Environ. Sci. Technol. 1999, 33, 3264-3270
Effects of Aluminum-Induced Aggregation on the Fluorescence of Humic Substances CHARLES M. SHARPLESS AND LINDA B. MCGOWN* Department of Chemistry, Duke University, Box 90346, Durham, North Carolina 27708
Aluminum-induced aggregates of terrestrial and aquatic humic acid standards from the International Humic Substances Society are shown to be fluorescent by means of a multiwavelength fluorescence anisotropy experiment in which the data was treated with a model for nonspherical particles. While aggregates of aquatic humic acids appear in the fluorescence signal at both short and long excitation wavelengths, aggregates of terrestrial humic acids are detected only at the long wavelength. Furthermore, the results indicate that emission obtained at longer excitation wavelengths is representative of smaller particles. At pH 4, the aquatic humic acids appear to exist in an extended conformation, whereas the terrestrial humic acids show less extension. The size and shape of the fluorescent particles display a complex dependence on Al concentration. Both enhancement and quenching of fluorescence are observed in the total luminescence spectra upon Al addition. However, quenching is shown to be the result of decreased humic acid concentration due to precipitation by Al rather than photophysical processes.
Introduction Humic substances (HS) are large, heterogeneous molecules formed by the decay of plant and animal biomass in aquatic and terrestrial systems (1, 2). There are three operational classes of HS: humic acids (soluble above pH 2), fulvic acids (soluble at all pH), and humins (insoluble) (1). Humic substances often form complexes with metals through carboxylic and phenolic groups with a wide range of pKa’s. These complexation reactions are an important determinant of the environmental fate of trace and heavy metals and have been studied by several techniques (3). There are a variety of sites of different binding strengths in HS, and the complexation isotherms can be explained by both continuum and multiple site binding models (4-8). The intrinsic fluorescence of HS provides a powerful means for studying their interactions with metals. Metal complexation can alter the fluorescence of HS (9-14), resulting in either quenching or enhancement depending on the metal cation (9-11, 14, 15). Advantages of using fluorescence to study complex samples such as HS include low detection limits and minimal sample perturbation and preparation. However, a potential disadvantage is that the fluorescence signal may not represent the entire sample. Indeed, the low fluorescence quantum yield of HS has led to an estimate that only one percent of the molecules are fluorescent (16). * Corresponding Author; phone: (919)660-1545; fax: (919)6601605; e-mail:
[email protected]. 3264
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Studies have shown that the optical properties of HS depend on molecular weight (17-22) and that the fluorescence in different spectral regions is associated with different types of ligand (14). Therefore, results of fluorescence studies will depend on the choice of excitation and emission wavelengths. In one study, gel permeation chromatography showed that the highest molecular weight fraction of an aquatic HS was not fluorescent at the wavelengths chosen for the study (17) and that this fraction was at least partially composed of aggregates of smaller molecules. Low molecular weight fractions are more fluorescent than high molecular weight fractions (22), raising the possibility of self-quenching in the latter. Dynamic quenching mechanisms have been proposed for HS-bound pyrene (23) and may be similar to self-quenching mechanisms in large HS aggregates. The present work investigates the relationship between fluorescence spectra and aggregation processes of humic acids (HAs). Specifically, steady-state fluorescence anisotropy was used to study whether cation-induced aggregates of HAs are fluorescent; if they are, then the anisotropy should detect a change in particle size upon aggregation. Previous investigations by other researchers used steady-state and timeresolved anisotropy in attempts to monitor conformational changes in solutions of fulvic acids as a function of concentration and pH (24, 25). The results were contradictory. We chose to use Al, which is a well-known and powerful coagulant of HS (26, 27) and is known to enhance the fluorescence of HAs (10, 11, 13, 28), indicating that ligands associated with the metal are fluorescent. To increase the rate of aggregation, we employed a heating step in the reaction between the HAs and Al. Production of stable aggregates (over 48 h) was verified by light scattering. Thus, our solutions contained both aggregates and metal-associated fluorophores. The anisotropy data was analyzed using a model for nonspherical particles to determine whether these aggregates contribute to the fluorescence signal. Our results show that in solutions of HAs, Al-induced aggregates are fluorescent.
