Anal. Chem. 1999, 71, 1491-1495
Articles
Subcritical Water Extraction Coupled to High-Performance Liquid Chromatography Yu Yang* and Bin Li
Department of Chemistry, East Carolina University, 205 Flanagan, Greenville, North Carolina 27858
Subcritical water has been successfully used as an environment-friendly extraction fluid for many classes of organic compounds. While only solvent trapping was used in previous subcritical water extractions, sorbent trapping has been tested for subcritical water extraction in this study. After subcritical water extraction, the sorbent trap (silica-bonded C18 column) was coupled to a HPLC system for analysis. The coupling technique of subcritical water extraction with HPLC eliminates the liquid-liquid extraction used in subcritical water extractions with solvent trapping and provides better sensitivity. Alkylbenzenes and polycyclic aromatic hydrocarbons were extracted from sand and contaminated soils and then analyzed using this new coupling technique. The effects of volume, temperature, and pressure of water on organic recoveries are also discussed in this paper. Water has a critical point of 374 °C and 218 atm. The high critical temperature and pressure make supercritical water very corrosive.1-4 Therefore, supercritical water is not a good extraction fluid for analytical purposes although supercritical water oxidation has been used to successfully destroy or degrade many kinds of chemical wastes.1-4 Compared to supercritical water, subcritical water is much less reactive and thus can be used in analytical chemistry. Another advantage of subcritical water is its widely tunable dielectric constant, surface tension, and viscosity, since they decrease significantly by raising water temperature under moderate pressures to keep water in the liquid state. For example, the dielectric constant of liquid water is lowered from 80 to ∼30 by raising temperature from ambient to 250 °C. Thus, the dielectric constant, surface tension, and viscosity of liquid water at 250 °C have values similar to those of methanol, ethanol, or acetonitrile (ACN) at ambient temperature.5-7 Obviously, high-temperature water be(1) Shaw, R. W.; Brill, T. B.; Clifford, A. A.; Eckert, C. A.; Franck, E. U. Chem. Eng. News 1991, (Dec 23), 26. (2) Thomason, T. B.; Modell, M. Hazard. Waste 1984, 1, 453. (3) Hirth, T.; Franck, E. U. Ber. Bunsen-Ges. Phys. Chem. 1993, 97, 1091. (4) Li, L.; Gloyna, E. F.; Sawicki, J. E. Water Environ. Res. 1993, 65, 250. (5) Haar, L.; Gallagher, J. S.; Kell, G. S. National Bureau of Standards/National Research Council Steam Tables; Hemisphere Publishing Corp.: Bristol, PA, 1984. (6) Melander, W. R.; Horvath, C. In High Performance Liquid Chromatographys Advances and Perspectives; Horvath, C., Ed.; Academic Press: New York, 1980; Vol. 2, pp 113-319. 10.1021/ac981186b CCC: $18.00 Published on Web 03/09/1999
© 1999 American Chemical Society
haves like an organic solvent. Recent studies have demonstrated that liquid water at elevated temperatures and pressures is an excellent extraction fluid for many organic compounds from solid matrixes.7-12 However, only solvent trapping has been used in previous work on subcritical water extractions (SBWE).8-10 A liquid-liquid extraction (e.g., methylene chloride-water extractant) has to be performed after the water extraction but prior to the GC analysis. This liquid-liquid extraction step makes the solvent trapping process more complicated. In addition, the sensitivity is limited for subcritical water extraction with solvent trapping, since only 1-2 µL out of a 3-5-mL sample can be injected into the GC. In this study, sorbent trapping was used to collect the extracted organic compounds during subcritical water extractions. After the extraction, the trap was connected to a HPLC system via two sixport valves. The HPLC calibration and subcritical water extraction can be performed simultaneously. The coupling technique of SBWE-HPLC eliminates the liquid-liquid postextraction that is used in subcritical water extractions with solvent trapping. Since the total mass of the collected species in the trap can be detected, the sensitivity of this approach is higher than that of the solvent trapping technique. Benzene, toluene, ethylbenzene, xylenes, and six polycyclic aromatic