Membrane filtration studies of aquatic humic substances and their metal species: a concise overview. Part 2. Evaluation of conditional stability constants by using ultrafiltration

The assessment of conditional stability constants of aquatic humic substance (HS) metal complexes is overviewed with special emphasis on the application of ultrafiltration methods. Fundamentals and limitations of stability functions in the case of macromolecular and polydisperse metal-HS species in aquatic environments are critically discussed. The review summarizes the advantages and application of ultrafiltration for metal-HS complexation studies, discusses the comparibility and reliability of stability constants. The potential of ultrafiltration procedures for characterizing the lability of metal-HS species is also stressed.     

Analytical fractionation of aquatic humic substances and their metal species by means of multistage ultrafiltration

The molecular-size fractionation of aquatic humic substances (HS) and their metal species by means of a novel sequential-stage ultrafiltration (UF) device equipped with five appropriate ultramembranes (1, 5, 10, 50 and 100 kD) is described. First of all, the concentration dynamics of macromolecules, particulary HS, during five-stage UF and its subsequent washing step has been modelled. Based on these results, the fractionation of aquatic HS (from ground and bog water) by means of multistage UF has been optimized for an analytical scale (10 ml sample, 1 mg/ml HS, 10 ml washing solution, pH 6.0). The molecular size-distribution of selected aquatic HS (BOC 1/2 from the DFG-Versuchsfeld Bocholt, VM 5 from Venner Moor, Germany) studied by five-stage UF exhibited strong systematic influences of the procedure used for their isolation. The molecular-size distribution of HS obtained by on-line UF and gel permeation chromatography (GPC) showed a satisfactory agreement in the range 1–50 kD. Moreover, when interrupting multistage UF for > 48 h a slow transformation in the HS samples has been found as gradually additional HS fractions of < 1 kD have been formed. Besides unloaded HS molecules, the molecular-size distribution of freshly formed metal species of HS (1.0 mg metal/g HS of Al(III), Cd(II), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), Zn(II), each) has been characterized by multistage UF as a function of pH-value, degree of loading and complexation time. Metal determinations as carried out by flame AAS, showed that considerable metal fractions in HS especially are present in molecules > 50 kD, which seemed to be rather acid-inert. With complexation times of < 2 days a transient shift of the molecular size distribution of both HS and their metal species (e.g., Al(III), Fe(III) to higher values (> 10 kD) has been found.     

Analytical fractionation of aquatic humic substances and their metal species by means of multistage ultrafiltration

The molecular-size fractionation of aquatic humic substances (HS) and their metal species by means of a novel sequential-stage ultrafiltration (UF) device equipped with five appropriate ultramembranes (1, 5, 10, 50 and 100 kD) is described. First of all, the concentration dynamics of macromolecules, particulary HS, during five-stage UF and its subsequent washing step has been modelled. Based on these results, the fractionation of aquatic HS (from ground and bog water) by means of multistage UF has been optimized for an analytical scale (10 ml sample, 1 mg/ml HS, 10 ml washing solution, pH 6.0). The molecular size-distribution of selected aquatic HS (BOC 1/2 from the "DFG-Versuchsfeld Bocholt", VM 5 from "Venner Moor", Germany) studied by five-stage UF exhibited strong systematic influences of the procedure used for their isolation. The molecular-size distribution of HS obtained by on-line UF and gel permeation chromatography (GPC) showed a satisfactory agreement in the range 1-50 kD. Moreover, when interrupting multistage UF for > 48 h a slow transformation in the HS samples has been found as gradually additional HS fractions of < 1 kD have been formed. Besides unloaded HS molecules, the molecular-size distribution of freshly formed metal species of HS (1.0 mg metal/g HS of Al(III), Cd(II), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), Zn(II), each) has been characterized by multistage UF as a function of pH-value, degree of loading and complexation time. Metal determinations as carried out by flame AAS, showed that considerable metal fractions in HS especially are present in molecules > 50 kD, which seemed to be rather acid-inert. With complexation times of < 2 days a transient shift of the molecular size distribution of both HS and their metal species (e.g., Al(III), Fe(III) to higher values (> 10 kD) has been found.     

