Chemiluminescent Detection of Amino-Acids using In-Situ Generated Ru(bpy)(3)(3+)

by Jackson, Warren Alan; Bobbitt II, Donald R.

A technique for the detection of underivatized amino acids based on the chemiluminescent (CL) reaction between in situ generated Ru(bpy)(3)(3+) and the amino acid is described. Glassy carbon was found to be an ideal material for the in situ generation of Ru(bpy)(3)(3+) from Ru(bpy)(3)(2+) in alkaline pH in the presence or absence of an organic modifier such as acetonitrile. The CL emission from the glassy carbon thin-layer flow cell used for the in situ studies was found to be dependent upon the concentration of Ru(bpy)(3)(2+) in the carrier buffer, the buffer flow-rate through the cell, and the applied potential at the working electrode. The detection scheme was applied to high performance ion-exchange chromatography (HPIEC) by utilizing a column packed with a polystyrene-divinylbenzene anion-exchange resin. The technique was found to provide a linear response over 3 orders of magnitude for leucine, a representative amino acid. Limits of detection (LOD) ranged from 100 fmol for proline to 22 pmol for serine at a S/N of 6. The CL-flow-injection analysis response of injected leucine was found to decay less than 8% over the course of 40 h of continuous electrolysis of Ru(bpy)(3)(2+) in the detection cell. The method is also shown to be applicable to the sensitive detection of PTH-glycine. Arguments toward the future miniaturization of the detection scheme in order to facilitate application to capillary electrophoresis and subsequent development of new protein sequencing methodologies will be presented.

Analytica Chimica Acta
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1873-4324; 0003-2670