Rice starch contains two types of glucose polymers, mainly linear amylose and hyper-branched amylopectin. Waxy rice has been characterised by the lack of amylose, the proportion of which being one of the most important parameters measured for rice quality. Germplasm collection work conducted in the Lao People’s Democratic Republic has previously demonstrated the diversity of this type of rice in terms of quality. The definition of a waxy rice variety is dependent on the meaning of amylose. The conventional method for determining amylose content has shown that waxy rice could have up to 5% amylose. However, including a 0% amylose standard in the standard curve causes the amylose content of these varieties to become 0-2%. In this work, the absence of amylose in waxy rice has been determined through three different approaches. Granule-bound starch synthase I (GBSS1), the enzyme which synthesises amylose, was not detected in waxy rice. Long linear chains associated with amylose were also not detected by size exclusion chromatography (SEC). The absence of these long chains affected functional properties of waxy rice, as indicated by the differences in viscosity curves between waxy and non-waxy rice. Moreover, these waxy varieties themselves exhibit differences in their viscosity curves, another indication of the diversity in coking properties in these varieties. A new approach, the ‘lnP(N) technique’, in analysing chain length distributions was applied to varieties with known mutations in two of the enzymes involved in the synthesis of amylopectin, and in determining gelatinisation temperature. It was determined that the presence of a novel feature, an interruption to linearity at DP 18-24, of the lnP(N) plot was found in rice samples with mutations in the alk gene, which codes for starch synthase (SS) IIa, and in samples with inactive branching enzyme (BE) IIb. Single nucleotide polymorphisms (SNPs) in the gene coding for SSIIa have been associated with lowered gelatinisation temperature. On the other hand, non-functionality of BEIIb changes the amylopectin structure such that gelatinisation temperature is increased. The novel feature of the lnP(N) plot is found when either or both SSIIa and BEIIb are non-functional. Waxy rice starch has hot-water-soluble (HWS) and insoluble (HWI) components. It has been confirmed that the soluble polysaccharides are structurally different from phytoglycogen, and are similar to amylopectin. Structural differences between the two fractions, which can account for their differences in solubility, were determined. At the level of the chain length distribution, the HWI fraction contained long chains not found in the HWS fraction. Considerable amounts of sucrose and glucose were found in the HWS fraction. At another level of structure, the degree of branching of the HWS components was higher than in the HWI fraction. On the other hand, the whole molecules of the HWS fraction were smaller than those of the HWI fraction. These structural differences between the two fractions potentially affected their physical behaviour, particularly solubility. The amount of leached material appears to be a property of the method, as varying cooking conditions changed the amount of HWS components. This amount reaches equilibrium at certain conditions, indicating the limited amount of the HWS material. On the other hand, the HWI component contains molecules that are insoluble in water, rather than molecules that solubilise slowly. Given the stability of the HWS fraction in solution, the ratio of the HWS fraction to the HWI fraction could potentially be used in measuring quality if the amount of the HWS fraction is variety-specific. However, the quantity of the HWS fraction appears to be a feature of the starch, rather than of the variety. Nevertheless, the fact that the soluble fraction is structurally and thermodynamically different from the insoluble fraction could presumably be grounds to classify the soluble component as a group of molecules distinct from amylopectin.

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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.

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.

