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Trace Organic Analysis

2/25/2002

Investigation of CE/LIF as a Tool in the Characterization of Sewage Effluent for Fluorescent Acids: Determination of Salicylic Acid

Sean Flaherty1,2*, Shelly Wark1,3*, Ginger Street1, John W. Farley1 and William C. Brumley4

1 Department of Physics, University of Nevada-Las Vegas, P.O. Box 454002, Las Vegas, NV
89154-4002
2 Present address: Gilbert Unified School District, 140 South Gilbert Road, Gilbert, AZ 85296
3 Present address: Lockheed Martin Aeronautics Company, P.O. Box 748, Fort Worth, TX 76101
4 U.S. Environmental Protection Agency, Environmental Sciences Division, P.O. Box 93478, Las Vegas, NV 89193-3478

Correspondence: Dr. John W. Farley, Department of Physics, Campus Box 454002, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, NV 89154-4002. (Tel: +702-895-3084; FAX: +702-895-0804; E-mail: farley@unlv.edu)

* Work performed while supported by NSF REU grant PHY-9802572.

Original citation:
S. Flaherty, S. Wark, G. Street, J. W. Farley, and W. C. Brumley, "Investigation of CE/LIF as a Tool in the Characterization of Sewage Effluent for Fluorescent Acidics: Determination of Salicylic Acid," Electrophoresis 23, 2327-2332 (2002).

ABSTRACT

The investigation of emerging contaminant issues is a proactive effort in environmental analysis. As a part of this effort, sewage effluent is of current analytical interest because of the presence of pharmaceuticals and their metabolites and personal care products. The environmental impact of these components is still under investigation but their constant perfusion into receiving waters and their potential effect on biota is of concern. This paper examines a tool for the characterization of sewage effluent using capillary electrophoresis/laser-induced fluorescence (CE/LIF) with a frequency-doubled laser operated in the ultraviolet (UV). Fluorescent acidic analytes are targeted because they present special problems for techniques such as gas chromatography/mass spectrometry (GC/MS) but are readily accessible to CE/LIF. As an example of the application of this tool, salicylic acid is determined near the 100 ng/L (7 x 10-10 M) level in sewage effluent. Salicylic acid is a metabolite of various analgesics. Relatively stable in the environment, it is a common contaminant of municipal sewage systems. Salicylic acid was recovered from freshly collected samples of the effluent by liquid-liquid extraction. Confirmation of identity was by electron ionization GC/MS after conversion of the salicylic acid to the methyl ester by means of trimethylsilyldiazomethane. CE/LIF in the UV has revealed more than 50 individual peaks in the extract and a background response that suggests a large and indeterminate number of additional compounds are present. These data together with complementary techniques provide information on the complexity and components in these effluent streams.

1.0 INTRODUCTION

As part of a proactive approach to environmental protection, emerging issues with potential impact on human health and the environment are the subject of ongoing investigation. One emerging area of environmental research concerns pharmaceuticals and personal care products (PPCPs) in the environment and their possible impact on biota and ecosystems [1]. The long term effects of constant perfusion of PPCPs into the aquatic environment via municipal sewage and other sources are presently unknown. Some compounds are known to have physiological effects on nontarget biota at extremely low concentrations (e.g., estrogens and estrogenic mimics and certain antidepressants) [1].

Among the possible target analytes are several compounds possessing chemical structures that are resistant to microbial degradation and/or capable of being bioaccumulated. Acidic metabolites of pharmaceuticals present one type of analyte that appears in the effluent of many publicly operated sewage treatment facilities. The subject of the present study is to examine alternative tools in the characterization of effluent, particularly the acidic components. Very polar components present special problems to preconcentration and separations because the techniques must often be tailored to the specific chemical or chemical class and often involve very polar solvents which are less suitable for GC/MS. Liquid chromatography/mass spectrometry (LC/MS) is often more applicable, but no universal separation approach or interface design works across a broad range of neutral and ionized analytes. Thus, the lack of a quasi-universal separation and detection approach comparable to GC/MS adds complexity to the analysis [2].

Capillary zone electrophoresis (CE) offers a particularly simple approach to the separation of anionic substances in aqueous buffers. However, detection limits have heretofore not been quite as low as typical techniques such as high performance liquid chromatography/diode array detection (HPLC/DAD) and GC/MS [3]. Using a laser operating in the ultraviolet (UV) region provides a unique tool for examining fluorescent acids in complex samples such as effluent but at environmentally relevant detection limits following typical sample handling (e.g., solid-phase extraction or liquid/liquid extraction with concentration) [4]. CE/LIF measurements with continuous UV laser excitation have included studies of groundwater migration [5], PNAs [6], and surfactants [7].

