Application of Sodium Dodecyl Sulfate Micellar Electrokinetic


Application of Sodium Dodecyl Sulfate Micellar Electrokinetic Chromatography (SDS MEKC) for the Rapid Measurement of Aqueous Phase 2,4,6-Trinitrotolue...
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Environ. Sci. Technol. 2000, 34, 2330-2336

Application of Sodium Dodecyl Sulfate Micellar Electrokinetic Chromatography (SDS MEKC) for the Rapid Measurement of Aqueous Phase 2,4,6-Trinitrotoluene Metabolites in Anaerobic Sludge: A Comparison with LC/MS CARL A. GROOM, SYLVIE BEAUDET, ANNAMARIA HALASZ, LOUISE PAQUET, AND JALAL HAWARI* Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2 Canada

The present study describes the application of sodium dodecyl sulfate micellar electrokinetic chromatography (SDS MEKC) for the fast analysis of TNT biotransformation products in the aqueous phase of anaerobic sludge cultures. SDS MEKC was performed using a HewlettPackard HP 3D CE capillary electrophoresis system with photodiode array detection. In a single sample injection the SDS MEKC method detected 2,4,6-trinitrotoluene (TNT), 2-hydroxylamino-4,6-dinitrotoluene (2-HADNT), 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4diaminonitrotoluene (2,4-DANT), 2,6-diaminonitrotoluene (2,6DANT), and 2,4,6-triaminotoluene (TAT). Analyte peaks were verified using visible/ultraviolet photodiode array spectra from commercial standards and periodic analyses using electrospray ionization liquid chromatography mass spectrometry (ES-LC-MS). A time course study constructed from the SDS MEKC data provided supporting evidence for the stepwise reduction of TNT to TAT. The SDS MEKC method was judged to be of practical value for the identification of polar and moderately polar TNT biotransformants in place of HPLC and LC-MS methods, with significant reduction in material expense and analysis time.

Introduction Micellar electrokinetic chromatography (MEKC) is a wellknown capillary electrophoretic method for the analysis of neutral compounds of low molecular weight. As the name implies, the methodology of MEKC is derived from electrokinetic chromatography and therefore makes use of processes including electroosmosis, electrophoresis, and chromatographic partitioning. Excellent descriptions of MEKC are provided by Terabe (1), Sepaniak et al. (2), and by Mazzeo (3). Much consideration has been given to the use of MEKC to support or replace existing HPLC methods. The underivatized fused silica capillaries used in MEKC are inexpensive compared to HPLC columns, and many different separation * Corresponding author phone: (514)496-6267; fax: (514)496-6265; e-mail: [email protected] 2330 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 11, 2000

selectivities can be obtained by altering the separation buffer composition within these capillaries. A single capillary can therefore be used to obtain separations based on size, charge, chirality, or hydrophobicity. Additionally, the electroosmotic transport mechanism produces resolutions equivalent to those of gas chromatography, and fast analysis times are obtained because short capillary lengths (50-65 cm) are used. The small capillary dimensions also significantly reduce the consumption of solvent and sample. The most commonly applied MEKC method makes use of sodium dodecyl sulfate (SDS), a surfactant which is added to the aqueous buffer of a capillary zone electrophoresis (CZE) system. SDS exhibits both hydrophilic and hydrophobic character and in aqueous buffer coalesces to form micelles with hydrophobic tails oriented toward the interior and polar heads pointed outward. For aggregation to occur the SDS must be dissolved in excess of its critical micelle concentration (CMC), which is 8.1 mM for 25 mM phosphate-borate buffer (pH 8.5) (1). Under the above conditions the SDS micelles have an average aggregation number of 62 and a mean diameter of 4 nm. The resulting micellar preparation exhibits the properties of a homogeneous solution and under isocratic conditions solubilizes hydrophobic compounds of molecular weight less than 5000. The SDS micelles do not only solubilize hydrophobic compounds but also have an affinity for molecules displaying a wide range of polarities (4). The objective of this work was to evaluate the applicability of SDS MEKC for the rapid analysis of compounds formed in the biological degradation of 2,4,6-trinitrotoluene (TNT) in anaerobic sludge. The inadequate disposal of TNT as waste from the munitions and defense industries is a significant environmental problem (5), and TNT is recognized for its toxic and mutagenic effects (6). There have been several attempts to biodegrade TNT (7-12), but the compound is found to undergo biotransformation rather than mineralization to carbon dioxide (13, 14). While the initial amine products 4-amino-2,6-dinitrotoluene (4-ADNT), 2-amino4,6-dinitrotoluene (2-ADNT), 2,4-diamino-6-nitrotoluene (2,4- DANT), and 2,6-diamino-4-nitrotoluene (2,6-DANT) have been identified using MEKC, HPLC, and GC-MS techniques, many of the remaining biotransformed products remain unidentified, and the fate of the explosive during and after remediation is unclear. A previous study (15) employing high performance liquid chromatography (HPLC), liquid chromatography-electrospray ionization mass spectrometry (ES-LC-MS), and solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) has revealed the transformation of TNT to 2,4,6-triaminotoluene (TAT) in 80% yield using anaerobic sludge. A biotransformation pathway for this process with the principal detected intermediates is depicted in Scheme 1. The subsequent polymerization and autoxidation of TAT was identified as a dominant factor limiting the mineralization of TNT. The simultaneous monitoring of TNT biotransformation with HPLC, SPME-GC-MS, and ES-LC-MS requires a significant investment in personnel and machine time. SDS MEKC appeared to be capable of resolving both polar and nonpolar analytes with little pretreatment, and this made the method attractive for the analysis of unstable intermediates. The majority of SDS MEKC applications have admittedly been focused on biochemical and pharmaceutical analyses (16), but the technique was demonstrated to resolve explosive mixtures (17) and some explosive detonation products (18). The method was also applied to the monitoring of various energetic contaminants and their corresponding metabolites in degradative bioprocesses (19), including 2,4,6-trinitro10.1021/es991233x CCC: $19.00

