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- Végvári, Ákos, et al.
(författare)
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A Hybrid Microdevice for Electrophoresis and Electrochromatography Using UV-Detection
- 2002
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 23:20, s. 3479-3486
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Végvári, Ákos, et al.
(författare)
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High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins
- 2002
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Ingår i: ChemBioChem. - 1439-4227 .- 1439-7633. ; 3:11, s. 1117-1125
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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