hydrocarbons (PAHs) were extracted from sand and contaminated soils. The effects of temperature, pressure, and volume of water on organic recoveries have been investigated. EXPERIMENTAL SECTION Reagents. Benzene, toluene, ethylbenzene, and xylenes (BTEX) were obtained from Fisher Scientific (Fair Lawn, NJ). Naphthalene, phenanthrene, pyrene, chrysene, benzo[a]pyrene, and benzo[ghi]perylene were purchased from Aldrich (Milwaukee, WI). HPLC grade methanol, acetonitrile, and deionized water (18 MΩ) were used as mobile phases for HPLC. A sample size of 10 µL was used for both subcritical water extraction (for spiking studies) and HPLC calibration. (7) Yang, Y.; Belghazi, M.; Hawthorne, S. B.; Miller, D. J. J. Chromatogr., A 1998, 810, 149. (8) Hawthorne, S. B.; Yang, Y.; Miller, D. J. Anal. Chem. 1994, 66, 2912. (9) Yang, Y.; Bøwadt, S.; Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1995, 67, 4571. (10) Yang, Y.; Hawthorne, Steven B.; Miller, David J. Environ. Sci. Technol. 1997, 31, 430. (11) Hageman, Kimberly J.; Mazeas, Laurent; Grabanski, Carol B.; Miller, David J.; Hawthorne, Steven B. Anal. Chem. 1996, 68, 3892 (12) Daimon, H.; Pawliszyn, J. Anal. Commun. 1996, 33, 421.
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Figure 1. Subcritical water extraction system with solid trap.
Samples. Ottawa sand (Fisher Scientific) was used for spiking studies. A gasoline-contaminated soil and a PAH-contaminated soil were collected locally. Subcritical Water Extraction with Sorbent Trapping. A homemade system for subcritical water extraction with solid trapping is shown in Figure 1. Ottawa sand was cleaned using methylene chloride followed by acetone and then dried in an oven at 100 °C for 1 h. The precleaned sand was packed into a stainless steel vessel (50 × 4.6 mm i.d., Keystone Scientific, Bellefonte, PA). A BTEX or a PAH mixture (10 µL) was spiked onto the sand for spiking studies. For the extraction of real samples, the contaminated soil was directly packed into the extraction cell (the void volume was filled with clean sand). The inlet of the loaded extraction cell was connected with an Isco 260 D syringe pump (Isco, Lincoln, NE) using a preheating coil and placed inside a Fisher Isotemp oven. The outlet of the cell was then connected to a solid trap packed with ODS (20 × 4 mm i.d., Keystone Scientific). The trap was placed inside an ice-water bath during the extraction. The outlet valve (HIP model 15-11AF1, HighPressure Equipment Co., Erie, PA) of the trap was closed and the system was pressurized to 15 atm to check for possible leaks of the system. Then the oven was heated to the desired temperature with both the inlet (V1) and the outlet (V2) valves of the extraction cell closed. After the desired extraction temperature was reached, the pump was set at either 400 atm (flow rate, 0.50.7 mL/min) in constant-pressure mode or at 0.6 mL/min (pressure, 90-110 atm) in constant-flow rate mode. The inlet and the outlet valves were opened and the extraction was performed. High-Performance Liquid Chromatography. A HewlettPackard (Avondale, PA) pump (series 1050) was used to deliver the mobile phase. A mixture of ACN and water (volume ratio of 4:3 for ACN-water) was used for BTEX separation in the spike studies. To have less interference, a ratio of 47:53 for ACN-water was used for BTEX separation from the gasoline-contaminated sample. For separation of PAHs from sand samples, a mobile phase with a volume ratio of 85:15 (methanol-water) was used for the first 5 min. Then 100% methanol was used as the mobile phase to shorten the analysis time. However, for separation of PAHs from the soil sample, a mixture of methanol and water (85: 15) was employed as the HPLC carrier for the entire analysis. A flow rate of 1 mL/min was used for both BTEX and PAH separations. Just before use, all of the mobile phases were purged using helium to remove dissolved gases. After a SBWE extraction, the trap was coupled to a HPLC system by using two Valco injectors (purchased from Keystone Scientific) as shown in Figure 2. The SBWE trap was connected to injector 2 as the sample loop, while the sample loop of injector 1492 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
Figure 2. Schematic diagram for the coupling of SBWE trap with HPLC.
