Molecular mass distribution and ¯uorescence characteristics of
dissolved organic ligands for copper(II) in Lake Biwa, Japan
F. Wu
a,b,*, Eiichiro Tanoue
aaInstitute for Hydrospheric-Atmospheric Sciences, Nagoya University, Nagoya 464-8601, Japan bThe State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry,
Chinese Academy of Sciences, Guiyang, People's Republic of China
Received 15 May 2000; accepted 24 October 2000 (returned to author for revision 16 October 2000)
Abstract
Molecular mass distribution and ¯uorescence characteristics of organic ligands for copper(II) in freshwater were, for the ®rst time, investigated using ultra®ltration, immobilized metal ion anity chromatography (IMAC) and three-dimensional excitation-emission matrix spectroscopy (3DEEM). Two types of humic-like ¯uorescence (Peaks A and B) were found in all molecular ligand fractions, while an additional type of protein-like ¯uorescence (Peak C) was only observed in the 0.1mm-GF/F molecular ligand fractions. The Ex/Em maxima of Peak A in the <5 kDa and 0.1mm-GF/F
molecular ligand fractions were blue-shifted (shift towards the shorter wavelength) relative to that in the 5 kDa±0.1mm
fractions. Contributions of total organic ligands were 1.6±4.0% of the bulk dissolved organic matter (DOM) in the original water, as determined by UV absorbance. In the molecular mass distribution of organic ligands, the relative contribu-tion of the fraccontribu-tion with the <5 kDa molecular masses was dominant (67±79%), while 17±30% of the total organic ligands were in the 5 kDa±0.1 mm fraction, leaving 3±6% in the 0.1mm±GF/F fraction. Fluorescence at the longer
excitation wavelength was more heavily weighted in the lower molecular organic ligands relative to that at the shorter excitation wavelength. The binding anity of organic ligands increased with their molecular weight. These results have signi®cance for further understanding the nature of organic ligands and their biogeochemical role in the aquatic environment.#2001 Elsevier Science Ltd. All rights reserved.
Keywords:Organic ligands; Metal speciation; Immobilized metal ion anity chromatography (IMAC); Three-dimensional excitation-emission matrix spectroscopy (3DEEM); Molecular mass distribution
1. Introduction
Naturally-occurring organic ligands have been stu-died extensively in terms of metal speciation in natural waters over the past decades (e.g. Tanoue and Mid-orikawa, 1995). Copper speciation is now recognized to be dominated by complexation with organic ligands (e.g. Donat and Bruland, 1995); more than 90% of cop-per was found to be complexed by organic ligands in freshwater (Mantoura et al., 1978). However, most studies
have only provided concentrations and conditional sta-bility constants of organic ligands, the fundamentals pertaining to the nature and chemical properties of organic ligands are poorly understood since they have never been isolated (Gordon et al., 1996; Midorikawa and Tanoue, 1996, 1998). Midorikawa and Tanoue (1996, 1998) reported chromophoric properties of dis-solved organic ligands for copper(II) in oceanic waters. To our knowledge, little is known about the character-istics of organic ligands in freshwater.
In freshwater, the distribution of molecular masses and copper complexing capacities of dissolved organic matter have been investigated. Ramamoorthy and Kushner (1975) reported that 89% copper-binding
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capacity was in the >45,000 Da fraction in the river water. In contrast, Smith (1976) noted that most of the carbon was distributed in the <1 KDa molecular frac-tions, but all molecular range factions had similar copper capacities. Other studies showed that Cu was mainly associated with colloidal materials (10 kDa±0.4 mm) in
coastal (Martin et al., 1995) and river waters (Ralph, 1976; Hart et al., 1992). Orlandini et al. (1990) and Salbu et al. (1987) reported that colloids had a domi-nant role in the aqueous chemistry of actinides as well as other trace metals. While controversial, these studies indicate that organic ligands may vary among various molecular mass fractions, which are poorly understood. Fluorescence, particularly three-dimensional excita-tion-emission matrix spectroscopy (3DEEM), has been widely used to obtain information on the nature of DOM in various environments (e.g. Coble et al., 1990; Mopper and Schultz, 1993; Coble, 1996). De Souza Sierra et al. (1994) and Donard et al. (1989) reported shifted emission maxima of DOM ¯uorescence in marine water relative to coastal and fresh water. Senesi (1990) reported dierences in ¯uorescence spectra of humic and fulvic acid of dier-ent origin. 3DEEM spectroscopy has been extensively used to compare ¯uorescent compounds between dierent environments e.g. fresh, coastal and marine waters, and surface and deep waters as well (Coble et al., 1990; Mopper and Schultz, 1993; Coble, 1996; Del Castillo et al., 1999). However, ¯uorescence characteristics of organic ligands have not been documented.
