide full automation, on-line coupling to detection systems, short sample preparation times and high-throughput. In this work a new technique for sample preparation that can be connected on-line to liquid chromatography (LC) and gas chromatography (GC) has been developed. Microextraction in packed syringe (MEPS) is a new solid-phase extraction (SPE) technique that is miniaturized and can be fully automated. In MEPS approximately 1 mg of sorbent material is inserted into a gas tight syringe (100-250 μL) as a plug. Sample preparation takes place on the packed bed. Evaluation of the technique was done by the determination of local anaesthetics in human plasma samples using MEPS on-line with LC and tandem mass spectrometry (MS-MS). MEPS connected to an autosampler was fully automated and clean-up of the samples took about one minute. In addition, in the case of plasma samples the same plug of sorbent could be used for about 100 extractions before it was discarded. A further aim of this work was to increase sample preparation throughput. To do that disposable pipette tips were packed with a plug of porous polymer monoliths as sample adsorbent and were then used in connection with 96-well plates and LC-MS-MS. The evaluation of the methods was done by the analysis of local anaesthetics lidocaine and ropivacaine, and anti-cancer drug roscovitine in plasma samples. When roscovitine and lidocaine in human plasma and water samples were used as model substances, a 96-plate was handled in about two minutes. Further, disposable pipette tips may be produced at low cost and because they are used only once, carry-over is eliminated.

La nanotechnologie est devenue un domaine clé de la technologie du XXIe siècle. L’important développement des approches pour la synthèse des nanoparticules (NPs) avec une composition, une taille et une forme désirées rend compte du potentiel de leur utilisation comme « blocs de construction » pour des structures de plus grande échelle. Cela permet d’envisager à la fois la fabrication de matériaux fonctionnels et de dispositifs directement à partir de colloïdes par approche ascendante et la conception de matériaux sur plusieurs échelles de grandeur. Le procédé utilise l'assemblage ou l'auto-assemblage de NPs et conduit à des matériaux avec des architectures différentes notamment 1D (bâtonnets), 2D (films) ou 3D (super-réseaux ou gels). Cependant, la plupart des assemblages 3D sont limités à l'échelle micrométrique et sont difficiles à contrôler. Pratiquement, la seule voie permettant la préparation de structures 3D macroscopiques à partir de NPs est la gélification et la préparation d'aérogels. Une voie alternative consiste à disperser les NPs dans une matrice, conduisant ainsi à un matériau composite massif, avec des NPs non agrégées distribuées de manière homogène.Le présent travail est consacré au développement de matériaux à partir de NPs d'oxydes métalliques (principalement Y3Al5O12: Ce et Li4Ti5O12) de différentes dimensions et pour diverses applications. La première partie de ce travail décrit la synthèse de NPs de YAG: Ce et de LTO par approche glycothermale. Dans le cas du YAG: Ce, les conditions de réaction ont été ajustées de façon appropriée pour obtenir des nanocristaux (NCs) non agrégés de quelques nanomètres. Des solutions colloïdales de différentes concentrations contenant de tels NCs ont été utilisées, pour la fabrication par la technique de « spin-coating », de films minces avec une épaisseur contrôlable. A l’inverse, la synthèse de LTO conduit à des NPs agrégées dans une structure hiérarchique très bénéfique pour les batteries au lithium. La grande surface spécifique et la porosité du matériau obtenu assurent en effet un échange efficace des ions lithium entre l'électrolyte et le matériau d'anode.Par ailleurs, les NCs de YAG: Ce ont été utilisés pour la préparation de matériaux monolithiques de grande taille avec une porosité et une transparence élevées. Pour cela, la solution colloïdale de NCs a été gélifiée par le changement brusque de la constante diélectrique du solvant de dispersion des NCs. Les gels ainsi obtenus ont été par la suite séchés de manière supercritique, donnant ainsi des aérogels à base de NPs de YAG:Ce, avec une porosité et une transparence élevées. La même approche s'est avérée appropriée pour d'autres systèmes à base de NPs de GdF3 ou de mélanges de NPS de YAG: Ce et de GdF3.Alternativement, les NPs de YAG: Ce ont été incorporées dans des aérogels de silice formant ainsi des aérogels macroscopiques robustes et hautement transparents présentant les propriétés des NPs incorporées. Ces aérogels composites ont été utilisés en tant que nouveaux types de capteurs pour les rayonnements ionisants de basse énergie dans les liquides ou les gaz. Leur porosité élevée permet un contact optimal entre l'émetteur radioactif et le scintillateur assurant ainsi une bonne récupération de l'énergie radioactive. / Nanotechnology has become a key domain of technology in XXI century. The great development of the synthetic approaches toward nanoparticles (NPs) with desired composition, size and shape expose the potential of their use as building blocks for larger scale structures. It allows fabrication of functional materials and devices directly from colloids by bottom-up approach, thus involving possibility of material design over several length scales. The process is referred to NPs assembly or self-assembly and leads to materials with varying architectures as for instance 1D (rods), 2D (films) or 3D (superlattices or gels). However most of 3D assemblies are limited to the micrometric scale and are difficult to control. Practically the only route allowing preparation of macroscopic 3D structures from NPs is their gelation and preparation of aerogels. As an alternative, NPs can be embedded in some matrix