24 Environmental Analysis


Instrument conditions HPLC conditions


Weigh 5 g soil homogenate into a 50 mL centrifuge tube Spike samples with 2000 µL spiking solution Add 5 mL water Add 10 mL CH3CN Add SampliQ QuEChERS AOAC salt packet Transfer 5 mL aliquot to SampliQ QuEChERS Dispersive SPE 15 mL tube Filter through a 0.45 um PVDF syringe filter Transfer 1 mL extract to an autosampler vial Samples are ready for HPLC-FLD analysis Figure 2: Flow chart of QuEChERS AOAC sample preparation procedure. Sample preparation


The soil sample was collected from the local botanical garden in Grahamstown, South Africa, air dried at ambient temperature and then sieved to obtain a homogeneous sample.


Extraction


A 5g sample of soil homogenate was placed into a 50mL centrifuge tube from the SampliQ QuEChERS AOAC Extraction kit. Samples were spiked with appropriate spiking solutions to yield the best working solutions for recoveries


and reproducibility studies. A 2000µL volume of spiking solution was added to all samples except the blank. Next, 5.0mL of water was added to the tube,


and the tube shaken vigorously for 1 min. A 10 mL amount of CH3CN was then added, followed by an Agilent SampliQ QuEChERS AOAC extraction


salt packet (p/n 5082-5755), which contained 6g of anhydrous MgSO4 and 1.5g of anhydrous NaOAc. The sample tubes were hand shaken vigorously for 1 min then further centrifuged at 4000rpm for 5min.


Dispersive-SPE Cleanup


A 6.0 mL aliquot of the upper ACN layer was transferred into a SampliQ QuEChERS AOAC Dispersive SPE 15 mL tube. This SPE tube contained 400


mg of PSA and 1200mg of anhydrous MgSO4. After one minute of shaking, the tubes were centrifuged at 4000rpm for 5 min. A 4 mL aliquot of the


extract was filtered through a 0.45µm PVDF syringe filter, then 1000µL extract was placed in an autosampler vial for HPLC-FLD analysis.


Results and Discussion Chromatographic analysis


The separation of the 16 PAHs was obtained on the Agilent ZORBAX Eclipse PAH column (4.6mm × 50mm, 1.8µm) by gradient elution with a binary system of acetonitrile – water. The chromatogram of the standard mixture is


presented in Figure 3. A chromatogram of the blank soil extract is presented in Figure 4 while overlay chromatograms of the spiked soil sample at level 1 (Table 3) are shown in Figure 5.


For detection and quantification, the fluorescence detector was set at varying emission wavelengths (Table 1) to accommodate the diverse absorption intensities of the PAHs. The overlays of Figures 3 and 5 are color- coded according to the chosen excitation and emission wavelengths. The dark blue portion of the chromatogram used the following excitation /emission wavelengths: 260-nm/352-nm; the red portion: 260-nm/420-nm and the light blue portion: 260-nm/440-nm. However, due to lack of a fluorophore, UV detection at 230nm was employed for acenaphthylene.


Figure 3: Overlay HPLC – FLD chromatograms of the standard mixture containing: 1. Nap 2. Acy 3. Ace 4. Flu 5. Phe 6. Ant 7. Fln 8. Pyr 9. BaA 10. Chr 11. BeP 12. BeA 13. BkF 14. DahA 15. BghiP 16. InP. The concentration of the PAHs was 1-mg/mL. The blue portion of the chromatogram used the following excitation/emission wavelengths: 260- nm/352-nm; the red portion: 260-nm/420-nm and the light blue portion: 260-nm/440-nm. However, due to lack of a fluorophore, UV detection at 230 nm was employed for acenaphthylene. Chromatographic conditions are shown in Table 1.


Figure 4: Chromatogram of the blank soil extract. Chromatographic conditions are shown in Table 1. The baseline chromatogram used the following excitation/emission wavelengths: 260-nm/352-nm. The other excitation/emission conditions showed no other interferences.


AET August/September 2010


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