1 was used for HPLC calibration. A HPLC column (ODS, 5 µm, 250 × 4.6 mm i.d. for BTEX; ODS2, 5 µm, 250 × 4.6 mm i.d. for PAHs) was connected to injector 2. The mobile phase entered injector 1 through port 4 and directly exited injector 1 through port 5 without sweeping the liquid sample loop of injector 1. Then, the mobile phase reached injector 2 through port 4 and swept the SBWE trap through port 3. After the mobile phase passed the trap, it reentered injector 2 through port 6 and flowed to the HPLC column through port 5. After separation, the collected species were detected by a LC-95 UV detector at 254 nm (PerkinElmer, Norwalk, CT). In this way, SBWE was simply coupled to HPLC. If deposits of the extracted compounds occurred in the tubing between the extraction cell and the trap (this could happen for analytes with low solubility in ambient water), both the tubing and the trap were connected to the HPLC system to recover the total mass extracted. The experimental setup for HPLC calibration mode is shown in Figure 3. Figure 3a shows the loading mode, and Figure 3b demonstrates the injection mode. It must be pointed out that the SBWE trap was disconnected from the HPLC system during HPLC calibration regardless of whether injector 1 was in the loading or the injection position. In this way, both SBWE and HPLC calibrations can be performed simultaneously. Sonication Extraction. Approximately 400 mg of gasolineor PAH-contaminated soils was placed in a 7-mL vial. Then, 3-5 mL of methylene chloride was added and the vial was sealed and wrapped with Parafilm. The sample vials were sonicated in a sonication bath for 12 h. Gas Chromatography. A HP 6890 GC/FID was used for analyzing sonication extracts of contaminated soils to verify the quantitation made by SBWE-HPLC. Sample was introduced into a HP capillary column (MS35, 30 m × 0.25 mm i.d., 0.25 µm film thickness) using the split mode. For BTEX, the initial temperature of 25 °C was maintained for 6 min. Then, the temperature was increased to 250 °C at 6 °C/min. The temperature was raised from 80 to 320 °C at 8 °C/min for PAH analyses. RESULTS AND DISCUSSION BTEX. The main concern of this coupling technique was whether a good focus of the extracted analytes in the trap could be achieved. In other words, whether peak broadening would occur after coupling SBWE with HPLC. Figure 4 shows the
a
b
Figure 5. SBWE-HPLC chromatogram of a gasoline-contaminated soil. Table 1. Volume Effect on BTEX Recoveries at 100 °C and 400 atm % recovery (% RSD)
Figure 3. Schematic diagram for HPLC calibration mode: (a) load mode; (b) inject mode.
Figure 4. Comparison of BTEX chromatograms obtained from the HPLC calibration (top) and the coupled SBWE-HPLC (bottom).
chromatograms obtained from the HPLC calibration (top) and the coupling of SBWE to HPLC (bottom). The subcritical water extraction was performed at 100 °C, 400 atm, and using 5 mL of water. The peak shapes of all BTEX components obtained after SBWE are the same as those of HPLC calibration (without SBWE) as shown in Figure 4. This demonstrates that the coupled system works well.
