Dansyl amino acids are determined using a tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)32+] chemiluminescence (CL) post-column reaction. The derivatized amino acids are separated on a reversed-phase column (Zorbax ODS) using a mobile phase containing 15% acetonitrile in 25 mM trifluoroacetate at pH 7.5. The eluted dansyl amino acids are combined at a mixing tee with 2.0 mM Ru(bpy)32+ CL reagent. The combined solution 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)32+ is oxidized to Ru(bpy)33+ which reacts with dansyl amino acids to emit light. CL intensities vary only slightly with CL reagent or with mobile phase flow-rates. CL intensities increase significantly with an increase in pH over the pH 6-8 region. A slight increase in CL intensity 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 μM (2 pmol injected).
Bibliographical noteFunding Information:
This work was funded, in part, by a grant from the Biotechnology Research and Development Corporation.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Organic Chemistry