Multi-Wavelength Molecular Fluorescence Spectrometry for

Environ. Sci. Technol. 1996, 30, 1565-1574

Multi-Wavelength Molecular Fluorescence Spectrometry for Quantitative Characterization of Copper(II) and Aluminum(III) Complexation by Dissolved Organic Matter JO ¨ R G L U S T E R , * ,† THOMAS LLOYD,‡ AND GARRISON SPOSITO Division of Ecosystem Sciences, 108 Hilgard Hall 3110, University of California, Berkeley, California 94720-3110

IAN V. FRY Department of Plant Biology, University of California, Berkeley, California 94720

Conditional stability constants and binding capacities are important parameters with which to estimate the biological availability of metal ions in aqueous solution in the presence of dissolved natural organic matter (fulvic acid, organic matter in natural waters or in aqueous extracts of forest litter). Determination of these parameters depends on analytical methods that can distinguish between free and organically bound metal ions. This speciation is difficult, mainly because natural organic matter typically is a complex mixture. In this paper, multi-wavelength molecular fluorescence spectrometry is evaluated prototypically as a method for the determination of stability constants and binding capacities for Cu(II) and Al(III) complexation by dissolved organic matter in a juniper leaf litter extract. Equilibrium ion exchange quantitation and electron spin resonance spectroscopy served as quantitative and qualitative reference methods, respectively. Three types of binding site for Cu and Al could be differentiated qualitatively by the reaction patterns of various wavelength regions of the total luminescence spectrum of the leaf litter extract in response to increasing metal ion addition. For both Cu (pH 6) and Al (pH 5), binding parameters for the two types of binding site forming the most stable complexes were deduced self-consistently from reactions evaluated at selected excitation/emission wavelength pairs.

0013-936X/96/0930-1565$12.00/0

 1996 American Chemical Society

Introduction Molecular fluorescence spectrometry (MFS) is a sensitive method for characterizing metal complexation by soluble humic substances (1, 2) and dissolved organic matter (DOM) in natural waters (3, 4) or in aqueous extracts of forest litter (5-7). Qualitative MFS studies typically have monitored changes in fluorescence intensity for a single excitation/ emission wavelength pair (EEWP) (8-12); changes in singletransect excitation, emission, or synchronous-scan spectra (2, 11, 13-17); or, most recently, changes in total luminescence spectra (TLS; multiple-transect excitation or emission spectra) (6, 7). Quantitative MFS studies (3, 1833) have been used to infer quasiparticle (34, 35) conditional stability constants (Kcqp) and binding capacities (Ltqp), with these two binding parameters usually determined following the mathematical approach introduced by Ryan and Weber (18). It assumes the formation of a single 1:1 quasiparticle complex (ML) and the existence of a linear relationship between the concentration of metal complexed by organic ligands and the change in fluorescence intensity. This latter assumption was questioned by Cabaniss and Shuman (29), who found a nonlinear relationship between fluorescence quenching and Cu(II) bound by an aquatic fulvic acid as measured by ion selective electrode potentiometry. This criticism remains controversial (36), however, and is perhaps vitiated by the fact that fluorescence quenching will be proportional to metal bound only if the true stoichiometry of the complexes represented as a 1:1 quasiparticle complex is 1:1 also. Otherwise, only a monotonic relationship is expected between quenching and metal bound. In all previous quantitative MFS studies, changes in fluorescence intensity upon metal complexation were monitored at a single EEWP, chosen usually at or near the maximum emission intensity in the TLS. The tacit assumption in this approach, that metal complexation is uniquely characterized by the use of a single EEWP, has apparently not been tested, although the question has been raised in connection with metal-induced fluorescence quenching and enhancement observed concurrently in synchronous-scan spectra of DOM (2, 16, 17). Luster et al. (37) have noted that metal complexation by DOM can provoke differing responses in different regions of a TLS, with the consequence that parametric characterization of metal complexes in terms of a quasiparticle model becomes dependent on which EEWP is selected for quantitative data analysis. The aim of the present study was to investigate this issue in detail for Cu(II) and Al(III) complexation by DOM in a model leaf litter extract (LLE). Copper(II) was chosen because it is the prototypical metal investigated in most fluorescence studies, and Al(III) was chosen because * Corresponding author fax: +411-739-22-15; e-mail address: [email protected]. † Present address: Swiss Federal Institute for Forest, Snow and Landscape Research, Zu ¨ rcherstrasse 111, CH-8903 Birmensdorf, Switzerland. ‡ Present address: Department of Environmental Engineering Science, Keck Laboratories 138/78, California Institute of Technology, Pasadena, CA 91125.

