Tracer-Grade Rhodamine WT: Structure of Constituent Isomers and

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Environ. Sci. Technol. 2001, 35, 4089-4096

Tracer-Grade Rhodamine WT: Structure of Constituent Isomers and Their Sorption Behavior DHARNI VASUDEVAN,* RYAN L. FIMMEN, AND ALEX B. FRANCISCO† Nicholas School of the Environment, Duke University, Durham, North Carolina 27708-0328

Rhodamine WT (RWT), a fluorescent xanthene dye, is often used as a conservative tracer in aquifer characterization and as a surrogate or sorbing tracer for contaminant fate and transport. Quantitative tracing employing RWT is confounded by the presence of two major fluorescent constituents in the tracer-grade mixture. Here, we have confirmed that the two constituents are isomers of RWT, elucidated their molecular structures and their percent mass distribution in the tracer-grade mixture, and examined their individual sorption behavior onto soil solids. The energyminimized geometry of the meta isomer indicates that it possesses a greater potential for (i) hydrophobic exclusion from bulk solution, (ii) electrostatic attraction to the solid phase, and (iii) surface complexation with surface-bound Al and Fe ions as compared with the para isomer. Hence, the meta isomer consistently sorbs to a higher extent onto the mineral phases examined. The para isomer has the potential to be a nearly conservative tracer, and the meta isomer has the potential to be a nonconservative tracer. To facilitate RWT use as a conservative tracer and comparison of tracer tests at different locations, we recommend modification of the RWT manufacturing process toward production of 100% of the para isomer. Alternatively, appropriately designed tests with tracer-grade RWT have the potential for simultaneous estimation of hydraulic parameters and contaminant fate and transport.

Introduction Rhodamine WT (RWT) is a fluorescent xanthene dye that has found widespread use as a water tracer due to its facile detection with a field fluorometer, even at concentrations as low as 0.1 ppb. In addition, RWT does not appear to present a carcinogenic or mutagenic hazard; nor is it acutely toxic at the recommended 2 mg/L concentration (1, 2). Although RWT is one of the more effective groundwater tracers, its use in quantitative tracing is often compromised by the following: (i) photochemical decay of dissolved RWT; (ii) RWT sorption to stream bed gravel, alluvial sediment, and organic matter; and (iii) chemical quenching of RWT fluorescence by dissolved aqueous constituents, including protons at pH values below 6 (3, 4). It is therefore not surprising that field studies of RWT fate and transport have yielded conflicting information with respect to RWT reactivity. Some studies * Corresponding author e-mail: [email protected]; phone: (919)613-8039; fax: (919)684-8741. † Current address: BBL-Sciences, Cranbury, NJ. 10.1021/es010880x CCC: $20.00 Published on Web 09/18/2001

