Analytical Procedure for Simultaneous Use of Seven Fluorobenzoates in Multitracer Tests by Ofer Dahan"? and Zev Ronen? Abstract ‘An analytical procedure allowing simultancous use of seven fluorobenzoate (FBA) isomers was developed and examined through column and field tracer experiments. The analytical method, hased on high performance liquid chromatography (HPLC), Included a reverse-phase separation method. This method was found to be very efficient for the FBA analysis, allowing accurate determination of seven FBAS in a single HPLC run, while avoiding analytical interference of other natural water constituents or pollutants. Consequently, this separation method allows the simultaneous use of seven isomers in multitracer tests in highly saline ‘or contaminated environments. The mobility properties of the FBAs were examined in percolation experiments through a column filled with a mixture of erushed chalk and common fracture filling and coating material, The FBAs behaved conservatively as com- pared to bromid. All seven FBA isomers were successfully used in a multitracer test in a field percolation experiment designed to identify fowpath along a fracture plane. Introduction Ground water flow is often investigated using chemical trac cers. Ideal tracers of water flow do not interact with the solid-phase co flowing-liquid ingredients, are not degradable or generated under the flow domain’s conditions, and are foreign in the flow envi- ronment (Davis et al, 1980). In mukitracer tess, where several trac cers need to be simultaneously applied, the set of tracers must have similar mobility properties. Moreover, itis essential to have an ana- lytical procedure that is capable of separately monitoring each tracer inthe flowing fluid and distinguishing between them and other preexisting constituents in the wate. Ip addition, the cost of the trac crs, a8 well as their measurement technique, must be reasonable. ‘A field percolation experiment conducted through fractures crossing an unsaturated chalk formation at the northem Negev Desert, Israel, required the use of tracers to identify flow trajecto- ries along che fracture plane. As a secondary objective of the per- ccolation experiment reported by Dahan et al. (1998, 1999, and 2000), the objectives ofthis research were to find a suite of tracers and a convenient analytical procedure suitable for multitracer per- ccolation field test. With the limitation and constraints previously rmentioned,, a range of commonly used tracers was examined for "The Seagram Center for Soil and Water Sciences, Faculty af Agricultural, Food and Envitonmental Quality Sciences, The Hebrew University of lersalem, PO. Box (2, Rehovot 76100, srael Current adress: Desert Reseach Institue, Division of Hydrologic Sciences, 2215 Raggio Pkwy, Reno, NV $9512; odahan@dri.edu “Blaustein Institute for Desert Research, and Depaetment of Goologicil and Enviroamental Sciences, Ben-Garion University of the "Negen, Sede Boker Campus 84990, Israel; zeevrone@lgumail.bgu.aeil Received March 2000, aecepted October 2000. possible use in the field percolation experiment. Through the range of common inorganic anion tracers, chloride and sulfate were rejected due to the natural high background inthe desert soils, bromide was rejected due to high industrial bromide pollution athe study ara, nitrate and nitrite re not steady and may be degradable or generated under eondition of the experiment, and iodide and fl cre are known tobe uneonservative and may be sorbed or retarded to some extent. Fluorescent dyes ten to have relatively wide exci tation and emission wavelengths that cause analytical interference in malitracr tests. Therefore only three fluorescent dyes ean be used and analyz simultaneously (Amino G aed, Lissamin FF, and Rhodamine WT) (Davis etal. 1987). Isotopic tracers suchas oxy~ gen-18 and deuterium were rejected due tothe high cos involved in large amounts of traced water needle for use ina field experi ment and the expensive analytical cost involved in large numbers of samples necded tobe analyzed. Yet, the ideal suite of tracers that ‘would be suitable fora muliracer test would be one capable of pro- viding the widest range of tracers that can be used and analyzed simultaneously. Fluorinated derivatives of benzoie acid (Nuorobenzoates or FBAs) form group of tracers that i especialy suitable for mati tracer tess, They have proven their usefulness as nonreactive water teacers in soil and ground water, and their mobility properties are similar 0 that displayed by bromide (Bowman 1984a,b; Bowman and Gibbens 1992; Pearson et al. 1992; Benson and Bowman 1994). However, recent studies have demonscated that in acidic soil, oF where igh conceatration of organic