Theory Fluorescence anisotropy (29-30) can be used to estimate molecular size because the rotational rate of a molecule is inversely dependent upon its size. Thus, a fluorophore excited with plane polarized light will have shifted the plane of polarization by the time it emits a photon. Experimentally, anisotropy, r, is defined by:
r ) (I|| - I⊥)/(I|| + 2I⊥)
(1)
in which I|| is the fluorescence intensity in the plane of the exciting light and I⊥ is the intensity perpendicular to that plane. For a sphere, the anisotropy is related to the rotational correlation time (φ) by the Perrin equation:
r ) ro/(1+(τ/φ))
(2)
Here, ro is the limiting anisotropy, i.e. the anisotropy that would be observed for a mixture of randomly oriented fluorophores in the absence of rotational diffusion, and τ is the fluorescence lifetime. The molar volume (V) may be calculated from φ by using the Stokes-Einstein relation, which assumes a spherical particle shape:
φ ) ηV/(RT)
(3)
in which η is the solution viscosity. A plot of 1/r vs T yields 10.1021/es981332v CCC: $18.00
1999 American Chemical Society Published on Web 08/07/1999
a line with slope proportional to φ. Deviations from linearity may be caused by several factors, such as the presence of multiple fluorophores with different φ and τ values, or nonspherical particles (31). Both factors should lead to a plot that is concave toward the T/η axis. In the special case of randomly oriented fluorophores in nonspherical particles, one may approximate the anisotropy as the sum of the anisotropies from two spherical particles with equal fluorescence intensities (31). The resulting equation is:
1/r ) (1/ro)[(2+(2τ/φ1))-1 + (2+(2τ/φ2))-1]-1
(4)
The justification for this approximation is that the random orientations of excitation and emission dipoles in an ellipsoidal molecule cause a large degree of uncoupling of the effects of the two rotational motions. The two rotational correlation times, φ1 and φ2, may each be expressed using eq 3.
Materials and Methods Materials. All chemicals were analytical grade and used as received. KCl (Mallinckrodt) was used as a background electrolyte. Dilute HCl (Mallinckrodt) and KOH (Fluka) were used to adjust sample pH. All water was distilled and deionized to a resistivity of at least 16 MΩ cm. HAs were obtained from the International Humic Substances Society and used as received. They include two terrestrial samples, Peat Humic Acid Reference (PHA) and Soil Humic Acid Reference (SHA), and two aquatic samples, Nordic Aquatic Humic Acid Reference (NHA) and Suwanee River Humic Acid Reference (SUA). Preparation of Humic Acid and Humic Acid-Aluminum Solutions. Stock solutions were prepared by dissolving ∼1 mg of HA in 10.0 mL of 0.05 M KCl, adjusting the pH to 8.5 and stirring for 5-6 h at which point no particulate matter could be observed. This solution was diluted to prepare 50.0 mL of 10 mg/L HA, which was then adjusted to pH 4.0 and allowed to stand overnight at room temperature. The solution was filtered the next day through a 0.45 µm syringe filter that had been washed with 20 mL of water. Aluminum stock solution was freshly prepared for each experiment by dissolving AlCl3‚6H2O (Aldrich) in 0.05 M KCl and then diluted to provide the desired Al concentrations for each HA, as follows: PHA (0, 5, 10, 12.5, and 100 µM); SHA (0, 1, 3, 5, and 100 µM); NHA (0, 10, 20, 30, and 200 µM); SUA (0, 20, 30, 40, and 200 µM). These concentrations were chosen to produce aggregation without causing a large increase in scattered light. Additions of Al to HA solutions were done with rapid stirring, and the pH of each solution was checked immediately afterward and adjusted if necessary. The solutions were then equilibrated at 50 °C for 2 h and allowed to re-equilibrate at room temperature for 2 h, followed by another pH check. The samples were then either allowed to stand for 24 h (anisotropy and lifetime measurements) or used immediately in a 3 day series of light scattering experiments. For all experiments, the samples were filtered through a 0.45 µm syringe filter immediately prior to measurements, which were made at 25 °C unless otherwise stated. Steady-State Fluorescence and Light Scattering Measurements. Measurements were made using a spectrofluorimeter (Spectronics, Inc., model 48000S) with a 450-W xenon arc lamp source and photomultiplier tube (PMT) detectors. Total Luminescence Spectra were collected as a set of emission spectra from 340 to 540 nm by varying the excitation wavelength from 290 to 490 nm. Excitation and emission wavelengths were varied in 4 nm steps, and the excitation and emission monochromator slits were set to 4 nm bandpass. Individual emission spectra also were collected. Cor-
