Application of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)-phenyl]oxalate to post-column chemiluminescence detection in high-performance liquid chromatography.

Bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) was used to examine the peroxyoxalate chemiluminescence (CL) reaction for the detection of fluorescent compounds. Some fluorescent compounds (perylene, eosine, rhodamine, Rose Bengal, fluorescein and umbelliferone) gave higher CL intensities as the proportion of water in the reaction medium increased to ca. 40%, whereas dansylalanine, 8-anilinonaphthalene-1-sulphonic acid, 7-nitrobenzo-2-oxa-1,3-diazol-4-ylproline and dihydronicotinamide adenine dinucleotide gave opposite results. The effects of temperature and time on the post-column reaction in reversed-phase high-performance liquid chromatography (HPLC) were investigated. Under the optimal conditions, the detection limit for dansylamino acids was at the sub-femtomole level. The advantage of using TDPO in HPLC is its stability in the presence of hydrogen peroxide [ca. 10% loss of activity per 8 h vs. 60% per 8 h for bis(2,4,6-trichlorophenyl) oxalate].