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Trace Organic Analysis

Revised 4/26/98

Capillary Electrophoresis/Laser-Induced Fluorescence Detection of Fluorescein as a Groundwater Migration Tracer

P. L. Ferguson,a A. H. Grange,b and W. C. Brumley*

* U.S. Environmental Protection Agency, National Exposure Research Laboratory, Environmental Sciences Division, P.O. Box 93478, Las Vegas, NV 89193-3478

J. R. Donnelly, Lockheed-Martin Environmental Services Group, 980 Kelly Johnson Drive, Las Vegas, NV 89119

John W. Farley, Department of Physics, University of Nevada-Las Vegas, Las Vegas, NV 89154

a This work was performed while P.L.F. held an internship with the UNLV Environmental Studies Program.

b This work was performed while A. H. G. held a National Research Council/NERL (CRD-LV) Senior Research Associateship.

Original citation:
W. C. Brumley, P. L. Ferguson, A. H. Grange, J. L. Donnelly, and J. W. Farley, "Applications of Capillary Electrophoresis/Laser-Induced Fluorescence Detection to Groundwater Migration Studies," J. Cap. Electrophoresis, 3, 295-299 (1996).

Running title:
Groundwater migration

Correspondence:
William C. Brumley, U. S. EPA, P. O. Box 93478, Las Vegas, NV 89193-3478; tel: 702-798-2684; fax: 702-798-2142; email: brumley.bill@epa.gov

Keywords:
Capillary zone electrophoresis, fluorescent dyes, groundwater tracers, laser-induced fluorescence, solid-phase extraction, fluorescein

Nonstandard abbreviations:
CZE, capillary zone electrophoresis; HPLC, high performance liquid chromatography; MT, migration time; QA/AC, quality assurance/quality control; RCRA, Resource Conservation and Recovery Act; ppq, parts per quadrillion; ppt, parts per trillion;

SUMMARY

Capillary electrophoresis (CE) has been applied to the determination of the groundwater migration tracer dye fluorescein based on laser-induced fluorescence (LIF) detection and compared to determinations obtained with traditional spectrofluorimetry. Detection limits of injected dye in the low ppt ranges have been accomplished with both CE/LIF based on the Ar ion laser and with a spectrofluorimeter. This approach was used for a real world problem in determining groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites by the Environmental Sciences Division in response to regional needs and as application of new analytical tools under development. Fluorescent dye was injected into source wells and then was determined in monitoring wells by extracting pads that adsorbed the dye or else by directly determining the dye in the water using solid-phase extraction (SPE) as a preconcentration technique. The approaches based on CE/LIF exhibits increased specificity over existing approaches due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water using solid-phase extraction and field-amplified injection techniques.

1.0 INTRODUCTION

Groundwater migration is an important parameter in determining the distribution and fate of environmental pollutants originating from various waste sites or in tracking plumes that result from specific sources [1-3]. The analytical problem can be approached by using various types of tracer compounds that can be determined very sensitively. Examples of such methodology include the use of fluorescent dyes or whiteners, radioactive tracers, and, historically, the use of microscopic organisms or solid particles such as chaff [2]. The Environmental Sciences Division is interested in developing new analytical tools that can be applied to a variety of environmental problems facing the U.S. EPA. Among these tools under investigation is capillary electrophoresis/laser-induced fluorescence detection (CE/LIF).

Fluorescent dyes are a convenient choice because of the ease of sensitive detection. Spectrofluorimetry, high performance liquid chromatography (HPLC)/UV or fluorescence detection, and capillary LC/fluorescence detection have been used [2-6]. For HPLC or capillary LC, the retention of dye analytes is enhanced using ion-pairing techniques. Logically, applications of capillary zone electrophoresis/laser-induced fluorescence (CZE/LIF) detection should be ideal for the determination of anionic (or cationic) dyes. In free-zone CE, the dyes are separated very simply on the basis of their mobilities in aqueous buffers. Among several reports on CE/LIF detection techniques are two recent papers based on detecting fluorescent dyes very sensitively [7,8]. Environmental applications of CE have been reviewed [9] and techniques of sample preparation for environmental analysis using CE have also been summarized [10].