 2001 American Chemical Society

found the fate and transport of RWT, tritium, and chloride to be similar and hence used RWT as a conservative tracer (5, 6), while others have established RWT’s nonconservative nature in the field as a result of sorptive losses onto particulates and soil solids (7-10). The nonconservative nature of tracer-grade RWT has inspired several studies on the use of RWT as a sorbing tracer or a surrogate for pesticide fate and transport (11-13). These studies have concluded that RWT can provide qualitative information on contaminant fate and transport; however, variability in the sorption behavior of RWT limits its use in quantitative prediction. Use of RWT in quantitative tracing is further confounded by the fact that tracer-grade RWT consists of two primary fluorescent compounds (assumed to be isomers of RWT) and other minor fluorescent constituents (14, 15). These potential isomers possess slightly different UV-vis absorption (15) and fluorescence emission spectra (16); hence, each compound has a distinct relationship between concentration and fluorescence or UV-vis absorption. Indirect evidence based on UV-vis and fluorescence spectroscopy suggests that the two primary fluorescent compounds are isomers of RWT distributed in a 40-60 or a 50-50% mass ratio (15, 16). However, neither estimate of the percent mass distribution has been confirmed with direct evidence. Shiau and coworkers (15) have suggested potential molecular structures for these compounds based on UV-vis spectroscopy. Since definitive structural assignments cannot be made on the basis of UV-vis spectra alone, these structures need to be confirmed. Careful laboratory studies of tracer-grade RWT interaction with model particles and soil samples have confirmed sorption onto soil organic matter, quartz (silica), clay silicates, and metal oxides (8, 11, 12, 16-19). These studies have shown that the extent of sorption varies as a function of aqueous RWT concentration, composition of the solid matrix, and aqueous phase characteristics including pH, ionic strength, and the ionic composition. Because a complex suite of factors influences RWT sorption, the solid-water distribution coefficient (Kd) for RWT cannot be adequately predicted by the traditional organic carbon-water distribution coefficient (Koc) and the organic carbon content of the soil/sediment (12). Instead, the pH-dependent partitioning to mineral surfaces should also be accounted for and quantified. Many of the above cited studies have evaluated tracer-grade RWT as a single solute, and little is known about the individual sorption behavior of the two primary constituents. The unique contribution of this study is an assessment of the similarities and differences in the structure and sorption behavior of the two primary fluorescent constituents in the RWT tracer-grade mixture. We hypothesize that the two fluorescent constituents are isomers of RWT whose structures differ only in the positioning of the phenyl-substituted carboxylate groups and that this difference is the cause of their distinct sorption behavior onto soil solids. The objectives of this study are (i) to evaluate whether the two primary fluorescent compounds are indeed structural isomers of RWT; (ii) to elucidate their respective structures and percent mass distribution in the tracer-grade mixture; and (iii) to understand their individual sorption behavior onto soil minerals and organic matter as function of time, pH, and aqueous RWT concentration. The impetus for this work comes from the need to improve the design and interpretation of RWT tracer tests, the estimation of aquifer dispersivity and hydraulic conductivity, and the prediction of contaminant transport using breakthrough curves of tracer-grade RWT. VOL. 35, NO. 20, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Materials and Methods Characterization of Tracer-Grade RWT. RWT was obtained from Turner Designs (Sunnyvale, CA). The tracer-grade mixture, as received, was at pH 8.0 and contained 21.33% active ingredient. Methanol, phosphoric acid, HCl, and NH4OH were purchased from Mallinckrodt; acetic acid and NaCl were from EMScience; NaOH was from Fisher Scientific; and methanol-d4 (CD3OD) was from Alfa Aesar. All stock solutions were prepared with deionized water (Milli-Q, Millipore Corp.), and all glassware was acid washed with 5 N HNO3 prior to use. Henceforth, RWT concentrations in the parts per million range are specified as milligrams per liter (mg/L), and NMR shifts are denoted in parts per million shift (ppm) from an internal reference. Chemical constituents of a 15 mg/L (active ingredient) aqueous solution of tracer-grade RWT were separated by HPLC (Hewlett-Packard 1100 series) with a Zorbax Eclipse XDB-C18 column. Mobile phases appropriate for the intended analytical techniques were utilized. Isocratic elution with 65% methanol/35% 0.023 M glacial acetic acid (pH 3.2) was used for the separation of constituents to be analyzed by mass spectroscopy. Isocratic elution with 65% methanol/ 35% 3.3 × 10-4 M H3PO4 (pH 3.2) was used for the separation of constituents to be analyzed by 1H NMR analysis. A diode array detector and a fluorescence detector (Hewlett-Packard 1100 series) were used to identify the UV-active and fluorescent constituents, respectively. In all cases, the mobile phase was pumped at 1 mL/min. The molecular masses of the two primary fluorescent constituents in the tracer-grade mixture were determined by mass spectroscopy. Following HPLC separation, the pH of the eluent samples was adjusted to 4, 5, and 7 using NH4OH, and molecular masses of the fluorescent constituents were acquired using electrospray ionization on a PE Sciex API 150EX instrument. Samples were introduced at 10 µL/ min and monitored in the range of 150-1000 Da. HPLC eluent fractions corresponding to the two likely isomers were repeatedly collected from over 50 separations of a 15 mg/L solution in order to obtain sufficient mass of each compound for NMR analysis. The pH of each sample was adjusted to approximately pH 8 (to match that of the tracer-grade mixture) by the addition of small aliquots of 0.5 N NaOH. Samples were lyophilized overnight to ensure complete water and methanol removal; the dried samples were dissolved in 0.6 mL of methanol-d4 to achieve a concentration of approximately 5.5 g/L and transferred to NMR sample tubes for analysis. 1H NMR spectrum of the two separated constituents and the tracer-grade mixture in methanol-d4 were acquired on a Varian 500 MHz spectrometer at 25 °C. 1H NMR spectra of the two separated constituents were used to determine the individual molecular structures and to identify characteristic chemical shifts and coupling constants. Subsequently, integrations of the individual isomer signals in 1H NMR spectrum of the tracergrade mixture were compared, and the percent mass distribution of the isomers in the tracer-grade mixture was ascertained. On establishing the molecular structure of the two compounds via 1H NMR, their energy-minimized groundstate geometries were computed using ab initio methods with the Hartree-Fock level of theory in combination with 6-31G* basis set (Gaussian 98, revision A.9) and visualized using CS Chem3D Pro (version 4.0, CambridgeSoft). Sorption Behavior. RWT sorption was evaluated on five representative model solids: (1) aluminum oxide, γ-alumina (Al2O3) (Degussa Corp., type C); (2) iron oxide, hematite (RFe2O3) (J. T. Baker); (3) fine grain sand, quartz (180-250 µm, Unimin Corp.) treated to remove organic carbon, inorganic carbon, and metal oxides using procedures outlined in Carter (20); (4) sand (no. 3) coated with iron oxide, hematite, (0.9 4090

