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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
supervisor
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 10:30:00
external identifiers
  • other:ISRN: LUNDL/NKAK-1048/1-220 (1999)
language
English
LU publication?
yes
additional info
Article: This thesis is based on the following papers, referred to in the text by their Roman numerals; Article: I) A study of a polysulfone membrane for use in an in-situ tunable microdialysis probe during the monitoring of starch enzymatic hydrolysates.N. Torto, T. Laurell, L. Gorton, G. Marko-VargaJ. Memb. Sci. 139 (1997) 239-248II) Optimal membrane choice for microdialysis sampling of oligosaccharides.N. Torto, J. Bång, S. Richardson, G. Nilsson, L. Gorton, T. Laurell,G. Marko-VargaJ. Chromatogr. A, 806 (1998) 265-278III) Poly (ethyline imine) coating to enhance hollow fibre membranes for microdialysis probes, submitted.N. Torto, M. Ohlrogge, L. Gorton, J. Van Alstine, T. Laurell, G. Marko-VargaIV) Maximising microdialysis sampling by optimising internal probe geometry.N. Torto, E. Mikeladze, L. Gorton, E. Csöregi, T. LaurellAnal. Commun. in pressV) Monitoring of enzymatic hydrolysis of ivory nut mannan using on-line microdialysis sampling and AEC with IPED.N. Torto, T. Buttler, L. Gorton, G. Marko-Varga, H. Stålbrand, F. TjerneldAnal. Chim. Acta 313 (1995) 15-24VI) On-line quantitation of enzymatic mannan hydrolysates in small-volume bioreactors by microdialysis sampling and CLC-IPED.N. Torto, G. Marko-Varga, L. Gorton, H. Stålbrand, F. TjerneldJ. Chromatogr. A, 725 (1997) 165-175VII) Monitoring of enzymatic hydrolysis of starch by microdialysis sampling coupled on-line to AEC and IPED using post-column switching.N. Torto, L. Gorton, G. Marko-Varga, J. Emnéus, C. Åkerberg, G. Zacchi, T. LaurellBiotechnol. Bioeng. 56 (1997) 546-554VIII) Electrochemical oxidation of mono- and disaccharides at fresh as well as oxidised copper electrodes in alkaline media.N. Torto, T. Ruzgas, L. GortonJ. Electroanal. Chem. 464 (1999) 252-258IX) Microdialysis introduction HPAEC/ionspray mass spectrometry for monitoring of on-line desalted carbohydrate hydrolysates.N. Torto, A. Hofte, R. van der Hoeven, U. Tjaden, L .Gorton, G. Marko-Varga,C. Bruggink, J. van der GreefJ. Mass Spectrom. 33 (1998) 334-341X) An automated system for carbohydrates analysis based on microdialysis, HPAEC, electrochemical detection and mass spectrometry.N. Torto, A. Cohen, L. Gorton, T. Laurell, R. A. M. van der HoevenLab. Robotics and Automation. 10 (1998) 361-367 Article: and a review paper as an appendix to the introduction Article: XI) Recent Trends in the Application of Microdialysis in Bioprocesses.N. Torto, T. Laurell, L. Gorton, G. Marko-VargaAnal. Chim. Acta 379 (1999) 281-305 previously published in Anal. Chim. Acta 374 (1998) 1-25. The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004)
id
d9b952c6-73f1-41dd-96f4-5f86a6df7d38 (old id 39552)
date added to LUP
2016-04-04 11:25:56
date last changed
2018-11-21 21:04:48
@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},
  language     = {eng},
  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},
}