Environ. Sci. Technol. 2002, 36, 2003-2007
Characterization of Sorption Sites on Pilayella littoralis and Metal Binding Assessment Using 113Cd and 27Al Nuclear Magnetic Resonance E L M A N E I D E V . M . C A R R I L H O , * ,†,‡ ANTONIO G. FERREIRA,† AND THOMAS R. GILBERT‡ Departamento de Quı´mica, Universidade Federal de Sa˜o Carlos - SP - Brazil, and Department of Chemistry and The Barnett Institute, Northeastern University, Boston, Massachusetts
Metal interactions with the cellular structures of the marine alga Pilayella littoralis have been investigated to better understand how biomaterials sorb dissolved metals. Algae metal binding capacity at pH 5.0 was 2000, 850, 430, and 560 µmol g-1 of dried material for AlIII, CuII, CdII, and CoII, respectively. Binding site characterization was assessed by 1H and 13C nuclear magnetic resonance spectroscopy. Also, Fourier Transform Infrared spectroscopy (FTIR) provided some information about the types of functional groups that appear to be present in the algal material. The results suggested the presence of carboxylate, ether, amino, and hydroxyl groups. Investigation of metal competition for the alga binding sites was performed using 27Al and 113Cd NMR spectroscopy, which proved to be a valuable technique for Al and Cd sorption assessment. Aluminum and Cu were efficiently sorbed by the alga sites, and the binding affinity order of these metals was AlIII > CuII > CdII > CoII.
Introduction Metal uptake by biological materials is believed to occur through accumulation processes involving functional groups found in the walls of the biological cells and is associated with biopolymers such as proteins, polysaccharides, and lignin. Amine, amide, carboxylate, hydroxyl, imidazole, sulfate, phosphate, thiol, and sulfhydryl groups are believed to be the functionalities responsible for metal uptake (1). This wide range of possible binding sites in biomasses indicates the great advantage of using biological materials as biosorbents. Therefore, the complete elucidation of the mechanisms by which metals are bound to biomasses plays an important role for the application of these materials in metal sorption and preconcentration. The knowledge of chemical structures of biosorbents is important to predict their affinities for metal ions, to choose new kinds of biomasses, and to improve their complexation properties. * Corresponding author present address: Embrapa Pecuaria Sudeste, Rod. Washington Luiz km 234, 13560-970 Sa˜o Carlos - SP, Brazil; phone: 55.16.2615611; fax: 55.16.2615754; e-mail: elmavm@ uol.com.br. † Universidade Federal de Sa ˜ o Carlos. ‡ Northeastern University. 10.1021/es0107834 CCC: $22.00 Published on Web 04/03/2002
2002 American Chemical Society
However, the identification of functional groups is rather a difficult task due to the complexity of these materials. Several strategies have been proposed to elucidate the functional groups responsible for the binding of different metals by biological materials. Competition for metal-binding sites on algae between metals and hydronium ions has been studied (2-5). Alkali and alkaline-earth metals were found to adsorb in an ion-exchange process based on electrostatic interaction (3). However, covalent bonding was involved in the uptake process for Cu in which protons were released (4). The nature of bonding between metallic ions and algal cell walls was evaluated by reporting data on proton displacement, pH titration, and ion exchange involving various elements and the alga (5). The authors investigated covalent and ionic bonding of metallic ions with nitrogen, carboxyl, and sulfate groups. Studies on metal binding by Datura innoxia indicated that carboxylate groups present on its cell walls belong to two different chemical moieties (6). The sorption affinity order observed in metal competition assessment was CuII > EuIII ≈ GdIII > CdII. Electrostatic and complexing interactions between this plant and AgI, NiII, SrII, or BaII were also investigated (7), and higher retention was found at pH e 5. Solutions containing carboxylate, amine, hydroxyl, sulfhydryl, oxalate, sulfate, and sulfonate functionalities have been utilized in 113Cd NMR spectroscopy to identify the moieties on D. innoxia cell walls responsible for chemical shifts due to interaction with CdII (8). It was found that carboxylate groups were the dominant functional groups responsible for the binding of CdII at pH e 5 in a 2:1 ratio. Diamine groups were also involved in CdII binding at pH 6.0. Aluminum binding to biomaterial derived from cell wall fragments of this plant has