La communication intercellulaire est essentielle pour le développement et la survie d'organismes multicellulaires. Dans le règne végétale, une des voies privilégiée pour la communication intercellulaire est la voie symplastique qui implique des canaux aux dimensions nanométriques connectant les cellules entre elles, leur permettant d'échanger directement photo-assimilats, miARN, protéines, oligoéléments etc. Observés pour la première fois en 1880 par le botaniste autrichien Eduard Tangl (Tangl 1880; Kohler & Carr 2006), ils ont longtemps été considérés comme de simples trous perméables permettant la diffusion de matériel cellulaire (Lee & Lu 2011; Oparka & Roberts 2001). Etant donné leurs taille nanoscopique, ce n'est que dans les années 1960, avec la démocratisation de la Microscopie Électronique en Transmission (MET) qui permet d'atteindre , que les premiers modèles ultrastructuraux sont établis (Lopez-Saez 1965; Robards 1970). Ils font état d'un canal d'environ 30 à 40 nm de diamètre avec un élément central cylindrique traversant le pore, appelé le desmotubule, connecté au Réticulum Endoplasmique des deux cellules (Figure 1 of our review Tilsner et al. 2016). Dans les années 1980 notre compréhension des plasmodesmes a quelque peu évolué et nous savons maintenant que ces structures ne sont pas de simples trous mais des structures membranaires très spécialisées et régulées (Lucas & Lee 2004; Faulkner & Maule 2011; Furuta et al. 2012). Le modèle ultrastructural actuel découle de la congrégation d'études ultrastructurale, physiologiques et pharmacologiques plus ou moins anciennes dépeignant une structure morphologiquement très souple et changeant de conformation au cours du développement. Les plasmodesmes peuvent réguler leur ouverture/fermeture par la constriction de leurs extrémités grâce à l'accumulation entre la membrane plasmique et la paroi végétale d'un polymère de sucre, la callose qui va pousser la membrane plasmique contre le desmotubule et en obstruer les entrées. Cette modulation permettrait majoritairement de réguler les flux intercellulaires qui impliquent les plasmodesmes. Cependant nos connaissances sur les remaniements membranaires prenant place durant le développement des plasmodesmes et sur la régulation de leur perméabilité sont encore imparfaites.La microscopie électronique en transmission, malgré l'ancienneté de la technique, est l'une des plus résolutive, largement utilisée en biologie. Avec l'amélioration des techniques de préservation d'échantillons, notamment les cryo- méthodes, elle permet d'atteindre à l'heure actuelle des résolutions inférieures à 5 nm en condition contrastée et inclus en résine et peut descendre en dessous du nanomètre pour la cryo-microscopie. Ce potentiel permet aisément l'étude des sous-compartiments cellulaires de l'ordre du µm tel que mitochondries, chloroplastes, noyaux etc. (Frey et al. 2002) mais permet également l'étude ultrastructurale précise de structures de l'ordre de la dizaine de nm (Beck et al. 2007; Al-Amoudi et al. 2007).En revanche, dans son utilisation classique, la microscopie électronique ne permet pas d'accéder à la troisième dimension de l'espace, rendant difficile l'interprétation de structure à l'architecture quelque peu compliquée. En effet, les images produites ne sont que des projections en deux dimensions d'objets en trois dimensions. Cela a mené au développement de la tomographie électronique en transmission (Crowther et al. 1970), méthode basée sur un concept mathématiques formulé par Johann Radon au XIXe siècle. Ce n'est que dans les années 2000 que la tomographie électronique a pris un essor significatif grâce au couplage avec des méthodes d'automatisation informatiques. / Plasmodesmata were first observed by Austrian botanist Eduard Tangl in 1880. He devoted himself to studying the anatomy and cytology of plants and his greatest discovery, of course, was the observation and first characterization of plasmodesmata (Tangl 1880, 1884 and 1885). Despite not having access to their ultrastructure, he observed thin striations (see front page engraving) between cotyledon cells of Strychnos nuxvomica and in the endosperm of seeds and described them as being conductive ducts. Already at the time, he was evoking the idea that these strands "unite them [the cells] to an entity of higher order", in other words formulating the first definition of a symplastic domain. lt is only in 1901 that Strasburger finally names these canals "plasmodesmata". His discovery led to a radical change in our conception of the plant entity and brought in new concepts such as the symplasm (Munch 1930) and transmembrane fluxes between cells, which are now being tackled with great interest by numerous research teams around the globe.Because of their size, plasmodesmata ultrastructure was not accessible until the advent of electron microscopy and they were long thought to be simple holes connecting plant cells one-another with no specific regulation. lt is only with the advent of electron microscopy and chemical fixation that botanists started to gain interest in this structure again. And even with these methods allowing the observation of structures down to several nanometers in size, there are still debates on the nature of the canal, its constituents and physiology (Lopez-Saez J. 1965, Robards A. 1970, Ding et al. 1992, Tilney et al. 1991, Overall and Gunning 1982, Schulz et al. 1995).Nowadays, with the advent of modern cryopreservation and three-dimensional electron tomography methods, great improvements are to be done in the understanding of the ultrastructure and physiology of these mysterious canals. More particularly by understanding the link between the membranous rearrangements taking place in these pores and the molecular transit regulation.My work has led us to view plasmodesmata as specialised Membrane Contact Sites (MCS). Hence, by analogy with MCS found in mammals, yeast and plants, this work embraces an original angle on the speculation of the composition and role of the desmotubule-plasma-membrane tethering complex. The work produced during my thesis allowed me to contribute to the publication of one review and two articles, which will constitute the introduction and two main sub-sections of the results chapter, respectively. The introductory review has been published in 2016 in Annual Review of Plant Biology. The first one is still under reviewing at Nature Plant and the other has been published in The Plant Cell journal in April 2015.

