Anal. Chem. lQ66, 60,301-305
membranes. This result may mean that complexed cation Kval'TPB- is more oil soluble than the simple K+TPB-. But there may also be some association between cations and anions. Nevertheless, the advantageous effect of added NaTPB on the potentiometric behavior of ionophore based electrodes is still useful, although limited in time. Registry No. DOS,122-62-3;o-NPOE, 37682-29-4; KTpCPB, 14680-77-4; NaTPB, 143-66-8; ETH 1001, 58801-34-6; PVC, 9002-86-2; K, 1440-09-1;Ca, 7440-70-2; valinomycin, 2001-95-8.
LITERATURE CITED Horvai, G.; Grif, E.; Tbth, K.; Pungor, E.; Buck, R. P. Anal. Chem. 1988, 58, 2735-2741. Tdth, K.; Grif, E.; Horvai, 0.; Pungor, E.; Buck, R. P. Anal. Chem. 1988, 58, 2741-2744. Buck. R. P.; Tbth, K.; Grif, E.; Pungor. E. J. Elechoanal. Chem. 1987, 223, 51-66. Lindner, E.: Niegreisz, Zs.; Pungor, E.; Buck, R. P., in preparation. Perry, M.; Lobei, E.; Bloch, R . J . Membr. Scl. 1976, 7 , 223-235. Kumlns, C. A.; London, A. J. folym. Sci. 1980, 46, 395-408. Oesch, U.; Simon, W. Anal. Chem. 1980, 52,692-700. Satchwill, T.; Harrison, D. J. J . Electroanal. Chem. 1988, 202, 75-81. Boles, J. H.; Buck, R. P. Anal. Chem. 1973, 4 5 , 2075. Morf, W. E.; Kahr, G.; Simon, W. Anal. Lett. 1974. 7 , 9. Morf, W. E.; Ammann, D.; Simon, W. Chlmk 1974, 28, 65. Kedem, 0.; Perry, M.; Bloch, R. IUPAC Internatlonal Symposium on Selective Ion-Sensitive Electrodes, Cardiff, 1973 paper 44. Craggs, A.; Moody, G. J.; Thomas, J. D. R. J . Chem. Educ. 1974, 51, 54 1-544.
30 1
(14) Meier, P. C.; Ammann, D.; Morf, W. E.; Simon, W. I n Medical and Biologlcel Appllcatlons of Electrochemical Devlces; Koryta. J., Ed.; Wliey: New York, 1980. (15) van den Berg, A.; van der Wai, P.; Ptasinski, D.; Sudhoiter, E. J. R.; Bergveld, P.; Reinhoudt, D. N. Anal. Chem. 1987, 59, 2827-2829. (16) Heifferich, F. Ionenaustauscher; Verlag Chemie: Weinheim, 1959; Band I. (17) Incz&Iy, J. Analytical Application of Ion-Exchangers ; Akaddmiai Kiadb: Budapest, 1966. (18) Ammann, D.; Morf. W. E.; Anker, P.; Meier, P. C.; Pretsch, E.; Simon, W. Ion-Sel. Electrode Rev. 1983, 5 , 3. (19) Armstrong. R. D., submitted for publication in Electrochim. Acta. (20) Ammann, D.;Pretsch, E.; Simon, W.; Lindner, E.; Bezegh, A,; Pungor, E. Anal. Chlm. Acta 1985, 171, 119-129. (21) Pretsch, E.; Wegmann, D.; Ammann, D.; Bezegh. A.; Dinten, 0.; Uubil, M. W.; Morf, W. E.; Oesch, U.; Sugahara, K.; Weiss, H.; Simon, W. In Recent Advances in the Theory and Application of Ion-Selectlve Electrodes h Physiology and Medlclne; Kessler, M., Harrison, D. K., Hoper, J., Eds.; Springer-Verlag, Berlin, 1985. (22) Meier, P. C.; Morf, W. E.; Laubii, M.; Simon, W. Anal. Chim. Acta 1984, 156, 1. (23) Tdth, K., unpublished resuits. (24) Lindner, E.; Tbth, K.; Pungor, E.; Behm, F.: Oggenfuss, P.; Welti, D. H.; Ammann, D.; Morf, W. E.; Pretsch, E.; Simon, W. Anal. Chem. 1984, 56, 1127.
