CHEMILUMINESCENT DETECTION OF AMINO-ACIDS USING IN-SITU GENERATED RU(BPY)(3)(3+)

A technique for the detection of underivatized amino acids based on th e 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(bp y)(3)(2+) in alkaline pH in the presence or absence of an organic modi fier such as acetonitrile. The CL emission from the glassy carbon thin -layer flow cell used for the in situ studies was found to be dependen t 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 wo rking 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 f ound to provide a linear response over 3 orders of magnitude for leuci ne, 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 le ss than 8% over the course of 40 h of continuous electrolysis of Ru(bp y)(3)(2+) in the detection cell. The method is also shown to be applic able to the sensitive detection of PTH-glycine. Arguments toward the f uture miniaturization of the detection scheme in order to facilitate a pplication to capillary electrophoresis and subsequent development of new protein sequencing methodologies will be presented.