rections were made for primary and secondary inner filtering effects as described elsewhere (32), but these corrections had no effect on the spectra since the total absorbances of the solutions were low. Light scattering measurements were made at 2, 24, and 48 h following sample preparation. A He-Ne laser (SpectraPhysics, model 127) provided incident light at 632.8 nm. Scattered light was selected through a monochromator at 633 nm with a spectral band-pass of 1 nm. These conditions essentially eliminated interference from HA fluorescence. Fluorescence Lifetimes. Fluorescence lifetimes were measured in the frequency domain (29) using a multiharmonic Fourier transform phase-modulation spectrofluorimeter (Spectronics, Inc., model 4850S). Phase-shift and demodulation data were collected simultaneously at 50 frequencies ranging from 4.1 to 205 MHz using a cross-correlation frequency of 25 Hz. Kaolin was used as a scattering source for a reference lifetime of 0 ns. Three replicates were run for each sample. The data were analyzed using nonlinear leastsquares analysis (Globals, Unlimited) and were fit to a three component model, which was the only model that consistently gave reasonable χ2 goodness-of-fit values. Other researchers have found that a three-component model adequately describes the fluorescence decay of HS (12, 24, 33), although each component probably represents several similar, unresolvable lifetime components. Fluorescence Anisotropy. Fluorescence anisotropy was measured using the 48000S spectrofluorimeter in the Tformat, in which the parallel and perpendicular polarization emission components are simultaneously detected in separate channels. Excitation was provided by either the 325 nm line from a He-Cd laser (Kimmon Co., IK Series) or the 457 nm line from an Ar+ laser (Orion Co., Innova 300). GlanThompson polarizers were placed in the excitation and emission channels to achieved the desired polarizations. An emission wavelength was selected using 420 or 500 nm interference filters (Orion, matched, 10-nm bandwidth), which were found to pass less than 0.01% of exciting light at both wavelengths. A correction factor for instrumental parameters (the G factor) was determined experimentally and applied to the anisotropy calculations following standard methods (29). Anisotropy was measured in triplicate as a function of temperature by varying the temperature of the water bath. The solutions were continuously stirred. Values of T/η for the solutions were approximated by the values for water. Sigmaplot software (Jandel Co.) was used for curve fitting of the anisotropy data. The rotational correlation times were averaged and standard deviations obtained by propagating the error in the individual correlation times. Fitting of the anisotropy data involves several considerations. First, an estimate must be made of the fluorescence lifetime in order to avoid complications from the use of the anisotropy equations for nonspherical molecules with multiple lifetimes. We used the average lifetime, 〈t〉, of each sample for the parameter τ in eq 4. The 〈t〉 was calculated as ΣRiτi2/ΣRiτi (29), in which Ri and τi are the fractional intensity contribution and lifetime of the ith lifetime component, respectively. Another option might be to use the best singleexponential fit to the data (25), but the χ2 values for such fits were unacceptable. The 〈t〉 were invariant with Al concentration within experimental error (Table 1). Another consideration is the decrease in fluorescence lifetime with increasing temperature. This results in each T/η point being skewed by a factor of τ/τo, where τo is the lifetime at a predetermined temperature (25), which in our case was 25 °C. To account for this, measurements of total fluorescence intensity were made as a function of temperature during the anisotropy experiments. The relation F/Fo ) τ/τo, where F is fluorescence intensity at temperature T VOL. 33, NO. 18, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Average Lifetimes of IHSS HAs and Their Aluminum Complexesa Lifetime (ns)
a
SHA
[AlCl3], µM 325/420 457/500
0 5.7 (0.8) 4.9 (0.1)
1 6.1 (0.7) 5.5 (0.2)
3 6.1 (0.6) 5.2 (0.2)
5 6.0 (0.6) 5.0 (0.1)
100 6.7 (0.4) 5.4 (0.6)
PHA
[AlCl3], µM 325/420 457/500
0 5.9 (0.7) 4.9 (0.3)
5 5.4 (0.2) 5.2 (0.3)
10 5.9 (0.9) 5.9 (0.6)
12.5 5.6 (0.3) 4.8 (0.8)
100 6.3 (0.8) 6.0 (0.4)
NHA
[AlCl3], µM 325/420 457/500
0 6.8 (0.3) 5.3 (0.1)
10 6.1 (0.4) 5.7 (0.2)
20 6.6 (0.4) 6.0 (0.3)
30 6.3 (0.7) 6.1 (0.3)
200 6.0 (0.6) 5.4 (0.1)
SUA
[AlCl3], µM 325/420 457/500
0 6.7 (0.2) 7.1 (0.6)
20 6.6 (0.2) 6.8 (0.4)
30 6.6 (0.7) 7.1 (0.6)
40 6.4 (0.3) 7.3 (0.1)
200 6.6 (0.3) 8.3 (0.8)
Standard deviations of triplicate runs in parentheses.
and Fo is the intensity at 25 °C, was used to estimate the lifetime at each temperature. Subsequently, each T/η point was multiplied by the factor Fo/F, to correct for the change in lifetime. The limiting anisotropy, ro, may be determined in vitreous solution (e.g., glycerol); however, this method may lead to erroneous values of the rotational correlation times for macromolecules in which segmental motion of the fluorophores contributes to the observed anisotropy (29). Thus, fitting of the anisotropy data to eq 4 involves three parameters: ro, φ1, and φ2. Allowing all three to vary yields large errors in the fitted values. To reduce the errors in the correlation times the following strategy was adopted. First, the data for one HA and its Al complexes at one wavelength were fit by allowing all three of the parameters to vary. The recovered ro parameters were averaged, and this average value was fixed for use in refitting the data, while allowing only the correlation times to vary. We justify this approach by noting that the values of ro obtained when allowing all three parameters to vary were very similar for a given HA at a given wavelength regardless of Al concentration.