creating bulk composite material, with homogenously distributed non-aggregated NPs.Therefore, this work is devoted to development of materials with different dimensionalities for various applications from metal oxides NPs (mainly Y3Al5O12:Ce and Li4Ti5O12). The first part describes the syntheses of YAG:Ce and LTO NPs by glycothermal approach. In the case of YAG:Ce, the reactions conditions were appropriately adjusted in order to obtain non-aggregated nanocrystals (NCs) of few nanometers. The colloidal solution containing such NCs with different concentration was used for fabrication of thin films with controllable thickness by spin-coating method. Contrary, the synthesis of LTO led to aggregated NPs with hierarchical structuration which was highly beneficial for Li-ion batteries. The large surface area and porosity ensured efficient exchange of Li ions between electrolyte and anode material. Furthermore, the YAG:Ce NCs were used for preparation of macroscopic monoliths with high porosity and transparency. For that reason, colloidal solution of NCs was gelled by the abrupt change of solvent dielectric constant. The gels were further supercritically dried yielding YAG:Ce NPs-based aerogels with high porosity and transparency. The same approach turned o be appropriate for other systems like GdF3 or hybrid aerogels of YAG:Ce and GdF3.Alternatively, YAG:Ce NPs were incorporated into silica aerogels forming robust macroscopic and highly transparent aerogels exhibiting properties of incorporated NPs. They served for novel type of sensors for low-energy ionizing radiation in liquids and gases. Their high porosity assured well-developed contact between radioactive emitter and the scintillator ensuring good harvesting of radioactive energy.

O uso de DNA plasmidial (pDNA) visando a aplicações terapêuticas tem aumentado nos últimos anos. A cromatografia aparece como a técnica de purificação mais comum para obtenção de amostras de pDNA com o elevado grau de pureza exigido. Porém, as resinas cromatográficas disponíveis apresentam ainda uma série de desafios, nomeadamente no desenvolvimento de ligantes específicos e matrizes capazes de acomodar este tipo de molécula. Relativamente à apuração de novos ligantes, alguns estudos têm mostrado o potencial do aminoácido arginina para estabelecer interações específicas e preferenciais com o pDNA. Por outro lado, resinas monolíticas surgem como suportes interessantes devido às suas excelentes propriedades de transferência de massa e altas capacidades de adsorção. Neste estudo, diferentes ligantes baseados em arginina (arginina, di-arginina e tri-arginina) foram imobilizados em resinas de agarose previamente ativadas. Um primeiro estudo de adsorção em batelada foi realizado a fim de avaliar e compreender os mecanismos envolvidos no processo de adsorção dos ácidos nucleicos pDNA e RNA em resina com o aminoácido arginina. Na sequência, apresentamos uma proposta inovadora para o uso de ligantes de arginina em resinas de agarose, em um único passo de purificação em modo negativo a seguir ao passo de concentração por isopropanol. A capacidade da resina para o pDNA foi substancialmente maior do que a obtida para o mesmo tipo de resina no modo positivo, com notória vantagem de capacidade no uso de di-arginina face a arginina com rendimentos próximos de 100% do plasmídeo carregado. Os ligantes di-arginina e tri-arginina foram também imobilizados em resinas monolíticas. Em comparação com o aminoácido arginina, a imobilização dos homopeptídeos nas resinas monolíticas levou ao aumento da capacidade de adsorção (cerca de 2,5 vezes superior) e especicificidade de interações, mostrando-se como uma estratégia promissora para processos de purificação de pDNA. / The use of plasmid DNA (pDNA) for therapeutic applications has increased in recent years. Chromatography appears as the most common purification technique to obtain samples of pDNA with the high degree of purity required. However, the available chromatographic resins still present a series of challenges, namely in the development of specific ligands and matrices capable of accommodating this type of molecule. Regarding the determination of new ligands, several studies have shown the potential of the arginine amino acid to establish specific and preferential interactions with the pDNA. On the other hand, monolithic resins appear as interesting approaches due to their excellent mass transfer properties and high adsorption capacities. In this study, different arginine based ligands (arginine and di-arginine) were firstly immobilized on activated agarose resins. The first part of the work describes the adsorption equilibrium of plasmid DNA adsorption process, as well as the interaction with its main impurity (RNA) on arginine supports in a batch format, in order to compare and gather crucial information about adsorption mechanisms involved in this type of affinity system. Then, a new use for chromatographic bead matrixes based on arginine ligands was proposed, working as an adsorption matrix pDNA purification in negative mode after isopropanol concentration of the sample. The arginine based supports capacity for pDNA under negative mode for pDNA was substantially higher than that obtained with the same type of resin in the conventional positive mode, with a notable advantage of using di-arginine with recovery yields near 100%. The homopeptides (di-arginine and tri-arginine) were also immobilized on functionalized monolithic resins (BIA Separations, Slovenia). Effectively, the immobilization of the arginine homopeptides made the monolithic resins more functional compared to the (mono)arginine based resin, exhibiting greater binding capacities (around 2,5 times higher) and interaction intensities, proving to be a promising strategy for purification processes of pDNA.

