The purification of pepsinogen from the stomach of red perch using aqueous two phase systems (ATPS) formed by polyethylene glycol (PEG) and salt at 4°C was optimized. Salt type, salt concentration, PEG molecular weight and PEG concentration had significant effects on total volume (TV), volume ratio (VR), enzyme activity (AE), protein content (CP), specific activity (SA), purification fold (PF) and recovery yield (RY). (NH4)2SO4 at 15% w/w concentration was selected as the optimum salt type and concentration. PEG 1500 at 18% w/w concentration was selected as the optimum PEG molecular weight and concentration. 15% (NH4)2SO4-18% PEG 1500, the optimal ATPS, was compared with ammonium sulfate fractionation (ASF). ATPS gave better partition of pepsinogen (SA of 5.40 U/mg, PF of 5.20 and RY of 86.6%) than ASF (SA of 2.55 U/mg, PF of 2.46, RY of 70.4%). / This is the electronic copy of partition of pepsinogen in aqueous two phase system method.

Mushroom (Agaricus bisporus) polyphenoloxidase (PPO) (EC 1.14.18.1) was isolated and purified using an aqueous two phase system composed of octyl phenol (ethyleneglycol) 7-8 ether (Triton X-114/TX-114). TX-114 is a non-ionic surfactant which thermoseparates in water and forms an aqueous two phase system with a surfactant-depleted top phase and a surfactant-enriched bottom phase. Critical micelle concentration (CMC) and cloud point of the surfactant are 0.17 mM and 22&ordm / C respectively. The partitioning behavior of mushroom PPO in water/TX-114 aqueous two-phase systems was studied and the effects of TX-114 concentration, ionic strength, pH, temperature and crude extract preparation on PPO partitioning were studied. PPO generally partitioned to the surfactant-depleted top phase / whereas many other hydrophobic proteins and molecules partitioned to the surfactantenriched bottom phase. When two-step ultrafiltration was used as a pretreatment, complete enzyme recovery was achieved with all studied TX-114 concentrations. Moreover, about 5 fold purification was achieved by using 8% TX-114. The purification increased to 10 fold by using polyvinylpolypyrolidone (PVPP) at pH 7.0 with a recovery of 72%. However changing pH from 7.0 to 6.0 increased the purification factor and enzyme recovery to 15 fold and 100%, respectively. Addition of potassium or sodium salts caused PPO molecules to partition in the surfactantenriched bottom phase. Finally, crude enzyme can be concentrated besides being purified and recovered by doing aqueous two-phase separation at room temperature.

Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.

Tobacco has long been considered as a host to produce large quantities of high-valued recombinant proteins. However, dealing with large quantities of biomass with a dilute concentration of product is a challenge for down-stream processing. Aqueous two-phase extraction (ATPE) has been used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. ATPE was studied for its applicability to recombinant protein purification from tobacco using egg white lysozyme as the model protein. Separate experiments with polyethyleneglycol(PEG)/salt/tobacco extract, and PEG/salt/lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two level fractional factorial designs were used to study the effects of factors such as PEG molecular weight, PEG concentration, the concentration of phase forming salt, sodium chloride concentration, and pH on protein partitioning. The results showed that PEG/sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. The binodial curve and tie line corresponding to the optimal condition for lysozyme recovery for the PEG 3400/sodium sulfate system were developed. The selectivity at the optimal condition was experimentally determined to be 47 with a lysozyme yield of 79.6 % with a purification factor of 10 and a concentration factor of 20. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco. / Master of Science

