ES&T FEATURES Fulvic acid: modifier of metal-ion chemistry This class of compounds, derived from the decay of plants and animals, is being studied for its role in the transport and toxicity of metal ions in soil and water
Robert A. Saar Geraghty & Miller, Inc. Syosset,N.Y. 11791 James H. Weber Chemistry Department University of New Hampshire Durham, N.H. 03824
Living biological systems are well ordered by the input of energy throughout any organism's lifetime. The result is that organisms are com posed of a fairly unchanging set of bi ological molecules, such as fatty acids, proteins, nucleic acids, and hormones, many of which are known to the last atom. Their reactivities can be repro duced in the laboratory. In contrast, the extreme ordering effects of energy are not present in an organism's wastes, or in its body after death. As degradation progresses, an increasing variety of organic structures can form. Many of the resulting com pounds are not fully characterized or named. Degradation compounds can be categorized as biopolymers, which are predominantly polysaccharides and polypeptides, and as geopolymers (humic substances), which are random polymers of a variety of biological monomers (7). Fulvic acid is the most hydrophilic of several classes of geo polymers (Figure 1); it is soluble at both high and low pH. Fulvic acid molecules, with atomic masses ranging from a few hundred to Feature articles in ES&T have by-lines, rep resent the views of the authors, and are edited by the Washington staff. If you are interested in contributing an article, contact the managing editor. 510A
Environ. Sci. Technol., Vol. 16, No. 9, 1982
thousands of atomic units, have a wide variety of aromatic and aliphatic structures bearing many oxygen-con taining functional groups, particularly — C O O H and — O H (2). These functional groups, which can be protonated and deprotonated in the pH range common in natural waters (pH 3-9), enable fulvic acid to behave as a polyelectrolyte ( J ) . The inability to characterize com pletely all fulvic acid structures does not prevent substantial progress in understanding the properties of these materials. Although particular mole
cules may show up in one batch of fulvic acid and not in another, the overall chemical properties of the two batches will be remarkably similar. Thus, fulvic acid can be investigated and described in terms of its group properties. Fulvic acid research does not fall neatly into the traditional disciplines of analytical, physical, organic, or bi ological chemistry, and yet it draws information and techniques from each. The vocabulary of traditional disci plines is used in fulvic acid work (for example, "functional groups" and
Origin of humic substances and relationships among them
Plants, animals, and microbes, and their wastes
Biopolymers Carbohydrates, proteins and fragments, fats, pigments
Decay processes
Τ
Geopolymers Humic substances
1 Humic acid Soluble in basic solution; insoluble in acid solution and in ethanol
Fulvic acid
Soluble in both acid and basic solution
0013-936X/82/0916-0510A$01.25/0
Humin
Soluble in neither acid nor basic solution
© 1982 American Chemical Society
FIGURE 2
Fulvic acid interactions with metal ions
H„FAg
M(H 0) m + Η
Competing metal ions; fulvic acid adsorption on clay minerals and other solids
PA9-1
M(HO) 2
"H_J,-H ,FA9
Key: j Best species, of those shown, forcomplexation.
and so on
Note: Fulvic acid and metal-ion species are arranged with those prevalent at low pH near the top and those prevalent at high pH near the bottom.
"conditional stability constants"), but the terms often take on new shades of meaning. Fulvic acid and metal ions
An important property of fulvic acid is its ability to form complexes with metal ions. Many of the oxygen-con taining functional groups, particularly carboxylic and phenolic moieties (4, 5), associate with metal ions, notably the alkaline earths (commonly Ca and Mg), and transition metals (for ex ample, Cu, Fe, Cd, Zn, V, and Ni). Whereas monovalent cations like Na and Κ can form weak electrostatic bonds with single anionic groups on fulvic acid, divalent metal ions may be complexed at two adjacent anionic sites, forming a chelate ring, an asso ciation that is generally much stronger than that formed by complexation through a single site. Many variables affect the strength of association between fulvic acid and metal ions. Figure 2 gives a greatly simplified view of the factors involved. The hydrogen ion concentration de termines which forms of the fulvic acid and metal ions are prevalent; different forms of these species have different tendencies to enter a complex. The most "eligible" species are indicated in the figure. Another way to look at the effect of pH on complexation is to consider that + H competes with metal ions for an ionic binding sites on fulvic acid, and OH~ competes with fulvic acid for the cationic metal ion. As the pH is raised, fulvic acid becomes more available for complexation, and the metal ion be comes less available. An intermediate
Ν
Ι Η ( O H J) } " " "
η— i
Adsorption on solids; inorganic complexes; other organic ligands
ZiEK
r mnpijonq·
'"11·"* Complexes of fulvic acid and so on and metal ions (various stoichiometries)