Results and Discussion Creation of Stable Aluminum-Induced Aggregates by Heated Reaction. A major goal of this study was to determine if Alinduced HA aggregates are fluorescent. Many reactions between HS and inorganic species may be described by multiple rate constants (34-39), one of which is usually low. Recent studies on magnesium complexation by HA indicate that there are two phases of this reaction, initial cationinduced intermolecular bridging (i.e., aggregation), followed by disaggregation and formation of stable, inner sphere complexes (40). The latter phase has also been proposed to explain the slow kinetics of copper and Al reactions with HS (34, 35). This reaction pattern would complicate the fluorescence anisotropy experiment. Thus, we sought conditions that would create aggregates that would be stable over a period of days. We observed that precipitation of HA occurred in solutions containing high-Al concentrations after several days at room temperature. We, therefore, employed a heating step during the solution preparation, followed by removal of the precipitate, to expedite the formation and removal of precipitate. Light scattering of the HA samples was measured to determine whether heating would produce stable aggregates and to decide which concentrations of Al should be used to avoid scattering artifacts in the anisotropy experiments. Since each sample was filtered through a 0.45 µm filter before measurement, only dissolved particles should remain in the solution. Formation and removal by filtration of precipitate 3266
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FIGURE 1. Light scattering from solutions of the HAs and their Al complexes: (a) PHA, (b) SHA, (c) NHA, (d) SUA; Symbols denote equilibration time: 2 h (circles); 24 h (triangles); 48 h (squares). that formed at the highest concentration of Al decreased the scattered light intensity (Figure 1). Blanks of Al without HA showed no increase in scattered light intensity over two days from which we concluded that the data corresponds to the HA-Al aggregates in solution. Heating the HA samples containing the chosen concentrations of Al produced aggregates that were stable over 48 h, demonstrating that slow reactions of HAs can be speeded up by using higher temperatures without compromising the experiment. Although an inverse dependence of particle size on temperature might be expected, this would yield convex curves in the Perrin plots (31). Since this was not observed, any such effect is negligible. Fluorescence Spectra. Total luminescence spectra of the HA with no Al and with the highest concentration of Al are shown in Figures 2 and 3. Excitation wavelength is on the vertical axis, emission wavelength on the horizontal axis, and the intensity is represented by contour lines. Excitation at shorter wavelengths (290-370 nm) produces a broad peak centered at ∼480 nm (terrestrial HA) or 440 nm (aquatic HA) with a shoulder at ∼400 nm. Excitation at longer wavelengths (∼430 nm) gives rise to a broad emission peak at 500 nm
FIGURE 2. Total luminescence spectra of terrestrial HAs and their Al complexes: (a) PHA (b) PHA + 100 µM Al, (c) SHA, (d) SHA + 100 µM Al.
FIGURE 3. Total luminescence spectra of aquatic HAs and their Al complexes: (a) NHA (b) NHA + 200 µM Al, (c) SUA, (d) SUA + 200 µM Al. (terrestrial HA) or 485 nm (aquatic HA), which is accompanied by a slight shoulder at longer excitation wavelengths. High-Al concentrations cause similar spectral changes for the aquatic and terrestrial HA, but the extents of the changes differ. For both, there is a decrease in emission intensity at most wavelengths but the emission shoulder at 400 nm at shorter excitation wavelengths increases in intensity. These changes are shown in more detail for PHA and SUA in Figure 4, which also show differences between terrestrial and aquatic HA at intermediate Al concentrations. The terrestrial HA show a continuous decrease in intensity at all wavelengths with Al, but the aquatic HA show enhanced emission intensity at many points in the spectra for the intermediate Al concentrations. Other researchers have seen enhancement and/or quenching of HS fluorescence by Al (10, 11, 13-15, 28). Enhancement of fluorescence by Al is a well-known phenomenon that is often used for determination
FIGURE 4. Emission spectra of PHA and SUA. (a) PHA, 325 nm excitation, (b) PHA, 457 nm excitation, (c) SUA, 325 nm excitation, (d) SUA, 457 nm excitation.