In this work, we applied CE/LIF detection to the determination of fluorescein as a tracer compound for groundwater migration, and compared the performance of CE/LIF with the more traditional spectrofluorimetry. Feasibility of determining dyes directly in the water through solid-phase extraction (SPE) preconcentration is also shown. To our knowledge, this is the first application of CE/LIF to groundwater migration using fluorescein dye [11]. Previously, we had reported studies of tinopal used as a groundwater tracer [12].

2.0 MATERIALS AND METHODS

2.1 Chemicals

All organic compounds were obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA) and Molecular Probes (Eugene, OR) unless otherwise specified. Other chemicals were from standard sources of supply, and all were used as received. Deionized water (18 mohm quality) was used for all aqueous solutions. Buffer solutions were freshly prepared at least weekly. Solutions of dye standards were prepared from solid dye and serially diluted.

2.2 Capillary Electrophoresis

A P/ACE Model 5000 Capillary Electrophoresis System (Beckman Instruments, Fullerton, CA, USA) was used for all electrophoretic determinations reported here. The instrument was fitted with a capillary 57 cm in total length (50 cm from the origin to the detector window) and 75 µm inside diameter. Detection was accomplished with an Ar ion laser operated at 488 nm emission and detection using a notch filter (488 nm) and a band pass filter (520DF20). Unless otherwise noted, electrophoresis was carried out at 30 kV. The temperature of the capillary was maintained at 25C. The capillary was equilibrated with running buffer for two minutes prior to beginning of an experiment, and washed for two minutes with alkali and water between runs. Migration times, peak widths, and peak areas were determined directly from peaks displayed by the data system or by processing software. Corrected peak areas, as computed by the instrumental software were further normalized by dividing them by the area of the peak of the internal standard which was erythrosin B. This computation was necessary to correct for the small variations in injection volumes resulting from the pressure injections typically of 5 sec duration (nominally 20 nL).

2.2.1 Calibration Curve

A regression analysis was carried out on the ratio of corrected areas (fluorescein corrected area divided by internal standard corrected area) versus the ratio of fluorescein concentration to internal standard concentration from 1 X 10-7 M to 1 X 10-10 M in half decade increments resulting in a 7-point calibration curve. Both unforced and forced through the origin regressions were considered and both resulted in correlation coefficients of 0.99. The equation of the line was used to calculate the concentrations of fluorescein in the samples and check standards run during the course of analysis. The concentration of fluorescein was calculable based on the known volume and concentration of internal standard added to the sample and the known volume/weight of the sample being analyzed.

2.2.2 Quality Assurance/Quality Control (QA/QC)

Each group of samples to be analyzed was bracketed before and after by a representative standard/internal standard QC sample to establish adherence to the calibration curve equation and MT variations due to changes in EO flow and therefore MT. Deviations greater than ±15% in agreement with the calibration curve results would be cause for rerunning of standards, construction of a new calibration curve, or replacement of the capillary.

The migration time variation on a given day/capillary was approximately ±5% in agreement with the standards of that day. The variation provides a rough window for anticipating the response of the internal standard and fluorescein. Using MT correction based on the internal standard, the expected and measured MT of the fluorescein peak fell within 0.3%. The position of both the internal standard and fluorescein could also be estimated from the position of the EO flow disturbance. The EO flow could be seen as a peak on the optical scale or can be monitored as a change in current as the injection plug exists the column. Further confirmation of the internal standard peak can be obtained by overspiking the sample with an additional aliquot of internal standard and observing the appropriate increase in peak area. Fluorescein itself could be confirmed by a similar procedure. In the samples reported in this work, no problems were encountered in identifying internal standard and analyte responses due to the low background level observed with CZE/LIF.

2.3 Groundwater Injection Studies

Four different dyes (Tinopal CBS-X, fluorescein (acid yellow 73), rhodamine WT, and eosin Y) were injected into four wells at a RCRA site and were monitored at three wells at a nearby Superfund site. Each dye (10 - 30 lbs) was injected with 2000 L of water resulting in a 10 mM concentration level for each dye. Thereafter, 8000 L of water was used to flush the dyes into the surrounding groundwater. Samples were taken before injection and for about two months afterward resulting in about 22 samples. Here, we report results only for the fluorescein tracer in a selection of 6 samples. Samples consisted of vial samples of water, "receptors", and 1-L water samples at the monitoring wells. Results are reported here for the extracts of the receptors and for SPE preconcentration of the water samples. The "receptors" consisted of fiberglass mesh filled with coconut charcoal and weighted to remain near the bottom of the well. The standard protocol called for 1 g of charcoal from the receptor to be extracted with 10 mL of a solution consisting of 5:3:2 (propanol:water:concentrated ammonium hydroxide). Results for fluorescein are reported as ppt-levels in the 10-mL extractant of the pads or ppt in the water when determined directly from a portion of the water sample.