vol, mL
benzene 101 µL/L
toluene 100 µL/L
ethylbenzene 100 µL/L
p-xylene 99 µL/L
2 5 10
94 (6) 91 (10) 83 (5)
100 (6) 100 (3) 99 (5)
91 (3) 96 (2) 99 (6)
94 (2) 97 (2) 102 (7)
Effect of Water Volume on BTEX Recoveries. The BTEX recoveries were determined using three different water volumes (2, 5, and 10 mL). As shown in Table 1, the recoveries of ethylbenzene and p-xylene were increased by increasing water volume because the solubilities of these two species in water are low and the extraction might be limited by solubility. Since toluene has higher solubility than ethylbenzene and p-xylene, the extraction was not limited by solubility, and thus no volume effect was observed. It is very interesting that benzene’s recovery was actually decreased by increasing water volume. Our speculation is that benzene’s solubility is so high that ambient water may elute the collected benzene from the trap during the extraction. This speculation was proven by performing the following experiments. When benzene was injected directly onto the solid trap and then pure ambient water was pumped through the trap (no analytical column) using a HPLC system, benzene started to elute after ∼3 mL of water passed through the trap. When the same experiment was performed for toluene, toluene was not eluted until ∼15 mL of water passed through the trap. The low RSDs in Table 1 demonstrate that this coupling technique is reproducible. Effect of Water Temperature on BTEX Recoveries. The temperature effect on BTEX recoveries was studied at 400 atm and using 5 mL of water. The temperatures used for this study were 50, 100, and 150 °C. The recoveries of BTEX range from 90 to 100% at all three temperatures. Therefore, no significant temperature effect was observed. Extraction of a Gasoline-Contaminated Soil. A gasolinecontaminated soil (∼30 mg) was extracted at 100 °C and ∼100 atm using 5 mL of water (at a constant flow rate of 0.6 mL/min). Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
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Table 2. Comparison of BTEX Concentrations Obtained by This Method and Sonication Extraction
Table 4. Temperature Effect on PAH Recoveries at 400 atm Using 10 mL of Water
concn, mg/g (% RSD)
benzene toluene ethylbenzene xylenes
SBWE-HPLC
sonication-GC
1.89 (31) 17.75 (25) 5.41 (18) 17.96 (21)
2.23 (32) 14.28 (27) 4.34 (24) 16.35 (27)
naphthalene phenanthrene pyrene chrysene benzo[a]pyrene benzo[ghi]perylene
concn µg/mL
100 °C
308 106 96 99 99 96
68 (18) 66 (23) 62 (22) 36 (13) 28 (15) 10 (68)
% recovery (% RSD) 150 °C 200 °C 250 °C 87 (7) 88 (5) 87 (11) 88 (14) 72 (13) 66 (41)
84 (6) 91 (5) 88 (6) 92 (8) 82 (17) 90 (14)
87 (6) 112 (11) 103 (23) 99 (12) 68 (19) 85 (4)
Table 3. Volume Effect of Water on PAH Recoveries at 150 °C and 400 atm concn, µg/mL naphthalene phenanthrene pyrene chrysene benzo[a]pyrene benzo[ghi]perylene
308 106 96 99 99 96
% recovery (% RSD) 5 mL 10 mL 15 mL 73 (9) 83 (7) 76 (11) 72 (17) 50 (22) 38 (32)
87 (7) 88 (5) 87 (11) 88 (14) 72 (13) 66 (41)
101 (8) 103 (9) 99 (12) 95 (14) 86 (20) 81 (25)
Figure 5 shows the HPLC chromatogram after subcritical water extraction. The concentrations of BTEX obtained by this method compared favorably with those of sonication extraction followed by GC analysis as demonstrated in Table 2. The lower concentration of benzene obtained by the SBWE-HPLC method was caused by the low recovery of benzene since some of the extracted benzene was eluted from the trap during SBWE extractions. Polycyclic Aromatic Hydrocarbons. PAH peak shapes were examined by comparing the chromatogram obtained after the coupling of SBWE-HPLC with that of HPLC calibration. As with BTEX, there is no peak broadening for all of the six tested PAHs after the coupling of the SBWE trap with HPLC. Effect of Water Volume on PAH Recoveries. Table 3 shows the PAH recoveries obtained using three different water volumes (5, 10, and 15 mL) at 150 °C. Since the solubilities of all of the PAHs are fairly low in water, increasing the water volume from 5 to 10 mL improved PAH recoveries at 150 °C. Further increasing the water volume to 15 mL resulted in quantitative recoveries for the low-molecular-weight PAHs. The elution study showed that naphthalene (the easiest one to elute among the PAHs) was not eluted from the trap using 30 mL of ambient water, demonstrating that the 5-15 mL of water used in this study did not cause any loss of the extracted PAHs from the trap. Effect of Water Temperature on PAH Recoveries. As discussed earlier in the introduction, temperature has the most significant influence on the polarity, surface tension, and viscosity of water. Therefore, extractions were performed at different temperatures to evaluate the temperature effect on PAH recoveries. Since the dielectric constant, surface tension, and viscosity of water are decreased by raising temperature, the PAH extraction efficiency was also significantly enhanced by increasing temperature as shown in Table 4. This was especially true for higher molecular weight PAHs. At low temperatures (e.g., 100 °C), the recoveries decreased when the molecular weight increased. For example, 68% of naphthalene was extracted at 100 °C, while only 10% of benzo[ghi]perylene was extracted at the same temperature. At 200 °C, all of the tested PAHs were almost quantitatively (g82%) extracted. 1494 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
Figure 6. SBWE-HPLC chromatogram of a soil sample containing PAHs.
