Chemical and reagents

A primary mixture standard solution with a concentration of 2000 µg/mL of each component of the 16 PAHs in a benzene/dichloromethane mix from AccuStandard was used. Deuterated internal standards (IS) containing naftalene-d₈, acenaphtene-d₁₀, phenantrene-d₁₀, chrysene-d₁₂, and perylene-d₁₂ each at 4000 µg/mL in dichloromethane were obtained from Hewlett-Packard.

The secondary mixture standard solution (20 µg/mL for each of the 16 PAHs) was prepared by the dilution of 1 mL of the PAH primary mixture to 100 mL with hexane. This solution was stored in a refrigerator in brown glass bottles and in the dark. A second solution (40 µg/mL) of IS was also prepared by dilution of 1 mL of a commercial solution of IS to 100 mL with hexane. Finally, calibration solutions were prepared by weight from different amounts of the secondary standard solution of the 16 PAHs, 40 µL of 40 µg/mL IS standard solution, being the final volume 1.5 mL. The PAH concentration of the calibration solutions ranged between 0.10 µg/mL and 1.95 µg/mL. These solutions were prepared at the moment of calibration.

High purity chromatographic quality hexane, acetone and dichloromethane were used. The validation of the methodology was carried out by the analysis of a certified soil (CRM-104-100 [LGC Promochem]) with PAH levels 0.77 and 24.8 mg/kg.

Instrumentation

A Hewlett-Packard (Palo Alto, CA, USA) model 6890 gas-chromatograph coupled to a Hewlett-Packard 5973 mass spectrometric detector working in single ion monitoring mode (SIM) were used for PAHs analytical separation and detection with an automatic injection system 7683 from Hewlett-Packard.

The GC separation column was a 30 m × 0.33 mm i.d. × 25 µm thin thickness DB-XLB (equivalent to 5 % phenyl, 95 % methylpolysiloxane) fused silica capillary column (Hewlett- Packard). An electronic pressure control was utilized to maintain a constant gas carrier helium flow of 1.0 mL/min throughout the oven program. The injector and detector port temperatures were 300 and 230 ºC, respectively. The oven program temperature was 60 ºC (hold 1.5 min), rate 8 ºC/min to 270 ºC (hold 10 min), and rate 5 ºC/min to 280 ºC (hold 13 min). Split mode was used.

The mass spectrometric detector was operated in electron ionization mode (EI) with ionization energy of 70 eV; the source and quadrupole temperatures were 230 and 150 ºC, respectively; the dwell time was 100 ms for all the ions.

A Milestone Ethos Sel was used for microwave-assisted organic extraction. This device is furnished with accessories for filtration (FilterEx-12), evaporation (EvaporEx-12) and software (Easywave), and the microwave oven incorporates a vacuum system (Milestone Vac 2000).

Several special materials for microwaves, e.g. Teflon pumps, microfilters, vials for filtration, weflon stirrers, etc., were provided for the Milestone Ethos Sel. A system for Soxhlet extraction (Selecta, Spain), a vacuum manifold (Whatman, Middlesex, England) and a rotatory evaporator (Heidolph, Nüremberg, Germany) were also used.

Separation of different PAHs by gas chromatography and mass spectrometry detection

By using a standard sample of PAHs containing the 16 USEPA pollutants, chromatographic separation was carried out with the internal standard (IS) technique in order to correct any possible instrumental drift. The deuterated homologous of PAHs (naftalene-d₈, acenaphtene-d₁₀, phenantrene-d₁₀, chrysene-d₁₂, and perylene-d₁₂) were used as IS. These compounds are not present in real samples, and they have physical and chemical properties similar to those analytes providing interference-free GC-MS signals.

The mass-spectrum of perfluorotributylamine was used for a wide mass-calibration range. Detection was carried out using the selection ion monitorization (SIM) mode, by measuring only the m/z ratio of the most abundant molecular fragments.

Table I shows retention times of the main and secondary ions of PAH and internal standards monitored, selected for each ion.

Extraction, filtration and preconcentration of PAHs from the soil samples procedure

The extraction of 16 PAHs from soil samples was carried out in the Milestone Ethos Sel microwave oven. One of the 12 vessels, with the same volume of solvent as the samples, was aimed at measuring the applied temperature. The vial of filtration belonging to each vessel was of use to test a microfilter, a performance disc (for microfilter scaling), a coated magnet on quartz and the base of the vial made from weflon. Two grams (2.0000 g) of sample soil were weighed and deposited in the filtration vial and then 25 mL of a hexane/acetone 1:1 mixture was added to the filtration vial allowing 10 mL of the mixture to remain in the extraction vessel. Then the extraction vessels were opened, the microwave power was set to 500 W, the stirring to 100 % and the system was started following the optimized program. A temperature of 120 ºC was selected in two steps, a first step from room temperature to 120 ºC for 10 min, and then temperature was maintained for another 10 min.

