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The kinetic interactions of metal ions with humic acids
Marine Chemistry, 36 (1991) 27-38
27
Elsevier Science Publishers B.Y., Amsterdam
The kinetic interactions of metal ions with humic acids Gregory R. Choppin and Sue B. Clarki Department of Chemistry, Florida State University, Tallahassee. FL 32306, USA (Received 23 May 1990; revision accepted II April 1991 )
ABSTRACT Choppin, G.R. and Clark, S.B., 1991. The kinetic interactions of metal ions with humic acids. Mar. Chern., 36: 27-38. Complexation of europium (III) and UO~+ by humic acid was found to be very fast « I m ). However, passage through ion exchange resin removed a fraction which decreased as the contact time increased over' a 2-3 day period. The dissociation kinetics for UO~+ -humate were described by an equation with five first order terms with rate constants ranging from 40.6 rnr ' to 5.64X10- 3 m -I. The percentage of UO~+ dissociating by the slower paths, comparable to that eluting with the humic acid through the ion exchange resin is assigned to internal site-bound cations (strong binding). The faster dissociating fraction corresponded roughly to the UO~+ removed by the cation exchange resin (weak binding). The time dependence ofthe weak to strong binding ratio was associated with change in the conformation of the macromolecular humic acid as the cations neutralized the charge repulsions of the anionic humate.
INTRODUCTION
Humic substances, which are collections of organic polyelectrolyte macromolecules are the principal organic components of soils and waters. Their concentration, average molecular weight and functional group constitution are dependent on many factors such as climate, pH, substrate topography and time. In surface waters, the dissolved organic carbon (DOC) ranges from 0.1 to 50 ppm (mg 1-1 C). In ocean waters, the DOC ranges from 0.5 to 1.2ppm, with the higher concentrations occurring in the surface waters. In addition, most waters contain an amount of suspended particulate organic matter which in the ocean equals about lO% of the DOC. In all waters, there also can be suspended inorganic material with organic coatings. A majority of the DOC as well as the particulate and sorbed organic matter falls within the classification of humic substances. 'Present address: Savannah River Laboratory, Aiken, SC 29808, USA.
0304-4203/91/$03.50 © 1991 Elsevier Science Publishers B.Y. All rights reserved.
28
G.R, CHOPPIN AND S.B. CLARK
Humic substances are subdivided by solubility. The fraction which is soluble at all pH values is called fulvic acid, that soluble above a pH of approximately 3.5 is humic acid and humin is the fraction insoluble at all pH values. For humic material from different locations, the spectrum of molecular weights, the nature of the carbon skeleton and the types, positions and relative numbers of functional groups vary widely, in part depending on the origin and age of the humic material. Acid-base titration, Carbon-13 nuclear magnetic resonance spectroscopy by the cross-polarization, magic angle spinning technique (CP /MAS ), and ultrafiltration provide data which allow definition of major operational characteristics ofthese materials. The diversity of functional groups allows binding of a large number of metal ions. Since these humic substances are ubiquitous, they can have significant roles in the geochemical speciation of metals. Not only can they retard metal ion migration (e.g. when the humics are sorbed to surfaces) or promote it (e.g. by metal complexation to soluble or colloidal humics ), these substances can also influence the oxidation state (e.g. PuO~+ is rapidly reduced to Pu4+ by humics) (Choppin, 1988). The binding constants to humic acids for the f-elernent cations (e.g. Eu3+, Th 4+, UO~+) are dependent on pH, reflecting increased ionization of the carboxylate binding groups. At pH 6 the values of the conditional binding constant, logj31l for tracer metal concentrations were found to be: Eu 3+, 11.5; Th 4+, 15.0; UO~+, 9.0 (Choppin, 1988). Increased ionization also induces changes (due to increased intergroup repulsions) in the conformation of the macromolecular structure. The role of such structural changes is not very well understood at present. The interaction of cations with anionic polyelectrolytes has been described by two different models. In one description, which uses the Poisson-Boltzmann equation, the metal ions associated with a polyelectrolyte molecule are considered site-bound (i.e. associated with specific binding groups of the polyelectrolyte) (Marinsky, 1976). The second model treats the metal ions as being mobile within a 'condensed' layer adjacent to the polyion surface (Manning, 1981). Little has been done to examine either the relationship between this condensation (or, territorial) binding and site binding or the extent to which one or the other exists in a particular system. Insight into metal ion interactions with the organic polyelectrolytes can be obtained from a study of the dissociation kinetics. When Sm3+ and Th4+ cations were added to solutions of humic acid, the cations were bound completely in less than a minute (Choppin and Cacheris, 1987a,b; Clark and Choppin, 1990). When these cations were dissociated from the humic acid, the kinetic expression could be resolved into five to seven first order pathways. The dissociation terms fall into two classes based on their dependence on pH and temperature and, primarily, their half-lives. In the first class, the dissociation rates fall in the time domain of seconds, while the second group
METAL ION INTERACTIONS WITH HUMIC ACIDS
29
have rates in the minutes to hours time span. The amount of metal dissociating by the longer lived pathways increased the longer the metal was allowed to stay bound to the humate prior to removal. Such variation in the relative populations of the dissociative pathways was observed for periods of metalhumate binding of less than 2 days but not for longer predissociative binding times. These kinetic studies have been extended to uranyl cation, UO~+ . We also report further binding studies on Eu 3 +. The effects of pH, metal ion loading and average molecular weight of the humic acid have been investigated. EXPERIMENTAL
Reagents
A stock solution of U0 2 (Cl0 4 h was prepared by dissolving U0 2 (N0 3 h' 6H 20 in concentrated HCl0 4 . After several evaporations to dryness and dissolution in 0.1 M HCl0 4 , the salt was diluted to volume with 0.1 M HCl0 4 and the solution was standardized by spectrophotometric measurement of uranyl (2=416 nm, <==7.82 crnr ' M- 1, (Rabinowitch and Belford, 1964». The preparation of other solutions followed the procedures in Clark and Choppin ( 1990). Unless otherwise noted, experiments were conducted with solutions at 0.1 M ionic strength (NaCl0 4 ) and buffered with 0.01 M acetic acid or MES (4-morpholine-ethane sulfonic acid). Humic acid
