DETECTION OF BIOMOLECULES BY CHEMILUMINESCENCE

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We have reported that the electrochemiluminescence reaction of a reducing agent with tris (bipyridine) ruthenium (III) (Ru(bpy)3+3) is more broad in scope and not just seen for a few compounds such as hydrazines or oxalate. In particular, mono-, di-, and tri-alkyl amines can be detected in the order indicated at nmole to pmole levels. To allow facile study of this fast (<1 sec) reaction, we have modified an HPLC fluorometer for post- column mixing. It is capable of detecting tripropylamine at 0.04 pmoles. Pharmaceuticals difficult to detect in the UV such as erythromycin and clindamycin-2-phosphate have been assayed easily at the ppb level. Three areas of study are proposed building on these completed projects. First, to permit faster start-up and more versatile operation, we plan to do on- line oxidation of Ru(bpy)3+2 to Ru(byp)3+3 using the ESA coulometric flow cell. Electrochemical pretreatment of the mobile phase to lower detection limits can also be done with another coulometric guard cell. To enhance chemiluminescence lifetime, cationic, anionic, zwitterionic, and nonionic surfactants will also be used to produce a micellar solution. Secondly, fundamental studies of Ru(bpy)3+3 chemiluminescence involving the mechanism of this reaction with both amines and sulfur compounds. Wide bore capillary GC will be used to quantify the suspected products. Finally, analytical applications using HPLC for the determination of biomolecules such as amino acids, peptides, and proteins are spelled out. In particular, secondary amino acids such as proline and hydroxyproline in proteins such as collagen will be focused on.