pH most favors complexation between fulvic acid and metal ions. Why be concerned with the inter action between fulvic acid and metal ions? There are many possible answers, of which three are offered here. First, many researchers have focused on the different biological availability or toxicities of complexed and uncomplexed metal ions (6-10). Metal ions such as Cu 2 + and Cd 2+ are known to be less toxic to aquatic organisms when they are part of complexes with fulvic acid or other ligands than when they are not complexed. Since it is easy to analyze for the total concentration of metal ions, one is tempted to correlate toxicity and other properties with this total. However, it is evident that in toxicity studies, complexed and hydrated species should be considered separately, as if they were different metal ions (11). Second, aquatic fulvic acid, as well as other fractions of dissolved and ad sorbed organic matter, can alter the geochemical mobility of metal ions (5, 12-14). Dissolved organic matter can release metal ions that had been ad sorbed on sediments, and organic matter adsorbed to sediments can se quester metal ions that are in solution. The stability constants for complexes between fulvic acid and metal ions like Cu 2 + and Pb 2 + are high, so that the fulvic acid can alter the metal-ion equilibria. Even the partitioning of cadmium, which is not complexed as strongly, is influenced by humic ma terials (15,16). Buffering capacity refers most often in chemistry to the regulation of hydrogeri'ion concentration. But, there
are other kinds of buffers: For exam ple, fulvic acid in natural water sys tems is a metal-ion buffer. As with all buffers, its capacity (in this case, the ability to complex metal ions) is lim ited. This limit defines what is called the complexing capacity of a water sample. Third, fulvic acid is important be cause it may change the ability of water treatment processes to remove metal ions. In a study of alum reac tivity with aqueous Cu 2+ , Cd 2+ , and Zn 2+ , fulvic acid increased the fraction of metal ions removed (17). Since alum coagulation can be important for removal of trace metals from a water supply, the changes in removal effi ciency caused by dissolved organic matter must be known. Analyzing for complexes A complete speciation scheme such as the one presented by Florence and Batley (77) includes metal ions that are free (fully hydrated) and those associated with various substances, including fulvic acid, in both the solid and solution phases. Of primary in terest is the solution phase which, by convention, includes all materials that pass through a 0.45-μπι filter. Many experimental factors can alter the results of speciation studies in volving fulvic acid and metal ions. Although the complexing properties of fulvic acid vary somewhat from sample to sample, the similarities are great enough so that findings in the world's literature overlap substantially (7J). Furthermore, fulvic acid derived from soils (SFA) has largely the same metal complexing properties as fulvic acid Environ. Sci. Technol., Vol. 16, No. 9, 1982
511A
TABLE 1
derived from water (WFA). Many of the experiments described here employed SFA because it is easier to obtain than WFA. Side-by-side studies of the two substances showed that the information obtained for SFA applies to WFA as well. The many variables listed in Table 1 can contribute to widely differing results for systems with fulvic acid and metal ions, which should otherwise be similar. The method of extraction can alter the fraction of organic matter that is obtained, or if the method is harsh, chemically change the fulvic acid. The concentration of fulvic acid appears to affect speciation, particularly for weakly bound metals like Cd 2 + (18). Ionic strength indicates the concentration of monovalent cations when a salt such as potassium nitrate is used. Ions like potassium can compete (if in
Factors that influence fulvic acid-metal ion complexation and stability constant determinations Source of fulvic acid Method of isolation Concentration of fulvic acid Ionic strength Temperature pH (affects both fulvic acid and metal ions) Method of analysis for complexes Method of data manipulation and stability constant calculation
high enough concentration) with divalent cations for complexation sites on fulvic acid molecules (19). Temperature is important because of its effect on the free energy of complexation. Hydrogen-ion concentration (pH), as indicated earlier, dictates the predominant forms of fulvic acid and metal ions and is, therefore, a key variable.
The method of speciation analysis can profoundly influence the results, because the various methods measure different aspects of the system and operate under different conditions. Finally, after all experimentation is done, the resulting data can be manipulated in different ways. Separation and nonseparation techniques are the two major types of analysis applied to speciation problems; commonly used methods are listed in Table 2. This table also shows the applicability, advantages, and disadvantages of the various methods. Separation of free and complexed metal ions can be done by chromatography, or with membranes that exclude the metal-ion complexes. Chromatographic techniques include liquid chromatography by size exclusion. Ultrafiltration and dialysis use membranes with small pores.
