Anal. Chem. 2003, 75, 761-767
Effect of Metal Ions on the Molecular Weight Distribution of Humic Substances Derived from Municipal Compost: Ultrafiltration and Size Exclusion Chromatography with Spectrophotometric and Inductively Coupled Plasma-MS Detection Kazimierz Wrobel,† Baki B. M. Sadi, Katarzyna Wrobel,† Juan R Castillo,‡ and Joseph A. Caruso*
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172
The effect of metal ions (Co, Cu, Ni, Pb, Zn) on the molecular weight distribution of humic substances (HSs) obtained from compost is studied. We believe this is the first of this type of study applied in this way to humic substances. Size exclusion chromatography is coupled with two on-line detection systems (spectrophotometric and ICPMS) to study the binding of metal ions by humic substances leached from compost. ICPMS provided highly specific, sensitive, and multielement analytical information that enabled obtaining direct experimental evidence for the participation of metal ions in molecular size distributions of humic compounds. The compost extract or its high molecular weight fraction (>5000) was put in contact with EDTA or citrate ions, thereby competing with HSs for binding metals. The experiments were carried out by varying the pH maintained by Tris-HCl or CAPS buffer (pH 8.0 and 10.3) and keeping the ionic strength constant. The elution profile of humic substances using UV/ visible detection was compared with those from ICPMS detection of Co, Cu, Ni, Pb, and Zn in the same chromatographic runs. The results obtained suggested that both bridging between small molecules and complexation/ chelation by individual molecules are involved in metal ion binding to humic substances. The use of ICPMS to study the role of metal ions in aggregation/disassociation of humic substances proposed in this work is promising. Coupling element-specific detection with SEC or other separation systems allows better understanding of the mobility and bioaccessibility of elemental species in the environment and further elucidation of the dissolved humic structure.
* To whom correspondence should be addressed. E-mail:
[email protected]. † On the leave from Instituto de Investigaciones Cientificas, Universidad de Guanajuato, L. de Retana No. 5, 36000 Guanajuato, Mexico. ‡ On leave from the Analytical Chemistry Department, Faculty of Sciences, University of Zaragoza, Zaragoza-50009, Spain. 10.1021/ac0261193 CCC: $25.00 Published on Web 01/16/2003
© 2003 American Chemical Society
Inductively coupled plasma-mass spectrometry (ICPMS) has proven to be a powerful analytical tool in different research areas1 owing to its unique combination of selectivity, sensitivity, wide linear dynamic range, nearly interference-free operation, and multielement capabilities. It has been widely used for trace element determinations in complex matrixes and as an elementspecific detector in speciation studies. Other possible applications, less explored so far, are the use of ICPMS to investigate the fate of elements in different chemical processes.2,3 In this study, the effect of metal ions (Co, Cu, Ni, Pb, Zn) on the molecular weight distribution of humic substances derived from compost is studied. To our knowledge, this is the first report on this approach for humic substances. The term humic substances (HSs) refer to the decay products derived from plant and animal tissues ubiquitous in waters, soils, and sediments. These are heterogeneous and polyelectrolytic organic compounds with a wide range of molecular weights (up to 100 000).4 HSs have been classified as biopolymers with a considerable number of similar units bound covalently. Furthermore, the arrangement of HSs into secondary, tertiary, and quaternary structures has been suggested to depend on weaker interactions occurring inside the individual polymeric molecule or between different molecules.5,6 The elucidation of the humic structure is necessary for better understanding its potential to interact with many natural and anthropogenic chemicals.7,8 Different operationally defined fractionation/separation schemes have been used for this purpose. The most common are followed by (1) Bacon, J. R.; Crain, J. S.; Van Vaeck, L.; Williams, J. G. J. Anal. At. Spectrom. 2000, 15, 1025-1053. (2) Taylor, H. E.; Huff, R. A.; Monaster, A. In Inductively Coupled Plasma Mass Spectrometry; Montaser, A., Ed.; Wiley-VCH: New York, 1998; pp 681-807. (3) Montes-Bayon, M.; Yanes, E. G.; de Leon, C. P.; Jayasimhulu, K.; Stalcup, A.; Shann, J.; Caruso, J. A. Anal. Chem. 2002, 74, 107-113. (4) MacCarthy, P.; Suffet, I. H. In Aquatic Humic Substances: Influence on Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry 219; American Chemical Society: Washungton DC, 1989; , pp xvii-xxx. (5) Benincasa, M. A.; Cartoni, G.; Imperia, N. J. Sep. Sci. 2002, 25, 405-415. (6) Parlanti, E.; Morin, B.; Vacher, L. Org. Geochem. 2002, 33, 221-236. (7) Conte, P.; Piccolo, A. Environ. Sci. Technol. 1999, 33, 1682-1690. (8) Wershaw, R. L. Soil Sci. 1999, 164, 803-813.
