2. Chapter Two Experimental Section This chapter describes the instrumentation, chemicals and procedures used throughout this work, unless specified otherwise in a particular chapter. 2.1 INSTRUMENTATION 2.1.1 Sample Preparation Techniques: MAE and SFE Microwave-assisted extraction was carried out using a MarsX (CEM, Matthews, NC, USA, 1200-Watt) laboratory microwave extraction system equipped with a solvent detector. The instrument is able to extract concurrently fourteen solid samples in PTFE extraction vessels under identical extraction conditions. It controls either pressure or temperature depending on which parameter reaches its control set point first. SFE in the dynamic mode was performed using a Jasco (Tokyo, Japan) PU-980 HPLC pump. A Trace ThermoQuest (Rodano, Italy) Series 2000 GC oven was used to produce the required critical temperature of CO2. A 10-ml stainless steel sample cell (Jasco) was installed in the GC oven. Methanol was added to the CO2 at intervals with a second PU980 HPLC pump. 43 2.1.2 Sample Measurement Systems: HPLC-UV, LC-MS and GC-ECD The HPLC system consisted of a Shimadzu (Kyoto, Japan) LC-6A pump, a Rheodyne (Cotati, CA, USA) 7010 injector equipped with a 20-µl loop, a Shimadzu SPD-6AV UVVIS detector and a Shimadzu C-R6A integrator. The LC-API-MS analyses were performed with a Thermo Separation gradient HPLC system (Model SCM 1000) coupled to a Finnigan MAT LCQ ion-trap mass spectrometer (all ThermoQuest, San Jose, CA, USA). The instrument was initially tuned based on a mixture of caffeine, L-methionyl-arginyl-phenylananyl-alanine acetate·H2O and a mixture of perfluoroalkoxycyclotriphosphazenes in both positive and negative ionization modes as suggested by the manufacturer. GC analysis was performed by a Hewlett-Packard (Palo Alto, CA, USA) 5890 Series II gas chromatograph equipped with a 63Ni electron-capture detector. Separations were conducted using a DB-5, 30 m × 0.32 mI.D. capillary column (0.25 µm stationary phase thickness) (J&W, Folsom, CA, USA). The carrier gas was purified nitrogen at a flow rate 1.5ml/min. 44 2.2 REAGENTS Analytical-grade PAHs (naphthalene and phenanthrene) were purchased from Supelco (Bellefonte, PA, USA) or Ultra-Scientific (North Kingston, RI, USA). The stock standard solutions were prepared in acetone at concentrations of 2.0 mg/ml for each compound and stored at - 4°C. Working solutions were prepared by diluting the stock solutions with acetone. Polychlorinated biphenyls (PCB-1242 and PCB-1248) were purchased as individual standard stock solutions containing a nominal concentration of 100 µg/ml in methanol from Ultra-Scientific. Atrazine (purity 98%) and simazine (purity 99%) were purchased from Supelco. Stock solutions (1000 µg/ml) were prepared by dissolving the solid standards in acetone and stored under refrigeration. Working solutions were obtained by diluting with acetone. The carbamates: propoxur (purity 99%), methiocarb (purity 99%), propham (purity 99.5%), thiuram (purity 98%) and chlorpropham (purity 99.5%) were supplied by ChemService (West Chester, PA, USA). The stock solutions containing each compound at 1000 µg /ml were prepared in methanol and diluted with the same solvent to obtain working solutions at various concentrations. They were stored at 4°C. 45 All solvents used in this study were either pesticide-grade or HPLC-grade and obtained from Fischer Scientific (Fair Lawn, NJ, USA). The water used (thereafter referred to as ultrapure water) was purified using a Milli-Q (Millipore, Bedford, MA, USA) water purification system. All the samples (solutions and extracts) were filtered through 0.45-m membranes (Millipore) and degassed in an ultrasonic bath before use. 2.3 PROCEDURES 2.3.1 Preparation of Water Samples Natural water samples were collected from local sites. They were filtered through a 0.45µm membrane (Millipore) to remove particulate matter before use. Freshly spiked water samples were prepared by adding an appropriate volume of spiking solutions into the natural water samples prepared above and ultrapure water. All the samples were degassed by an ultrasonic bath before LC-MS analysis. 2.3.2 Preparation of Soil Samples 2.3.2.1. Blank soils Blank soils, collected from local sites were air-dried, pulverized and sieved through a 60mesh sieve. In order to remove possible traces of PAHs, PCBs, triazines, carbamates and other organic contaminants, 100 g of each of the soil sample was immersed in 200 ml 46 each of methanol, acetone, dichloromethane and n-hexane for at least 24 hr sequentially. The treated soil was spread out on a tray and air-dried for hr in a fume hood to remove as much solvent as possible. Finally, it was determined that there were no detectable levels of the target analysts in soil samples before spiking. 2.3.2.2. Freshly spiked soils Freshly spiked soil samples were prepared by adding an appropriate volume of spiking solutions into the soils. To ensure that the analytes were well distributed, a reasonable amount of acetone was added to moisten the soil and careful agitation was performed. These standards were prepared 10-14 days prior to soil analysis. 2.3.2.3. Aged spiked soils Aged spiked soil samples were obtained by storing the above spiked soil in bottles in a dry, dark location for 60 days. It was assumed that the contaminants were uniformly distributed in the sample and that, because the soil still retained residual moisture throughout the storage period, any analyte-matrix interactions would have occurred, over the weathering period, to a similar extent to those in real contaminated soil with similar properties. 47 2.3.3 Preparation of Biological Samples Goldfish, tortoises (Trachemys scripta elegans), and green alga (Sea lettuce: Ulva lactuca) were purchased in a local market. In order to remove possible trace organic contaminants prior to preparation, all of them were washed in fresh running water and then rinsed with deionized water. The fish samples were grinded in a mortar and pestle with liquid nitrogen until a homogenous fine powder was obtained. In order to meet the fortification standards for recovery and analytical precision, a portion of each fish sample was added an appropriate volume of standard solution, thereby obtaining the required levels of target analytes. To ensure that the analytes were well distributed, enough methanol was added to just moisten the samples that were then stirred. After freeze-drying for 24 hours, the fine powders were accurately weighed and placed in polyethylene flasks. Tortoises were removed from their shells after being submerged in liquid nitrogen, and tissues were then crushed and lyophilized. Spiked tortoise samples were prepared in a similar manner as the fish. The remainders of the fish and tortoise samples were stored at -20 °C for later analyses. All samples were analyzed as freeze-dried powders. Sea lettuce was crushed until a homogeneous mass was obtained. Spiked samples used for recovery determination were prepared via the addition of standard stock solutions to the homogenized specimen (30 g) that was then left for hour to allow the spiked 48 solution to penetrate the material. The samples were then immediately freeze-dried for 24h, and finally stored in polyethylene bottles after being weighed accurately. No sieving was performed before analysis. To assess possible contamination from sample preparation, together with each series of samples, blanks were made to ensure there were no detectable levels of the target analytes in each freeze-dried blank samples before spiking. 49 . running water and then rinsed with deionized water. The fish samples were grinded in a mortar and pestle with liquid nitrogen until a homogenous fine powder was obtained. In order to meet the. possible traces of PAHs, PCBs, triazines, carbamates and other organic contaminants, 100 g of each of the soil sample was immersed in 20 0 ml 46 each of methanol, acetone, dichloromethane and n-hexane. levels of the target analysts in soil samples before spiking. 2. 3 .2. 2. Freshly spiked soils Freshly spiked soil samples were prepared by adding an appropriate volume of spiking solutions into