TABLE 2
Techniques for investigating complexes of fulvic acid and metal ions Technique
Applicability (which metal)
Adsorption problems
What measured
Shift equilibrium
Ionic strength
Separation Liquid chromatography (including reversed phase and gel filtration)/ atomic absorption spectrometry (AAS)
Any AAS metal
Total metal-ion and free metal-ion concentrations in various eluted fractions
Possibly
Possibly
No restrictions
Ultrafiltration (UF)/AAS
Any AAS metal
Total metal-ion and free metal-ion concentrations
Yes—on membrane and ultrafiltration cell; varies depending on metal
Possibly
No restrictions
Equilibrium dialysis/AAS
Any AAS metal
Total metal-ion and free metal-ion concentrations
Possibly (on membrane)
Possibly
~ 0 . 0 0 1 M or greater
Electron paramagnetic resonance spectroscopy (EPR)
EPR-active metals (e.g., V, Cu, Mn, Fe)
Various forms of metal ion
No
No
No restrictions
ion-selective electrode potentiometry
Cd, Cu, Pb, Ca
Hydrated metal-ion concentration
Possibly
No
~ 0 . 0 1 M or greater
Hydrogen-ion potentiometry
Any
Increase in hydrogen ion concentration from which complexation is inferred
No
No
No restrictions
Anodic stripping voltammetry
Cu, Cd, Pb, Zn
Hydrated and ASVlabile metal ions
Yes
Possibly
~ 0 . 0 1 M or greater
Fluorescence
Paramagnetic metal ions like Cu
Uncomplexed fulvic acid
No
No
No restrictions
Nonseparation
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Environ. Sci. Technol., Vol. 16, No. 9, 1982
Nonseparation techniques that distinguish between free and complexée metal ions in situ include voltammetry and potentiometry. Fluorescence, also a nonseparation technique, measures the concentration of free ligand. Separation techniques. These methods have two major pitfalls: adsorption of species on membranes or chromatographic materials and the possibility of shifting equilibria. Although the losses of metal ions (20) and organic matter (21) have been studied, the adsorption problem for membranes and chromatographic materials is nevertheless generally ignored. The adsorption of metal ions on purified dialysis membranes is apparently minimal in solutions of high ionic strength (22, 23). The complexation equilibrium probably shifts during chromatographic separations. Figura
Advantages
Disadvantages
Can use unmodified natural water samples
Not particularly applicable for stability constant calculations
Faster than equilibrium dialysis; no ionic strength or metalion restrictions
Adsorption; possible incomplete separation; special UF cell needed
No metal-ion restrictions; can use unmodified natural water samples
Possible adsorption and contamination by membrane; possible incomplete separation
Provides information on types of complexes
Only EPR-active metals; high detection limit
Rapid titration
Few metal ions; possible adsorption on electrode; high detection limit
No ionic strength or metal-ion restrictions
Extreme care needed during titration
Very low metal-ion concentration can be measured
Few metal ions; adsorption; possible equilibrium shift
An alternative view Applicable to few of complexation; metal ions very sensitive; can use unmodified natural waters
and McDuffie (24) describe this problem in their use of Chelex chelating ion-exchange resin to separate various metal ions from natural water samples. The chief advantage of separation techniques is found in the wide range of metal ions that can be measured, generally by means of atomic absorption or inductively coupled plasma spectrophotometry. The nonseparation methods, except for hydrogen-ion potentiometry (25), are each applicable to only a few metal ions. Chromatography and ultrafiltration. Two recent papers on reversedphase liquid chromatography demonstrate that some C u 2 + in swamp (26) and estuarine (27) waters j s associated with organic fractions. Gel filtration chromatography, which retards free metal ions, demonstrates that iron occurs in complexes in a variety of natural water samples (28) and that free and complexed metal ions exist in solutions of isolated humic and fulvic acids (29). Ultrafiltration studies demonstrate that a variety of metal ions, including C d 2 + , P b 2 + , C u 2 + , and iron, are retained on membranes with pores between approximately 0.4 μπι and 0.02 μιη (30). Since that is the "dissolved" range, the results suggest that the metal ions are complexed to aquatic organic matter. A major difficulty in interpreting ultrafiltration data is that no membrane has pores small enough to separate completely dissolved metal complexes from metal ions adsorbed on colloidal particulate matter. Metal species between 0.4 and 0.2 μπι prob ably include both metal complexes and adsorbed metal ions. Equilibrium dialysis (ED). The ED experiment is set up with two solutions: a solution with metal ions and ligands outside bags made of dialysis mem branes and a solution with neither constituent inside the bags (31). Ide ally the membranes will allow only hydrated metal ions to pass through to the inside. Thus, one can measure free metal ion inside the bag and total metal ion (hydrated and complexed) outside it. A plot of free metal ion vs. total metal ion will show a sharp endpoint ( C L ) if the conditional stability con stant Κ is sufficiently large. This C L value indicates the quantity of ligands or complexing groups in the solution. A recent paper (22) describes tests of
this method with titrations of 6.25 μΜ ethylenediaminetetraacetic acid by C u 2 + and C d 2 + ; the experimental C L values were within 2.4% of the theo retical 6.25-μΛ/ value. Initial experiments in our laboratory with soil-derived fulvic acid (SFA) demonstrated that C u 2 + and C d 2 + penetrate the dialysis membrane, whereas SFA does not (22). Experi ments were performed with 0.001 M KNO3, an ionic strength similar to that of fresh water. Titrations of 10 m g / L SFA with C u 2 + and C d 2 + showed that the titration endpoint C L depended on the pH and on which metal ion was used: C L increased from pH 5 to 8 and is higher for C u 2 + than for C d 2 + (Table 3). Earlier ion-selec tive electrode studies (18, 32) indi cated these trends. After the completion of ED experi ments with SFA, this approach was extended to seven freshwater samples from southeastern New Hampshire (33). Equilibrium dialysis does not require sample modification such as addition of a supporting electrolyte, so the samples had only to be filtered through a 0.4-μιη polycarbonate membrane before titration with C u 2 + or C d 2 + . Base was added to maintain the original pH. The measured C L values (Table 3) are generally larger for C u 2 + (1-15 μΜ) than for C d 2 + ( 0 - 1 0 μΜ) as we expected from ex periments with isolated SFA (18, 22, 32). Nonseparation techniques. Unfor tunately, adsorption occurs with all separation techniques in which a membrane, chromatographic material, solid electrode, or mercury electrode touches a solution containing humic matter. This is a problem that ultra filtration, equilibrium dialysis, and chromatography (separation tech niques) share with two nonseparation techniques: ion-selective electrode potentiometry and voltammetry. In contrast, the problem of shifting equilibrium is more common with separation techniques (particularly chromatography) than with nonsep aration ones, although the equilibrium can be shifted during voltammetry experiments. Nonseparation ap proaches cover relatively few metal ions. For example, voltammetry is commonly used for C u 2 + , C d 2 + , P b 2 + , and Z n 2 + . The applicability of other nonseparation techniques is shown in Table 2.
