High-performance liquid chromatography gradient formation

FIGURE 3 4-Nitrosophenol formation from the reaction of peroxynitrite (ONOO ), nitric oxide (NO), or both (added simultaneously) with phenol at pH 6.0, 7.4, and 8.0. All reactions contained 2 mM phenol and 0.1 mM DTPA (to minimize redox-active metals) in a final volume of 2 ml of 0.1 M potassium phosphate at the indicated pH at 37"C. ONOO", nitric oxide (210 jU.1 of 1.9 mM solution), or both were added simultaneously to rapidly stuxed solutions. The final pH was measured. Reaction products were measured by high-performance liquid chromatography (8-cm C-18, 100 mM ammonium formate, pH 3.5, linear gradient from 5% to 80% acetonitrile over 10 min). Detection at 280 and 365 nm (nitroso and nitro adducts absorb at 365 nm) was used to identify and quantify reaction products relative to authentic standards. 

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