Unfractionated humic acid was extracted from sediments from Lake Bradford in Florida and characterized as reported by Torres (1979). A portion of this humic acid was fractionated by size using ultrafiltration. During ultrafiltration, extreme care was used to ensure constant experimental conditions (Wheeler, 1976; Buffle et al., 1978). The stock solutions contained less than 150 mg I-I humic acid at pH 7.0 (buffered with 0.01 M Ntris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, TES), and 0.1 M (NaCl) ionic strength. An Amicon No. RG5 (51) fiberglass reservoir, pressurized to approximately 50 psi with N 2 and connected to an Amicon No. 8050 (65 ml) ultrafiltration cell with constant stirring was used. The humate fractions were those retained above the Amicon XM300 membrane (approximate size > 300 000 daltons) and the fraction that eluted through the Amicon YM I00, but was retained above the Amicon XM50 (approximate size 50 000-100 000 daltons). All humic acid samples were characterized by potentiometry to determine the carboxylic group capacity and by UVjvisible and 13C NMR. The poten-
30
G.R. CHOPPIN AND S.B. CLARK
tiometry measured pH changes of the humic acid solutions (0.01 g HA in 100 ml) under N 2 from pH 3.7 to 10.2 during titrations with CO 2-free NaOH (0.10 M). A rebuilt (On-Line Instrument Systems) Cary-14 interfaced to a Zenith 248 computer was used to measure the absorbance of a 250 mg 1-1 solution of each humate fraction (buffered to pH 7.0 with 0.01 MTES,,u=O.l M NaC1) at 465 nm and at 665 nm. The ratio of the absorbances (i.e. the £4/£6 ratio) is related to the degree of aromatic character (Konova, 1966; Schnitzer and Khan, 1972). Carbon-13 NMR spectra were collected on solid humic acid samples with an IBMjBruker WP 200 SY instrument employing cross-polarization with magic angle spinning (Wilson et al., 1981). The spectra were divided into three regions of interest: the carboxylate carbons (160-185 ppm), the olefinic carbons (100-160 ppm), and the aliphatic carbons (10-90 ppm). The area under the NMR spectrum for each region of interest was obtained by integration and normalized by dividing by the total spectral area. Binding studies
The extent and rate of Eu3+ binding to each humate fraction was studied using ultrafiltration and ion exchange. An Amicon No. 8010 (10 ml) ultrafiltration cell containing a YM2 (1000 dalton) membrane was loaded with a solution of humic acid. While stirring, an aliquot of Eu 3 + spiked with radiotracer europium-152 was added. A small portion of the solution in the cell was withdrawn for measurement of the europium-152 (to confirm the Eu concentration). The cell was immediately pressurized to 20 psi to cause filtration. After filtering, a sample of the filtrate was collected and analyzed for humic acid by spectrophotometry to ascertain complete removal of the humic acid. The filtrate and the prefilter sample were analyzed for europium-152 using a well-type NaI detector with a single channel analyzer. Ion exchange was used to monitor changes in binding as a function of the length of time Eu3+ was in contact with the humate. Cation exchange resins have been shown to separate rapidly and quantitatively uncomplexed lanthanide ions from their complexes (Choppin and Williams, 1973). Eu3+ spiked with europium-152 was added to a solution of humic acid. At fixed time intervals after the addition, aliquots were withdrawn and passed through a 1.2 em (i.d.)X5.7 em column of Dowex 50X4, 100-200 mesh resin. Europium-humate complexes were eluted rapidly using 5 ml of acetate buffer ( < 30 s elution time); the Eu3+ retained by the resin was eluted with 0.1 M Hel. Each eluant fraction was counted for europium-ISz.
METAL ION INTERACTIONS WITH HUMIC ACIDS
31
Dissociation studies The rate of UO~+ dissociation from the humic acid samples was determined using a ligand exchange technique (Choppin and Cacheris, 1987a,b). After equilibrating the metal-humate complex for a fixed amount of time, arsenazo(III) was added to the solution. The arsenazo(III) forms more stable complexes with UO~+ than does humic acid, thereby preventing the reformation of the UO~+ -humate complex. The intrinsic rate ofarsenazo complexation is very rapid relative to the dissociation rate of the humate complex. Consequently, the observed rate of arsenazo complexation is the rate ofmeta1 dissociation from the humic acid. Alteration of the concentration of arsenazo was found not to affect the rate of humate dissociation, indicating no catalysis by the arsenazo of the dissociation of the humate complex. The rate of UO~+ -humate dissociation was determined by following the growth of the arsenazo complex spectrophotometrically (Amax = 645 nm for UOz-arsenazo ). The slower dissociation processes (t 1/2> 0.5 min) were monitored using a conventional Milton Roy Spectronic 1201 spectrophotometer, while the more rapid dissociations were measured with a DurrumGibson D-109 stopped-flow spectrophotometer interfaced to an IBM PC XT. The dissociation kinetics were measured for samples in which the loading (mEq UO~ + as a percentage of total milliequivalence of carboxylate groups of humic acid) was 2.5, 5, and 9.5%. Above 10% loading, precipitation occurred. To study the different size humic acid samples, 5%loading was used.
Data analysis All calculations were performed on a Zenith ZF-158-42 PC. To determine the number of first order components in the dissociation, analysis by the kinetic spectrum method (Choppin and Cacheris, 1987a,b) was used. This technique uses the change in absorbance and J absorbance, with time. The utility of this approach had been shown for Cu2+ dissociation from humic acid (Olsen and Shuman, 1983). This method utilizes a unitless distribution function, H(k,t), defined by H(k t) = 15 2 (J absorbance) _ 15 (J absorbance) 15 On t) z J On t) ,
where the first term is the second derivative of the variation of absorbance with time and the second term is the first derivative. A plot of H(k,t) vs. In time yields a curve with a maxima for each first order process, the position of which provides an estimate of the half-life for that process. The resolved area of each peak is related to the percentage of metal ion dissociation with that lifetime. The kinetic spectra were calculated using a cubic spline smoothing calcu-
32
G.R. CHQPPIN AND S.B. CLARK
lation (Caceci and Cacheris, 1984). Since the domain of the kinetic spectrum is In t, small errors in peak position correspond to large errors in rate constants. Therefore, the kinetic spectrum analysis was used to obtain preliminary estimates of the rate constants and the percentage dissociation by each process. These values were used as the initial estimates in a simplex non-linear regression fit of the spectral data to obtain the half-lives and populations of the decay paths. RESULTS
Humate characterization Table 1 gives the results of the characterization studies of the fractionated humate samples plus the data of Torres (1979) for the unfractionated humic acid. The sizes for the two fractions relate to the membranes used in ultrafiltration, A/D indicates the ratio of the amounts of aliphatic to olefinic carbon determined by 13C NMR.