Environ. Sci. Technol., Vol. 16, No. 9, 1982
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TABLE 3
Total ligand concentration (C L ) for 10 mg/L soil fulvic acid (SFA) solutions and some New Hampshire fresh waters as determined by equilibrium dialysis concentration was 50 μΜ. This ag gregation occurs well before the ap proximately 400 μπιοί of carboxylic SFA a 5 16.2 8.0 and phenolic complexation groups are SFA a 6 24.1 20.3 saturated with Pb 2 + , which indicates SFA a 7 28.7 24.3 that the solid-phase, lead-fulvate ag Portsmouth Reservoir 6 6.3 8.6 0 gregates should be able to do two Lamprey River" 10.7 6.6 0.4 things: physically adsorb and chemi c Oyster River" cally complex additional Pb 2 + ions. 1.1 7.3 However, because Κ values should Exeter River b 2.1d 7.4 4.3 6 reflect only solution-phase complexa Durham Reservoir 7.4 5.0 3.1 tion, data for solutions without ag Drew Pond b 6.4 11.9 9.7 gregates were used. As in the Cu 2+ Barrington Swamp 6 5.7 15.1 2.0 (32) and Cd2+ (18) studies, Κ in Ref. 22. creased with pH. For example, Κ val * Ref. 33. ues for Pb 2+ -SFA complexes are Microbes coated dialysis membrane. 1 X 104 at pH 4.0, and 2 Χ 106 at pH 6. Incomplete titration because of malachite formation. These ISE studies demonstrate that under the same conditions, Cu 2+ - and Electron paramagnetic resonance It was found that increased pH re Pb2+-fulvate complexes have similar {EPR). EPR can provide many types sulted in higher Κ values, and more Κ values, whereas those of Cd2+-fulof information for complexes that Cu 2+ bound to SFA or WFA sites. vate are about 100 times lower. Cu 2+ contain paramagnetic species. In one Also, the average Κ value increased as and Pb 2 + differ in that Pb2+-fulvates study, EPR showed that SFA could more ligand was added during the ti aggregate at much lower M 2+ /fulvic reduce vanadium(V) to vanadium(IV) tration. This result was expected, be acid mole ratios; Cd 2+ is even less ef (34). In another vanadium study, EPR cause fulvic acid is a mixture of ligands fective than Cu 2+ in aggregating fulvic allowed measurement of the distance that have different affinities for metal acid (45). between complexing sites, the condi ions such as Cu 2+ . Typical Κ values Differential pulse anodic stripping tional stability constants for those sites, are between 1 X 106and 10 Χ 106. The voltammetry (DPASV). Researchers and the aggregation of vanadyl-SFA extent of Cu 2+ binding can be related commonly use DPASV (ES&T, Vol. complexes (35). Senesi and co-workers to the total acidity (carboxyl plus 16, No. 2, p. 104A) experiments to found both strong and weak Fe 3+ sites phenol content) of SFA (13.4 meq/g) determine metal-ion speciation in on humic and fulvic acids (36). The and WFA (10.5 meq/g). natural water samples, because the EPR results yielded information on For complexation of Cd 2+ by SFA technique can measure very low con site symmetries and on the resistance and WFA, average Κ values decrease centrations of metal ions—down to to reduction of Fe 3+ at the two types of during titration with metal ion titrant, about 1 Χ ΙΟ"9 Μ (11). Despite its sites. Lakatos and co-workers (37) and increase with increasing pH (18). popularity, DPASV has several observed EPR signals in systems con Also, the total binding of Cd 2+ by drawbacks. DPASV experiments, like 2+ 2+ taining humic acid and Mn , Cu , fulvic acid increased with increasing those with ISE, need a supporting V 0 2 + , Mo(VI), Mo(V), and Cr 3 +. pH. These results are similar to those electrolyte, and work only for certain for the Cu 2+ work (32). The major metal ions, including Pb 2 + , Cu 2+ , Ion-Selective Electrode (ISE). The new finding of the Cd 2+ study was that Cd2+, and Zn2+. detection limits of ion-selective elec Κ also depends on total fulvic acid trodes are too high to measure the DPASV has two additional major concentration. For example, at pH 6, drawbacks that limit its effectiveness concentration of free metal ion in most Κ for complexes of SFA and Cd 2+ in natural water matrices. First, unmodified samples of natural water. decreases from 2.9 X 104 to 1.2 X 104 DPASV disturbs the equilibrium be Moreover, ISE experiments cannot be as the SFA concentration increases tween free and complexed metal ions: done at very low ionic strength. How from 19 to 360 mg/L. This depen Dissociation of metal complexes often ever, ion-selective electrodes are widely dence necessitates titration with a occurs during the plating