Simultaneous determination of Ca, Cu, Ni, Zn and Cd binding strengths with fulvic acid fractions by Schubert’s method

Analytica Chimica Acta 402 (1999) 169–181

Simultaneous determination of Ca, Cu, Ni, Zn and Cd binding strengths with fulvic acid fractions by Schubert’s method G.K. Brown a,∗ , P. MacCarthy b , J.A. Leenheer c b

a U.S. Geological Survey, 3215 Marine St., Boulder, CO 80303, USA Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401, USA c U.S. Geological Survey, Arvada, CO 80002, USA

Received 1 March 1999; received in revised form 1 July 1999; accepted 7 July 1999

Abstract The equilibrium binding of Ca2+ , Ni2+ , Cd2+ , Cu2+ and Zn2+ with unfractionated Suwannee river fulvic acid (SRFA) and an enhanced metal binding subfraction of SRFA was measured using Schubert’s ion-exchange method at pH 6.0 and at an ionic strength (µ) of 0.1 (NaNO3 ). The fractionation and subfractionation were directed towards obtaining an isolate with an elevated metal binding capacity or binding strength as estimated by Cu2+ potentiometry (ISE). Fractions were obtained by stepwise eluting an XAD-8 column loaded with SRFA with water eluents of pH 1.0 to pH 12.0. Subfractions were obtained by loading the fraction eluted from XAD-8 at pH 5.0 onto a silica gel column and eluting with solvents of increasing polarity. Schuberts ion exchange method was rigorously tested by measuring simultaneously the conditional stability constants (K) of citric acid complexed with the five metals at pH 3.5 and 6.0. The log K of SRFA with Ca2+ , Ni2+ , Cd2+ , Cu2+ and Zn2+ determined simultaneously at pH 6.0 follow the sequence of Cu2+ > Cd2+ > Ni2+ > Zn2+ > Ca2+ while all log K values increased for the enhanced metal binding subfraction and followed a different sequence of Cu2+ > Cd2+ > Ca2+ > Ni2+ > Zn2+ . Both fulvic acid samples and citric acid exhibited a 1 : 1 metal to ligand stochiometry under the relatively low metal loading conditions used here. Quantitative 13 C nuclear magnetic resonance spectroscopy showed increases in aromaticity and ketone content and decreases in aliphatic carbon for the elevated metal binding fraction while the carboxyl carbon, and elemental nitrogen, phosphorus, and sulfur content did not change. The more polar, elevated metal binding fraction did show a significant increase in molecular weight over the unfractionated SRFA. ©1999 Elsevier Science B.V. All rights reserved. Keywords: SRFA; Schubert’s ion-exchange method; Metal binding

1. Introduction The binding or complexing of metal ions by fulvic acid in natural waters and soils are an important factor in metal toxicity, bioavailability and transport. Complexation can drastically change the biolog∗ Corresponding author. Tel.: +1-303-541-3010; fax: +1-303-4472505 E-mail address: [email protected] (G.K. Brown)

ical and physiochemical properties of the trace metal species [1,2,3]. Due to the ubiquitous nature of fulvic acids in soils and water, they are important natural complexing agents of metal ions. The molecular functional groups responsible for the observed metal binding have been the subject of many studies and several functional group assemblages have been suggested including citric acid [4,5], weakly acidic enols [6], malonate groups [7], salicylic acid [8], and aromatic o-dicarboxylic acids [9]. Despite this work, there is

0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 5 3 0 - 9

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still a poor understanding of the nature of metal binding due to the possibility of many functional groups being present at once. It is unclear what functional groups or functional group assemblages are responsible for strong metal binding or if they are common throughout fulvic acid molecules. The study of fulvic acid–metal interactions in natural systems is complicated by the heterogeneous nature of the fulvic acid molecules and the competitive effects of multiple metals which are typically present. One method of decreasing the heterogeneity of the fulvic acid material is to fractionate it, thereby grouping molecules having similar polarities, hyphobicities, functional group content, etc. In this experiment, we have fractionated and subfractionated a fulvic acid, selecting for the subfraction that exhibits elevated Cu2+ metal binding. We then measured and compared; (1) the metal binding conditional stability constants (K) for five metals (Ca2+ , Ni2+ , Cd2+ , Cu2+ , Zn2+ ), (2) carbon functional group type, (3) the elemental composition (S, N, P, O, H and C), and (4) the molecular weight, of both the high metal binding subfraction and the unfractionated fulvic acid starting material. A first goal of this work was to fractionate a fulvic acid by differences in hydrogen bonding and polar functional group content on XAD-8 resin by eluting with water of increasing pH (pH gradient fractionation). To further decrease the heterogeneity, a subfractionation was performed on the XAD-8 pH gradient fractions that exhibited elevated Cu2+ binding strengths as determined by copper ion selective electrode potentiometry (ISE). This second fractionation was performed on silica gel, eluting with organic solvents of increasing polarity resulting in fractionation by molecule polarity. The relative binding strengths of the silica gel fractions were also measured by Cu2+ ISE and the fraction (PH5-SGAP) eluted with a 25 : 75 (v/v) mixture of acetonitrile : propanol showed the highest binding strengths. This elevated metal binding subfraction (PH5-SGAP) and the unfractionated fulvic acid (SRFA) were selected for conditional stability constant determinations by Schuberts method. The experimental pH chosen for both Cu2+ ISE and Schuberts method was 6.0 which is a typical environmental pH, but still low enough to avoid metal oxide or hydroxide formation [10].