VOL. 30, NO. 5, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of its central role in the environmental chemistry of acidic soils (38). Juniper leaf litter was selected from a sample set including the leaf litter of various dominant tree species from different forested regions in the United States and Europe that are under multiple environmental stresses. The TLS of its aqueous extract exhibited a particularly pronounced reaction upon titration with the two metals, and therefore, it is a good choice with which to evaluate the potential of multi-wavelength MFS. The experimental approach included the equilibrium ion exchange method of Luster et al. (39) as a quantitative reference for the determination of Cu(II) and Al(III) binding parameters and electron spin resonance (ESR) spectroscopy as a qualitative reference for evaluating the structural significance of Cu(II) binding parameters.

Experimental Section and Data Analysis General. Chemicals were reagent grade, and water was purified by a Milli-Q water system (Millipore). Before each use, glassware and plasticware were soaked for about 24 h in 3 M HNO3, followed by rinsing with doubly-deionized water. Leaf Litter Extract (LLE). Samples were collected from a thin organic soil surface layer beneath Utah juniper (Juniperus osteosperma) in Coconino County, northern Arizona (elevation, 1900 m; exposure, SW). The residues of the branches covered by tiny leaves were separated from the bulk samples and divided into younger (beige) and older (greyish brown) material. These samples were dried at 40 °C and ground to a 40-mesh size. A suspension of 2.5 g of the younger material in 100 g of water in a polypropylene bottle was purged with nitrogen and mixed for 20 h on a horizontal shaker. It was filtered immediately through Whatman no. 2 and Millipore HV (0.45 µm) filters. The filtrates (pH ) 4.34 ( 0.04 and DOC ) 2900 ( 200 mg/kg) were further purified by elution over a strongly acidic cation exchange resin (BioRad AG 50W-X8, 20-50 mesh) in H+ form. Fluorescence Titrations. Sample solutions were prepared in low-density polyethylene bottles as follows. An amount of LLE to give a final concentration of 25 mg/kg DOC was added to 80 g of 0.01 mol/kg KClO4. An amount of a 0.01 or 0.001 M Cu(ClO4)2 or Al(ClO4)3 stock solution to give the final total concentration of Cu or Al was added while continuously adjusting pH to 6 (Cu titrations) or 5 (Al titrations). Finally, the solutions were made up to 100 g with 0.01 mol/kg KClO4 adjusted to pH 6 or 5, respectively. After equilibration of about 20 h, pH was adjusted, and fluorescence was measured in quartz cells (path length 1 cm) on a Perkin Elmer MPF-66 spectrometer. Excitation and emission monochromator entrance and exit slit widths were set to 5 nm. Total luminescence spectra were recorded as a series of emission or synchronous scan spectra. Surface plots of TLS were generated from the data with Graf Tools software (version 3.3.; Graphical Analysis System), contour plots with Deltagraph Professional software (version 2.0.1; Delta Point). For measurements made at a single EEWP, the average of three readings was used for data analysis. Changes in fluorescence intensity with increasing total Cu concentration, [Cu]t, at selected EEWP were analyzed mathematically using essentially the method of Ryan and Weber (18). Quasiparticle conditional stability constants, Kc, and binding capacities, Lt (dropping the superscript qp for simplicity),

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were determined by nonlinear regression analysis of a plot of I/Iref vs [Cu]t using eq 1:

I Iref

)1+

(

)

ICuL 1 -1 {1 + KcLt + Kc[Cu]t Iref 2KcLt

x(1 + KcLt + Kc[Cu]t)2 - 4Kc2Lt[Cu]t}

(1)

