Environ. Sci. Technol. 1997, 31, 2656-2664
Determination of EDTA, NTA, and Other Amino Carboxylic Acids and Their Co(II) and Co(III) Complexes by Capillary Electrophoresis CHRISTA S. BU ¨ RGISSER* AND ALAN T. STONE Department of Geography and Environmental Engineering, The Johns Hopkins University, Baltimore, Maryland 21218
EDTA and NTA were widely used in the processing of nuclear materials and in the production of nuclear energy. Their in-plant chemical transformations and post-disposal chemical and biological transformations generated many additional amino carboxylic acid compounds. Toxic and radioactive metals are frequent co-contaminants of these chelating agents. Some of the resulting metal ion-chelating agent complexes, most notably 60CoIIIEDTA, resist decomposition and adsorb poorly onto subsurface minerals, thereby facilitating migration. This study sought to separate and detect free amino carboxylic acids and their Co(II) and Co(III) complexes in a single run without prior pretreatment using capillary electrophoresis (CE). The electrolyte filling the CE capillary contained 25 mM phosphate buffer (pH 7). Equilibrium calculations performed for this electrolyte identified predominant cobalt species and helped explain the CE findings. Co(III) complexes are thermodynamically (and kinetically) more stable than corresponding Co(II) complexes and are readily separated and detected. Co(III) complexes of EDTA, NTA, and IDA were synthesized and analyzed. Two different diastereomers of CoIIIIDA2 were found and could be separated. Co(II)-amino carboxylate complexes range from ones that are not thermodynamically stable in the capillary and therefore not detectable to those that are highly stable in the capillary and yield good linearity.
Introduction Inorganic and organic ligands exert a profound effect on the speciation and biogeochemistry of naturally-occurring and contaminant-derived metal ions. Increasing ligand concentrations can shift the distribution of metal ions between dissolved, sorbed, and precipitated phases; can change the redox potential of metal ion redox couples, and can alter bioavailability and migration rates of the metal ions. The synthetic multidentate chelating agent EDTA (ethylenediaminetetraacetic acid) has been used for recovering radioactive metals from contaminated surfaces in nuclear reactors. Mixed wastes containing both 60Co and EDTA from such cleaning activities were disposed in shallow landfills (1-3). CoIIEDTA adsorbs onto aluminum and iron (hydr)oxides (4-6). The extremely stable CoIIIEDTA complex adsorbs poorly onto subsurface minerals, resulting in facilitated transport of radioactive 60Co (7, 8). In order to evaluate * Corresponding author present address: Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-8600 Du ¨ bendorf, Switzerland; fax: +41-1-823-5028; e-mail address:
[email protected].
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the potential for subsurface migration, it is important to detect and analytically distinguish CoIIEDTA and CoIIIEDTA. In addition to EDTA, commercial nuclear wastes contain a complex mixture of hydrophilic compounds generated by in-plant chemical transformations (9). Amino carboxylic acids like the ones included in this study have been found in such mixtures. EDTA and NTA (nitrilotriacetic acid) have also been extensively used in a wide variety of industrial, pharmaceutical, and agricultural applications. Other amino carboxylic acids can accumulate as breakdown products of EDTA and NTA as a result of biological activity (10), thermal degradation at elevated temperatures (11, 12), and photodegradation (13). Technical-grade EDTA contains a wide variety of impurities, some of them present in significant concentrations, which are also present whenever EDTA is used. Capillary electrophoresis (CE) is a highly efficient technique for the separation and determination of both organic and inorganic cations and anions (14-16). In CE, analytes migrate through a capillary immersed in a electrolyte solution on applying high voltage. The electrophoretic mobility is a function of the ion charge of the analyte and its hydrodynamic radius. The advantages of CE compared to high-performance liquid chromatography (HPLC) or ion chromatography (IC) are its higher efficiencies, simpler chemistry, faster separation time, ease of automation, and smaller