Schubert’s ion-exchange method has been used for conditional stability constant (K) determinations since its conception in the late 1940s [11]. Under the proper experimental conditions, it not only yields log K values but also the metal to ligand ratios of the complexes are formed. Before any stability constant determinations on the fulvic acid materials were made, Schuberts method was rigorously proved using the well characterized ligand citric acid with the five metals at both pH 3.5 and 6.0 and compared to calculated log K values. Stability constants were then determined and compared for both the unfractionated (SRFA) and the highly fractionated (PH5-SGAP) fulvic acid samples at pH 6.0. Finally, comparisons were made between SRFA and PH5-SGAP for elemental N, S, P, O, H and C content determined by combustion methods [12], as well as functional group C type determined by liquid state 13 C nuclear magnetic resonance (NMR) spectroscopy. In related studies, 13 C-NMR, 1 H-NMR, and FT-IR spectrometry, elemental, titrimetric and molecular weight determinations were made to derive a structural model for fraction PH5-SGAP [13]. Conditional stability constants were also determined from the Cu2+ ISE titration data for all fractions and subfractions as well as standard compounds [14].

1.1. Theoretical Schubert’s method has been used in metal-humate log K determinations for many divalent metals as well as some trivalent metals [15–21] and has been shown to be applicable to simultaneous, three-metal determinations [22]. Schubert’s ion-exchange method for determining log K values involves measuring the distribution coefficients (λ, and λ0 ) of a metal ion between a cation-exchange resin and solution phase, in both the presence and absence of a complexing agent such as fulvic or citric acid. A typical metal binding determination was performed by holding the amount of resin and ligand constant while increasing the amount of metal added. The use of Schubert’s ion-exchange method for the determination of conditional stability constants is applicable for all stoichiometries, but when the complex is mononuclear with respect to the metal, and the slope of the properly plotted data is an integer value, then the intercept will be equal to the log conditional stability constant. For the system to

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work properly, the following conditions must be met [11,15,23,24]: 1. A constant ionic strength is maintained by a large excess of bulk electrolyte. 2. The total concentration of the metal cations is negligible compared to the concentration of the complex-forming ligand. 3. The pH of solutions must be constant. 4. The cation exchange resin must be previously saturated with the cation component of the bulk electrolyte. 5. There is no absorption on the resin of ligand or ligand–metal complexes. 6. The experiment is carried out on the linear portion of the ion-exchange isotherm by having a small metal concentration compared to resin exchange sites and ligand concentration. 7. The equilibration temperature of the systems must be constant. Under these conditions, the distribution coefficient, λ0 , between the resin and solution phases for the metal ion Mn + , in the absence of a complexing agent, is defined by the equilibrium ratio: λ0 =

[Mr ] [M]

(1)

where [M] is the free metal ion concentration in solution and [Mr ] is the concentration of metal on the resin, reported here as the number of millimoles of metal per gram of resin (corrected for moisture). The slope of the plot of [Mr ] versus [M] at different loadings of metal, Mn + , will give λ0 . In the presence of a complexing agent in solution, the distribution coefficient λ, is defined by the equilibrium ratio: λ=

[Mr ] [Mc ] + [M]

(2)

where [Mc ] is the concentration of complexed metal ion in solution, reported here as moles of metal per liter of solution. Combining Eqs. (1) and (2) gives: [Mc ] m[Mm Ln ] λ0 −1= = λ [M] [M]

(3)

where m is the number of metal ions combined per complexed molecule, and n is the number of ligands (or binding sites), L, per complexed molecule. By

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measuring the quantity, [M] + [Mc ], for the equilibrium determination of λ0 , [Mr ] is calculated by difference. When the complexes in solution conform to a discrete model (for instance a 1 : 1 or 1 : 2 ratio of ligand to metal), it is then (and in other special cases) possible to further interpret the distribution data yielding conditional stability constants [24]. If the system consists of a single binary polynuclear metal–ligand complex: mM + nL ⇔ Mm Ln

(4)

where Mm Ln is the only complex species that forms, T , is the overall thermodynamic stability constant, βmn defined as: aM L [Mm Ln ] γM L T = mm nn = × mm nn (5) βmn aM aL [M]m [L]n γM γL where ax and γ x are the activity and activity coefficient of species x, respectively. The charges on species are ignored for convenience. Since all experiments are carried out at constant pH, ionic strength and temperature, the activity coefficients of the species remain essentially constant, so that, γMm Ln (6) m γ n = constant (C) γM L Rearranging Eq. (5) and substituting Eq. (6), we have βmn =

[Mm Ln ] βT = mn m n [M] [L] C

(7)

where β mn is the apparent or conditional stability constant. Substituting from Eq. (3) yields: λ0 − 1 = mβmn [M]m−1 [L]n (8) λ Taking the logarithm of both sides:   λ0 − 1 = log m + log βmn + (m − 1)log[M] log λ +n log[L] (9) When a single mononuclear complex (m = 1) is present, Eq. (9) simplifies to:   λ0 (10) − 1 = log βmn + n log[L] log λ Eq. (10) is the basic equation used to determine conditional stability constants if the complexes

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are mononuclear as pointed out by several authors [15,25,26]. But the ratio of complexed metal to free metal ions in the solution for which λ is measured is always given by [(λ0 /λ) − 1] regardless of the number or nature of the complexes formed in solution [23].

