Environ. Sci. Technol. 2007, 41, 1983-1988
Continuous Catalytic Hydrogenation of Polyaromatic Hydrocarbon Compounds in Hydrogen-Supercritical Carbon Dioxide TAO YUAN,† ANICK R. FOURNIER,‡ RAYMOND PROUDLOCK,‡ AND W I L L I A M D . M A R S H A L L * ,† Department of Food Science and Agricultural Chemistry, MacDonald Campus of McGill University, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9, and Preclinical Services, Department of Genetic Toxicology, Charles River Laboratories, 22022 Transcanadienne, Senneville, Quebec, Canada H9X 3R3
A continuous hydrogenation device was evaluated for the detoxification of selected tri-, tetra-, or pentacyclic polyaromatic hydrocarbon (PAH) compounds {anthracene, phenanthrene, chrysene, and benzo[a]pyrene (B[a]P)} by hydrogenation. A substrate stream in hexane, 0.05-1.0% (w/v), was mixed with hydrogen-carbon dioxide (H2-CO2, 5-30% v/v) and delivered to a heated reactor column (25 cm × 1 cm) containing palladium supported on gamma alumina (Pd0/γ-Al2O3) that was terminated with a capillary restrictor. The flow rate from the reactor, ∼800 mL min-1 decompressed gas, corresponded to 4 mL min-1 fluid under the operating conditions of the trials. Reaction products were recovered by passing the reactor effluent through hexane. At 90 °C, the anthracene or phenanthrene substrate was hydrogenated only partially to octahydro and dodecahydro species and contained only a minor quantity of totally hydrogenated products. For substrates with increasing numbers of fused aromatic rings, the hydrogenation efficiency was decreased further. However, at an increasing temperature (90-150 °C) and increasing mobile phase flow rate (20.68 MPa corresponding to 2100 mL min-1 decompressed gas), B[a]P and chrysene were hydrogenated, virtually totally, to their corresponding perhydro analogues (eicosahydrobenzo[a]pyrenes and octadecahydrochrysenes), respectively. That this approach might be useful for decontaminating soil extracts was supported by companion in vitro trials in which the substrate and products were assayed for mutagenic activity with five bacterial strains that are auxotrophic for histidine (Salmonella typhimurium TA98 , TA100, TA1535, and TA1537) or tryptophan (Escherichia coli WP2 uvrA), using the bacterial reverse mutation assay (modified Ames test). Generally, substantial increases in revertant colony counts were not observed with any of the strains following exposure to the hydrogenation products in the absence or presence * Corresponding author phone: (514) 398-7921; fax: (514) 3987977; e-mail:
[email protected]. † McGill University. ‡ Charles River Laboratories. 10.1021/es062194+ CCC: $37.00 Published on Web 02/15/2007
2007 American Chemical Society
of the 10 or 30% S9 mix, which is consistent with the loss of mutagenic activity from these hydrogenation products.
Introduction The polycyclic aromatic hydrocarbons (PAHs) represent a class of organic compounds that consist of two or more fused aromatic rings. PAHs are ubiquitous in the natural environment and originate from two main sources: natural (biogenic and geochemical) and anthropogenic (1). It is the latter source of PAHs that is the major cause of environmental pollution and hence the focus of many remediation programs. PAHs occur naturally in fossil fuels such as coal and petroleum but are also formed during the incomplete combustion of organic materials such as coal, diesel, wood, and vegetation (2, 3). Point sources can represent industrial processes such as coal liquefaction and gasification during coke production (4). As examples, creosotes and coal tar, which are byproducts of coking, can contain appreciable quantities of PAHs (up to 85% for creosote) (1). Because of their carcinogenic and mutagenic properties, PAHs have long been regarded as environmental priority pollutants (5) that require metabolic activation to electrophilic intermediates (6-8) and subsequent covalent adduct formation with cellular DNA to elicit their adverse biological activity (9, 10). Although several different activation pathways have been identified, strong evidence points to the prominent role of bay- and fjord-region dihydrodiol epoxides as ultimate mutagenic and carcinogenic metabolites (11) of PAH compounds. The mutagenic activity of selected PAH compounds containing four or more fused rings has long been recognized, and benzo[a]pyrene (B[a]P) is considered one of the most potently carcinogenic of all known PAHs (12). In contrast to mammals, fewer studies have been performed on marine organisms to determine the mechanism of polycyclic aromatic hydrocarbon-induced toxicity (13). Despite the fact that PAHs can be biodegraded, their persistence in soil, as measured in terms of half-life, can exceed 8 years (14). Point sources of PAH contamination are the most significant