Binding of the protoxin and toxin proteins ofBacillus thuringiensis subsp.kurstaki on clay minerals

The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (M_r=132 kDa) and toxins (M_r=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na₂CO₃, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni.     

Adsorption/Desorption of toluene on clay minerals

Adsorption/desorption of toluene on montmorillonite, illite, and kaolinite was studied using the batch equilibrium method. The isotherms measured fit the Freundlich equation (r² >0.95). Montmorillonite adsorbed more toluene than illite or kaolinite; the adsorption of toluene on illite and kaolinite was not significantly different. Adsorption of toluene by montmorillonite showed an exponential increase as the ratio of toluene to clay was increased from 5 to 100. The rate studies showed that 62% of the adsorption was completed within 6 h. A rapid desorption was observed initially, followed by slow desorption after 1 h. The desorption rate decreased as the time of adsorption was increased. Almost all of the adsorbed toluene was extracted with water from the clay when the adsorption time was 0.1 h, but only 61% of the toluene could be desorbed when the adsorption time was 24 h.     

Effects of Temperature,pH and Salt Concentrations on the Adsorption of Bacillus subtilis on Soil Clay Minerals Investigated by Microcalorimetry

Adsorption of microorganisms on minerals is a ubiquitous interfacial phenomenon in soil. Knowledge of the extent and mechanisms of bacterial adsorption on minerals is of great agronomic and environmental importance. This study examined adsorption of Bacillus subtilis on three common minerals in soils such as kaolinite, montmorillonite and goethite under various environmental conditions. Isothermal titration calorimetry (ITC) was used to investigate the effects of temperature (20, 30, and 40°C), pH (5.0, 7.0, and 9.0) and KNO₃ concentration (0.001, 0.01, and 0.1 mol L^?1) on the adsorption by direct measurement of enthalpies. The results revealed that the adsorption process in all the mineral systems were exothermic, with the enthalpy changes (ΔH_ads ) ranging from ?52 to ?137, ?33 to ?147, and ?53 to ?141 kJ kg^?1 (dry weight of adsorbed bacteria) for kaolinite, montmorillonite, and goethite, respectively. No obvious dependence of ΔH_ads on temperature was observed. The heat release for all the systems generally declined with pH and decrease of salt concentration, which can be explained by the variations of hydrophobicity and electrostatic force with pH or salt concentration. The largest decrease was found for goethite among the three minerals from pH 5.0 to 7.0, suggesting that electrostatic attraction may play a more important role in bacterial adsorption on this mineral. The ΔH_ads values for all the minerals became nearly the same at pH 9.0, indicating that the same force probably hydrophobicity governing the adsorption for the minerals in alkaline environment. It is assumed that acidic or saline soils and the associated environments favor the adsorption of B. subtilis on clay minerals. In addition, the negative enthalpies expressed as kJ kg^?1 (carbon) revealed an energy flow into the environment accompanied by the carbon adsorption on the minerals in soil.     

Spectroscopic investigation of the structure of protoxin protein isolated from Bacillus thuringiensis contacted with minerals

Effects of minerals on the conformational changes of protoxin isolated from Bacillus thuringiensis were investigated by circular dichroism and fluorescence spectroscopy. Contact of the protoxin with attapulgite, montmorillonite and kaolinite for 3 h resulted in no significant changes in the spectra of circular dichroism and a slight decrease in the fluorescence intensity. There were significant changes in spectra of circular dichroism of protoxin after desorption in comparison to the native protoxin. The fluorescence intensity of protoxins desorbed from minerals retained 77.5, 63.7 and 60.4% of intensity of native protoxin, respectively. The influential extent of desorption on the secondary structure was higher than that of contact.     

Interaction between ATP,metal ions,glycine, and several minerals

Summary The adsorption of ATP and ADP on montmorillonite, kaolinite, and A1(OH)₃ was studied as a funtion of pH and, for montmorillonite and kaolinite, as a funtion of the ionic composition of the system. The three minerals exhibit different adsorption charcteristics. Mg²⁺- and Zn²⁺-montmorillonite adsorb ATP and ADP more than Na⁺-montmorillonite, presumably because of complex formation. In kaolinite, the effect of these divalent cations is small. Pure ATP decomposes upon heating, and the rate of the decomposition is accelerated by the presence of glycine. Drying and heating glycine to 70°C under vacuum in the presence of ATP results in abiotic peptide formation with yields up to 0.25%. This peptide formation also occurs when kaolinite or montmorillonite is added to the system. The presence of kaolinite, Mg²⁺-or Zn²⁺-koalinite, or Mg²⁺-montmorillonite results in a reduction in the rate of the ATP decomposition in the abiotic peptide synthesizing system. These results suggest that one role for clays and metal ions in chemical evolution may have been the stabilization of nucleotides during prebiotic peptide synthesis.On Leave from the Hebrew University of Jerusalem, Israel     