Unfractionated humate studies Using ultrafiltration, it has been found that when Ln 3+ in amounts equal to 5%of carboxylate capacity of the humic acid sample was mixed with humic acid solution and immediately (in less than 1 min) filtered, the metal ion was completely retained with the filtered humic acid (Clark and Choppin, 1990). This was interpreted as indicating very rapid, complete binding of the metal cations. When Eu3+ (also 5% loading) was added to a humic acid solution and the solution was passed through a column of cation exchange resin 15 min after mixing, only 4% of the Eu3+ eluted with the humic acid (Clark and Chopp in, 1990). Increasing the time to 48 h between mixing and passage through the resin (i.e. the contact time) increased the amount eluted to 40%. TABLE I Characterization of humate samples
A/O'
Size fraction (daltons) ~300000
50000-100000 Unfractionated
Carboxylate Capacity (mEq g-I)
6.15 6.81 6.94
1.9 1.9 1.6
3.52±0.22 6.89 ±0.56 3.86±0.03 b
"Ratio of aliphatic to olefinic carbon as calculated from the areas in the aliphatic and olefinic regions of the 13C NMR spectra. "Torres (1979).
METAL ION INTERACTIONS WITH HUMIC ACIDS
33
These studies have been repeated for a series of contact times with the results shown in Fig. 1 for both 2.5 and 5% loading. The simplest interpretation of the ultrafiltration and ion exchange resin studies is that the cations immediately bind, largely by condensation or by binding to surface sites. With time, changes in the macromolecular conformation occur within the humate which result in an increasing fraction of the metal ions being retained in interior sites. It is tempting to interpret the ion exchange data as related to strong binding (the fraction which elutes with the humic acid) and weak binding (the fraction which is removed from the humic acid by the resin). When the eluant sample of europium + humic is allowed to stand for between 28 (2.5% loading) and 48 h (5% loading) and then passed through the resin column, from only 30 (5%) to 40% (2.5%) was eluted. This is evidence that the 'weak' and 'strong' binding is an equilibrium distribution and is reversible since the percentage of europium (III) eluted for a solution after a 2 day mixing is the same as that eluted when a 'stripped' solution is allowed to stand 2 days before being passed again through the column. When bound UO~+ (5% loading) was dissociated from the humic acid using the arsenazo(III) competitive ligand method, the analysis of the kinetic spectrum showed five distinguishable first order paths (Figs. 2 and 3). The calculated rate constants and the percentage of UO~+ dissociating by each pathway are given in Table 2. The percentage of UO~+ dissociating by each process was dependent on the length of time the metal ion was in contact with the humate prior to initiating dissociation. Analysis of the dissociation kinetics following very short binding times (5 min or less) showed that most of the UO~+ dissociated via the two short-lived pathways. Increasing the predissociative binding time caused an increase in the percentage of dissociation by the three longer lived processes. For predissociative contact times longer than 2 days, no further 301::J
7
201-
::J
W
a.!2 101- 10
2
Fig. 1. Percentage of europium (III) eluting as humic acid complex as a function of the preelution contact time of europium(III) and humic acid: (Eu(III)] =2.50X 10- 6 M (D); [Eu(IlI}J=5.00XIO- 6 M (e); [-COi]=1.00xlO- 4 Eq/l-I; 1=0.10 M (NaCI0 4 ) ; pH=4.2.
34
G,R. CHOPPIN AND S,B.CLARK 2,50,---------------.
N~
2,00-
i
Q
2:S
g-
1,501-
~
1,00/---
r
!
o~
a
I
o
3
2
4
5
Ln Time (5) Fig, 2. Kinetic spectrum for fast UO~+ dissociation processes from the humic acid complex: [UO~+]=9.97XlO--6M; [-C0 2 ofHu]=2.00XI0- 4Eql-'; (Arsenazo (III)]=1.25XIO- 5 M; contact time e cS h; pH=4.22; T=295 K.
2.001-
a c
0 00
a
°CXbeeP'cP""q,~0
N
Q
a a a a a
1,501-
X
o
0"0&' 00
0
0,592
2
I
4
I
6
8
Ln Time (rn) Fig, 3, Kinetic spectrum for slow UO~+ dissociation processes from the humic acid complex: (UO~+]=9,97XlO-6M; [-C0 2 ofHu]=2.00XlO- 4Eql-l; [Arsenazo (III)] = 1.25X 10- 5 M; contact time=48 h; pH=4.22; T=295 K. TABLE 2 Rate constants and percentage dissociation for UO~+ decomplexation by humic acid Rate constant kl
k2
k3 k4 k5
= (6,76±0.11) X10- 1 S... I =(40.6±0.7) m" = (10,03 ± 0,26) X 10-- 2 s-I = (6,02±0.16) m- I = (7.83±0.75) X 10- 1 m- I = (6.98±0.63) X 10- 2 m>' = (5.64±0,62) X 10- 3 m- I
% Dissociation
16.02±3.01 21.43 ± 5.63 15,61±4.67 15.75±4.83 10.36±2.69
T=25°C, 1=0,10 M (NaCI0 4 ) , pH 4.2.
changes were observed in the percentages of metal ion dissociating by each process. To establish a constancy in the dissociation kinetics with Sm3+ and Th 4 + 2 days were also required (Choppin and Cacheris, 1987a,b; Clark and Choppin, 1990).
METAL ION INTERACTIONS WITH HUMIC ACIDS
35
The kinetics for UO~+ dissociation were found to be dependent on pH and temperature. As the pH of the medium was increased from 4.2 to 5.2, the dissociation rates decreased as shown in Figs. 4 and 5. It is probable that this is due to the increased ionization of carboxylate groups of the humic acid as the pH increases. The resultant increase in net negative charge on the humic macromolecule would retard cation dissociation. For all pathways, increasing temperature increased the rate of dissociation; however, the longer lived dissociation processes were noticeably more sensitive to temperature changes than the shorter lived processes. Such differences between the shorter and longer lived processes were noted also in dissociation studies of Th4+ from humic acid (Choppin and Cacheris, 1987a,b). The effect of altering lanthanide loading on to humic acid was studied. Ultrafiltration data showed that in the range of 2.5-9.5% loading, complexation was very fast and complete. When Eu3+ in excess of 10% of the carboxylate capacity of the humic acid was added, coagulation and precipitation occurred. Increasing the loading increased the amount of binding time required to reach an equilibrium between the 'strong' and 'weak' binding in the
0-
I
·~.7-o- - - - - , , . L , - - - - - ; : - L ; : - - - - _ ; : _ '
5.5
5.0
4.5
pH
Fig. 4. Variation of'log k) and log k2 with pH for fast processes of uranyl dissociation from humic acid: [UO~+ I =9.97x 10- 6 M; [-C0 2 of Hul =2.00X 10- 4 Eq 1-1; [Arsenazo (III) I = 1.25 X 10- 5 M; contact time=48 h; T=295 K.
