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True Monoliths as Separation Media: Homogeneous Gels for Electrophoresis and Electrochromatography in the Capillary and Microchip Modes
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed.

The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. N,N’-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, i.e., a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power.

A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2002. , p. 46
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 747
Keywords [en]
Biochemistry, capillary electrophoresis, capillary electrochromatography, gel electrophoresis, agarose, polyacrylamide, b-cyclodextrin, DNA, peptide, glutathione, drug enantiomers, microchip
Keywords [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-2584ISBN: 91-554-5395-3 (print)OAI: oai:DiVA.org:uu-2584DiVA, id: diva2:161919
Public defence
2002-10-04, Lecture Hall B21, BMC, Uppsala, 10:00
Opponent
Available from: 2002-09-13 Created: 2002-09-13Bibliographically approved
List of papers
1. An Approach to Ideal Separation Media for (Electro)Chromatography
Open this publication in new window or tab >>An Approach to Ideal Separation Media for (Electro)Chromatography
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1998 In: J. Cap. Elec., Vol. 5, no 1-2, p. 13-26Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89956 (URN)
Available from: 2002-09-13 Created: 2002-09-13Bibliographically approved
2. A New Easy-to-Prepare Homogeneous Continuous Electrochromatographic Bed for Enantiomer Recognition
Open this publication in new window or tab >>A New Easy-to-Prepare Homogeneous Continuous Electrochromatographic Bed for Enantiomer Recognition
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2000 In: Electrophoresis, Vol. 21, no 15, p. 3116-3125Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89957 (URN)
Available from: 2002-09-13 Created: 2002-09-13Bibliographically approved
3. Stable Homogeneous Gel for Molecular-Sieving of DNA Fragments in Capillary Electrophoresis
Open this publication in new window or tab >>Stable Homogeneous Gel for Molecular-Sieving of DNA Fragments in Capillary Electrophoresis
2002 In: J Chromatogr. A, Vol. 960, no 1-1, p. 221-227Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89958 (URN)
Available from: 2002-09-13 Created: 2002-09-13Bibliographically approved
4. High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins
Open this publication in new window or tab >>High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins
2002 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 3, no 11, p. 1117-1125Article in journal (Refereed) Published
Abstract [en]

The tripeptide glutathione is a prominent intracellular constituent that provides protection against genotoxic and carcinogenic electrophiles and is also a component of several biological signal substances. Glutathione conjugates, free glutathione, and glutathione disulfide contain charged amino acid residues, which contribute to solubility in aqueous media. However, the amphipathic nature of glutathione conjugates and the small differences that may distinguish the S substituents, pose analytical problems in their resolution. The present study demonstrates how homologous S-alkyl and S-benzyl conjugates of high structural similarity can be efficiently resolved by capillary electrophoresis. Inclusion of beta-cyclodextrins in the buffer or in a polyacrylamide gel affords baseline separation of the analytes. The separation methods described are applicable to enzyme assays in vitro and to the identification and quantification of glutathione conjugates of importance in toxicology and physiology. The contribution of beta-cyclodextrin to the separation is primarily based on interactions between its hydrophobic cavity and the S-alkyl and S-benzyl groups of the analytes.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89959 (URN)10.1002/1439-7633(20021104)3:11<1117::AID-CBIC1117>3.0.CO;2-E (DOI)12404638 (PubMedID)
Available from: 2002-09-13 Created: 2002-09-13 Last updated: 2017-12-14Bibliographically approved
5. A Hybrid Microdevice for Electrophoresis and Electrochromatography Using UV-Detection
Open this publication in new window or tab >>A Hybrid Microdevice for Electrophoresis and Electrochromatography Using UV-Detection
2002 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 20, p. 3479-3486Article in journal (Refereed) Published
Abstract [en]

We have designed a new class of microdevices composed of a supporting plastic (polyvinyl chloride, PVC) plate integrated with a groove for a piece of fused silica capillary (the separation channel), a slit for on-tube detection, an "islet" for the application of sample, electrode vessels and platinum electrodes. The design permits electrophoretic, electrochromatographic and chromatographic separations with on-tube UV detection. The efficient heat dissipation allows relatively high field strengths. This article is the first one dealing with microdevices where polymer solutions are replaced by homogeneous gels. A new type of gels synthesized from acrylamide and 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) as a cross-linker was employed for electrophoresis and electrochromatography. 2-Acrylamido-2-methylpropanesulfonic acid was added to the monomer solution to create a high electroendosmotic flow in electrochromatographic runs. These gels have excellent electrochromatographic and electrophoretic properties for low-molecular-weight compounds and DNA, as shown previously, namely high resolution combined with high stability. The unique cross-linker can be used for specific interaction with the alkyl and phenyl groups. The tripeptide glutathione (gamma-L-glutamyl-L-cysteinyl-glycine) and its benzyl conjugates were selected as model compounds to study the resolving power of the gel because they are difficult to separate by free zone electrophoresis. The limit of detection (LOD) for S-benzyl-glutathione was determined (ca. 7 microM). Run-by-run reproducibility was high (the separation factor of glutathione in the gel was 0.3 with 2.5% coefficient of variation, CV). Neutral compounds (acetone, acetophenone, propiophenone and butyrophenone) were separated electrochromatographically in the gel. The influence of organic solvent (acetonitrile) on the electroendosmotic mobility was similar to that in reversed-phase separations, although the separation mechanism is different. ATP, ADP and AMP were separated in less than 10 s by free-zone electrophoresis.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89960 (URN)10.1002/1522-2683(200210)23:20<3479::AID-ELPS3479>3.0.CO;2-W (DOI)12412115 (PubMedID)
Available from: 2002-09-13 Created: 2002-09-13 Last updated: 2017-12-14Bibliographically approved
6. Electrophoretic Analysis with UV-Detection of Peptides, Proteins, DNA and Virus in a Hybrid Microdevice Using Various Separation Media
Open this publication in new window or tab >>Electrophoretic Analysis with UV-Detection of Peptides, Proteins, DNA and Virus in a Hybrid Microdevice Using Various Separation Media
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-89961 (URN)
Available from: 2002-09-13 Created: 2002-09-13 Last updated: 2010-01-13Bibliographically approved

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