In this eq, I is the fluorescence intensity at a given [Cu]t and Iref the reference fluorescence intensity. The latter is the fluorescence intensity at the beginning of the analyzed range of a plot of I/I0 vs [Cu]t, with I0 being the fluorescence intensity of the metal-free LLE. Equation 1 is valid for both enhancement and quenching relative to Iref. The relative fluorescence intensity of the Cu-saturated complex, ICuL/ Iref, is a third parameter in eq 1 that can be determined by nonlinear regression analysis. Often, three-parameter nonlinear regression analysis of eq 1 using a simplex algorithm has led to unreasonably small values for Lt (20, 21, 28). The number of fitting parameters for eq 1 can be reduced to two by using a constant value for ICuL/Iref estimated from nonlinear regression analysis of a plot |I/ Iref - 1| vs [Cu]t using the equation:

|

I

Iref

| |

-1 )

|

ICuL - 1 (1 - e-R[Cu]t) Iref

(2)

with fitting parameters |ICuL/Iref - 1| and R. With Kc and Lt as the only remaining fitting parameters in eq 1, reasonable values for Lt can then be obtained. Titrations with Ion Exchange Quantitation. Suspensions of 60 mg of air-dried strongly acidic cation exchange resin (BioRad AG 50W-X8, 63-150 µm, K+ form) in 27 g of a solution containing 0-1.33 mmol/kg Cu(ClO4)2 in 0.0111 mol/kg KClO4 were equilibrated in polycarbonate culture tubes at pH 6 for 12 h while stirring at a rate of 300 rpm. Then 1.94 g of LLE (DOC ) 387 mg/kg) was added, and the suspensions were equilibrated for 60 h. During equilibration, pH was checked periodically and adjusted with dilute HClO4 or KOH. As soon as pH did not change more than 0.1 unit within 8 h, the solutions were made up to 30 g with water. The suspensions were filtered through 5- and 0.4µm polycarbonate membrane filters (Poretics and Nuclepore, respectively). After measuring their fluorescence as described above, the filtrates were acidified to pH 2 and analyzed for total Cu by inductively coupled plasma optical emission spectrometry (Perkin-Elmer Plasma 40). Free Cu2+ concentrations ([Cu]f) were calculated from total concentrations with the help of a separately recorded Cu(II) adsorption isotherm on the ion exchange resin. Concentrations of organically bound Cu(II) ([Cu]b) were calculated as the difference between [Cu]t and [Cu]f. Details of the data analysis are given by Luster et al. (39). Electron Spin Resonance Spectra. Copper(II) complexes of the LLE were prepared as for the fluorescence measurements. After equilibration of about 20 h, the dilute preparations were concentrated 100-fold using a rotary evaporator with a water bath temperature of 40 °C. ESR spectra of the frozen solutions were recorded at 85-90 K on a Bruker 200ER spectrometer operating at X-band frequency with 100-kHz magnetic field modulation and equipped with an Oxford Instruments ESR-900 cryostat. The spectrometer settings were 12 dB attenuation (12.5 mW power), 0.63 mT modulation amplitude, 9.38 GHz frequency, and 50 ms time constant. Nine 200-mT scans

FIGURE 1. Equilibrium ion exchange data of Cu(II) complexation by a juniper LLE (DOC ) 25 mg/kg) at pH 6 and µ ) 0.01 M; data points represent mean values of two replicates; fitting parameters and curves are deduced from nonlinear regression analysis of the data.

centered at 300 mT were averaged and processed to determine g-values (N,N-diphenylpicrylhydrazyl as gmarker) and hyperfine-coupling constants (A) using computer-scope programs from R. C. Electronics.