-
0
-0---11--0--0_ k l
+
+ 4.5
5.0
5.5
6.0
pH
Fig. 5. Variation of the log k, with pH for the slow processes of U01+ dissociation from humic acid: [UO~+ I =9.97X 10- 6 M; [-C0 2 of Hul =2.00X 10- 4 Eq 1-'; [Arsenazo (III) I = 1.25 X 10- 5 M; contact time=48 h; T=295 K.
36
G.R. CHOPPIN AND S.B. CLARK
TABLE 3
Summary of dissociation results No. fast processes
No. slow processes
% Fast dissociation
1112 of
Humic acid (un fractionated) 2.S%load 1.0±0.S
3
3
22.83 (m)
S% load
2.0±0.S
3
3
9.S%load
3.S±0.5
3
4
55.28 ±S.13 66.34 ±6.27 65.71 ±6.20
3
3
2.97 (h)
3
3
64.23 ±6.83 71.88 ±6.47
Metal loading
Equilibrium binding time (days)
Humic acid (fractionated, 5% load) ;<; 300000 daltons 3.0±0.5 SOOOO-IOOOOO daltons
2.0±0.5
slowest process
41.56 (m) 8.12 (h)
47.54(m)
ion exchange elution measurements. Also, the percentage of 'strongly bound' Eu3+ decreased with loading and an additional long-lived dissociation pathway appeared in the kinetic spectrum at high humate loading. These results are summarized in Table 3. These results must be assessed with some uncertainty, as the variable loading has a number of possible and largely unknown effects on the humic acid macromolecules and the covariability which must be present may not be properly reflected in the parameters measured.
Fractionated humic acidstudies The carboxylate capacities of the size-fractionated humate samples differed. However, we conducted our studies using the same carboxylate concentrations in terms of milliequivalence carboxylate per milliliter solution and with 5% loading of lanthanide ions. Ultrafiltration studies confirmed rapid, complete binding, while ion exchange again demonstrated changes in the labilities of the complexes with increasing binding time. Table 3 shows that the contact time required for equilibrium in the 'stronger' and 'weaker' binding increases as the size of the humic macromolecule increases. The half-life for dissociation by the slowest process (the strongest binding?) is also greater for the larger molecular weight fraction. These observations are consistent with slower conformational change. The percentage bound by the faster dissociating modes is smaller for the lower molecular weight fraction, although the uncertainties in the data in Table 3 are large. The fact that the carboxylate capacities (Table 1) differed by a factor of almost 2 had no significant effect at 5%loading.
METAL ION INTERACTIONS WITH HUMIC ACIDS
37
DISCUSSION
The results are consistent with a model in which metal ions bind rapidly and largely in a non-specific manner to humic acid macromolecules. This cationic binding perturbs the electrostatic interactions as well as hydrogen bonding, etc. between the functional groups which results in changes in the conformation of the flexible humic macromolecules. Such changes open the polymer structure and allow greater migration of the metal ions to specific binding sites which induces further conformational changes. Such effects are also related to the size of the humic macromolecules. Eventually, an equilibrium would be established between the cations which can be described as more weakly bound (faster dissociation kinetics) and those more strongly bound (slower dissociation kinetics). These studies have shown that there is such an equilibrium since, when the 'weakly' bound fraction is removed (by ion exchange), it is restored by release of metal ions from the strong sites over a period of time equivalent to that required to reach the initial ratio of weakto-strong binding by metal addition to the humic acid. It is also possible that acid-catalyzed dissociation could explain the decrease in the rates of uranyl dissociation with increasing pH. However, it has been shown that conformational effects with increasing pH affect many properties of polyelectrolytes associated with a more open structure. It would be quite difficult to interpret [H+] dependencies of the rate constants in such simple terms as acid catalysis which would be accompanied by large conformational changes. The small rate of a fraction of the dissociation processes indicates that kinetics as well as thermodynamics of metal-humic interactions may need to be included in geochemical models of metal ion migration. ACKNOWLEDGMENT
This research was supported by a grant from the US Department of Energy-Office of Basic Energy Sciences Division of Chemical Sciences.
REFERENCES Buffle, J., Delassy, P. and Haerdi, W., 1978. Anal. Chim. Acta, 101: 339-357. Caceci, M.S. and Cacheris, W.P., 1984. Byte, 9: 340-362. Choppin, G.R., 1988. Radiochim. Acta, 44/45: 23-28. Choppin, G.R. and Cacheris, W.P., 1987a. J. Less-Common Met., 122: 551-554. Choppin, G.R. and Cacheris, W.P., 1987b. Radiochim. Acta, 42: 185-190. Choppin, G.R. and Williams, M.K.R., 1973. J. Inorg. Nuc!. Chern., 35: 4255-4269. Clark, S.B. and Choppin, G.R., 1990. In: D.C. Melchior and R.L. Bassett (Eds.), Chemical Modeling of Aqueous Systems II, Am. Chern. Soc. ACS Symp. Ser. No. 416, pp. 519-525.
38
G.R. CHOPPIN AND S.B. CLARK
Konova, M.M., 1966. Soil Organic Matter. Pergamon Press, Oxford. Manning, G.S., 1981. J. Phys, Chem., 85: 870-877. Marinsky, I.A., 1976. Coord. Chern. Rev., 19: 125. Olson, D.L. and Shuman, M.S., 1983. Anal. Chern., 55: 1103-1107. Rabinowitch, E. and Belford, R.L., 1964. Spectroscopy and Photochemistry of Uranyl Compounds. MacMillan, New York. Schnitzer, M. and Khan, S.U., 1972. Humic Substances in the Environment. Marcel Dekker, New York. Torres, R.A., 1979. Ph.D. Dissertation, The Florida State University. Wheeler, I.R., 1976. Limnol, Oceanogr., 21: 846-852. Wilson, M.A., Barron, P.F. and Gillam, A.H., 1981. Geochim. Cosmochim, Acta, 45: 17431750.