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mg of Fe/g of sand) using procedures outlined in Gu et al. (21); and (5) sand (no. 3) coated with humic acid (Aldrich Co.) (∼0.01% C) using procedures outlined in Laor et al. (22). The mineralogy of these solids was determined by X-ray diffraction (XRG 3000, Philips Analytical Xray), and the N2 BET surface area was measured using a Gemini 2360 surface area analyzer (Micromeritics Corp.). Thirty milliliter glass amber vials with Teflon septa, wrapped in aluminum foil, served as batch reactors for most sorption experiments; 500-mL brown HDPE bottles (Nalgene) were used only for evaluating the sorption kinetics of the sand-based solids. A predetermined mass of the test solid was added to deionized water to achieve a final loading of 10 g/L for the pure-phase metal oxides and a final loading of 500 g/L for the three sands. The ionic strength of the suspension was set to 10-2 M using NaCl. The suspension was allowed to equilibrate for 30 min and then spiked with a known concentration of tracer-grade RWT (0-7 mg/L, active ingredient), and the pH was set using small amounts of NaOH/HCl. Reactors with pure-phase metal oxides were continuously agitated using stir bars, while orbital or endover-end shakers were used for the sands to ensure minimal particle shearing. After a suitable equilibration period (0-24 h), aliquots of the supernatant were removed and filtered (0.22 µm membrane filter). Chemical constituents in the filtered aliquot were separated by HPLC (as previously described), and the concentrations in the supernatant were determined using UV absorbance at 256 nm (Diode Array Detector, Hewlett-Packard 1100 series). The pH of the reactors was measured following equilibration and recorded as the pH of the experiment. For each sorption experiment, a reactor containing a solid-free (blank) solution was prepared, equilibrated, and filtered in the same manner as a reactor containing a solid suspension. The compound concentration in the blank solution served as the initial concentration. The mass sorbed was calculated from mass balance by subtracting the concentration recovered in the supernatant from the initial concentration. Since RWT shows a slow but measurable rate of photolysis under natural conditions (23), all reactors were kept in the dark for the duration of the experiment. Sorption of the two primary constituents (isomers) on all five test solids was first measured as a function of time (0-24 h) at pH 6 in reactors with an initial RWT concentration of 3 mg/L to determine the time to equilibrium sorption. Then, sorption was measured as a function of (i) pH (pH 4-8) in reactors with an initial RWT concentration of 3 mg/L following 24 h equilibration and (ii) dissolved RWT concentration (1-7 mg/L) at pH 6 following 24 h equilibration. Replicate sorption experiments were performed for selected conditions. To ensure that decreases in aqueous RWT concentration were due to sorption and not degradation, desorption of previously sorbed RWT was tested by addition of 10-3 M NaF or extraction with methanol.

Results and Discussion Characterization of Tracer-Grade RWT. Tracer-grade RWT is separated by HPLC/DAD analysis into three UV-active compounds, of which only two exhibit fluorescence. Mass spectroscopy of the two fluorescent compounds both yield parent ion peaks of 487 Da. We, therefore, conclude that the two primary fluorescent compounds in the tracer-grade mixture are indeed structural isomers of RWT. For convenience of discussion, the isomer that elutes first from our C18 column is henceforth referred to as isomer 1, while the isomer that elutes next is referred to as isomer 2. This nomenclature is consistent with our previous work (16). A molecular mass of 487 Da corresponds to a molecular formula of C29H31O5N2 and the structure depicted in Figure 1. This formula is consistent with previously published structures that correspond to the sodium and chloride salts

FIGURE 1. Structure of RWT corresponding to a molecular mass of 487 Da. The A-B-C ring system without the diethylamino groups depicts xanthene. The isomers differ in the positioning of the carboxylate groups on the phenyl substitutent (D). a, sites of protonation/deprotonation. b, H(1), H(2), and H(3) correspond to H1, H2, and H3, respectively, in Figure 2.

TABLE 1. 1H NMR Data for Two Principle Fluorescent Constituents within Tracer-Grade RWT proton assignment

Separated Isomers isomer 1 (para)

isomer 2 (meta)

1 2 3 4 5 6

Chemical Shift in Methanol-d4 (ppm) 7.31 6.99 6.90 8.06 8.18 7.78

7.31 6.99 6.89 8.70 8.12 7.22

J(1-2) J(2-3) J(4-5) J(4-6) J(5-6)

Coupling Constants (Hz) 9.56 2.35 8.11 nda 1.50

9.57 2.52 1.57 nda 7.78

Isomers in the Tracer-Grade Mixture proton isomer 1 isomer 2 assignment (para) (meta) 4 5 6

Relative Peak Integrations 0.90 0.90 0.94

0.9 1.0b nac

a Coupling not observed. b Peak area manually set to 1. c Peak area is qualitatively similar as evident from Figure 2; due to peak overlap with an impurity in the tracer-grade mixture, accurate integration was not possible.

of the fully deprotonated species, C29H29O5N2Na2Cl, possessing a molecular mass of 566.5 Da (11, 12, 15, 19). The structure of the xanthene ring system (rings A-C in Figure 1) including the diethylamino functional group has been previously established and agreed upon (4, 12, 15, 19). Using 1H NMR, we elucidated the position of the dicarboxylate (-COOH) functionality present on the lower phenyl ring (ring D). Isomer Structure. We used chemical shifts, coupling patterns, and the corresponding coupling constants from 1H NMR spectra to establish the relative positioning of protons and the -COOH groups on the lower phenyl ring of each isomer. This same approach is used to verify established structural details of the diethylamino groups and the aromatic xanthene ring system. Chemical shifts and coupling constants of protons in the aromatic region of the NMR spectra for the individual isomers are listed in Table 1, and the NMR spectra of the aromatic protons in the tracer-grade mixture (containing both isomers) are shown in Figure 2. The aromatic protons present on rings A and C are related by a C2 axis of symmetry through the center of the xanthene system. Since