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spectrometric characterization.9-12 However, interpretation of the experimental results has not been straightforward, because these are affected by experimental conditions applied that may lead to possible changes in the humic structure.8,9 Several mechanisms have been proposed to describe the aggregation of humic substances.13 The random coil model helps to explain the different behavior of HSs observed in size exclusion chromatography or in ultrafiltration, depending on the concentration in solution, the acidity, and the ionic strength. According to this model, humic macromolecules behave like flexible linear polymers in diluted neutral solutions, where they become densely coiled into a globular conformation with increasing concentration, ionic strength, and acidic media.14-16 In another approach, intermolecular aggregation is described as the formation of micellelike or membranelike structures where the hydrophilic constituents are at the exterior surfaces in contact with the solution.17-19 This model helps to interpret the interactions of dissolved HSs with hydrophobic organic matter. The effect of HSs on the fluorescence quenching of nonpolar organic compounds was explained by their entrapment inside the micelles.19-21 The results obtained by studying aggregation of the humic substances in the presence of mineral and organic acids under various pH and ionic strengths were suggested as supportive of the micellelike model.7,22 More recently, it has been suggested that high molecular size HS structures might be formed by self-assembling of the relatively small and heterogeneous molecules through weak forces such as dispersive interactions and hydrogen bonding.8,23,24 In the work by Andre et al.,25 the authors proposed that metal ions might play a crucial role in such aggregation of low molecular weight ( Ni(II) > Co(II) > Pb(II) > Cd(II) > Cr(III) . (Mn(II), Mo(VI), Zn(II)).27,30,32 The possible effect of metal ions on the aggregation of humic and fulvic acids has been mentioned in few studies, and the data obtained to date seem to support the mechanism of aggregation suggested by Andre.25 Using scanning electron microscopy, Chen et al.29 observed varying shape and size of humic and fulvic acids in the presence of Cu(II), Al(III), Fe(II), and Fe(III) at different pH values. Enhanced aggregation with increasing amounts of Cd(II) bound to humic acids was found in experiments with atomic force microscopy.33 The possible effect of this metal ion on the aggregation of humic substances was also studied by NMR and light scattering measurements at pH values of 4 and 6.5.34 The influence of magnesium bromide on the fluorescence and surface tension of diluted HS aqueous solutions was examined, and the results obtained were interpreted in terms of enhanced pseudomicelle formation upon the addition of magnesium ion.21 Steady-state fluorescence anisotropy was used for studying aluminum-induced aggregation of humic substances.33 Both fluorescence enhancement and fluorescence quenching were observed after addition of aluminum, the former effect being ascribed to the formation of Al-induced aggregates and the latter to loss of material by precipitation. Most relevant to the study reported here is that the techniques used in the above-mentioned reports did not provide direct information on metal ion distributions. The elucidation of metal ion interactions with humic substances has become an important issue, since it would help in understanding their mobility in the environment as well as bioaccessibility to plants and animals.27 The suggested metal ion-induced aggregation of humic substances also might be important for bioaccessibility of xenobiotics such as persistent organic pollutants (POPs).35 Moreover, the presence of certain compounds (citric, tartaric, lactic, malic and oxalic acids, hydroxamate siderphores, (26) Chen, Y.; Stevenson, F. J. In The Role of Organic Matter in Modern Agriculture; Chen, Y., Avnimelch, Y., Nijhoff, M., Eds.: Martinus Nijhoff: Boston, 1986; p 73. (27) Evangelou, V. P.; Marsi, M. Plant Soil 2001, 229, 13-24. (28) Bhandari, S. A.; Amarisiriwardena, D.; Xing, B. In Understanding Humic Substances. Advanced Methods, Properties and Applications; Ghabbour, E. A., Davis, G., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 1999; pp 203-222. (29) Liu, C.; Huang, P. M. In Understanding Humic Substances: Advanced Methods, Properties and Applications; Ghabbour, E. A., Davies, G., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 1999; pp 87-99. (30) Sadi, B. B. M.; Wrobel, K.; Wrobel, K.; Castillo, J. M.; Caruso, J. A. J. Environ. Monit. 2002, 4, 1010-1016. (31) Spark, K. M.; Wells, J. D.; Johnson, B. B. Aust. J. Soil Res. 1997, 35, 89101. (32) Pandey, A.; Pandey, S. D.; Misra, V. Ecotoxicol. Environ. Saf. 2000, 47, 195-200. (33) Charles, M. S.; Linda, B. M. Environ. Sci. Technol. 1999, 33, 3264-3270. (34) Dixon, A. M.; Larive, C. K. Appl. Spectrosc. 1999, 53, 426A-440A. (35) Arnold, C. G.; Ciani, A.; Muller, S. R.; Amirbahman, A.; Schwarzenbach, R. P. Environ. Sci. Technol. 1998, 32, 2976-2983.
