Anal. Chem. 1986, 58, 1839-1844
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Determination of Selected Azaarenes in Water by Bonded-Phase Extraction and Liquid Chromatography Thomas R. Steinheimer*
IJ.S. Geological Survey, WRD, 5293 Ward Rd., Mail Stop 407, Aruada, Colorado 80002 Martin G . Ondrus
Department of Chemistry, University of Wisconsin-Stout, Menomonie, Wisconsin 54751
A method for the rapld and simple quantttatlve determination of quinollne, lsoqulnollne, and five selected three-rlng azaarenes in water has been developed. The azaarene fraction Is separated from its carbon analogues on noctadecyl packing material by elution with acidified water/acetonltrlie. Concentration as great as 1000-fdd Is achieved readily. Instrumental analysls involves high-speed IIquM chromatography on flexlble-walled, wide-bore columns with fluorescence and ultraviolet detection at several wavelengths employing filter photometers in series. Method-valldatlon data Is provided as azaarene recovery effklency from fortified samples. Dlstllled water, river water, contaminated ground water, and secondary-treatment effluent have been tested. Recoveries at part-per-bllllon levels are nearly quantitative for the three-rlng compounds, but they decrease for qulndlne and Isoqulnollne.
Azaarenes are nitrogen analogues of polycyclic aromatic hydrocarbons (PAHs). Unlike the PAHs, relatively little information is available concerning the physical and chemical properties or the extent of food and water contamination related to this class of compounds. With the exception of quinoline, isoquinoline, and some of their methyl derivatives, azaarenes are high-melting solids with extremely low water solubilities. They are usually found in water in association with sediment ( I , 2). Azaarenes are believed to be formed during combustion or pyrolysis processes involving fossil fuels. The source of the nitrogen in the formation of azaarene molecules is uncertain, but some comes from the nitrogen content of the fossil fuel. Several investigators (3-5) have suggested that coal-burning processes are a major source of azaarenes, resulting from the high nitrogen content of the coal. A variety of environmental sample types have been studied with respect to azaarene content. Composition of automobile exhaust (6),industrial effluents (7), urban suspended particulate matter (8, 9 ) , coal-derived products ( l o ) ,and fresh and saline surface water sediments ( 1 , 2, 1 1 ) have been investigated. Several two- and three-ring parent and alkylated azaarenes have been detected in cigarette smoke (12). However, azaarenes in water have been reported infrequently, and then under somewhat unique circumstances. High concentrations (1-100 mg/L) of quinoline and higher molecular weight azaarenes have been reported in wastewaters and raw waters of coking and coal conversion processes (7,13). As a class, azaarenes may be significant contributors (along with PAH compounds) to the biological activity of fossil fuel combustion products (14). Quinoline and its seven monomethyl derivatives have been found to be carcinogens (15). Certain azaarenes are even more carcinogenic than the most active PAH (16). Underground in situ conversion of oil shale, which is being studied as a possible method for recovering shale oil from the vast deposits of oil shale in the western United States, could 0003-2700/86/0358-1839$01.50/0
contaminate important ground water resources with azaarenes, in a part of the country where pure water is already in short supply. Oil shale from the eastern United States, which is lower in organic content than oil shale from the western United States, probably will require concentration of the organic portion by some process such as froth flotation prior to surface retorting. Although water is abundant in the eastern United States, considerable surface and ground water contamination from process waters may occur. The high nitrogen content of shale oil increases the likelihood of azaarene contamination. Since 1978, seven municipal wells in the Minneapolis-St. Paul, MN, area have been closed because of seepage of creosote oils (which are denser than water) from a creosote-treatment facility in St. Louis Park, MN. These wells were found to be contaminated with PAHs (19, and more recently azaarenes were identified (18). A case history of this situation is detailed in a recent survey of hazardous-waste problems in America (19). Azaarenes have been determined by thin-layer chromatography (201,paper chromatography (211,electrophoresis (22), room-temperature phosphorescence (23),and conventional liquid chromatography on adsorption (10,24,25) and ionexchange (26)packings. High-pressure liquid chromatographic (HPLC) separation of standard mixtures by reversed-phase partition