FIGURE 5. Emission spectra of dissolved material (solid line) and precipitated material (dashed line) at 100 µM Al and pH 9 for PHA and 200 µM Al and pH 9 for SUA: (a) PHA, 325 nm excitation, (b) PHA, 457 nm excitation, (c) SHA, 325 nm excitation, (d) SHA, 457 nm excitation. of this metal (41). However, there is no known mechanism for direct fluorescence quenching by Al. Possible mechanisms include self-quenching of HA in aggregates and precipitation of fluorophore-containing molecules. To test the latter hypothesis, we prepared HA samples with the highest Al concentration to induce precipitation. These were reacted as usual and then filtered to collect the dissolved material. The filter was washed with 0.05 M KCl at pH 4 and then back flushed with 0.05 M KCl at pH 11 to dissolve the precipitate. The pH of both filtrate and precipitate was adjusted to 9. Emission spectra of PHA and SUA in Figure 5 show that the precipitate contains mostly fluorophores that emit at longer wavelengths regardless of excitation wavelength. These are the same wavelengths at which VOL. 33, NO. 18, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 6. Perrin plots for PHA, SUA and their Al complexes at different excitation/emission wavelengths: (a) PHA, 325/420 nm; (b) SUA, 325/420 nm; (c) PHA, 457/500 nm; (d) SUA; 457/500 nm. Symbols denote increasing aluminum concentration in the following order: O, 0, ), 4, 3. intensity is decreased for all of the HA, most likely due to Al-induced precipitation. Thus, the simplest explanation for any decrease in intensity is loss of fluorophores to precipitation as opposed to fluorescence quenching. This is supported by the anisotropy results that show a decrease in particle size at high-Al concentrations. Fluorescence Anisotropy. Even when care is taken to eliminate excitation light from reaching the detectors (as is the case here), scattering of exciting and emitted light can still lead to a decrease in the measured anisotropy (29). Energy transfer can also lead to low anisotropy values (29), but this is not likely here since the solutions are dilute and have relatively low absorbance. Thus, we concerned ourselves only with the possibility of scattered light artifacts. Measurements of fluorescein in aqueous 50% glycerol in which scattered light intensity was varied by addition of small amounts dextran (500 kD) indicated that a value of 2.5× the original scattered light intensity (2.5S°) was the limit above which artifacts were produced. With scattering up to 2.5S°, the Perrin plots (data not shown) yielded a molar volume for fluorescein that was within 5-10% of crystallographic values (42). With higher scattering, the slopes increased due to scattering depolarization. Hence, Al concentrations were chosen to allow a maximum of 2.5S° for the anisotropy experiments. Perrin plots for the HA at 325/420 nm and at 457/500 nm are concave, as shown for PHA and SUA in Figure 6. At 325/ 420 nm at 25 °C, all four HA showed increased anisotropy with Al. A precipitate formed at the highest Al concentration; filtration was used to remove the precipitate, and the anisotropy dropped below its original value. This occurred at 457/500 nm for all HA, except SHA, which showed a continuous increase in anisotropy; also, the anisotropy of PHA and NHA at the highest Al concentration decreased but not below the original values. It is important to be aware of the assumptions made in fitting the data. The main assumption is that the emission dipoles are randomly oriented with respect to the ellipsoidal axes. Given the heterogeneous nature of HA, this condition should be satisfied for the sample as a whole. For each size fraction, there will be molecules with various dipole orientations; the net effect is akin to using a fluorescent tag that can adopt any orientation on the target molecule. This allows the description of the anisotropy in terms of two spheres 3268
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FIGURE 7. Rotational correlation times of terrestrial HAs and their Al complexes at 325/420 nm (b) and 457/500 nm (4): (a) O1 for PHA, (b) O1 for SHA, (c) O2 for PHA, (d) O2 for SHA. since the emission dipoles have equal probability of orientation along either ellipsoidal axis. Combined with the assumption that the fluorescence intensities are equal for different molecules (i.e., the humic molecules contain similar fluorophores), this leads directly to eq 4 as the appropriate steady-state model. Another assumption is that the average fluorescence lifetime, 〈t〉, of a sample may be used as the fluorescence lifetime term in the model. This is a practical limitation on the interpretation of anisotropy data because it assumes that the fluorescence lifetimes are equally distributed across the various particle sizes so that 〈t〉 represents the average fluorophore in each size fraction. Recent literature suggests that there is a slight association of longer lifetimes with