2. Materials and methods 2.1. Fulvic acid fractionation The fulvic acid fractions were obtained from a two-step, tandem fractionation scheme applied to a quantity of unfractionated fulvic acid from the Suwannee river, GA [27,28]. The first fractionation, pH gradient, was performed by sorbing 40 g of fulvic acid on XAD-8 resin and sequentially eluting with deionized water of increasing pH from 1.0 to 13.0, generating 19 fractions. A stepwise pH increase of 0.5 from pH 1.0 to 6.0 and a 1.0 pH increase up to pH 13.0 was used. A sub-fractionation was performed on the XAD-8 pH gradient fraction eluted at a pH of 5.0 using silica gel and eluting with organic solvents of increasing polarity. A final cleanup was performed on all fractions and sub-fractions by acidification and re-adsorption on XAD-8 resin, followed by passage through a macroporous cation exchange resin (AG MP-50, 20–50 mesh, hydrogen form) previously cleaned and hydrogen saturated with 500 ml of 10% HClO4 (J.T. Baker 1 ). The fractions were then freeze-dried in the acid form. The Cu2+ , Cd2+ , Ni2+ , Zn2+ and Ca2+ metal content of both fulvic acid samples was determined by ICP-AES, and was found to be below detection limits. Other studies of Suwanee river fulvic acid also report very low metal concentrations [29]. 2.2. Standardization of NaOH titrants A 1 l volume of 0.0223 M NaOH was prepared from reagent grade NaOH and standardized against potassium hydrogen phthalate (KHP) in triplicate. The standardized NaOH solution was stored at 4◦ C, in a polyethylene bottle, and under nitrogen. This solution 1 The use of trade names is for information only and does not constitute endorsement by the U.S. Geological Survey.

was used to standardize a citric acid solution as well as for standardizing aqueous metal Stock I solutions.

2.3. Aqueous metal standard solutions Aqueous standard solutions of each metal ion were prepared from each metal’s nitrate salt. All metal salts used were reagent grade and were from Mallinckrodt except for Ca(NO3 )2 which was from J.T. Baker. The individual metal solutions were standardized in triplicate by means of proton exchange on AG-MP50 analytical grade macroporous cation exchange resin that was hydrogen saturated with 3 N HCl and rinsed with DI water [30]. Each divalent metal solution was then passed through the resin, followed by a DI rinse. The effluent and rinse (containing two protons per divalent metal ion) were collected, pH titrated with NaOH, and the metal ion concentrations calculated. The five Stock I metal solutions were then stored in Teflon FEP containers. A mixed, five metal solution was prepared and used as the metal ion source for all instrumentation standards as well as for the metal ion source in both the isotherm solutions, and citric or fulvic acid equilibrium exchange solutions.

2.4. Purification and standardization of citric acid Initial attempts at a mass balance cross-check in the measurement of distribution coefficients (λ0 ) between the metals and the cation exchange resin indicated a significant outside source of calcium and to a lesser extent copper and zinc. The sources of the low level metal contamination were from the citric acid, the filter paper, the nitric acid, and probably the sodium nitrate. The contamination problems were solved by switching to Ultrex II HNO3 (J.T. Baker, Lot J06543, Phillipsburg, NJ, 08865), Suprapure NaNO3 (EM Science, Lot 34355, Gibbstown, NJ, 08027), and by substituting 50 ml Gooch filtering crucibles with fritted disks (40–60 ␮m) for filter paper. The citric acid, made from reagent grade citric acid monohydrate (J.T. Baker), was cleaned at a pH of 1.95 by cation exchange using AG-MP50 resin. The cleaned citric acid solution was standardized in triplicate by titrating with the previously standardized NaOH solution.

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2.5. Inductively coupled plasma atomic emission spectroscopy Measurement of free or complexed + free metal concentrations of the filtrate sample solutions were performed on a Perkin Elmer Optima 3000 inductively coupled plasma atomic emission spectrometer (ICP-AES). A five point standardization curve, including a blank, was used to calibrate the instrument prior to analysis and the instrument was recalibrated approximately every 20 samples. Approximately every fifth sample, a calibration standard was analyzed to monitor the analysis results of the ICP-AES. The calibration standards were also acidified with Ultrex nitric acid to a concentration equivalent to that in the samples. The detection limits were determined [31] to be 21, 1, 0.4, 6 and 50 ng/l for Ca, Cd, Cu, Ni and Zn, respectively. A background matrix interference problem was observed with citric acid containing samples, which was overcome by spiking the standards and blanks with citric acid. 2.6. Fulvic acid characterization Quantitative 13 C NMR spectra of the fulvic acid samples were obtained on a Varian XL-300 NMR spectrometer using operating conditions previously described [32]. The samples (about 200 mg) were dissolved as potassium salts in 2 ml of D2 O at pH 8 using l M KOH. The elemental carbon, hydrogen, oxygen, phosphorus, nitrogen and sulfur determinations (ash corrected, dried sample basis) were performed by Huffman Laboratories, Wheat Ridge, CO, with methods optimized for fulvic acids [12]. The molecular weight determinations, also performed at Huffman Labs, were made by vapor pressure osmometry in tetrahydrofuran. 2.7. Schuberts experiment exchange resin The cation-exchange resin used in Schuberts log K determinations was AG 50W-X8, Analytical Grade resin, 100–200 mesh, hydrogen form, from Bio-Rad Laboratories (Richmond, CA.), having an exchange capacity of 4.9 milliequivalents per dry gram. About 250 g of the resin was prepared by transferring to a glass column, and sequentially rinsing with 2 l of