been investigated using solid-state 27Al NMR spectroscopy (9). Carboxylate groups have been determined to be the functionalities responsible for AlIII binding at pH 3.5 and at pH 5.0. The presence of an additional octahedral Al-binding site was also suggested at the higher pH. An interesting approach for binding site elucidation has been the modification of five nonliving algal species by esterifying their carboxyl groups using acidic methanol (10). CopperII, AlIII, and AuIII binding abilities of the modified algae were determined and compared to unmodified algal biomasses. The methanol-modified biomass showed major decreases in CuII and AlIII binding, while AuIII binding slightly increased, indicating that carboxyl groups on the algal cells were responsible for a great portion of CuII and AlIII binding and that they played an inhibitory role in AuIII binding. Watkins et al. (11) used X-ray absorption to investigate the nature of AuI and AuIII binding by Chlorella pyrenoidosa. They found strong evidence for ligand-exchange reactions involving gold-sulfur and gold-nitrogen bonds on the cell. Majidi et al. (12) examined metal interaction with the alga Stichhococcus bacillaris by 113Cd NMR and suggested that Cd uptake by this alga involved interaction with multiple carboxylic groups. The dynamic response of this alga to the exposure of ZnII, MnII, CdII, and CuII has also been examined by using 31P NMR and exhibited a metal adsorption equilibrium after 3 min of exposure (13). Metal affinities of freshwater blue-green algae were characterized, and sorbed elements were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (14). Other methods such as potentiometric and conductometric titrations have been used to determine strongly and weakly acidic groups in biological material (15). The brown alga Pilayella littoralis is a filamentous freeliving form, widely distributed in the north shore beaches of VOL. 36, NO. 9, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Massachusetts, U.S.A. This alga washes up on the shore of Nahant Bay beaches in irregular shapes all year long. At low tides, dense mats up to 30 cm thick are formed during the summer and the fall. Its proliferation has been causing undesirable odors stemming from H2S produced during its decomposition. To find an efficient and promising use for this abundant natural source, Carrilho and Gilbert (16) have studied the ability of this alga to sorb metal ions. This biomass was used in batch tests with synthetic solutions to study its metal uptake properties, including metal binding capacity, pH dependence of metal uptake, and the kinetics of metal sorption. Sorption of AlIII, CdII, CoII, CrVI, CuII, FeIII, NiII, and ZnII occurred within the first 5 min of exposure, and the metals were bound to the algae at pH 5.5. These metals were desorbed with diluted HCl and determined by ICP-OES. The authors have also proposed the use of a silicaimmobilized algae column for AlIII, CoII, CuII, and FeIII preconcentration from lake water samples (17). Utilizing an online flow injection - ICP-OES approach, sorption efficiency ranged from 86 to 90%. Accuracy was assessed by using certified reference material and graphite furnace atomic absorption spectrometry as a comparison technique. This paper describes characterization of the metal binding sites on the alga P. littoralis by nuclear magnetic resonance (NMR) spectroscopy. Fourier Transform Infrared spectrometry (FTIR) also provided some useful information about the types of functional groups which may be present in the investigated alga. The results suggested the presence of carboxylate, ether, amino, and hydroxyl groups. Metal interaction with this alga and sorption sites competition among metals were assessed by 27Al and 113Cd NMR.
Experimental Section Apparatus. A 10-channel PLASMA-SPEC (Leeman Labs Inc., Lowell, U.S.A.) inductively coupled plasma optical emission spectrometer was used for metal determination. The plasma was operated at a nominal applied power of 1.0 kW. The nebulizer and coolant gas (argon) flow rates were 0.4 and 12 L min-1, respectively, while sample uptake rate was 0.9 mL min-1. Algae suspensions were centrifuged with a Centrifuge Model IEC HN-S (Damon, Needham Heights, U.S.A.) operating at 2000 rpm. Infrared spectra of the algal biomass were recorded on a Michelson 102 (BOMEN, Quebec, Canada) FTIR spectrometer. Nuclear magnetic resonance experiments were performed on DRX400 and ARX200 (Bruker Analytik GmbH, Rheinstetten, Germany) spectrometers with 9.4-T and a 4.7-T narrow-bore superconducting magnets, respectively.