Salicylic acid (2-hydroxybenzoic acid) is the chief metabolite of various analgesics including aspirin [1]. It is a common component of sewage effluent [8, 9, 10] and its recovery has been discussed there. Compounds closely related to salicylic acid are the subject of current U.S. EPA methods including the use of acid/base-neutral fractionation approaches. Benzoic acid is an EPA target analyte and other benzoic acid derivatives are used as herbicides (e.g., dicamba). A number of other drugs or metabolites possess a structure involving an aromatic hydroxyl group suggesting fluorescence detection may be useful in their determination. Generally, the presence of nitro or halogen groups quenches much or all of the fluorescence [3]. Thus, compounds must be examined individually to determine optimum excitation/emission wavelengths for detection with due regard to matrix or background levels as well.

Actually, little is known about the complete content of municipal sewage effluent with regards to very polar substances. A number of pharmaceuticals and their metabolites have been targeted, but characterization studies are needed to obtain a more complete picture of potential risk associated with components in the effluent.

HPLC/DAD provides an alternative and more general detection for acidic compounds provided adequate detection limits can be reached. LC/MS/MS has been chosen to provide highly specific quantitative data for sewage effluent [1, 9, 10].

GC/MS analysis of such acids is facilitated by conversion to a volatile ester. Methyl esters are particularly convenient for electron ionization MS and are readily made by reaction of the substrate acids with diazomethane. However, diazomethane, explosive and allergenic, is a hazardous reagent to work with and is typically used immediately after its generation. The use of diazomethane in this manner is set forth in a U.S. EPA Method [11]. Salicylic acid itself has been determined in the same manner using diazomethane by Ternes et al. [10] and Hignite and Azarnoff [8]. At higher levels, such as in pharmacokinetic studies, salicylic acid may be determined directly by HPLC/DAD without derivatization [12].

The diazomethane derivative, trimethylsilyldiazomethane (TMSDM), is a convenient alternative to diazomethane and exhibits many of the reactions of diazomethane, including the reaction with carboxylic acids to yield methyl esters. TMSDM chemistry has been reviewed [13, 14]. The carbon of the ester methyl group produced by reaction with TMSDM is derived from the carbon which bears the diazo group. Nevertheless, the presence of methanol is necessary to bring about conversion to the methyl ester. We also note that TMSDM has also been used in conjunction with two U.S. EPA methods, 552.2 [15] and 515.2 [16].

In this work we present the application of CE/LIF to the characterization of sewage effluent. As a specific example component, salicylic acid is determined by CE/LIF based on liquid/liquid extraction from sewage effluent, and then confirmed by GC/MS as the methyl ester.

2.0 MATERIALS AND METHODS

2.1 Chemicals

(a) Trimethylsilyldiazomethane, 2.0 M solution in hexanes. (Aldrich Chemical Co., Milwaukee, WI).
(b) 2-hydroxybenzoic acid, sodium salt (sodium salicylate). 97%, (Aldrich Chemical Co.).
(c) Sodium hydroxide. 98.9%, (Fisher Scientific, Pittsburgh, PA).
(d) Hydrochloric acid. ACS grade, 36.5-38%(Fisher Scientific).
(e) Methylene chloride. Capillary GC/GC-MS grade (Fisher Scientific).
(f) Methanol.ACS/HPLC grade (Fisher Scientific).
(g) Acetone. AR grade (Fisher Scientific).
(h) Sodium tetraborate decahydrate. ACS grade (Aldrich).
(i) PCB standards. Research grade (Chem Service). Individual congeners were received in 2 mL ampules at 1 痢/mL concentration.
Solutions
(a) 6N NaOH. 100 mL batches were freshly prepared from 24.3 g of the solid NaOH made up to 100 mL with deionized water.
(b) Borate Buffer. 20 mM borate buffer was prepared from 0.1907 g of sodium tetraborate decahydrate and dissolved in deionized water and diluted to 100 mL.
Other Materials
(a) Vials Silanized via high-temperature treatment with hexamethyldisilazane, 12 x 32-mm wide mouth, screw cap with PTFE/silicone cap liners. (Alltech Associates, Inc., 2051 Waukegan Rd, Deerfield, IL 60015).

