Analysis of Polycyclic Aromatic Hydrocarbons in Soil with AgilentSampliQ QuEChERS AOAC Kit and HPLC-FLD


An HPLC-Florescence Detection (FLD) method was developed and validated for the


determination of sixteen polycyclic aromatic hydrocarbons (PAHs) in soil. The analysed


PAHs include naphthalene (Nap), acenaphthylene (Acy), acenaphthene (Ace),


fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fln), pyrene (Pyr),1,2- benza[a]anthracene (BaA), chrysene (Chr),


benzo[e]pyrene (BeP), benzo[e]acenaphthylene (BeA), benzo[k]fluoranthene (BkF), dibenzo[a,h]anthracene (DahA),


benzo[g,h,i]perylene (Bghi)P and indeno[1,2,3- cd]pyrene (InP). The method employs aquick, easy, cheap, effective, rugged and safe (QuEChERS) multiresidue sample


preparationprocedure adopted from the Association of Analytical Communities (AOAC) Official method 2007.01 for extraction and cleanup. The analytes were separated onan Agilent ZORBAX Eclipse PAH column (4.6 mm × 50


mm, 1.8 µm) by gradient elutionwith a binary system of acetonitrile - water with subsequent fluorescence detectionset at appropriate excitation and emission wavelengths. The analyte recoveries ranged from 86.0% to 99.2% with


relative standard deviations ranging from 0.6% to 1.9% at three different fortification levels. The limits of detection and quantification ranged from 0.005 to 0.78 and 0.02 to 1.6 ng/g, respectively.


Figure 1: Chemical structures for the polycyclic aromatic hydrocarbons used in the study.


Polyaromatic hydrocarbons, or poly- nuclear aromatic hydrocarbons (PAHs), are fused ring aromatic compounds classified by the number of carbon rings as well as their carcinogenicity.


The two and three ring PAHs are non- Column Flow rate


Column temperature Injection volume Mobile phase Gradient


Agilent ZORBAX Eclipse PAH C18 4.6 × 50 mm, 1.8 µm 0.8 mL/min


18 °C 5 µL


A = Deionized H2O B = CH3CN T (min)


0


1.5 7


13 Detection


Wavelengths: Time (min)


0 – 5 (dark blue) 0 – 14 (red) 0 – 14 (light blue)


% B 60 60 90


100


UV at 230 nm (Acy) and varying fluorescence excitation (Ex) and emission (Em) wavelengths


Ex/Em


wavelengths (nm) 260/352


260/420 260/460 Table 1: HPLC Conditions Used for Separation of PAHs PAH detected


Nap, Ace, Flu, Phe, Chr


Ant, Pyr, BeP, DahA, BghiP


Fln, 1,2- BaA,BeA, BkF, InP


carcinogenic, while several of the four, five and six ring PAHs are carcinogenic. The four ring PAHs, chrysene and benzo- [a]anthracene, the five ring PAHs, benzo[a]pyrene, benzo[b]fluoranthene, benzo [k] fluoranthene and dibenzo [a,h] anthracene, and the six ring PAH, indeno [1,2,3-cd] pyrene, are carcinogenic PAHs. Benzo [a] pyrene is the most potent carcinogen among the PAHs [1].


The US-EPA and EU lists sixteen of these


PAHs as hazardous compounds [2]. Generally PAHs are lipophilic compounds that show a high affinity for organic matter and their determination in soil always requires powerful extraction techniques to release the strongly sorbed contaminants from the soil material [3]. Several extraction methods (soxhlet, liquid-liquid or solid phase extraction) for sample preparation of soil have been investigated and most of these involved an eva- poration step which leads to the loss or low recoveries of the volatile PAHs such as naphthalene [4].


The AOAC QuEChERS method has been widely applied in the analysis of pesticides in food since it was introduced by United States Department of Agriculture (USDA) scientists [5].


More recently, the technique has branched out into new application areas outside of food safety. In general, there are two major steps: extraction and dispersive SPE cleanup. The method uses a single step buffered acetonitrile extraction while simultaneously salting out water from the aqueous sample using anhydrous magnesium sulfate (MgSO4) to


induce liquid-liquid partitioning. After removing an aliquot from an organic layer, for further cleanup, a dispersive solid phase extraction (dSPE) is conducted using a combination of primary secondary amine (PSA) sorbent


to remove fatty acids from other components and anhydrous MgSO4 to reduce the remaining water in the extract. Other sorbents, such as graphitized carbon black (GCB), may be added to remove pigments and sterol, or C18 to remove lipids and waxes.


This application note presents a method for the analysis of PAHs at trace levels in soil with HPLC-Fluorescence detection (FLD). The HPLC methods are useful for PAH analysis since UV and fluorescence detection offer enhanced selectivity over other techniques such as GC with flame ionization detection [6]. The method includes sample preparation with SampliQ AOAC Buffered Extraction kit (p/n 5982-5755) and SampliQ AOAC Fatty Dispersive SPE 15 ml kit (p/n 5982-5156). Chemical structures of the PAHs in this study are shown in Figure 1.


Experimental Reagents and Chemicals


All reagents were analytical or HPLC grade. Acetonitrile (CH3CN) and PAHs were purchased from Sigma-Aldrich (St. Louis, MO, USA). The water used was


from a MilliQ system (Milford, Mass, USA). The mobile phase was filtered through a Whatman membrane filter (47 mm diameter and 2 µm pore size).


Standard Solutions


Standard stock solutions (1 mg/mL) were prepared by dissolving 10 mg of the desired PAH in 10 mL CH3CN and stored at –20°C. All working solutions were prepared fresh daily by serial dilution with CH3CN.


Equipment and Material


The analysis was performed on an Agilent 1200 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump and a fluorescence detector (FLD) set at varying excitation and emission wavelengths (Table 1). The selection of the excitation and emission wavelengths for fluorescence detection was based on the optimum responses for the various PAHs. Since acenaphthylene does not fluoresce, UV detection at 230nm was used. Separation of the compounds was achieved


on an Agilent ZORBAX Eclipse PAH column (4.6 mm × 50 mm, 1.8 µm), p/n 959941-918. The data was processed by HPLC 2D Chemstation software.


Extraction and cleanup were achieved with Agilent SampliQ Buffered QuEChERS AOAC Extraction kit, p/n 5982-5755 and SampliQ QuEChERS AOAC Dispersive SPE kit, p/n 5982-5058 (Agilent Technologies).


August/September 2010 AET


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