RECEIVED for review June 24, 1987. Accepted September 24, 1987. Support from the Hungarian Academy of Sciences and NSF (under Grants CHE8406976 and INST-8403331) is gratefully acknowledged.
Liquid Chromatographic Determination of Nitrilotriacetic Acid, Ethylenediaminetetraacetic Acid, and Related Aminopolycarboxylic Acids Using an Amperometric Detector Jihong Dai' and George R. Helz*
Chemistry and Biochemistry Department, University of Maryland, College Park, Maryland 20742
An amperometric detector empioylng a carbon-paste electrode is used to determine aminopoiycarboxylic aclds, Inciudlng nltrliotriacetic acld (NTA) and ethyienediamlnetetraacetic acld (EDTA), after liquld chromatographlc separation on a reversed-phase column with an aqueous trlchioroacetk acld mobile phase at pH lower than 2. The aminopoiycarboxylic acids are dlrectly oxidlzed at the detector electrode wlthout Involving an lntermedlate species. Glycine, hninodlacetic acld, common amino aclds, cltrk acld, and fulvlc acids do not interfere with the determination of NTA and EDTA. The low moblie-phase pH limits Interference from metal lons In natural waters. Where such interference occurs, a stronger chelatlng reagent [e.g. dlethylenetriamlnepentaacetic acid (DTPA)] can be used to suppress it. NTA and EDTA In aqueous samples, including wastewater treatment plant influent and effluent, can be determlned without prlor sample preparation. The minimum detectable amounts are 0.1 ppm for NTA and 0.15 ppm for EDTA with a precision of less than 7% relative standard deviation.
Aminopolycarboxylic acids, such as ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid ("FA), are widely 'Present address: B d Polar Research Center, Ohio State University, Columbus, O g 43210.
used in industry and agriculture. Their use in commercial detergents, specialized cleaning reagents, and a variety of other products, including foods, results in their ultimate release to the environment ( 1 , 2 ) . Their strong chelating capacity may play an important role in the distribution and transportation of metals in the aquatic environment. These chelating agents can enhance the levels of dissolved heavy metals by both releasing them from sediments (I,3) and inhibiting removal through precipitation ( 4 ) . They are synthetic products with undetermined health and toxicological effects (5, 6 ) . It has been suggested that they may constitute a source of nutrient nitrogen for aquatic algae (7), although this has been questioned (8). A number of analytical methods exist for NTA and EDTA. Among them, the most widely used for environmental purposes are the highly sensitive gas chromatographic methods, originally developed by Aue et al. (9) and subsequently improved and modified by others (10-15). However, the GC methods are limited by long analysis time and various interferences (16). Recently, in efforts by researchers to develop analytical methods adequate for environmental applications, liquid chromatography (LC) techniques have been exploited, especially for EDTA and NTA (17-24). So far, these techniques have been based on detection by ultraviolet (UV) absorption of complexes formed between the analyte ligand and a metal ion in the mobile phase. The reported detection limits by the LC-W methods are from 1to 10 FM. However, UV detection
0003-2700/88/0360-0301$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
