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The HPLC system consisted of two Rainin HPX pumps (10 ml head), aRheodyne injector (model 7125) with a 20-ul loop, an LDC FluoromonitorIII, and a Hewlett-Packard recorder-integrator (model 3390A). The column used for the quantitation of EQ was a reversed-phase C18 (5 um 4 mm ID x 15 cm) (SGE, Austin, Tex.) protected with a guard column filled with Perisorb RP 18 (Upchurch Scientific). The mobile phase was acetonitrile-10 mM ammonium phosphate, monobasic, containing 1 mM tetrabutyl-ammonium hydrogen sulfate and adjusted to pH 5.9 with solid ammonium hydrogen carbonate (7:3, v/v) and the flow rate was 0.9 ml/min.The ammonium phosphate buffer was prepared every 5 d. The analyses were performed at ambient temperature; the detector range was set at 20-100 and the injection volume ranged 0.5-20ul according to the concentration of EQ in the sample. EQ HCI was prepared as described (Kim, 1985) by converting the available dark oily EQ (a gift from Monsanto) into crystalline EQ HCI and was recrystallized several times from methanol-acetonitrile to give a colorless analytical sample (mp 192-193¡ãC). The standard solution was prepared weekly in methanol (10 mg/ml) and immediately diluted with acetonitrile to 0.1 ug/ml and kept in the dark at room temperature. [ Last edited by zl0921516 on 2012-5-31 at 14:55 ] |
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