2.4 SPE Sample Handling

Fluorescein was isolated from spiked DI water samples or groundwater samples using SPE with styrene-divinylbenzene (SDVB) extraction disks. The disks were prepared following the manufacturer's directions by soaking in 10 mL acetone and then pulling the solvent through the disk. The process was repeated with 10 mL methanol and then water without letting the disk become dry. Samples were then added, adjusted to pH 5.0, and pulled through at 25 mm Hg vacuum. The disks were dried for 2 min and then eluted twice with 6 mL of methanol. The methanol eluant was concentrated as necessary with a gentle stream of nitrogen with gentle warming to achieve a recovered concentration within the detection limits of the CE/LIF technique.

2.5 Spectrofluorimetry

Fluorescence emission spectra were recorded using a Spex Fluorolog 2, which is a double monochrometer instrument (excitation and emission) with 450 W Xe lamp (Instruments S. A., Edison, NJ). Groundwater studies used maximum sensitivity (1-cm cells) with 5.0-mm slits (8.5-nm bandpass) and a synchronous scan (1 sec/nm, 1 sec integration time) with a Stokes shift of 15 nm (optimized for the visible fluorescent dye tracers). Other reported data used 1.25 mm slits (2.13 nm bandpass).

3.0 RESULTS AND DISCUSSION

3.1 Migration Studies

Fluorescent dyes were injected into wells at the RCRA site, and monitoring wells located at the nearby Superfund site were sampled regularly by means of water samples and by receptors placed in the monitoring wells. Fluorescein was found to have an absorption maximum near 488 nm in DI water. The fluorescence emission maximum of fluorescein for excitation at 488 nm was found to be near 520 nm so that LIF and spectrofluorimetry were operating in a roughly equivalent manner. The Raman band of water at an excitation of 488 nm does not contribute significantly to the emission detection of fluorescein, and therefore this band contributes minimally to the background signal.

The "standard" protocol involved spectrofluorimetry with synchronous scans. The concentrations of the dyes in the groundwater were so low that only dyes adsorbed onto pads could be detected (in the eluting solvent system). Preconcentration using SPE is also considered later in this work. Based on 1 g charcoal extracted with 10 mL of the extracting solvent, results are reported as ppt in the 10-mL extract. Table 1 gives results by spectrofluorimetry, CE/LIF, and corrected migration times for Fluorescein in environmental samples compared with a standard run on that day. The agreement between spectrofluorimetry and CE/LIF is fairly good considering that the former is estimated by peak height against a fairly high background level. An example of this is Figure 1 for a standard and a sample.

Table 1. Quantitation of Fluorescein in Groundwater Samples (extracts of receptors).

Sample
ppt Spectro-fluorimetry in extract of receptor
ppt CE/LIF in extract of receptor
1
90
95
2
200
153
3
90
180
4
100
62
5
200
147
6
30.0
NDa
a N/D = not detected

Image of chromatograph - contact brumley.william@epa.gov for further information

Figure 1. Spectrofluorimetry results using a synchronous scan with a difference of 15 nm for fluorescein standard at 20 ppt.

The power of coupling a separation with fluorescence detection is an obvious advantage of CE/LIF.

3.2 Separation Conditions

A free-zone CE separation using a 40 mM borate buffer was chosen for fluorescein. With this system, fluorescein gave a migration time of about 4 min. Figure 2 reveals a typical response for injections of 1 X 10-7 M fluorescein and 1 X 10-5 M internal standard, having been buffered at 10% of the running buffer concentration.

Image of chromatograph - contact brumley.william@epa.gov for further information

Figure 2. CE/LIF electropherograms for fluorescein (MT=4.02 min) standard at 1.0 X 10-7 M; internal standard erythrosin B (MT=3.67 min)

Two impurities in the internal standard that also migrated earlier than fluorescein further bracketed the fluorescein response, yielding great certainty about its migration time (or corrected migration time).