In previous studies, immobilized metal ion anity chromatography (IMAC) was successfully used to iso-late organic ligands in oceanic waters, the conditional stability constant (LogK0
CuL) was determined to be 7±10 by ¯uorescence quenching titration (Midorikawa and Tanoue, 1996, 1998). In this study, by a combination of a tangential ¯ow ultra®ltration technique, IMAC and 3DEEM spectroscopy, we report the molecular mass distribution and ¯uorescence characteristics of organic ligands for copper(II) and their contribution to the bulk DOM in Lake Biwa in terms of UV absorbance and ¯uorescence. 3DEEM patterns and relative binding anity are discussed.
2. Methods and materials
Samples were collected in Lake Biwa, the largest lake and freshwater resource in Japan, in June 1999. Sampling sites are shown in Fig. 1. Water samples were ®ltered through a GF/F glass-®ber ®lter (Whatman, Maidstone, UK) immediately after sampling. The ®ltrate was sub-jected to fractionation in terms of molecular mass dis-tribution with a tangential ¯ow ultra®ltration system (Minitan II system, Millipore Co. Ltd) with Durapore (0.1
mm pore size) and Biomax (cuto membrane, molecular
size 5 kDa) membranes successively. About 15±20 l of
original water was concentrated to 200±400 ml in each molecular mass fraction, namely, 0.1mm-GF/F and 0.1 mm±5 kDa. The <5 kDa fraction was not concentrated.
The system was strictly pre-cleaned following the man-ufacturer's instructions.
GF/F ®ltrates and molecular mass fractions were subjected to IMAC; the procedure has been reported in detail elsewhere (Midorikawa and Tanoue, 1996, 1998) and is only summarized here. The sample was passed through the IMAC column (20 cm16 mm i.d. Phar-macia) at a rate of 4.0 ml/min for non-concentrated samples, and 0.4 ml/min for concentrated samples. Twenty millilitres of chelating Sepharose1fast ¯ow gel
(Pharmacia, Uppsala, Sweden) was used as the solid matrix for IMAC, and the column was charged with 0.02 M copper solution before sample loading. The organic ligands adsorbed to copper (II) on the column were eluted with mobile solution (0.01 M HCl and 0.1 M NaCl) at a rate of 0.8 ml/min. The eluants were monitored by UV absorbance at 254 nm (Shimadzu, MPS-2400, UV-vis multipurpose).
Additional aliquots of samples were subjected to UV absorbance and dissolved organic carbon (DOC) mea-surement without IMAC for the quanti®cation of the bulk DOC. DOC concentration was measured by high tem-perature catalytic oxidation using potassium hydrogen phthalate as a standard. After the water sample was acid-i®ed with HNO3and DIC was removed by bubbling with pure air for 15 min, 200ml of sample was injected into
the TOC analyzer (TOC 5000A, Shimadzu Co. Ltd).
System and pure water (Milli Q TOC, Millipore Co. Ltd) blanks were on average of 2±4 and 6mM C, respectively.
