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Development of Micro Liquid Separation Techniques using Electrospray Ionisation Mass Spectrometry in the Analysis of Polar Compounds and Proteins/Peptides
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Electrospray ionisation (ESI) coupled to mass spectrometry (MS) is one of the most important detection techniques for chemical analysis of small drugs as well as large biomolecules in life science today. In this thesis, aspects on improved compatibility between liquid based separation systems and mass spectrometric detection were investigated regarding buffers, sample preparation and analysis of polar compounds as well as peptides and protein digests for enhanced ESI-MS performance.

Capillary electrophoresis (CE) coupled to ESI-MS detection, was evaluated using both a sheath flow interface and a sheathless design. The separation of peptides and small, polar compounds was optimised for both CE-ESI interfaces. The effect of sheath liquid composition was also studied with the aim to improve sensitivity in the ESI-MS detection.

Polar compounds were retained and separated by capillary ion-pair chromatography coupled to ESI-MS detection. Since commonly used ion-pairing reagents are detrimental to the ESI process they were effectively removed before the ionisation by the use of a trapping column after the separation. Alternatively, the ion-pairing reagents were exchanged to volatile constituents.

A method for peptide mapping by liquid chromatography (LC)-ESI-MS was developed for lactate dehydrogenase. The method was further enhanced to involve the proteolysis on-line to the LC-ESI-MS. No manual sample handling was then needed and the total analysis time decreased from 7 to 1.5 hours. The amount of information was also shown to increase in the on-line system.

Finally, the on-line concept was extended to an innovative interface for direct coupling of a pumped liquid flow to an electroosmotically driven flow. This provided a valve-free sample transfer between capillary LC and CE, aiming towards increased peak capacity per unit time for the analysis of complex peptide samples.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2003. , p. 46
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 808
Keywords [en]
Analytical chemistry
Keywords [sv]
Analytisk kemi
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-3304ISBN: 91-554-5534-4 (print)OAI: oai:DiVA.org:uu-3304DiVA, id: diva2:162357
Public defence
2003-03-21, B21, Uppsala University, Uppsala, 10:15
Opponent
Available from: 2003-02-25 Created: 2003-02-25 Last updated: 2011-12-09Bibliographically approved
List of papers
1. Reversed-phase ion-pair chromatography coupled to electrospray ionisation mass spectrometry by on-line removal of the counter-ions
Open this publication in new window or tab >>Reversed-phase ion-pair chromatography coupled to electrospray ionisation mass spectrometry by on-line removal of the counter-ions
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1999 In: J Chromatogr A, Vol. 854, no 1-2, p. 155-162Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90126 (URN)
Available from: 2003-02-25 Created: 2003-02-25Bibliographically approved
2. Optimization of capillary electrophoresis conditions for coupling to a mass spectrometer via a sheathless interface
Open this publication in new window or tab >>Optimization of capillary electrophoresis conditions for coupling to a mass spectrometer via a sheathless interface
2000 (English)In: J Mass Spectrom, Vol. 35, no 7, p. 919-924Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90127 (URN)
Available from: 2003-02-25 Created: 2003-02-25 Last updated: 2011-05-09
3. Studies of signal suppression in liquid chromatography–electrospray ionization mass spectrometry using volatile ion-pairing reagents
Open this publication in new window or tab >>Studies of signal suppression in liquid chromatography–electrospray ionization mass spectrometry using volatile ion-pairing reagents
2001 In: J Chromatogr A, Vol. 937, no 1-2, p. 41-47Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90128 (URN)
Available from: 2003-02-25 Created: 2003-02-25Bibliographically approved
4. Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis
Open this publication in new window or tab >>Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis
2003 In: J Am Soc Mass Spectrom, Vol. 14, no 2, p. 236-240Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90129 (URN)
Available from: 2003-02-25 Created: 2003-02-25Bibliographically approved
5. Miniaturized on-line proteolysis-packed capillary LC-MS for peptide mapping of lactate dehydrogenase
Open this publication in new window or tab >>Miniaturized on-line proteolysis-packed capillary LC-MS for peptide mapping of lactate dehydrogenase
In: J Chromatogr AArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-90130 (URN)
Available from: 2003-02-25 Created: 2003-02-25Bibliographically approved
6. On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface
Open this publication in new window or tab >>On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface
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2003 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 11, p. 1723-1729Article in journal (Refereed) Published
Abstract [en]

A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the electrophoretic separation was also developed. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoamperometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spectrometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE column inlet meet in a two-level cross (95 μm ID) in the PDMS structure, enabling independent flow characterization.

Keywords
Capillary electrophoresis, Flow injection, Hyphenation, On-column electrode, Poly(dimethylsiloxane)
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-94267 (URN)10.1002/elps.200305362 (DOI)
Available from: 2006-04-07 Created: 2006-04-07 Last updated: 2017-12-14Bibliographically approved
7. Development of a Poly(dimethylsiloxane) Interface for On-Line Capillary Column Liquid Chromatography: Capillary Electrophoresis Coupled to Sheathless Electrospray Ionization Time-of-Flight Mass Spectrometry
Open this publication in new window or tab >>Development of a Poly(dimethylsiloxane) Interface for On-Line Capillary Column Liquid Chromatography: Capillary Electrophoresis Coupled to Sheathless Electrospray Ionization Time-of-Flight Mass Spectrometry
2003 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 75, no 20, p. 5461-5467Article in journal (Refereed) Published
Abstract [en]

An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI−MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC−CE−TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 μL LC injection of 25 μg/mL peptide standard.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-90132 (URN)10.1021/ac030117g (DOI)
Available from: 2003-02-25 Created: 2003-02-25 Last updated: 2017-12-14Bibliographically approved

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