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Cd NMR Binding Studies of Cd-Fulvic Acid Complexes: Evidence of Fast Exchange
C Y N T H I A K . L A R I V E , * ,† AISLING ROGERS,† MARTHA MORTON,† AND W . R O B E R T C A R P E R * ,‡ Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, and Department of Chemistry, Wichita State University, Wichita, Kansas 67260
The binding of Cd2+ to Suwannee River fulvic acid is studied using potentiometric methods and 113Cd NMR spectroscopy. The potentiometric results indicate 6.4 and 1.2 mequiv/g carboxylic and phenolic acid groups for the Suwannee River fulvic acid standard. At pH 6.4, the fraction of of the carboxylic acid groups that are deprotonated is 0.89. The observed 113Cd NMR chemical shift changes with constant Cd2+ concentration and varying fulvic acid concentration at pH 6.4 are consistent with the fast exchange model. Analysis of the 113Cd NMR chemical shift data using a Scatchard plot yields a conditional binding constant of 6.7 × 102 mL/mg. A number-average apparent molecular weight of 800 g/mol, gives a value of 0.9 for the fraction of the fulvic acid carboxylate sites that are bound to cadmium and a cadmium-fulvic acid complex formation constant of 5.4 × 102 M-1.
Introduction Humic substances play an important role in water quality since they are the major constituents of dissolved organic carbon. A detailed understanding of the cation binding properties of humic substances has particular importance for developing long-term solutions to the problems of waste storage and remediation as well as the more fundamental characterization of metal ion transport and bioavailability (1). The metal ion complexation properties of humic and fulvic acids are complex, because of both the heterogeneity of the samples and the polyelectrolyte nature of these mixtures. The predominate metal coordination sites in aquatic humic and fulvic acids are the carboxylate groups because of their prevalence. However, since humic samples have a high degree of variability in their composition, sulfur-, nitrogen-, and phosphorus-containing functional groups are also possible ligands, although they are generally less prevalent than the carboxylate moieties. Cadmium complexation by humic and fulvic acids has been examined primarily using ion-selective electrodes and * Authors to whom correspondence should be addressed; (C.K.L.) e-mail address
[email protected]. † University of Kansas. ‡ Wichita State University.
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by ultrafiltration (2-10). Fluorescence spectroscopy has also been used to characterize the interactions of fulvic acid with metal ions by measuring the quenching of the fulvic acid fluorescence as a function of the complexation of a paramagnetic metal ion such as Cu2+ (11, 12). However, this method is not amenable for the study of diamagnetic ions such as Cd2+. In this paper, we describe the results of an NMR study of the binding of 113Cd2+ to the International Humic Substance Society Suwannee River fulvic acid standard. 113Cd is an excellent metal ion probe due to its ability to form complexes with many different conformations and ligand numbers. Furthermore, the 113Cd chemical shift covers a wide range from 750 to -200 ppm, and the nature of the coordinating ligands and their coordination numbers are strongly reflected in the chemical shift (13, 14). In particular, ligands that bind through oxygen cause increased shielding of the Cd nucleus while ligands that bind through nitrogen produce deshielding of the Cd nucleus. Finally, ligands which bind via sulfur produce very large deshielding (downfield chemical shifts). Therefore, the 113Cd chemical shift in solutions of cadmium-containing coordination compounds is reflective of the directly bonded heteroatom of the ligand. Although 1H and 13C NMR have been used extensively for the characterization of humic and fulvic acids, NMR has not been widely used in the examination of metal ion complexation by naturally occurring organic matter. We have identified one report of the use of 113Cd NMR to study the binding of cadmium to two soil humic acid samples (15). In these experiments, varying amounts of soil humic acid were added to solutions containing equal amounts of Cd2+. The soil humic acid samples were much less soluble than the aquatic fulvic acid standard used in this study. The amount of cadmium complexed by the soil humic acids was determined by measuring the decrease in the amount of free 113Cd in solution using NMR spectroscopy. Only the free 113Cd2+ was detected in this study, and no evidence for chemical exchange between free and bound cadmium was detected since the bound cadmium was removed from solution by precipitation as the humic acid complex. The measured chemical shift value of 113Cd is dependent on the free and complexed chemical shifts as well as the exchange rate between free 113Cd and 113Cd in the fulvic acid complex. Under the conditions of fast exchange, one observes a single cadmium resonance with a chemical shift that is a statistical average of the chemical shift values for the free and complexed species (16). It is possible to add one complexing agent in excess until the chemical shift value remains essentially constant, at which point it is assumed that the final chemical shift value is due completely to the complex. This allows the evaluation of the binding site constant from the chemical shift data even for a sample as complex and heterogeneous as fulvic acids.