Published 2000 by the Am. Chem. Soc. Published on Web 04/14/2000

SCHEME 1. Constructed Pathway for the Biotransformation of TNT in Anerobic Sludgea

a The principal detected intermediates in the reductive pathway are as follows: 2,4,6-trinitrotoluene (TNT), 2-hydroxylamino-4,6-dinitrotoluene (2-HADNT), 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4diaminonitrotoluene (2,4-DANT), 2,6-diaminonitrotoluene (2,6-DANT), and 2,4,6-triaminotoluene (TAT). At neutral pH and in the presence of the sludge consortia TAT was observed to form 2,2′,4,4′-tetraamino-6,6′-azotoluene (2,2′,4,4′-TA-6,6′-azoT) and 2,2′,6,6′-tetraamino-4,4′-azotoluene (2,2′,6,6′-TA-4,4′-azoT) with subsequent polymerization.

toluene (TNT) and its biotransformation products 2-ADNT, 4-ADNT, 2,4-DANT, and 2,6-DANT in wastewater samples (20). For these reasons SDS MEKC was further applied for the analysis of TNT biotransformants in anaerobic sludge cultures. The rapidity and simplicity of the method were clearly demonstrated, and the method was observed to be advantageous for monitoring the unstable intermediates 2-hydroxylamino-4,6-dinitrotoluene (2-HADNT), 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT), and 2,4,6- triaminotoluene (TAT).