3.3 Detection Levels Achieved in DI Water and Receptors

With a molecular weight of the disodium salt as 376, fluorescein at 1.00 X 10-9 M corresponds to 376 parts per trillion (ppt). Typical quantitations are illustrated for each of six separate 1-g samplings of the "pads" or receptors and are given in Table 1 (sample 2 was not run by CE/LIF). The response from a sample is shown in Figure 3.

Image of chromatograph - contact brumley.william@epa.gov for further information

Figure 3. Sample quantitated at 62.0 ppt fluorescein (MT=3.58) in the receptor pad extract with internal standard erythrosin B (MT=3.30); predicted MT=3.59 min based on MT of internal standard.

Fluorescein was clearly detected in the samples down to the detection limit of the technique which is estimated at 1 X 10-10 M at a S/N of 10. The separation has eliminated the fluorescent background evident in the spectrofluorimetric results, and this enabled the CE/LIF technique to detect fluorescein below the screening level of spectrofluorimetry. This level probably represents a background level of fluorescein in groundwater.

The detection of tracer dye helped to indicate that groundwater was migrating from one site (RCRA) to the other (Superfund) based on the arrival of fluorescein in one of the monitoring wells at the Superfund site. The findings of spectrofluorimetry and CE/LIF were in qualitative agreement. The greater specificity provided by CE/LIF affords increased confidence in the findings relative to spectrofluorimetry.

3.4 SPE of fluorescein in groundwater

The SPE technique potentially allowed the concentration detection limit to be extended to 10-14 M in spiked water samples with a 250 mL volume used for isolation. Concentration of the eluted isolate would result in the ability to detect fluorescein at 10-14 M or 3.76 ppq in water. For practical reasons, recoveries of spiked DI water samples were limited to 10-10 M to 10-12 M and were found variable, ranging from about 40 to 100% and averaged about 67%. In order to achieve a blank recovery prior to working on real samples or low level spikes, a new filtration apparatus was used in a laboratory where fluorescein had not been used. For the purposes of this study, levels determined to be below 1 X 10-12 M are not considered to be reliable because of the difficulty of consistently achieving reagent blank background levels below 10-13 M.

Figure 4 illustrates the response of a groundwater sample preconcentrated by SPE and quantitated at 342 parts per quadrillion (ppq).

Image of chromatograph - contact brumley.william@epa.gov for further information

Figure 4. Sample quantitated at 0.342 ppt fluorescein (MT=3.97)in the water sample (preconcentrated by SPE) with internal standard (MT=3.63); predicted MT=3.98 based on MT of internal standard.

Evidence for the ability of CZE to separate background responses is clear. This level of fluorescein represents approximately our lowest limit of reliable detection based on reagent blank background levels.

Table 2 gives the concentrations of fluorescein found in water samples collected at the time of the pad removal. Concentrations indicate that the dye is present in water at several orders of magnitude below the levels found in the extracts of the pads. The pads have, therefore, effected a preconcentration similar to that achieved by SPE, as anticipated. There is, however, no clear way to relate pad recoveries to concentrations in water since an unknown volume of water flows in contact with the pads during their residence in the well. The SPE of the water sample is a snapshot of the fluorescein level in the water flowing through the well at that sampling. To our knowledge, this is the first attempted determination of these levels of concentration in real samples of groundwater migration studies by CE/LIF. The data indicate that the background level of fluorescein in the groundwater was below 10-12 M (or 376 ppq). Quantitations below 10-12 M are considered unreliable as indicated previously because of the difficulties in keeping reagent blanks at nondetectable concentrations or below 10-13 M. These data need to be confirmed with new samples, controls, and performance standards because of the long holding time before analysis by CE/LIF. The levels of fluorescein in vial samples taken at the same time and determined by spectrofluorimetry in a timely manner were below the detection limits of that technique (ca 5 ppt). Vial samples run directly by CE/LIF also indicated that fluorescein was below detection limits (ca. 10-10 M). Thus, preconcentration with CE/LIF determination appears to have promise in determining the limiting background levels for tracer studies. This results from the separation effected by CE in eliminating background contributions from the sample. Further studies would be needed to confirm levels below 10-12 M with proper assurances of prompt analysis, laboratory blanks, and reproducibility.

Table 2. Quantitation of Fluorescein in Groundwater Samples (250 mL volumes).