is critically dependent upon metal-ligand complexing, since free ligands such as NTA and EDTA do not strongly absorb in the ultraviolet and visible regions. Because many metal ions can compete for the chelating ligands, miscellaneous metal ions present in samples cause interference ( 1 4 2 4 ) . Another reported analytical problem with a UV detector is the limited linear range of the calibration curves (22, 23). Several electroanalytical techniques have also been utilized for the determination of NTA, EDTA, and related compounds. Usually a metal species is employed which is reduced or oxidized at a mercury-based electrode. When the metal species is complexed by NTA and EDTA, its redox characteristics are changed from those of the uncomplexed metal species. Thus NTA (25,26), EDTA, and related compounds (27) can be indirectly determined with high-sensitivities. On the other hand, EDTA and its analogues are oxidizable electrochemically at platinum and carbon electrodes (28-31). However, it appears that no effort has been made to use the electrochemical (EC) detection technique in liquid chromatography (LC-EC) for the direct detection of this group of compounds. In this work a carbon-paste electrode is used as the LC-EC detector a t which the aminopolycarboxylic acids, including NTA and EDTA, are amperometrically oxidized and consequently determined. An acidic mobile phase is used both to maximize hydrophobicity of the analytes and to destablize interactions between the ligands and cations present in the sample. Separation is then accomplished on a reversed-phase column. Our purpose is to develop an LC-EC method for aminopolycarboxylic acids in general and for EDTA and NTA in particular, with special emphasis on potential environmental applications. EXPERIMENTAL SECTION Apparatus. The liquid chromatograph is a BAS (Bioanalytical systems; West Lafayette, IN) LC-303 system with an LC-3 amperometric detector. The electrode material, carbon paste, was homemade with paraffin oil and a special grade graphite powder, UCP-1-M, from Ultra Carbon (Bay City, MI). Procedures of fabricating carbon-paste electrodes are as described by Cheng et al. (32). This electrode has geometric area of ca. 0.03 cm2. Reference electrodes of Ag/AgCl in 3 M KC1 (BAS RE-1) were used, and potentials mentioned in this paper are measured against this reference. Two analytical columns were used in this work column 1 is a Biophase ODS column (5 pm, 250 X 4.6 mm) from BAS; column 2 is a PRP-1 poly(styrene-divinylbenzene)column (10 pm, 250 X 4.1 mm) from Hamilton (Reno, NV). Cyclic voltammetry was performed on a PAR 173 polarographic analyzer from Princeton Applied Research, EG&G (Princeton, NJ), with a Houston Instruments XY recorder. Reagents and Chemicals. NTA and the disodium salt of EDTA were obtained from J. T. Baker Chemical Co.; 1,2-diaminocyclohexane-N,N,”,”-tetraacetic acid (DCTA) was from Sigma Chemical Co. and diethylenetriaminepentaacetic acid (DTPA) from Alfa Products. They were used without further purification. DL-Amino acids were purchased from Sigma. High-purity (99.8%) trichloroacetic acid was a product of J. T. Baker and all other chemicals used were of reagent grade. Distilled water used for preparing mobile phases and samples was run through a Millipore Milli-Q water system and then through a membrane filter of 0.2-rm pore size. Crystalline solids of NTA, EDTA, DTPA, organic acids, and amino acids were dissolved in Milli-Q water to prepare stock solutions. Standards of NTA, EDTA, and DTPA were diluted from the stock solutions for daily use. Mobile-Phase Preparation. To prepare a mobile phase with pH lower than 2.5, trichloroacetic acid was dissolved in water in sufficient quantity to give the desired pH. This solution was then filtered again and degassed under vacuum. For mobile phases above pH 2.5, mixtures of acetic acid and HCl were used and the resulting solutions were also filtered and degassed. Trichloroacetic acid, acetate, and phosphate salts were added to water to prepare solutions of pH 1-9 for cyclic voltammetry tests.
I
T
I r A
I 1
-
c0
0.5
1.0
Potential V vs Ag AgCl
Cyclic voltammetry of NTA and EDTA: 0.10 mM NTA and EDTA solutions at pH 4.0; carbon-paste electrode; scan rate = 100 mVls: dashed line indicates blank response. Figure 1.