Sorption and desorption of copper by and from clay minerals

Summary The sorption of Cu by different clay minerals from dilute CuSO₄ solutions was studied as a function of pH. It was found that Cu sorption increased with increasing the equilibrium pH. The low sorption at low pH values was attributed to the competition effect of H⁺ ions and the release of octahedral Mg, Fe and Al from the 2: 1 minerals in acid solutions. The higher sorption of copper at higher pH values was related to the absence of H⁺ ions and to the increase in the pH-dependent negative charge in kaolinite and gibbsite.The desorption of copper using solutions of 1 N NaCl at different pH values indicated that kaolinite and montmorillonite released large amounts of their adsorbed Cu even at high pH values. Vermiculite, on the other hand, exhibited a strong retention of Cu against extraction with NaCl. Oven-drying had no significant effect on the desorption characteristics of the Cu-saturated clays.The Ca-Cu exchange isotherms on montmorillonite were studied at two different pH values. The isotherms indicated a preference of Ca at the lower pH (pH 3.5), with K = 0.931 and G = +41.0 cal./mole. At the higher pH (5.2) the isotherms indicated a preference of Cu with K = 1.282 and G = –141.0 cal./mole. The difference was attributed to the competition of H⁺ at low pH.     

Bt毒素在三种矿物表面的吸附与解吸

研究了Bt库斯塔克亚种(kurstaki）毒素（65 kDa）在高岭土、针铁矿和氧化硅表面的吸附和解吸特性.结果表明：在磷酸盐缓冲体系（pH 8）中,3种矿物的等温吸附曲线均符合Langmuir方程（R²>0.9661）,它们对Bt毒素的吸附顺序为：针铁矿﹥高岭土﹥二氧化硅.矿物对Bt毒素的吸附1 h就基本达到了吸附平衡.在pH 6～8范围内,针铁矿、高岭土和二氧化硅对Bt毒素的吸附量随pH值的升高而降低.10 ℃～50 ℃范围内,针铁矿和氧化硅对Bt毒素吸附量随温度升高有所下降（8.39％和47.06％）,高岭土对Bt毒素吸附则略有升高（5.91％）.红外光谱分析显示,Bt毒素被矿物吸附后结构仅有微小变化.被矿物吸附的Bt毒素不易被去离子水解吸,水洗3次总解吸率为28.48%～42.04%.     

EFFECT OF DISSOLUTION AND DEGRADATION OF BAClLUlS THURlNGIENSlS PARASPORAL CRYSTALS ON TOXICITY TO COTTON BOLLWORM*

Abstract The effect of dissolution and buffer pH values on the proteolysis of Bacillus thuringiensis subvar. kurstaki HD-1 parasporal crystals were studied by SDS-PAGE. Dissolution made crystal much easier to be digested by larval Helicoverpa armigera midgut proteases; the rate of proteolysis of protoxin was faster than that of the crystal. Incubated with larval midgut juice, bovine trypsin and chymotrypsin for 17 h at 30°C, the proteolytic process of crystals deepened as the pH of buffer rose from 6. 9 to 10.5; the complete degradation of crystals occurred at pH 10.5, and the active domains were similar, with molecular weight of 60.5±1.26Kd, 61.6±1.16Kd, and 61.7±2.0Kd respectively. Bioassay results suggested that incubation in alkaline buffer could improve the toxicity of crystals to H. armigera, proteolysis gave no further modification to the toxicity and the toxicity was not significantly different among the products from proteolysis by larval midgut juice, bovine trypsin and chymotrypsin, Whether diets with different pH would affect the toxicity of crystals to H. armiger were also tested. Diets made by 0. 01mol.L^-1 Tris-HC1 buffer, pH 8.0 and 0.01mol*L^-1 glycine-NaOH buffer, pH 10.0, all significantly increased the toxicity of crystals and no difference between them was observed.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Toxicity of ammonia and nitrite to yellow catfish (Pelteobagrus fulvidraco)

