Chelex-100 Column Research Articles

Evaporation-free tandem-column separation for Sr from geological samples with high Rb/Sr ratios

A novel tandem column method was devised for effective purification of Sr from geological samples characterized by relatively high Rb/Sr ratios, employing a strategic sequence of columns of AG50-X12 cation resin, Chelex-100 chelating resin and AG50-X8 cation resin. The process initiates with the AG50-X12 column to remove most of the matrix elements, followed by the Chelex-100 column to remove the residual Rb under alkaline conditions despite its inability to adsorb alkali metals such as Na, K, and Rb. The Sr fraction was eluted directly into the Chelex-100 column from the AG50-X12 column by a NH3-NH4Cl solution. The final purification through the AG50-X8 column to eliminate residual NH4Cl ensures the effective removal of matrix elements (such as K, Na, Li, Ti, Al, Fe, Mg, Mn and Ca) and Rb. The Chelex-100 resin acts comparatively to a similar Sr-specific resin for Rb and Sr. This method proved its efficacy and precision by aligning the Sr isotopic ratios from a range of reference materials, (JR-1, JR-2, JB-2, BCR-2, BHVO-2, W-2, GSP-2, CRPG Mica-Mg, CRPG Mica-Fe and NIST SRM 987) with their published values, demonstrating its reliability for Sr isotope analysis in complex geological matrices.

Effect of Various Natural Dissolved Organic Carbon on Copper Lability and Toxicity to the Tropical Freshwater Microalga Chlorella sp.

This study adds further critical information to the limited body of knowledge on the ameliorative ability of Australian dissolved organic carbon (DOC), reinforcing the importance of DOC source and concentration as significant factors controlling the risk copper poses to organisms in freshwater systems. The ameliorative ability of five unstudied DOCs on the chronic toxicity of copper to the tropical alga Chlorella sp. was compared. Sensitivity to copper varied dramatically; effect concentrations at the 50 percent effect level (EC50) increased by up to 22-fold in the high DOC treatment compared to controls and more than 2-fold between DOCs at the same concentration. The analytical techniques, diffusive gradients in thin films (DGT) and Chelex column, were used to understand whether differences in copper toxicity could be explained by copper lability. Labile copper mirrored the trends seen in the toxicity tests; lability decreased with increasing DOC concentration and varied between DOCs at the same concentration. The equilibrium model, WHAM VII, was also used to better understand the role of the free copper ion on the toxicity observed. Disagreement between EC50 values derived using the WHAM-predicted free Cu2+ concentrations and agreement between DGT-labile and the maximum dynamic concentration ( cmaxdyn) suggest free copper is not the sole contributor to toxicity and that the source of the specific DOCs also plays a role.

Diffusive Milli-Gels (DMG) for in situ assessment of metal bioavailability: A comparison with labile metal measurement using Chelex columns and acute toxicity to Ceriodaphnia dubia for copper in freshwaters

Fluctuations in concentrations of bioavailable metals occur in most natural waters. In situ measurements are desirable to predict risks of adverse effects to aquatic organisms. We evaluated Diffusive Milli-Gels (DMG), a new in situ passive sampler, for assessing the bioavailability and toxicity of copper in waters exhibiting a wide range of characteristics. The performance was compared to an established Chelex-column method that measures labile copper concentrations by discrete sampling, and the ability to predict acute toxicity to the cladoceran, Ceriodaphnia dubia. The labile copper concentrations measured by the DMG and Chelex-column methods decreased with increasing dissolved organic carbon (DOC) (1.9–15 mg L−1) and hardness (21–270 mg CaCO3 L−1 hardness), with 20–70% of total dissolved copper being present as labile copper. Toxicity decreased with increasing DOC and hardness. Strong linear relationships existed between the EC50 for C. dubia and DOC, and when the EC50 was related to either the labile copper concentrations measured by DMG (r2 = 0.874) or the Chelex column (0.956) methods. The study demonstrates that the DMG passive sampler is a relevant tool for the in situ assessment of environmental risks posed by metals whose toxicity is strongly influenced by speciation.

Trace elements determination in seawater by ICP-MS with on-line pre-concentration on a Chelex-100 column using a ‘standard’ instrument setup.

Trace element determination in seawater is analytically challenging due to the typically very low concentrations of the trace elements and the potential interference of the salt matrix. A common way to address the challenge is to pre-concentrate the trace elements on a chelating resin, then rinse the matrix elements from the resin and subsequently elute and detect the trace elements using inductively coupled plasma mass spectrometry (ICP-MS). This technique typically involves time-consuming pre-treatment of the samples for ‘off-line’ analyses or complicated sample introduction systems involving several pumps and valves for ‘on-line’ analyses. As an alternative, the following method offers a simple method for ‘on-line’ analyses of seawater by ICP-MS. As opposed to previous methods, excess seawater was pumped through the nebulizer of the ICP-MS during the pre-concentration step but the gas flow was adjusted so that the seawater was pumped out as waste without being sprayed into the instrument. Advantages of the method include:•Simple and convenient analyses of seawater requiring no changes to the ‘standard’ sample introduction system except from a resin-filled micro-column connected to the sample tube. The ‘standard’ sample introduction system refers to that used for routine digest-solution analyses of biota and sediment by ICP-MS using only one peristaltic pump; and•Accurate determination of the elements V, Mn, Co, Ni, Cu, Zn, Cd and Pb in a range of different seawater matrices verified by participation in 6 successive rounds of the international laboratory intercalibration program QUASIMEME.