environmental concern. Although the affected areas can be relatively small in size, the contaminant concentration at these sites is often high and associated with co-contaminants such as benzene, toluene, ethylbenzene, and xylene (BTEX) compounds, heavy metals, and aliphatic hydrocarbons, which can hinder/compromise bioremediation efforts. Soils can be contaminated with concentrations that can range up to 300 g kg-1 PAHs (15). In general, the higher the molecular weight of the PAH molecule, the higher the hydrophobicity and toxicity, and the longer the environmental persistence of the molecule (16). Previous research (17) on catalytic batch hydrogenations of selected PAH compounds has demonstrated that it was feasible to achieve rapid and complete hydrogenation with relatively mild conditions (0.42 MPa H2, 90 °C). Hydrogenations of Fused Rings. There has been extensive research on the catalytic hydroprocessing of lower molecular weight PAHs (predominantly anthracene and phenanthrene) (18-21). The product distribution of PAHs hydrogenation is highly dependent on catalyst type, temperature, and solvent. Hydrogenation processes are believed to be stepwise, but intermediates with partially hydrogenated rings are often not detected (22). For hydroaromatics, the temperature above which dehydrogenation is favored over hydrogenation decreased with an increasing number of fused rings. For phenanthrene, the crossover temperature was determined VOL. 41, NO. 6, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Structures of selected tri-, tetra-, and pentacyclic polyaromatic hydrocarbon (PAH) compounds. to be ∼400 °C (23). The intramolecular rearrangement of hydroaromatic compounds can take two forms, corresponding to the process of ring contraction (i.e., tetrahydrophenanthrene to its methylcyclopentanic isomer over sulfided Ni-Mo/Al2O3 at 430 °C) (24) or the process of ring shift (interconversion of anthracene derivatives and phenanthrene derivatives) that has been observed in the presence of a variety of catalysts at 200-300 °C (25), in acid at 482 °C (26), thermally induced equilibration (27), or in the presence of AlCl3 (28). Supercritical CO2 as a Reaction Medium. The use of scCO2 frequently is considered to be an ideal phase because of its mild critical properties (TC ) 31 °C, PC ) 7.4 MPa), nontoxicity, non-flammability, modest cost, and a lack of restrictive regulations. Molecular hydrogen is completely miscible with near-critical CO2 (29) and can result in a very high initial rate of reaction. Some 1400 mol of formic acid has been produced from CO2 per mol of catalyst per hour (30). The same reaction under identical conditions but in liquid organic solvents is much slower, principally because of the results of decreased diffusion rates and the limited solubility of H2 in most organic solvents. Solvent polarity of the reaction medium can be fine-tuned with ease in the near-critical region (generally, 1.05-1.2 TC) (31) by simply changing the pressure. To achieve suitable substrate solubility, the reaction medium can be modified by adding an inert cosolvent. Moreover, the reactants/products can be separated readily from the reaction medium by pressure reduction. Because aromatic hydrogenations are highly exothermic (32), reactions are favored, and selectivities can be increased by operation at lower temperatures. However, for hydrogenations on a larger scale, some means of heat dissipation can become necessary. The heat capacities of scCO2 can also be pressure-tuned to be more liquid-like (33) and to minimize product hold up. For porous solid catalysts, product selectivity can also be optimized to mitigate pore-diffusion limitations and decrease the accumulation of coke forming precursors that can inactivate catalytic sites. Catalytic inactivation has been reported during hydrogenations over Pto/Al2O3 in near-critical CO2 (34) that might have resulted from the possible formation of one or more possible surface species including formates, carbonates, and/or CO in the presence of CO2 + H2. The objective of the current study was to evaluate/ optimize a device for the continuous hydrogenation of selected -three to five fused ring PAHs under mild temperatures and pressures. It was anticipated that with these conditions, hydrogenation (rather than dehydrogenation) would predominate and that hydrocracking reactions and/ or rearrangements would be minimized. Concurrently, it was projected that PAH compounds would be detoxified by dearomatization if hydrogen was included in the scCO2 mobile phase. This process would resemble processing at near or scCO2 in the presence of a gaseous organic solvent (hexane) as the reaction medium. As a consequence, trials were performed in the presence of a large excess of catalyst in a hexane-hydrogen-scCO2 atmosphere.