these protons are magnetically equivalent and display identical NMR signals, they are not uniquely enumerated here (Table 1; Figures 1 and 2). On the basis of the chemical shifts and coupling constants of the D ring protons of the individual isomers (Table 1), we infer that the -COOH groups in isomer 1 are para to each other while the -COOH groups in isomer 2 are meta to each other. The proposed structures of the D rings are depicted in Figure 2. The fundamental structural difference between the two isomers is the positioning of a single -COOH group. Evidence supporting this structural assignment is provided below. For isomer 1, the chemical shifts for protons labeled 4i, 5i, and 6i are within 0.4 ppm of each other, indicating that the strong electron-withdrawing effect of the carboxylate groups acts uniformly on all three protons. It is, therefore, likely that each proton is both ortho and meta to a carboxylate group (Figure 2). The coupling patterns and magnitude of the coupling constants further confirm this structural assignment (Table 1): (1) proton 5i is coupled to 4i with a coupling constant of 8.11 Hz, a characteristic value for ortho proton coupling (24); (2) proton 5i is also coupled to 6i, with a coupling constant of 1.5 Hz characteristic of meta proton coupling; and (3) coupling of protons 4i and 6i is not observed, typical of para coupling. For isomer 2, protons labeled 4ii, 5ii, and 6ii resonate within a 1.5 ppm range, indicating that the carboxylate groups have a more nonuniform effect on the magnetic environment of ring D protons. The chemical shift for proton 4ii (8.70 ppm) is the most downfield, indicating that it is strongly deshielded by both carboxylate groups. Therefore, proton 4ii is likely to be ortho substituted with respect to both carboxylate groups. The chemical shifts for proton 5ii and 6ii are 8.12 and 7.22 ppm, respectively, and indicate that proton 5ii is ortho to one -COOH and that proton 6ii is meta to both -COOH groups. Furthermore, these shifts are consistent with those observed for protons of m- and p-benzene dicarboxylic acids (25). As previously detailed for isomer 1, the coupling patterns and coupling constants for the three isomer 2 protons (4ii, 5ii, and 6ii; Table 1) further support our structural assignment. We find that the chemical shifts for protons 4i-ii, 5i-ii, and 6i-ii observed in the spectra of the individual isomers are nearly identical to those observed in the spectrum of the tracer-grade mixture (fall within 0.02 ppm). Given that these six proton resonances fall within a 1.5 ppm window, it is indeed fortunate that all peaks are clearly resolved in the NMR spectra of the tracer-grade mixture. Protons 1-3 on the xanthene ring (Figure 1) are shifted upfield of protons 4-6 with one exception (Figure 2). On the basis of constants characteristic of ortho, meta, and para coupling, we find that proton 1 is ortho to 2 and para to 3, while proton 2 is meta to 3. Furthermore, we find that NMR signals for protons 1-3 are nearly identical for isomers 1 and 2, indicating an identical xanthene system in both isomers (Table 1). It is noteworthy that protons 1-3 possess double the integration values of protons 4-6 in the individual samples of isomers and four times the integrations of 4-6 in the tracer-grade mixture, thereby confirming the magnetic equivalency of the protons on the A and C rings. Protons on the diethylamino groups are represented by a single triplet-quartet pattern typical of simple ethyl groups (data not shown). The methylene protons (-CH2-) resonate at 3.64 ppm (quartet), and the terminal methyl protons (-CH3) are found at 1.28 ppm (triplet) with a coupling constant of 6.8 Hz. The presence of a single triplet-quartet in the spectrum of the tracer-grade mixture confirms that the diethylamino protons on the A and C rings are magnetically equivalent as expected from the inherent C2 axis of symmetry through the center of the xanthene system. Therefore, we VOL. 35, NO. 20, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. 1H NMR spectrum of tracer-grade RWT; the aromatic region is displayed. Spectra of the individual isomers were used to ascertain the positioning of the carboxylate groups. Protons 1-3 are depicted in Figure 1. conclude that the structure of A-B-C ring system and the diethylamino groups is identical in the two isomers. Furthermore, our structural assignment of protons on the xanthene ring system and the diethylamino groups is consistent with previously published structures (4, 12, 15, 19). Our conclusion that both isomers possess one carboxylate that is positioned ortho to the xanthene system is consistent with suggestions by Shiau and co-workers (15) based on UVvis spectra of the two isomers. Our unique contribution based on 1H NMR spectroscopy is conclusive evidence that isomer 1 (henceforth referred to as the para isomer) has parasubstituted carboxylate groups, which are in turn ortho- and meta-positioned with respect to the xanthene ring, and that isomer 2 (henceforth referred to as the meta isomer) has meta-substituted carboxylate groups, which are ortho and para to the xanthene ring. Percent Mass Distribution of Isomers in Tracer-Grade Mixture. The NMR spectra of the two isomers differ only in the signals for the protons 4-6. By employing this established difference and integrating the NMR resonances representing these protons in the tracer-grade mixture, we find that the relative integrations of the peaks representing protons 4i and ii, 5i and ii, and 6i and ii are similar (Table 1). We, therefore, conclude that the two isomers are present in equal abundance in the tracer-grade mixture in a 50-50% mass distribution. This distribution is consistent with our previous speculations (16) and is assumed in our quantification of RWT sorption (presented below). Other batches of tracer-grade RWT need to be subject to a similar analysis to establish whether the manufacturing process consistently produces a 50-50% mass distribution of the two isomers or if this distribution is unique to our RWT sample obtained from Turner Design. Sorption of RWT Isomers. We observe that each RWT isomer has a distinct pattern of sorption, with the meta isomer sorbing to a greater extent than the para isomer onto all test solids, under all experimental conditions (Figures 3 and 4; Table 2). Both isomers achieve sorption equilibrium relatively rapidly (30 min-2 h) on all the solid phases examined. Hence, a 24 h equilibration time is used for experiments aimed at evaluating the effect of pH and aqueous RWT concentration on the extent of sorption. On the basis of our replicate experiments, we find that the error in the fraction sorbed is (0.5-6%. 4092