matters present, the FBAs may not display ideal conservative behavior and may sorb or be retarded Jaynes 1994; Seaman 1998). Nevertheless, for many hydrological applications, FBA tracers are stil usefl ‘Although atleast 18 FBA isomers or derivatives exist, only about 13 are commercially available. FBA concentrations are typ- 306 Vol. 39, No. 3—GROUND WATER—May-June 2001 (pages 366-370)‘Tracer 1 ‘Tracer 2 Tracer3 Tracer 4 2,6-DFBA —2,3,6-IFBA_—2,3- DFBA es oO a0 Oo of & or ‘Tracer 7 2,3,4,5-TeFBA TracerS Tracer 6 3,5-DFBA O-TrFMBA QM Bey Wee ' a F © F F Or Figure 1. Molecular structure of the selected seven fluorobenzoate isomers. ‘Table 1 ‘The 13 Fluorobenzoate (FBA) Isomers Examined in This Study Retention Time from the Reverse-Phase HPLC Column (ea) FBA, iia Peak Final 246DiFBA, 64 66 691 2.3,6-THEBA, 137 154 18 2S-DIFBA 731 165, 8 235,6-TeIRFBA 183 801 a4 23-DIFBA 2 au Sat 245-THFBA 9.08 9.9 on 34DIFBA 942 958 9.96, 23.4-THFBA ost 974 ros 35-DiEBA 9.65 oat 10.22 PemaFBA on 939 lous (O-THF-methy-BA 10.09 1027 ost 234-TetraFBA 10.96 us as: 09 126 1.68 ‘leas desig he ee FBAS eat ais wah, ically measured using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (Bowman 1984p; Bowman and Gibbens 1992; Benson and Bowman 1994), or ion chro- ‘matography (IC) with conductivity detection (Pearson et al. 1992}, In both methods, FBAs are separated on a strong anion-exchange ‘column. It appears that by using these methods, a maximum of only six FBAs may be clearly separated and distinguished, The main dis- advantage ofthe anion-exchange separation method i that it does ‘not completely separate the FBAS from the natural inorganic anions (eg, BrCl, NO,”, NOx", or SCN>), and they typically co-elute ‘nthe same time range as the FBAs (Bowman 1984b; Bowman and Gibbens 1992; Pearson et al. 1992; Benson and Bowman 1994), ‘Therefore, these natural anions tend to interfere with the FBA, analysis and limit the number of isomers that can be simultaneously analyzed. Consequently, analytical methods that rely on anion- ‘exchange separation method may be inapplicable if tracers are applied on salty soils (such as desert areas), coastal regions, agri- ‘cultural areas or in contaminated environments (such as landfills, etc). Reverse-phase chromatography is not based on simple ion exchange but involves adsorption-desorption reactions between the compounds and the stationary phase. These reactions are con- trolled by the chemical solubility inthe mobile phase. Ionized com- pounds like FBAs will become less soluble in the aqueous phase if the pH is below the pKa ofthe compound. This mechanism allows increased retention of the acids on the reverse-phase columa, Modifying the concentration of the organic phase in the mobile phase allows gradual elution ofthe acids and hence improved separation, Inorganic anions, however, will not be retained by the column under these conditions. In this research, we will integrate several existing techniques to provide a new approach that enables the simultaneous use and analysis of seven FBAS while avoiding the interference problem ‘caused by natural water ingredients and pollutants, Results of lab- ‘oratory and field tracer experiments demonstrate the use of the ‘method and its efficiency. Material and Methods Analytical Procedure ‘The analytical FBA procedure was based on high-performance Jiguid chromatography (HPLC) (Kontron, Milan). The instrument consisted of a model 422 pump, a model 425 gradient former, a ‘model 465 auto-sampler and a model 440 diode array detector (DAD). The data were acquired and integrated by 450-MT2 data- system software. The FBAS were separated on a Supeleosil LC-18 (25 em long on 4.6 ID) analytical column (Supelco, Bellefonte, Pennsylvania). The mobile phase gradually changed from 75% 0.05 M H,PO, with 25% acetonitrile as organic modifier to SO% of | each component in a 10-minute linear gradient. The flow rate was 1.5 mL/min and the detection was at a wavelength of 222 nm. An external standard method was used for quantification, ‘Seven FBA isomers were selected out of 13 examined inthis, study (Table 1 and Figure 1). All 13 examined FBAS had been pre- viously investigated and applied as tracers for water movements by other researchers (Bowman 1984a,b; Bowman and Gibbens 1992; Pearson et al. 1992; Benson and Bowman 1994). The selection of the seven isomers (boldface in Table 1) was based on the FBA sep- aration obtained by the aforementioned analytical procedure, to ‘ensure thatthe chromatograms display no overlap among the trac- fers and no interference with other environmental constituents ‘Table 1 displays the retention time on the chromatogram of each of | the 13 examined FBAs. The FBAS were obtained from Aldrich Chemical Co, Inc. (Milwaukee, Wisconsin) with a chemical purty areater than 97%. They were first dissolved in 0.5 N NaOH to Achieve concentrated solutions (10 g/L), and then diluted to achieve ‘concentrations required both forthe column and field experiments. (0. Dahan, Z, Ronen GROUND WATER 39, n0. 