smaller size fractions of natural organic matter (43), but this has yet to be demonstrated for a purified HA fraction. The 〈t〉 are given in Table 1 for the different Al concentrations. With the exception of SUA, 〈t〉 was shorter at the longer wavelength peak than at the shorter wavelength peak. Also, 〈t〉 at the shorter wavelength peak was generally shorter for the terrestrial HA than for the aquatic HA. The fitted rotational correlation times (φ1 and φ2) at each wavelength are shown graphically as a function of Al concentration in Figures 7 and 8. The two correlation time model was found to adequately describe the data since inclusion of a third correlation time gave values for φ3 very close to those for φ2 and increased the errors in the fitted parameters. For all of the HA, φ1 is on the order of 2 ns at 325/420 nm and, with the exception of SUA, φ1 is shorter at 457/500 nm. Other researchers have reported rotational relaxation times of 2 ns (correlation times of 0.7 ns) for soil fulvic acids (24, 25). Larger values are expected for HAs, which tend to be larger than fulvic acids (2, 22, 44). The φ2 is different for the two HA classes: at 325/420 nm it is ∼10 ns for the terrestrial HA and ∼40 ns for aquatic HA. Like φ1, φ2 is also a function of wavelength, in this case for all samples and again is shorter at 457/500 nm. Al causes similar changes in the correlation times within a given class of HA, but the two classes differ. At 325/420 nm, φ1 of the terrestrial HA shows no change prior to precipitation and then decreases. At 457/500 nm, φ1 increases gradually
Another observation is that φ1 yields smaller molecular weights at 457/500 nm than at 325/420 nm, except for SUA, indicating that fluorescence from the smaller fractions is more heavily weighted at the longer excitation wavelengths. To our knowledge, no one has investigated this possibility, although there are conflicting reports in the literature concerning the relation between molecular weight and emission wavelength at a given excitation wavelength (15, 19). The relative values of φ1 and φ2 indicate that the aquatic HA exhibit a higher degree of extension than the terrestrial HA, although both appear to be ellipsoids. The aquatic HA have larger φ2 values, and given the similarity of aquatic and terrestrial φ1 values, this indicates a greater axial ratio for the aquatic samples. The axial ratios were estimated from φ1 and φ2 using Perrin’s equations (45) to be ∼7:1 for the aquatic HA and ∼3:1 for the terrestrial HA. This agrees with other results that suggest that both aquatic and terrestrial HAs can exist in an extended conformation (46-48).
FIGURE 8. Rotational correlation times of aquatic HAs and their Al complexes at 325/420 nm (b) and 457/500 nm (4): (a) O1 for NHA, (b) O1 for SUA, (c) O2 for NHA, (d) O2 for SUA.
TABLE 2. Molar Volumes ( 1 Standard Deviation and Molecular Weights for IHSS HAs Calculated from O1 Results SHA PHA NHA SUA
325/420 nm Molar Volume MW 4,800 ( 300 8,400 4,900 ( 300 8,600 5,200 ( 300 10 200 5,300 ( 300 10 400
457/500 nm Molar Volume MW 990 ( 30 1,700 2,700 ( 300 4,700 2,200 ( 300 4,300 5,400 ( 300 10 600
with increasing Al and then decreases to near its original value. The φ2 of PHA increases gradually with Al at both wavelength pairs. The φ2 of SHA stays relatively constant at low Al concentrations and then increases. For the aquatic HA, φ1 increases with Al concentration at both wavelength pairs prior to precipitation and then decreases. At 325/420 nm, φ2 steadily decreases with increasing Al. At 457/500 nm, the trends in φ2 are similar to those in φ1. To interpret these results, we note two points concerning rotational correlation times of ellipsoids. First, for a prolate ellipsoid, φ1 (the correlation time for rotation about the major axis) is for all axial ratios very close to the value that would be obtained for a sphere of equal volume (45) and, therefore, is indicative of particle size. Second, the difference between φ1 and φ2 is a measure of the axial ratio, i.e. the extent of elongation of the molecules. With these points in mind, we observe that φ1 is similar for all four samples at 325/420 nm. At 457/500 nm, φ1 is approximately the same as at 325/420 nm for SUA, smaller for PHA and NHA, and much smaller for SHA. While we cannot directly determine molecular weights from our measurements, estimates may be made by using published data on specific volumes of HAs. The molecular weights were determined at the two wavelength pairs using the values 0.57 cm3/g for the terrestrial HA and 0.51 cm3/g for the aquatic HA (21). The results (Table 2) at 325/420 nm are in good agreement with other reports for terrestrial HA, but the aquatic HA appear to be about 3-4 times larger than expected (18, 21, 44). The source of this discrepancy is not yet determined. It is unlikely due to hydration, which would require that the aquatic HA hold roughly three× their volume (1.5× their mass) of water.