Bromelina é um nome coletivo para enzimas proteolíticas encontradas no talo, fruto e folhas do abacaxi (Ananas comosus Merr). A bromelina possui propriedades anti-inflamatórias, de debridamento, entre outras. Para a produção da bromelina deve-se, preferencialmente, usar resíduos do abacaxi, visto que os produtos do fruto têm aplicação comercial. Este trabalho teve como objetivo a extração de bromelina a partir de cascas de abacaxi através de sistema de duas fases aquosas (SDFA), e sua aplicação em hidrogel polimérico. Foram realizados estudos de estabilidade da bromelina comercial, em que se observou maior estabilidade em pH 5,0 com menor perda da atividade relativa em todas as temperaturas estudadas (20, 30, 40 e 50°C). O estudo da extração da bromelina em SDFA formado por polietileno glicol (PEG) e ácido poliacrílico (PAA) (com auxílio da análise de variância de parâmetros como rendimento, fator de purificação e coeficiente de partição) proporcionou rendimento de 335% e fator de purificação de 25,8. Os hidrogéis poliméricos à base de PEG estudados apresentaram-se flexíveis, com pouca elasticidade e taxa de absorção superior a 1000%. Hidrogel carreado de bromelina pelo método de turgescência proporcionou a maior liberação da enzima, assim como a maior atividade (80% da bromelina liberada em 24 h e 278 ± 89 U/mL). / Bromelain is a collective name for the proteolytic enzymes found in the stem, fruit and leaves of pineapple (Ananas comosus Merr.). Bromelain possesses anti-inflammatory properties, debridement, among others. For bromelain production one should preferably use the waste materials, whereas pineapple fruit products have commercial application. This study aimed to extract bromelain from pineapple peels using aqueous two-phase system (ATPS), and its application in polymeric hydrogels. Stability studies of commercial bromelain were performed, which found greater stability at pH 5.0 with minor loss of relative activity at all temperatures studied. The study of bromelain extraction in ATPS composed by polyethylene glycol (PEG) and poly acrylic acid (PAA) (with assistance of variance analysis of parameters such as yield, purification factor and partition coefficient) showed yield 335% and purification factor of 25.8. The PEG-based hydrogels studied presented flexibility, low elasticity and swelling ratio higher than 1000%. Hydrogel containing bromelain, loading by embedding (solvent sorption) method, yielded the highest enzyme release, as well as the highest activity (80% bromelain released over 24 h and 278 ± 89 U / mL).

A partição das enzimas glicose-6-fosfato desidrogenase (G6PD) e uroquinase (UK) em sistemas micelares de duas fases aquosas foi investigada, teórica e experimentalmente. Inicialmente, a partição de G6PD em sistema micelar de duas fases aquosas composto pelo tensoativo não-iônico C10E4 (óxido de n-deciltetraetileno) foi estudada. Observou-se uma partição governada primariamente por interações repulsivas estéricas, do tipo \"volume de exclusão\", sendo a G6PD recuperada preferencialmente na fase inferior do sistema, pobre em micelas, resultando em valores de KG6PD inferiores a 1,0. Os tensoativos catiônicos CnTAB (brometos de alquiltrimetilamônio, n = 8, 10 e 12) foram adicionados ao tensoativo C10E4, de modo a formar sistemas micelares mistos (não-iônico/catiônico) de duas fases aquosas e atrair a G6PD de carga efetiva negativa para a fase superior, rica em micelas positivamente carregadas. Os coeficientes de partição obtidos nos sistemas C10E4/CnTAB/tampão foram no mínimo 2,5 vezes maiores que os correspondentes no sistema C10E4/tampão mantendo-se o volume de exclusão constante. De uma forma geral, o sistema