Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO‐CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO‐CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4‐dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5 – 7 and pH 3 – 4, respectively. Dipyridamole and benzydamine hydrochloride (anti‐inflamatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.