Materials and Methods Chemicals. Sodium tetraborate, boric acid, methanol, and acetic acid were obtained from Aldrich Canada (Oakville ON). Analytical standards of 2-ADNT, 4-ADNT, 2,4-DANT, 2,6DANT, 2-HADNT, 4-HADNT, and TNT were purchased from AccuStandard Inc (New Haven, CT). A technical grade (about 95% pure) of 2,4,6-triaminotoluene hydrochloride (TAT‚3HCl) was obtained from Chemservice (Westchester, PA). TNT for use in microcosms was obtained from the Department of VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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National Defense (Valcartier, PQ). Sodium phosphate dibasic, sodium phosphate monobasic, sodium acetate, sodium chloride, ammonium carbonate, and hydrochloric acid were supplied by Fisher Canada (Montreal, PQ). Sodium dodecyl sulfate was obtained from Fluka chemicals (Neu-Ulm, Switz.). CE grade NaOH and phosphoric acid were obtained from Hewlett-Packard (Mississauga, ON). Capillary Electrophoresis System for SDS MEKC. Separations were performed using a commercially available Hewlett-Packard (HP) 3D CE capillary electrophoresis instrument interfaced with a HP Vectra personal computer running HP Chemstation software.The HP 3D CE was fitted with a HP G-1600-31232 fused silica bubble capillary with a total length of 64.5 cm and an effective length (inlet to detection window) of 56 cm. The HP bubble capillary internal diameter of 50 µm was expanded at the detection window to obtain a path length of 150 µm. The voltage was set at 30 kV and the temperature at 25 °C. Samples were injected by applying 50 mbar pressure to the capillary inlet for 5 s. The separation buffer was composed of 2.5 mM sodium tetraborate and 12.5 mM boric acid (pH 8.5) containing 50 mM sodium dodecyl sulfate. Absorbance was monitored at a wavelength of 225 nm. Unless otherwise indicated the separation time was 8 min with post-conditioning flushes of the capillary after each run in the following sequence: methanol (0.5 min), 0.1 N NaOH (0.5 min) and running buffer (3 min). The total analysis time was therefore 12 min. The operating current under the above conditions was typically 22 µAmpere. SDS MEKC Reproducibility Tests and Method Calibration. A standard solution of TNT (5 mg/L) and six of its metabolites (2-ADNT, 4-ADNT, 2,4-DANT, 2,6-DANT, 2-HADNT, 4-HADNT, 5 mg/L each) was prepared by combining the appropriate volumes of each analytical standard to a single vial containing separation buffer. The sample was injected repeatedly for a total of 25 analyses using the abovedescribed method. The peaks were then integrated, and the migration times and area counts of each analyte were determined. In the case of TAT which is known to oxidize readily, a separate aqueous solution of TAT‚3HCl (25 mg/L) was stabilized in Na2S2O3 (4.5%), and the analysis time was shortened to 5 min without post-conditioning. Standard calibrations for the analytes were run at five levels (0.5, 1, 5, 10, 25 mg/L) with quadrupulate sampling followed by linear regression and the determination of relative standard deviations (21). Anaerobic Microcosm Preparation and Sampling for TNT Metabolites. Wastewater anaerobic sludge was obtained from a nearby food manufacturer (Champlain Industries; Cornwall, ON) and stored at 4 °C when not in use. On average the sludge contained 8 g of volatile suspended solids per kg wet weight (g VSS/kg) and possessed a zero reduction potential (0 Eh) at the time of innoculation. Serum bottles (100 mL) were charged with deionized water (34 mL), anaerobic sludge (5 mL), and a mineral salt medium (1 mL) composed of 2.0 g/L KH2PO4, 3.0 g/L K2HPO4, 30 g/L NaHCO3, 35 g/L KHCO3, and 30 g/L Na2SO4. Molasses (3.3 g/L) served as a carbon source and TNT (50 mg/L) as the nitrogen source for the degrading microorganisms. The headspace in each microcosm was flushed with nitrogen gas to maintain anaerobic conditions and then sealed with butyl rubber septa and aluminum crimp seals to prevent the loss of volatile metabolites. The serum bottles were stored in a New Brunswick scientific G24 Environmental Incubator Shaker with temperature set at 35 °C and agitation at 60 rpm. The shaker was covered with aluminum foil to protect the microcosm contents from photolysis. Sample removal to monitor the formation and disappearance of metabolites was carried out at 2 h intervals for the first 12 h of the experiment followed by intervals of approximately 12 h until 2332