Results and Discussion Copper(II) Complexation Parameters by Equilibrium Ion Exchange. A plot of [Cu]b/[Cu]f vs [Cu]b is shown in Figure 1. It exhibits a characteristic shape, convex towards the x-axis and leveling out at [Cu]b/[Cu]f ≈ 1.0 for [Cu]b > 5 µmol/kg. This shape indicates strong binding at small Cu/ ligand and very weak binding at larger Cu/ligand, the latter possibly the result of binding sites being created with increasing Cu loading. Nonlinear regression analysis of this plot provided conditional stability constants, Kc, and binding capacities, Lt, for two 1:1 Scatchard quasiparticles, Cu(L1) and Cu(L2) (40). One set of binding parameters was determined by analyzing the entire titration curve and another by analyzing the part at low [Cu]b only (see Figure 1). The former set of binding parameters thus can be assumed to characterize all forms of organically bound Cu, and the latter to represent strongly bound Cu only. Total Luminescence Spectra. In the absence of Cu, the TLS of the LLE consisted of two major peaks with maxima at the EEWP 281/311 and 323/448 (wavelength in nm) and a shoulder around EEWP 280/360 (Figure 2a,b). The addition of 1.5 µmol/kg Cu (Cu/C molar ratio ) 1/1390) led to the appearance of a new peak with a maximum at EEWP 503/538 (Figure 2c,d). The rest of the spectrum remained largely unchanged. Upon further Cu(II) addition (cf. Figure 2e,f for [Cu]t ) 30 µmol/kg and Cu/C ) 69), the entire TLS, including the newly formed peak, was quenched. The relative quenching, however, was not uniform (Figure 2f). The peak at EEWP 323/448 was quenched much less than the peak at EEWP 281/311 or the shoulder around EEWP 280/360. The most pronounced quenching occurred for the low-fluorescence region at high excitation wavelengths (λex > 370 nm), except for a small region around EEWP 440/460. A peak at a similar EEWP as that of the major peak of the juniper LLE (323/448) has been described as the maximum fluorescence peak in almost all previous studies on the fluorescence of fulvic acid and DOM in natural waters or in LLE where this information is available (3, 18-20,

22-24, 29, 33, 41). The TLS of one surface water sample (42) and the intensity increase in synchronous scan spectra of various LLE from λex ) 320 to 300 nm (16, 17) are the only indications in the literature for the existence of a peak with a maximum at an EEWP similar to the other major peak of the juniper LLE (281/311). Since this wavelength region of the TLS is not included in most previous studies, the existence of such a peak for other DOM remains possible. The existence of a fluorescing Cu complex at small Cu/C may be unique to the juniper LLE, however. In order to study different regions of the TLS (Figure 2f) in more detail, relative intensities were plotted either vs [Cu]t (Figure 3a,d,g,j) or vs [Cu]b as calculated from [Cu]t using the binding parameters from ion exchange quantitation. The relationship of changes in fluorescence intensity to all forms of bound Cu was examined using [Cu]b calculated with the parameters obtained from analysis of the entire ion exchange titration curve (Figure 3b,e,h,k), whereas the relationship with strongly bound Cu only was examined using [Cu]b calculated with the parameters deduced from the low [Cu]b part of the ion exchange titration curve (Figure 3c,f,i,l). (1) EEWP 503/538 (representing region A in Figure 2f): The fluorescence intensity increased dramatically up to [Cu]t ) 1.5 µmol/kg (Figure 3a), with the increase being proportional to [Cu]b up to 1 µmol/kg (Figure 3b,c). Then the intensity decreased proportionally to strongly bound Cu for [Cu]t g 4 µmol/kg (Figure 3c). (2) EEWP 383/458 (region B): In this region, the fluorescence showed a small increase up to [Cu]t ) 1.5 µmol/kg, then remained almost constant up to [Cu]t ) 4 µmol/kg (Figure 3d,e,f), and finally decreased sharply and proportionally to weakly bound Cu for [Cu]t g 15 µmol/kg (Figure 3e). (3) EEWP 323/448 (region C): The fluorescence increased slightly up to [Cu]t ) 1.5 µmol/kg (Figure 3g,h,i) and then decreased proportionally to strongly plus weakly bound Cu (Figure 3h). (4) EEWP 281/311 (peak in region D) and 277/352 (shoulder in region D): A very small increase in fluorescence was observed up to [Cu]t ) 0.75 µmol/kg, followed by a sharp decrease (Figure 3j,k,l). Quenching of the peak was almost proportional to strongly bound Cu over the entire titration range, whereas quenching of the shoulder was linear only up to [Cu]t ) 15 µmol/kg (Figure 3l). Three types of binding site, forming very strong, strong, or weak complexes with Cu, thus can be inferred for the juniper LLE from the MFS data. At very small Cu/C, complexation by binding sites (type 1) forming very strong complexes leads to a marked increase of the fluorescence intensity in region A and a slight increase in regions B and C. Upon further Cu addition, complexation by binding sites (type 2) forming strong complexes causes a marked quenching of region D, of the newly formed peak in region A, and, to a lesser extent, of region C. Additional weak Cu complexation (type 3 sites) leads to further quenching in region C, with the same proportionality factor as complexation by type 2 sites, and to marked quenching of the fluorescence in region B. Copper(II) Complexation Parameters by MFS. Conventional MFS data analysis based on eq 1 requires a monotonic change of fluorescence intensity with increasing [Cu]t, which is proportional to [Cu]b. Such a change starting at [Cu]t ) 0 is observed mainly at EEWP 503/538 and, to