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deionized water, 2 l of 2 M HCl, 2 l of 2 M NaOH and finally 2 l of deionized water. The resin was converted to the sodium form by passing 2 l of 2 M NaNO3 , and rinsed with 2 l of deionized water. The cleaned resin was air dried to a constant weight, and stored in an air-tight polyethylene container. The percent moisture was determined at 100◦ C for 3 h. 2.8. Establishing metal/ion-exchange isotherms (␭0 ) Two different ion-exchange isotherms were measured, one at pH 3.5 and the other at pH 6.0. The pH 3.5 ion-exchange isotherm was necessary to compare citric acid complexation results against published data as a test of experimental and analytical procedures. The pH 6.0 isotherm was used for all fulvic acid metal complexation studies as well as one experimental measurement of citric acid complexation. The amount of resin used always represented at least a 1000 times excess of exchange capacity over the amount of total metal used to assure compliance with Schubert’s experimental conditions. For each determination, a series of five 200.0 ml volumes of mixed metal solutions were prepared in acid rinsed (dilute nitric acid) polypropylene volumetric flasks as follows. Varying amounts of the mixed metal Stock II solution were added to the volumetric flasks along with 20.0 ml of a 1.0 M Suprapure NaNO3 solution. The volumes were brought up to approximately 185 ml and the pH was adjusted to either 3.5 or 6.0 with the addition of 0.1 N NaOH or 0.1 N Ultrex HNO3 . After the pH of the solutions were adjusted, the volumes were brought up to 200.0 ml with deionized water. The sample sets were prepared by adding accurately weighed, 1 gm weights of the cleaned, Na+ saturated, AG 50W-X8 cation exchange resin to 250 ml polycarbonate centrifuge tubes. Volumes of 50.0 ml of the prepared mixed metal solutions were added to the polycarbonate containers, and shaken on a shaker table for 3 h. The samples were heat insulated from the shaker table by adding 1 in. of Styrofoam to the bottom. The samples were removed from the shaker table and the exchange resin was removed by filtration through acid rinsed, 50 ml, Gooch fritted crucibles. The supernatant of each sample was collected in 100 ml acid rinsed, high density polyethylene

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bottles, acidified to pH 2 with nitric acid, and stored at 4◦ C until analysis by ICP-AES. 2.9. Determination of conditional stability constants of metal–ligand complexes in a mixed-metal system The ion-exchange procedure used to determine stability constants for ligands and divalent cations was similar to the procedures used to establish the distribution coefficients (λ0 ) between mixed-metal cations and AG 50W-X8 resin. Here, though, the amount of metal was held constant while the amount of ligand is increased. For a typical series of ligand ion-exchange experiments, five 200 ml volumes of mixed metal solutions were prepared in acid-rinsed polypropylene volumetric flasks as follows: 4.0 ml of the mixed metal solution, varying amounts of the ligand solution and 20.0 ml of a 1.0 M Suprapure NaNO3 solution were added to the 200.0 ml volumetric flasks. The volumes were brought up to approximately 185 ml and the pH was adjusted to the target pH and the volumes were brought up to 200.0 ml. 50 ml volumes of the multimetal plus ligand solutions were then added to 250 ml polycarbonate centrifuge tubes containing accurately weighed, 1 gm weights of the cleaned, Na+ saturated, AG 50W-X8 cation exchange resin. The sample sets were shook for 3 h, and filtered, collecting the filtrate in polyethylene bottles and acidifying to pH 2 with HNO3 . Zhang [22], under similar experimental conditions, showed equilibrium was reached well before 2 h, while others [16,17,20,33] have shown that equilibrium conditions were reached within an hour. The filtered solutions were then analyzed by ICP-AES.

Fig. 1. Triplicate isotherms (λ0 ) of Ca2+ in a five metal system at pH 3.5 and ionic strength of 0.10 (NaNO3 ) showing the limits of linearity in stability constant determinations. Isotherms of Cu2+ , Zn2+ , Ni2+ and Cd2+ metals displayed similar features. The total free metal in solution for any stability constant determination never exceeded 7.5 × 10−6 M.

the upper limits of linearity of λ0 for total Ca2+ , Ni2+ , Cd2+ , Cu2+ , or Zn2+ were performed at pH 3.5. The initial isotherm for Ca2+ is shown in Fig. 1and indicates that the maximum concentration of metal which still allows for operation within the linear region of the plot is approximately 1.3 × 10−5 M. The total amount (sum) of free metal in solution for any stability constant determination reported here never exceeded 7.5 × 10−6 M. The unit of mmole/g was adopted in all calculations for the concentration of metal sorbed on the resin. Keeping well below these estimates of the upper limits of linearity of λ0 , three complete, independent measurements of ␭0 at pH 3.5 and 6.0 in a mixed metal system were made. The isotherms for Ca2+ are shown in Figs. 2 and 3. Table 1 summarizes the mean results of three individual determinations of λ0 for the five metals at pH 3.5 and 6.0. 3.2. Mass balance cross-checks

3. Results and discussion 3.1. Measurement of distribution coefficients, ␭0 , at pH 3.5 and 6.0 for AG 50W-X8 Resin The distribution coefficients, λ0 , between AG 50W-X8 resin and solution phases for a five-metal system are defined as the slope of the line regressed through an isotherm plotted as [MR ] on the ordinate versus [M] on the abscissa. Initial measurements of

A series of isotherm experiments were performed to assure that there were no losses of metal from solution due to adsorption on labware used, or additions of metal from outside sources. The five metals in solution were equilibrated with the AG 50W-X8 resin as usual and then filter separated. The metal on the resin was then desorbed using 3.0 N HNO3 . The metal in the supernatant and that desorbed from the resin was analyzed by ICP-AES. Triplicate recovery results were greater than 97% for all five metals.