27
Elsevier Science Publishers B.Y., Amsterdam
The kinetic interactions of metal ions with humic acids Gregory R. Choppin and Sue B. Clarki Department of Chemistry, Florida State University, Tallahassee. FL 32306, USA (Received 23 May 1990; revision accepted II April 1991 )
ABSTRACT Choppin, G.R. and Clark, S.B., 1991. The kinetic interactions of metal ions with humic acids. Mar. Chern., 36: 27-38. Complexation of europium (III) and UO~+ by humic acid was found to be very fast « I m ). However, passage through ion exchange resin removed a fraction which decreased as the contact time increased over' a 2-3 day period. The dissociation kinetics for UO~+ -humate were described by an equation with five first order terms with rate constants ranging from 40.6 rnr ' to 5.64X10- 3 m -I. The percentage of UO~+ dissociating by the slower paths, comparable to that eluting with the humic acid through the ion exchange resin is assigned to internal site-bound cations (strong binding). The faster dissociating fraction corresponded roughly to the UO~+ removed by the cation exchange resin (weak binding). The time dependence ofthe weak to strong binding ratio was associated with change in the conformation of the macromolecular humic acid as the cations neutralized the charge repulsions of the anionic humate.
INTRODUCTION
Humic substances, which are collections of organic polyelectrolyte macromolecules are the principal organic components of soils and waters. Their concentration, average molecular weight and functional group constitution are dependent on many factors such as climate, pH, substrate topography and time. In surface waters, the dissolved organic carbon (DOC) ranges from 0.1 to 50 ppm (mg 1-1 C). In ocean waters, the DOC ranges from 0.5 to 1.2ppm, with the higher concentrations occurring in the surface waters. In addition, most waters contain an amount of suspended particulate organic matter which in the ocean equals about lO% of the DOC. In all waters, there also can be suspended inorganic material with organic coatings. A majority of the DOC as well as the particulate and sorbed organic matter falls within the classification of humic substances. 'Present address: Savannah River Laboratory, Aiken, SC 29808, USA.
0304-4203/91/$03.50 © 1991 Elsevier Science Publishers B.Y. All rights reserved.
28
G.R, CHOPPIN AND S.B. CLARK
Humic substances are subdivided by solubility. The fraction which is soluble at all pH values is called fulvic acid, that soluble above a pH of approximately 3.5 is humic acid and humin is the fraction insoluble at all pH values. For humic material from different locations, the spectrum of molecular weights, the nature of the carbon skeleton and the types, positions and relative numbers of functional groups vary widely, in part depending on the origin and age of the humic material. Acid-base titration, Carbon-13 nuclear magnetic resonance spectroscopy by the cross-polarization, magic angle spinning technique (CP /MAS ), and ultrafiltration provide data which allow definition of major operational characteristics ofthese materials. The diversity of functional groups allows binding of a large number of metal ions. Since these humic substances are ubiquitous, they can have significant roles in the geochemical speciation of metals. Not only can they retard metal ion migration (e.g. when the humics are sorbed to surfaces) or promote it (e.g. by metal complexation to soluble or colloidal humics ), these substances can also influence the oxidation state (e.g. PuO~+ is rapidly reduced to Pu4+ by humics) (Choppin, 1988). The binding constants to humic acids for the f-elernent cations (e.g. Eu3+, Th 4+, UO~+) are dependent on pH, reflecting increased ionization of the carboxylate binding groups. At pH 6 the values of the conditional binding constant, logj31l for tracer metal concentrations were found to be: Eu 3+, 11.5; Th 4+, 15.0; UO~+, 9.0 (Choppin, 1988). Increased ionization also induces changes (due to increased intergroup repulsions) in the conformation of the macromolecular structure. The role of such structural changes is not very well understood at present. The interaction of cations with anionic polyelectrolytes has been described by two different models. In one description, which uses the Poisson-Boltzmann equation, the metal ions associated with a polyelectrolyte molecule are considered site-bound (i.e. associated with specific binding groups of the polyelectrolyte) (Marinsky, 1976). The second model treats the metal ions as being mobile within a 'condensed' layer adjacent to the polyion surface (Manning, 1981). Little has been done to examine either the relationship between this condensation (or, territorial) binding and site binding or the extent to which one or the other exists in a particular system. Insight into metal ion interactions with the organic polyelectrolytes can be obtained from a study of the dissociation kinetics. When Sm3+ and Th4+ cations were added to solutions of humic acid, the cations were bound completely in less than a minute (Choppin and Cacheris, 1987a,b; Clark and Choppin, 1990). When these cations were dissociated from the humic acid, the kinetic expression could be resolved into five to seven first order pathways. The dissociation terms fall into two classes based on their dependence on pH and temperature and, primarily, their half-lives. In the first class, the dissociation rates fall in the time domain of seconds, while the second group
METAL ION INTERACTIONS WITH HUMIC ACIDS
29
have rates in the minutes to hours time span. The amount of metal dissociating by the longer lived pathways increased the longer the metal was allowed to stay bound to the humate prior to removal. Such variation in the relative populations of the dissociative pathways was observed for periods of metalhumate binding of less than 2 days but not for longer predissociative binding times. These kinetic studies have been extended to uranyl cation, UO~+ . We also report further binding studies on Eu 3 +. The effects of pH, metal ion loading and average molecular weight of the humic acid have been investigated. EXPERIMENTAL
Reagents
A stock solution of U0 2 (Cl0 4 h was prepared by dissolving U0 2 (N0 3 h' 6H 20 in concentrated HCl0 4 . After several evaporations to dryness and dissolution in 0.1 M HCl0 4 , the salt was diluted to volume with 0.1 M HCl0 4 and the solution was standardized by spectrophotometric measurement of uranyl (2=416 nm, <==7.82 crnr ' M- 1, (Rabinowitch and Belford, 1964». The preparation of other solutions followed the procedures in Clark and Choppin ( 1990). Unless otherwise noted, experiments were conducted with solutions at 0.1 M ionic strength (NaCl0 4 ) and buffered with 0.01 M acetic acid or MES (4-morpholine-ethane sulfonic acid). Humic acid