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FIGURE 2. Surface and contour plots of the TLS of a juniper LLE (DOC ) 25 mg/kg) at pH 6 and µ ) 0.01 M (a and b) and of its reaction upon the addition of 1.5 µmol/kg Cu(II) (c and d) and 30 µmol/kg Cu(II) (e and f).

a lesser extent, at EEWP 383/458 and 323/448 (Figure 3a,d,g). Data analysis at these EEWP for [Cu]t ) 0-1.5 µmol/kg and Iref ) I0 leads to logKc ≈ 7 and Lt ≈ 1 µmol/kg (Table 1). Of

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these parameters characterizing the very strong type 1 binding sites, those deduced at EEWP 503/538 can be considered most reliable.

FIGURE 3. Change of the TLS of a juniper LLE (DOC ) 25 mg/kg) at pH 6 and µ ) 0.01 M upon titration with Cu(II) at selected EEWP; mean values of two replicates are shown with error bars indicating the standard deviation under the assumption of no error for I0 (omitted in panels a, d, g, and j for better clarity); for information on the calculation of [Cu]b see text; for the binding parameters used to calculate the fitting curves in panels a, d, g, and j see Table 1; the lines in panels c, e, h, and l indicate the linear relationships mentioned in the text.

The decrease in intensity upon further Cu addition can be analyzed in terms of binding parameters characterizing the strong and/or weak complexes (type 2 and type 3 binding sites) under the assumption that quenching begins when the type 1 sites are saturated. For each titration point, [Cu]t is corrected by calculating Cu bound by type 1 sites (using log Kc and Lt from data analysis at EEWP 503/538), then subtracting this value from [Cu]t. Data analysis begins at the titration point with maximum fluorescence intensity Imax (EEWP in regions A-C: [Cu]t,uncorr. ) 1.5 µmol/kg, Figure 3a,d,g; EEWP in region D: [Cu]t,uncorr. ) 0.75 µmol/kg, Figure 3j), with Iref ) Imax. As a consequence of different behavior with respect to proportionality of I/Iref to [Cu]b, the results obtained at different EEWP differ greatly (Table 1). The binding parameters obtained for type 1 sites from data analysis at EEWP 503/538 were assumed to characterize

quasiparticle Cu(L1) as inferred from the ion exchange data. They were then combined with each of the parameter sets obtained from analysis of the quenching at different EEWP, assumed to characterize quasiparticle Cu(L2) as inferred from the ion exchange data. A good fit of the first part of the ion exchange data ([Cu]b e 5 µmol/kg) was obtained by combination with the parameter sets from data analysis at EEWP 281/311 and 340/375 (Figure 4). At both EEWP, the same approximately linear relationship between quenching and strongly bound Cu was observed (shown for EEWP 281/311 in Figure 3l). A good fit of all ion exchange data was obtained by combination with the parameter set from analysis at EEWP 323/448 (Figure 4) where a linear relationship between quenching and both strongly and weakly bound Cu was found (Figure 3h). Combination with all other parameter sets, i.e., with those determined


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TABLE 1

Conditional Binding Parameters for Cu(II) Complexation by a Juniper LLE as Determined by MFS at Different EEWP region in TLS (Figure 2f)

λex

λem

A

503

538

B

383

458

C

323

448

D D D

340 281 277

375 311 352

[Cu]t (µmol/kg)

log Kc

Lt (µmol/kg)

ICuL/I0

0-1.5 1.5-40 4-40 0-1.5 1.5-40 0-1.5 1.5-40 0.75-40 0.75-40 0.75-40

7.47 5.83 5.61 6.99 5.47 7.21 4.79 5.88 5.76 5.83

1.1 9.0 4.1 0.8 20.2 0.6 20.9 4.0 4.9 9.6

3.170 0.085 0.036 1.076 0.516 1.070 0.600 0.663 0.733 0.671

fit-signature in Figure 3 ‚‚‚ s --‚‚‚ s ‚‚‚ s s ---

TABLE 2

Conditional Binding Parameters for Cu(II) and Al(III) Complexation by a Juniper LLE at µ ) 0.01 M As Determined by Multi-Wavelength MFS Mz+

pH

Cu2+ (this study)

