Bis(2,4-dinitrophenyl) Oxalate [Chemiluminescence reagent for the determination of fluorescent compounds by HPLC and FIA]

Chemiluminescence is the phenomenon of luminescence emission when the excited state of a chemical species which occurred by a chemical reaction reverts to the ground state. Bis(2,4,6-trichlorophenyl) Oxalate (TCPO) [A5302], Bis(2,4-dinitrophenyl) Oxalate (DNPO) [A5303] are drawing attention in the aspects of high quantum yields and stability. The emission mechanism is shown below.¹⁾

1) Produce an active intermediate (1,2-dioxetanedione) through the reaction with H₂O₂.
2) The intermediate excites a fluorescent substance from the ground state to an excited state.
3) The fluorescent substance in the excited state returns to the ground state with the emission of light.

Based on this chemiluminescence reaction, the quantifications of H₂O₂ and glucose,²⁾ light emitting detection of dansyl amino acid on TLC,³⁾ and quantification of polycyclic aromatic hydrocarbons⁴⁾ have been reported.

This chemiluminescence reaction was applied to the detection part of HPLC, and 10 fmol level dansyl amino acid was successfully separated and quantified,⁵⁾ which compared to the conventional method using fluorescence, showed more than 1 order (50 times) higher sensitivity. Furthermore, Imai and co-workers have developed the assay system of dansyl amino acid making use of the DNPO-H₂O₂ luminous reaction which has been applied to flow-injection analysis.⁶⁾ The detection limit of dansyl alanine is 5 fmol.