Experimental Procedures Chemicals. Anthracene, phenanthrene, chrysene, B[a]P, (Figure 1), 9-aminoacridine (9AC), 2-aminoanthracene (2AA), dimethyl sulfoxide (DMSO), 2-nitrofluorene (2NF), 4-nitroquinoline N-oxide (NQO), and sodium azide (NaAz), all ACS 1984
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reagent grade, were obtained from Sigma-Aldrich Co., Oakville, ON. Palladium supported on alumina (Pd0/γ-Al2O3, 5%, w/w) was purchased from Alfa-Aesar, Ward Hill, MA (catalog number 11713), and organic solvents were obtained from Fisher Scientific, Ottawa, ON. Hydrogen gas and carbon dioxide were purchased from MEGS, Montre´al, QC. Gaseous mixtures were prepared by over-pressuring a suitable quantity of H2 with CO2 within a high pressure cylinder. After mixing thoroughly, the gaseous mixture was further pressurized to the desired conditions with a diaphragm pump. Reactor and Operation. The reactor consisted of a 25 cm × 0.46 cm (inner diameter) HPLC column containing palladium supported on gamma alumina (Pd0/γAl2O3) that was terminated with a capillary restrictor. In operation, the substrate (2.5 mg in hexane) was injected as a pulse or pumped (0.05 mg min-1) continuously for up to 3.5 h and entered the reactor via a tee fitting. The reactor had been pre-equilibrated to 90 °C in a water bath. Hydrogen in supercritical carbon dioxide (5-30% v/v) served as the mobile phase. The reaction products were collected from the exit of the restrictor by trapping in hexane. Separate fractions, corresponding to 10 or 15 min of cumulative trapping of effluent, were collected sequentially during 220 min. The course of hydrogenations was monitored by assaying the hexane traps using gas chromatography with mass spectrometric or flame ionization detection (FID). GC Analysis. GC-MS was performed on a Varian model 3900 gas chromatograph fitted with a model 8400 autosampler and a model 2100T MS detector. The DB-5 capillary column (30 m × 0.25 mm i.d.; 0.25 µm film thickness) was eluted with helium at 1.0 mL min-1. After an initial hold of 1 min at 50 °C, the column was ramped, at 10 °C min-1, to 300 °C and held for a further 3 min prior to cool down. The temperature of the injector, transfer line, and detector was maintained at 250, 250, and 150 °C, respectively. Preliminary identification of the eluting components was performed by comparing the experimental mass spectra with spectra catalogued in the National Institute of Standards and Technology (NIST) or the Saturn mass spectral libraries. Quantification of the sample was performed on a HP 5890 gas chromatograph with FID under similar chromatographic conditions as stated previously. Bacterial Reverse Mutation Assay. Bacterial Strains. Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 and Escherichia coli strain WP2 uvrA were originally supplied by Moltox Inc., Boone, NC and were characterized and maintained as previously recommended (35, 36). S9 Mix. Phenobarbital/5,6-benzoflavone-induced male Sprague-Dawley rat liver fractions were supplied by Moltox Inc. The S9 mix contained either 10 or 30% by volume of the S9 fraction and the following sterile cofactors: 8 mM magnesium chloride, 33 mM potassium chloride, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate, and 4 mM nicotinamide adenine diphosphate. Positive Controls. 9AC, 2AA, BaP, 2NF, and NQO were formulated in DMSO, and NaAz was formulated in water. Positive control (9AC, 2AA, BaP, 2NF, NQO, and NaAz) solutions were prepared in advance as frozen solutions that were thawed on the day of use. All formulations of the reference articles (chrysene and B[a]P) and hydrogenated products (octadecahydrochrysene, dodecahydrochrysene, eicosahydrobenzo[a]pyrene, and tetradecahydrobenzo[a]pyrene) were prepared freshly on the day of dosing, just prior to use. Preincubation Assay. A total of 0.05 mL or 0.1 mL of the test solution, 0.1 mL of fresh bacterial culture (containing >109 viable cells), and 0.5 mL of phosphate buffer 0.2 M pH 7.4 or S9 mix (10 or 30% v/v) were mixed in a sterile container. The S9 mix/buffer, bacteria, and treatment were incubated for 30 min with shaking (180 rpm) at 37 °C. Molten top agar
FIGURE 2. Variations in the cumulative clearance of anthracene from the reactor with pressure of the supercritical carbon dioxide eluting fluid (15.84-20.85 MPa, open symbols) or with the flow rate of added hexane (0.1-1.5 mL min-1, solid symbols). (ca. 2 mL) supplemented with biotin and minimal histine and tryptophan was added to the samples, and the solution was mixed and overlaid onto a minimal glucose plate. The plates were inverted and incubated at 37 °C for 48-72 h. Evaluation and Interpretation of Results. Sterility, negative/vehicle, and positive control tests were included in each experiment. Triplicate plates were evaluated at each experimental point. Revertant colonies were routinely counted using an automated colony counter (ProtoCOL by Synbiosis, Cambridge, UK). The background lawn was evaluated using an inverted microscope. Toxicity was identified by a substantial reduction in the integrity of the lawn or a marked reduction in the number of revertants as compared to the vehicle control (fold response < 0.6). A positive mutagenic response was defined as a dose related increase in revertant colony numbers to at least twice the concurrent untreated control levels (1.5× for strain TA100) provided that mean values lay outside the historical control range.