6

Al3+ (37)

5

Lt (mol/ log Kc [kg C])

EEWP (nm/nm)

type 1 type 2

7.47 5.82

0.045 0.18

types 2 and 3 type 1

4.79 8.05

0.84 0.023

type 2

5.79

0.43

503/538 340/375 281/311 323/448 322/357 310/460 390/425

binding site

FIGURE 4. Equilibrium ion exchange data of Cu(II) complexation by a juniper LLE (DOC ) 25 mg/kg) at pH 6 and µ ) 0.01 M; data points represent mean values of two replicates; fitting parameters and curves are deduced from data analysis of the change in fluorescence intensity of the juniper LLE TLS at selected EEWP upon Cu addition.

at EEWP where no linear relationship between quenching and [Cu]b was observed, did not lead to good fits of the ion exchange data. In all these cases, [Cu]b was overestimated. An extended data analysis of the quenching at EEWP 503/538 was based on the perfectly linear relationship between I/I0 and strongly bound Cu for [Cu]t g 4 µmol/kg. A new Iref was defined as the intensity at the intersection of the extrapolations of the two linear parts of the plot of I/I0 vs strongly bound Cu, as indicated in Figure 3c. Data analysis for [Cu]t g 4 µmol/kg, using the newly defined Iref and correcting [Cu]t as described above, led to binding parameters similar to those deduced from the quenching at EEWP 281/311 (Table 1). Thus, if [Cu]t is corrected for complexation by type 1 sites, parameters for the type 2 sites can be obtained from the quenching of the peak centered at EEWP 281/311 or the region around EEWP 340/375 (Table 2). Only by an extended data analysis can parameters for type 2 sites be deduced from the quenching of the type 1 complex fluorescence in region A. A parameter set characterizing both type 2 and type 3 sites can be obtained from the quenching of the peak in region C (Table 2). No convincing way was found to deduce parameters for type 3 sites (weak Cu complexes) from the marked quenching in the region around EEWP 383/458 (Figure 3e). Electron Spin Resonance Spectra. Figure 5 shows ESR spectra of 100-fold concentrated preparations of juniper

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FIGURE 5. ESR spectra of Cu(II) complexes of a juniper LLE; gain was 1 × 106 (Cu/C ) 1/2080 and 1/1040) and 2.5 × 105 (Cu/C ) 1/260 and 1/52), respectively; the vertical scale of the left-hand part of the spectra is enlarged by a factor of 10 with respect to the scale of the right-hand part; positions of the hyperfine-split parallel signals 1-3 (Table 3) are indicated.

LLE complexes with initial Cu(II) concentrations of 1, 2, 8, and 40 µmol/kg (Cu/C ) 1/2080, 1/1040, 1/260, and 1/52, respectively). All spectra exhibit an anisotropic signal around g ) 2, typical for Cu(II) in a square planar or tetragonally distorted octahedral ligand field. The position of the perpendicular component does not depend on Cu/C

TABLE 3

ESR Spectral Parameters for Cu(II) Complexes of a Juniper LLE signal 1

signal 2

signal 3

Cu/C

no. of spectra

g|

A| (cm-1 × 104)

g|

A| (cm-1 × 104)

g|

A| (cm-1 × 104)