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For all RWT-solid combinations, we observe that sorption of both isomers decreases with an increase in pH (Figure 3a-d). Both isomers show a similar pattern of pH-dependent sorption onto sand, iron oxide-coated sand, and humic acidcoated sand: a near linear decrease in sorption with an increase in pH (Figure 3c,d). The two isomers, however, exhibit different pH-dependent sorption behaviors onto the pure-phase oxides (Figure 3a,b): the meta isomer shows a sorption plateau from pH 4 to pH 6 followed by a decrease in sorption between pH 6 and pH 8, while the para isomer exhibits a near linear decrease in sorption between pH 4 and pH 8. For the experimental conditions employed in this study, we find that the sorption of the meta isomer onto sand and iron oxide-coated sand shows a linear increase with increase in aqueous RWT concentration; however, sorption onto humic acid-coated sand is nonlinear (Figure 4; Table 2). In general, sorption of the para isomer onto all sand-based solids is small or negligible (Figure 4c,d; Table 2). For the concentration range examined, both RWT isomers exhibit linear isotherms for sorption onto the pure-phase oxides (Figure 4a,b; Table 2). It is important to note that the extent of isomer sorption (on a mg of RWT/g of solid basis) on the pure-phase metal oxides is 2 orders of magnitude higher than that onto the sand-based solids. Furthermore, both isomers show a slightly higher extent of sorption onto aluminum oxide as compared with iron oxide (Figures 3a,b and 4a,b) and a higher extent of sorption onto the humic and iron oxide-coated sands as compared to the cleaned sand (Figures 3c,d and 4c,d). Upon accounting for the surface area of the solids (i.e., examining Kd values on a L/m2 basis), these differences are considerably less significant; however, iron oxide exhibits a higher affinity for RWT isomers than the aluminum oxide (Table 2). Although we are able to desorb the RWT isomers using methanol, we are unable to quantify the precise extent of desorption due to the analytical difficulties of resolving the two isomers in a methanol-water mixture. We are convinced that RWT loss from bulk solution is due to sorption; microbial or abiotic degradation can be excluded as equilibration times were 24 h or less. Furthermore, loss of RWT due to photolysis can be excluded because our reactors were kept in the dark for the duration of the experiment and under these conditions our blank (solid-free) solutions of RWT did not show a

FIGURE 3. RWT isomer sorption as a function of pH for (a) para isomer sorption onto 10 g/L iron oxide (b) and aluminum oxide (0); (b) meta isomer sorption onto 10 g/L iron and aluminum oxides; (c) para isomer sorption onto 500 g/L clean sand (4), humic acid-coated sand ([), and iron oxide-coated sand (O); (d) meta isomer sorption onto 500 g/L clean, humic acid-coated, and iron oxide-coated sand. Experimental conditions: [RWT isomer]initial ) 3 mg/L, 10-2 M NaCl, and 24 h equilibration time. decrease in RWT isomer concentrations over the course of the experiments. Effect of Isomer Structure on the Extent of Sorption. On the basis of compound structure, we expect the RWT isomers to interact with soil solids via hydrophobic exclusion from aqueous solution, electrostatic attraction to oppositely charged surfaces or surface sites, and complexation of carboxylate groups on RWT with surface-bound Al and Fe atoms (26, 27). The arguments presented below illustrate that the meta isomer (isomer 2) sorbs to a significantly higher extent (by 1 order of magnitude) than the para isomer (isomer 1) because the molecular structure and physical-chemical properties of the meta isomer facilitate all of the abovementioned interactions to a significantly greater extent. pKa and log Kow of the tracer-grade mixture have been previously measured to be 5.1 and -1.33, respectively (1, 15). However, the pKa and Kow (and Dow) values of individual isomers are currently unknown. On the basis of 2D structures, Kow values of the two isomers are not expected to be very different. However, under dissolved phase conditions where the two isomers are expected to possess similar charge, the para isomer (isomer 1) elutes significantly ahead of the meta isomer (retention times of 3.1 and 4.5 min, respectively) on our C18 HPLC column, suggesting that the meta isomer is considerably more hydrophobic. The energyminimized geometries depicted in Figure 5 show that the phenyl (D) ring in both isomers is nearly orthogonal to the xanthene ring system. Thus, the carboxylate groups of the para isomer are situated on either side of the plane formed by the xanthene ring system, while the meta isomer has one carboxylate along the plane and one above the plane. This geometric arrangement of the carboxylate groups in the meta isomer likely constitutes a larger hydrophobic area about