sample and reagent requirements. One of the disadvantages of CE is it’s relatively low sensitivity. With UV/VIS detection, CE sensitivity is in the milligram per liter range. Recently, papers have been published on the analysis of CoII and CoIIIEDTA with IC (17) and a column/oxidation separation methodology followed by atomic absorption spectrophotometric measurement (18), yielding detection limits of 10-5-10-6 M. Nowack at al. (19) presented a HPLC method for the determination of various metal-EDTA complexes down to concentrations of 10-8 M, where EDTA was complexed with Fe(III) and detected as a FeIIIEDTA complex. EDTA and NTA can be measured by GC in concentrations down to 10-9 M (20, 21). All these methods are rather specific and time-consuming, and the free EDTA or other amino carboxylic acids as well as their metal complexes cannot be detected using the same analytical method. Few reports are available regarding the separation of aliphatic amino carboxylic acids with CE. Methods have been developed for amino acids (22-24) and carboxylic acids such as citrate, oxalate, ascorbate, and lactate (25-29). Degradation products of NTA in a desulfurization process have been determined (30) and preliminary studies of the enhanced detection of EDTA through the addition of heavy metals have been reported (31). By coupling CE with metal complex formation, the separation and detection limits for metal ion analysis have been enhanced (32). The use of EDTA as a chelating agent in CE has been reported for the determination of alkali and alkaline-earth metal ions (33, 34) and of Cr(III), Fe(III), Cu(II), Co(II), Ni(II), and Pb(II) (35, 36). Speciation investigations through the simultaneous detection of free chelating agents and complexes or separation of complexes in different metal oxidation states to date have not been published. This work is concerned with the speciation of solutions containing EDTA, NTA and related amino carboxylic acids, and the metal ions Co(II) and Co(III). The development of an analytical method is an important first step, which will allow us in proceeding work to evaluate the potential for forming dissolved complexes via ligand-assisted and reductive dissolution of Co(III)-containing mineral surfaces. CE is used to separate different free and cobalt-bound amino carboxylic acids prior to detection by direct UV spectrophotometry. CE
S0013-936X(97)00080-1 CCC: $14.00
1997 American Chemical Society
TABLE 1. Amino Carboxylic Acids with Their Abbreviations Used in This Study, in Order of Their Migration Times, and Their Speciation Calculated for CE Analysis Condition amino carboxylic acid (HnL)
log K (max first three)a
abbrev
oxalic acid (H2L) formic acid (HL) nitrilotriacetic acid (H3L) ethylenediaminetetraacetic acid (H4L)
OX FOR NTA EDTA
4.27, 1.25 3.75 10.13, 2.94, 2.01 11.01, 6.32, 3.12
diethylenetriaminepentaacetic acid (H5L)
DTPA
11.55, 9.5, 4.92
acetic acid (HL) ethylenediaminetriacetic acid (H3L) glyoxylic acid (HL) N-(2-hydroxyethyl)ethylenediaminetriacetic acid (H3L)
AC ED3A GLYOX HEEDTA
4.76 3.46 10.3, 5.82, 2.88
iminodiacetic acid (H2L) methyliminodiacetic acid (H2L) N-(2-hydroxyethyl)iminodiacetic acid (H2L) ethylenediimino-N,N′-diacetic acid (H2L)
IDA MeIDA HEIDA EDDA
9.79, 2.84, 1.8 10.01, 2.59, 1.9 9.11, 2.4, 1.6 10.04, 6.76, 2.36
glycine (HL) ethylenediaminemonoacetic acid (HL)
GLY EDMA
9.78, 2.35 10.05, 6.67, 1.6
complexb L2LHL2HL3H2L2H2L3HL4LHL2- d LHL2H2LHLHLHLHLH2L0 HL0 HL0 H2L+
speciationb (%)
detection limit (µM)c
100 100 100 93 7 98 1 100
5 20 10 50
100 96 4 100 100 100 67 33 100 64 36
10 10 2 20 100 10 10 10 20 30 100
a Thermodynamic stability constants (K) at 25 °C and I ) 0 (44). b Speciation calculated with HYDRAQL (43) for 1 mM amino carboxylic acid at CE analysis conditions (25 mM PO43-, 40 mM Na+, pH ) 7). c Detection limits of amino carboxylic acids found in CE measurements (pH ) 7) at 185 nm. d Estimated predominant protonation form of ED3A at CE analysis conditions.
involves injecting the aqueous sample into a capillary electrolyte of dissimilar chemical composition. For this reason, it will be important to characterize the thermodynamic stability and kinetic lability (rate of ligand exchange) of metalligand complexes within the capillary electrolyte and to establish whether breakdown of metal-ligand complexes occurs within the time scale of measurement.