step. There used to measure the complexation of concentrated Cd 2 + solution and fore, the measured stripping current is metal ions to isolated humic matter in maintenance of a fairly constant SFA composed of two parts: the diffusion solutions with metal-ion concentra concentration. In contrast, the Cu 2+ - current caused by dissolved, hydrated tions higher than those usually found SFA system gives the same results for metal ions, those that were not part of in the environment (4, 38-41). either Cu 2+ or SFA titrant. complexes; and the kinetic current, An early project carried out by this research group employed an ISE to Pb 2 + binding to SFA and WFA by which arises from metal ions that have study Cu 2+ binding to water-derived ISE (44) was also studied. An impor just dissociated from complexes. A fulvic acid (WFA) and SFA in 0.1 M tant discovery was that even small measure of only the concentration of KNO3 (32). Because the total con amounts of Pb 2 + caused aggregation hydrated metal ions requires that the centrations of ligand and metal ion and (formation of solids) in fulvic acid so kinetic current be subtracted from the the concentration of free metal ion are lutions, as indicated by light scattering stripping current. This differentiation known, one could calculate conditional measurements. For example, aggre is theoretically and experimentally stability constants (K values) based on gation occurred in a 32 mg/L solution difficult. Second, humic matter ad the Scatchard binding model (42, 43). of SFA at pH 4 and 5 when the Pb 2 + sorbs on the mercury electrode, makCL,MM
Sample
pH
a
c
d
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Environ. Sci. Technol., Vol. 16, No. 9, 1982
Cu*+
Cd*+
ing it difficult to interpret results (46, 47). Our recent paper (48) described a DPASV method that overcame the problems of kinetic currents and fulvic acid adsorption. We titrated 10, 20, and 40 m g / L S F A solutions in 0.1 M K N 0 3 with C u 2 + at p H 5 and 6, and measured the total stripping current, which was recorded along with the total amount of metal ion added. The data were computer fitted to an equa tion developed by S h u m a n and Cromer (49). One computer program calculates, among other things, the total ligand concentration in the solu tion ( C L ) , and the conditional stability constant (K); a second program cal culates stripping currents with the ki netic current removed, as well as cor rected C L and Κ values. The contribution from kinetic cur rent proved to be substantial for titra tions of SFA by C u 2 + . For example, in the titration of 20 m g / L SFA at pH 6, the C L values increased by 22% as a result of the kinetic-current correction. That is, the uncorrected data under estimated the total ligand concentra tion by 22%. The problem of fulvic acid adsorp tion onto the mercury electrode is overcome by in situ calibration curves. Calibration curves done in .the absence of fulvic acid lead to erroneous values for hydrated (free) C u 2 + concentra tions. The correct concentrations for free C u 2 + allow calculation of Κ by any desired means of data treatment. Fluorescence spectrometry (FS). Humic matter and natural water samples fluoresce (22,50, 51 ) , but the fluorescence of organic ligands is quenched by complexation to para magnetic metal ions, that is, those with unpaired electrons (52, 53). There fore, the intensity of humic matter fluorescence decreases during titration by such metal ions. FS experiments have several ad vantages over other methods. First, fluorescence is measurable in solutions with concentrations of dissolved or ganic carbon even lower than those found in many samples of natural water (detection limit is less than 1 m g / L ) . Second, FS experiments, un like DPASV and ISE, need no sup porting electrolyte. Third, unlike all other methods discussed here, FS measures the concentration of free li gands, rather than that of free or total metal ions. This third advantage re