micelar misto C10E4/C10TAB/tampão forneceu o melhor KG6PD = 7,7, com um rendimento de G6PD na fase superior de 71%. A adição de ligantes de afinidade (inibidores enzimáticos) ao sistema de duas fases aquosas C10E4/tampão para purificação de UK e G6PD também foi estudada. Não foram observadas diferenças significativas no perfil de partição da UK na presença ou não do ligante p-aminobenzamidina, o qual apresentou partição uniforme entre as duas fases do sistema, sendo obtidos valores de KUK ~ 0,70 em ambos os casos. Para a G6PD, um pequeno incremento no KG6PD foi observado na presença dos ligantes de afinidade cibacron blue e procion red, porém resultando ainda em valoresfnferiores a 1. Portanto, a utilização de inibidores enzimáticos livres em solução como ligantes de afinidade para purificação de UK e G6PD em sistema micelar C10E4/tampão de duas fases aquosas não resultou em diferenças significativas no perfil de partição das enzimas. Por fim, um sistema de purificação em dois estágios, sendo o primeiro constituído de sistema micelar não-iônico (C10E4) de duas fases aquosas e o segundo de sistema micelar misto (C10E4/ C10TAB) de duas fases aquosas foi aplicado para a purificação de G6PD presente em homogeneizado celular de Saccharomyces cerevisiae, sendo obtido um rendimento final de G6PD de 59%, com um fator de purificação de 1,3. / The partitioning of the hydrophilic enzymes glucose-6-phosphate dehydrogenase (G6PD) and urokinase (UK) in two-phase aqueous micellar systems was investigated, both experimentally and theoretically. Initially, the partitioning of G6PD in two-phase aqueous micellar system composed of the nonionic surfactant C10E4 (n-decyl tetra(ethylene oxide) was studied. It was observed a partitioning behavior governed primarily by repulsive, steric, excluded-volume interactions, which drove the enzyme preferentially to the bottom, micelle-poor phase, resulting in KG6PD values smaller than one. The cationic surfactants CnTAB ((alkyltrimethylammonium bromide, n = 8, 10, and 12) were mixed wíth the nonionic surfactant C10E4 to form two-phase aqueous mixed (nonionic/cationic) micellar systems. The measured G6PD partition coefficients in the C10E4/CnTAB/buffer systems were at least 2.5 times larger than those in the corresponding C10E4/buffer system, clearly demonstrating that the net negatively charged G6PD was attracted electrostatically to the top, mixed micelle-rich phase, containing a greater number of positively-charged C10E4/CnTAB mixed micelles. Overall, the two-phase aqueous mixed (C10E4/C10TAB) micellar system yielded the highest KG6PD = 7.7, with a G6PD yield in the top phase of 71%. The addition of affinity ligands (enzyme inhibitors) to the two-phase C10E4 /buffer systems was studied for UK and G6PD purification. No significant differences in the UK partitioning behavior was observed in the presence or not of the ligand p-aminobenzamidine, since the ligand was found to partition evenly between the two phases of the system. Partition coefficient values of about 0.70 were obtained in both cases. For the G6PD, the partition coefficient was found to be slightly higher in the presence of either cibacron blue or procion red as affinity ligand, however still smaller than one. Therefore, the use of competitive inhibitors free in solution as affinity ligands for UK and G6PD purification in two-phase C10E4 /buffer systems did not provide significant differences in the enzymes partitioning behaviors. Finally, a two-step purification process, combining a two-phase aqueous nonionic (C10E4) micelar system, followed by a two-phase aqueous mixed (C10E4/C10TAB) micellar system, was employed for the purification of G6PD present in a Saccharomyces cerevisiae cell homogenate. It was obtained a G6PD final yield of 59%, and a purification factor of 1,3.