on CISbonded-silica packings, using acetonitrile/ water gradient elution, is governed primarily by ring number and water solubility (27). Work done recently has demonstrated the separation of azaarenes by isocratic reversed-phase HPLC on C8 and CISpackings. Because azaarenes have some basic character contributed by the ring nitrogen, the chromatography is considerably more difficult than analogous PAH separations. Protonation of the nitrogen in weakly acidic and neutral solutions results in poor resolution and dramatic peak tailing with many normal- and reversed-phase columnpacking materials (28). Techniques for the quantitative determination of azaarenes in water involve a combination of extraction and concentration, preferably done in a single step, followed by instrumental analysis employing gas or liquid chromatography (GC or LC) (29-32). Solid-surface sorption techniques have proven more useful and less time-consuming than liquid-liquid partition approaches for isolation of an azaarene fraction. Materials that have been used successfully include completely synthetic sorbents such as XADs (33)and chromatographic-grade silicas modified by chemical derivatization. Chromatographic detectors that have been used include photometric devices for LC and the mass spectrometer for capillary GC. We report our results on the rapid estimation of two- and three-ring azaarenes in a variety of water samples by bonded-phase extraction and reversed-phase LC. The names and structures of those compounds investigated are listed in Table I. The procedure, which takes advantage of the basicity of azaarenes to separate them from PAHs, is convenient and rapid, with preinstrumental cleanup and enrichment accom0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
Table I. Nomenclature and Structures of Azaarenes Selected for Study named as derivative of polycyclic aromatic hydrocarbon
common name
1-azanaphthalene
quinoline
2-azanaphthalene
isoquinoline
structure
9,lO-diazaphenanthrene benzo[c]cinnoline: 5,6-
4-azafluorene 10-azaanthracene 10-azaphenanthrene
acridine: benzo[b]quinoline phenanthridine: benzo [c]quinoline
4-azaphenanthrene
benzo[h]quinoline
9-azafluorene
carbazole
& ti
plished in minutes. The method can be used to detect part-per-trillion concentrations in relatively pure water samples, and it also can be used to determine azaarenes in complex, highly contaminated waters containing PAHs and other organics that might be expected to provide significant interference. EXPERIMENTAL SECTION Equipment. The HPLC system (Waters Chromatography Division, Millipore Corp., Milford, MA) consisted of M6000A and M45 pumps, Model 660 solvent programmer, and RCM-100 radial compression module, with a Model 440 dual-channel absorbance detector with 254-nm filter, an extended-wavelengthmodule with lamps and fitem for 229- and 214-nm measurements, and a Model 420-AC fluorescence detector with 254-nm excitation and 425-nm emission filters. Separations were performed on an 8-mm-i.d. radial compression Nova-PAK 5-fimspherical particle CIScolumn. Chromatograms and the M6000A pressure signal were recorded on Houston Instruments (Austin, TX) dual-pen recorders. The extended-wavelength module permits absorbance measurements to be made at one wavelength only, so consecutive injections were required for recording chromatograms first at 229 nm and then at 214 nm, following replacement of appropriate lamps and filters. Materials. Baker Analyzed HPLC acetonitrile (J. T. Baker Chemical Co., Phillipsburg, NJ) was used. Aqueous ammonia and phosphoric, sulfuric, and hydrochloric acids were reagent grade. Reagent grade ketones and aldehydes and (2,4-dinitrophenyl)hydrazine (2,4-DNP) were purchased from Eastman Kodak Co. (Rochester, NY) and used as received to prepare the phenylhydrazones investigated as potential internal standards. Azaarenes were obtained from Ultra Scientific (Hope, RI). Pure reference compounds of greater than 98% purity were used as received. Water used for HPLC was treated with a Continental (Millipore Corp., Bedford, MA) carbon adsorption bed and a Continental Model 3011 reverse-csmosis (Ro)unit. This RO water was further purified with cation and anion deionizing beds and a four-bowl Millipore Milli-Q unit. The Milli-Q system was allowed to recycle for at least 15 min prior to withdrawing water. HPLC analysis of this water following 250-fold trace enrichment produced a flat base line, indicating absence of organic impurities that might interfere under the experimental conditions of this study. Each