Experimental Section Chemicals and Solutions. The fulvic acid used in this research was a standard fulvic acid sample that was isolated from the Suwannee River, Georgia, obtained from the International Humic Substances Society (IHSS). The isolation and characterization of this material are described in
S0013-936X(96)00084-3 CCC: $12.00
1996 American Chemical Society
detail elsewhere (17). 113CdO (93.4% enriched) was purchased from Cambridge Isotopes. All solutions for NMR analysis were prepared in D2O (99.96 atom %, Sigma). Concentrated solutions of DCl and NaOD, purchased from Isotec, were diluted in D2O and used to adjust the solution pH of the NMR samples. For the pH titrations, potassium hydrogen phthalate (Fisher, certified ACS grade) was used to standardize the sodium hydroxide (Fisher, certified ACS grade) titrant solution. pH Titrations. The acidic nature of the fulvic acid sample was determined by pH titration. Since the acidity of fulvic acid is mostly due to the presence of carboxylic acid and phenolic functional groups, titration with NaOH allows the determination of the total number of these groups per milligram of sample available for complexation by cadmium metal ions. All solutions were prepared from boiled and cooled, distilled, deionized water to avoid any contribution to acidity from dissolved carbon dioxide. All titrations were carried out in triplicate under a controlled N2 atmosphere in a polyethylene glove bag, and the sample solutions were bubbled with N2 gas before analysis. Ionic strength effects were not controlled in these experiments, and activity effects were not taken into consideration. A combination pH electrode (Fisher 13-620-288) and a Fisher Acumet 10 pH meter were used for these studies. The electrode was calibrated daily using aqueous pH 1.00, 4.00, 7.00, and 10.00 buffers (Fisher). NaOH (0.0982 N) was delivered to the sample solution in 10-, 20-, and 50-µL aliquots. The autopipets used for titrant delivery were calibrated and, based on an average of 12 replicate measurements, were found to deliver 9.8, 24.4, and 48.8 µL of solution, respectively. Titrant volumes recorded were adjusted accordingly. The titrant was standardized in duplicate against the primary standard, potassium acid pthalate (KHP), immediately prior to each use using standard quantitative analytical techniques. The fulvic acid solutions were prepared by dissolving 10.0 mg in 2.0 mL of CO2-free water. A blank was prepared containing 2.0 mL of CO2-free water. The volumes of NaOH used in the titration of the sample were corrected by subtraction of the volume required to titrate the blank. Prior to titration, the pH of the sample was adjusted to 2 using 1 µL of 1 M HCl. No attempt was made to control or correct for ionic strength effects. Preparation of Samples. All 113Cd solutions were diluted from a 0.0875 M stock solution prepared by dissolving 113CdO in a small volume of in DCl (35%) and diluting the solution to 1000 µL using D2O. Samples containing 4.38 mM 113Cd2+ and fulvic acid in the range 2.25-50.0 mg/mL were prepared by mixing the required volumes of the cadmium and fulvic acid stock solutions and dilution to a total volume of 500 µL with D2O. All pH measurements in D2O solution were corrected for the deuterium isotope effect using the relationship pD ) pH meter reading + 0.4 (18). For simplicity, the corrected pD values for D2O solutions are referred to as pH throughout. The pH of each D2O solution was adjusted to 6.4 (i.e., a pH meter reading of 6.0) using concentrated NaOD. pH measurement of the samples prepared for NMR analysis were made using a 3-mm glass pH microelectrode (Ingold) and a Fisher Acumet 10 pH meter at ambient temperature. The electrode was calibrated daily using aqueous buffers as described above. NMR Studies. The NMR studies were carried out using a Bruker AM-500 MHz spectrometer, equipped with a broad
FIGURE 1. Potentiometric titration of Suwannee River fulvic acid sample. The solution pH vs the volume (in mL) of 0.0982 N NaOH added to the solution is plotted for three separate trials.
band multinuclear probe and operated at a spectral frequency of 110.94 Hz. Chemical shifts are reported relative to 1 M Cd(ClO4)2 used as an external reference. The spin-lattice relaxation time, T1, of the Cd(ClO4)2 reference solution was 65 s measured using the standard inversion recovery method. The relaxation times of the fulvic acidcomplexed Cd are much less than this value (approximately 1 s) but were not precisely determined due to the low concentrations and low signal-to-noise of the spectra obtained for these solutions. Typically, 8 000-20 000 scans were recorded using a 3-µs (40°) pulse and a 0.85-s repetition time. The data files were transferred to a Silicon Graphics Indigo workstation and processed using Felix 2.30 (Biosym). The cadmium free induction decays were apodized by multiplication with a decaying exponential equivalent to 50-Hz line broadening. Baseline distortions were corrected by fitting selected baseline points to a fifth order polynomial.