the molecule, which in turn may allow for a higher potential for hydrophobic exclusion from bulk solution and a higher extent of sorption. In addition to one site of permanent positive charge (iminium cation, R1dN+sR2R3), both isomers possess three sites for protonation/deprotonation: one amine group (R1NH+-R2R3) and two carboxylate groups (Figure 1). Predictions of isomer pKa values (28) suggest that both isomers possess three similar macroscopic pKa values: 2.8, 3.6, and 4.4 (meta) and 2.8, 3.5, 4.4 (para). Speciation plots based on these pKa values (Figure 6) suggest that both isomers possess a net +2 charge (iminium cation and cationic amine) below pH 2.5, have either a net +1 charge (iminium cation, cationic amine, one anionic carboxylate), or exist as zwitterions (iminium cation and one anionic carboxylate and/or iminium cation, cationic amine, and two anionic carboxylates) between pH 2.5 and pH 4.7 and a net -1 charge (iminium cation and two anionic carboxylates) above pH 4.7 (Figure 6). Therefore, nonspecific electrostatic attraction (resulting from net charge) to oppositely charged surfaces is expected to be relatively similar for both isomers. However, electrostatic attraction to individual positively charged surface sites is most likely greater for meta isomer because the positioning of the carboxylate groups allows for a more focused region of negative charge density (Figure 5). With respect to surface complexation via the carboxylate groups, para positioning promotes a greater inductive electron-withdrawing effect rendering these groups less suited for electron donation to the surface-bound metal atom. This same inductive effect is not as pronounced in the meta position, thus favoring complexation to surface metal ions. It must be noted that the para and meta isomers may compete for the same sorption sites. Therefore, it is likely VOL. 35, NO. 20, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. RWT isomer sorption as a function of aqueous concentration (sorption isotherms) for (a) para isomer sorption onto 10 g/L iron oxide (b) and aluminum oxide (0); (b) meta isomer sorption onto 10 g/L iron and aluminum oxides; (c) para isomer sorption onto 500 g/L clean sand (4), humic acid-coated sand ([), and iron oxide-coated sand (O); (d) meta isomer sorption onto 500 g/L cleaned, humic acid-coated, and iron oxide-coated sand. Experimental conditions: pH 6, 10-2 M NaCl, and 24 h equilibration time.

TABLE 2. Solid-Water Distribution Ratios (Kd) for RWT Isomer Sorptiona para isomer solid matrix

surface area (m2/g)

pHzpcb

iron oxide (hematite: R-Fe2O3) aluminum oxide (γ-alumina: γ-Al2O3) cleaned sand (quartz: SiO2) iron oxide-coated sand

8.8

8-8.5

humic acid-coated sand

105 0.14 ∼0.14 ∼0.14

Kd (L/g) [Kd′ (L/m2)]c

2-3

2.0E-02 [2.3E-03] 5.6E-02 [5.3E-04] nad

na

nad

na

nad

8-9

meta isomer

R2 0.77 0.96

Kd (L/g) [Kd′ (L/m2)] 1.3E-01 [1.5E-02] 4.5E-01 [4.3E-03] 1.8E-04 [1.3E-03] 9.0E-04 [6.7E-03] nonlinear

R2 0.94 0.89 0.91 0.90

Kd values are obtained from linear regression of data shown in Figure 4. b From refs 33 and 34. c Kd′ (L/m2) obtained using Kd (L/g) and surface areas listed. d Sorption low to negligible (RWT lost from solution is e5%). a

that the para isomer may exhibit a greater extent of sorption in the absence of the meta isomer, i.e., in experiments employing single isomers standards as opposed to the tracergrade mixture. Effect of pH. The general decrease in RWT isomer sorption with increase in pH (4-8) is due to a decrease in the potential for sorption via hydrophobic exclusion, electrostatic attraction, and surface complexation. As pH increases, deprotonation of the -COOH groups on the phenyl (D) ring increases the number of charged atoms on the RWT molecule resulting in decreased compound hydrophobicity. Additionally, at higher pH (greater than 6) the RWT molecules possess a net negative charge, and the oxide surfaces also begin to acquire more negative charge resulting in electrostatic repulsion and decreased sorption (Figure 6). Because sand (quartz) is negatively charged over the entire pH range examined, electrostatic repulsion between RWT and the sand surface 4094

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becomes important at pH values greater than 4.5. Furthermore, at pH values greater than 6, any likely surface complexation of RWT is reduced due to competition between RWT -COOH groups and hydroxyl ions (OH-) in solution for sorption sites on metal oxides. As mentioned earlier, the pH envelope for meta isomer sorption on metal oxides has a unique sorption plateau between pH 4 and pH 6 followed by a decrease in sorption. This plateau is likely due to the fact that the meta isomer is structurally better suited for surface complexation via the exchange of a surface hydroxyl group by a -COOH group at low pH values where OH- release is favorable. At higher pH values (above 6), hydroxyl ions in solution effectively compete with the meta isomer for the surface metal ions (26). Effect of Solid-Phase Properties on the Extent of Sorption. The higher extent of RWT isomer sorption onto metal oxides as compared to sand-based solids (Figure 4),

FIGURE 5. Energy-minimized ground-state geometries of RWT (a) para and (b) meta isomers computed using ab initio methods (Gaussian 98, revision A.9) and visualized using CS Chem3D Pro (version 4).

