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Chromatographic detection using tris(2,2'-bipyridyl)ruthenium(III) as a fluorogenic electron-transfer reagent
S J Woltman1, W R Even, S G Weber
1Chevron Science Center, University of Pittsburgh, Pennsylvania 15260, USA.
Analytical Chemistry
|April 30, 1999
Summary
A new method uses non-fluorescent ruthenium complex (M(III)) as a reagent for sensitive detection of oxidation-susceptible analytes in chromatography. This redox-based fluorescence detection offers an alternative to electrochemical methods with potential for very low detection limits.
Area of Science:
- Analytical Chemistry
- Electrochemistry
- Biochemistry
Background:
- Tris(2,2'-bipyridyl)ruthenium (M(II)) is fluorescent, but its oxidized form (M(III)) is not.
- The M(III) form is a potent oxidizing agent.
- Chromatographic detection methods can be enhanced by postcolumn reagent addition.
Purpose of the Study:
- To develop a novel postcolumn fluorogenic detection method for chromatography.
- To utilize the redox properties of Tris(2,2'-bipyridyl)ruthenium for analyte detection.
- To establish a sensitive detection method for peptides containing oxidizable amino acid residues.
Main Methods:
- A non-fluorescent M(III) ruthenium complex was used as a postcolumn reagent.
- The M(III) reagent was generated online using a porous carbon electrode.
- Analyte oxidation of the M(III) reagent was detected via fluorescence of the generated M(II).
- The method was applied to the detection of dynorphin A and its fragments.
Main Results:
- The method successfully detected peptides containing tyrosine and tryptophan residues.
- Tyrosine transferred approximately one electron, and tryptophan transferred approximately four electrons to the M(III) reagent.
- Dynorphin A and tyrosine-containing fragments were detected at 100 nM (14 pmol) using laser-induced fluorescence.
- A mass detection limit of 80 fmol was estimated for tyrosine-containing fragments.
Conclusions:
- A novel redox-based fluorescence detection scheme was demonstrated for chromatographic analysis.
- This method provides an alternative to electrochemical detection, free from surface fouling.
- The technique offers potential for extremely low detection limits, particularly for peptides with oxidizable residues.