It should be pointed out that deposits of the extracted species in the tubing between the extraction cell and the trap could occur during extractions at higher temperatures (e.g., above 100 °C), especially for nonpolar analytes with poor water solubilities (like PAHs). If this is the case, both tubing (between the extraction cell and the collection trap) and the trap should be connected to the HPLC system for quantitative analysis. Effect of Water Pressure on PAH Recoveries. The extraction and collection efficiency was determined using two different pressures (100 and 400 atm) to evaluate the pressure effect on PAH recoveries. Extractions were performed at 150 °C using 10 mL of water. Our results show that the PAH recoveries are almost identical from extractions at 100 and 400 atm, demonstrating that water pressure does not affect PAH extraction efficiency. Since the recoveries of the higher molecular weight PAHs were low (64-72%) at the temperature used (150 °C), greater RSDs (2141%) resulted for benzo[ghi]perylene and benzo[a]pyrene. Extraction of PAH-Contaminated Soils. Although the spiked PAHs were quantitatively extracted from sand at 200 °C, previous work with supercritical CO2 showed that organic contaminants are more difficult to extract from real samples.13 Therefore, 250 (13) Burford, M. D.; Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1993, 65, 1497.
Table 5. Comparison of PAH Concentrations Obtained by This Method and Sonication Extraction concn, µg/g (%RSD)
phenanthrene pyrene
SBWE-HPLC
sonication-GC
9.46 (13) 11.28 (16)
10.75 (18) 13.42 (30)
°C (tougher extraction condition) was used to extract PAHs from a soil sample (∼40 mg). The extractions were performed at a constant flow rate of 0.6 mL/min (∼100 atm). The SBWE-HPLC chromatogram of this soil sample is shown in Figure 6. Only phenanthrene and pyrene were detected in this soil sample. As demonstrated in Table 5, the PAH concentrations obtained by this method are similar to those obtained by sonication extraction followed by GC analyses. The extraction of real samples at higher temperatures could be more difficult to perform than the extraction of spiked samples because of plugging caused by complex sample matrixes. In this case, replaceable frits of the trap are desirable. CONCLUSIONS Subcritical water extraction was successfully coupled (off-line) to a HPLC system using a solid trap and two six-port LC injectors. The peak shapes obtained after the coupling of SBWE-HPLC are
the same as those of HPLC calibration, demonstrating no peak broadening occurred using this coupling technique. The recoveries of organic compounds with low solubility in ambient water (e.g., ethylbenzene, p-xylene, and PAHs) were slightly increased by increasing water volume. However, water volume had no effect on toluene recovery, while benzene recovery actually decreased with increasing water volume. Since all of the BTEX were quantitatively extracted at lower temperatures, no temperature effect was observed for the extraction of BTEX. But the extraction efficiency of PAHs was significantly improved by raising water temperature. This was especially true for high-molecular-weight PAHs. The extraction pressure did not affect extraction and collection efficiency. The concentration of BTEX and PAHs from contaminated soils obtained using this coupling technique compared favorably with that obtained by sonication extraction followed by GC analyses. An on-line coupling system of SBWEHPLC is under investigation. ACKNOWLEDGMENT The authors thank Hewlett-Packard Co. for instrument loans. Technical help from Clinton Eaton is also appreciated. Received for review October 29, 1998. Accepted February 6, 1999. AC981186B
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