On-line fractionation and characterization of aquatic humic substances by means of sequential-stage ultrafiltration

A five-stage tangential-flow ultrafiltration (UF) device equipped with advanced membrane filters (molecular weight cut-off: 1, 5, 10, 50 and 100 kDalton) of the polyethersulfone type is described and applied for the analytical on-line fractionation of a series of aquatic humic substances (HS) originating from surface or groundwaters. Fractionation patterns of HS (6 fractions each) evaluated by this UF device exhibit their particular dependence on the HS concentration, the pH-value and the salt content of the sample (10 ml) to be analyzed. Fundamental parameters (e.g., washing volume) governing the molecular-size fractionation of HS by means of multistage UF are discussed, too. The fractionation of an aquatic reference HS (BOC 3/9.5) by means of the above UF procedure reveals considerable differences preferably characterized by the UV-VIS absorption ratio E₃₅₀/E₄₅₀ and metal complexing capacity (Cu(II)) of the produced fractions. Moreover, molecular spectroscopy investigations (FTIR, ¹H-NMR) of the fraction series of this HS indicate that carbohydrate substructures (preferably found in fractions >50 k Dalton) and aromatic ones (preferably in fractions <5 k Dalton) are unevenly distributed.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Analytical fractionation of aquatic humic substances by metal affinity chromatography on iron(III)-coated cellulose

The chromatographic fractionation of aquatic humic substances (HS) onto iron(III)-coated cellulose (Cell-Fe(III)) as a metal-loaded adsorbent is described, analogously to the separation principle of the well-established metal affinity chromatography (MAC). For that purpose the sorption of HS from different aquatic origin on that collector was characterized by their kinetics and equilibrium distribution coefficients Kd. Based on Kd values of 10³ to 10⁴,mL/g, and fast sorption kinetics a preparative HPLC procedure, using stepwise increased pH-values (pH 8–12.5, borate buffer) as an eluent, was developed for the fractionation of dissolved HS (up to 7 fractions of different amount). The fractions obtained by this MAC procedure from selected aquatic HS samples were different in their Cu(II) complexation capacity, absorbance ratio E_{265 nm}/E_{365 nm} and Fourier transform infrared spectra. Received: 14 June 1999 / Revised: 6 August 1999 / Accepted: 10 August 1999     

Analytical fractionation of aquatic humic substances by metal affinity chromatography on iron(III)-coated cellulose

The chromatographic fractionation of aquatic humic substances (HS) onto iron(III)-coated cellulose (Cell-Fe(III)) as a metal-loaded adsorbent is described, analogously to the separation principle of the well-established metal affinity chromatography (MAC). For that purpose the sorption of HS from different aquatic origin on that collector was characterized by their kinetics and equilibrium distribution coefficients Kd. Based on Kd values of 10³ to 10⁴,mL/g, and fast sorption kinetics a preparative HPLC procedure, using stepwise increased pH-values (pH 8–12.5, borate buffer) as an eluent, was developed for the fractionation of dissolved HS (up to 7 fractions of different amount). The fractions obtained by this MAC procedure from selected aquatic HS samples were different in their Cu(II) complexation capacity, absorbance ratio E_{265 nm}/E_{365 nm} and Fourier transform infrared spectra. Received: 14 June 1999 / Revised: 6 August 1999 / Accepted: 10 August 1999     

Analytical fractionation of labile metals in selected groundwater humic substances by means of chelating ion-exchanger