Mobile-phase flow was maintained at 0.8 or 1.0 mL/min. Samples of 50 pL were injected via a filled loop. RESULTS AND DISCUSSION Cyclic Voltammetry. The cyclic voltammetry of NTA, EDTA, DTPA, and DCTA was performed in buffered solutions with pH from 1 to 9. Typical cyclic voltammograms of EDTA and NTA at a carbon-paste electrode are shown in Figure 1. The oxidation waves at high potentials are characteristically irreversible. Similar behavior is found at a glassy-carbon electrode. As described by Petak and Vydra (30),the oxidation waves depend strongly upon solution pH. EDTA yields a well-defined, reproducible, single wave at pHs below 4.0. Above this pH a second wave appears at higher potentials in the voltammogram. For NTA the single voltammetric wave is reproducible throughout the pH range (1-9). In acidic media (below pH 3) both DTPA and DCTA yield multiple anodic waves. The first waves appear a t approximately the same potentials as those of EDTA and NTA, with half-wave potentials ranging from 0.86 to 0.91 V at pH 4.0 and 0.97 to 1.04 V at pH 1.9. Johnson et al. proposed a reaction mechanism for EDTA oxidation on Pt (31) in which formation of a free radical leading to decarboxylation was suggested to be the primary electron-transfer process. According to the proposed reaction scheme all aminoacetic acids are candidates for a similar electrochemical oxidation. However, when glycine and iminodiacetic acid were studied with cyclic voltammetry, we found no voltammetric waves within the potential (-0.2 to 1.5 V) and pH (1-3) ranges investigated. For our purposes, the absence of a voltammetric response is an advantage, because biogenic amino acids could be a source of serious interference if they were electrochemically active. Additional experiments confirm that the carbon-paste electrode detector selectively responds only to NTA- and EDTA-type compounds (see later discussion). Chromatograpy. A chromatogram of NTA, EDTA, and DTPA is shown in Figure 2. Capacity factors are 0.52, 2.80, and 4.10, respectively. DCTA does not elute from the column when the mobile-phase pH is below 2.5. Further discussion will not include DCTA, since most of the chromatography was done in mobile phases of pH 2 or below to avoid metal-ligand complexation. In Figure 3 chromatographic peak heights are plotted against the detector potential. It can be seen that the sensitivity of the detector increases substantially with the increase in potential. However, at potentials beyond 1.35 V the base line or residual current becomes excessively high. In practice, we have usually set the detector potential between 1.10 and 1.30 V.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988 8.0
3
,
5 0
,
303 1.0
\
\
2
*
0
100
140
180
1 2 80
2 20
0
300
340
0
380
PH
Figure 4. EDTA retention time (+) vs mobile-phase pH. Solid line represents equilibrium fraction (a)of uncharged EDTA (H,Y), computed by using K, values from ref 36; retention obtained on column 1.
I
0
5
10
I
15
T I M E , min
Flgure 2. Chromatogram of 50 pM NTA (l),50 p M EDTA (2), and 50 pM DTPA (3). Column 2 conditions were as follows: mobile-phase pH, 1.58; flow rate, 1.0 mL/min; detector potential, 1.20 V.
i
/ J/
I
Detector Potential, V vs. Ag/AgCI
Figure 3. Sensitivity vs detector potential: (+) 10 pM NTA, (A)20 and (X) 50 pM DTPA; column 1 conditions, mobilaphase pH, 1.70.
pM EDTA,
The retention of the three analytes is strongly influenced by the mobile-phase pH; the retention times decrease with increasing pH from 1.5 to 3.0. The retention t i m e p H profiles resemble the plots of the equilibrium fractions of the respective uncharged species as functions of pH (Figure 4), suggesting that the uncharged species are primarily responsible for the retention by the hydrophobic stationary phase. For EDTA, the mobile-phase pH can be as high as 3.0 without serious loss of retention, but the practical pH range extends only up to 2.0, above which the detector performance (reproducibility, sensitivity) deteriorates significantly. For NTA, the capacity factor is only 0.40 a t pH 1.8, and it decreases rapidly with increasing pH. The retention time of DTPA decreases similarly with increasing pH. More importantly, the DTPA detection limit increases with pH. Above pH 1.8, the detection limit deteriorates to approximately 100 pM. In routine analysis of natural water samples, we have used only mobile phases of pH 1.5-1.8 to obtain maximum retention for NTA and EDTA, because there are some nonretained, electroactive compounds from which the analytes must be separated (see Figure 5 and related discussion). The lower limit of the mobile-phase pH is set by the stability of the chemically bonded silica-based stationary phase.
