Microdialysis Sampling, Electrochemical Detection and Mass Spectrometry of Carbohydrates in Monitoring Bioprocesses

Abstract: This thesis addresses; (1) the microdialysis experiment, application and methodology to maximise its performance when sampling from enzymatic bioprocesses, (2) electrochemical detection and electrochemical oxidation of carbohydrates on Au as well as Cu electrodes in alkaline solutions, and (3) coupling and maximising the performance of a microdialysis sampling based high performance anion exchange chromatography-integrated pulsed electrochemical detection-mass spectrometry system. Studies were carried out to investigate the effect of temperature, analyte as well as biomatrix complexity, analyte concentration, permeability factors, protein(enzyme)-membrane interaction and membrane material on the performance of microdialysis sampling. The performance data, as indicated by the extraction fraction, was correlated with membrane characteristics as reflected by their SEM micrographs in the dehydrated state. The performance of the membranes appeared not to be significantly affected by continuously sampling at 90°C, for up to 24 h. All membranes showed some degree of interaction with enzymes (and biomatrix), and the subsequent decrease in performance was associated with the so-called Andrade effect. A method for the in-situ modification of microdialysis membranes by surface coating with PEI, in view to coimmobilise PEI-PEG so as to suppress membrane-protein interaction was developed. Microdialysis sampling, continuous flow as well stopped flow modes were employed for sampling during the characterisation of hydrolytic properties of enzymes and enzyme substrates. The IPED monomer and molar unit sensitivity was investigated for manno- and maltooligasaccharides. As the results indicated to a homologous series dependent response, electrochemical oxidation studies were carried out on fresh as well as modified Cu RDE in order to evaluate n and k values, and compare these to those obtained for Au. Electrochemical oxidation mechanism for monosaccharides on Cu, was found to be different from that reported on Au. The oxidation of hexoses proceeded with a passage of 12 electrons, but that of disaccharides, appeared to be oxide dependent as 2-18 electrons were observed. MS was used for the qualitative evaluation of saccharides during the enzymatic hydrolysis of wheat starch. A membrane desalting device, employing water as the regenerant was used to desalt up to 600 mM Na+ from the chromatographic effluent.