Results and Discussion Reactor Characterization. The influence of pressure (15.8624.13 MPa) or added solvent on the course of reaction and clearance of products was evaluated for the hydrogenation of 2.5 mg of anthracene (Figure 1), introduced as a pulse to the reactor and monitored for 60 min. Six samples, each representing cumulative 10 min fractions of reactor eluate, were collected. At 90 °C and a pressure of 15.86, 17.93, 20.68, or 24.13 MPa scCO2 (flow rate 4 mL min-1), the substrate was partially hydrogenated to octahydroanthracene or totally hydrogenated to perhydroanthracene. A comparison of the
open symbols of Figure 2 indicates that the substrate was cleared more rapidly at higher pressures (20.68 and 24.13 MPa) than at lower pressures (15.86 and 17.93 MPa), consistent with increased substrate solubility in the mobile phase. However, the flow rate of the mobile phase would be predicted to have increased slightly with increased pressure since the length of the capillary restrictor was not varied between trials. In companion trials (90 °C, 24.13 MPa), the scCO2 mobile phase was mixed/diluted with hexane (0.11.5 mL min-1) prior to entry into the reactor, but the added cosolvent had only a minimal effect on the rate of clearance (Figure 2, solid symbols vs open symbols) of substrate. Hydrogenations. The hydrogenation of phenanthrene (Figure 1) delivered at 0.1 mg min-1 in the presence of various concentrations of H2 (5, 16, or 30%) in scCO2 (4 mL min-1) at 90 °C and 20.68 MPa was also investigated (Table 1). The principal products of phenanthrene hydrogenation were perhydrophenanthrenes (six isomers detected), octahydrophenanthrenes, and a minor quantity of biphenyls detected only in the presence of the 30% H2/scCO2. Phenanthrene hydrogenation was more efficient in the presence of the increased H2 content in the scCO2. Perhydrogenated isomers accounting for some 26% of the product distribution for the 5% H2 mixture were dominant (∼60%) for the 16% of H2 mixture and were the only appreciable products (g90%) for the 30% H2 mixture once quasi-equilibrium conditions had been established within the reactor (traps 6-8). On the other hand, equilibrium conditions were established considerably more slowly for the 30% than for the 16% or for the 5% H2 mixture. Products were not detected in the first trap, and the repeatability although uneven in the second trap was improved for subsequent traps. Because phenanthrere hydrogenation standards were not available commercially, quantitation by FID was performed on the basis of phenanthrene. For trials performed with decreased substrate loading (0.05 mg min-1, 16% H2/scCO2, 24.13 MPa delivered at 4 mL min-1), the hydrogenation of anthracene, phenanthrene, chrysene, and B[a]P was in all cases incomplete; only partial hydrogenation was observed. Whereas for the tricyclic substrates (anthracene and phenanthrene), perhydrogenated species were the dominant products, for B[a]P, perhydrogenated products (20H-B[a]p) were minor, and for chrysene, they were not observed. Moreover, despite some indication of increased variability among replicates, there was no indication that equilibrium conditions were being approached with these operating conditions. However, an increased flow rate of the mobile phase (10 mL min-1 16% H2/scCO2) coupled with increasing reactor operating temperature (150, 200, or 250 °C) proved to be more efficient for hydrogenations of B[a]P (Table 2). For these trials, 14 successive traps of reactor eluate were collected (each representing 15 minutes of cumulative reactor effluent).