HPLC solvent and solvent mixture was degassed by vacuum filtration through a fresh 0.45-pm hydrophilic Durapore Millipore filter. These membrane filters, made from a modified dihalogenated fluoroalkane having chemical resistance similar to Teflon, contain no surfactants or coatings, and water extractables are less than 0.5% by weight. Mobile Phase. Milli-Q water was pH adjusted by adding 1.0 mL of phosphoric acid to 1 L and titrating to pH 6.5 by dropwise addition of 14 M aqueous ammonia. This buffering procedure allowed us to readily vary the pH to determine at what point protonation begins to reduce the quality of the chromatographic separation. We chose ammonium phosphate because it is transparent to UV radiation and is less corrosive to stainless steel than buffers containing sodium ion. Citrate buffers were avoided because citrate is a chelating agent and may hasten dissolution of silica. The desired 42:58 (v/v) acetonitrile/water ratio was achieved through solvent programmer pump control or through premixing the solvents and pumping with the M6000A. The mixed solvent had a measured pH of 7.2. Best results were realized by recycling the premixed mobile phase overnight at 1.0 mL/min to allow column equilibration. Stock and Standard Solutions. Stock 50 fig/mL solutions were prepared in glass-stoppered volumetric flasks, which then were sealed with parafilm and stored at -10 "C. Since detection sensitivity was affected to some degree by the makeup of the solvent matrix, standards were prepared with solvent composition as near to the sample composition as possible, by diluting the appropriate volume of stock solution with acetonitrile, water, or a mixture of acetonitrile and water. Internal Standard. A series of (2,4-dinitrophenyl)hydrazones was prepared from aliphatic aldehydes and ketones in an effort to find a satisfactory internal standard. One milliliter of aldehyde or ketone was added to a mixture containing 10 mL of ethanol and 10 mL of 2,4-DNP reagent (4 g of 2,4-DNP, 20 mL of H2S04, 20 mL of H20, and 100 mL of ethanol). The resulting yellow precipitate was filtered, washed, recrystallized from aqueous ethanol, and air-dried. Stock 50 pg/mL solutions were prepared, as with the azaarenes. These acetonitrile solutions were stable for several months stored at -10 "C. The recrystallized solid remained stable for 1year or more. However, standards in water exhibited noticeable hydrolysis of the 2,4-DNP derivative in a matter of days, thus necessitating preparation on the day they were to be used. Trace Enrichment and Sample Cleanup. A water sample with volume up to 2 L (depending on concentration factor desired) was filtered through a Whatman No. 1 filter paper to prevent clogging of the extraction cartridge. A Waters CISSep-PAK was activated by passing through 10 mL of acetonitrile, followed by 10 mL of purified water. The filtered water sample then was passed through the cartridge, using air pressure or a 60-mL disposable syringe, at flow rates up to 200 mL/min. Following trace enrichment, the Sep-PAK was connected to a 2-mL glass Luer-Lock syringe, and the unit was suspended in a small test tube. The apparatus was centrifuged (1500 rpm) to ensure removal of most of the residual water from the cartridge packing. Exactly 2.00 mL of eluting solvent, acetonitrile, was pipetted into the syringe barrel, forced through the Sep-PAK into a clean test tube, and centrifuged as before. The final sample extract was then membrane filtered (if necessary) through a Swinney apparatus containing a 0.2-pm-porosity nylon membrane. A 20-pL aliquot was used for instrumental analysis. In contaminated aqueous systems in which high concentrations of PAHs were suspected, the procedure was modified to remove interferences. After trace enrichment, the Sep-PAK was rinsed with 2 mL of purified water, followed by 2 mL of 25:75 (v/v) acetonitrile/water containing 5 mL/L 14 M aqueous ammonia, and finally by another 2 mL of purified water. The cartridge was attached to a 2-mL syringe, centrifuged, and then eluted as before, using 25:5:70 (v/v) acetonitrile/concentrated HCl/water. The eluate was neutralized by adding three drops of 14 M aqueous ammonia and filtered (if necessary), and 20 pL was injected into the HPLC. RESULTS AND DISCUSSION In combination with fluorescence detection, HPLC has proven to be a powerful tool for the separation and deter-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
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Table 11. Response Factors and Ratios for the Eight Nitrogen Heterocycles Investigated response factors, cm/ng" compd
X = 214 nm
X = 229 nm
X = 254 nm
fluorescence
quinoline isoquinoline benzo[c]cinnoline 4-azafluorene
1.67 f 0.04 2.21 f 0.11 0.308 f 0.022 0.452 f 0.045 0.211 f 0.038 0.364 f 0.038 0.245 f 0.017 0.270 f 0.030
1.53 f 0.04 0.228 f 0.008 0.557 f 0.006 0.132 f 0.003 0.225 f 0.002 0.226 f 0.003 0.320 f 0.004 0.367 f 0.004
0.122 f 0.002 0.101 f 0.004 1.12 0.01 0.278 f 0.003 0.575 f 0.007 0.571 f 0.006 0.167 f 0.001 0.157 f 0.002