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termination of the experiments after 50 h. The samples were filtered through Millex FG 0.2 µm filters (Millipore, Boston, MA) and analyzed using the SDS MEKC method described above. Control microcosm experiments were conducted using the above-described preparations without the addition of TNT. HPLC Analysis. Solutions of TAT were analyzed using a Waters HPLC system fitted with a Waters pump (Waters Chromatography Division, Milford, MA) and connected to a WISP auto injector (Model 717; Millipore). Samples were injected (20 µL volume) into a Supelcosil ABZ+ column (25 cm by 4.6 mm; particle size; 5 µm) held at 35 °C (Temperature Control Module Model TCM: Millipore) using an initial mobile phase of 0.025 M phospate buffer (pH 7.0) containing 1% methanol and 0.5% w/w octanesulfonic acid as ion pairing reagent. The mobile phase methanol concentration was varied linearly as follows: (i) 1% to 20% from 0 to 6 min, (ii) constant at 20% from 6 to 12 min, (iii) 20% to 60% from 12 to 15 min, (iv) constant at 60% from 15 to 25 min, (v) 60% to 1% from 25 to 28 min. A programmable UV-vis multiwavelength detector (Model 490; Millipore) was set to scan from 200 to 350 nm for peak identification with chromatograms extracted at 219 nm. LC-MS. LC-MS was performed on a Micromass Platform II benchtop single-quadropole mass detector fronted by a Hewlett-Packard 1100 series HPLC system. The chromatographic conditions used were a C18 LC column (25 cm by 4.6 mm; 5-µm diameter particles) and acetonitrile-water gradient programmed from 30 to 80% (vol/vol), using a flow rate of 1 mL/min with a postcolumn split of 5:95. The mobile phase acetonitrile concentration was varied linearly with time as follows: (i) 30% to 40% from 0 to 20 min, (ii) 40% to 80% from 20 to 30 min, (iii) constant at 80% from 30 to 32 min, (iv) 80% to 30% from 32 to 34 min, (v) constant at 30% from 34 to 40 min. Analyte ionization, a process which produces mainly the deprotonated molecular ion (M-H)-, was achieved in the negative electrospray ionization mode (ES-) by using a probe tip potential of 3.0 kV and a skimmer voltage of 30 V. The temperature of the electrospray ionization capillary was maintained at 90 °C. The mass spectrum was typically scanned at a rate of 1 s/100 Daltons (Da). The total ion current was acquired between 40 and 500 Da, which was followed by extracting the deprotonated molecular ion (M-H)- of the suspected metabolite. In the case of TAT, 2,4-DANT, and 2,6-DANT, analyte ionization was achieved by using positive electrospray ionization (ES+), a process which produces mainly the protonated molecular ion (M + H)+.

Results and Discussion Method Reliability. An acceptable level of in-house reproducibility must be established before any separation method can be applied to the analysis of complex samples. To evaluate the reliability of the available SDS MEKC apparatus, standard solutions (5 mg/L) for 2-ADNT, 4-ADNT, 2,4-DANT, 2,6DANT, 2-HADNT, 4-HADNT, and TNT were repeatedly injected and monitored for variability with respect to migration time and peak area. The tendency for TAT to degrade in H2O necessitated an individual trial using 25 mg/L TAT with Na2S2O3. Relative standard deviations and capacity factors for all eight standards are presented in Table 1. For all tested compounds (except TAT) the migration times were constant with relative standard deviations less than 1%, while the relative standard deviations with respect to area were around 2%. The migration times for TAT were constant but despite the inclusion of 4.5% Na2S2O3 the peak areas were observed to linearly decrease with time. Accordingly linear regression was performed, and the standard deviation of residuals from the fitted line about the centroid value was reported as a measure of variability (21). Calibration data using quadrupulate fresh samples for the eight standards

TABLE 1. Reproducibility of TNT Metabolites Standards (5 mg/L) Using SDS MEKCa migration time (min) compd TATb 2,6-DANT 2,4-DANT TNT 2-HADNT 4-HADNT 2-ADNT 4-ADNT a

Abs: 225 nm.

b

peak area (mAU-s)

mean

SD

RSD %

mean

SD

RSD %

capacity factor K′

2.953 3.965 4.197 4.792 4.966 5.277 5.780 6.013

0.0325 0.0264 0.0485 0.0326 0.0414 0.0428 0.0523 0.0592

1.10 0.667 1.154 0.668 0.8354 0.8116 0.9048 0.9852

39.33 9.995 12.72 11.02 5.409 4.383 18.37 18.42

1.1123 0.2956 0.2809 0.380 0.2318 0.0517 0.2775 0.2002

2.83 2.95 2.21 3.45 4.29 1.18 1.5 1.09

0.189 0.785 0.989 1.630 1.858 2.324 3.300 3.882

Concentration ) 25 ppm.

TABLE 2. Calibration Area Counts (mAU-s) vs Concentration (ppm) for TNT biotransformant standardsa

a

compd

equation (y ) a + bx)

N

R2

linear range (ppm)

TAT 2,6-DANT 2,4-DANT TNT 2-HADNT 4-HADNT 2-ADNT 4-ADNT

area ) 0.0646 + 1.6167 concn area ) 0.0533 + 1.0372 concn area ) 0.0602 + 1.3782 concn area ) 0.0593 + 1.5290 concn area ) 0.0558 + 1.1925 concn area ) 0.0708 + 0.9896 concn area ) 0.1164 + 2.6663 concn area ) 0.1230 + 2.8577 concn