1/2080 1/1040 1/520 1/260 1/52

3 3 5 4 5

2.359 ( 0.002 2.355 ( 0.002 2.348 ( 0.003 2.349 ( 0.003 2.335 ( 0.003

143 ( 2 147 ( 1 158 ( 1 155 ( 5 154 ( 1

2.299 ( 0.003 2.300 ( 0.001 2.299 ( 0.001 2.294 ( 0.003 2.306 ( 0.003

173 ( 4 174 ( 3 174 ( 3 179 ( 2 176 ( 1

2.259 ( 0.003 2.249 ( 0.006 2.263 ( 0.002

185 ( 3 197 ( 3 185 ( 3

and is located at g⊥ ) 2.066 ( 0.001 (n ) 22). The parallel component in all spectra is composed of three signals that can be interpreted as arising from Cu(II) in different binding environments. The corresponding g|-values and hyperfine-coupling constants A| are listed in Table 3. Up to Cu/C ) 1/260, a signal centered at g| ≈ 2.30 with A| ≈ 175 × 10-4 cm-1 is dominant. Such a signal has been observed for Cu(II) complexes with fulvic acid (43-47) and leaf litter (48, 49), being attributed to a square planar inner-sphere complex with four O ligand atoms. A signal arising from Cu(II) in a stronger ligand field, with g| ≈ 2.26 and A| ≈ 190 × 10-4 cm-1, is apparent in the spectra only up to Cu/C ≈ 1/500. Such a signal in the spectra of Cu(II) complexes of fulvic acid and soil fungal polymers has been attributed to a mixed 2O/2N ligand environment (44, 45, 48, 50). A signal at g| ≈ 2.35 with A| ≈ 155 × 10-4 cm-1, typical for Cu weakly bound to O functional groups and water molecules (51), is small at low Cu/C but becomes predominant at Cu/C ≈ 1/50. Molecular Significance of Cu(II) Complexation Parameters. At Cu/C 1/250, ESR spectroscopy suggests weak Cu binding by O ligands, including water. Region C of the TLS, assigned to simple phenolic compounds, and region B are quenched. The latter region includes fluorescence of highly conjugated phenols (e.g., coumarins) and ketones (e.g. quinones) (56). The conditional stability constant (log Kc ) 4.8) deduced from quenching at EEWP 323/448 (region C), which characterizes complexation at intermediate and high Cu/C, is not typical for the inner-sphere Cu(II) complex of any particular model compound. Thus, the weakest Cu(II) binding may be caused by outer-sphere complexes involving ketonic, phenolic, and probably carboxylic functional groups. Al(III) Complexation by the Juniper LLE. The TLS of the juniper LLE at pH 5 (Figure 6a,b) was almost identical to that at pH 6 except for higher intensity of the peak at EEWP 281/311. Small Al(III) additions did not lead to a particularly pronounced response of a single region as in the case of Cu(II). Upon addition of 50 µmol/kg Al (Al/C ) 1/42), the peak at EEWP 323/448 shifted to 330/410 and increased drastically in intensity (Figure 6c). Because of this shift, I0 was quenched in region C* of the TLS and enhanced in region D*, with a maximum enhancement at EEWP 330/380 (Figure 6d). In addition, the appearence of new peaks at EEWP 420/440 and EEWP 515/565 led to a marked increase of I0 in regions B* and A* (Figure 6d). For region E*, with the peak at EEWP 281/311 and the shoulder around EEWP 280/360, only a very slight quenching was observed. In a qualitative TLS study on aqueous pine LLE (6), high Al additions caused a wavelength shift and an intensity increase of peaks around EEWP 320/450 that were similar to those in the present investigation. Both enhancement (2, 16, 17, 25) and quenching (17, 24, 32, 33) of fulvic acid or DOM fluorescence upon the addition of Al have been described in qualitative single-transect spectra and quantitative single-EEWP studies. In these studies, the regions of fluorescence quenching and enhancement were similar to those found for the juniper LLE. In a detailed investigation, using plots of relative fluorescence intensity at selected EEWP vs [Al]t and [Al]b


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FIGURE 6. Surface and contour plots of the TLS of a juniper LLE (DOC ) 25 mg/kg) at pH 5 and µ ) 0.01 M (a and b) and its reaction upon the addition of 50 µmol/kg Al(III) (c and d).

(37), three types of Al(III) binding site could be distinguished for the juniper LLE. At very small Al/C, complexation by type 1 binding sites led to a slight enhancement of the entire TLS. Upon further Al addition, complexation by type 2 binding sites caused the increase in regions A* and B* (Figure 6d). Weak Al complexation by type 3 binding sites was responsible for the marked fluorescence enhancement in region D* and the quenching in region C* (Figure 6d). Comparison with equilibrium ion exchange as a reference method showed that binding parameters for type 1 binding sites could be determined by data analysis of the initial increase in regions C* and D* and parameters for type 2 sites by analysis of the enhancement in region B* (37). Comparison reveals that the conditional stability constants for type 1 and 2 binding sites are very similar for both Cu(II) and Al(III) complexes of the juniper LLE, whereas the binding capacities differ (Table 2). Fluorescence data on model compounds (17, 56-58) suggest that phenolic ligands are involved in Al complexation by the juniper LLE. For a mixture of catechol and gentisic acid, a λex shift of the maximum fluorescence intensity from 320 to 340 nm (λem ) 500 nm) was observed upon Al addition (17). Highly conjugated phenolic compounds, like 1-hydroxyanthraquinones (57, 58) or the related