3DEEM ¯uorescence spectroscopy was recorded with a ¯uorescence spectrophotometer (Hitachi, Model F-4500). Wavelengths ranged from 230 to 400 nm for excitation (5 nm bandwidth), and from 250 to 600 nm for emission (2 nm bandwidth). A procedural blank was prepared by passing the mobile solution through a blank copper-loaded column. The blank EEM was sub-tracted to remove possible column contamination and water Raman scattering of samples. Instrumental cor-rection was made according to the manufacturer's instructions. MatlabTMwas used to obtain the 3DEEM surface and contour plots in which Ex/Em maxima can be identi®ed. Fluorescence intensity was calibrated to be evaluated in quinine sulfate units (QSU); 1 QSU=1mg/l
of quinine sulfate monohydrate in the solution of 0.05 M H2SO4at excitation/emission (Ex/Em) 350/450 nm.
3. Results and discussion
3.1. Molecular mass distribution of organic ligands
IMAC chromatograms, shown in Fig. 2, from various molecular mass fractions generally exhibited one major and resolved peak, as determined by UV absorbance at 254 nm. No obvious peak was observed in the blank column, and no signi®cant change in the absorbance at 254 nm was detected when water samples were loaded without copper immobilization (data not shown). This implies that the eluted organic ligands were reacting with copper immobilized on the column, not from adsorption onto the solid matrix of the IMAC gel, nor from elution of the IMAC gel itself. These IMAC chro-matograms are similar to those in oceanic waters (Mid-orikawa and Tanoue, 1996, 1998), and we anticipate that similar organic ligands have been isolated in fresh-waters. Thus, eluants with high UV absorbance were collected as organic ligand fractions for further char-acterization. The IMAC elution patterns will be further discussed in a later section.
The DOC and UV absorbance of fractioned DOM and isolated organic ligands are listed in Table 1. As deter-mined by DOC concentration, the relative abundance of the <5 kDa molecular fraction ranged from 54 to 69% of the total DOC, the 5 kDa±0.1 mm fraction from 30 to
43%, and 0.1mm-GF/F fraction from 1 to 2% (Table 1),
indicating that most DOC was in the <5 kDa molecular mass fraction. As determined by UV absorbance, the <5 kDa fraction accounted for 57±85% of the total DOM in the original water, which was slightly higher than those (54±69%) as determined by DOC.
Carlson et al. (1985) reviewed the ultra®ltration pro-cedure in seawater. It is necessary to examine the mass balance in assessing the validity of any ultra®ltration
system. The proportion of total absorbance of all frac-tions to the original GF/F ®ltrate was 89±109% for DOC, 84±111% for DOM, and 86±107% for organic ligands (Table 1). These recoveries are well in agreement with previous reports in oceanic waters (e.g. 80±118%; Carlson et al., 1985; Carlson et al., 1985; Guo et al., 1994). Our procedure using two broad-size membranes (5 kDa, 0.1mm) is proved to be able to quantitatively
estimate molecular mass distribution of DOC, DOM and organic ligands.
The relative DOC abundance of the <5 kDa fraction (54±69%) appears to be lower than that in oceanic waters of recent studies, i.e. the <1 kDa fraction accounted for 50±78% in seawater (Carlson et al., 1985; Guo et al., 1994; Guo and Santschi, 1996; Buesseler et al., 1996). In other related studies of freshwater, Ralph (1976) reported that only about 33% of DOC was in the <1 kDa fraction. Martin et al. (1995) showed that about 43% of DOC was in the <10 kDa fraction in the Lena River. It appears that DOC in the terrestrial environment may contain a higher proportion of high molecular mass fractions than in the oceanic environ-ment. Table 1 also shows that ratios of UV absorbance
Fig. 2. Elution patterns of organic ligands from GF/F ®ltrate
(&) and molecular mass fractions of the <5 kDa (*), 5 kDa±
at 254 nm to DOC concentrations were higher in the low (<5 kDa) and high (>0.1 mm) molecular mass
fractions than those in the intermediate (0.1mm±5 kDa)
fractions: the molecular mass distribution shifted in both directions (to lower and higher molecular frac-tions) for UV absorbance, as compared to DOC.