Materials and Methods Chemicals. The amino carboxylic acids employed in this study and the abbreviations used to refer to them are listed in Table 1; all are available as analytical reagent-grade free acids or sodium salts. Most of the acids were purchased from Aldrich. EDMA was purchased from TCI America, and OX was from Baker. ED3A was synthesized in Battelle Pacific Northwest Laboratories (Richland, WA). Solutions were prepared from distilled, deionized water (Millipore Corp., MA). Solutions of Co(II) complexes were prepared by mixing different quantities of cobalt(II) chloride hexahydrate (Baker) with the amino carboxylic acids and equilibrating for at least 1 day prior to analysis in CE to ensure formation of the Co(II) complexes in thermodynamic equilibrium. Solutions were prepared in water and 25 mM phosphate buffer solution (pH 7). A standard solution of CoIIIEDTA (Figure 1) was prepared from K[CoIIIEDTA]‚2H2O, which was synthesized according to the procedure of Dwyer et al. (37). The addition of H2O2 to a heated (90 °C) aqueous solution of CoIIEDTA and potassium acetate yields CoIIIEDTA; on cooling and addition of excess methanol, deep purple K[CoIIIEDTA]‚2H2O crystals precipitate. The crystals were purified with repeated methanol-water recrystallization, followed by a final drying at 70 °C. CoIIINTA was synthesized according to the procedure of Mori et al. (38). Two different forms of CoIIINTA are distinguished, an R- and a β-form (Figure 1). R-CoIIINTA converts into β-CoIIINTA by heating or acidifying with acetic acid. For the synthesis of R-CoIIINTA, cobalt(II) chloride hexahydrate was added to NTA in a potassium bicarbonate solution. Addition of H2O2 on an ice bath yields blue, fine precipitates of R-K[Co(NTA)(OH)(H2O)]‚2H2O. The product was purified through recrystallization in potassium acetate
FIGURE 1. Simplified representations of [CoIIIEDTA]- (in sexidentate coordination), geometrical isomers of [CoIIINTA(OH)]- (r- and β-isomers as proposed by Mori et al. (38)) and the three possible geometrical isomers of [CoIIIIDA2]- (u-fac-, mer- and s-fac-isomer). under short heating and washed with cold water and ethanol. The reddish β-CoIIINTA was obtained by acidifying a solution of R-CoIIINTA with acetic acid (in a 10 times higher concentration). Co(III) complexes with IDA were prepared according to Hidaka et al. (39). Two terdentate molecules of IDA can form an octahedral complex with Co(III). This complex provides three possible geometrical isomers: s-fac, mer, and u-fac (Figure 1). Hidaka et al. (39) concluded from absorption
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spectra and optical rotatory dispersion curves that their synthesis procedure yields the u-fac isomer (called cis(N) isomer by Hidaka et al. (39)) and the s-fac isomer (called trans(N) isomer). In basic solutions, u-fac is isomerized to the s-fac isomer (40). The mer isomer is not very stable in water and exists as a transient labile intermediate in the isomerization reaction of u-fac to s-fac. The mer isomer was not synthesized for this study. u-fac-CoIIIIDA2 was prepared by adding cobalt(II) chloride hexahydrate to a solution of IDA in sodium hydroxide. Taking this solution, adding H2O2 on an ice bath, and storing over night in a refrigerator yields reddish violet crystals of u-fac-K[Co(IDA)2]‚2.5H2O. The crystals were purified by repeated recrystallization from water, washed with cold water and ethanol, and dried in the air. s-fac-Co(III)IDA2 was prepared by adding cobalt(II) chloride hexahydrate to a solution of IDA in sodium hydroxide. After addition of H2O2 on a water bath of 80 °C and heating for 1 h, brown yellow needles of s-fac-K[Co(IDA)2]‚2H2O formed on cooling. The product was recrystallized from hot water, washed with ethanol, and dried at room temperature. Capillary Electrophoresis Analysis. The solutions were analyzed with the Quanta 4000E capillary electrophoresis instrument (Waters, Milford, MA). The mode applied was capillary zone electrophoresis. Bare fused silica capillaries of 75 µm inner diameter and 60 cm length to the detector (68 cm total length) were used (Polymicro Technologies, Phoenix, AZ). A solution of 25 mM phosphate buffer (pH 7, with a few measurements at pH 8) and 0.5 mM tetradecyl trimethylammonium bromide (TTAB, Aldrich) was prepared as carrier electrolyte. Electroosmotic