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tetracycline (56), form fluorescent Al complexes with fluorescence maxima in the range of region A*. Comparison of the condititional stability constants found for the Al complexes of the juniper LLE with data on model compounds (54) indicates an important contribution of aliphatic R-dicarboxylic acids to type 2 binding sites.

Conclusions A prototypical study on a juniper LLE leads to the following conclusions on the potential of multi-wavelength MFS as a method for the quantitative characterization of Cu(II) and Al(III) complexation by natural organic matter: (1) Different reaction patterns of the TLS upon metal ion addition allow qualitative distinction of several types of binding site for both Cu and Al complexation, if the MFS is paired with a quantitative reference method and/or the reaction patterns are sufficiently different. (2) The binding parameters for two types of binding site that form the most stable complexes can be deduced from the change of fluorescence intensity at selected EEWP, if the reaction patterns linked with the two types and the stabilities of the corresponding complexes are sufficiently different.

(3) The conditional stability constants deduced from the change of the MFS reflect the molecular structure of the corresponding types of binding site. Thus, the change of the TLS upon titration with Cu or Al may offer complete information on the complexation behavior of the ligand mixture including nonfluorescing ligands. This is remarkable given the fact that, in the case of fulvic acid, only about 1% of the molecules are believed to fluoresce (59). Because of its operational advantages, multi-wavelength MFS would be a method of choice for the routine characterization of the metal complexation behavior of natural organic matter. This is possible, however, only if the correct sets of binding parameters are always obtained at the same EEWP, at least for large categories of similar organic matter. Only then is an independent reference method such as equilibrium ion exchange no longer necessary. The fact that similar spectra with a relatively small number of different peak types have been described for fulvic acid, DOM in natural waters, and other LLE, all of which seem to react similarly upon addition of Cu or Al, argues in favor of juniper LLE as a good model. For example, in almost all quantitative Cu(II) studies using classical data analysis (18), a conditional stability constant between 104 and 105, as determined in the present study, has been deduced from the quenching of a peak around EEWP 320/ 450 (3, 18-21, 23). However, the reaction of the TLS at low Cu/C or low Al/C, which is clearly separated from the reactions at higher metal/C ratios and thus greatly facilitates the distinction of different types of binding site, may be unique to very few types of natural organic matter.

MFS TLS

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)

Acknowledgments

(22)

This research was supported by U.S. NSF Grant CES8896204 as well as funds of the Swiss Federal Institute for Forest, Snow and Landscape Research, Birmensdorf, Switzerland. The authors thank Dr. F.-R. Chang for suggesting the method of reducing the number of variables described in the Experimental Section and Dr. N. Senesi for critically reviewing the interpretation of the ESR spectra. Gratitude is expressed to U. Beutler for providing some control measurements.

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[Al]t, [Cu]t [Al]b, [Cu]b [Cu]f DOC DOM EEWP ESR I I0 ICuL Iref LLE Kc Lt λex, λem

(24) (25) (26) (27) (28) (29) (30) (31)

Symbols total concentration of Al and Cu, respectively concentration of organically bound Al and Cu, respectively concentration of free, i.e., uncomplexed Cu dissolved organic carbon (concentration) dissolved organic matter excitation/emission wavelength pair electron spin resonance fluorescence intensity fluorescence intensity of metal free DOM fluorescence intensity of Cu(II) saturated DOM reference fluorescence intensity leaf litter extract conditional stability constant metal binding capacity excitation and emission wavelength, respectively

molecular fluorescence spectrometry total luminescence spectrum/spectra

(32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46)

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Received for review July 18, 1995. Revised manuscript received November 22, 1995. Accepted December 17, 1995.X ES950542U X

Abstract published in Advance ACS Abstracts, March 1, 1996.

Multi-Wavelength Molecular Fluorescence Spectrometry for

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