2.2 Sample handling

Three 4-liter samples were collected from the sewage effluent. The contents of each 4-liter bottle were treated in parallel fashion following identical procedures. First, in order to remove base/neutrals, each was basified to pH 12-14 with 6 N aqueous NaOH. The liquid-liquid extraction used here follows the protocol of an EPA Method [17]. After basification each 4-liter sample was extracted three times with 400 mL of dichloromethane. The aqueous phases were then acidified to pH 1 with concentrated hydrochloric acid and again extracted three times with 400 mL of dichloromethane. The dichloromethane extracts from these acidified solutions were combined, dried over anhydrous sodium sulfate and concentrated down to a volume of 5.1 mL after solvent exchange with acetone. Concentration was achieved using a refluxing apparatus consisting of a large round bottom flask, boiling chips (Teflon), and a three-ball Snyder column; this ensured that all surfaces were continuously bathed with condensing liquid during concentration. Of the resulting concentrate, 100 無 was subjected to analysis by CE/LIF following addition of 10 無 of buffer and fluorescent internal standard; a separate 100 無 was subjected to GC/MS following addition of internal standard (PCB #19) and derivatization with TMSDM.

2.3 Derivatization with TMSDM

Derivatizations with TMSDM were carried out in the commercially silanized vials. For the standard response the vial was charged with 1.0 mL of an acetone solution. The 1.0 mL comprised acetone and 10 無 amount of a stock solution of sodium salicylate (MW 160.1) in DI water (0.0224 g in 100 mL, yielding a concentration of 1.4 x 10-3 M). On top of the 1.0 mL in each vial was added 25 無 of the 2.0 M TMSDM reagent and 100 無 of methanol. A 50 無 aliquot of internal standard was added that consisted of polychlorobiphenyl congeners #19, 54, 104, 155, 184, and 204 that was 200 pg/無 in each congener. These congeners do not occur in commercial Aroclors and are the most highly orthosubstituted congeners of each chlorination level; they cover a large retention time range and therefore are suitable as a general internal standard mix. To a 100 無 aliquot of sewage extract in acetone was added 2.5 無 of TMSDM, 10 無 of methanol, and 5 無 of the PCB internal standard. After thorough mixing, the homogenous reaction mixtures were allowed to stand at ambient temperature for two hours.

2.4 GC/MS Analysis

GC/MS analysis was carried out directly on the reaction vial contents on an Agilent Technologies 6890 GC/5973 MSD. A 30-m x 0.25-mm ID HP 5MS column with a 0.25 痠 film was used. The temperature program was 46.0 min long and ramped as follows: 60 °C to 150 °C @ 10.00 deg/min (9 min); 150 °C to 250 °C @ 4.00 deg/min (25 min); 250 °C to 300 °C @ 10.00 deg/min (5 min); maintained at 300 °C until 46.00 min total.

Injections were 2 無 and pulsed-splitless mode was used. The carrier gas was helium at a flow of 1.0 mL/min with pressure programming and the instrument was operated in EI mode. The retention times of the methyl esters of salicylic acid and internal standard (PCB #19) were 9.45 and 18.57 min, respectively. The m/z values monitored (dwell time 25 milliseconds, resulting EM voltage 2035.3) were, respectively: salicylic acid methyl ester - 152.0; 121.0; 120.0; 92.0; PCB#19 - 257.90. An additional suite of ions were monitored for 13 acidic herbicides and for the other PCB internal standard congeners.

2.5 CE/LIF Analysis

All samples, buffers, and standards were filtered through 0.2-痠 pore size 25 mm diameter syringe filters. In the case small samples, 5 mm-diameter syringe filters were used. Buffers and rinse solutions were prepared from deionized 18 M water (Millipore quality). The optical bench has been described elsewhere [5]. Briefly, this laboratory-built instrument consists of the Coherent (Santa Clara, CA) 300 FReD laser, two high reflective mirrors, a focusing objective (fused silica optics), a capillary holder, a focusing lens for the fluorescent light at 90 deg to the beam, a slit to reduce stray fluorescence before the photomultiplier, a current amplifier, and a data system. The CE experiment requires a high voltage supply as well. A reversible polarity 30 kV 1.5 mA power supply was employed (Glassman High Voltage, High Bridge, NJ). For excitation at 244 nm using 5 mW power, two optical filters were used in series (390DF70, Omega Optical, Battleboro, MA) as the bandpass filters for fluorescent light (center frequency 390 nm with 50% attenuation at 35 nm). Capillaries were prepared from bulk fused silica obtained from PolyMicro Technologies (Phoenix, AZ) with reported work using 58 cm capillaries (window 8 cm from end). Running buffer was 20 mM borate buffer. Capillaries were rinsed initially each day with 5 min pressurized rinses of 0.1 N NaOH, DI water, and running buffer. Between each run the capillary was rinsed for 2 min with each of the rinsing solutions. Injections were by gravity for 3 to 20 sec at 25 cm height. All reported work used capillaries of 0.075 mm ID with 18 kV as the separation voltage.