Results Determination of the Number of Titratable Functional Groups. Because fulvic acids are by nature heterogeneous samples, apparent equilibrium constants calculated using ligand concentrations in units such as molarity are not really meaningful. An alternative approach is to determine the total number of ionizable functional groups, such as carboxylate groups per mass unit of the ligand (19). The results of the potentiometric pH titrations of the Suwannee River fulvic acid standard are shown in Figure 1. The total number of carboxylate groups per milligram of sample was determined by subtracting the volume of NaOH required to achieve a sample pH of 8 from the total volume of NaOH added in the titration (carboxylate + phenolic acid groups). Our results are 6.4 mequiv/g of carboxylic acid groups and 1.2 mequiv/g of phenolic acid groups for the Suwannee
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TABLE 1 113Cd NMR
Chemical Shift Measurements of Cd-Fulvic Acid Complexes fulvic acid (mg/mL)
δobs (ppm)
Cd (bound)
Cd (free)
2.25 3.0 7.5 10 15 50
25.36 18.72 0.06 -2.66 -7.43 -11.90
1.65 2.08 3.41 3.61 3.94 4.25
2.73 2.30 0.97 0.77 0.44 0.13
River fulvic acid standard. These results are in good agreement with a previous report of 6.1 mequiv/g of carboxylic acid groups and 1.2 mequiv/g of phenolic acid groups for this standard (20). At pH 6.4, the fraction of the carboxylic acid groups (RCOO-) that is deprotonated is 0.89, decreasing to 0.82 at pH 6.0. Metal Ion-Fulvic Acid Binding Constants. The ability of 113Cd NMR to give widely separate chemical shifts for various types of functional groups will enable the simultaneous determination of metal ion complex formation constants for different binding sites. In typical fast exchange NMR studies of equilibrium constants, the observed chemical shift, δobsd, represents an average of the free chemical shift, δF, and the bound (metal ion complex) chemical shift, δC, (16). For a n:1 complex, the observed chemical shift, δobsd, is a weighted average of δF and δC as follows:
δobsd ) [Cdbound]δC/[Cdtotal] + [Cdfree]δF/[Cdtotal]
(1)
If the total Cd concentration is held constant and the fulvic acid concentration is varied, then
K ) [Cdbound]/[Cdfree][nFo - Cdbound]
(2)
where Fo refers to the total fulvic acid concentration and n is the number of cadmium binding sites per fulvic acid molecule. Equation 2 can be rearranged into a Scatchard equation
[Cdbound]/[Cdfree][Fo] ) nK - K[Cdbound]/[Fo]
(3)
so that a plot of [Cdbound]/[Cdfree][Fo] vs [Cdbound]/[Fo] will yield K and n from the slope (-K) and intercept (nK), respectively. In this study, the total cadmium concentration, Cdtotal, was maintained at 4.38 mM throughout while varying the fulvic acid concentration from 2.25 to 50 mg/mL at pH 6.4. The observed chemical shifts relative to Cd(ClO4)2 at 25 °C are given in Table 1. By definition from eq 1, as the concentration of the fulvic acid approaches infinity (while the Cd concentration is constant), the observed chemical shift is due only to that of the pure complex (16). As a result, a plot of observed chemical shift vs reciprocal fulvic acid concentration yields the chemical shift of the pure complex at a value of 1/[fulvic acid] ) 0, the y intercept. A plot of measured chemical shift vs reciprocal fulvic acid concentration gives an intercept of -13.6 ppm for the chemical shift of the “pure” complex. In a secondary plot, the observed chemical shift was fitted to a polynomial expansion of reciprocal fulvic acid concentration, yielding a value of 48.3 ppm for the chemical shift of free 113Cd. These chemical shift values were then substituted into eq 4 to calculate values for bound and free Cd as shown in
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FIGURE 2. Scatchard plot of the data in Table 1. The apparent binding constant and number of binding sites can be determined from the slope and intercept of the line. The cadmium concentration was held constant at 4.38 mM while the fulvic acid concentration was varied from 2.25 to 50 mM.