Reagents and Samples Distilled, deionized water of 18 MΩ-cm resistivity was prepared using a MilliQ system (Millipore, Bedford, U.S.A.) and was used to prepare all solutions. All analytical solutions were buffered at pH 5.0 in 5 mmol L-1 CH3COOK prepared from analytical reagent grade potassium acetate (Fisher Scientific, Atlanta, U.S.A.). The pH of all solutions was adjusted by dropwise addition of nitric acid or ammonium hydroxide solution. Diluted hydrochloric acid 0.12 mol L-1 (Fisher Scientific) was used for algae prewash. Buffer standard solutions at pH 4 and 7 were used to calibrate the pH meter prior analysis. Reagent grade nitric and perchloric acids (Fisher Scientific) were used for acid digestion of algae samples. Working solutions of 10 g L-1 of AlIII, CdII, CoII, and CuII were prepared from reagent grade Al2(SO4)3, CdCl2, CoCl2‚ 6H2O, and CuCl2, respectively. The reagent 3-trimethylsilyl2,2,3,3-tetradeuteropropionic-acid, sodium salt (TSPd4) (Aldrich, Milwaukee, U.S.A.) was used in the NMR experiments. Algae samples were separated from sedimentary and other biological matter, dried, ground to approximately 150-µm 2004
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particle size, and washed with 0.12 mol L-1 HCl to remove naturally sorbed metals. These samples were then rinsed with 5 mmol L-1 CH3COOK at pH 5.0 and oven dried at 80 °C for approximately 1 h. All glassware and plasticware were washed with diluted neutral cleaning solution, soaked overnight in 10% nitric acid, and rinsed 3 times with distilled deionized water before use. Metal Binding Capacity. The algae binding capacity for Al, Cd, Co, and Cu was assessed by repeatedly suspending 50 mg of this biomass in fresh 5-mL aliquots of 5 mg L-1 of AlIII, CdII, CoII, or CuII at pH 5.0. The mixtures were agitated for 5 min and centrifuged. After reaching saturation, the metal-loaded algae samples were washed in acetate buffer at pH 5.0 to remove unretained metals. Three replicates were run for each metal. The algae were oven dried and digested in HNO3/HClO4 mixture at 50-80 °C, and the metals in the digestates were determined by ICP-OES. Special caution was required to handle the perchloric acid due to its explosive potential in the presence of organic matter under heating. FTIR and NMR Assessment. Dried P. littoralis samples were analyzed in the FTIR for identification of functional groups. Algae pellets (1%) were prepared in potassium bromide, pressed, and kept under vacuum before analysis. Suspensions of 100 mg of P. littoralis in 1 mL of D2O/NaOD solution were used in the 1H and 13C NMR experiments. Deuterated water was used to achieve homogenization of the magnetic field, and TSPd4 was the external reference. To investigate any possible denaturing of the algae binding sites possibly caused by the acid wash, 1H spectra of the diluted HCl after algae rinsing were obtained by presaturating the HOD signal. Aluminum and Cd sorption on the algae was investigated by 27Al and 113Cd NMR spectroscopy. Samples of P. littoralis (100 mg) were suspended in 1 mL of a solution containing 10 g L-1 of Al2(SO4)3 or CdCl2 at pH 5.0 in 5 mmol L-1 potassium acetate buffer. The pH of each of the solutions was adjusted by dropwise addition of nitric acid or ammonium hydroxide solution. Metal Competition Assessment by NMR Spectroscopy. First, a 113Cd NMR spectrum was obtained in the DRX400 using 10 g L-1 CdCl2 solution at pH 5.0 as a reference standard. After adding the alga to the metal solution, a new spectrum was obtained to observe the metal complexation on the algae sorption sites. This spectrum was then later compared with that obtained after the addition of a second metal to the CdII-alga suspension to evaluate possible competition among the metal ions studied. In these cases, 10 g L-1 CoII, CuII, or AlII at pH 5.0 were added to CdII-alga suspension. Cadmium would be displaced from its site on the alga if the competitor metal exhibited higher affinity for the occupied binding sites. For each metal added, a new free and bound Cd spectrum were obtained. The same procedure was used in the 27Al NMR experiments. In this case, the ARX200 spectrometer was used. The reference solution for this metal was 10 g L-1 Al2(SO4)3 at pH 5.0. Competition for aluminum sorption sites was also evaluated with CdII, CoII, and CuII.