It is claimed that degradation of the alkyl-bonded phase occurs below pH 1.5 and above pH 8 (33). Snyder and Kirkland (34) advise against use of mobile phases with pH lower than 2. In this work, no significant deterioration in column performance, judged by retention times and peak widths, was seen in the course of 4 months when the Biophase ODS column was subject to daily use of mobile phases of pH 1.5-2.0. However, the pressure needed to maintain a constant flow rate gradually increased. Recent development (33)of the chemically more inert macroporous poly(styrene-divinylbenzene) phases (PRP-1)allows mobile-phase pH as low as 1 to be used with reversed-phase columns. Our experience with the Hamilton PRP-1 column indicates that it satisfactorily withstands the use of strongly acidic mobile phases. Precision and Sensitivity. Calibration curves (peak height vs concentration) for all three compounds are linear throughout the concentration range (0-100 pM)studied. At 10 pM, standard deviations from multiple (six to eight) injections are 3-4%. From the formula given by Stojek and Osteryoung (27),the detection limits were calculated to be 0.5 pM (5 X M), or 0.10, 0.15, and 2.0 ppm for NTA, EDTA, and DTPA, respectively, under the conditions specified in Figure 3. As can be expected, the detector sensitivity depends strongly on the mobile-phase pH. Above pH 1.8 the detector sensitivity at a given potential decreases significantly with pH for EDTA and DTPA. No effort was made to study the effect of mobile-phase pH on detector response above pH 3.0. Method Specificity. In the determination of NTA and/or EDTA in aqueous samples with gas chromatography, several naturally occurring compounds, such as iminodiacetic acid, citric acid, and oxalic acid, were reported to interfere (10, 16, 35). In this work, 1.0 mM solutions of iminodiacetic acid, citric acid, glycine, gluconic acid, and oxalic acid were tested with cyclic voltametry; none exhibited voltammetric waves or peaks from 0.0 to 1.4 V. These solutions, along with 1.0 mM solutions of eight common amino acids (alanine, aspartic acid, glutamic acid, leucine, serine, phenylalanine, lysine, and valine) were injected into the chromatograph. The carbon-paste electrode detector was found not to respond to any of the above compounds at mobile-phase pH 1.5-1.8. We also prepared a 10 mg/L solution of fulvic acids, which had been extracted from the water of a pond in an oak forest by a published method (36); no detector response was observed after injection of the fulvic acid solution. These results indicate that the electrochemical detector is highly selective to NTA and EDTA type compounds (tertiary aminopolyacetic acids). NTA and EDTA can be detected without potential interference from biogenic amino acids and miscellaneous organic acids that are known to be present in natural waters and wastewaters.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
~~~
~
Table I. Effects of Metal Ions on the Detection of NTA and EDTA" original peakb NTA
metal
[M"+l,r M
MgZc or Ca2+ cuzc
0 100 1000 0 10 50 100 1000 0 10 50 100 900 0 100 1000 0 10 20 50 100 150 200 500 1000 0 10 50 100 900
Fe3+
EDTA Mg2+ or Ca2+ cu2+
Fe3'
t,
At
zp
Table 11. Masking of Cu(I1) and Fe(II1) by DTPA in Determining EDTA"
second peakb At I*
t,
3.22 0.39 27 NP' 3.21 0.39 26 3.21 0.40 25 NP 3.22 0.39 27 3.21 0.38 28 3.23 0.39 28 3.23 0.39 26 3.24 0.40 27 NP 3.22 0.39 28 3.20 0.41 19 3.15 0.52 15 3.14 0.53 14 3.14 0.55 10 NP 7.84 0.65 21 7.81 0.62 21 7.85 0.65 20 NP 7.84 0.65 21 7.78 0.76 18 NP 7.70 0.82 15 NP 7.70 overlapping 7.00 overlapping 7.70 2 6.20 0.78 12 7.70 2 5.43 0.54 16 1 5.28 0.42 24 7.70 5.08 0.36 32 NP 4.99 0.30 36 NP 7.84 0.65 2 1 NP 7.84 0.61 10
NP NP NP
Samples: 20 pM solutions of NTA and EDTA in Milli-Q water. Conditions: column 2; mobile-phase pH, 1.62; flow rate, 1.0 mL/ min; injection volume, 50 pL; detector potential, 1.20 V. * t , = retention time in minites; At = half-peak width in minutes (t, and At can be measured to i0.03 min). I , = peak height in nanoamperes. 'NP = no peak.