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Table 1 Experimentally determined distribution coefficients (λ0 ) of metal ions at pH 3.5 and 6.0 on AG 50W-X8 resina Metal ion pH Ionic strength λ0

Fig. 2. Triplicate isotherms (λ0 ) of Ca2+ in a five metal system at pH 3.5 and ionic strength of 0.10 (NaNO3 ). The resin used is AG 50W-X8. Isotherms for Cu2+ , Zn2+ , Ni2+ , and Cd2+ metals displayed similar features. Mean R2 coefficients of all metals are all better that 0.997. Slopes are equal to λ0 (Table 1) and were used in all pH 3.5 stability constant determinations (see Eq. (10)).

s

Intercept 3.1 × 10−4

s

Ni2+ Cu2+ Zn2+ Cd2+

3.5 3.5 3.5 3.5 3.5

0.1 0.1 0.1 0.1 0.1

2843 1411 1405 1336 1933

80 28 27 30 29

2.4 × 10−4 1.4 × 10−4 1.6 × 10−4 2.3 × 10−4

1.7 × 10−4 3.8 × 10−5 5.4 × 10−5 8.7 × 10−5 3.2 × 10−5

Ca2+ Ni2+ Cu2+ Zn2+ Cd2+

6.0 6.0 6.0 6.0 6.0

0.1 0.1 0.1 0.1 0.1

2848 1499 1430 1421 2053

60 3.4 × 10−4 8 5.7 × 10−5 10 5.7 × 10−5 30 6.9 × 10−7 43 −8.5 × 10−6

9.4 × 10−5 6.3 × 10−5 1.2 × 10−4 6.9 × 10−6 3.0 × 10−5

Ca2+

a All values are a mean of three individual determinations in a mixed metal system. One standard deviation values (s) are given for λ0 and the intercept.

Table 2 Log K (intercepts) and metal-to-ligand ratios (slopes) for five metals in a mixed-metal/citric acid system at pH 3.5 and 6.0 with µ = 0.10a

Fig. 3. Triplicate isotherms (λ0 ) of Ca2+ in a five metal system at pH 6.0 and ionic strength of 0.10 (NaNO3 ). The resin used is AG 50W-X8. Isotherms for Cu2+ , Zn2+ , Ni2+ , and Cd2+ metals displayed similar features. Mean R2 coefficients of all metals are all better that 0.998. Slopes are equal to λ0 (Table 1) and were used in all pH 6.0 stability constant determinations (see Eq. (10)).

3.3. Calculation of log conditional stability constants of metal–citric acid complexes at pH 3.5 and 6.0 Citric acid was chosen as the standard ligand material for testing Schubert’s method. The log conditional stability constants for organic ligands at a given pH are not normally available but they can be approximated using Eq. (11) [34]. log KApparent = log(α1 β1 + α2 β2 + α3 β3 + α4 β4 + · · · + αi βi )

(11)

Here, α i is the relative amount of each solute species present at a particular pH, ionic strength, and temper-

Metal ion

pH

Slope

Log K measured

Mean R2

log K Calculated

Ca2+ Ni2+ Cu2+ Zn2+ Cd2+

3.5 3.5 3.5 3.5 3.5

0.796b 0.963b 0.998b 1.009b 0.915b

1.34b 2.51b 3.04b 2.30b 1.59b

0.977 0.974 0.980 0.978 0.978

1.34 2.59 2.93 2.22 1.42

Ca2+ Ni2+ Cu2+ Zn2+ Cd2+

6.0 6.0 6.0 6.0 6.0

1.095c 0.991c 1.217c 0.978c 1.032c

3.66c 5.80c 6.55c 5.20c 3.76c

0.958 1.000 0.947 0.999 0.919

3.00 5.20 5.70 4.78 3.56

a Values are determined from Fig. 4a and b. The measured log K values at pH 3.5 and 6.0 are compared to calculated log K values of citric acid with each metal. b Value of a single determination. c Mean value of three individual determinations.

ature, and β i is the individual stability constant of the species present. The α values were calculated using pKa values obtained at 25◦ C and ionic strength (µ) of 0.1 from Martell and Smith [35]. Values for β i between citric acid and the metals Ca, Ni, Cu, Zn, and Cd, at 25◦ C and µ = 0.1 [35], were substituted into Eq. (11), yielding calculated citric acid log conditional stability constants which are summarized in Table 2.