3366-370 (367Table 2 Mineralogical Composition of the Crushed Chalic ‘and Fracture Coating and Filling Materials Used for the Solid Misture in the Column Experiment Constitent wi Chat 2 Manganese oxides (exert) 2 “Type coming of etre was xpsun 1 A secondary mineral commonly found ig mail in ace Cetesie B ‘secondary miners commonty ean lig ati acts eid Unsomotated Tiling ia s Commonly found in dissolution channels inthe festare voids Cntins que (30-358), ake (00-20%, elspa 10%), ops CE (00-15%) ioe (5) nd tay (1-258 contig of tie (600), palyporsite (305), and ‘incr amuns of kknite Column Experiment ‘The laboratory column experiment was designed to evaluate the performance ofthe selected isomers as water tracers for the field ‘racer experiments, to be conducted in unsaturated fractured chalk inthe nonthern Negev Desert, Israel, The tracers stock solution for ‘the column experiments was prepared by mixing all seven FBAS in tap water (650 mg/L TDS, 150 mg/L alkalinity, 250 mg/L. Cl, 45, mg/L. SO,. 21 mg/L, NO,) to a final concentration of 50 mg/l. each; 100 mg/L of Br was added (as KBr) to the stock solution as reference tracer, to allow evaluation ofthe FBAs' mobility prop- erties inthe chalk medium, The pH ofthe solution was neutralized by phosphoric acid and by the large buffer capacity of the tap water to 7.54, Note thatthe chalk Zeta potential is negative, 283 mV at pH 7.6, as measured on crushed chalk by a Zeta meter (Malvern Instrument, United Kingdom), excluding the possibility {or opposite charge between the chalk and FBAs. ‘The concentrations of Br and other anions were measured using ion chromatography (Dionex 45001) with chemical sup- pression, with a mobile phase of 1:1 (yoWWol) 1.8 mM Na,CO, and 1.8 mM NaHCO, at a flow rate of 2 mL/min, Prior to the ion analysis, the samples were filtered through a filter meant to remove ‘organic substances, such as FBAS, while allowing inonganic ions to pass through (ilter model IC-RP, Alltech, Deerfield, llinois). ‘transparent Plexiglas column (420 mm long and 45 mm LD.) was filled with 568 grams of ai-dried, crushed chalk and fracture fill- ing and coating materials (aggregate size of 0.5 to 2 mm), typical ‘ofthe fractures intersecting the chalk inthe study area (Table 2). The unsaturated field conditions were simulated by dripping water ‘onto the top of the vertically placed column, The water gravita- tionally drained through the column and seeped out frely from its bottom. The dripping rate into the column was eontrolled by a flow valve and kept steady and low (-3.3 mL/min) to avoid the generation ofa free-water phase on top ofthe column and to pre serve unsaturated flow conditions. Water content in the packed column during the test was gravimetrically determined to be 59%. ‘The saturated fraction of the pore volume, hereafter referred to as 1 wetted pore volume (1 WPV), was equivalent to 315 mL of 368 0. Dahan, Z. Ronen GROUND WATER 39, no. 3: 366-370 a0 4 S sé 250 “Ee « 2 3 i= ‘les a ey io i z iS Zz 2 a «| 00 18 30 45 69 75 99 195 mo BS ‘Time (min) Figure 2. HPLC chromatogram of seven Nuorobenzoate isomers in a reverse-phase separating column. water. The column experiment started by percolating tap water into the column for a period of ~24 hours to achieve steady-flow conditions (i., equal inflow and outflow fluxes). Then the pereo- lating water was replaced with the tracer stock solution to start the tracer test, Following the dripping of 1 WPV of tracer solution onto the top of the column, it was replaced by tap water to stat the trac= ers flushing process. The outflowing solutions were quantitatively collected for analysis in an averaged sequence of every eight min. utes (corresponds 10 ~0.08 WPV), in glass vials, and stored under refrigeration. The samples where analyzed by HPLC for FBAs concentration within 24 hours from the sampling time, Field Experiment "The experimental setup used in the field is described in detail in Dahan ot al. (1998). The results ofthe field experiment using the seven EBA isomers as tracers are presented in Dahan ea (1999), ‘A brief description ofthe experimental concep follows to demon- strate the usefulness ofthe FAs in a multtracer test. ‘The experimental setup was designed for a detailed invest gation of water flow trough a discrete natural fracture. It was composed ofa ste of percolation ponds installed sequentially along a fracture exposed on the surface ofa rock ledge, and a