When the extraction program was finished and temperature was lower than 40 ºC (after approximately 20 min), the filtration vessels were opened and the filtration was carried out using the FilterEx-12 system by connecting the vacuum pump. After filtration, the vials were coupled to the EvapEx-12 evaporation system and this set was introduced into the oven. Then, the microwave power was tuned to 135 W, while a vacuum was applied simultaneously. In 14.5 min the volume was reduced to 2 mL. This extract was cleansed by solid phase extraction (SPE) using two coupled silica gel cartridges, previously conditioned with hexane. The PAHs were retained there and they were eluted by 5 mL (two times) of a hexane/acetone 7:3 mixture. This extract was pre-concentrated again to 2 mL by evaporation (applying the vacuum commercial system to 135 W of power during 12 min). After the adequate addition of internal standards and apposite dilutions (1 preconcentrate:1 hexane) dilution for naphthalene, acenaphthylene, acenaphthene, fluorene, and dibenzo(a,h)anthracene determination; (1 preconcentrate:10 hexane) dilution for phenanthrene, anthracene, pyrene, venzo(a)anthracene, indene(1,2,3-cd)pyrene, and venzo(g,h,i)perylene determination; and (1 preconcentrate:50 hexane) dilution for fluorantene, chrysene, venzo(b,k)fluorantene, and venzo(a)pyrene determination, the resulting treated and diluted sample solutions were injected in the gas chromatograph.

Optimization of the microwave-assisted solvent extraction

The optimum experimental parameters for the extraction of PAHs by MASE were investigated by using a sample soil contaminated with these pollutants.

Extraction temperature

Temperatures of 80, 100, 115, 120, 120* and 130* ºC were selected according to the literature (^{López-Ávila et al. 1994}, ^{Barnabas et al. 1995}, ^{Chee et al. 1996}). The asterisks mean that in such cases the applied temperature program was carried out as follows: a first step from room temperature to final temperature (10 min) and then the final temperature was maintained for 10 min. The rest of the parameters (extraction time, volume of the solvent and solvent) were maintained constantly.

Table II shows the concentrations for the 16 studied PAHs according to the applied temperatures with the exception of dibenzo(a,h)anthracene. As can be seen, for PAHs containing 2-4 aromatic rings, better extraction conditions were obtained by applying the extraction program in two steps at 120* ºC, being 100 ºC the best option for PAHs with 5-7 aromatic rings. In subsequent studies, we selected a temperature of 120* ºC in two steps, as a compromise solution. This temperature allows for the extraction of higher amounts of most PAHs, especially the most volatile ones (with lower molecular mass), which can be found in soils at lower concentrations.

Extraction time

A extraction temperature of 120 ºC (*) in two steps was selected (first, from room temperature to 120 ºC in 10 min for all cases, and a second step at 120 ºC for 5, 10, 15 and 20 min), and the same parameters referred in the previous paragraph were maintained constantly. The obtained results are shown in table III. As can be seen, similar considerations as those formulated in the study of temperature can be applied: for compounds having 2-4 aromatic rings in their molecule, the optimum extraction time was 20 min at 120 ºC, but for PAHs with 5-7 aromatic rings, the time was 15 min, although differences of concentrations for both times are not significant as has been mentioned in the literatures. ^{Budzinski et al. (1999)} and ^{Letelier and Budzinski (1999b)} have in mind the same criterion for the study of the temperature. A compromise solution of 20 min as extraction time in the second step was selected.

Volume of solvent

The volume of solvent used for MASE is another parameter that has an effect on the extraction of PAHs from soil samples. In order to analyze its influence on the extraction of analytes by using an hexane/acetone 1:1 mixture as extraction solvent, several volumes (20, 25, 30, and 35 mL) of such mixture were used under two conditions: (1) the volume of solvent must be enough to cover the soil sample and (2) 10 mL of this volume must be added to the extraction vessel to secure the direct contact of the sensor with the solution, and the rest 10, 15, 20 and 25 mL should be added to the filtration vial. The capacity of the vials (26 mL) limits the use of a higher volume. Obviously, for the reduction to a final volume of 2 mL by using microwave energy (135 W) and vacuum together, different time lapses must be applied: 12 min for 20 mL, 14.5 min for 25 mL, 17 min for 30 mL and 20 min for 35 mL. During this study, the rest of the parameters remained constant: 2.0000 g of contaminated soil, 120 ºC as extraction temperature in two steps: 20 min as extraction time, 500 W for the microwave power supply, stirring 100 % and 20 min as a final cooling step. Table IV shows the concentrations of PAHs found on the sample soil when different volumes of solvent mixture were used for the extraction. Again, slight differences in concentration values for different volumes of solvent were observed, thus it can be said that the volume of solvent is not an important factor for the extraction of PAHs from soil samples, as has been previously mentioned (^{Barnabas et al. 1995}, ^{Chee et al. 1996}). For subsequent experiments, a volume of 25 mL of a hexane/acetone 1:1 mixture, added into the filtration vial, was selected as the optimum alternative for the majority of PAHs.