Table 1. A Scatchard plot of the calculated values (Figure 2) gave n ) 1.1 and K ) 6.7 × 102 mL/mg for Suwannee River fulvic acid at pH 6.4.
[Cdbound] ) [Cdtotal](δCdfree - δobs)/(δCdfree - δCdcomplex) (4) Carboxylic Acid Binding Sites. The upfield chemical shift of the pure complex (-13.6 ppm) indicates that Cd is binding to carboxyl groups (13, 14), matching our results from the potentiometric titrations. In support of this interpretation is the observation that the strong acid characteristics (pKa e 3.0) of Suwannee River fulvic acid can be separated into 57% aliphatic carboxyl groups and 43% keto acid and aromatic carboxyl group structures (21). In particular, an important fraction of carboxyl groups in Suwannee River fulvic acid includes cyclic aliphatic R-ester and R-ether structures (22). Finally, carboxylate groups are the primary binding sites for Ca2+ and Cd2+ in peat humic acid (23) and in aqueous fulvic acid at pH 6.0 (24), similar to our observations with Suwannee River aqueous fulvic acid in this report. Concentration of Binding Sites. Our potentiometric titrations (Figure 1) indicate that there are approximately 6.4 mequiv of carboxyl groups/g of fulvic acid. Using a number-average molecular weight of approximately 800 g/mol for the Suwannee River standard material, there are on average 4-5 carboxylic acid ligands per fulvic acid molecule (25). At pH 6.4, the effective number of deprotonated carboxylate binding sites should be approximately four per fulvic acid molecule. However, considering the large variation in component molecular weights for this fulvic acid, a number of different binding motiffs are possible. The value of 1.1 for n, determined from the
Scatchard plot, is the number of moles of Cd2+ binding sites per 1000 g of fulvic acid. Using the number-average molecular weight of approximately 800 g/mol, this translates into 0.9 for the fraction of the fulvic acid carboxylate sites that are bound to cadmium. Although there are four-five carboxylate groups per fulvic acid molecule, under the conditions of our experiments, on average, a 1:1 cadmiumfulvic acid complex is formed. Metal Ion Stability Constants and Multiple Binding Sites. Studies of metal ion complexation by humic acids in estuaries has only recently been a subject of systematic investigations, and these are primarily concerned with Cu2+. In an early study (26), the coordination of copper(II) ions in soil and water fulvic acids was investigated, and two classes of binding sites were identified with stability constants of 1 × 106 and 8 × 103 over the pH range of 4-6 (25). Luminescence spectroscopy was used to investigate metal binding sites on fulvic acid obtained from air-dried Okchun Metamorphic belt (Korea) topsoil samples. Both 1:1 and 1:2 carboxylate moieties were identified using deconvoluted 7Fo f 5Do excitation spectra of Eu(III). The weaker binding species was quite abundant and showed a rapid increase from pH 2.9 to pH 6.3 (27). The apparent Cd-fulvic acid complex formation constant of 6.7 × 102 mL/mg obtained at pH 6.4 is reasonable in view of weaker stability constants obtained for Cd2+ vs Cu2+ reported previously (24). We do not observe a second Cd2+ binding site as determined by NMR in this sample; however, this is possibly due to the limited Cd2+ conccentration range used in this study (24, 27). Fast Exchange. Finally, there is clear evidence that fast exchange exists relative to the NMR time scale. The change in observed chemical shift with changing concentration is clear evidence for such exchange and contrasts sharply with 113Cd studies of binding to proteins (28, 29) and in soil humic acids (15) where there is little if any evidence for such exchange. The fact that such exchange occurs in fulvic acids suggests that a competitive binding study with other metals will yield additional equilibrium constants.
Acknowledgments This work was supported by NSF Grants CHE-9524514 (C.K.L.) and CHE-9524865 (W.R.C.). The authors wish to especially thank Dr. E. Michael Thurman of the U.S. Geological Survey for many helpful discussions.
(4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
(18) (19) (20)
(21) (22) (23) (24) (25)
(26) (27) (28) (29)
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Received for review January 25, 1996. Revised manuscript received April 17, 1996. Accepted April 29, 1996.X ES9600845 X
Abstract published in Advance ACS Abstracts, July 1, 1996.
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