FIGURE 6. Schematic illustrating RWT and test solids net charge as a function of pH based on RWT pKa values stated in the text and mineral pHzpc values listed in Table 2. Note: At pH ) pKa, the protonated and deprotonated forms are at equal concentrations; at pH ) pHzpc, the number of positively charged sites is equal to the number of negatively charged sites. despite a 50-fold lower solids loading, is due to the greater surface area of metal oxides (Table 2) and the higher potential for RWT-oxide interactions. Metal oxides afford both positive and negatively charged sites for interaction with anionic carboxylate and cationic iminium and amine (N+/NH+) groups of RWT, respectively. However, only a weak electrostatic attraction to the sands (quartz) can occur via interaction between cationic iminium/amine groups and the negatively charged quartz surface, assuming favorable steric factors. Furthermore, Al and Fe surface metal ions are known to complex carboxylate groups of organic molecules (2931). Therefore, higher surface area and a higher potential for favorable electrostatic interactions and surface complexation via the carboxylate groups likely afford a higher extent of RWT isomer sorption onto the metal oxides. Differences in the extent of sorption onto iron and aluminum oxides are likely due to differences in site density and oxide mineralogy. The higher extent of meta isomer sorption onto the humic acid-coated sand than onto the cleaned sand is due to a increased fraction of organic carbon on the solid surface, which is expected to favor RWT-surface interactions. Likewise, the increased extent of sorption onto the iron oxidecoated sands is likely due to increased electrostatic attraction and surface complexation to iron oxide coatings. The distinct pattern of pH-dependent meta isomer sorption onto the metal oxides is not evident for the iron oxide-coated sands most likely due to the fact that only a 0.9 mg of Fe/g of sand coating was achieved. The near negligible sorption of the para isomer onto sand-based solids at pH 6 (Figure 4) may be due to limited hydrophobic exclusion of the para isomer

and electrostatic repulsion between the negatively charged quartz surface and the negative charges on both sides of the phenyl ring. Implications for RWT Use as a Tracer. Several researchers have pointed to inconsistencies in the literature regarding RWT fate and transport. For example, studies that use RWT as a conservative tracer and find the breakthrough behavior similar to tritium and/or chloride observe incomplete mass recovery (5, 32). Furthermore, Bencala and others (7) show that RWT concentrations were as low as 45% of that expected based on chloride data. Several column studies have found that RWT breakthrough curves were not of the conventional sigmoidal shape, instead a two-step breakthrough curve was often observed with the first-step plateau at C/Co ∼0.4-0.5 (12, 15, 19). Breakthrough curves evaluated for shorter time periods found a single plateau at C/Co ∼0.4-0.5 (11). Our findings of (i) a 50-50% mass distribution of the two isomers, (ii) the near conservative behavior of the para isomer (on account of its low sorption potential onto soil solids), and (iii) the nonconservative nature of the meta isomer (on account of its high sorption potential onto soil solids) explain the 40-50% mass recovery when conservative behavior is observed. Our results also suggest that subsurface hydraulic parameters and contaminant fate and transport determined from field and laboratory studies that treat tracer-grade RWT as a single solute and use a field fluorometer for the measurement of RWT concentration have to be treated with caution. Field fluorometers are calibrated to measure fluorescence at specified emission and excitation settings suited for tracer-grade RWT. As we have previously established (16), the distinct sorption behavior of the two isomers is likely to result in their chromatographic separation in the subsurface. Because these separated compounds have a distinct relationship between concentration and fluorescence and because their mass distribution in the tracer-grade mixture is not accounted for, RWT concentration determined by the fluorometer and the related hydraulic parameters/contaminant transport characterization is likely to be erroneous. Although sample collection in the field followed by laboratory analysis of the concentration of separated isomers is not as simple as direct fluorometer use, it allows for improved quantitative tracing when using the tracer-grade mixture. It must be noted that all solutes used as groundwater tracers react to some degree with the subsurface resulting in a limited degree of nonconservative behavior. Since RWT has several well-documented advantages over other groundwater tracers (4) and was originally intended to function as a conservative water tracer, we recommend that the RWT manufacturing process be modified to produce 100% of the intended conservative isomer, the para isomer. Although VOL. 35, NO. 20, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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separation can be achieved in the laboratory using preparatory columns, an easily available single isomer RWT standard will facilitate the comparison of tracer tests conducted by different investigators. On the basis of our characterization of individual isomer sorption behavior, we find tracer tests using the currently available tracer-grade mixture in combination with discrete sampling and laboratory analysis of individual isomer concentrations provide a potential approach for combined hydraulic parameter estimation and contaminant fate and transport modeling. In a single column or field test, near conservative transport can be established from the breakthrough of isomer 1 (para), while the breakthrough curve of isomer 2 (meta) can be used to evaluate the fate and transport of contaminants of related sorption behaviors. Additionally, such tests may assist in the characterization of mixed contaminant plumes.