The 96‐h LC₅₀ (median lethal concentration, LC₅₀) tests were conducted using four different sizes of yellow catfish Pelteobagrus fulvidraco to provide primary information on the sensitivity of this species to elevated ammonia and/or nitrite, and to determine if the sensitivity is mediated by size under the same conditions. The results showed that 96‐h LC₅₀ of fish weighing 0.034 ± 0.002, 0.296 ± 0.049, 3.52 ± 0.95 and 32.96 ± 5.75 g to total ammonia nitrogen‐N was 24.96, 35.85, 47.44 and 68.79 mg L^?1, respectively; un‐ionized ammonia nitrogen‐N was 0.34, 0.49, 0.65 and 0.94 mg L^?1 in test conditions of pH 7.42 and 23°C; and that nitrite nitrogen‐N was 69.06, 97.23, 133.61 and 196.05 mg L^?1 in test conditions of pH 7.58 and 23°C, respectively. The NOEL (No Observable Effect Level) of fish (body weight from 0.03 to 30 g) to ammonia and nitrite was 2.25–6.22 mg L^?1 total ammonia nitrogen‐N, 0.03–0.10 mg L^?1 un‐ionized ammonia nitrogen‐N in test conditions of pH 7.42 and 23°C, and 6.27–17.68 mg L^?1 nitrite nitrogen‐N in test conditions of pH 7.58 and 23°C, respectively. These results indicate that the susceptibility of this fish to total ammonia or nitrite was reduced with increasing size, and that a dose‐dependent relationship might exist between them. The 96‐h LC₅₀ and NOEL of different sizes of fish to total ammonia, un‐ionized ammonia and nitrite would be important to know for water quality standards in yellow catfish aquaculture.     

Evaluation of the Adsorption of Mono- and Polytungstates onto Different Types of Clay Minerals and Pahokee Peat

Detailed knowledge about the fate and transport of tungsten in soils is critical to understanding and effectively addressing tungsten behavior in the environment. Recent studies have shown that tungsten anions may polymerize (depending upon concentration, pH, and aquatic geochemistry) in aquatic and soil systems. However, to date, of all soluble tungstate species only monotungstates have been scrutinized to a fair extent in adsorption studies. There is a lack of information evaluating adsorption mechanisms of mono- and polytungstates onto clay minerals. The objective of this work is to investigate the adsorption behavior of monotungstates (sodium tungstate, Na₂WO₄) and polytungstates (sodium metatungstate, 3Na₂WO₄·9WO₃) onto different types of clay minerals (montmorillonite, kaolinite, illite) and an organic adsorbent (Pahokee peat). Batch equilibrium experiments as a function of concentration (adsorption isotherms) and pH (adsorption envelopes) were performed to provide information about mono- and polytungstate adsorption onto clays and Pahokee peat. Adsorption equilibrium data for mono- and polytungstates onto different types of clay minerals and Pahokee peat were modeled with Freundlich and Langmuir isotherms. The adsorption affinity of clays and Pahokee peat for monotungstates follows the order: Pahokee peat>kaolinite>montmorillonite>illite; for polytungstates, the order is as follows: kaolinite>Pahokee peat>montmorillonite>illite. Results of this study suggest that the charges of the clay mineral surface, tungsten species, and solution pH are the main factors controlling tungsten adsorption. Moreover, polymeric tungsten species (i.e., metatungstate) appear to be more mobile in the environment than monomeric tungstate.     

The adsorption and release of tylosin by clays and soils

Summary The uptake and release of tylosin by Wyoming bentonite, Ca⁺⁺ montmorillonite, illite (Fithian Illinois) and kaolin (china clay) was studied. The adsorption capacities of bentonite and montmorillonite for tylosin were found to be 190 and 65 g mg^–1 respectively, while the adsorption capacities of illite and kaolinite were found to be 22 and 6.5 g mg^–1. 7.6 and 7.4% respectively of the tylosin adsorbed by bentonite and montmorillonite, and 30 and 25% respectively of the tylosin adsorbed by illite and kaolinite, was released in phosphate buffer.The presence of tylosin in two different soils amended with tylosin fermentation waste was also studied, and the effect of temperature on tylosin release was examined in one of the soils. The concentration of tylosin in the soil water was found to be related more to the concentration of tylosin fermentation waste than to the incubation temperature. The amounts of tylosin present in the initial leachates of the two soils, a modified John Innes compost and a limestone waste, were found to be in excess of the minimum inhibitory concentrations for susceptible bacteria. It is hypothesised that in these particular soils if the tylosin is not broken down by microbial action it could affect susceptible populations of bacteria.     