Results and Discussion Metal Binding Capacity and FTIR Assessment. An investigation on the pH dependence of metal accumulation by P. littoralis has been performed in earlier work by Carrilho and Gilbert (16). In the present work, all metal solutions used in the NMR and FTIR experiments were prepared at pH 5.0. At this pH, alga binding capacity for AlIII, CuII, CdII, or CoII was 2000, 850, 430, and 560 µmol g-1 of dried material, respectively. These results were correlated with formation constants (logKf) reported for metals and phthalate, acetate, and EDTA (18, 19). This study was an attempt to compare these ligands with some specific functional groups in the algae structure and to evaluate their different abilities to complex with AlIII,
CuII, CdII, or CoII. High values of logKf have been reported for metal ions with EDTA, which indicates their strong affinity for its carboxylate groups. Similarly, we suggest that carboxylate groups in the algae may play an important role in metal sorption. Acetate and phthalate also exhibit the ability to complex with metal ions. However, the use of acetate as a metal buffer did not affect metal binding by the algae. In addition, the strong affinity of EDTA with metal ions, indicated by its high values of log Kf, implies that acetate would not compete with carboxylate sites on the algae and that the metals are preferentially bound as free metal ions rather than as metal-acetate complexes. Although the use of (2-N-morpholino)ethanesulfonic acid (MES) buffer has been suggested due to its inability to complex with metal ions (6-9), acetate buffer has also been used in metal sorption studies (10, 20-22). Copper, Zn, and Hg, present in solution as acetates, have been efficiently sorbed in algal biomass as free metal ions (20). The FTIR spectra (not shown) obtained in this experiment provided some information to help on the partial elucidation of the functional groups present in the algae. The results showed an absorption at 3450 cm-1 which is believed to be due to hydrogen bonding to different groups such as O-H and N-H. The spectra also exhibited absorptions at approximately 2930 and 2850 cm-1 suggesting the occurrence of C-H stretching. Absorptions at 1730 and 1650 cm-1 were observed and could be assigned to conjugated CO2H and CdO, respectively. Another absorption at 1540 cm-1, taken together with the 1450 cm-1, may be a carboxylate anion site. The spectra also exhibited absorption in lower wavelength regions. Even though FTIR cannot provide conclusive evidence, the absorptions observed in the IR spectra suggest the presence of functional groups such as SdO, P-O-C, P-OH, C-N, or P-O. 1 H and 13C NMR Spectra of the Algae. 1H NMR experiments were performed with a sweep width of 9057 Hz and a 3.6 s acquisition time. A signal was found in the spectra (not shown) of 0.12 mol L-1 HCl algae rinsing solution, which is attributed to the presaturated hydrogen from HOD molecules of the D2O added to the washing acid. No other signals were observed. This suggests that no detectable changes in the alga structure occurred as evidenced by the 1H NMR spectrum. However, in such a complex NMR spectrum, highly acidic label groups possibly present in the alga may not be detectable after complete hydrolysis by the acid treatment. The algal material might change with the acid wash without having its binding sites denatured. However, from the 1H NMR spectrum one can suggest that diluted hydrochloric acid apparently does not remove functional groups from the algae backbone. This evaluation was important since this acid was extensively used in the algae prewash steps. The 1H NMR spectrum of P. littoralis is depicted in Figure 1. A characteristic region in the spectrum observed between δ 7.2 and 6.4 ppm is due to aromatic C-H. The peak at δ 4.8 ppm is typical of olefin, and the region between δ 4.0 and 3.0 ppm is assigned to hydrogen on methyl, methylene, and methine carbons bound to oxygen from sugar-like components, ether or methoxyl groups. It is also believed that hydrogens bound to nitrogen from amide are present in this region. According to Francioso and co-workers (23) the chemical shifts from δ 3.4 and 1.5 ppm are assigned to hydrogens on carbons R to electronegative functional groups such as carbonyl, carboxyl, and aromatic rings. The chemical shifts from δ 0.9 to δ 1.5 are from hydrogens on carbons of aliphatic methyl and methylene groups. The intense peak at δ 8.2 ppm can be attributed to the formate ion (23). As expected, the alga contains a variety of functional groups that can represent the sites involved in the metal sorption process. This investigation was supported by obtaining the
FIGURE 1. 1H NMR spectrum of dried P. littoralis. Algae suspension was prepared with 100 mg of P. littoralis in 1 mL of D2O/NaOD solution.