compart- ‘mental sampler installed ina horizontal borehole drilled along the plane ofthe same fracture, ~ I m below the percolation ponds. Water percolating from the various ponds through the fracture exposed at their bottom was collected by the various cells ofthe compartment sampler in the horizontal borehole below. The water in each pond on land surface was individually tagged by one of the seven FBA isomers and the water samples collected by the compartmental sampler were analyzed for the entre tracer suite. Consequently, the FBAs’ composition detected in cach sampler cell identified the source ponds that contributed water to each particular sampler cell through the fracture flow network, thus, allowing flowpath delin- cation along the fracture plane.500 50 < + a y = 4.0975x - 0.4408 8 = ee : +4087 Fe = 0.9994 ce Fe = 0.9999 ma) $ 200 . $ 20 2 . a 100 . 10 oat 0 1 o 2 40 60 8 100 0 20 40 «©6080 100 Concentration (mg/L) Concentration (mg/L) Figure 3. Calibration curves for 26-Diuorobenzole acid. a _ RESULTS AND DISCUSSION : |-+ nso Figure 2is an HPLC chromatogram of the tracer stock solu- 2 | <-ssenwea tion used forthe column experiment. The peaks ofthe seven FBA “ soma ‘isomers appeared on the chromatogram between sx and 12 minutes, 0 | cosastwne and both their height and area (under each peak) ae linearly cor. ome related with the isomer concentrations. Figure 3 shows calibration go aaa jan curves for2,6-DIFBA (the first peak in Figure 2), demonstrating the je “trenterer®* near correlation between the tracer concentration and the area and oaeetem eight ofits peak on the chromatogram, Similar calibration curves Wena pore volume Figure 4, Breakthrough curves of the seven fuorobenzoate isomers and Br in a column containing erushed chalk mixed with common fracture filing and coating materials, 45 1s Absorbance units (mAbs) 30 Too 15 60 75 90 105 120 135 ‘Time (min) 30 45 Figure 5. HPLC chromatogram of a water sample from a multi- tracer test fleld experiment. The five major peaks represent the presence of five uorobenzoate isomers in the water sample at con- centrations correlated to thei relative peak areas. were obined for ll sven FBAs with near regression coefficients (2) ranging from 0.970 to 0.999. The detection limit achieved by this method reached a low concentration of about 0.5 mg/L of ‘each of the FBAS used. ‘The chromatogram (Figure 2) shows thatthe HPLC reverse- pphase separation method provided good separation of the seven FBAs, as they appear as seven peaks. The chromatogram displays no significant overlaps ofthe FBA peaks’ are Although a slight overlap exists on the tiling of 3.5-DIFBA and O-TriF-methy!- BA, in general, these two tracers were distinguishable up 10 100- fold dilution (maximum concentration used 50 mg/L, and detection limit 0.5 mg/L). Therefore, that sight overlap was considered as insignificant and the two tracers could still be used as flow tajee- tory markers The chromatogram aso shows that there is no inter ference with te other anions existing inthe water or leached out from the roek, as these anions appear on the chromatogram as peak noise in the first two minutes. Good separation was also obtained for samples collected during the field tracer tests. It should bbe noted that during the field tracer tests, the percolating tracer slu- tion leached als from both the soi and the chalk and reached salin- ites exceeding 6500 mg/L TDS (Dahan et al. 1999; Dahan etal. 2000) with no significant influence on the chromatogram. Mean travel time was calculated as 90 minutes (equivalent to 1 WPV) through the timing differences between the racers breakthrough and fushing processes. Apparent, this travel time (exposure of the trac- ers to the geological material for possible sorption/etardation) ‘was found to be ofthe same order or higher than the travel times found in the fracture percolation field experiments The effluents draining out of the column were quantiatively collected and analyzed for FBA and Br concentrations using the HPLC reverse-phase separation method. Figure 4 displays the breakthrough curves of all seven isomers and Br trough the col (0. Dahan, Z. Ronen GROUND WATER 39, no. 3: 366-370-369umn during the percolation experiment. These curves, displaying almost identical breakthroughs, show that all FBA isomers exhib- ited the same mobility properties as well as conservative behavior, similar to Br. Indeed, mass balance calculation for all tracers, including Br, resulted in an average recovery of 99.9% with a standard deviation of 3% ‘The tracers were introduced to the top of the column at 0 \WPV and the tracers Nushing process started as they reached | WPV. ‘The tracers were fist detected atthe column outta about 05 WPY, ‘whereas travers fishing was frst detected there at about 1.5 WPV (Figure 4). The complete tracer flashing process was reached at ahout2.7 WPY (Figure 4), The symmetry of timing between the ini- tial breakthrough of the tracers at the coluran outlet (0.5 WPV ater tracers were introduced into the top of the column) and the tracer flushing process’ appearance at the column outlet (0.5 WPV ater the leaching process was initiated) suggests that none ofthe tracers was significantly retarded, The tiling observed during the last stages ofthe tracer flushing process (from I.