phenolic acids, polymeric phenols, 2-ketogluconic acid, etc.) that can compete for the metal ions as chelating agents36 could affect the aggregation of humic substances, hence the mobility of different pollutants possibly entrapped in their structure. In a previous study, carried out in this laboratory, size exclusion chromatography (SEC) with spectrophotometric (UV/visible) and ICPMS detection was used for the characterization of elements binding to humic substances derived from municipal compost.30 In this coupled technique, SEC provided a relatively simple and convenient means for studying apparent molecular weight distribution of humic substances, while element-specific detection assured highly selective and specific information for individual elements from one chromatographic run.28,30 In this study, the effect of metal ions (Co, Cu, Ni, Pb, Zn) upon the molecular weight distribution of humic substances from the same source was investigated. The high molecular weight fraction of compost extract was put in contact with EDTA or citrate ions, thereby competing with HSs for binding metals (the concentration, ionic strength, and pH were kept constant). After ultrafiltration, the samples obtained were analyzed by on-line SEC-UV/visibleICPMS. The results indicate that coupling a chromatographic separation with element-specific detection is a promising analytical tool for studying the aggregation of humic substances in the presence of metal ions. EXPERIMENTAL SECTION Samples and Reagents. Compost samples were derived from urban solid waste from Spain and were provided by the Analytical Chemistry Department, University of Zaragoza, Zaragoza, Spain. The commercial standard solutions containing 1000 mg/L Co, Ni, Cu, Zn, and Pb in 2% HNO3 were from Spex CertiPrep, Inc. (Metuchen, NJ). A 1 mg/L standard mixture of individual elements of the above standards was prepared in 0.01 M TrisHCl (Sigma) buffer containing 0.01 M NaCl (Sigma), pH 8.0. The following Sigma reagents were used: sodium hydroxide, 3-cyclohexyloamino-1-propanesulfonic acid (CAPS), Na2EDTA, citric acid trisodium salt, and hydrochloric acid. Deionized water (18.2 MΩ‚ cm) was used throughout (NanoPure, Barnstead, Boston, MA). SEC-UV/Visible-ICPMS System. An Agilent (Agilent Technologies, Palo Alto, CA) series 1100 high-performance liquid chromatographic system was used, equipped with an autosampler, a diode array detector (DAD), and Chemstation (UC electronics shop). The chromatographic column was Superdex HR 10/30 Peptide (Amersham Biosciences, Inc., Piscataway, NJ). Calibration of the column was accomplished with a standard mixture of myoglobin (17 000), lysozyme (14 400), substance P (1350), and (Gly)6 (360), showing in this range of a good linear response for the log of molecular weight versus retention time (r2 ) 0.9914).30 An Agilent 7500s inductively coupled plasma-mass spectrometer was coupled on-line, the effluent being introduced to ICPMS through the concentric nebulizer. The instrumental operation conditions are given in Table 1. Extraction of Humic Substances from Compost. A volume of 10 mL of 0.1 M sodium hydroxide was added to the dry compost (1 g); the mixture was stirred for 30 min at room temperature and centrifuged for 10 min at 3000g (a model Chermle Z 230 (36) Stevenson, F. J. Humus Chemistry: Genesis, Composition, Reactions, 2nd ed.; John Wiley & Sons: New York, 1994.