Formation of 1-Deoxy-d-fructose, 1-Deoxy-d-sorbose, 1-Deoxy-d-tagatose and 1-Deoxy-erythrulose by Cell-free Extracts of Bacteria and Actinomycetes

In order to investigate the effect of the spacer in pepstatin-Sepharose on adsorption and elution of acid protease (AcP) in raw shoyu (unpasteurized soy sauce), a homologous pepstatin-aminoalkyl agarose series, (pepstatin-NH₂(CH₂)_n-Sepharose), that varied as to the length of the hydrocarbon chains was synthesized. When raw shoyu containing many kinds of proteases was subjected to affinity chromatography on these pepstatin-C_n-Sepharoses (n = 2, 4, 6, 8, 10 and 12), all of them adsorbed AcP. With increasing length of the spacer up to 6, more and more AcP became adsorbed onto the pepstatin-C_n-Sepharose, whereas with decreasing length of the spacer, more and more AcP was eluted with 0.05 M acetate buffer (pH 3) containing 2 M urea. The AcP was purified in one step from raw shoyu and did not have any carboxypeptidase activity. Some properties of the major component of the eluted AcPs were as follows: molecular weight, 6.7 × 10⁴, on gel filtration with TSK-G3000SW, optimum pH for activation of trypsinogen, 3.5, optimum pH for hydrolysis of hemoglobin, 2.75, and the Ki value toward pepstatin, 1.0 × 10⁸ m.     

Methacryloylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of ferritin

A new metal-chelate adsorbent utilizing 2-methacryloylamidohistidine (MAH) was prepared as a metalchelating ligand. MAH was synthesized using methacryloly chloride and histidine. Monosize nanospheres with an average diameter of 450 nm were produced by emulsion polymerization of 2-hydroxyetylmethacrylate (HEMA) and MAH. Then, Fe³⁺ ions were chelated directly onto the monosize nanospheres. Mon-poly(HEMA-MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and elemental analysis. Fe³⁺ chelated monosize nanospheres were used in ferritin adsorption from an aqueous solution. The maximum ferritin adsorption capacity of Fe³⁺-chelated mon-poly(HEMAMAH) nanospheres was 202 mg/g at pH 4.0 in acetate buffer. The non-specific ferritin adsorption on the monpoly( HEMA-MAH) nanospheres was 20 mg/g. The adsorption behavior of ferritin could be modeled using both Langmuir and Freundlich isotherms. The adsorption capacity decreased with increasing ionic strength of the binding buffer. High desorption ratios (> 95% of the adsorbed ferritin) were achieved with 1.0 M NaCl at pH 7.0. Ferritin could be repeatedly adsorbed and desorbed with the Fe³⁺-chelated mon-poly(HEMA-MAH) nanospheres without significant loss of adsorption capacity.     

Transient electric birefringence of T-even bacteriophages. II. T4B in the presence of tryptophan and T4D.

Measurements of electric birefringence, sedimentation velocity, and biological adsorption rate are used to study the properties of bacteriophage T4B in the presence of excess tryptophan. The adsorption rate determined in borate buffer pH 9 (at 25°C) increases from 0.003 × 10^?8 ml min^?1 (0.025 M) to 0.130 × 10^?8 ml min^?1 (0.150 M). The Kerr coefficient, rotational diffusion coefficient, and the sedimentation coefficient of the phage are also dependent on buffer concentration and reach plateau values above 0.12 M given by K_sp = ?(275 ± 18) × 10^?9 OD^?1 cm² statvolt^?2, D_25,w = 133 ± 4 sec^?1, and s_20,w = 818 ± 11 S. From a comparison of electric birefringence measurements of T4B and T4D it is concluded that T4D and T4B (in the presence of excess tryptophan) exhibit a similar hydrodynamic behavior. The change in physical parameters is solely due to a shift in fiber configuration. At high buffer concentrations the fibers make an angle of approximately 3π/4 with the sheath and the permanent dipole moment is about 200,000 D. This dipole moment is roughly ten times as large as that of a phage particle with nonextended fibers. This difference may be due to a change in hydrodynamic center upon fiber extension or to the presence of positive charges on the fiber tips, or both. At intermediate buffer concentrations the phage population behaves as if it were monodisperse. Probably not all six fibers are extended under such conditions.     

Adsorption and separation of proteins by a smectitic clay mineral

The adsorption of proteins by a smectitic clay mineral was investigated. The clay used in this study is a mixture of montmorillonite and amorphous SiO₂. Due to the high porosity the montmorillonite units are accessible for protein adsorption. The amorphous silica prevents the montmorillonite from swelling and allows column packing. Protein adsorption was performed at different pH under static conditions. Furthermore, static capacities were determined. The material reveals high adsorption capacities for proteins under static conditions (270–408 mg/g), whereby proteins are mainly adsorbed via electrostatic interactions. The Freundlich isotherm is suggested as an adsorption model. For desorption a pH shift was found to be most effective. Binding and elution of human serum albumin and ovalbumin were tested under dynamic conditions. Dynamic capacities of about 40 mg/g for ovalbumin at 764 cm/h were found. The clay mineral provides suitable properties for the application as cost-efficient, alternative separation material.     