flow is caused by the creep of the counterions along the charged wall of the capillary. Addition of TTAB to the electrolyte changes the direction of the electroosmotic flow in the capillary (25, 41). TTAB adsorbs at the silica wall of the capillary, making the wall charge positive and therefore reversing the direction of the electroosmotic flow. When using a negative power supply (defining the location of the anode on the detection side), electroosmotic flow generated a net fluid movement toward the detector, resulting in shorter migration times for the anions. The application of TTAB has another advantage in our analysis. Metal ions and free amino carboxylic acids would adsorb to the most reactive silanol sites if the sites were not already occupied by adsorbed TTAB. Samples were injected in hydrostatic mode at 10 cm for 30 s. A run current of 50 µA (which resulted in a run voltage of about 19 kV) with negative power supply at 25 °C constant temperature was chosen. The constant current method was found to give better reproducibility as compared to the constant voltage method (42). Joule heating is avoided at these CE analysis conditions. Between runs, the capillary was washed with 0.1 M potassium hydroxide, followed by distilled, deionized water to eliminate residual contaminants in the capillary. Direct UV photometric detection was used with 185 and 254 nm optical filters. A mercury lamp provides a high energy output at these wavelengths and allows operation at the low wavelength of 185 nm. Fixed-wavelength instruments with atomic vapor lamps offer a higher sensitivity for detection below 254 nm than variable-wavelength detectors (14). For the vast majority of compounds, absorption coefficients increase in the direction of lower wavelengths. Using 185 nm improves detectability for a large number of analytes. For detection at this low wavelength, solvent transparency is a major issue. Unlike HPLC and IC, the carrier electrolyte in CE is mostly water with much lower salt concentrations, and the detection path length is much shorter. Therefore, the solvent transparency is usually not an issue in CE. Since a great number of potential contaminants also absorb at 185 nm, the carrier electrolyte should be as pure as possible. Constituents in the sample other than the analytes are also detected at 185 nm. Fortunately, the good
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separation power of CE usually makes it possible to separate out the peaks caused by non-analytes. An alternative method for measuring compounds that do not absorb light above 200 nm is the indirect detection method. In this procedure, a compound capable of absorbing light and having the same charge as the analyte is added to the carrier electrolyte. As the analyte migrates through the capillary, it displaces the light-absorbing compound in the carrier electrolyte and yields a negative peak as it passes through the detector. This method was tested using the same analytical conditions but at 254 nm and with a solution of 5 mM chromate (Mallinckrodt; sulfuric acid added to pH ) 7.3) and 0.5 mM TTAB as carrier electrolyte. Results from the injection of 100 µM NTA, AC, IDA, HEIDA, and EDDA gave 50% less sensitivity, less efficient separation, and greater sensitivity to changes in sample medium composition as compared to the direct detection method at 185 nm with phosphate as carrier electrolyte. All the remaining experiments were therefore performed in direct detection mode. Peak identification for each analyte was carried out by spiking with the known standard. Because anions elute in order of decreasing charge to size ratios, relative migration times can be estimated by comparing charge to size ratios of the different analytes. Running the samples at different detection wavelengths and comparing the absorbance was found to be a very useful tool for the identification of unknown peaks. Quantification was done by calculating the ratio of peak area over migration time. This value is more accurate than peak area alone. For equal concentrations and detector response, peaks areas increase with longer migration time. Peak heights remain constant, but peaks get wider because the later eluting solutes move through the detector more slowly. The detection limit was defined as three times the signal to baseline noise ratio.