Summary Trace metals remaining in humic substances (HUS) after their acidic isolation (XAD 2) from aquatic sources may preferably be bound in inert form. In the present study, the reactivity (lability) of such a trace metal fraction (e.g. Cu, Fe, Mn, Zn) in selected groundwater HUS (BOC 1 and 3 from the DFG Versuchsfeld Bocholt, FRG) is characterized by its different separation behaviour towards a chelating ion-exchanger (e.g. Hyphan) using a time-controlled sequential batch procedure (96 h). Under standardized conditions, the kinetics and the degree of the ion-exchange reaction serve as parameters for the operational evaluation of metal lability in the above mentioned HUS. Surprisingly, according to the above ion-exchange procedure, about 50% of the total Fe, 81 (95)% Cu, 65 (97)% Mn, 69 (97)% Ni and 82 (95)% Zn in BOC 1 (BOC 3) proved to be bound in labile form. However, 20 to 30% of the labile metal fraction only react following very slow first order kinetics (half-time 24 h). In contrast, trace metals freshly bound to BOC 1 are quantitatively recovered by the collector Hyphan within 1 to 2 h, with the exception of Fe. Moreover, in this way HUS samples can be purified for molecular spectroscopy investigations (e.g. NMR, ESR, fluorescence) which are highly sensitive to metal interferences.     

Labile/inert metal species in aquatic humic substances: an ion-exchange study

Summary An ion-exchange procedure has been developed for the analytical fractionation of metals (e.g. Al, Co, Cu, Fe, Mn, Ni, Pb, Zn) forming labile/inert complexes with aquatic humic substances (HS) isolated (XAD 2, XAD 8, ultrafiltration) from bog, forest, ground and lake water. Using 1-(2-hydroxyphenylazo)-2-naphthol groups immobilized on cellulose (Cellulose HYPHAN) as chelating collector (batch and column procedure, resp.) for reactive metal fractions in dissolved HS, the kinetics and the degree of separation (referred to the total metal content) serve for the operational characterization of the metal lability. According to the separation kinetics (96 h), mostly the reactivity order Mn>Zn>Co>Pb>Ni>CuAl>Fe is observed for the above metals in HS, resulting in recoveries of >98% for Mn and Zn, but strongly varying for the other metals (e.g., 44–95% Cu, 18–84% Fe). By means of cellulose HYPHAN four metal fractions (e.g. Cu) can be distinguished kinetically: (a) about 50% of Cu freshly complexed with HS are directly exchanged (2nd order kinetics, k=0.275 1 · mol^–1 · s^–1) followed by (b) a less labile fraction (20–30%) of 1st to 2nd order exchange; (c) a hardly reactive fraction (5–10%) revealing uniform half times t_1/2 of 25 h closes the Cu exchange from HS. Moreover the Cu fraction (d), being exchange-inert in HS, amounts to 5–10% and increases by slow transformation processes of the formed HS/Cu species.     

Lability of heavy metal species in aquatic humic substances characterized by ion exchange with cellulose phosphate

Labile metal species in aquatic humic substances (HSs) were characterized by ion exchange on cellulose phosphate (CellPhos) by applying an optimized batch procedure. The HSs investigated were pre-extracted from humic-rich waters by ultrafiltration and a resin XAD 8 procedure. The HS-metal species studied were formed by complexation with Cd(II), Ni(II), Cu(II), Mn(II) and Pb(II) as a function of time and the ratio ions to HSs. The kinetics and reaction order of this exchange process were studied. At the beginning (<3 min), the labile metal fractions are separated relatively quickly. After 3 min, the separation of the metal ions proceeds with uniform half-lives of about 12-14 min, revealing rather slow first-order kinetics. The metal exchange between HSs and CellPhos exhibited the following order of metal lability with the studied HSs: Cu > Pb > Mn > Ni > Cd. The required metal determinations were carried out by atomic absorption spectrometry.     

Labile complexes of trace metals in aquatic humic substances: Investigations by means of an ion exchange-based flow procedure

