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2. Experiment

II.1. Reagents and Chemicals

All chemicals used are of analytical grade unless otherwise specified. Acetic acid, acetonitrile, ascorbic acid, citric acid monohydrate, lead(tetra)oxide, polytetrahydrogen phosphate dihydrate, etc. were purchased from Merck (Darmstadt, Germany). Ammonium hydroxide 25% (M/V), methylene chloride, methanol (HPLC grade), sodium acetate, chlorite monoblock-toluenesulfonic acid (P-Zone), obtained from Japan Tobacco Company Baker (Phillipsburg, NJ, USA). Brilliant green and N, N, N9, N9-tetramethyl-1,4phenylenediamine hydrochloride (TMPD) were obtained from Eidia (Steinheim, Germany), and from diatomaceous earth Acros (Geel, Belgium). N, N-di

was purchased from Rathburn (Walkerburn, UK). Leucoplasts - malachite green and 1 p entanesulfonic acid (sodium salt) were made from Sigma (St. Louis, MO, USA) and malachite green oxalic acid (vetrenal), from Riedel-Hae¨n (under seelze, german many). Aromatic sulfonic acid bonded SPE columns (3 ml; 500 mg) were purchased from Japan Tobacco Company Baker. Water Quality Reporting Quality (Millipore, Bedford, USA).

II.2. Instruments

Aliquots into screw-cap polypropylene tubes (50 ml and 6 ml) by Sarstedt (Nimbrecht, Germany). A series of SPE cartridges equipped with 75 ml reservoir power adapters were processed simultaneously using a vacuum manifold (Trully Baker). Samples were sun-dried using an evaporator manifold, manufactured by Pierce (Rockford, IL, USA).

Two.5. Liquid Chromatography-Mass Spectrometry

For LC-MS analysis, 10 ml aliquots are injected continuously on the HPLC system with two HPLC pumps (PE200 series, Applied Biosystems), than autowas sampling rate (PE 200 series, Applied Biosystems) and an API-365 mass spectrometer detector (Applied Biosystems). Credit-mass spectrometry was controlled by the Analyst software package (version 1.1). The HPLC column was a Phenomenex Lunar C (under 5032 mm, Phenomenex, Torrance, USA) with a guard column (SecurityGuard C18 4*2 mm, Phenomenex). The mobile phase was a mixture of 50 mM ammonium acetate, pH 4.4, and acetonitrile (2:3, v/v), flowing at 200 ml/min. Before the eluent is introduced into the ionization chamber, for her detection of analytical results, the effluent is passed through a post-column reactor filled with lead(tetra)nitrogen oxide and diatomaceous earth (see Section 2.4).

The MS is equipped with an electrospray interface operating at an ionization voltage of 5500 V and a

source temperature of 400 8C. The entrance, declustering and focusing potentials were set to 29, 40 and 180 V, respectively. Tandem mass spectrometry analysis, using multiple reaction monitoring (MRM method) mode. Collision Energy (CE) is optimized for trace element determination of each product ion. The following traces were monitored: m/z 329.3 → m/z 165.0 (CE 75 V), m/z 329.3 → m/z 208.0 (CE 55 V), m/z 329.3 → m /z 313.3 (CE 45 V) and the internal standard m / z 385.0 → m / z 341.0 (CE 50 V) (Figure 1).