20 20 20 20 20 20 20 20

0.996 0.994 0.996 0.999 0.999 0.995 0.999 0.999

0.211-25 0.209-25 0.205-25 0.154-25 0.177-25 0.214-25 0.079-25 0.074-25

Abs: 225 nm.

are presented in Table 2 with regression equations, r 2 values, and indications for linear range as determined by three times the standard deviation of the baseline noise (0.07 mAU-s). Observation of TNT Metabolites in Anaerobic Microcosm Samples. Following characterization of the SDS MEKC methods reliability and precision with repect to the desired biotransformation products, the method was applied to the monitoring of TNT biotransformation in anaerobic sludge. Sample preparation was minimal with the culture fluid being drawn and then filtered through a sterile 0.22 µm cartridge before immediate injection into the CE system without dilution. Peak identification was assisted by comparison of the sample peak photodiode array spectra to a spectral library of 100 mg/L reference standards and by spiking in standard solutions to the microcosm samples. An electropherogram for a sludge sample taken 5 h after innoculation with a corresponding biological control is shown as Figure 1. As indicated in Figure 1, the analytes TAT (1), 2,6-DANT (2), 2,4-DANT (3), TNT (4), 2-HADNT (5), 4-HADNT (6), 2-ADNT (7), and 4-ADNT (8) are observed within 8 min after injection using the MEKC method, with baseline separation for each component. Table 3 provides the performance characteristics for the MEKC method with resolution for each of the components. The order of separation for the metabolites was similar to that observed for the corresponding C18 liquid chromatography mass spectrometry method (22). This confirmed that the earlier components were separating chromatographically by their partition coefficients within the SDS micelles rather than by electrophoretic migration within the borate aqueous phase. For compounds of low molecular weight and little charge, the capacity factors K′ (Table 3) are defined as the ratio of the analyte amount residing in the micelles to that found in the mobile aqueous phase. The optimal K′ is equal to (tmc/to)1/2 where tmc and to are the respective micellar and electrosmotic migration times. K′ is related to the thermodynamic distribution coefficient by the ratio of the partial volumes of the micelles and the aqueous phase. The capacity factor for a given analyte therefore increases linearly with surfactant concentration. Decent separations usually require that the K′ values for each analyte

FIGURE 1. Electropherogram (absorbance at 225 nm) for an aerobic sludge microcosm sample 5 h after innoculation with 50 ppm TNT (upper trace) and control microcosm sample lacking TNT (lower trace): EOF electroosmotic front, 1: TAT, 2: 2,6-DANT, 3: 2,4-DANT, 4: TNT, 5: 2-HADNT, 6: 4-HADNT, 7: 2-ADNT, 8: 4-ADNT, 9 Sudan III, A: molasses components. Electrophoretic conditions: 2.5 mM sodium tetraborate/12.5 mM boric acid (pH 8.5) with 50 mM sodium dodecyl sulfate. Separation voltage 30 kV. Resultant current 24.2 µAmperes. Pressure injection for 5 s at 50 mbar. vary between 0.5 and 10. In viewing Table 3 one can assume that a better resolution for TAT is possible through the addition of SDS, as it would increase the partial volume of the micelles and the total amount of TAT residing in the pseudostationary phase. Mussenbrock and Kleibohhmer (17) reported the use of sodium laureth sulfate, a surfactant with greater lipophilicity than SDS due to the placement of ethoxy groups in its aliphatic chain, to be advantageous for the separation of polar compounds such as TAT. However, in view of the excellent selectivity and short run times obtained, no optimization of the buffer composition was attempted. 2-HADNT and 4-HADNT bear a slight negative charge at the separation pH (8.5) that imparts an electrophoretic mobility with resulting longer migration time. Electrophoretic VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Diode array absorption spectra for peaks in Figure 1 (solid lines) with normalized library spectra from commercial standards (broken lines) for (a) 2-HADNT, (b) 4-HADNT, (c) TAT, and (d) hydroxylamino-amino-nitrotoluene (standard not available).