The molecular mass distribution of organic ligands has not been well studied. All molecular mass distribu-tions of organic ligands were estimated based on the measurements of copper(II) dissolved in the solution fractionated in terms of molecular mass distributions without direct measurement of any properties of organic ligands. Hart et al. (1992) and Ralph (1976) reported that most of the copper-complexing capacities were found in the >1 kDa fraction of DOM in freshwater. Martin et al. (1995) found that the >10 kDa molecular fraction of DOM had higher copper-complexing capa-cities than the <10 kDa fractions in coastal water.
For direct measurements of organic ligands, a few molecular mass distributions have been reported in
oceanic waters, and yet no data are available in fresh-water. Midorikawa and Tanoue (1998) reported that the <1 kDa fraction of organic ligands accounted for 49± 62% and 63±76% of total organic ligands in Equatorial Paci®c and North Paci®c water, respectively. Gordon et al. (1996) reported that 41% of one ligand class from estuarine water was in the <1 kDa fraction. Our results show that the relative contribution of organic ligands in the low molecular weight (<5 kDa) fraction was again dominant (67±79%), while 17±30% of the total organic ligands was in the 5 kDa±0.1mm fraction, leaving 3±6%
in the 0.1mm±GF/F fraction in Lake Biwa (Table 1).
The contribution of organic ligands varied among various molecular mass fractions. For the GF/F ®ltrate, organic ligands accounted for 1.6±4.0% of the bulk DOM in the original water (Table 1). In seawater less than 30% of the DOM has been biochemically identi®ed so far; most conventional isolation methods such as XAD resins can recover 5±10% of the total DOM (Ben-ner et al., 1992). Our IMAC ligands may only contain
Table 1
Mass balance of the bulk DOM, DOC, and organic ligands of various molecular mass fractions of lake water in Lake Biwa, as monitored in terms of DOC and UV absorbance
DOC DOM Organic ligands
Samples Fractions mM Ca Recoveryb
(%)
Absorbance at 254 nma
(10ÿ4cmÿ1)
Recoveryb
(% )
Abs/DOCc
(arb)
Absorbance at 254 nma
(10ÿ4cmÿ1)
Recoveryb
(%)
Proportion of ligand relative to DOM (%)
Station B
2.5 m GF/F ®ltrate 104.3 100 160 100 3.2 100 2.0 0.1mm±GF/F 2.1 (2) 10 (7) 3.5 0.1 (3) 1.0
5 kDa±0.1mm 40.1 (43) 50 (36) 0.8 0.9 (30) 1.8
<5 kDa 50.5 (54) 80 (57) 1.1 2.0 (67) 2.5 Sum of three fractions 92.7 (100) 89 140 (100) 88 3.0 (100) 94
20 m GF/F ®ltrate 92.0 100 150 100 2.7 100 1.8 5kDa- GF/F 38.4 (41) 50 (36) 0.9 0.6 (21) 1.2 < 5kDa 56.3 (59) 90 (64) 1.1 2.3 (79) 2.5 Sum of three fractions 94.7 (100) 103 140 (100) 93 2.9 (100) 107 2.1 70 m GF/F ®ltrate 88.8 100 132 100 2.1 100 1.6 0.1mm- GF/F 0.9 (1) 4.0 (3) 3.0 0.1 (6) 2.5
5kDa- 0.1mm 28.0 (30) 18 (12) 0.4 0.3 (17) 1.7
< 5kDa 65.3 (69) 125 (85) 1.2 1.4 (78) 1.1 Sum of three fractions 94.1 (100) 106 147 (100) 111 1.8 (100) 86
Station A
2.5 m GF/F ®ltrate 106.9 100 190 100 7.6 100 4.0 0.1mm±GF/F 2.3 (2) 10 (6) 3.0 0.2 (3) 2.0
5 kDa±0.1mm 47.2 (40) 30 (19) 0.5 1.7 (23) 5.7
<5 kDa 67.2 (58) 120 (75) 1.3 5.5 (74) 4.6 Sum of three fractions 116.7 (100) 109 160 (100) 84 7.4 (100) 97
a The value is estimated as that in the original water. The values in parentheses denote the percentage of each fraction to the sum of
all fractions.
the most reactive component of DOM in the original water.