Unfractionated humic acid was extracted from sediments from Lake Bradford in Florida and characterized as reported by Torres (1979). A portion of this humic acid was fractionated by size using ultrafiltration. During ultrafiltration, extreme care was used to ensure constant experimental conditions (Wheeler, 1976; Buffle et al., 1978). The stock solutions contained less than 150 mg I-I humic acid at pH 7.0 (buffered with 0.01 M Ntris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, TES), and 0.1 M (NaCl) ionic strength. An Amicon No. RG5 (51) fiberglass reservoir, pressurized to approximately 50 psi with N 2 and connected to an Amicon No. 8050 (65 ml) ultrafiltration cell with constant stirring was used. The humate fractions were those retained above the Amicon XM300 membrane (approximate size > 300 000 daltons) and the fraction that eluted through the Amicon YM I00, but was retained above the Amicon XM50 (approximate size 50 000-100 000 daltons). All humic acid samples were characterized by potentiometry to determine the carboxylic group capacity and by UVjvisible and 13C NMR. The poten-
30
G.R. CHOPPIN AND S.B. CLARK
tiometry measured pH changes of the humic acid solutions (0.01 g HA in 100 ml) under N 2 from pH 3.7 to 10.2 during titrations with CO 2-free NaOH (0.10 M). A rebuilt (On-Line Instrument Systems) Cary-14 interfaced to a Zenith 248 computer was used to measure the absorbance of a 250 mg 1-1 solution of each humate fraction (buffered to pH 7.0 with 0.01 MTES,,u=O.l M NaC1) at 465 nm and at 665 nm. The ratio of the absorbances (i.e. the £4/£6 ratio) is related to the degree of aromatic character (Konova, 1966; Schnitzer and Khan, 1972). Carbon-13 NMR spectra were collected on solid humic acid samples with an IBMjBruker WP 200 SY instrument employing cross-polarization with magic angle spinning (Wilson et al., 1981). The spectra were divided into three regions of interest: the carboxylate carbons (160-185 ppm), the olefinic carbons (100-160 ppm), and the aliphatic carbons (10-90 ppm). The area under the NMR spectrum for each region of interest was obtained by integration and normalized by dividing by the total spectral area. Binding studies
The extent and rate of Eu3+ binding to each humate fraction was studied using ultrafiltration and ion exchange. An Amicon No. 8010 (10 ml) ultrafiltration cell containing a YM2 (1000 dalton) membrane was loaded with a solution of humic acid. While stirring, an aliquot of Eu 3 + spiked with radiotracer europium-152 was added. A small portion of the solution in the cell was withdrawn for measurement of the europium-152 (to confirm the Eu concentration). The cell was immediately pressurized to 20 psi to cause filtration. After filtering, a sample of the filtrate was collected and analyzed for humic acid by spectrophotometry to ascertain complete removal of the humic acid. The filtrate and the prefilter sample were analyzed for europium-152 using a well-type NaI detector with a single channel analyzer. Ion exchange was used to monitor changes in binding as a function of the length of time Eu3+ was in contact with the humate. Cation exchange resins have been shown to separate rapidly and quantitatively uncomplexed lanthanide ions from their complexes (Choppin and Williams, 1973). Eu3+ spiked with europium-152 was added to a solution of humic acid. At fixed time intervals after the addition, aliquots were withdrawn and passed through a 1.2 em (i.d.)X5.7 em column of Dowex 50X4, 100-200 mesh resin. Europium-humate complexes were eluted rapidly using 5 ml of acetate buffer ( < 30 s elution time); the Eu3+ retained by the resin was eluted with 0.1 M Hel. Each eluant fraction was counted for europium-ISz.
METAL ION INTERACTIONS WITH HUMIC ACIDS
31
Dissociation studies The rate of UO~+ dissociation from the humic acid samples was determined using a ligand exchange technique (Choppin and Cacheris, 1987a,b). After equilibrating the metal-humate complex for a fixed amount of time, arsenazo(III) was added to the solution. The arsenazo(III) forms more stable complexes with UO~+ than does humic acid, thereby preventing the reformation of the UO~+ -humate complex. The intrinsic rate ofarsenazo complexation is very rapid relative to the dissociation rate of the humate complex. Consequently, the observed rate of arsenazo complexation is the rate ofmeta1 dissociation from the humic acid. Alteration of the concentration of arsenazo was found not to affect the rate of humate dissociation, indicating no catalysis by the arsenazo of the dissociation of the humate complex. The rate of UO~+ -humate dissociation was determined by following the growth of the arsenazo complex spectrophotometrically (Amax = 645 nm for UOz-arsenazo ). The slower dissociation processes (t 1/2> 0.5 min) were monitored using a conventional Milton Roy Spectronic 1201 spectrophotometer, while the more rapid dissociations were measured with a DurrumGibson D-109 stopped-flow spectrophotometer interfaced to an IBM PC XT. The dissociation kinetics were measured for samples in which the loading (mEq UO~ + as a percentage of total milliequivalence of carboxylate groups of humic acid) was 2.5, 5, and 9.5%. Above 10% loading, precipitation occurred. To study the different size humic acid samples, 5%loading was used.
Data analysis All calculations were performed on a Zenith ZF-158-42 PC. To determine the number of first order components in the dissociation, analysis by the kinetic spectrum method (Choppin and Cacheris, 1987a,b) was used. This technique uses the change in absorbance and J absorbance, with time. The utility of this approach had been shown for Cu2+ dissociation from humic acid (Olsen and Shuman, 1983). This method utilizes a unitless distribution function, H(k,t), defined by H(k t) = 15 2 (J absorbance) _ 15 (J absorbance) 15 On t) z J On t) ,
where the first term is the second derivative of the variation of absorbance with time and the second term is the first derivative. A plot of H(k,t) vs. In time yields a curve with a maxima for each first order process, the position of which provides an estimate of the half-life for that process. The resolved area of each peak is related to the percentage of metal ion dissociation with that lifetime. The kinetic spectra were calculated using a cubic spline smoothing calcu-
32
G.R. CHQPPIN AND S.B. CLARK
lation (Caceci and Cacheris, 1984). Since the domain of the kinetic spectrum is In t, small errors in peak position correspond to large errors in rate constants. Therefore, the kinetic spectrum analysis was used to obtain preliminary estimates of the rate constants and the percentage dissociation by each process. These values were used as the initial estimates in a simplex non-linear regression fit of the spectral data to obtain the half-lives and populations of the decay paths. RESULTS
Humate characterization Table 1 gives the results of the characterization studies of the fractionated humate samples plus the data of Torres (1979) for the unfractionated humic acid. The sizes for the two fractions relate to the membranes used in ultrafiltration, A/D indicates the ratio of the amounts of aliphatic to olefinic carbon determined by 13C NMR.