sults in a direct measurement of C L . The major disadvantage of FS is that it is very effective only with strongly binding, paramagnetic metal ions, such as C u 2 + . SFA and W F A fluorescence spectra exhibit a broad, featureless emission peak at 445-450 nm upon excitation at 350 nm (50). Two requirements must be met before FS is used for binding studies with fulvic acid and metal ions: Uncomplexed metal ions must not quench fluorescence, and metal-ion quenching of fluorescence must be proportional to metal-ion complexa tion. In initial studies (50) we demon strated that the first requirement was met by observing that at pH 1.4, at which divalent metal ions are not complexed to fulvic acid, its fluores cence is unquenched, even at very high concentrations of metal ions. As for the second requirement, we showed with the model ligand salicyclic acid (ohydroxybenzoic acid) that the per centage of C u 2 + bound (calculated from the known stability constant) and the percentage of fluorescence quenched are equal during titration by C u 2 + at p H 6. These two preliminary experiments encouraged us to use flu orescence as a probe to study metal ion complexation by fulvic acid. Accordingly, we titrated 32 m g / L solutions of S F A and W F A with C u 2 + , P b 2 + , C o 2 + , and N i 2 + at various p H values (50). For any specified métal ion/fulvic acid mole ratio, the percentage of fluorescence quenched for each metal ion increased as p H increased. This trend agrees with complexation studies done by ISE (18, 32, 44, 45), which demonstrated increased Κ values (and hence, increased com plexation) at higher pH values. The effectiveness of metal ions in quenching FA fluorescence is: C u 2 + > P b 2 + > Co 2 + « Ni 2 + > Cd 2 + · Cu 2 + and P b 2 + form strong fulvic acid complexes with similar Κ values, but paramagnetic C u 2 + is much more ef fective than diamagnetic P b 2 + in quenching fluorescence. The weakly bound paramagnetic ions C o 2 + and N i 2 + quench some fluorescence, and the weakly bound and diamagnetic C d 2 + ion has no effect on fluorescence intensity. Finally, we demonstrated with FS and ISE titrations that Cu 2 + and P b 2 + binding to fulvic acid is proportional to fluorescence quenching.
Our recent results (54) extend the earlier work by describing a quantita tive method for determining micromolar C L values of fluorescing ligands for metal ions. We initially titrated 36-μΜ solutions of the model com pound L-tyrosine with C u 2 + to test an equation and a curve-fitting program that we developed. The observed and calculated C L value of 33 μΜ is close to the 36-μΜ known concentration. The average Κ value of 5.8 Χ 10 4 at pH 6 agrees well with the first condi tional stability constant (K) of 5.9 X 10 4 (55). We also found that the re sidual fluorescence, after complexes have been formed, was only 2.6 ± 1.7% of the original fluorescence exhibited by the free ligand. Experiments with 10 m g / L (16 μΜ) solutions of S F A at p H 5, 6, and 7 yielded C L values of approxi mately 20 μΜ. Earlier ISE titrations (32) also showed that, on average, there was more than one complexing site per average SFA molecule (42). The conditional stability constant Κ increased with increasing pH, a result seen with the other techniques. In contrast to the L-tyrosine trials, the residual fluorescence for SFA was substantial, about 20%. This relatively high residual may occur because the fluorescence efficiency of the complex is about 20% that of the free ligand. Alternatively, it is possible that the residual fluorescence is attributable to nonbinding fluorescent molecules in the S F A mixture. In this case, the binding material, as in the L-tyrosine experiments, would be quenched al most completely. Fluorescence spectrometry has several advantages, including the re quired detection limit, for determining micromolar complexing capacities of natural organic matter. It gives results that are comparable to those from DPASV, ISE, and dialysis/atomic absorption experiments. Fluorescence, which differentiates free and bound ligand, is an excellent complement to the other techniques for measuring complexing capacity, which distin guish between free and bound metal ions. Furthermore, FS is fairly rapid and requires no supporting electro lyte. Measurement of solution scattering with the FS instrumentation gives valuable information on aggregation and precipitate formation; this is im portant, because the usual goal in Environ. Sci. Technol., Vol. 16, No. 9, 1982
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TABLE 4
A comparison of methods for calculation of the conditional stability constant Κ for complexes containing fulvic acid and copper(ll) or cadmium(ll)a Log Κ Cadmium(ll)
Copper(li) pH
Buffle method"
Scatchard method
4.0
4.0
Strong sites: 5.6 Weak sites: 4.0
3.0
3.2
5.0
4.9
Strong sites: 6.0 Weak sites: 4.1
3.5
3.8
6.0
6.0
Strong sites: 6.3 Weak sites: 3.8
3.6
4.1
7.0
4.0
4.3
8.0
4.4
4.6
a b
Ref. 58. Ref. 56.