Effect of Metal Ions. Because NTA and EDTA are strong chelators, metal cations present in the samples conceivably could alter the separation and detection properties of these compounds. Four cations, Ca(II), Mg(II), Cu(II), and Fe(III), were selected for study. The first two of these were selected because they are the principal cations associated with aminopolyacetic acids in natural waters (5,37),and the last two were selected because they form particularly strong complexes. Samples containing 20 pM NTA or EDTA with variable amounts of each of the cations were prepared and the resulting chromatograms were obtained. The experimental data are summarized in Table I. Note that the highest concentrations tested exceed those usually found in natural waters. Martin and Meybeck (38) give the average dissolved river water concentrations as follows: Ca, 360 pM; Mg, 160 pM; Cu, 0.36 pM; Fe, 0.72 pM. Wastewaters may contain higher concentrations, e.g. 3 pM Cu and 20 pM Fe (39, 40). No change was found in the chromatographic peaks of NTA and EDTA at concentration levels of Ca(I1) or Mg(I1) up to 1000 pM. Therefore Ca(I1) and Mg(I1) are not likely to interfere in the determination of the analyks in natural waters. Addition of Cu(I1) up to 1000 pM changes the NTA peak negligibly. The presence of Fe(II1) causes a slight fronting in this peak. Nonetheless, the peak area remains useful for quantitation a t Fe(II1) concentrations up to 1000 pM. The EDTA peak is modified by both Cu(I1) and Fe(II1). This peak, a t 7.8 min, is not affected by an equal molar or less Cu(I1) concentration, but an excess of Cu(I1) causes the appearance of a second peak a t 5.1 min. With increasing Cu(II), the new peak grows at the expense of the original peak, and at a molar ratio of approximately 2 5 1 the original EDTA
Cu(I1) Fe(II1)
EDTA Deak height, nA 50 pM DTPA added
[MI pM
no DTPA
0.0 5.0 10.0 0.0 2.5 5.0 10.0 20.0
5.2 f 0.5 two peaks
two peaks 5.2 f 0.5 3.0 i 0.5 no peak no peak no peak
5.2 f 0.5 5.2 f 0.5 (one peak) 5.1 f 0.5 (one peak) 5.2 0.5 4.8 f 0.5 4.7 f 0.4 3.0 f 0.3 2.4 f 0.3
*
"Conditions: samples: 5 pM EDTA in Milli-Q water. Column 2: mobile-phase pH, 1.65; flow rate 1.0 mL/min; injection volume 50 pL; detector potential 1.20 V; t r , ~ d T A= 7.37 min; tr,pppA = 10.06 min. Table 111. Recovery of Spiked NTA and EDTA in Natural Waters and Wastewaters" sample water source raw sewage'
treated effluent'
fresh riverd estuarine' mixed estuarine'
amt added
amt recovered
RSD,* %
20.0 NTA 100 NTA 5.0 EDTA 20.0 EDTA 20.0 NTA 100 NTA 5.0 EDTA 20.0 EDTA 20.0 NTA 20.0 EDTA 20.0 NTA 20.0 EDTA 5.0 NTA 20.0 EDTA 100 EDTA
18.0 103 3.5 17.8 20.4 104 4.9 21.7 21.1 19.9 17.0 17.4 4.2 20.1 96.8
3 3 6 2 4 2 2 3 3 3 7 3 4
All concentrations in micromolars. * RSD = relative standard deviation from three replicate measurements. Fort Meade Wastewater Treatment Plant, Fort Meade, MD. Susquehanna River at Port Deposit, MD. e Chesapeake Bay, MD. IBack River at Baltimore, MD; 1 km from estuarine outfall of a secondary wastewater treatment plant. See ref 42 for detailed description of Back River environment.