As internal standard for CE/LIF work, 7-hydroxycoumarin-4-acetic acid was chosen due to its fluorescence in the UV and relatively long migration time. A 6-point calibration curve was obtained from solutions that ranged from 1.12 X 10-5 M (1.79 mg/L) to 2.24 X 10-7 M (3.59 痢/L) in aqueous sodium salicylate and nominally 1 X 10-5 M (2.20 mg/L) in the internal standard; solutions were maintained at 2 mM borate (10% of the running buffer concentration). The curve was constructed from ratios of corrected areas (due to on-column detection) of salicylate to corrected areas of the internal standard response versus the corresponding amount ratios. A linear plot resulted with a weighted linear regression r2 of 0.99.

3.0 RESULTS AND DISCUSSION

3.1 CE/LIF Characterization; Determination of Salicylic Acid in Effluent

CE/LIF using the 244 nm excitation line from the frequency-doubled laser exhibits about the same sensitivity (for fluorescent compounds studied here) on a mass/volume injection basis as does GC/MS albeit at a smaller overall injection volume and amount on-column. For example, GC/MS responses for 1 to 50 pg/無 per component allow typical detection of individual compounds. In the case of CE/LIF where the analyte is present in the 10-7 to 10-8 M concentration range, actual amounts injected and detected may typically involve 1 to 30 fg/nL where 1 to 20 nL total volume is injected. Normal sample handling capabilities thus afford a sample within the capabilities of the CE/LIF detection with typical sample preconcentration of about 1000-fold for sub 痢/L detection limits. In addition, the normal selectivity of fluorescence detection provides more specificity than absorption detection by excluding responses from nonfluorescent components in a complex sample.

Figure 1 illustrates the response of salicylic acid and the internal standard at migration times of 6.68 and 7.78 min, respectively.

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

Figure 1. CE/LIF (ex 244 nm) of salicylate (2.24 X 10-6 M, 6.65 min) and 7-hydroxycoumarin-4-acetic acid (7.78 min) internal standard. Separation conditions: 0.075 mm ID X 58 cm capillary (window 8 cm from end); running buffer was 20 mM borate buffer (pH 9.2); separation voltage 18 kV.

The signal to noise suggests a detection limit of about 5.6 X 10-8 M (7.7 痢/L) for salicylic acid (see Figure 2, where the S/N = 16 at 2.24 X 10-7 M or 31 痢/L).

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

Figure 2. CE/LIF (ex 244 nm) of salicylate (2.24 X 10-7 M, 6.55 min) with S/N = 16. Separation conditions: 0.075 mm ID X 58 cm capillary (window 8 cm from end); running buffer was 20 mM borate buffer (pH 9.2); separation voltage 18 kV.

The extract of the effluent provided the electropherogram of Figure 3.

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

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

Figure 3. CE/LIF (ex 244 nm) of extract of effluent with salicylate at 6.65 min and internal standard at 7.80 min (EO flow at 4.26 min) (A) and expanded view of migration time region from 6.6 to 8.2 min (B). Separation conditions: 0.075 mm ID X 58 cm capillary (window 8 cm from end); running buffer was 20 mM borate buffer (pH 9.2); separation voltage 18 kV.

The large peak at 4.26 min represents the EO flow and neutral components as well as anionic substances migrating very close to the EO flow. The anions with significant mobilities fall into a bimodal distribution with numerous individual components (at least 50) and with a continuous-looking distribution forming the background. The large peak at 7.80 min is the internal standard and salicylate appears at 6.68 min. The expanded part of Figure 3 provides a clearer look at the migration time window of salicylate. The response from salicylate (MW 137.1) was quantitated at 97 ng/L (7.1 x 10-10 M) in the effluent using the calibration curve and the internal standard response.

The significance of the result consists partially in the relatively clear presence of individual components and also in the indication of a large number of components that are fluorescent. The result thus provides a CE/LIF characterization of effluent at 244 nm. Fluorescence is, obviously, not confined to hydroxylated aromatic compounds. For example, an amino function attached to an aromatic ring is also fluorescent at the excitation of 244 nm. The migration time of 4-aminobenzoic acid is, for example, 6.31 min for a migration time of IS of 7.80 min.