3.2. Fluorescence characteristics of organic ligands
Fig. 3 shows 3DEEM surface and contour plots of organic ligands from various molecular mass fractions. Two Ex/Em maxima can be observed in these 3DEEMs, one from excitation in the visible region at 310±320 nm (Peak A, hereafter), and one from excitation in the UV region at around 250 nm (Peak B, hereafter). Peak A had an emission maximum at 378±430 nm, and Peak B an emission maximum at 428±446 nm. Ex/Em maxima of Peak A and B identi®ed in 3DEEMs of various molecular ligands are summarized in Table 2. For peak A, in the 3DEEM contour plots (Fig. 3), the emission maxima wavelength remained constant with increas-ingly longer or shorter excitation wavelength, indicating that peak A may be from a single ¯uorophore. The contour plots for Peak B, however, had an elliptical shape, with emission maxima slightly increased with excitation wavelength (Fig. 3). This may indicate that Peak B was derived from a mixture of similar ¯uor-ophores, thus Em maximum wavelength at Ex=250 nm was chosen to measure ¯uorescence intensity for Peak B
in this study. These peaks are similar to previous reports for DOM ¯uorescent substances in aquatic environ-ments (e.g. Mopper and Schultz, 1993; Coble, 1996; Del Castillo et al., 1999), and are usually referred to as humic-like ¯uorescence (Mopper and Schultz, 1993).
Peaks A and B existed in organic ligands from all molecular mass fractions (Table 2). The positions of Ex/ Em maxima of Peak A in the <5 kDa and 0.1mm-GF/F
molecular ligands were similar for each sample, around Ex/Em 310±315/394±414 nm, while Ex/Em maxima of Peak A from the 5 kDa±0.1mm (intermediate) ligands
were around 315±320/402±430 nm. Comparison of var-ious molecular ligands in each respective sample (Table 2) clearly suggests that Ex/Em maxima of Peak A in both low and high molecular ligands were blue-shifted relative to those in the intermediate molecular ligands except for surface water of Stn A (Table 2), where only Em max-ima were blue-shifted. No signi®cant shift of Peak B was observed between various molecular fractions.
Shifts of Ex/Em maxima of organic ligands between various molecular mass fractions have not been repor-ted. Ex/Em maxima may depend mainly on the mole-cular chemical composition and structure, while other factors such as pH, temperature, metal ions and solvents were reported to mainly aect ¯uorescence intensity and
Table 2
Ex/Em maxima in 3DEEMs of organic ligands in various molecular mass fractions, and their ratios of ¯uorescence intensity to UV absorbance at 254 nm (FL/At)
Peak A Peak B Peak C Samples Fractionsa UV
absorbance (cmÿ1)
Ex/Em maxima (nm)
FL/Atb
(10)
Ex/Em maxima (nm)
QSU/Atb
(10)
Ex/Em maxima (nm)
FL/Atb
(10 QSU) A/Bc
Station B
2.5 m GF/F ®ltrate 0.102 310/392 250/428 270/306 1.5
0.1mm±GF/F 0.054 310/394 1.9 250/440 3.6 280/316 4.6 0.5
5 kDa±0.1mm 0.279 320/430 1.9 250/446 2.9 ± 0.6
<5 kDa 0.088 315/400 3.5 250/428 5.2 ± 0.7 20 m GF/F ®ltrate 0.165 320/382 250/434 ± ±
5 kDa±GF/F 0.059 320/392 4.6 250/444 5.3 ± ± 0.9 <5 kDa 0.139 315/378 9.8 250/426 4.7 ± 2.1 70 m GF/F ®ltrate 0.076 320/378 250/432 280/308 1.0
0.1mm±GF/F 0.041 315/402 0.4 250/332 0.6 265/318 1.0 0.7
5 kDa±0.1mm 0.118 320/408 3.0 250/434 4.0 ± 0.8
<5 kDa 0.286 ± ± 250/436 0.6 ± ±
Station A
2.5 m GF/F ®ltrate 0.142 315/390 250/432 280/318 0.7
0.1mm±GF/F 0.032 315/406 2.6 250/436 7.0 280/318 3.3 0.4
5 kDa±0.1mm 0.195 315/424 2.1 250/436 3.5 ± 0.6
<5 kDa 0.135 315/414 3.0 250/440 4.4 ± 0.7
a Molecular mass fractions measured are the same as those for DOC and UV absorbance in Table 1. b The values are ratios of QSU to the UV absorbance at 254 nm in organic ligands.