D 3
Buffle method"
1: 1 method"
Ref. 32. Ref. 18.
measuring complexing capacity and stability constants is an understanding of solution-phase equilibria. By care fully considering both fluorescence intensities and scattering, one can distinguish between metal-ion com plexation, which is a solution-phase process, and adsorption, which occurs only when a solid phase is present. Work is continuing in our laboratory on the application of fluorescence to natural water samples. Modeling Various models have been used to explain complexation of metal ions by fulvic acid. The Scatchard method (42, 43) applies to a model in which fulvic acid has distinct classes of sites, each with a common ability to complex metal ions. The calculation method described by Buffle and co-workers (56) applies to a system with two types of complexes: one average fulvic acid molecule and one metal ion (1:1 com plex) and two average fulvic acid molecules and one metal ion (2:1 complex). A simpler model system than either of these is the one in which only 1:1 complexes are postulated. More complex models are also avail able (4, 57). Certain experimental conditions and different metal ions or samples of ful vic acid may favor one model over the others. A complete discussion of vari ous models and their merits would be tedious and, possibly, not helpful. Table 4 shows results of stabilityconstant calculations for fulvic acid complexes with strongly bound Cu 2 + and weakly bound Cd 2+ ions (58). For each metal ion, the stability constants have been determined by applying two different calculation schemes to the same data set. The results for the two calculation methods are quite similar, 516A
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Environ. Sci. Technol., Vol. 16, No. 9, 1982
indicating, perhaps, that the two models in each case are about equally appropriate (or inappropriate). The two sets of results for either metal ion would work about equally well if in corporated into more complex models that include both inorganic and or ganic factors affecting metal-ion speciation. Avoiding pitfalls Complexes between metal ions and naturally occurring organic matter can be studied, but the commonly used methods have pitfalls. Data must be interpreted with attention to possible shifts in equilibrium during measure ment, adsorption of organic matter on electrodes and membranes, and the appearance of a solid phase if aggre gation occurs. In addition, the re searcher needs to consider such factors as isolation procedures, as well as the need to modify natural water samples when trying to draw conclusions about environmental processes from the re sults of laboratory experiments. Acknowledgment This work was supported in part by Of fice of Water Resources Technology Grant B004-NH through the University of New Hampshire Water Resources Research Center, and National Science Foundation Grants O C E 77-08390 and O C E 7910571. Prior to publication, this article was read and commented on for suitability as an ES& Τ feature by Russell F. Christman, Chairman, Department of Environmental Science and Engineering, University of North Carolina, Chapel Hill, N . C . 27514, and John Ertel, Department of Oceanog raphy, WV-10, University of Washington, Seattle, Wash. 98105.
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(2) Liao, W.; Christman, R. F.; Johnson, J. D.; Millington, D. S.; Hass, J. R. Environ. Sci. Technol. 1982, 16, 403-10. (3) Burch, R. D.; Langford, C. H.; Gamble, D. S. Can. J. Chem. 1978, 56, 1196-1201. (4) Gamble, D. S.; Underdown, A. W.; Langford, C. H.;Anal. Chem. 1980,52, 1901-8. (5) Mantoura, R. F. C ; Dickson, Α.; Riley, J. P. Estuarine Coastal Mar. Sci. 1978, 6, 387-408. (6) Jenne, Ε. Α.; Luoma, S. N. In "Biological Implications of Metals in the Environment," Proceedings 15th Annual Hanford Life Sci ences Symposium, Richland, Wash., Sept. 29-Oct. 1, 1975; pp. 110-43. (7) Guy, R. D.; Kean, A. Water Res. 1980,14, 891-99. (8) Babich, H.; Stotzky, G. Adv. Appl. Mi crobiol. 1978, 23, 55-117. (9) Wilson, D. E. Limnol. Oceanogr. 1978, 23, 499-507. ( 10) Baccini, P.; Suter, U. Schweiz. Z. Rydrol. 