Bromelina é um nome coletivo para enzimas proteolíticas encontradas no talo, fruto e folhas do abacaxi (Ananas comosus Merr). A bromelina possui propriedades anti-inflamatórias, de debridamento, entre outras. Para a produção da bromelina deve-se, preferencialmente, usar resíduos do abacaxi, visto que os produtos do fruto têm aplicação comercial. Este trabalho teve como objetivo a extração de bromelina a partir de cascas de abacaxi através de sistema de duas fases aquosas (SDFA), e sua aplicação em hidrogel polimérico. Foram realizados estudos de estabilidade da bromelina comercial, em que se observou maior estabilidade em pH 5,0 com menor perda da atividade relativa em todas as temperaturas estudadas (20, 30, 40 e 50°C). O estudo da extração da bromelina em SDFA formado por polietileno glicol (PEG) e ácido poliacrílico (PAA) (com auxílio da análise de variância de parâmetros como rendimento, fator de purificação e coeficiente de partição) proporcionou rendimento de 335% e fator de purificação de 25,8. Os hidrogéis poliméricos à base de PEG estudados apresentaram-se flexíveis, com pouca elasticidade e taxa de absorção superior a 1000%. Hidrogel carreado de bromelina pelo método de turgescência proporcionou a maior liberação da enzima, assim como a maior atividade (80% da bromelina liberada em 24 h e 278 ± 89 U/mL). / Bromelain is a collective name for the proteolytic enzymes found in the stem, fruit and leaves of pineapple (Ananas comosus Merr.). Bromelain possesses anti-inflammatory properties, debridement, among others. For bromelain production one should preferably use the waste materials, whereas pineapple fruit products have commercial application. This study aimed to extract bromelain from pineapple peels using aqueous two-phase system (ATPS), and its application in polymeric hydrogels. Stability studies of commercial bromelain were performed, which found greater stability at pH 5.0 with minor loss of relative activity at all temperatures studied. The study of bromelain extraction in ATPS composed by polyethylene glycol (PEG) and poly acrylic acid (PAA) (with assistance of variance analysis of parameters such as yield, purification factor and partition coefficient) showed yield 335% and purification factor of 25.8. The PEG-based hydrogels studied presented flexibility, low elasticity and swelling ratio higher than 1000%. Hydrogel containing bromelain, loading by embedding (solvent sorption) method, yielded the highest enzyme release, as well as the highest activity (80% bromelain released over 24 h and 278 ± 89 U / mL).

A procedure for the preparation of spinach leaf mitochondria was developed. The procedure combines differential centrifugation, partition in dextran- polyethyleneglycol two-phase system and Percoli density gradient centri- fugation. The different steps separate the material mainly according to size, surface properties and density, respectively. No chlorophyll was present in the final mitochondrial preparation and the mitochondria were also markedly enriched relative to peroxisomes and microsomes as esti­mated from the recovery of marker enzymes. The latency of enzyme activities was used to study the apparent intactness of the mitochondrial membranes. These measurements showed that both the inner and outer mitochondrial membranes were more than 90 % intact. The mitochondria were also functionally intact since the coupling between respiration and oxidative phosphorylation was retained. The purity of the preparation made it possible to study cytochromes from leaf mitochondria. The cytochrome content of stalk and leaf mitochondria was measured in order to compare mitochondria from photosynthesizing and non-photosynthesizing tissue. The measurements were performed by difference spectroscopy both at room temperature and at liquid nitrogen temperature. Qualitatively the cytochrome content in mitochondria from stalks and leaves was identical. Quantiatively leaf mitochondria contained,on a protein basis, only half the amount of the different cytochromes as compared to stalk mitochondria. The relative content of the different cytochromes was, however, similar suggesting that the composition of the respiratory chain was the same. The photorespiratory conversion of glycine to serine takes place