Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/3232
Título: Understanding ion-exchange adsorption mechanism under overloaded conditions
Autor: Silva, Gonçalo Fradique Lopes da
Orientador: Cabral, Ana Cristina Mendes Dias
Palavras-chave: Cromatografia de troca iónica
Microcalorimetria de fluxo
Carboximetil celulose
Albumina de boro bovino
Data de Defesa: Jun-2013
Resumo: Ion-exchange chromatography (IEC) is a powerful and widely used separation technique in the biotechnological industry. The greatest challenge of any chromatographic technique is predicting the adsorptive behaviour of biomolecules onto the chromatography resin. This investigation attempts to examine the complexity of protein adsorption onto ion-exchangers and the role of nonspecific effects in the establishment of the adsorptive process. Flow microcalorimetry (FMC) and adsorption isotherms measurements were used to illustrate lysozyme adsorption mechanism on carboxymethyl cellulose (CMC) at both absence and presence of salt (NaCl 50mM) at pH 5. FMC results show that under all the studied conditions the adsorptive process is, as expected in ion exchange, enthalpy driven. Direct correlation between microcalorimetry data and isotherm measurements is observed. Under linear protein concentrations, protein adsorption occurs in the same extension regardless salt concentration. However, when isotherm levelling point is reached, lysozyme reorientation in the presence of salt seems to be the leading mechanism to further adsorption. Under overloaded conditions in the presence of salt, with increasing surface concentration, as a new layer of protein molecules is formed, an expected decrease in the net heat of adsorption is observed, consistent with an energetic equilibrium towards the formation of the new layer. FMC experiments and isotherm measurements were also performed for Bovine Serum Albumin (BSA) adsorption onto Toyopearl® GigaCap Q-650M. The results showed a high overall exothermic process. Secondary adsorption of BSA to the surface, resulting from its alteration of conformation seems to be present. Also, at high protein surface concentrations, high repulsive interactions may occur. All these results confirm that FMC is a powerful technique to illustrate protein adsorption mechanisms in ion-exchange.
URI: http://hdl.handle.net/10400.6/3232
Designação: Dissertação apresentada à Universidade da Beira Interior para a obtenção do grau de Mestre em Bioquímica
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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