1979,41, 291-314. (11) Florence, T. M.; Batley, G. E. CRCCrit. Rev. Anal. Chem. 1980, 9, 219-96. (12) Nissenbaum, Α.; Swaine, D. J. Geochim. Cosmochim. Acta 1976, 40, 809-16. ( 13) Jackson, K. S.; Skippen, G.B.J. Geochem. Explor. 1978, 10, 117-38. (14) Jackson, K. S.; Jonasson, I. R.; Skippen, G. B. Earth Sci. Rev. 1978, 14, 97-146. (15) Gardiner, J. Water Res. 1974, 8, 23-30. (16) Gardiner, J. Water Res. 1974, 8, 157164. (17) Truitt, R. E.; Weber, J. H. Water Res. 1979, 13, 1171-77. (18) Saar, R. Α.; Weber, J. H. Can. J. Chem. 1979,57, 1263-68. (19) Gamble, D. S. Can. J. Chem. 1973, 51, 3217-22. (20) Truitt, R. E.; Weber, J. H. Anal. Chem. 1979,5/, 2057-59. (21 ) Buffle, J.; Dcladoey, P.; Haerdi, W. Anal. Chim. Acta 1978, 101, 339-57. (22) Truitt, R. E.; Weber, J. H. Anal. Chem. 1981, 53, 337-42. (23) Hart, B. T.; Davies, S. H. R. Aust. J. Mar. Freshwater Res. 1977, 28, 397-402. (24) Figura, P.; McDuffie, B. Anal. Chem. 1980,52, 1433-39. (25) Stevenson, F. J. Soil Sci. 1977, 123, 10-17. (26) Lee, J. Water Res. 1981, 15, 507-9. (27) Mills, G. L.; Quinn, J. G. Mar. Chem. 1981, 10, 93-102. (28) Crerar, D. Α.; Means, J. L.; Yuretich, R. F.; Borcsik, M. P.; Amster, J. L.; Hastings, D. W.; Knox, G. W.; Lyon, Κ. Ε.; Quiett, R. F. Chem. Geol. 1981, 33, 23-44. (29) Mantoura, R. F. C.; Riley, J. P. Anal. Chim. Acta 1975, 78, 193-200. (30) Laxen, D. P. H.; Harrison, R. M. Water Res. 1981, 15, 1053-65. (31) Guy, R. D.; Chakrabarti, C. L. Can. J. Chem. 1976,54,2600-11. (32) Bresnahan, W. T.; Grant, C. L.; Weber, J. H. Anal. Chem. 1978, 50, 1675-79. (33) Truitt, R. E.; Weber, J. H. Environ. Sci. Technol. 1981,15, 1204-8. (34) Wilson, S. Α.; Weber, J. H. Chem. Geol. 1979, 26, 345-54. (35) Templeton, G. D., Ill; Chasteen, N. D. Geochim. Cosmochim. Acta 1980, 44, 741-52. (36) Sencsi, N.; Griffin, S. M.;Schnitzer, M.; Townsend, M. G. Geochim. Cosmochim. Acta 1977,47,969-76. (37) Lakatos, B.; Tibai, T.; Meisel, J. Geoderma 1977,79,319-38. (38) Takamatsu,T.;Yoshida,T.SO,/.SW. 1978, 725, 377-86. (39) Giesy, J. P., Jr.; Briese, L. Α.; Leversee,
"G. J. Environ. Geol. 1978, 2, 257-68. (40) Cheam, V.; Gamble, D. S. Can. J. Soil Sci. 1974,54,413-17. (41 ) Brady, Β.; Pagenkopf, G. Κ. Can. J. Chem. 1978,56,2331-36. (42) Sposito, G. Environ. Sci. Technol. 1981, 15,396-403. (43) Scatchard,G./4nn. N.Y.Acad. Sci. 1949, 57,660-72. (44) Saar, R. Α.; Weber, J. H. Environ. Sci. Technol. 1980,14, 877-80. (45) Saar, R. Α.; Weber, J. H. Geochim. Cosmochim. Acta 1980,44, 1381-84. (46) Cominoli, Α.; Buffle, J.; Haerdi, W. J. Electroanal. Chem. 1980, 110, 259-75. (47) Benes, P.; Koc, J.; Stulik, K. Water Res. 1979,13, 967-75. (48) Bhat, G. Α.; Saar, R. Α.; Smart, R. B.: Weber, J. H. Anal. Chem. 1981, 53, 227580. (49) Shuman, M. S.; Cromer, J. L. Environ. Sci. Technol. 1979,13, 543-45. (50) Saar, R. Α.; Weber, J. H. Anal. Chem. 1980,52,2095-2100. (51) Stewart, A. J., Wetzel, R. G. Limnol. Oceanogr. 1980, 25, 559-64. (52) Chen, R. F. In "Biochemical Fluorescence: Concepts"; Chen, R. F.; Edelhoch, H., Eds.; Marcel Dekker: New York, 1976; Vol. 2, Chapter 13. (53) Parker, C. A. "Photoluminescence of So lutions with Applications to Photochemistry and Analytical Chemistry"; Elsevier: Am sterdam, 1968. (54) Ryan, D. K.; Weber, J. H. Anal. Chem. 1982, 54, 986-90. (55) Smith, R. M.; Martell, A. E. "Critical Stability Constants. Volume 1: Amino Acids"; Plenum Press: New York, 1974; p. 31. (56) Buffle, J.; Greter, F.-L.; Haerdi, W. Anal. Chem. 1977,49,216-22. (57) Hunston, D. L. Anal. Biochem. 1975,(55, 99-109. (58) Saar, R. A. Ph.D. Dissertation, University of New Hampshire, Durham, 1980.
Robert A. Saar (left) is a senior scientist with the groundwater consulting firm of Geraghty & Miller, Inc., where he pre pares monitoring programs for ground water contamination studies and interprets the resulting chemical data. He studied at the University of New Hampshire during 1975-80, and was a member of the Man hattan College Chemistry Department faculty during 1980-81. James H. Weber (right) is a professor of chemistry at the University of New Hampshire. He lectures on general and inorganic chemistry. His research group does organometallic chemistry as well as environmental coordination chemistry.
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CIRCLE 8 ON READER SERVICE CARD Environ. Sci. Technol., Vol. 16, No. 9, 1982 • 517A