in the mitochondria and involves oxidative decarboxylation of glycine. The ability to oxidize glycine via the respiratory chain was present in spinach leaf mitochondria, but absent in mitochondria prepared from roots, stalks and leaf veins from the same plants. This confirmed the specific localization of the glycine oxidizing activity to photosyntheticaliy active tissue, as suggested by studies with other plant material. The conversion of glycine to serine is a complex reaction depending on the combined action of two enzymes: glycine decarboxylase and serine hydroxymethyltransferase. The effect of inhibitors on the serine hydroxy­methyl transferase activity and the rate of the glycine bicarbonate exchange reaction associated with glycine decarboxylase was studied. These reactions represent partial steps in the conversion of glycine to serine and the aim was to investigate the site of inhibition for the different inhibitors, namely, isonicotinyl hydrazide (a pyridoxa!phosphate antagonist), amino- acetonitrile, glycinehydroxamate (glycine analogues) and cyanide. The results showed that these inhibitors had a complex pattern of inhibition. The same inhibitor affected more than one site and often with an apparently different mechanism. It was, however, found that aminoacetonitrile at low concentrations specifically inhibited glycine decarboxylase and that cyanide specifically inhibited serine hydroxymethyltransferase. / digitalisering@umu

Data de aprova??o retirada da vers?o impressa do trabalho. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-01-04T19:50:32Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) edgard_ronny_delgado_huamani.pdf: 5898825 bytes, checksum: 78eee370de5fed297b3181e82d98f63d (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-01-17T18:40:25Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) edgard_ronny_delgado_huamani.pdf: 5898825 bytes, checksum: 78eee370de5fed297b3181e82d98f63d (MD5) / Made available in DSpace on 2018-01-17T18:40:25Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) edgard_ronny_delgado_huamani.pdf: 5898825 bytes, checksum: 78eee370de5fed297b3181e82d98f63d (MD5) Previous issue date: 2017 / Neste trabalho, Carbon Dots (CD) oxidados foram preparados a partir de rea??es de desidrata??o/oxida??o ?cida da celulose, e posteriormente desoxigenados em meio alcalino e rea??o com sulfato de hidrazina. As caracteriza??es dos CD foram feitas com Microscopia eletr?nica de Transmiss?o, FTIR, titula??o potenciom?trica, Resson?ncia Magn?tica Nuclear de 13C e espectroscopia UV-Vis e fluoresc?ncia. Os resultados obtidos com estas caracteriza??es mostraram que a amostra oxidada apresenta um tamanho m?dio aproximadamente de 2,7 nm com uma significativa quantidade de grupos funcionais oxigenados ?cidos, dos quais 63% s?o ?cidos carbox?licos. Ap?s as rea??es de modifica??o superficial foi verificado que em meio alcalino ocorre somente uma elimina??o de nanoestruturas mais oxidadas, enquanto que na rea??o com hidrazina ocorre a redu??o de alguns grupos carbox?licos e ep?xidos com forma??o de grupos funcionais hidrazonas. Como consequ?ncia da modifica??o superficial, as propriedades ?pticas dos CD s?o alteradas significativamente. Com a redu??o, o band gap diminui e a energia da emiss?o aumenta, deslocando-se para a regi?o azul do espectro eletromagn?tico. Uma vez caracterizados, a parti??o de todos os CD preparados foi investigada em diferentes Sistemas Aquosos Bif?sicos (SAB), nos quais foram avaliados os efeitos dos c?tions e ?nions dos sais, dos pol?meros e do pH inicial do sistema no coeficiente de parti??o (K). Em adi??o foi avaliado o efeito da modifica??o superficial das nanopart?culas. A rela??o do K com comprimento da linha de amarra??o (CLA), o par?metro termodin?mico dos SAB foi estudado para todos os sistemas. Os resultados obtidos mostraram que a melhor separa??o ocorreu com o sistema PEO1500+sulfato de l?tio+?gua em pH=3 e usando os Carbon Dots reduzidos com hidrazina. O valor do K para este sistema foi de 64,4. Por outro lado, o menor valor de K foi 0,79, foi obtido para o sistema PEG1500+tartarato de s?dio+?gua em pH=6 e usando Carbon Dots oxidados. As an?lises detalhadas das fases superior e inferior de alguns destes sistemas com espectroscopia de fluoresc?ncia