c Values are ratios of Peaks A and B ¯uorescence.
quantum eciency (Hall and Lee, 1974; Coble, 1996). Bue et al. (1982) observed that emission and excitation maxima wavelengths of DOM ¯uorescence were shifted to longer wavelength with increasing DOC in river waters. They attributed this shift to the formation of aggregates due to high DOC concentrations. Matthews et al. (1996) reported that Ex/Em maxima of Aldrich humic acid did not change signi®cantly or shift in the 10±100 ppm concentration range. Our data (Table 2) show that the concentrations of organic ligands, as indicated by UV absorbance, were not related to Ex/ Em maxima in each molecular ligand of each sample, indicating the shifts of Ex/Em maxima observed may not result from a concentration dierence. Hayase and Tsubota (1985) reported that the Ex/Em maxima in sedimentary fulvic acid were blue-shifted relative to humic acid, and were independent of molecular weight. Previous studies showed that excitation maxima of natural organic matter were blue-shifted as the mole-cular weight decreased (Ewald et al., 1988; Senesi, 1990), which is not consistent with our results. Thus our data suggest that IMAC organic ligands may be dierent from the ¯uorescent humic and fulvic compounds.
Another interesting dierence between various mole-cular ligands is that an additional Ex/Em maximum in the UV region (Peak C, hereafter) was observed in 3DEEMs from the 0.1mm-GF/F and GF/F fractions in
the surface and deep water (Fig. 3 and Table 2). The Ex/ Em maxima of Peak C were 265±280/306±318 nm. This observed Peak C is similar to the protein-like ¯uores-cence reported in seawaters (e.g. 270/320 nm, Determann et al., 1994; 270/320 nm, Mopper and Schultz, 1993; 280/ 325±335 nm, Coble et al., 1990), and also in algal bloom and higher productive waters (Traganza, 1969; Petersen, 1989). Protein-like ¯uorescence was, therefore, believed to be associated with the growth of living organisms. Since phytoplankton, bacterioplankton and picophyto-plankton can be included in the 0.1mm-GF/F molecular
fractions, our results may indicate that the protein-like ¯uorescence of organic ligands may be due to these liv-ing particles. This can be substantiated by previous stu-dies that phytoplankton and bacteria were two main sources of protein-like ¯uorescence (Determann et al., 1996, 1998).
Fluorescence distributions for Peaks A and B in var-ious molecular organic ligands are shown in Table 3. Mass balance is again satisfactory, i.e. recoveries are 81± 102%. For humic-like ¯uorescence, it was again domi-nant in the <5 kDa molecular ligands of all samples, accounting for 77±91% and 59±86% of the total ¯uo-rescence for Peaks A and B, respectively. For Peak A, the ratios of ¯uorescence intensity to UV absorbance (FL/At) increased with decreasing molecular weight, suggesting that the distribution shifted to the lower molecular weight fractions for Peak A, as compared to UV absorbance. This is consistent with previous reports
that ¯uorescence intensity or FL/Atincreased with the decreasing molecular weight (Stewart and Wetzel, 1980; Ewald et al., 1988; Senesi, 1990). However, it is not the case for Peak B, FL/Atdid not obviously increase with decreasing molecular weight. The ratios of ¯uorescence intensity of Peak A to B increased with decreasing molecular weight. Peak C only occurred in the high molecular weight fractions. This indicates that ¯uores-cence at the longer excitation wavelength was more heavily weighted in the lower molecular organic ligands relative to that at the shorter wavelength and UV absorbance, while ¯uorescence at the shorter excitation wavelength was more weighted in the high molecular ligands. For humic-like ¯uorescence, Peak A was pre-ferentially tied to lower molecular organic ligands, and Peak B to the higher molecular ligands.