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 / Doctor of Agricultural Science / Kyoto University / DFAM

[pt] A filtração profunda de suspensões particuladas ocorre em muitos processos industriais e ambientais, como filtração de água e contaminação do solo. Na indústria petrolífera, a filtração profunda ocorre próximo ao poço injetor durante a injeção de água, causando redução de injetividade. A captura de partículas no meio poroso pode ser causada por diferentes mecanismos físicos (exclusão pelo tamanho, forças elétricas, gravidade (sedimentação), etc.). No caso do mecanismo de exclusão pelo tamanho, quanto maiores forem as partículas e menores forem os poros, mais intensa será a captura. Conseqüentemente, maior será o dano à formação. Entretanto, o modelo tradicional não considera as distribuições de tamanho de partículas e de poros. Assumindo que as partículas são capturadas pelo mecanismo de exclusão pelo tamanho, foram deduzidas as equações básicas para o transporte de suspensões particuladas no meio poroso considerando as distribuições de tamanho de poros e de partículas. Apenas o fluxo de água via poros acessíveis transporta partículas, ou seja, as partículas não podem acessar poros menores do que elas. No presente trabalho, os efeitos da redução do fluxo de partículas e da inacessibilidade devido ao fluxo seletivo de diferentes tamanhos de partículas são incluídos no modelo estocástico para a filtração profunda. As soluções analíticas obtidas mostram um comportamento físico mais realístico do que o previsto pelo modelo tradicional. O modelo de medição (concentrações totais) obtido difere substancialmente do modelo tradicional para a filtração profunda. Vários dados experimentais foram tratados, mostrando boa concordância e validando o modelo proposto. Um sistema de equações estocásticas para modelar a formação do reboco externo foi proposto e soluções analíticas foram obtidas, permitindo tratar a filtração profunda e a formação do reboco externo, utilizando o mesmo formalismo matemático. / [en] Deep bed filtration of water with particles occurs in several industrial and environmental processes like water filtration and soil contamination. In petroleum industry, deep bed filtration occurs near to injection wells during water injection, causing injectivity reduction. It also takes place during well drilling, sand production control, produced water disposal in aquifers, etc. The particle capture in porous media can be caused by different physical mechanisms (size exclusion, electrical forces, bridging, gravity (sedimentation), etc.). In case of size exclusion mechanism, the larger are the particles and the smaller are the pores, the more intensive is the capture and the larger is the formation damage. Nevertheless, the widely used traditional model does not account for particle and pore size distributions. Considering that particles are captured due to size exclusion mechanism, we derived basic equations for transport of particulate suspensions in porous media, accounting for particle and pore radii distributions. Particles are carried by water flowing through the accessible pore space only, i.e. particles cannot access smaller pores. In the current work, the effects of porous space accessibility and particle flux reduction due to selective flow of different size particles are included into the stochastic deep bed filtration model. The particle and pore ensembles for analytical solutions of the derived system show more realistic physics behaviour than that of the traditional model. Averaging of the derived stochastic equations leads to a new deep bed filtration model that significantly differs from the classical deep bed filtration system. Treatment of several experimental data shows good agreement between the laboratory and modelling data and validates the proposed model. The derived stochastic model has been extended to model formation of external filter cake by particles from the injected polydispersed suspension, allowing treating both deep bed filtration and external filter cake formation in the framework of the same system of governing equations.

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.

No description available.

No description available.