Unfractionated humate studies Using ultrafiltration, it has been found that when Ln 3+ in amounts equal to 5%of carboxylate capacity of the humic acid sample was mixed with humic acid solution and immediately (in less than 1 min) filtered, the metal ion was completely retained with the filtered humic acid (Clark and Choppin, 1990). This was interpreted as indicating very rapid, complete binding of the metal cations. When Eu3+ (also 5% loading) was added to a humic acid solution and the solution was passed through a column of cation exchange resin 15 min after mixing, only 4% of the Eu3+ eluted with the humic acid (Clark and Chopp in, 1990). Increasing the time to 48 h between mixing and passage through the resin (i.e. the contact time) increased the amount eluted to 40%. TABLE I Characterization of humate samples
A/O'
Size fraction (daltons) ~300000
50000-100000 Unfractionated
Carboxylate Capacity (mEq g-I)
6.15 6.81 6.94
1.9 1.9 1.6
3.52±0.22 6.89 ±0.56 3.86±0.03 b
"Ratio of aliphatic to olefinic carbon as calculated from the areas in the aliphatic and olefinic regions of the 13C NMR spectra. "Torres (1979).
METAL ION INTERACTIONS WITH HUMIC ACIDS
33
These studies have been repeated for a series of contact times with the results shown in Fig. 1 for both 2.5 and 5% loading. The simplest interpretation of the ultrafiltration and ion exchange resin studies is that the cations immediately bind, largely by condensation or by binding to surface sites. With time, changes in the macromolecular conformation occur within the humate which result in an increasing fraction of the metal ions being retained in interior sites. It is tempting to interpret the ion exchange data as related to strong binding (the fraction which elutes with the humic acid) and weak binding (the fraction which is removed from the humic acid by the resin). When the eluant sample of europium + humic is allowed to stand for between 28 (2.5% loading) and 48 h (5% loading) and then passed through the resin column, from only 30 (5%) to 40% (2.5%) was eluted. This is evidence that the 'weak' and 'strong' binding is an equilibrium distribution and is reversible since the percentage of europium (III) eluted for a solution after a 2 day mixing is the same as that eluted when a 'stripped' solution is allowed to stand 2 days before being passed again through the column. When bound UO~+ (5% loading) was dissociated from the humic acid using the arsenazo(III) competitive ligand method, the analysis of the kinetic spectrum showed five distinguishable first order paths (Figs. 2 and 3). The calculated rate constants and the percentage of UO~+ dissociating by each pathway are given in Table 2. The percentage of UO~+ dissociating by each process was dependent on the length of time the metal ion was in contact with the humate prior to initiating dissociation. Analysis of the dissociation kinetics following very short binding times (5 min or less) showed that most of the UO~+ dissociated via the two short-lived pathways. Increasing the predissociative binding time caused an increase in the percentage of dissociation by the three longer lived processes. For predissociative contact times longer than 2 days, no further 301::J
7
201-
::J
W
a.!2 101- 10
2
Fig. 1. Percentage of europium (III) eluting as humic acid complex as a function of the preelution contact time of europium(III) and humic acid: (Eu(III)] =2.50X 10- 6 M (D); [Eu(IlI}J=5.00XIO- 6 M (e); [-COi]=1.00xlO- 4 Eq/l-I; 1=0.10 M (NaCI0 4 ) ; pH=4.2.
34
G,R. CHOPPIN AND S,B.CLARK 2,50,---------------.
N~
2,00-
i
Q
2:S
g-
1,501-
~
1,00/---
r
!
o~
a
I
o
3
2
4
5
Ln Time (5) Fig, 2. Kinetic spectrum for fast UO~+ dissociation processes from the humic acid complex: [UO~+]=9.97XlO--6M; [-C0 2 ofHu]=2.00XI0- 4Eql-'; (Arsenazo (III)]=1.25XIO- 5 M; contact time e cS h; pH=4.22; T=295 K.
2.001-
a c
0 00
a
°CXbeeP'cP""q,~0
N
Q
a a a a a
1,501-
X
o
0"0&' 00
0
0,592
2
I
4
I
6
8
Ln Time (rn) Fig, 3, Kinetic spectrum for slow UO~+ dissociation processes from the humic acid complex: (UO~+]=9,97XlO-6M; [-C0 2 ofHu]=2.00XlO- 4Eql-l; [Arsenazo (III)] = 1.25X 10- 5 M; contact time=48 h; pH=4.22; T=295 K. TABLE 2 Rate constants and percentage dissociation for UO~+ decomplexation by humic acid Rate constant kl
k2
k3 k4 k5
= (6,76±0.11) X10- 1 S... I =(40.6±0.7) m" = (10,03 ± 0,26) X 10-- 2 s-I = (6,02±0.16) m- I = (7.83±0.75) X 10- 1 m- I = (6.98±0.63) X 10- 2 m>' = (5.64±0,62) X 10- 3 m- I
% Dissociation
16.02±3.01 21.43 ± 5.63 15,61±4.67 15.75±4.83 10.36±2.69
T=25°C, 1=0,10 M (NaCI0 4 ) , pH 4.2.
changes were observed in the percentages of metal ion dissociating by each process. To establish a constancy in the dissociation kinetics with Sm3+ and Th 4 + 2 days were also required (Choppin and Cacheris, 1987a,b; Clark and Choppin, 1990).
METAL ION INTERACTIONS WITH HUMIC ACIDS
35
The kinetics for UO~+ dissociation were found to be dependent on pH and temperature. As the pH of the medium was increased from 4.2 to 5.2, the dissociation rates decreased as shown in Figs. 4 and 5. It is probable that this is due to the increased ionization of carboxylate groups of the humic acid as the pH increases. The resultant increase in net negative charge on the humic macromolecule would retard cation dissociation. For all pathways, increasing temperature increased the rate of dissociation; however, the longer lived dissociation processes were noticeably more sensitive to temperature changes than the shorter lived processes. Such differences between the shorter and longer lived processes were noted also in dissociation studies of Th4+ from humic acid (Choppin and Cacheris, 1987a,b). The effect of altering lanthanide loading on to humic acid was studied. Ultrafiltration data showed that in the range of 2.5-9.5% loading, complexation was very fast and complete. When Eu3+ in excess of 10% of the carboxylate capacity of the humic acid was added, coagulation and precipitation occurred. Increasing the loading increased the amount of binding time required to reach an equilibrium between the 'strong' and 'weak' binding in the
0-
I
·~.7-o- - - - - , , . L , - - - - - ; : - L ; : - - - - _ ; : _ '
5.5
5.0
4.5
pH
Fig. 4. Variation of'log k) and log k2 with pH for fast processes of uranyl dissociation from humic acid: [UO~+ I =9.97x 10- 6 M; [-C0 2 of Hul =2.00X 10- 4 Eq 1-1; [Arsenazo (III) I = 1.25 X 10- 5 M; contact time=48 h; T=295 K.