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standard deviation and are not significantly different from one. Other authors have also reported that citric acid will form a predominately 1 : 1 stochiometry with the metals of interest here [4,5,23]. A comparison of the log K values determined by Schubert’s method versus our calculated values yield an average percent difference of 4.2 at pH 3.5 and 10.9 at pH 6.0. Based upon the closeness of fit of the Schubert’s log K values to the calculated log K values at both pH for citric acid, log K determinations were made on the unfractionated fulvic acid (SRFA) and it’s subfraction (PH5-SGAP). 3.5. Experimentally determined log K constants of metal–fulvic acid complexes in a mixed metal system at pH 6.0

3.4. Measured log K constants of metal–citric acid complexes at pH 3.5 and 6.0

Using the same methodology as that used for citric acid, the conditional stability constants and metal-to-ligand ratios for the two fulvic acid samples were determined. Figs. 5 and 6 show the plots of log (λ0 /l − 1) versus log LFulvic for Cu2+ , Ni2+ , Zn2+ , Cd2+ and Ca2+ in a mixed-metal/fulvic acid system for SRFA and fraction PH5-SGAP at pH 6.0 and an ionic strength of 0.1. Each plot is of three replications of the five point determinations under identical conditions. The log K values and metal : ligand ratios are given in Table 3. The slope value has significance only when it is an integer (or reasonably close to an integer) within

Eq. (10) was used to calculate the log K (y intercept) and the metal : ligand ratio (slope) of citric acid complexed with the five metals using Schubert’s ion-exchange method. Fig. 4a shows the experimental results of three individual determinations at pH 3.5. The resulting measurements of the log K values for citric acid by Schubert’s method at both pH 3.5 and 6.0 are summarized in Table 2 and are compared to the log K values calculated. The linearity of each determination for all metals is very high as evidenced by the high R2 values (multiple correlation coefficient squared). According to Eq. (10), the slopes of these plots give the composition of the complexes, which are all close to unity indicating a 1 : 1 metal : ligand ratio with the exception of Ca2+ at pH 3.5 (0.796) and Cu2+ at pH 6.0 (1.217). These slopes have a high

Fig. 5. Schuberts plots of log (λ0 /l − 1) vs. log LFulvic using Eq. (10) for five metals in a mixed-metal/unfractionated fulvic acid (SRFA) system at pH 6.0 and ionic strength of 0.10 from three individual determinations. Slopes are determined by four concentrations of ligand. The slopes represent the metal to ligand ratio and the Y-axis intercept represent log K.

Fig. 4. Schubert plots of the ratio of complexed metal to free metal (log(λ0 /l − 1)) vs. ligand concentration (log Lcitric ) for five metals in a mixed-metal/citric acid system at an ionic strength of 0.10 for three individual determinations; (a) pH 3.5 and (b) pH 6.0.

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Table 3 Comparison of conditional log K values and metal : ligand ratios from Schubert’s plots for five metals in a mixed-metal/fulvic acid system at pH 6.0 and ionic strength of 0.10a Metal ion

Sample

Metal : ligand ratio

Conditional log K

Mean R2

Log K increase

Ca2+ Ni2+ Cu2+ Zn2+ Cd2+

SRFA SRFA SRFA SRFA SRFA

1.159 0.984 1.095 0.957 0.964

3.02 3.80 5.24 3.77 4.42

0.991 0.996 0.995 1.000 0.996

– – – – –

Ca2+ Ni2+ Cu2+ Zn2+ Cd2+

PH5-SGAP PH5-SGAP PH5-SGAP PH5-SGAP PH5-SGAP

1.037 1.075 0.980 1.002 0.966

4.10 4.05 5.47 4.06 4.50

0.993 0.993 0.992 0.990 0.992

1.08 0.25 0.23 0.29 0.08

a Values are the mean from three individual determinations and the correlation coefficient, R2 , is the fit of the three individual determinations together.

Fig. 6. Schuberts plots of log (λ0 /l − 1) vs. log LFulvic using Eq. (10) for five metals in a mixed-metal/metal binding fraction (PH5-SGAP) system at pH 6.0 and ionic strength of 0.10 from three individual determinations. Slopes are determined by four concentrations of ligand. The slopes represent the metal to ligand ratio and the Y-axis intercept represent log K.

the limits of experimental error in measurement [11,23,24]. In all cases of fulvic acid binding in this work, there was a 1 :1 stoichiometric increase in the amount of metal bound with the increase in ligand concentration as measured in units of milliequivalents of acid per gram. A 1 : 1 ligand to metal stoichiometry has been reported with fulvic acids from a variety of sources [2,15,20,22]. The log K results of our Schubert’s ion-exchange analysis (Table 3), indicate that there is an increase in log K for all the metals for the stronger metal binding fraction PH5-SGAP over the unfractionated SRFA. This across-the-board increase could be associated with an increase in more site specific binding in the fraction PH5-SGAP due to an increase in homogene-

ity of the fraction, a change in the distribution of the type of metal binding sites, or an increase in the number of coordination sites per metal ion. Ca2+ showed the largest log K increase (1.08), while Cu2+ , Zn2+ , and Ni2+ exhibit similar log K increases (0.23, 0.29, and 0.25). For Ca2+ , the change represents a 10-fold increase in binding strength over the original material which we suspect is due to an enrichment of PH5-SGAP in carboxyl groups with ether or hydroxyl groups which are located ␣ to carboxyl groups. Evidence for ‘clustered’ carboxyl, hydroxyl, and ether functional groups comes from a comparison of 13 C-NMR and 1 H-NMR chemical shifts of the two fractions [13]. Carboxylic acids that are closely associated with α ether oxygen(s) have been shown to be especially good Ca2+ complexers [36,37]. It is reasonable to suspect that the higher polarity of PH5-SGAP is due to close associations of carboxylic acids or oxy-carboxylic acids forming a polyanion. A direct effect of carboxylic or oxy-carboxylic acid groups closely associated with each other (clustered) on a molecule, is the oligoelectrolyte effect, where enhancement of complexation is augmented by the long-range coulombic attraction generated by neighboring anionic sites. The enhanced charge associated with a polyelectrolyte can affect covalence and ionic bonding with more or less polarizable cations. Ca2+ , with no d-orbital electrons, is non-polarizable and non-polarizing and hence, low in contributing to covalent bonding. This effect increases the bond strength of Ca2+ with a polyanion through ionic bonding. The reverse effect can be seen with d-orbital metals