TABLE 1. Mass Balance in Successive Trapsa Observed for Phenanthrene (0.1 mg min-1) Hydrogenations at 20.68 MPa/90 °C for Various Concentrationsb of H2 in scCO2c H2 (%)
products
2
3
4
5
6
7
8
5%
octahydrophenanthrene perhydrophenanthrene sum octahydrophenanthrene perhydrophenanthrene sum biphenyl octahydroanthracene perhydroanthracene sum
13 ( 34 25 ( 8 38 ( 6 7 ( 41 26 ( 9 33 ( 24 0 4(2 12 ( 43 17 ( 31
60 ( 13 26 ( 6 86 ( 8 28 ( 4 56 ( 4 84 ( 7 4(2 4(2 22 ( 7 31 ( 5
77 ( 9 27 ( 12 104 ( 4 33 ( 28 61 ( 1 94 ( 11 5 ( 0.7 4(3 34 ( 15 43.5 ( 12.1
77 ( 10 27 ( 8 104 ( 5 31 ( 44 70 ( 9 100 ( 1 5 ( 0.5 4(4 68 ( 10 77 ( 9
78 ( 2 26 ( 2 103 ( 1 13 ( 2 55 ( 4 95 ( 5 5(3 4(3 95 ( 7 105 ( 7
79 ( 5 26 ( 2 105 ( 3 9(8 52 ( 0.1 88 ( 0.7 5 ( 0.8 5(6 102 ( 5 111 ( 5
81 ( 4 25 ( 4 106 ( 4 9(7 61 ( 2 97 ( 3 5(3 5(6 108 ( 3 117 ( 3
16% 30%
a
Each representing 15 min of continued collection of reactor effluent in hexane. b 5, 16, or 30% (v/v). c Delivered at 4 mL min-1.
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TABLE 2. Mass Balance in Successive Traps for the Hydrogenation of Benzo[a]pyrenea in 16% H2-CO2, 24.13 MPa, Delivered at 10 mL min-1 to the Reactor Maintained at 150, 200, or 250 °C conditions (°C/mg min-1)
3
a
4
5
6
7
59 ( 115 84 ( 40 110 ( 7 105 ( 4 88 109 104 107 29 ( 86 69 ( 17 95 ( 13 98 ( 9 57 ( 16 104 ( 6 106 ( 16 100 ( 17
150/0.05 150/0.1 200/0.05 250/0.05
0.1 or 0.05 mg/min.
b
8
9
10
11
12
13
14
103 ( 5 104 ( 3 104 ( 16 98 ( 6 108 ( 2 105 ( 9 105 ( 11 107 104 103 104 106 103 107 102 ( 7 97 ( 13 95 ( 8 102 ( 2 92 ( 13 97 ( 8 94 ( 9 105 ( 14 103 ( 23 104 ( 8 104 ( 20 104 ( 15 105 ( 14 103 ( 19
110 ( 4 106 NPb NP
NP ) not performed.
TABLE 3. Yields of Minor Productsa That Were Observed in Successive Traps for the Hydrogenation of Anthracene, Phenanthrene, or Chrysene During 3.25 h of Continued Operation substrate
product
1
2
3
4
5
6
7
8
9
10
11
12
13
anthracene phenanthrene chrysene
bicyclohexyl bicyclohexyl dodecahydro
0.85 0.67 6.58
0.78 1.28 8.57
0.81 1.30 8.16
0.69 1.27 4.14
0.79 1.19 NDb
0.80 1.26 ND
0.76 1.16 ND
0.73 1.41 ND
0.90 1.32 ND
0.64 1.19 ND
0.78 1.36 ND
0.74 1.16 ND
0.85 1.06 ND
a
Mol %.
b
ND: none detected (less than 0.05 mol %).
With these operating conditions, only perhydrogenated products were observed (seven isomers detected). Although the mass balances for effluent were somewhat inefficient during the equilibration phase of the runs (traps 1-4), in subsequent traps, the variability among replicate runs was decreased, and the mass balance was improved. In subsequent trials, the quantity of influent substrate was increased while maintaining the reactor temperature at 150 °C. For anthracene or phenanthrene, the influent in hexane was added to the reactor at 0.1 mg min-1 and for B[a]P at 0.05 mg min-1, and for chrysene, the influent rate was 0.025 mg min-1. For all four compounds, the initial traps were characterized by less than complete recovery of substrate during the equilibration phase of the reactor operation. However, later traps were repeatable and provided virtually complete hydrogenation. In all cases, the only products present in appreciable quantities were totally hydrogenated. Other products that were detected in these trials are summarized in Table 3. The tricyclic substrates (anthracene and phenanthrene) generated traces (