Results and Discussion HYDRAQL Speciation Calculations. Protonation level, molecular charge, and other attributes of each aqueous species affect migration rates during electrophoresis. Once a sample has been injected, analyte speciation can be altered by chemical reaction with the carrier electrolyte medium. For this reason, speciation calculations were performed using the equilibrium program HYDRAQL (43) under chemical conditions that might be encountered in the carrier electrolyte: 0.50 mM Co(II), 1.00 mM Cl-, 1.00 mM amino carboxylic acid ligand, 25.0 mM PO43-, and 40.0 mM Na+ at pH 7.0. For comparison, calculations were also performed at lower pH values. Thermodynamic stability constants were taken from Martell et al. (44) and were corrected to a ionic strength I ) 0, when necessary, using the Davies equation. Results from the speciation calculations are shown in Tables 1-3 and are discussed in the following sections. CE Analysis of Free Amino Carboxylic Acids. Free amino carboxylic acids do not absorb much light in the UV region above 200 nm. At 185 nm, however, they show good absorbance and can therefore be directly analyzed with CE. An electropherogram resulting from the injection of all 15 amino carboxylic acids used in this study is shown in Figure 2. The predominant protonation level of each free ligand, calculated using HYDRAQL for the CE conditions employed, are given in Table 1. All the acids except EDMA and GLY exist mainly in a anionic form at pH 7. FOR, AC, GLYOX, IDA, MeIDA, HEIDA, and EDDA all have the same negative charge of (-1) but can be nicely separated according to their different hydrodynamic radii. FOR is the smallest molecule and therefore eludes most rapidly. AC and ED3A exhibit the same migration time, so their peaks merge. OX, NTA, ED3A, and HEEDTA all carry a (-2) charge and elute relatively early (log K values for ED3A are not available, but its charge can be inferred from related amino carboxylic acids). OX eludes first because of its small molecular size. EDTA is detected
FIGURE 2. Electropherogram of 15 amino carboxylic acids (0.1 mM each) at 185 nm. CE electrolyte: 25 mM phosphate/0.5 mM TTAB (pH 7). 1, OX; 2, FOR; 3, NTA; 4, EDTA; 5, DTPA; 6, AC; 7, ED3A; 8, GLYOX; 9, HEEDTA; 10, IDA; 11, MeIDA; 12, HEIDA; 13, EDDA; 14, GLY (see inset); 15, EDMA (see inset); S, system peak. just after NTA. Despite its higher charge, the bigger molecular size causes it to migrate slower than NTA. EDMA and GLY are neutral species at pH 7. Due to electroosmotic flow, neutral species are also transported through the capillary, but only very slowly. Therefore, GLY and EDMA were detected as relatively broad peaks at late migration times. The detection limits of the acids obtained by the CE technique employed are listed in Table 1. Most of the amino carboxylic acids have a detection limit of 10-20 µM. EDTA has a slightly lower detection limit. EDTA causes the baseline to rise, thereby broadening the peak. This may be caused by EDTA adsorbing to the capillary wall more than the other amino carboxylic acids. HEEDTA appears in a broader and split peak. CE electropherograms collected with a phosphate carrier electrolyte buffer at pH 8 show no splitting of the peak, and the detection limit could be improved to 50 µM. At pH 8, nearly 100% of the complex is in the monoprotonated form, whereas at pH 7 only 96% is in this form (see speciation calculation in Table 1). The presence of the diprotonated form of HEEDTA at the lower pH might cause the peak to split. EDMA is transported through the capillary very slowly because it carries no charge at pH 7. Therefore, it appears as a broad peak with a lower detection limit. If other compounds are present in the sample solution in high concentrations, the amino carboxylic acids are more difficult to detect. For example, if the concentration of the background electrolyte exceeds 0.01 M, lower detection limits are achieved. For quantitative analysis, standards must therefore be made up in the solution matrix, or internal standards have to be used. Peaks of anions such as chloride, nitrate, or sulfate do not interfere directly with the measurements since they exhibit much lower migration times in CE analysis. Speciation Calculations for Co(II) Complexes with Amino Carboxylic Acids. Phosphate, which is used to maintain constant pH in the CE carrier electrolyte, forms relatively strong complexes with Co(II) and might be able to exchange with amino carboxylic acids complexed to Co(II). HYDRAQL calculations were performed in order to determine the equilibrium speciation of Co(II) within the CE carrier electrolyte. Table 2 shows the results of speciation calculations for 0.5 mM Co(II) (and 1.0 mM Cl-) in the carrier electrolyte medium
TABLE 2. Co(II) Speciation in Carrier Electrolyte at CE Analysis Conditions ligand
equationa
PO43- (L3-) HL/H‚L H2L/H2‚L H3L/H3‚L CoHL/Co‚H‚L NaL/Na‚L NaHL/Na‚H‚L NaH2L/Na‚H2‚L OH- (L-) HL/H‚L CoL‚H/Co CoL2‚H2/Co CoL3‚H3/Co CoL4‚H4/Co Co2L‚H/Co2 CoL2(s)‚H2/Co Cl- (L-) CoL/Co‚L
log Kb
complex
12.38 19.57 21.72 15.42 0.82 13.23 19.8 14.00 -9.7 -18.8 -31.5 -46.3 -11.0 -13.1 0.5
HPO42H2PO4H3PO40 CoHPO40 NaPO42NaHPO4NaH2PO40 H2O Co(OH)+ Co(OH)20 Co(OH)3Co(OH)42Co2(OH)3+ Co(OH)2(s) CoCl-
speciationc (%)
74