Sample
ppq CE/LIF
Conc (10-12 M) Fluorescein
A
478
1.27
B
256
0.68
C
262
0.70
D
1810
4.81
E
873
2.31
F
1570
4.19

3.5 Variations in Migration Times

Under free-zone electrophoresis, variations in migration times (MTs) of target ions are principally due to variations in the EO flow. Corrections to this variation were carried out12 and corrected migration times for fluorescein generally agreed within 0.3% for predicted versus observed MTs. Variations outside this range may be due to a number of factors or could indicate that the peak assignment is questionable. Some examples of corrected MTs are indicated in captions of Figures 3 and 4 relative to a standard run that day (Figure 2).

3.6 Advantages/Disadvantages of CE/LIF Versus Spectrofluorimetry

CE/LIF offers greater specificity in determining dyes and eliminating background signal levels. The ease of sample handling is very similar for the two techniques. Availability of a CE instrument and proper laser excitation sources may be current drawbacks for some laboratories. Consideration of using CE/LIF as a confirmatory technique for spectrofluorimetry is recommended. Spectrofluorimetry is capable of high throughput with about 5 min per sample required compared with about 10 min per sample for CE. An advantage of commercial CE instrumentation is the ability to use an autosampler to run samples unattended. Spectrofluorimetry is a straightforward technique that can be used with minimal training and is recommended for screening purposes. Cost of instrumentation actually favors the CE/LIF system in this case. Other dyes are accessible via a variety of laser sources, some of which are HeNe lasers that are relatively inexpensive.

4.0 CONCLUSING REMARKS

CE/LIF detection offers a sensitive, specific approach to groundwater migration studies involving fluorescent tracer dyes. The analytical problem of detecting fluorescent tracer compounds (ionic analytes) in water samples is perfectly matched to separations involving free-zone CE. The detection limits afforded by CE/LIF are as good or better than those of traditional spectrofluorimetry, while providing increased specificity because of the separation based on ion mobilities. Future studies of groundwater migration should be planned with this capability in mind. Choice of tracer dyes can be based on the availability of lasers matched to the absorption maximum of appropriate dyes.

NOTICE

The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development, funded and performed the research described here. This work has been subjected to the Agency's peer review and has been approved as an EPA publication. The U.S. Government has the right to retain a non-exclusive, royalty-free license in and to any copyright covering this article. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

5.0 REFERENCES

  1. Smart, P. L., & Laidlaw, I.M.S., Water Resources Res. 1977, 13:15-33.
  2. Davis, S. N., Campbell, D. J., Bentley, H. W., & Flynn, T. J., Ground Water Tracers, 1985, Natl. Water Well Assoc., Worthington, OH; Cooperative agreement CR-810036,U. S. Environmental Protection Agency, Robert S. Kerr Environmental Research Laboratory, Ada, OK 74820.
  3. Aulenbach, D. B., Bull, J. H., & Middlesworth, B.C., Ground Water, 1978, 16:149-157.
  4. Laane, R.W.P.M., Manuels, M. W., Staal, W. Water Res., 1984, 18:163-165.
  5. Van Soest, R.E.J., Chervet, J.P., Ursem, M., Suijlen, J.M., LCGC International, 1996, September, 586-593.
  6. Jones, T. L., Master's Thesis: The detection and evaluation of fluorescent brighteners as organic tracers in alkaline groundwaters, Department of Chemistry, University of Nevada-Las Vegas, Las Vegas, NV, December, 1991.
  7. Timperman, A. T., Khatib, K., Sweedler, J. V., Anal. Chem., 1995, 67:139-144.
  8. Chen, D. Y., Dovichi, N. J., Anal. Chem., 1996, 68:690-696
  9. Brumley, W. C., LCGC, 1995, 13:556-568.
  10. Brumley, W. C., J. Chromatogr. Sci., 1995, 33:670-685.
  11. Ferguson, P., Undergraduate Thesis: The Use of Spectrofluorimetry and Capillary Electrophoresis/Laser-Induced Fluorescence For The Determination of Fluorescent Dyes in Groundwater Migration Studies, Department of Environmental Studies, University of Nevada-Las Vegas, NV, December, 1997.
  12. Brumley, W. C., Ferguson, P. L., Grange, A. H., Donnelly, J. L., Farley, J. W., J. Cap. Electrophoresis, 1996, 3:295-299.

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