$ 102.7 WPY) is, ausibuted to tracer’ ffasion i and out of imile zones as rock ageregates. Figure 5 isa chromatogram of a water sample collected during the field experiment, where all seven isomers were used for tagging the various percolation ponds located onthe facture inlet at land su face. The water sample was trapped in one of the sampler cells located down along the fracture in the horizontal borehole. This hro- ‘matogram, representing a single HPLC analysis, displays five peaks representing five FBA isomers identified in that water sample. The presence of five isomers in this water sample implies that this par- ticular sampler cell (wapping water from a particular segment ofthe facture) eceved water that had drained along he facture fom ive percolation ponds, thereby suggesting converging flow from these ‘ponds into tis one sampler cel. The variable peak sizes reflect the different water contibations from each ofthe five ponds. Visually, this chromatogram reveals that during a particular stage in the experiment, his ection ofthe fracture received most ots War from {wo souree ponds tagged bythe frst and second peaks onthe chro- ‘matogram (2,6-DiFBA and 2,36-THFBA, respectively). Smaller ‘water volumes were contributed from ponds tagged bythe thicd and fifth peaks on the chromatogram 2,3-DiFBA and O-Tri-methy!- BA. respectively). A minor amount of water atived from the ponds tagged by the fourth peak onthe chromatogram (2.4.5-THEBA), The chromatogram shows that this particular sampler cell received no water from ponds tagged by the remaining two FBAs, 2345. ‘TetrFBA and 3,5-DiFBA. This chromatogram is just one example ofthe hundreds produce for water samples collected during the field experiment, which allowed the establishment ofall breakthrough courves of al tracers in ll sampler ells during the field malitzacer test, While this example chromatogram displays five FBA peaks, rep- resenting the connection of a particular sampler cell to five perco- lation ponds, other chromatograms displayed Hinks between the percolation ponds ad ther coresponding sampler ells, ranging from ‘single peak to a maximum of seven (representing one and seven ddonor ponds, respectively). Analysis of all the chromatograms ‘enabled an accurate delineation of the flow trajectories along the frac- ture plane. The hydrological implications ofthe field tracer exper ment are provided in Dahan etal. (1999) Conclusions ‘The HPLC analytical method combined with a reverse-phase separation method allows the simultaneous use and analysis of 370 0. Dahan, Z Ronen. GROUND WATER 39, no. 3: 366-370 seven FBA isomers as seven tracers for water flow. The reverse [hase separation provides an efficient technique to separate the FBAS from naturally occurring anions or contaminants when these isomers ‘are used as tracers in saline or polluted ground water systems. The ‘column experiment showed that under the experimental condi- tions, all seven FBAS have identical mobility properties and are as ‘conservative as Br. The field experiment demonstrated how these FBAS can be efficiently used as markers for flow trajectories, Where a multtracer testis needed. Moreover, the entire range of BAS can be analyzed ina single HPLC analysis, thereby signifi- cantly reducing the high analytical cost typically involved in multi- tracer experiments, Acknowledgments ‘This study was funded by the Israel Science Foundation, the Ministry of the Environment, and the Ramat-Hovay Industrial Council. We would like to thank Ronit Nativ from Hebrew University of Jerusalem and Eilon Adar from the Ben Gurion University ofthe Negev for their generous support, We would like to thank David Sabatini, Tim Strutmann, and an anonymous reviewer for thei constructive comments, Editor’s Note: The use of brand names in peer-reviewed papers is for identification purposes only and does not constitute endorse- ‘ment by the authors, their employers, or the National Ground ‘Water Association References Benson, C-E, and R'S. Bowman, 1994, Ti and tetrafluorobenzoates as ‘nonreactive traces in sol and ground water. Sil Sc. Soc. Am. 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