DETERMINATION OF DANSYL AMINO-ACIDS USING TRIS(2,2'-BIPYRIDYL)RUTHENIUM(II) CHEMILUMINESCENCE FOR POSTCOLUMN REACTION DETECTION IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Dansyl amino acids are determined using a tris(2,2'-bipyridyl)rutheniu m(II) [Ru(bpy)(3)(2+)] chemiluminescence (CL) post-column reaction. Th e derivatized amino acids are separated on a reversed-phase column (Zo rbax ODS) using a mobile phase containing 15% acetonitrile in 25 mM tr ifluoroacetate at pH 7.5. The eluted dansyl amino acids are combined a t a mixing tee with 2.0 mM Ru(bpy)(3)(2+) CL reagent. The combined sol ution passes through a flow cell, which has a dual platinum electrode at a potential of 1250 mV. On the surface of the electrode Ru(bpy)(3)( 2+) is oxidized to Ru(bpy)(3)(3+) which reacts with dansyl amino acids to emit light. CL intensities vary only slightly with CL reagent or w ith mobile phase flow-rates. CL intensities increase significantly wit h an increase in pH over the pH 6-8 region. A slight increase in CL in tensity is obtained with increasing acetonitrile in the mobile phase. The working curve for dansyl-derivatized glutamate covers three orders of magnitude with a detection limit of 0.1 mu M (2 pmol injected).