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(Cu2+ , Cd2+ , Ni2+ , and Zn2+ ) which are polarizable and polarizing due to increasing nuclear charge and availability of d electrons. The net effect of these bonding differences is to increase the log K for Ca2+ over the d-orbital metals and change the log K sequence from Cu2+ > Cd2+ > Ni2+ > Zn2+ > Ca2+ for SRFA to Cu2+ > Cd2+ > Ca2+ > Ni2+ > Zn2+ for the more polar subfraction, PH5-SGAP.

3.6. Comparison of measured log K constants with literature values The log K values found in the literature are determined over a wide of experimental conditions including pH, ionic strength, ligand concentration, metal loading, temperature, and methods of determination. These differences make comparisons of the log K values determined here difficult. Also confounding relative comparison of log K values is the wide array of fulvic acid materials used, each exhibiting their own distinct polyfunctional nature and metal binding characteristics. Some of the log K values found in the literature are determined at high metal concentrations so that the log K value given is an average value of a wide distribution of discrete log K values. Many authors report a decrease of log K as pH decreases, presumably due to competition with protons at acidic binding sites. Hart and Jones [38], using ISE copper potentiometry, determined log K values for natural organic ligands from Magela Creek system in Northern Australia to range from 5.7 to 5.8 at an ionic strength of 0.1 M KNO3 and pH 6.0. The ligand concentrations ranged from 93.4 to 58.5 ug/l and the copper additions were from 1.0 × 10−6 to 7.0 × 10−4 . Shuman and Woodward [2], using a procedure similar to amperometric titration, determined log K values of copper-organic chelates in several fresh water samples at controlled pH. The log K constants ranged from 4.5 to 5.7 at pH 6.5, with dissolved organic carbon concentrations from 14 to 39 mg/l. They found evidence of a 1 : 1 metal-to-ligand complexation ratio for all fresh water samples with their low copper loading experiments. Varney et al. [39], using copper ISE, reported a log K values for Cu2+ binding with aquatic fulvic acid of 6.63 at a pH of 6.22, ionic strength of 0.1 M NaClO4 and fulvic acid concentration of 29.1 mg/dm3 . Their metal binding data indicated a 1 : 1 ligand to

copper metal ratio under their experimental conditions. Schnitzer and Hansen [20], using Schubert’s ion-exchange equilibrium method, reported log K values of 3.3 and 4.0 for Cu2+ , 3.2 and 4.2 for Ni2+ , 2.7 and 3.3 for Ca2+ , and 2.2 and 3.6 for Zn2+ , respectively, at a pH of 3.0 and 5.0. The ligand was a fulvic acid originating from the Bh horizon of the Armadale podzol profile. Liu and Ingle [40], using a two-column ion-exchange method, reported log K values of 6.78 between copper and Willamette river fulvic acid at a pH of 6.8, using a single ligand model. Using a modified ion-exchange technique, Ardakani and Stevenson [41] determined log conditional stability constants of Zn2+ –humic acid complexes (five different humic acids) to range between 3.13 and 5.13 at a pH of 6.5 and an ionic strength of .25 N KCl. They found 1 : 1 relationships between ligand and metal. Meelu and Randhawa [18] also determined log conditional stability constants between five humic acids and Zn2+ at a pH of 6.0. They report a range of log K values between 2.67 and 6.61 using Schubert’s ion-exchange method with an ionic strength of 0.1 N NaCl. Zhang [22] using very similar conditions to those used here but at a significantly lower pH of 3.5, reported Schubert’s metal : ligand ratios of 1 : 1 for Cu2+ , Ni2+ and Zn2+ and log K values of 3.49, 3.25, and 2.94, respectively, for Suwannee river fulvic acid in a mixed-metal systems. McKnight and Wershaw [42] performed ion-selective potentiometric titration on Suwannee river fulvic acid and reported two binding sites for Cu2+ . Their first conditional stability constant for the first binding site had a log K of 5.9 at a pH of 6.0, ionic strength of 10−3 , and a fulvic acid concentration of 1.4 × 10−6 moles per milligram of carbon. 3.7. Comparison of carbon functional groups Carbon functional group information from 13 C NMR analysis on the two fulvic acids are given in Table 4 and show the effects of the fractionation schemes employed here, directed towards producing a strong metal binding fraction. Fraction (PH5-SGAP) shows a 5.5% increase in aromatic carbon (110–160 ppm) while the aliphatic carbon (0–62 ppm) decreased 7.4%. This is consistent with McKnight et al. [43] who found that as the aromatic content of fulvic acid

G.K. Brown et al. / Analytica Chimica Acta 402 (1999) 169–181 Table 4 Carbon distribution from fulvic acid samples