3.3. Binding anity of organic ligands
To examine the relationship between the anity of organic ligands to copper(II) and their molecular weight, the IMAC elution patterns of respective mole-cular mass fraction were studied. As shown in Fig. 2, organic ligands of molecular weight less than 5 kDa were eluted faster than those of the intermediate mole-cular weight, and organic ligands of the high molemole-cular weight were eluted last. Since the stronger ligands may be retained longer in the IMAC column, and would thus be eluted later (Midorikawa and Tanoue, 1998), our observations suggest that the strength of organic ligands is in the order: 0.1mm±GF/F> the 5 kDa±0.1mkDa>
the less than 5 kDa organic ligands. The results are consistent with previous reports in seawater (Mid-orikawa and Tanoue, 1998).
At present we have little information about the com-position or structure of the organic ligands, The reasons why the higher molecular masses had higher anity for copper(II) are unclear. Our direct observation of organic ligands in various molecular fractions may bring some new insights. (1) Proteins and oligopeptides were reported to be a constituent of DOM of high molecular masses containing primary amines in sea-water (Lee and Bada, 1975; Tanoue et al., 1996). Pro-tein-like ¯uorescence may be derived from free aromatic amino acids or protein constituents in organic ligands. Our observation that protein-like ¯uorescence was detected in the higher molecular mass (0.1mm±GF/F)
Tanoue, 1998). Experiments demonstrated that exudates from certain phytoplankton and bacteria, two strong sources for protein-like ¯uorescence, are strong Cu che-lators (McKnight and Morel, 1980; Determann et al., 1998). Therefore, it may be possible that high molecular organic ligands containing protein-like ¯uorescence may be more related to strong ligands. (3) High molecular weight ligands may have a higher apparent surface potential than lower molecular weight ligands, and have an enhanced ability to bind metal ions, as reported in humic substances (Green et al., 1992). (4) Organic molecules of high molecular weight may have structural advantage for metal complexations, e.g. forming con-formation among the functional groups, and providing more and stronger binding sites.
It is noteworthy that the 5 kDa±0.1 mm molecular
ligands in this study may be from colloidal (1 nm±1mm).