Orientador: Carla Beatriz Grespan Bottoli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T03:14:53Z (GMT). No. of bitstreams: 1 Sato_MarianaRobertoGama_D.pdf: 4595333 bytes, checksum: 8882cf8d1d6576b19fd550c9d1d84f8b (MD5) Previous issue date: 2014 / Resumo: O desenvolvimento da cromatografia líquida capilar (CLC), como uma alternativa à cromatografia líquida de alta eficiência, tem se destacado no âmbito da miniaturização, uma tendência geral em instrumentação analítica para separações cromatográficas. O objetivo deste trabalho foi desenvolver e caracterizar fases estacionárias monolíticas para uso em CLC, empregando monolitos poliméricos orgânicos baseados em metacrilatos hidrofóbicos e hidrofílicos. O preparo das fases estacionárias monolíticas foi realizado in situ, ou seja, o recheio foi moldado no interior das colunas capilares de 150 µm de diâmetro interno. Foram empregados os monômeros butil metacrilato, lauril metacrilato e trimetilolpropano trimetacrilato no preparo de fases estacionárias monolíticas para separações no modo fase reversa; o polietileno glicol dimetacrilato foi empregado no preparo de fases estacionárias para separações no modo HILIC. As condições de preparo das fases estacionárias monolíticas foram comparadas e envolveram a aplicação de diferentes métodos de iniciação da reação de polimerização, como iniciação por radiação UV e iniciação térmica. O simples entrecruzamento de um monômero multifuncional e a polimerização controlada via radical livre também foram aplicados como métodos de preparo dos materiais monolíticos. As fases estacionárias monolíticas foram caracterizadas por técnicas físicas, como microscopia eletrônica de varredura e porosimetria por sorção de nitrogênio; as colunas preparadas foram avaliadas cromatograficamente pela técnica de CLC. As fases estacionárias monolíticas mostraram eficiência cromatográfica de até 47000 pratos m-1 para a separação de alquilbenzenos no modo fase reversa e de até 45000 pratos m-1 para a separação de acrilamidas no modo HILIC / Abstract: The development of capillary liquid chromatography (CLC), as alternative to high performance liquid chromatography, has emerged in miniaturization studies, a common trend in analytical instrumentation for chromatographic separations. The goal of this work was to develop and characterize monolithic stationary phases for use in CLC, using organic polymer monoliths based on hydrophobic and hydrophilic methacrylates. Stationary phases were prepared in situ by molding of the material inside the capillary columns of 150 µm of internal diameter. The monomers butyl methacrylate, lauryl methacrylate and trimethylolpropane trimethacrylate was employed to prepare monolithic stationary phases for separations by reversed phase; polyethylene glycol was used to prepare monolithic stationary phases for separations by HILIC mode. The preparation conditions were compared and involved the application of different initiation methods for polymerization reactions, such as initiation by UV radiation and initiation by heating. The single crosslinking of multifunctional monomer and controlled free-radical polymerization were also applied as preparation methods of monolithic materials. The monolithic stationary phases were characterized by physical techniques as scanning electronic microscopy and nitrogen sorption porometry; the columns prepared were chromatographic evaluated by CLC. Stationary phases showed chromatographic efficiency up to 47000 plates m-1 for the separation of alkylbenzenes in reversed phase mode and up to 45000 plates m-1 for the separation of acrylamides by HILIC mode / Doutorado / Quimica Analitica / Doutora em Ciências

Ce manuscrit est consacré à l'élaboration de phases stationnaires monolithiques organiques pour lesmicrosystèmes électrochromatographiques plastiques. La partie bibliographique situe d'abord l'intérêt destechniques électrocinétiques en microsystème. Ensuite, l'état de l'art sur l'utilisation des monolithes dans cestechniques séparatives est présenté en portant une attention particulière à la description du processus dephotopolymérisation. La fonctionnalisation de surface du copolymère d'oléfine cyclique (COC) est ensuitedétaillée afin d'envisager l'ancrage du monolithe aux parois du canal.La partie expérimentale a porté tout d'abord sur le développement et la caractérisation de deux monolithesacrylates dédiés à la chromatographie à polarités des phases inversées. Le travail expérimental s'est ensuiteorienté vers le développement d’un nouveau procédé permettant la synthèse et l’ancrage du monolithe dansles microcanaux en COC, ceci en une seule étape et à partir des photoinitiateurs de type I. Ce protocolepermet l'obtention d'efficacités intéressantes qui restent cependant inférieures à celles obtenues en capillaire.Des adaptations des conditions d'irradiation ont