mostraram que os SAB s?o capazes, al?m de particionar, separar as nanopart?culas com diferentes propriedades ?pticas, as quais est?o diretamente associadas ?s propriedades superficiais e tamanho de part?culas. Por fim, n?s avaliamos a atividade catal?tica foto-Fentom dos CD na degrada??o do corante ani?nico ?ndigo de carmim e os resultados mostraram que os CD apresentam atividade fotocatal?tica neste sistema. Devido ? alta parti??o verificada para o sistema PEG1500+ sulfato de l?tio+?gua, este foi usado para avaliar a potencialidade de seu uso em cat?lise de transfer?ncia de fase usando CD como fotocatalisador e o corante ?ndigo de carmim como mol?cula modelo. Apesar da complexidade e da dificuldade de caracteriza??o dos produtos da foto-oxida??o no SAB, os resultados obtidos indicam que o sistema testado pode ser usado em rea??es de transfer?ncia de fase fotocatalisadas. Como conclus?o final, acredita-se que os resultados apresentados, em especial, a parti??o/sele??o das nanopart?culas com distintas propriedades ?pticas s?o de extrema import?ncia para o desenvolvimento de novas e eficazes aplica??es dos CD. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / In this work, oxidized Carbon Dots (CD) were prepared by a dehydration/oxidation reaction of cellulose and subsequently deoxygenated in alkaline medium and reaction with hydrazine sulfate. The CD characterizations were made with Transmission Electron Microscopy, FTIR, potentiometric titration. 13C Nuclear Magnetic Resonance, UV-Vis and fluorescence spectroscopy. The results obtained with these characterizations showed that the oxidized sample has an average size of 2.7 nm with a significant amount of acid oxygenated functional groups, of which 63% are carboxylic acids. After the superficial modification reactions, it was verified that in alkaline conditions only one elimination of more oxidized nanostructures occurs, whereas in the reaction with hydrazine the reduction of some carboxylic groups and epoxides with formation of hydrazones functional groups. As a consequence of the surface modification, the optical properties of the CD are significantly changed. The band gap decreases and the emission energy increases, shifting to the blue region of the electromagnetic spectrum. Once characterized, the partitioning of all CD prepared was investigated in differente Aqueous two-phase systems (ATPS), in which the effects of the cations and anions of salts, of the polymers and the initial pH of the system in the partition coefficient (K) were evaluated. In addition, the surface modification effect of the nanoparticles was evaluated. The relationship of K with Tie Line Length (TLL), the thermodynamic parameter of the ATPS was studied for all systems. The results showed that the best separation occurred with the system PEO1500 + lithium sulfate + water at pH = 3 and using the reduced carbon dots with hydrazine. The K value for this system was 64,4. On the other hand, the lowest K value was 0,79, it was obtained for the system PEG1500 + sodium tartrate + water at pH = 6 and using oxidized carbon dots. The detailed analysis of the upper and lower phases of some of these systems with fluorescence spectroscopy showed that the ATPS are capable, in addition to partition, to separate the nanoparticles with different optical properties, which are associated directly to the surface properties and particle size. Finally, we evaluated the photo-Fentom catalytic activity of the CD in the degradation of the anionic anion dye indigo carmine and the results showed that the CD present photocatalytic activity in this system. Due to the high partition found for the PEG1500 + lithium + water sulfate system, it was used to evaluate the potentiality of its use in phase transfer catalysis using CD as a photocatalyst and the indigo carmine dye as a model molecule. Despite the complexity and difficulty of characterizing the photo-oxidation products in ATPS the results indicate that the system tested can be used in photocatalysed phase transfer reactions. As a final conclusion, we believe that the results presented, in particular, the partitioning / selection of nanoparticles with different optical properties are of extreme importance for the new and effective applications of CD.