3.2 GC/MS Confirmation of Salicylate

Salicylic acid in a sewage effluent was recovered by liquid-liquid extraction in accordance with EPA methods. After recovery, the salicylic acid was converted to the methyl ester using TMSDM for electron ionization GC/MS confirmation of identity. A suite of ions was monitored for a number of acidic analytes including salicylic acid methyl ester. Figure 4 presents a mass spectrum (constructed from the monitored ions) of a standard and of the sample with the agreement in relative abundance and with previously published spectra confirming the identification together with additional support from identical retention times (9.45 min).

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

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

Figure 4. GC/MS mass spectrum of standard of methyl salicylate at 9.46 min (A) and peak at 9.45 min (B) from extract of effluent. Separation conditions: 20 m x 0.18mm ID DB-XLB column (J & W, Folsum, CA, USA), temperature program (60, 1 min; 60-150 @ 10 degC/min; 150-250 degC @ 4 degC/min; 250-300 @10 degC/min), splitless injection (injector 250 degC); 1.0 mL He/min flow.

4.0 CONCLUDING REMARKS

This work demonstrates the facile determination of salicylic acid as a sewage effluent contaminant down to the 100 parts per trillion level. The salicylic acid may be recovered from the effluent by liquid-liquid extraction in accordance with EPA methods. After concentration by solvent evaporation, the salicylic acid is determined by CE/LIF directly. It can also be conveniently converted to the methyl ester by means of commercial TMSDM, followed by EI GC/MS confirmation of identity. To the best of our knowledge, this work represents the first report of the use of CE/LIF to characterize sewage effluent and to determine salicylic acid in this manner.

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 nonexclusive, 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. Daughton, C. D., Ternes, T. A., Environ. Health Perspect. 1999, 107, suppl 6, 907-938. (available at: http://www.epa.gov/nerlesd1/bios/daughton.htm)
  2. US EPA Method 8270 (Revision 3-December, 1996). (This and other EPA Methods may be accessed in pdf format at site http://www.epa.gov/epaoswer/hazwaste/test/main.htm)
  3. Sovocool, G. W., Brumley, W. C., Donnelly, J. R.Electrophoresis, 1999, 20:3297-3310.
  4. Brumley, W. C., J. Chromatogr. Sci., 1995, 33, 670-685.
  5. Brumley, W. C., P. L. Ferguson, A. H. Grange, J. R. Donnelly, and J. W. Farley, J. Cap. Elec., 1996, 3(6):295-299.
  6. Yan, C., R. Dadoo, H. Zhao, R. N. Zare, and D. J. Rakestraw, Anal. Chem., 67 (1995) 2026.
  7. Kok, S. J., Hoornweg, G., de Ridder, T., Brinkman, U., Velthorst, N. H., Gooijer, C., J. Chromatogr. A 1998, 806:355-360.
  8. Hignite, C., Azarnoff, D. L., Life Sciences, 1977, 20:337-342.
  9. Stumpf, M., Ternes, T. A., Haberer, K., Seel, P., Baumann, P. W., Vom Wasser 1996, 86, 291-303.
  10. Ternes. T. A., Wat. Res. 1998, 32:3245-3260.
  11. US EPA Method 8151A (Revision 1-December, 1996).
  12. McMahon, G. P., Kelly. M. T., Anal. Chem. 1998, 70:409-414. (An abbreviated version of this work may be accessed at http://www.iscpubs.com/pubs/abl/articles/b0002/b0002mcm.pdf)
  13. Shiori, T., Aoyama, T., Trimethylsilyldiazomethane-A Versatile Synthon for Organic Synthesis in A. Dondoni, ed., Adv. In the Use of Synthons in Organic Chemistry 1993, 1:51-101.
  14. Hashimoto, N., Aoyma, T., Shiori, T. Chem. Pharm. Bull. 1981, 29:1475-1478; Aoyama, T., unpublished results.
  15. Pawlecki-Vonderheide, A. M., Munch, D. J., Munch, J. W. J. Chromatogr. Sci. 1997, 35,293-301.
  16. US EPA Method 515.2 (Revision 1.1-1995) listed in 40 CFR 141.24(e) which references EPA Report 600/R-95-131 that contains the detailed method.
  17. US EPA Method 3510C (Revision 3-December, 1996), SW846 CH 4.2.1, available at: http://www.epa.gov/epaoswer/hazwaste/test/main.htm

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Author: William C. Brumley
email: William C. Brumley


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