Adsorption of Ni2+ and Cu2+ using Bio-Mineral: Adsorption Isotherms and Mechanisms

Heavy metal pollution has become one of the most serious environmental pollution problems. This study aimed to determine the adsorption and desorption characteristics of Ni²⁺ and Cu²⁺ by bio-mineral which was induced by Bacillus subtilis, and to explore the effect of pH on adsorption characteristics. The results showed that the Langmuir model gave a better fit to the experimental data than the Freundlich model, which demonstrated the adsorption was of a single-molecule layer form. The maximum adsorption capacities of the bio-mineral for Ni²⁺ and Cu²⁺ were determined as 67.114 mg/g and 69.930 mg/g, respectively. The desorption rates of Ni²⁺ and Cu²⁺ were very low, especially for Ni²⁺ which was almost 0. Besides, the bio-mineral maintained high adsorption capability for metals ions within a wide pH range (pH ≥ 3). It did not show any new phases after adsorption of Ni²⁺ and Cu²⁺ tested by FTIR, indicating that the bio-mineral and heavy metal ions might mainly physically be adsorbed. The bio-mineral has a larger internal and external specific surface area, pore volume and colloidal properties which are beneficial for the adsorption of metals ions, but shows limits in desorption. This study provides a theoretical basis for the utilization of bio-mineral and opens a new perspective for the remediation of heavy metals pollution.     

Adsorption isotherms of lanthanum to soil constituents and effects of pH,EDTA and fulvic acid on adsorption of lanthanum onto goethite and humic acid

Abstract

La³⁺ adsorption isotherms to five soil constituents (quartz, feldspar, kaolinite, goethite and humic acid) are studied. EDTA, fulvic acid and pH effects are also investigated on the adsorption of lanthanum by goethite and humic acid because of their relative importance in affecting metal environmental behavior. Adsorption isotherms of La³⁺ to five constituents show differences in adsorption capacity and in shape in the studied range of La³⁺. These constituents can be classified according to their adsorption capacity: humic acid > goethite ≈ kaolinite > feldspar ≈ quartz. pH increase could promote humic acid and goethite adsorption of La³⁺ while EDTA could reduce the adsorption by these two adsorbents. Fulvic acid can reduce humic acid adsorption but has less effect on adsorption by goethite.     

Optimization of flow rate,initial metal ion concentration and biomass density for maximum removal of Cu2+ by immobilized Microcystis

The potential of alginate-immobilized Microcystis packed in a column for maximum removal of Cu²⁺ at different flow rates, biomass, and initial metal ion concentration was assessed in a continuous flow system. Although Cu²⁺ removal did occur at all the flow rates tested, it was maximum (54%) at 0.75-ml min⁻¹ flow rate, 30 μg ml⁻¹ initial metal ion concentration and 0.016 g biomass. Cu²⁺ removal was influenced by inlet metal ion concentration and biomass density. An increase in the biomass concentration from 0.016 to 0.128 g resulted in an apparent increase in percentage removal but the Cu²⁺ adsorbed per unit dry wt. declined. When the flow rate (0.75 ml min⁻¹) and biomass density (0.064 g) were kept constant and the inlet metal ion concentration was varied from 10 to 150 μg ml⁻¹, a 68% removal of Cu²⁺ was obtained at 50 μg ml⁻¹ initial concentration in a time duration of 15 min. The metal-laden columns were efficiently desorbed and regenerated following elution with double distilled water (DDW) (pH 2) (89%). This was followed by 1 mm EDTA > 1 mm NTA > 0.1 mm EDTA > 1 mm HCl > 1 mm HNO₃ > 5 mm CaCl₂ > DDW (pH 7.0) > 1 mm NaHCO₃ > 1 mm CaCl₂. Of the total (2.83 mg) adsorbed Cu²⁺, 1.89 mg (67%) was desorbed by DDW (pH 2) within the first 20 min of elution time. Thereafter the desorption rate slowed down and only 22% (0.632 mg) desorption was obtained in the last 20 min. In contrast to water pH 2, the desorption of Cu²⁺ by 1 mm EDTA was very slow, the maximum being 8% after 40 min of elution. This revised version was published online in July 2006 with corrections to the Cover Date.