Table 1. Instrumental Operating Conditions for Size Exclusion Chromatography and UV/Visible and ICPMS Detection Chromatographic Separation Parameters column Superdex HR 10/30 Peptide mobile phase I 10 mM CAPS buffer, pH 10.3 mobile phase II 10 mM Tris-HCl buffer, pH 8.0 flow rate 0.7 mL/min injection volume 100 µL Detection Parameters UV/visible for humic 254-600 nm substances 59Co, 60Ni, 63Cu, 66Zn, 208Pb ICPMS for elements forward power 1300 W dwell time 200 ms nebulizer gas flow: carrier gas 0.89 L/min makeup gas 0.24 L/min sample introduction Meinhard nebulizer
centrifuge, National Labnet Co., Woodbridge, NJ). The supernatant was filtered through a 0.2-µm cellulose syringe filter, and the volume was adjusted to 20 mL. The solution obtained (100 µL) was injected into the SEC-UV/visible-ICPMS system (Table 1, mobile phase I without or with addition of Na2EDTA, 10 mM). Fractionation by Ultrafiltration. Two aliquots of compost extract (3 mL) were transferred to the ultrafiltration unit (Ultrafree-4 centrifugal filter and tube, cutoff 5000, Biomax, Millipore Corp., Bedford, MA) and were centrifuged at 2000g until ∼200 µL samples of the initial solutions were left as supernates. The high molecular weight (HMW) fractions were combined and washed. To do so, the above procedure was repeated three times, each time the volume was brought to ∼2 mL with Tris-HCl buffer, and the samples were centrifuged. The final volume of the HMW fraction was adjusted to 10 mL with the same buffer. The aliquots of 0.8 mL were transferred to three 1.5-mL Eppendorf tubes (Fisher Scientific, Pittsburgh, PA). Then, 0.4 mL of Tris-HCl buffer was added to the first tube, 0.4 mL of Na2EDTA (0.2 M in TrisHCl buffer) to the second tube, and 0.4 mL of sodium citrate (0.2 M in Tris-HCl buffer) to the third tube. To keep ionic strength constant, sodium chloride was added to Tris-HCl buffers in the first two tubes (final concentrations respectively, 1.2 and 0.3 M). After mixing, the solutions were transferred to an ultracentrifuge filter (MW 5000) and centrifuged at 2000g until 200 µL was left. The high molecular weight fraction was washed three times by adding 0.8 mL of Tris-HCl buffer to each filter unit and centrifuging. Final volume of each of the solutions was brought to 1 mL with this same buffer. The 100-µL aliquots were injected into the SEC-UV/visible-ICPMS system (Table 1, mobile phase II). RESULTS AND DISCUSSION In this study, ICPMS detection was coupled with size exclusion chromatography to obtain direct experimental evidence of the participation of metal ions in the molecular size distribution of dissolved humic compounds. The source of humic substances was compost derived from municipal wastes. The extraction conditions were investigated and discussed in a previous study.30 In this study, sodium hydroxide was used as the extracting agent instead of sodium pyrophosphate to avoid any structural changes in the Analytical Chemistry, Vol. 75, No. 4, February 15, 2003
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Figure 1. SEC-UV/visible chromatograms of humic substances from compost obtained using two detection wavelengths: 254 (1, 2) and 400 nm (3, 4). Mobile phase was CAPS buffer, pH 10.3, without (1, 3) and with (2, 4) addition of Na2EDTA (details in Table 1). In the upper part, the elution of MW standards is marked.