Colloids have been reported to have extreme reactivity and strong binding capacity with trace metals (Martin et
al., 1995), and participate in equilibria involving aggrega-tion and disaggregaaggrega-tion (Orlandini et al., 1990), but their ¯uorescence characteristics are unknown. Our results show that Peak A was red-shifted in the 5 kDa±0.1mm
molecular ligands relative to the lower and higher ligands, probably just re¯ecting their special ¯uorescence char-acteristics. Changes in excitation maxima indicate a change in chemical composition and/or structures (Coble, 1996). A red shift in emission maxima can be caused by a decrease between the ground state and the ®rst excited state. Structural and compositional changes that could result in the red-shifted emission maximum can include the following factors: (1) an increase in the number of aromatic rings, and of conjugated bonds in a chain structure; (2) an increase of certain functional groups such as hydroxyl and amine (Senesi, 1990). Those changes can also be responsible for the reported characteristics of colloids such as reactivity and binding anity, as discussed above. In this connection, our
Table 3
Mass balance of ¯uorescence of organic ligands from various molecular mass fractions of lake water in Lake Biwa, as monitored in terms of UV absorbance and ¯uorescence
Peak Ab,c Peak Bb,d
Samples Fractionsa Fluorescence
(arb)
Recoverye
(%)
FL/Atf
(104cm)
Fluorescence (arb)
Recoverye
(%)
FL/Atf
(104cm)
A/Bg
Station B
2.5 m GF/F ®ltrate 7.5 100 7.7 100
0.1mm±GF/F 0.2 (3) 1.0 0.2 (3) 1.0 0.9
5 kDa±0.1mm 1.2 (19) 0.6 1.7 (22) 0.7 0.9
<5 kDa 4.8 (77) 1.1 5.9 (76) 1.1 1.0 Sum of three fractions 6.2 (100) 83 7.8 (100) 101
20 m GF/F ®ltrate 1.4 100 3.8 100
5 kDa±GF/F 0.2 (15) 0.7 1.3 (41) 2.0 0.4 <5 kDa 1.1 (85) 1.1 1.9 (59) 0.7 1.4 Sum of three fractions 1.3 (100) 93 3.2 (100) 84
70 m GF/F ®ltrate 5.2 100 3.3 100
0.1mm±GF/F 0.1 (2) 0.3 0.2 (8) 1.3 0.3
5 kDa±0.1mm 0.4 (8) 0.5 0.4 (15) 0.9 0.5
<5 KDa 4.8 (91) 1.2 2.0 (77) 1.0 1.2 Sum of three fractions 5.3 (100) 102 2.6 (100) 79
Station A
2.5 m GF/F ®ltrate 10.9 100 11.7 100
0.1mm±GF/F 0.1 (1) 0.3 0.3 (3) 1.0 0.3
5 kDa±0.1mm 0.9 (10) 0.4 1.3 (11) 0.5 0.9
<5 kDa 7.8 (89) 1.2 9.8 (86) 1.2 1.0 Sum of three fractions 8.8 (100) 81 11.4 (100) 98
a Molecular mass fractions measured are the same as those for DOC and UV absorbance in Table 1.
b The value is given as that in the original water. The values in parentheses denote the percentage of each fraction to the sum of all
fractions.
c and d Peak A and B ¯uorescence is measured using emission maxima ¯uorescence at Ex 315 and 250 nm, respectively. e Recovery is expressed as a proportion of the sum for all fractions relative to the value for the GF/F ®ltrate.
®nding may be indicative of structural and composi-tional characteristics of the colloidal organic ligands.
4. Conclusions and implications
This study demonstrates the advantage of combined ultra®ltration, IMAC and 3DEEM techniques in char-acterizing organic ligands for copper(II). The results indicate the unique chromophoric characteristics of organic ligands from various molecular mass fractions, and their relation with binding anity. The molecular distribution of organic ligands is similar to that of dis-solved organic matter in previous studies, but with dif-ferent ¯uorescence characteristics. Although the IMAC organic ligands may be weak ligands in terms of stability constants, as reported in oceanic waters (Midorikawa and Tanoue, 1996, 1998), these ligands may play an important role in the mobilization and transport of nutrients and pollutants, and soil weathering in terrestrial ecosystems (Mantoura et al., 1978; Tegen and Dorr, 1996; Li and Shuman, 1997; Kalbitz and Wennrich, 1998). Thus, our ®ndings have signi®cance for further understanding the nature, dynamics and biogeochemical role of organic ligands in aquatic environments.
Acknowledgements
The authors are grateful to Drs. Yoshioka, Susuki, Saino, Masuzawa and Konohiro (Nagoya University), and Drs. Takahashi and Hayakawa (Lake Biwa Research Institute) for their discussion and assistance in sampling and use of laboratory instruments. This research was supported by the Grant-in-Aid for Scien-ti®c Research for IGBP from the Ministry of Education, Science, Sports and Culture, Japan, Grant-in-Aid for JSPS fellowship and partly by the Lake Biwa Research Institute.
Associate EditorÐS.G. Wakeham
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