FIGURE 2. 13C NMR spectrum of (a) 100 mg of dried P. littoralis suspended in 1 mL of D2O/NaOD solution and in single metal solutions of 10 g L-1 of (b) CdII, (c) CuII, or (d) AlIII at pH 5.0. 13C
NMR spectra of the algae. Figure 2a shows the 13C NMR spectrum of the algae suspension. Due to the structural complexity of the alga and its low solubility in D2O, the spectrum exhibited poor resolution and sensitivity at the conditions used (number of scans, 71 000). However, four main regions can be observed in the spectrum. The broad resonance from 10 to 50 ppm can be attributed to methyl, methylene, and methine groups in alkyl chains with possible contributions of carbons from amino acids (∼40 ppm). The signal at 55 ppm is assigned to O-CH3 groups, and the intense broad resonance between 60 and 80 arises from carbohydrate (24). The broad resonance between 100 and 150 is attributed to aromatic compounds, and the strong peak between 165 and 195 is assigned to carbonyls mostly from carboxylic groups. The results from 1H and 13C NMR studies indicated VOL. 36, NO. 9, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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that the main functionalities present in P. littoralis samples were carboxylate, ether, amine, and hydroxyl groups which can potentially be involved in metal sorption by this alga. Assessing Metal Sorption and Metal Competition by NMR. The 13C NMR spectra of metal-loaded P. littoralis were obtained to investigate the sorption of metals on the algae sites. Figure 2 shows the spectra before (a) and after (b, c, and d) loading the algae with AlIII, CuII, or CdII, respectively. Since the signal-to-noise in the carbon spectrum in Figure 2a could be improved by increasing the number of scans, the new spectra b, c and d, obtained after the addition of Al, Cu, and Cd, were processed with approximately 235 000 repetitions. This number of scans greatly improved the sensitivity and resolution of the new spectra. The binding of these metal ions caused the carboxylate shifts between 160 and 200 ppm in a to become essentially one type of carboxylate at 180 ppm exhibited in b, c, and d. This indicates the strong affinity of AlIII, CuII, and CdII for these functional groups as previously discussed in the metal binding capacity section. A similar behavior seems to take place for most of the remaining regions. The resonance at about 130 ppm in Figure 2a, possibly assigned to phenolic carbons, is greatly attenuated in the alga-Cd spectrum and completely eliminated in the alga-Al and alga-Cu spectra. Similarly, the peak at about 55 ppm (spectrum a) previously attributed to carbohydrates seems to shift to about 60 ppm in the alga-Cd spectrum and disappears in the corresponding Al and Cu spectra. Despite the fact that carboxylates have been determined to be the responsible functionalities for CdII (8, 12) and AlIII (9, 10) binding to different biomasses, the results with P. littoralis indicate that phenolic and carbohydrate groups can also be involved in the retention of these metal ions. It may also be possible to have different functional groups working in tandem to adsorb metal. However, it has been reported that at 10 g L-1 CdII (the concentration used in the P. littoralis studies) the carboxyl groups greatly dominate over all other functional groups, and signals other than the Cd-carboxyl hardly are observed (12). Studies on the complexation of CdII with several fulvic acids using 113Cd NMR indicated that this metal ion predominantly binds to this humic substance through oxygen components, in particular carboxylate moieties (25). A quantitative study of acid group structures in fulvic acid concluded that they contain several strongly acidic sites, identified as carboxyl groups associated with aromatic and aliphatic carboxylic acids (26, 27). Since Al and Cd exhibit higher natural abundance and receptivity than Co and Cu (28), 113Cd and 27Al NMR were used to investigate metal sorption on the algae. A 5 mm broad-band probe was tuned to observe the CdCl2 signal at 88.74 MHz at 298 K. The 113Cd NMR spectra were obtained with a 90° pulse of 12.0 µs, acquisition time of 0.4 s, sweep width 75 188 Hz, recycle delay 1 s, and 15 000 transients. They were processed with zero-filling and exponential multiplication (line broadening, LB ) 3 Hz). The Al2(SO4)3 signal was observed using a 5 mm probe tuned at 52.15 MHz at 298 K. A 90° pulse with 6.0 µs duration, recycle delay of 1 s, acquisition time 0.13 s, sweep width 71 429 Hz, and 64 transients were used to obtain the spectra, which were processed with zero-filling and exponential multiplication (LB ) 10.0 Hz). Figures 3a and 4a show the NMR spectra of the aqueous complexes of CdCl2 and Al2(SO4)3, respectively. They contain a single resonance absorption for each metal and are used as the chemical shift references in 113Cd and 27Al NMR experiments. The NMR spectra of CdCl2 and Al2(SO4)3 algae suspensions are shown in Figure 3b and 4b, respectively. The singlets shown at 0.0 ppm correspond to the residual CdCl2 and Al2(SO4)3 in the solutions, and the broader resonances at approximately 50 ppm upfield and 75 ppm 2006