The lability/inertness of heavy metals bound in aquatic humic substances (HS) has been characterized by means of ligand exchange with cellulose-immobilized triethylenetetramine-pentaacetic acid (TETPA) applying a flow system. On the basis of high metal distribution coefficients, K_d of 10³ to 10⁴ (ml/g) on cellulose TETPA even in slightly acidic HS solutions, labile and inert metal fractions in HS are characterized by their different kinetics and degree of phase exchange in small TETPA columns. For traces of metals bound to dissolved HS, the lability order Cd Mn(II)>Zn>Pb>Co>Ni>Cu is revealed. Systematic variation of environmentally relevant parameters shows the strong influence of the pH value and the ratio of metal loading/complexing capacity on the metal lability in HS. Surprisingly, in the case of freshly formed HS/Ni and HS/Cu complexes, slow transformation processes occur which lower their initial lability by one order of magnitude and supposedly increase their thermodynamic stability.Dedicated to Prof. Dr. F. Huber, Department of Chemistry, University of Dortmund, on the occasion of his 65th birthdayOn leave from Department of Analytical Chemistry, Institute of Chemistry, UNESP, Campus de Araraquara, CEP 14800-900, C. P. 355-Araraquara, SP, Brasil     

Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron(III)-loaded ion exchangers

The analytical fractionation of aquatic humic substances (HS) by means of immobilized metal-chelate affinity chromatography (IMAC) on metal-loaded chelating ion exchangers is described. The cellulose HYPHAN, loaded with different trivalent ions, and the chelate exchanger Chelex 100, loaded to 90% of its capacity with Fe(III), were used. The cellulose HYPHAN, loaded with 2% Fe(III), resulted in HS distribution coefficients Kd of up to 10(3.7) mL/g at pH 4.0 continuously decreasing down to 10(1.5) at pH 12, which were appropriate for HS fractionation by a pH-depending chromatographic procedure. Similar distribution coefficients Kd were obtained for HS sorption onto Fe(III)-loaded Chelex 100. On the basis of Fe-loaded HYPHAN both, a low-pressure and high-pressure IMAC technique, were developed for the fractionation of dissolved HS applying a buffer-based pH gradient for their gradual elution between pH 4.0 and 12.0. By coupling the Chelex 100 column under high-pressure conditions with an inductively coupled plasma mass spectrometer an on-line characterization of HS metal species could be achieved. Using these fractionation procedures a number of reference HS were characterized. Accordingly, the HA (humic acids) and FA (fulvic acids) studied could be discriminated into up to 6 fractions by applying cellulose HYPHAN, significantly differing in their Cu(II) complexation capacity but hardly in their substructures assessed by conventional FTIR. In the case of using Chelex 100 exchanger resin two major UV active HS fractions were obtained, which significantly differ in their complexation properties for Cu(II) and Pb(II), respectively.     

Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron(III)-loaded ion exchangers

The analytical fractionation of aquatic humic substances (HS) by means of immobilized metal-chelate affinity chromatography (IMAC) on metal-loaded chelating ion exchangers is described. The cellulose HYPHAN, loaded with different trivalent ions, and the chelate exchanger Chelex 100, loaded to 90% of its capacity with Fe(III), were used. The cellulose HYPHAN, loaded with 2% Fe(III), resulted in HS distribution coefficients K_d of up to 10^3.7 mL/g at pH 4.0 continuously decreasing down to 10^1.5 at pH 12, which were appropriate for HS fractionation by a pH-depending chromatographic procedure. Similar distribution coefficients K_d were obtained for HS sorption onto Fe(III)-loaded Chelex 100. On the basis of Fe-loaded HYPHAN both, a low-pressure and high-pressure IMAC technique, were developed for the fractionation of dissolved HS applying a buffer-based pH gradient for their gradual elution between pH 4.0 and 12.0. By coupling the Chelex 100 column under high-pressure conditions with an inductively coupled plasma mass spectrometer an on-line characterization of HS metal species could be achieved. Using these fractionation procedures a number of reference HS were characterized. Accordingly, the HA (humic acids) and FA (fulvic acids) studied could be discriminated into up to 6 fractions by applying cellulose HYPHAN, significantly differing in their Cu(II) complexation capacity but hardly in their substructures assessed by conventional FTIR. In the case of using Chelex 100 exchanger resin two major UV active HS fractions were obtained, which significantly differ in their complexation properties for Cu(II) and Pb(II), respectively.     