peak completely disappears. In contrast to the behavior with Cu(II), Fe(II1) causes suppression of the EDTA peak. The peak is reduced in proportion to the amount of Fe(II1) added and disappears when [ Fe(111)] exceeds [EDTA]. In many samples interferences from dissolved cations will be negligible owing to low concentrations of the cations. In extreme cases, where dissolved cations interfere, DTPA can be used to suppress the interference. Table I1 shows the results of experiments in which DTPA was added to solutions containing EDTA and Fe(II1) or Cu(I1). As can be seen, the EDTA peak was largely restored. At higher Fe(II1) concentrations, the release of EDTA by the stronger chelating agent, DTPA, is incomplete but reproducible. Further increasing the concentration of DTPA releases EDTA more completely, but above 50 pM the DTPA peak begins to interfere with the EDTA peak. In routine use of this method for EDTA, we recommend that samples be run both with and without DTPA spikes in order to detect possible metal ion interferences. NTA and EDTA in Natural Waters and Wastewaters. In order to explore the possibility of unanticipated interferences in natural waters, samples from several sources (Table 111) were collected for analysis with the proposed LC-EC method. With the exception of the river water, which was refrigerated before analysis, all the samples were analyzed on the same day when they were collected. All samples were filtered (0.2 pm) prior to injection into the chromatograph.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
T I
2
0
the EDTA peak, is affected. To reduce the sulfide peak, we added reagent grade hydrogen peroxide (H202)to samples containing sulfides and NTA. When a 1.0 mM excess of HzOz was added, the sulfide peak was decreased to noninterfering levels.
lOnA
1
5
1
10
305
I5
T I M E , min
Flgure 5. Chromatogram of 10 pM NTA (1) and 10 pM EDTA (2) In the effluent from a wastewater treatment plant. Column 2 conditions were as follows: mobile-phase pH, 1.58; detector potential, 1.20 V. Sample was not treated with hydrogen peroxide.
Filtering not only protects the column from fouling but also eliminates potentially interfering cations that might be released from particles in the acidic mobile phase. Tests of NTA and EDTA recovery show that there is no significant loss due to filtering. Neither NTA nor EDTA was found in any of the samples in Table I11 above the detection limit of the method (5 X lo-’ M). This was expected because detergents containing NTA are currently not marketed in the sampled areas. In spikerecovery experiments, small amounts of NTA and EDTA were added to both the raw sewage and the treated effluent of a wastewater treatment plant. After filtering, the spiked wastewater samples were analyzed by using the LC-EC method. Shown in Figure 5 is one of the chromatograms obtained. This chromatogram illustrates the excellent selectivity of the electrochemical detector. Only one extraneous peak not associated with one of the analytes is observed. Similar recovery testa were conducted in various river waters and estuarine seawater. The results are presented in Table 111. Reproducibility, indicated by the standard deviations in the right-hand column, was excellent. The recoveries were mostly between 85% and loo%, but only 70% in one case. Standards for these experiments were prepared in Milli-Q deionized water. Low recovery appears to be associated with smaller analyte concentrations. The worst recovery (70%) occurred in a raw sewage matrix. In this case recovery may have been reduced by partitioning of the analytes onto suspended particles. Such problems could be controlled by the use of the standard addition method of calibration, definitely an advisable approach when analyzing very complex samples, such as wastewaters. As can be seen in Figure 5, there is a positive detector response to some materials that are present in the natural water and wastewater samples and are not retained or are weakly retained by the column. Since NTA elutes early from the column, the peak from these nonretained materials may cause difficulty in NTA quantitation. Generally, this potentially interfering peak was found to be smaller in the treated effluent samples than in the raw sewage samples and was found to be much smaller in surface estaurine and river water samples. In the case of the estuarine and river waters, determination of micromolar NTA was completely unaffected by this peak. We investigated a few compounds as the possible source of the interfering peak. We believe that hydrogen sulfide (H,S) could be responsible, at least partially, for the peak, especially in the raw sewage. At concentrations over 0.1 mM a sulfide peak tails so strongly that the NTA peak, and even
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RECEIVED for review December 10,1986. Resubmitted June 17, 1987. Accepted September 24, 1987.