13 C

179

NMR quantitative spectra of unfractionated (SRFA) and fractionated, strong metal binding (PH5-SGAP)

Sample

% C-aliphatica 0–62 ppm

% C-heteroaliphaticb 62–90 ppm

% C-anomericc 90–110 ppm

% C-aromaticd 110–160 ppm

% C-carboxyle 160–190 ppm

% C-ketonef 190–220 ppm

SRFA PH5-SGAP

33.6 26.2

10.1 9.7

5.3 6.5

23.3 28.8

22.2 22.0

5.4 6.7

7.4

0.4

1.2

5.5

0.2

1.3

% change a

Aliphatic — methyl, methylene, and methine carbon (C–C, some C–N and C–S). Hetero-aliphatic — carbohydrate, ether, ester, alcohol carbon (C–O, some C–N). c Anomeric — aromatic, acetal, ketal anomeric carbon (hemiacetal carbohydrate). d Aromatic — olefinic and aromatic carbon. e Carboxyl — carboxylic acid and esters carbon. f Carbonyl — ketone, aldehydes and quinone carbon. b

Table 5 Elemental analysis and molecular weight for the enhanced metal binding fraction, PH5-SGAP and unfractionated SRFAa Elemental contents and molecular weight Carbon (% Mass) Hydrogen (% Mass) Oxygen (% Mass) Sulfur (% Mass) Phosphorus (% Mass) Nitrogen (% Mass) Molecular Weight (Daltons)

PH5-SGAP 54.1 4.9 41.8 0.6 <0.1 0.7 956

SRFA

Relative % difference

53.8 4.3 40.9 0.6 <0.1 0.7 781

0.6 13.0 2.2 0.0 0.0 0.0 20.1

a

These results are reported on a dried sample basis and corrected for ash content. Molecular weight data by vapor-pressure osmometry in tetrahydrofuran.

increased, the potential for metal binding increased. There is a smaller increases (1.3%) in ketone or quinone carbon for fraction PH5-SGAP (Thorn [44] showed that aldehydes are not present). Surprisingly, the carboxyl carbon (160–190 ppm) content for fraction PH5-SGAP remained the same as its source, SRFA.

3.8. Comparison of molecular weight The molecular weight of PH5-SGAP is 20.1% higher (relative % difference) than SRFA. Higher molecular weight fractions of Suwannee river humic acid have been shown to have a higher apparent surface potential than lower molecular weight fractions (oligoelectrolyte effects) [45]. Assuming similar surface potential characteristics for SRFA, the enhancement in metal binding (especially for Ca2+ ) observed with PH5-SGAP could be a response to a higher surface potential.

3.9. Comparisons of elemental makeup Table 5 gives C, H, O, S, P, and N% mass values for both SRFA and PH5-SGAP samples along with relative % differences. Only hydrogen content shows a significant difference between fractions, and is probably related to the shift in aromatic carbon to aliphatic carbon functional group content (Table 4) seen in PH5-SGAP. Of particular interest is the lack of change in elemental N, S and P. A survey of critical stability constant data of nitrogen, sulfur and phosphorus containing compounds indicate there are functional group assemblages containing these elements that can have very high log K values with metals.

4. Conclusions The applicability of Schubert’s ion exchange method to a five metal system was tested. The method generated log K values very close to calculated log K

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values for a citric acid ligand at two different pHs with excellent reproducibility. The metal to citric acid ratio was found to be 1 : 1 in determinations at both pH 3.5 and 6.0. This result agrees with observations in the literature. Log K determinations at pH 6.0 for a highly fractionated, strong metal binding fulvic acid (PH5-SGAP) and the unfractionated fulvic acid starting material (SRFA) showed log K increases between 0.2 and 0.3 log units for Cu2+ , Ni2+ , and Zn2+ , while Ca2+ showed the largest log K increase of 1.08 log units for PH5-SGAP. Cd2+ showed a small increase in log K of about 0.1. These values indicate that enhancement of metal binding in PH5-SGAP is more pronounced for metals not having d-orbitals. Both PH5-SGAP and SRFA demonstrated a 1 : 1 metal:ligand stochiometry under Schubert’s experimental conditions of low metal loading. A comparison of fulvic acid log K values determined here agreed with other published log K values determined for similar fulvic acid materials. Significant functional group and elemental make-up differences between the highly fractionated PH5-SGAP and its parent material, SRFA, were discovered. A comparison of quantitative 13 C NMR spectra show increases of 5.5% aromatic carbon and 1.3% ketone carbon for PH5-SGAP over SRFA. In contrast, aliphatic carbon in PH5-SGAP decreases by 7.4% compared to SRFA. The similar % carboxyl carbon (22.2 versus 22.0) in the elevated metal binding constant sample might be deceiving and incorrectly lead to conclusions that carboxyl carbon has no effect on metal binding. Rather, this could indicate a grouping of the carboxyl’s into a more polar arrangement of close proximity to each other. A significant increase in molecular weight for the PH5-SGAP fraction was found, while elemental nitrogen, sulfur, and phosphorus remained the same as before fractionation.

Acknowledgements The authors wish to thank Dr. Tom Wildeman and Dr. Patrick MacCarthy from the Colorado School of Mines for use of their ICP-AES equipment and expertise. We also thank Dr. Robert Wershaw (USGS) for his assistance in the acquirement of the NMR spectral data presented here.

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