Aqueous Phase Partitioning has a long history of applications to the analytical characterisation of biomolecules. However process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The main aim of this work was to explore the proposition that partition in Aqueous Two Phase Systems (ATPS) can be used as an analytical tool to detect protein isoforms and to assess the applicability of the method in clinical assays and for quality control in bioprocessing through examination of several analytical problems. The work also examined the development of automated methods of system preparation and sampling techniques to determine the partition coefficient in ATPS. The study demonstrated that the geometrical form of the phase diagram co-existence curve was of crucial importance since this directly affected the accuracy with which systems of defined Tie Line Length and Mass Ratio could be constructed. The TLL %Bias (accuracy) of a theoretical system range in the PEG1000-(NH4)2SO4 system at shorter TLL (12.2) was in the range +80.6% to -100% while at a longer TLL (53.1) the %Bias (accuracy) was reduced to +0.1% to -1.9%. At the same time the MR %Bias (accuracy) at shorter TLL (12.2) was in the range +59.5% to -21.3% while at the longer TLL (53.1) this was reduced to +2.7% to -2.6%. By contrast in the PEG8000-Dextran500 system the TLL %Bias (accuracy) at shorter TLL (13.1) was in the range +3.7% to -4.12%, while at a longer TLL (31.1) the range was +0.74% to -0.67%. The MR %Bias (accuracy) at the shorter TLL (13.1) was in the range +3.6% to -3% while at the longer TLL (31.1) the range was +1.1% to -1.4%. This illustrated that it is more difficult to work with a high degree of accuracy (e.g. %Bias <5%) close to the critical point in PEG-salt systems than in PEG-dextran systems. Two different approaches were taken to examine analytical phase partitioning. In the first approach the structure of the isoforms of a model protein (ovalbumin) were altered enzymatically. Analytical methods involving Strong Anion-Exchange chromatography were developed and applied to the separation of the ovalbumin isoforms. Removal of the phosphorylated groups (dephosphorylation of ovalbumin) was undertaken using alkaline phosphatase and de-glycosylation was attempted using neuraminidase and Endo-glycosidase F. However, both enzymatic approaches to deglycosylation were unsuccessful. Dephosphorylated isoforms were successfully produced and characterised. After partitioning in ATPS a clear difference was demonstrated between the behaviour of the native and dephosphorylated forms of ovalbumin. The mean % recovery in a PEG-salt ATPS was 99.8% (± 3.59) for the naive protein and 75.6% (± 4.03) for the dephosphorylated form. On the other hand, in a PEG3350-Dextran500 system, where solubility was maintained, a significant difference in the partition coefficient (K) of native and dephosphorylated ovalbumin was found. K for native ovalbumin was 0.85 while the partition coefficient of the dephosphorylated ovalbumin was 0.61. Analysis of covariance (ANCOVA) indicated that the regression coefficients of the respective partition isotherms were significantly different (p value < 0.05). In a second approach to examine analytical phase partitioning, chemical modification of a specific target surface amino acid of another model protein (serum albumin) was used to determine the degree of conjugation of the protein and also to determine its oxidative state. The method examined the reactivity of a free surface thiol to a wide range of labels ( (a) 2-methylsulfonyl-5-phenyl -1,3,4 oxidiazole reagent, (b) N-Ethylmaleimide (NEM) reagent, (c) 5, 5’-dithiobis (2-nitrobenzoate)(DTNB) (Ellman’s reagent), (d) N-pyrenylmaleimide (NPM) reagent, (e) Fluorescein-5-maleimide (F-5-M) Reagent). Only DTNB was found to modify the surface free thiol of serum albumin in a highly specific and quantitative manner. In the course of the development of a partitioning assay for surface free thiols of serum albumin significant oxidative properties were found to be associated with poly(ethylene glycol) PEG solutions and several attempts were made to find an oxidatively safe partitioning system by including antioxidants and by removal of contaminants by freeze drying. PEG3350-Dextran500 was found to provide an oxidatively safe environment for the development of a partitioning assay for the determination of albumin free thiols. A phase partitioning assay system capable of quantitatively resolving protein associated free thiols and low molecular weight thiols from a mixture of the two was developed. Correlation coefficients (R2) for the regression of experimentally determined protein free thiols in the presence of different levels of added LMW free thiol on the known addition of protein ranged from 0.77 to 0.83. The results demonstrated that the assay could quantify and distinguish both types of thiol in a simple two-step procedure.