Quantification of partial structures of aquatic humic substances by 1H-NMR under WATR conditions

Aquatic humic substances (HS) isolated from surface water, leakage water and ground water have been investigated by ¹H-NMR. The overlapping HOD signal was eliminated by adding ammonium chloride and applying the multiecho method CPMG (Carr, Purcell, Meiboom, Gill) under WATR conditions (water attenuation by transverse relaxation) permitting quantification of partial structures of HS. The proportion of carbohydrates/alcohols/ethers decreases and the proportion of alkyl moieties increases with increasing water or soil depth and thus microbial diagenesis. Also, increasing deoxygenation of aromatic substituents is observed with increasing water or soil depth. In some cases, elimination of the NMR signal of HOD is accompanied by the appearance of another HOD signal which is slightly shifted and much smaller in intensity; this signal probably results from water strongly bound by hydrogen bonding within the HS macromolecules.     

Approach combining on-line metal exchange and tangential-flow ultrafiltration for in-situ characterization of metal species in humic hydrocolloids

This paper deals with the development and optimization of an analytical procedure using ultrafiltration and a flow-injection system, and its application in in-situ experiments to characterize the lability and availability of metal species in humic-rich hydrocolloids. The on-line system consists of a tangential flow ultrafiltration device equipped with a 3-kDa filtration membrane. The concentration of free ions in the filtrate was determined by atomic-absorption spectrometry, assuming that metals not complexed by aquatic humic substances (AHS) were separated from the complexed species (M–AHS) retained by the membrane. For optimization, exchange experiments using Cu(II) solutions and AHS solutions doped with the metal ions Ni(II), Mn(II), Fe(III), Cd(II), and Zn(II) were carried out to characterize the stability of the metal–AHS complexes. The new procedure was then applied in-situ at a tributary of the Ribeira do Iguape river (Iguape, São Paulo State, Brazil) and evaluated using the ions Fe(III) and Mn(II), which are considered to be essential constituents of aquatic systems. From the exchange between metal–natural organic matter (M–NOM) and the Cu(II) ions it was concluded that Cu(II) concentrations >485 μg L^?1 were necessary to obtain maximum exchange of the complexes Mn–NOM and Fe–NOM, corresponding to 100% Mn and 8% Fe. Moreover, the new analytical procedure is simple and opens up new perspectives for understanding the complexation, transport, stability, and lability of metal species in humic-rich aquatic environments.     

Investigations on the conditional kinetic and thermodynamic stability of aquatic humic substance-metal complexes by means of EDTA exchange, ultrafiltration and atomic spectrometry

The conditional metal availability and the kinetic stability of humic substance-metal species in humic-rich waters (e.g. bog water) was characterized by means of EDTA exchange. For this purpose a combined procedure consisting of time-controlled ligand exchange by EDTA, species differentiation by a fast single-stage tangential-flow ultrafiltration (TF-UF) technique (cut-off 1 kDa) and sensitive atomic spectrometry methods (e.g. AAS, ICP-OES, TXRF) was developed. The kinetics and the yield of the EDTA exchange served as operational parameters for assessing the kinetic stability and EDTA availability of HS-metal species, respectively. Considerable fractions of natural HS-metal species studied were shown to be EDTA-inert (e.g. 31% of the total Fe, 44% of the total Al) even after long reaction times (48 h), in contrast to artificial ones formed in solutions of isolated HS. Moreover, the conditional thermodynamic stability of HS-metal complexes formed by successive loading of an aquatic reference HS (HO14) with a number of heavy metal ions (e.g. Cr(III), Cu(II), Fe(III), Mn(II), Zn(II)) was also evaluated discriminating the free metal concentrations by means of TF-UF. In addition, from the loading isotherms obtained conditional complexation capacities could be derived for the studied HS exhibiting the order Fe(III)>Cu(II)>Cr(III)>Co(II)>Mn(II).     

