ROLE OF ELECTRON-DONATING WITHDRAWING CHARACTER, PH, AND STOICHIOMETRY ON THE CHEMILUMINESCENT REACTION OF TRIS(2,2'-BIPYRIDYL)RUTHENIUM(III) WITH AMINO-ACIDS

A postcolumn chemiluminescent technique for the detection of underivatized amino acids using electrogenerated tris-(2,2'-bipyridyl)ruthenium(III) is described. The reaction chemistry has been investigated, and it has been shown that the electron-withdrawing/donating character of the R group attached to the alpha-carbon of the amine influences the chemiluminescent efficiency of the reaction. Stoichiometric studies conducted on four amines produced a mole ratio of 2:1, ruthenium: amine. At optimum reaction conditions the relative intensities of the primary amino acids tested varied by a factor of 55, with serine yielding the lowest chemiluminescence and leucine yielding the highest chemiluminescence. Detection limits for serine and leucine have been calculated to be 135 and 3 pmol, respectively, at a S/N of 3. Linearity has been demonstrated over 2 orders of magnitude for Leu. The ability to implement this system for postcolumn chemiluminescence detection of amino acids following a protein digest has been demonstrated.