We now consider the effect of Al upon the rotational correlation times and the question of whether aggregates are fluorescent. There is no change in φ1 with Al at 325/420 nm for the terrestrial HA, suggesting that aggregates in these samples do not fluoresce in this spectral region, although this is not the only possible explanation, as discussed below. In contrast, Al increases φ1 of the aquatic HA at this wavelength pair, which indicates detection of fluorescent aggregates. For both terrestrial and aquatic HA, aggregates are detected at 457/500 nm. At the highest Al concentration, the terrestrial HA show a decrease in φ1 (i.e., particle size), indicating precipitation of high molecular weight material, as would be expected under these conditions (49-51). In combination with the emission spectra of the precipitate (Figure 6), this provides evidence that higher molecular weight HA fractions fluoresce primarily at the longer emission wavelengths. The aquatic samples show little change in size at either wavelength pair after precipitation. For SUA, the final particle size is similar to the original size. However, NHA shows detectable aggregates at both wavelength pairs even after precipitation. This suggests that the larger particles do not precipitate as easily in aquatic HA as in terrestrial HA, perhaps due to increased charge and hydrophilicity. The absence of detectable aggregates in terrestrial HA at 325/420 nm is intriguing. Perhaps fluorophores monitored at 325/420 nm in these samples undergo self-quenching when aggregated. Alternatively, larger molecules may coil up, reducing their size and, hence, their contribution to the anisotropy; concurrently, smaller particles will be aggregating, offsetting this loss of polarization. Coiling has been suggested by other researchers to explain various results (46-48) and appears to have been observed directly for larger fractions of a fulvic acid using fluorescence polarization (20). In the case of smaller, more hydrophilic particles, their size would restrict the amount of coiling that they could undergo, while their higher charge would tend to keep their Al complexes dissolved. This would explain the Al-dependent increase in φ1 at 457/500 nm for the terrestrial HA if the fluorescence in this region represents smaller particles. We conclude that Al-induced aggregates of HA do fluoresce. Decreased intensity in the fluorescence spectra upon Al addition may be due to HA self-quenching in larger aggregates but is at least partially due to loss of material to precipitate. The lack of detection of fluorescent aggregates at 325/420 nm in the terrestrial HA samples could be the result of coiling processes or quenching in aggregates, but there is no way to distinguish between the two from these experiments. Finally, the differences and trends in particle size at 325/420 nm and 457/500 nm indicate that different ligand types are observed in different spectral regions and VOL. 33, NO. 18, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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highlights the need to investigate multiple wavelengths when using fluorescence to study HS.
Acknowledgments This work was supported by the Office of Exploratory Research of the United States Environmental Protection Agency (Grant R822251-01) and by a Duke University Integrated Toxicology Training Grant Fellowship (to C.M.S.) from the National Institutes of Health, National Institute of Environmental Health Sciences (Grant 5T32-ES07031-15).
Literature Cited (1) MacCarthy, P.; Suffet, I. H. In Aquatic Humic Substances: Influence In Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry Series 219; American Chemical Society, Washington D. C., 1989; pp xvii-xxx (Introduction). (2) Senesi, N. In Organic Substances in Soil and Water: Natural Constituents and Their Influences on Contaminant Behavior, Beck, A. J., Jones, K. C., Hayes, M. H. B., Mingelgrin, U., Eds.; The Royal Society of Chemistry, Cambridge, 1993; pp 74-77 (3) Saar, R. A.; Weber, J. H. Environ. Sci. Technol. 1982, 16, 510A517A. (4) Mathuthu, A. S.; Marinsky J. A.; Ephraim, J. H. Talanta 1995, 42, 441-447. (5) Lead, J. R.; Hamilton-Taylor, J.; Hesketh, N.; Jones, M. N.; Wilkinson, A. E.; Tipping, E. Anal. Chim. Acta 1994, 294, 319327. (6) Yoon, T. H.; Moon, H.; Park, Y. J.; Park, K. K. Environ. Sci. Technol. 1994, 28, 2139-2146. (7) Nederlof, M. M.; De Wit, J. C. M.; Van Rlemsdijk, W. H.; Koopal, L. K. Environ. Sci. Technol. 1993, 27, 846-856. (8) Susetyo, W.; Dobbs, J. C.; Carreira, L. A.; Azarraga, L. V.; Grimm, D. M. Anal. Chem. 1990, 62, 1215-1221. (9) Ryan, D. K.; Weber, J. H. Anal. Chem. 1982, 54, 986-990. (10) Cabaniss, S. E. Environ. Sci. Technol. 1992, 26, 1133-1139. (11) Tam, S.-C.; Sposito, G. J. Soil Sci. 1993, 44, 513-524. (12) Cook, R. L.; Langford, C. H. Anal. Chem. 1995, 67, 174-180. (13) Esteves da Silva, J. C. G.; Machado, A. A. S. C. Appl. Spec. 1996, 50, 436-443. (14) Luster, J.; Lloyd, T.; Sposito, G.; Fry, I. V. Environ. Sci. Technol. 1996, 30, 1565-1574. (15) Philpot, W. D.; Vodacek, A. Remote Sens. Environ. 1989, 29, 51-65. (16) Seitz, W. R. Trends Anal. Chem. 1981, 1, 79-83. (17) Peuravuori, J.; Pihlaja, K. Anal. Chim. Acta 1997, 337, 133-149. (18) Chin, Y.-P.; Aiken, G.; O’Loughlin, E. Environ. Sci. Technol. 1994, 28, 1853-1858. (19) Green, S. A.; Morel, F. M. M.; Blough, N. B. Environ. Sci. Technol. 1992, 26, 294-302. (20) Lakshman, S.; Mills, R.; Fang, F.; Patterson, H.; Cronan, C. Anal. Chim. Acta 1996, 321, 113-119. (21) Reid, P. M.; Wilkinson, A. E.; Tipping, E.; Jones, M. N. Geochim. Cosmochim. Acta 1990, 54, 131-138. (22) Thurman, E. M. Organic Geochemistry of Natural Waters; Martinus Nijhoff/Junk: The Hague, The Netherlands, 1985; pp. 273-361. (23) Engebretson, R. R.; von Wandruszka, R. Environ. Sci. Technol. 1994, 28, 1934-1941.