Elucidation of aquatic humic partial structures by multistage ultrafiltration and two-dimensional nuclear magnetic resonance spectrometry

An on-line wavelet transform (WT) algorithm was proposed and successfully used in processing the photoacoustic (PA) signal. By investigating the spectra of three rare earth compounds, it was indicated that after on-line decomposition by WT, the noise in the PA spectra was cleanly removed, and the resolved information in the overlapping peaks was easily retrieved. The effect of different sampling rate on resolved results was also investigated. The experimental results showed that the position of peaks after decomposition agreed with the corresponding intramolecular energy transition bands, which demonstrated that WT is a useful and efficient tool for the study of energy bands of rare earth compounds using PA spectroscopy. Received: 7 July 1998 / Accepted: 14 September 1998     

Membrane filtration for the continuous fractionation of species and macromolecules: Component distribution in the Arzhaan spring water

An improved device was proposed for the fractionation and preconcentration of particles and macromolecules. The device provides new means for the separation of water components. In the proposed method, a tangential flow of the test solution is passed through a series of successive membranes with decreasing pore size. The method was applied to the fractionation of low-mineralized water (Ty va Arzhaanes) with low concentrations of trace components.     

Size fractionation of aquatic colloids and particles by cross-flow filtration: analysis by scanning electron and atomic force microscopy

The suitability of the combined application of environmental scanning electron microscopy (ESEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the evaluation of the ability of cross-flow filtration (CFF) to perform adequate size fractionation of freshwater colloids and particles was examined. ESEM and SEM imaging provided reference images of the CFF-generated fractions and, in estimating the experimental cut-off diameter of the membrane, provided evidence that separation was not consistent with nominal pore sizes of the membranes. However, analysis of the images showed that size distribution of CFF-generated fractions and the estimated cut-off diameter of the membranes were dependent on the advantages and limitations of the two imaging techniques. With both ESEM and SEM, best estimates of size cut-offs were lower than the nominal pore size of the membrane in the case of 0.45 μm membranes, but roughly accurate in the case of 0.1 μm pore size membranes. The results also suggested that the effectiveness of CFF may benefit from a pre-separation step using a minimally perturbing technique such as split thin-flow fractionation. AFM demonstrated the presence of colloids smaller than 50 nm in all fractions including the retentates, showing that CFF fractionation is not fully quantitative and not based on size alone. The results indicate that previous studies investigating trace element partitioning using CFF may need re-evaluation as the importance of particles and large colloids may be over-estimated.     

Transformations of metal species in ageing humic hydrocolloids studied by competitive ligand and metal exchange

Transformations of metal species (particularly Al, Ca, Fe, Mg, Mn, Zn) in ageing humic hydrocolloids were studied, applying a competitive ligand and metal exchange approach. For this purpose, metal-containing hydrocolloids, freshly collected from humic-rich German bog lake waters (Hohlohsee (HO), Black Forest; Venner Moor (VM), Muensterland; Arnsberger Wald (AW), Northrhine-Westfalia) and conventionally pre-filtered through 0.45 m membranes, were subjected on-site to an exchange with EDTA and Cu(II) ions, respectively, as a function of time. EDTA complexes gradually formed, metal fractions exchanged by Cu(II) (as well as free Cu(II) concentrations) were operationally discriminated by means of a small time-controlled tangential-flow ultrafiltration unit (nominal cut-off: 1 kDa). Metal and DOM (dissolved organic matter) fractions obtained this way were determined off-site using instrumental methods (AAS, ICP-OES, carbon analyzer). After weeks of storage, the collected hydrocolloids were studied again by this approach. The EDTA availability of colloid-bound metals (particularly Al and Fe) exhibited different ageing trends, dependent on the sample (VM: decrease of Fe availability (98–76%), HO: increase of Fe availability (76–82%)). In contrast, the Cu(II) exchange equilibria of colloid-bound metals revealed merely low availability of Al (16–38%) and Fe (5–11%) towards Cu(II) ions, also dependent on ageing effects. In particular, the conditional copper exchange constants K_ex obtained from the exchange between Cu(II) ions and available metal species (such as Ca, Mg, Mn, Zn) exhibited a strong decrease (by a factor of 2–100) during sample storage, indicating considerable non-equilibria complexation of these metal ions in the original bogwaters studied on-site.