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

Torto, Nelson LU (1999)
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... (More)
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. (Less)
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author
opponent
  • Prof Lunte, Susan M.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
monosaccharides, membrane, IPED, ion spray, probe internal geometry, extraction fraction, electrochemical oxidation, electrochemical detection, disaccharides, desalting, carbohydrates, bioprocess monitoring, Cu, APCI, Au, MS, oligosaccharides, PAD, PEI, Analytical chemistry, Analytisk kemi
pages
220 pages
publisher
Department of Analytical Chemistry, Lund University
defense location
Lecture Hall B
defense date
1999-05-21 10:30
external identifiers
  • other:ISRN: LUNDL/NKAK-1048/1-220 (1999)
language
English
LU publication?
yes
id
d9b952c6-73f1-41dd-96f4-5f86a6df7d38 (old id 39552)
date added to LUP
2007-10-14 17:29:24
date last changed
2016-09-19 08:45:10
@phdthesis{d9b952c6-73f1-41dd-96f4-5f86a6df7d38,
  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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.},
  author       = {Torto, Nelson},
  keyword      = {monosaccharides,membrane,IPED,ion spray,probe internal geometry,extraction fraction,electrochemical oxidation,electrochemical detection,disaccharides,desalting,carbohydrates,bioprocess monitoring,Cu,APCI,Au,MS,oligosaccharides,PAD,PEI,Analytical chemistry,Analytisk kemi},
  language     = {eng},
  pages        = {220},
  publisher    = {Department of Analytical Chemistry, Lund University},
  school       = {Lund University},
  title        = {Microdialysis Sampling, Electrochemical Detection and Mass Spectrometry of Carbohydrates in Monitoring Bioprocesses},
  year         = {1999},
}