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FIGURE 3. 113Cd NMR spectra of (a) 10 g L-1 CdII solution and (b) 100 mg of dried algae suspended in 1 mL of 10 g L-1 of CdII in the presence of either (c) CoII, (d) CuII or (e) AlIII. downfield are attributed to sorbed Cd and Al on the algae cells, respectively. The broadening of the chemical shift of sorbed metal ions may indicate a variety of similar sites rather than a specific site. The competition for CdII binding sites was assessed by adding CoCl2‚6H2O, CuCl2, or Al2(SO4)3 solutions to the CdCl2algae mixture. The results of this study are also depicted in Figure 3. When Co (Figure 3c) was added to Cd-loaded algae, a slight change in the spectrum was observed. However, in Figure 3d the Cd-algae peak is completely lost indicating that Cu efficiently displaces Cd from its binding site(s) on the algae. When Al is added to the Cd-algae suspension, a small amount of bound Cd still remains in the alga site(s) (Figure 3e). As stated elsewhere (12) the Cd-algae shifts indicate the presence of dominant sorption sites responsible for the accumulation of specific metals. In other words, sorption of Al, Cu, and Cd may preferably occur in the same specific sites of the alga, which may not be the case as for Co. Since Co exhibited greater binding capacity than Cd and its formation constant with EDTA (19) is slightly higher than that for Cd, it can be suggested that Co may bind more strongly to different sites in the algae. According to the results assessed by 113Cd NMR, the decreasing sorption affinity order CuII > AlIII > CdII > CoII can be suggested. All results for AlIII binding sites competition studies are shown in Figure 4. When CuCl2, CoCl2‚6H2O, or CdCl2 was added to Al-algae mixture, no decrease in the bound Al signal
NMR spectroscopy provided important information to support the use of this biomass as an efficient biosorbent.
Acknowledgments We appreciate the help from Alvicle´r Magalha˜es and Alexandre Schefer with the NMR studies and all suggestions from Dr Christine Piggee. E.N.V.M.C acknowledges the financial support from Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo - FAPESP (Process 98/07268-7).
Literature Cited
FIGURE 4. 27Al NMR spectra of (a) 10 g L-1 Al2(SO4)3 solution and (b) 100 mg of dried algae suspended in 1 mL of 10 g L-1 Al2(SO4)3 in the presence of either (c) CuII, (d) CoII, or (e) CdII. was observed (Figure 4 (parts c-e, respectively)). This indicates that none of these metal ions were able to remove AlIII from the occupied algae site(s). This suggests that AlIII is more tightly bound to the algae sites than any of the other studied metals. It can also be observed in Figure 4 that the intensities of bound Al increase in the presence of the other metals. Apparently, while the process of addition of the second metal to the Al-alga suspension is taking place, remaining free Al, still present, continues to bind the algae and consequently the Al-algae peak increases. Since no competition for Al binding sites was observed in the presence of these elements, the incubation time for each added metal was increased to 24 h in order to study the kinetic of metal sorption and possible chemical shifts in longer incubation times. Al NMR spectra in the presence of Cd, Co, or Cu, obtained at an extended exposition time (not shown), were compared to those in Figure 4, and a slight increase in the bound Al signals was observed. The 113Cd and 27Al NMR studies indicate the binding affinity order AlIII, CuII > CdII > CoII which apparently can be correlated with the results from metal binding capacity assessed by ICP-OES. The investigation on chemical functionalities responsible for metal ion binding in the alga P. littoralis by 27Al and 113Cd
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Received for review March 23, 2001. Revised manuscript received December 26, 2001. Accepted January 28, 2002. ES0107834
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