humic substances that might be caused by the latter.37 The extract obtained was analyzed by SEC-UV/visible-ICPMS. The mobile phase was a CAPS buffer (Table 1, mobile phase I), which was used to prevent any drastic changes of pH in the alkaline extract.30 In the second chromatographic run, Na2EDTA was added to this mobile phase (final concentration 10 mM) in order to verify how this potent metal chelating agent affects the apparent molecular weight distribution. It should be noted that alkaline conditions assured better solubility of humic substances during extraction and also favored the metal ion complexation by EDTA. In Figure 1, typical UV/visible chromatograms of compost extract are presented that were obtained with the two mobile phases. It is observed that the elution profile of humic substances changed in the presence of a chelating agent. The absorbance in the region of higher molecular weight compounds became lower, and the contribution of compounds eluting with higher retention times increased. This could possibly be ascribed to dissociation of humic substances caused by removing metal ions from their structure. The peak area under the chromatogram obtained with the mobile phase containing EDTA was essentially the same as in the absence of it (99.2 ( 0.7%) when the detector was set at 254 nm. Using 400 nm for detection, the peak area after addition of EDTA to the mobile phase was lower compared to that obtained in the absence of chelating agent (92 ( 2%). This comparative evaluation suggests the complete recovery of humic substances from the column with the use of two mobile phases. It also confirms that the size of the compounds present in the extract was diminished after EDTA addition. The difference in peak areas observed at 400 nm was in agreement with earlier reports that the molecular weight of humic compounds affected their absorption spectra.7,22 This effect is graphically presented in Figure 2. The UV/visible chromatogram of compost extract was registered in the wavelength range 254600 nm (DAD), the ratio (A254/Ai) between the absorbance measured at 254 nm (A254) and the absorbance at higher wavelength (Ai, i ) 314, 354, 394, 434, 474, and 514 nm, respectively) was calculated, and these values were plotted against (37) Hayes, H. B.; Graham, C. L. In Humic Substances. Versatile Components of Plants, Soils and Water; Ghabbour, E. A., Davis, G., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 2000; pp 91-.
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Figure 2. Retention time versus the absorbance ratio A254/Ai (i ) 314, 354, 394, 434, 474, 514 nm) from the chromatogram of humic substances obtained with mobile phase containing CAPS buffer and Na2EDTA (Table 1).
retention time (directly related to apparent molecular weight). It can be observed that, with increasing wavelength, the absorbance in the elution region of lower molecular weight compounds clearly decreased. The chromatographic effluent was introduced on-line to ICPMS for detection of the five metal ions (Cu, Co, Ni, Pb, Zn). These metals were selected based on their affinity for humic substances27,30,32 and high stability constants for their complexes with EDTA. It was observed that the ICPMS elution profiles (Figure 3) followed those of humic substances in the absence of EDTA (Figure 1). In the presence of complexing agent the elution of Co, Cu, Ni, Pb and Zn containing species was moved toward higher retention times, indicating formation of compounds with lower apparent molecular weight (Figure 3, ICPMS chromatogram for Cu not shown). The retention times of these smaller metal species matched those observed when a mixed standard solution of metal ions was analyzed (in the absence of compost extract, mobile phase I with EDTA). These results show that metal ions were removed from humic compounds during the disassociation observed with EDTA. This observation supports the mechanism proposed by Andree et al.25 for metal-induced aggregation of humic substances. It can be expected that, in addition to humic substances, the compost extract used in the above experiments contained a variety of low molecular weight compounds. To obtain further evidence on the role of metal ions on the aggregation of humic compounds into HMW structures, ultrafiltration of the compost extract was carried out before the addition of complexing agent. The HMW fraction of extract (>5000) was incubated with EDTA or citrates, again ultrafiltered (5000 cutoff) and analyzed by SEC-UV/visibleICPMS. The alkalinity of the HMW fraction was lowered to pH 8.0 using Tris-HCl buffer. The same buffer was used as the mobile phase for SEC (mobile phase II, Table 1). To avoid possible negative effects of changing ionic strength to the humic structure, this parameter was kept constant by addition of sodium chloride (details given in the Experimental Section). Typical size exclusion chromatograms obtained with tandem on-line detection systems, are presented in Figure 4. The solutions were ultrafiltered before chromatographic separation, so they should contain only the compounds with molecular weights higher than 5000. Similar elution profiles of HMW humic substances can be observed in
Figure 3. Size exclusion chromatograms of compost extract from Figure 1 with ICPMS detection for lead (m/z 208), nickel (m/z 60), zinc (m/z 66), and cobalt (m/z 59): (s) in the presence and (- -) in the absence of EDTA in the mobile phase I (Table 1).
Figure 4a and in Figure 1 (retention time below 20 min), indicating that switching the mobile phase from CAPS buffer (pH 10.3) to Tris-HCl buffer (pH 8.0) and increasing the ionic strength of the solution cause only minor changes in the molecular size distribution of humic substances (>5000; the relation between the first two peaks in Figure 1 and Figure 4a is noteworthy). Furthermore, the addition of two complexing agents caused a decrease of total area under the chromatogram, with more pronounced effect in the presence of EDTA. This indicates that the molecular size of humic substances was reduced (compare with Figure 1) and the lower molecular size fractions (