3270
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 18, 1999
(24) Lochmu ¨ ller, C. H.; Saavedra, S. S. Anal. Chem. 1986, 58, 19781981. (25) Lapen, A. J.; Seitz, W. R. Anal. Chim. Acta 1982, 134, 31-38. (26) Hundt, T. R.; O′Melia, C. R. J. Am. Water Works Assoc. 1988, 80, 176-186. (27) Dempsey, B. A.; Ganho, R. M.; O′Melia, C. R. J. Am. Water Works Assoc. 1984, 76, 141-150. (28) Patterson, H. H.; Cronan, C. S.; Lakshman, S.; Plankey, B. J.; Taylor, T. A. Sci. Total Environ. 1992, 113, 179-196. (29) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: 1983; pp 52-151. (30) Weber, G. Biochem. J. 1952, 51, 145-155. (31) Weber, G. Adv. Protein Chem. 1953, 8, 425-459. (32) Mobed, J. J.; Hemmingsen, S. L.; Autry, J. L.; McGown, L. B. Environ. Sci. Technol. 1996, 30, 3061-3065. (33) Kumke, M. U.; Abbt-Braun, G.; Frimmel, F. H. Acta Hydrochem. Hydrobiol. 1998, 26, 73-81. (34) Rate, A. W.; McLaren, R. G.; Swift, R. S. Environ. Sci. Technol. 1993, 27, 1408-1414. (35) Plankey, B. J.; Patterson, H. H.; Cronan, C. S. Anal. Chim. Acta 1995, 300, 227-236. (36) Shuman, M. S. Environ. Sci. Technol. 1992, 26, 593-598. (37) Plankey, B. J.; Patterson, H. H. Environ. Sci. Technol. 1987, 21, 595-601. (38) Olson, D. L.; Shuman, M. S. Anal. Chem. 1983, 55, 1103-1107. (39) Fukushima, M.; Nakayasu, K.; Tanaka, S.; Nakamura, H. Anal. Chim. Acta 1995, 317, 195-206. (40) Engebretson, R. R.; Von Wandruska, R. Environ. Sci. Technol. 1998, 32, 488-493. (41) Lobinski, R.; Marczenko, Z. In Comprehensive Analytical Chemistry Vol. XXX, S. G. Weber, Ed., Elsevier Science: Amsterdam, 1996; pp. 277-288. (42) Osborn, R. S.; Rogers, D. Acta Crystallog. B 1975, 31, 359-362. (43) Kumke, M. U.; Tisneau, C.; Abbt-Braun, G.; Frimmel, F. H. J. Fluorescence 1998, 8, 309-318. (44) Beckett, R.; Jue, Z.; Giddings, J. C. Environ. Sci. Technol. 1987, 21, 289-295. (45) Perrin, F. J. Phys. 1934, VII, 5, 497-511. (46) Ghosh, K.; Schnitzer, M. Soil Sci. 1980, 129, 267-276. (47) Murphy, E. M.; Zachara, J. M.; Smith, S. C.; Phillips, J. L.; Wletsma, T. W. Environ. Sci. Technol. 1994, 28, 1291-1299. (48) Chin, Y.-P.; Gschwend, P. M. Geochim. Cosmochim. Acta 1991, 55, 1309-1317. (49) Aiken, G. R.; Malcolm, R. L. Geochim. Cosmochim. Acta 1987, 51, 2177-2184. (50) Tambo, N.; Kamei, T.; Itoh, H. In Aquatic Humic Substances: Influence In Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry Series 219; American Chemical Society, Washington D. C., 1989; pp. 453-471. (51) Kim, J. S.; Chian, E. S. K.; Saunders: F. M.; Perdue, E. M.; Giabbai, M. F. In Aquatic Humic Substances: Influence In Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry Series 219; American Chemical Society, Washington D. C., 1989; pp. 473-497.
Received for review December 22, 1998. Revised manuscript received June 18, 1999. Accepted June 23, 1999. ES981332V