TABLE 3. Separation Characteristics of TNT Biotransformant Peaksa Identified from the MEKC Analysis of an Anaerobic Sludge Microcosm Sample 5 h after Inocculation peak

migration time (min)

capacity K′

absolute effic N

resolution R

EOF TAT 2,6-DANT 2,4-DANT TNT 2-HADNT 4-HADNT 2-ADNT 4-ADNT

2.78 3.346 3.680 3.680 5.191 5.435 5.901 6.658 7.021

0 0.340 0.562 1.157 2.193 2.603 3.598 6.250 8.446

91 232 79 700 94 744 123 859 81 484 94 994 117 035 131 356

6.446 10.93 26.9 6.138 9.25 16.66 7.943

a

Abs: 225 nm.

mobility as a result of weak acid character has been noted for separations of nitro and aminophenolic compounds using CZE (23). To the best of our knowledge this is the first reported detection of 2-HADNT, 4-HADNT, and TAT in the biodegradation of TNT using SDS MEKC. Supporting DAD spectra with corresponding spectra from authentic commercial standards for the three compounds are shown in Figure 2 parts a-c. 2334

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The peak apex sample spectrum for TAT (Figure 2 part c) differed from its reference spectrum in possessing a slight shoulder at 240 nm. This was suspected to have resulted from the in-peak oxidative dimerization of TAT, as the sample SDS MEKC analyses did not employ the 4.5% Na2S2O3 antioxidant applied to TAT reference samples. The small unlabled peak observed at 4 min in Figure 1 was tentatively identified as an isomer of hydroxylamino-amino-nitrotoluene by its (ES-) LC-MS spectra, with a deprotonated molecular ion (M - H)- at m/z 182 (Figure 3). The DAD spectra for the tentative hydroxylamino-amino-nitrotoluene intermediate is included as Figure 2 part d. While the method was observed to possess excellent selectivity, peak efficiency was unexceptional, varying from 79 000 absolute theoretical plates for 2,6-DANT to 131 000 for 4-ADNT. The peak efficiencies for MEKC methods are routinely reported to be around 100 000. The most commonly cited cause of low peak efficiency in MEKC is axial distortion due to the high rate of electroosmosis and this was considered acceptable in view of our desire for short run times. Other causes of low peak efficiency include sample matrix viscosity, micelle diameter polydispersity, poor thermodynamic exchange between phases, and relatively long hydrodynamic injection times. For practical purposes the main implication of low peak efficiency is the number of compounds that can

FIGURE 3. Mass spectrum of hydroxylamino-amino-nitrotoluene intermediate [M - H ] m/z ) 182, as determined using negative electrospray (30 kV) LC-MS.

FIGURE 4. Electropherogram (225 nm) for an aerobic sludge microcosm sample 47 h after innoculation with 50 ppm TNT: EOF: electroosmotic front. 1 and 2: tetraamino-azonitrotoluene isomers. S: Sudan III marker. Electrophoretic conditions: 2.5 mM sodium tetraborate/12.5 mM boric acid pH 8.5 with 50 mM sodium dodecyl sulfate. Separation voltage 30 kV. Resultant current 24.2 µAmperes. Pressure injection for 5 s at 50 mbar.

FIGURE 5. Time course for an anerobic sludge microcosm showing the biotransformation of 2,4,6-TNT to 2,4,6-TAT. Part a: l 2,6-DANT, + 2-HADNT, X 4-HADNT, u 2-ADNT, m 4-ADNT. Part b: l 2,4-DANT, + TNT, X TAT. Error bars indicate relative standard deviations (N ) 4).

be identified in a single complex sample. The migration time window for MEKC is defined as the maximum possible contact time for an analyte between the mobile and micellar pseudostationary phases and is determined by the relative mobilities of the electroosmotic front and the SDS micelles themselves. In viewing Figure 1 the EOF was observed to have a migration time (teof) of 2.79 min, and the micellar migration time (tmc) was observed to be 7.85 min using Sudan III as a hydrophobic marker. The separation window was therefore observed to be 5.77 min or 0.55 when conventionally defined as teof/tmc. A peak efficiency of 79 000 implies that within a 5.77 min time window a complex mixture of only 40 ideally evenly distributed peaks could be resolved to baseline separation. The separation of hydrophobic species becomes particularly difficult, as the pseudostationary behavior of the micelles causes resolution to decrease as a hyperbolic function of capacity factor. The implications of this phenomenon are apparent in viewing Figure 4, where the second of two large hydrophobic peaks is observed to be incompletely resolved from the Sudan III marker (Figure 4 peak 2). The photodiode array spectra obtained from these peaks indicated the presence of azotoluene derivatives, the most abundant being 2,2′,4,4′-tetraamino-6,6′-azotoluene and 2,2′,6,6′-tetraamino-4,4′-azotoluene as determined previously by (ES+) LC-MS data (15). Verification of the these metabolites will require the acquisition of authentic standards. The most common method employed to improve both resolution and efficiency in MEKC is to reduce the electroosmotic flow via the addition of methanol, modification of pH, or through the use of capillary coatings. All of these