Choice of solvent

Acetone has been recommended as a more suitable solvent than hexane for the extraction of PAHs from soil samples (^{Barnabas et al. 1995}). The acetone molecule has a permanent dipole that can induce dipole-dipole interactions with numerous π-type electrons from PAHs. On the other hand, hexane is a non-polar solvent, which means that it is unable to generate such interactions. We have analyzed whether there is a difference between using a mixture of hexane/acetone and acetone alone for the extraction. Once the previous parameters were optimized, the results obtained by using the hexane/acetone mixture were compared with those obtained by using acetone with the same purposes. In this case, the constant parameters were 20 min as extraction time, 500 W for the microwave power supply, stirring 100 % and 20 min as a final cooling step. The results obtained are shown in table V, where we can verify that the mixture hexane/acetone is more suitable for PAHs extraction from soil samples, which agrees with prior research (^{López-Ávila et al. 1994}, ^{Chee et al. 1996}).

Analytical performance characteristics

To evaluate the linearity of detector responses after MASE-GC-MS, standard solutions containing all 16 PAHs in a range of concentrations between 35 and 2000 ng/mL were prepared. The results are summarized in table VI. For all PAHs, the responses were linear over the range tested, with regression coefficients better than 0.9940 (n = 3). The linear range was established by plotting the quotient area of the analytic/area of IS vs. the quotient concentration of analyte/concentration of IS. The limits of detection (LOD) and limits of quantification (LOQ) were calculated from equations defining calibration lines by measuring (by triplicate) three solutions containing 16 PAHs in very low concentrations (35, 155 and 254 ng/mL) with the ^{Long and Winefordner (1985)} criterion. The values found were 10-32 ng/mL for LOD and 32-108 ng/mL for LOQ, which shows that quantification should be possible for PAHs at levels found in soils and sediments (^{Popp et al. 1997}, ^{Saim et al. 1997}, ^{Schantz et al. 1997}). Repeatability, which was evaluated for two levels of concentrations (155 and 531 ng/mL), with and without IS, was satisfactory with standard relative deviations (RDS) between 1% and 6% if IS was employed.

The analytical advantages of the MASE-GC-MS procedure were previously evaluated in samples of polluted soil collected near a coke oven by comparing the observed results with those obtained for the PAHs of such samples previously extracted by using the classical Soxhlet method recommended by the ^USEPA(1995). In this latter method, the soil amount was 5.0000 g, the volume of the solvent used was 350 mL of a 1:1 mixture of hexane/acetone, and the extraction time was 18 hours at the rate of 3-4 cycles per hour. The cleaning and pre-concentration of extracts were carried out by rotoevaporation-SFE-rotoevaporation. Then the cleaned extracts were analyzed by GC-MS. The results obtained for both PAHs extraction methods under scrutiny (MASE and Soxhlet) in mg of PAH/kg of soil are shown in table VII. As can be seen, both extraction techniques provided similar values for the majority of PAHs with the exception of chrysene, benzo(b)fluoranthene and benzo(k)fluoranthene.

Validation of the MASE method and real sample application

In order to validate the proposed method, the new set-up was used to extract PAHs from the CRM 104-100 soil. The reference material came from Elisabeth River in the Chesapeake Bay, VA, USA, containing 16 PAHs (the values for benzo(k,b)fluoranthene and dibenzo(a,h)anthracene are only recommended). These two compounds of the reference material were extracted by both MASE and Soxhlet techniques.

Table VIII shows the certified values for different PAHs with their confidence limits; concentrations (mg/kg) of the pollutants obtained by the MASE and Soxhlet extraction methodologies; and recovery values for the different PAHs with final determination by GC-MS. In both cases, MASE and Soxhlet, we found that our values fall into the confidence limits established for the reference material. However, the recovery values obtained for PAHs turned out to be higher than those obtained with MASE (anomalous values were observed for fluoranthene (-147 %) and benzo(g,h)perylene (-124 %). Nevertheless, once clean extracts were obtained for the analysis by GC-MS, it was necessary to handle the extracts at three dilution levels: (1 preconcentrate:1 hexane) dilution for naphthalene, acenaphthylene, acenaphthene, fluorene, and dibenzo(a,h)anthracene determination; (1 preconcentrate:10 hexane) dilution for phenanthrene, anthracene, pyrene, venzo(a)anthracene, indene(1,2,3-cd)pyrene, and venzo(g,h,i)perylene determination; and (1 preconcentrate:50 hexane) dilution for fluorantene, chrysene, venzo(b,k)fluorantene, and venzo(a)pyrene determination. The necessary dilutions are the main drawback of the GC-MS determination, but they are necessary following both PAHs extraction methods.

Figure 1 shows the chromatogram of PAHs contained in the CRM104-100 soil extract using MASE as the extraction technique and 1:10 dilution.

Fig. 1 Chromatogram from the CRM 104-100 soil extract after Microwave Assisted System Extraction (Dilution 1:10)