Acknowledgments Funding from the National Institute of Environmental Health under Grant 1P42-ES-10356-01 and the National Science Foundation under Grant BES-9984489 is gratefully acknowledged. We sincerely thank Dr. Antony Ribeiro for help with 1H NMR analysis conducted at the Duke NMR Spectroscopy Center, which was established with funds from NSF, NIH, NC Biotechnology Center, and Duke University; Chris Goss for help with the NMR interpretation; Gerardo Andres Cisneros and Dr. Weitao Yang for guidance and access to Gaussian 98; Drs. Zbigniew Kabala and Doug Sutton for the stimulating discussions and critiques; Dr. George Dubay for mass spectral analysis; and Ellen M. Cooper and Wes Willis for solids characterization.

Literature Cited (1) Smart, P. L. NSS Bull. 1984, 46, 21-33. (2) Field, M. S.; Wilhelm, R. G.; Quinlan, J. F.; Aley, T. J. Environ. Monit. Assess. 1995, 38, 75-96. (3) Wilson, J. F. Techniques of Water-Resources Investigations of the United States Geological Survey; U.S. Geological Survey: Washington, DC, 1968; Vol. 3, pp 1-31. (4) Smart, P. L.; Laidlaw, I. M. S. Water Resour. Res. 1977, 13 (1), 15-33. (5) Aulenbach, D. B.; Bull, J. H.; Middlesworth, B. C. Ground Water 1978, 16 (3), 149-157. (6) Pang, L.; Close, M.; Noonan, M. Ground Water 1998, 36 (1), 112-122. (7) Bencala, K. E.; Rathbun, R. E.; Jackman, A. P.; Kennedy, V. C.; Zellweger, G. W.; Avanzino, R. J. Water Resour. Bull. 1983, 19 (6), 943-950.

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(8) Aldous, P. J.; Smart, P. L. Ground Water 1988, 26 (2), 172-178. (9) Everts, C. J.; Kanwar, R. S.; Alexander, E. C.; Alexander, S. C. J. Environ. Qual. 1989, 18 (4), 491-498. (10) Ptak, T.; Schmid, G. J. Hydrol. 1996, 183 (1-2), 117-138. (11) Everts, C. J.; Kanwar, R. S. J. Hydrol. 1994, 153, 53-70. (12) Sabatini, D. A.; Austin, T. A. Ground Water 1991, 29 (3), 341349. (13) Kanwar, R. S.; Baker, J. L.; Singh, P. J. Environ. Sci. Health, A 1997, 32 (7), 1907-1919. (14) Hofstraat, J. W.; Steendijk, M.; Vriezekolk, G.; Schreurs, W.; Broer, G. J. A. A.; Wijnstok, N. Water Res. 1991, 25 (7), 883-890. (15) Shiau, B. J.; Sabatini, D. A.; Harwell, J. H. Ground Water 1993, 31 (6), 913-920. (16) Sutton, D. J.; Kabala, Z. J.; Francisco, A. B.; Vasudevan, D. Water Resour. Res. 2001, 37 (6), 1641-1656. (17) Trudgill, S. T. Hydrol. Processes 1987, 1, 149-170. (18) Di Fazio, A.; Vurro, M. Adv. Water Resour. 1994, 17 (6), 375378. (19) Kasnavia, T.; Vu, D.; Sabatini, D. A. Ground Water 1999, 37 (3), 376-381. (20) Carter, M. R. Soil Sampling and Methods of Analysis; Lewis Publishers: Ann Arbor, MI, 1993. (21) Gu, B.; Mehlorn, T. L.; Liang, L.; McCarthy, J. F. Geochim. Cosmochim. Acta 1996, 60, 2977. (22) Laor, Y.; Framer, W. J.; Aochi, Y.; Strom, P. F. Water Res. 1998, 32 (6), 1923-1931. (23) Tai, D. Y.; Rathbun, R. E. Chemosphere 1988, 17 (3), 559-573. (24) Friebolin, H. Basic One- and Two-Dimensional NMR Spectroscopy, 2nd ed.; VCH Publisher: Berlin, 1993. (25) Pouchert, C. J.; Behnke, J. The Aldrich Library of 13C and 1H FT-NMR Spectra, 1st ed.; Aldrich Chemical Co.: Milwaukee, WI, 1993. (26) Stumm, W. W. Chemistry of the Solid-Water Interface; John Wiley and Sons: New York, 1992. (27) Schwarzenbach, R.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry; John Wiley and Sons: New York, 1993. (28) SPARC on-line calculator. http://ibmlc2.chem.uga.edu/sparc/ style/welcome.cfm. (29) Biber, M. V.; Stumm, W. Environ. Sci. Technol. 1994, 28 (5), 763-768. (30) Evanko, C. R.; Dzombak, D. A. Environ. Sci. Technol. 1998, 32 (19), 2846-2855. (31) Tejedor-Tejedor, M. I.; Anderson, M. A. Langmuir 1986, 2, 203. (32) Sinton, L. W.; Finlay, R. K.; Pang, L.; Scott, D. M. Water, Air, Soil Pollut. 1997, 98, 17-42. (33) Schindler, P. W.; Stumm, W. Aquatic Surface Chemistry; John Wiley: New York, 1987. (34) Sposito, G. The Chemistry of Soils; Oxford University Press: New York, 1989.

Received for review April 20, 2001. Revised manuscript received August 2, 2001. Accepted August 2, 2001. ES010880X