methods increase resolution at the expense of longer run times. SDS MEKC Time Study: Metabolic Pathway of TNT Biotransformation. A time course constructed from MEKC data for a microcosm starting with 50 mg/L TNT is presented as Figure 5 parts a and b. The phosphate within the culture medium and the generation of methane and carbon dioxide (at least initially) resulted in a strict anaerobic environment under slight positive pressure with the pH remaining at 7.3. As shown in Figure 5, TNT was observed to disappear in less than 15 h with the rapid accumulation of 4-HADNT and 2-HADNT. The formation of 4-HADNT preceded that of 2-HADNT, and the subsequent conversion of 4-HADNT to 4-ADNT also occurred at a faster rate than the conversion of 2-HADNT to 2-ADNT. Accordingly, the observed concentrations of 2,4-DANT were at all times greater than 2,6DANT, and 2,4-DANT was observed to persist in the culture for over 30 h. The formation of TAT was observed to begin 5 h after innoculation and accumulated to a concentration of 30 mg/L for the duration of the experiments. The accumulation of TAT to constant levels and the late appearance of azo-toluene derivatives (Figure 4) provided supporting evidence for TAT formation and subsequent polymerization to dead-end polymeric products (15). The direct polymerization and humification of mono or diaminotoulenes to the solid phase, a phenomenon reported to occur in oxidative environments (24), must also be considered, but the elevated level of TAT and highly anaerobic conditions appear to exclude this possibility. According to Preuss and Rieger (25), anaerobic sludge consortia transform 2,4,6-TNT through the reduction of one or more aryl nitro groups via VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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nitroso and hydroxylamino intermediates to form arylamines. The initial reduction products are 2-nitroso-dinitrotoluene (2-NO-DNT) and 4 nitroso-dinitrotoluene (4-NO-DNT) which then are quickly reduced to 2-HADNT and 4-HADNT, with the formation of 4-HADNT being favoured. 2-ADNT and 4-ADNT were subsequently formed with little indication of the formation of dihydroxylamino intermediates. The cycle of reduction is repeated for each nitro group to generate 2,4-DANT and 2,6-DANT, with predominance of 2,4-DANT. The reduction of 2,4-DANT was reported to be the rate limiting step in the overall reduction of TNT to TAT. The SDS MEKC method did not detect any nitroso intermediates but was successful in detecting 2-HADNT and 4-HADNT, and the observed time course generally supports the abovedescribed biotransformation. Previous LC-MS analyses also did not detect any nitroso intermediates in anerobic sludge (15), although some transient intermediates were detected using fungi to degrade TNT (22). It is likely that these structures do not accumulate in anaerobic sludge to concentrations above the limits of detection for either method. Applicability of SDS MEKC. Using the SDS MEKC method analytes previously detected using two different analytical methods (and three sample injections) were now observable from one with a considerable savings in time. This is not to say, however, that an equivalent amount of information was obtained, as the additional dimension provided from mass detection is invaluable for the indentification of novel contaminants or intermediates. The SDS MEKC method was not optimal for the analysis of hydrophobic species arising from the polymerization of TAT. However, the methods short analysis time and minimal pretreatment facilitated the rapid monitoring of the unstable transformants 2-HADNT, 4-HADNT, and TAT and reduced the time requirement for the construction of time courses for the biotransformation of TNT to form TAT. The SDS MEKC method was therefore considered to be of practical use for the rapid monitoring of TNT and its initial polar and moderately polar biotransformation products and as a complement to existing LC-MS methods. The SDS MEKC method is currently being applied to the analysis of soil washings from a contaminated waste site and to the analysis of hexahydro-1,3,5,-trinitro-1,3,5triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX) consuming anerobic microcosms.

Literature Cited (1) Terabe, S. Micellar Electrokinetic Chromatography; Beckman monograph 266924; Beckmann Instruments Inc.: Fullerton, CA, 1986.

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Received for review November 3, 1999. Revised manuscript received February 25, 2000. Accepted March 1, 2000. ES991233X