alors été apportées pour la synthèse de ces monolithes àl’intérieur des microsystèmes afin d'obtenir des efficacités de l’ordre de 250 000 plateaux/m. Pour terminer,les domaines d'applications et la longévité du dispositif ont été illustrés par la séparation de différentesfamilles de solutés (PAH, mycotoxines, catécholamines, acides aminés, amines biogènes) et l'analysed'échantillons réels tels que les vins (pour déterminer la teneur en certaines amines biogènes : histamine ettyramine). / This manuscript is dedicated to the synthesis of organic monolithic stationary phases in plasticelectrochromatographic microchips. The bibliography part shows the benefit of electrokinetic techniques formicrosystems. After an overview on the use of monoliths in separation techniques, the manuscript is focusedon the description of photopolymerization process. Finally, a detailed review of the different protocols offunctionnalization of cyclic olefin copolymer (COC) surface to allow anchoring of the monolith to thechannel walls is produced.The experimental study firstly deals with the development and characterization of two acrylate monolithsdedicated to the reversed phase mode in chromatography. Then, the experimental part focus on a new onestepmethod for the simultaneous synthesis and anchoring of organic monolith inside COC microchipchannel, thanks to the use of type I photoinitiators. This protocol improves efficiencies which neverthelessstay below those obtained in capillary. Further, the adjustment of the irradiation conditions allows to reachup to 250 000 plates/m. Finally, the field of applications and the durability of microsystem have beenillustrated by the separation of different kinds of solutes (HAP, mycotoxins, catecholamines, biogenicamines) and the analysis of real matrices such as wine samples (for the quantitative determination of twobiogenic amines : histamine and tyramine).

Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride (AETAC) and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion exchanger and quaternary amine as a strong anion exchanger, respectively. An alternative weak anion exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. The dynamic binding capacities of the three monoliths were comparable or superior to values that have been reported for various other monoliths. Chromatographic performances were also similar to those provided by a modified poly(GMA-co-ethylene glycol dimethacrylate) monolith. Separations of standard proteins were achieved under gradient elution conditions using these monolithic columns. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, laborious surface modifications were avoided and the PEGDA crosslinker improved the biocompatibility of the monolithic backbone. Protein separations by capillary size exclusion chromatography (SEC) require a monolith that is biocompatible, has sufficient pore volume, has the appropriate pore size distribution, and is rigid. Most polymer monoliths have not possessed a biomodal pore-size distribution, i.e., especially with one distribution in the macropore region and the other in the mesopore region. Furthermore, non-specific adsorption of proteins in these stationary phases has persisted as a major unresolved problem. To overcome these difficulties, a porous poly[polyethylene glycol methyl ether acrylate (PEGMEA)-co-PEGDA] monolith which can resist adsorption of both acidic and basic proteins when using an aqueous buffer without any organic solvent additives was developed. Based on this biocompatible monolith, surfactants were introduced as porogens with the hope of significantly increasing the mesopore volume within the polymer. Two types of surfactants were studied, including poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO and Brij. Pore size distributions were examined using a well-defined molecular weight range series of proteins and peptides by inverse size exclusion chromatography, which indicated relatively large volume percentages of mesopores and micropores. The two new monoliths demonstrated different SEC behaviors, low nonspecific adsorption of proteins, and high mechanical rigidity. High density lipoprotein (HDL) is a heterogeneous class of lipoprotein particles with subspecies that differ in apolipoprotein and lipid composition, size, density, and charge. In this work, I developed a new capillary SEC method for size separation of native HDL particles from plasma using a capillary packed with BioSep-SEC-4000 particles, Three major sizes of HDL particles were separated. Additionally, capillary SEC and capillary strong anion-exchange chromatography of non-delipidated HDL were accomplished using poly(PEGMEA-co-PEGDA) and poly(AETAC-co-PEGDA) monoliths. These new LC methods using packed and monolithic stationary phases provided rapid separation of HDLs and excellent reproducibility.