-
0
-0---11--0--0_ k l
+
+ 4.5
5.0
5.5
6.0
pH
Fig. 5. Variation of the log k, with pH for the slow processes of U01+ dissociation from humic acid: [UO~+ I =9.97X 10- 6 M; [-C0 2 of Hul =2.00X 10- 4 Eq 1-'; [Arsenazo (III) I = 1.25 X 10- 5 M; contact time=48 h; T=295 K.
36
G.R. CHOPPIN AND S.B. CLARK
TABLE 3
Summary of dissociation results No. fast processes
No. slow processes
% Fast dissociation
1112 of
Humic acid (un fractionated) 2.S%load 1.0±0.S
3
3
22.83 (m)
S% load
2.0±0.S
3
3
9.S%load
3.S±0.5
3
4
55.28 ±S.13 66.34 ±6.27 65.71 ±6.20
3
3
2.97 (h)
3
3
64.23 ±6.83 71.88 ±6.47
Metal loading
Equilibrium binding time (days)
Humic acid (fractionated, 5% load) ;<; 300000 daltons 3.0±0.5 SOOOO-IOOOOO daltons
2.0±0.5
slowest process
41.56 (m) 8.12 (h)
47.54(m)
ion exchange elution measurements. Also, the percentage of 'strongly bound' Eu3+ decreased with loading and an additional long-lived dissociation pathway appeared in the kinetic spectrum at high humate loading. These results are summarized in Table 3. These results must be assessed with some uncertainty, as the variable loading has a number of possible and largely unknown effects on the humic acid macromolecules and the covariability which must be present may not be properly reflected in the parameters measured.
Fractionated humic acidstudies The carboxylate capacities of the size-fractionated humate samples differed. However, we conducted our studies using the same carboxylate concentrations in terms of milliequivalence carboxylate per milliliter solution and with 5% loading of lanthanide ions. Ultrafiltration studies confirmed rapid, complete binding, while ion exchange again demonstrated changes in the labilities of the complexes with increasing binding time. Table 3 shows that the contact time required for equilibrium in the 'stronger' and 'weaker' binding increases as the size of the humic macromolecule increases. The half-life for dissociation by the slowest process (the strongest binding?) is also greater for the larger molecular weight fraction. These observations are consistent with slower conformational change. The percentage bound by the faster dissociating modes is smaller for the lower molecular weight fraction, although the uncertainties in the data in Table 3 are large. The fact that the carboxylate capacities (Table 1) differed by a factor of almost 2 had no significant effect at 5%loading.
METAL ION INTERACTIONS WITH HUMIC ACIDS
37
DISCUSSION
The results are consistent with a model in which metal ions bind rapidly and largely in a non-specific manner to humic acid macromolecules. This cationic binding perturbs the electrostatic interactions as well as hydrogen bonding, etc. between the functional groups which results in changes in the conformation of the flexible humic macromolecules. Such changes open the polymer structure and allow greater migration of the metal ions to specific binding sites which induces further conformational changes. Such effects are also related to the size of the humic macromolecules. Eventually, an equilibrium would be established between the cations which can be described as more weakly bound (faster dissociation kinetics) and those more strongly bound (slower dissociation kinetics). These studies have shown that there is such an equilibrium since, when the 'weakly' bound fraction is removed (by ion exchange), it is restored by release of metal ions from the strong sites over a period of time equivalent to that required to reach the initial ratio of weakto-strong binding by metal addition to the humic acid. It is also possible that acid-catalyzed dissociation could explain the decrease in the rates of uranyl dissociation with increasing pH. However, it has been shown that conformational effects with increasing pH affect many properties of polyelectrolytes associated with a more open structure. It would be quite difficult to interpret [H+] dependencies of the rate constants in such simple terms as acid catalysis which would be accompanied by large conformational changes. The small rate of a fraction of the dissociation processes indicates that kinetics as well as thermodynamics of metal-humic interactions may need to be included in geochemical models of metal ion migration. ACKNOWLEDGMENT
This research was supported by a grant from the US Department of Energy-Office of Basic Energy Sciences Division of Chemical Sciences.
REFERENCES Buffle, J., Delassy, P. and Haerdi, W., 1978. Anal. Chim. Acta, 101: 339-357. Caceci, M.S. and Cacheris, W.P., 1984. Byte, 9: 340-362. Choppin, G.R., 1988. Radiochim. Acta, 44/45: 23-28. Choppin, G.R. and Cacheris, W.P., 1987a. J. Less-Common Met., 122: 551-554. Choppin, G.R. and Cacheris, W.P., 1987b. Radiochim. Acta, 42: 185-190. Choppin, G.R. and Williams, M.K.R., 1973. J. Inorg. Nuc!. Chern., 35: 4255-4269. Clark, S.B. and Choppin, G.R., 1990. In: D.C. Melchior and R.L. Bassett (Eds.), Chemical Modeling of Aqueous Systems II, Am. Chern. Soc. ACS Symp. Ser. No. 416, pp. 519-525.
38
G.R. CHOPPIN AND S.B. CLARK
Konova, M.M., 1966. Soil Organic Matter. Pergamon Press, Oxford. Manning, G.S., 1981. J. Phys, Chem., 85: 870-877. Marinsky, I.A., 1976. Coord. Chern. Rev., 19: 125. Olson, D.L. and Shuman, M.S., 1983. Anal. Chern., 55: 1103-1107. Rabinowitch, E. and Belford, R.L., 1964. Spectroscopy and Photochemistry of Uranyl Compounds. MacMillan, New York. Schnitzer, M. and Khan, S.U., 1972. Humic Substances in the Environment. Marcel Dekker, New York. Torres, R.A., 1979. Ph.D. Dissertation, The Florida State University. Wheeler, I.R., 1976. Limnol, Oceanogr., 21: 846-852. Wilson, M.A., Barron, P.F. and Gillam, A.H., 1981. Geochim. Cosmochim, Acta, 45: 17431750.