The determination of the isotopic composition of Cu and Zn in seawater

The stable isotope compositions of Zn and Cu in natural materials are newly available for measurement with the advent of multiple-collector inductively coupled mass spectrometry (MC–ICPMS). Although the oceans are prime scientific targets, no progress has been made as yet because of considerable analytical challenges involving the low concentrations of these elements in seawater. We present a procedure which allows isotopic analysis of Zn and Cu isotope compositions of seawater samples at least 1 L in size by MC ICPMS. The main difficulty in analysing Zn and Cu isotopes from seawater samples is their low concentrations (nM/kg) in a matrix with much higher concentrations of other elements. We have surmounted these problems using a procedure that involves pre-concentration using either a BioRad Chelex®100 column or co-precipitation, followed by further purification and separation of Cu and Zn on an anion column. The major advantage of co-precipitation is that the blank is low and allows the precise isotopic analysis of even the most Zn-depleted surface ocean samples. Zn samples have been analysed using a double spike in order to correct for analytical mass discrimination. This approach has been tested using standard-doped seawater samples that had previously been stripped of their metal contents using the Chelex column. The approach is highly successful and yields δ 66Zn = − 0.02 ± 0.05‰ ( n = 5) relative to the standard dopant. For multiple analyses of a large sample of the English Channel we obtain a result for δ 66Zn of 0.35 ± 0.08‰ ( n = 11) for Chelex extraction and 0.31 ± 0.04‰ ( n = 5) for co-precipitation. For both Cu and Zn, correction for instrumental mass discrimination using standard-bracketing requires some care to overcome artefacts-related sample introduction into the mass spectrometer. A depth profile from the NE Pacific is also presented. Measured concentrations are very similar to those reported elsewhere. Cu and Zn isotopes in the upper 400 m of the water column are anti-correlated and may reflect the dominance of scavenging for Cu and biological recycling for Zn.

A rapid Chelex column method for the determination of metal speciation in natural waters

A simple, rapid Chelex resin column method has been developed for the determination of metal speciation in natural water samples. A water sample (pH 6–8.2) was pumped through a small plug of the Ca-form of Chelex 100 at a flow rate of 48 ± 4 mL/min. The flow regime was optimised to give the shortest possible contact time (0.25 s) but at the same time ensured quantitative uptake of inorganic metal standards onto the resin. Labile metal was calculated as the difference between influent and effluent metal concentrations, measured by some form of atomic spectrometry. In tests with six metal-spiked natural freshwater samples (10 μg/L added metal, pH 7.0–8.2), the labile fraction measured ranged from 87 to 98%, 32 to 64%, 12 to 78%, 38 to 91% and 63 to 89% for cadmium, copper, lead, nickel and zinc, respectively. Application of the method to copper-contaminated waters showed that the results provided a conservative estimate of the fraction inhibiting growth of a copper-sensitive bacterium, thus proving to be a useful measure of bioavailability. Tests on a range of model metal complexes showed that the measured labile metal fraction was inversely proportional to log K, and controlled by the kinetics of metal complex dissociation. Iron-containing colloids, present in natural freshwaters were not retained by the column. The method therefore discriminates trace metal species on the basis of both size and kinetics.

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

Custom-made interface between electrothermal vaporiser and inductively coupled plasma-mass spectrometer: determination of nickel by isotope dilution in plant samples

ICP-mass spectrometer Perkin Elmer Elan 6000 was adapted for ETV analysis by coupling with GBC GF 3000 graphite furnace atomiser. Both instruments were connected by a 25 cm long PTFE tubing of 6 mm i.d. and a custom-made interface. The apparatus was applied to nickel determination using isotope dilution technique. The efficiency of analyte transfer through the interface was approximately 20%, however the incomplete transfer did not influence the blank values. Measured isotope ratio was very stable, whereas the magnitude of ion signal showed a little shift. CRM samples were analysed after microwave decomposition and by slurry technique as well. Obtained results were compared with those of pneumatic sample nebulisation after nickel separation on Chelex column. All tested techniques gave accurate results with comparable precision. However, the slurry technique allowed significant decreasing of the blank values and the limit of detection. The duration of analysis run can also be substantially shortened from several hours to a few minutes.

Determination of zinc in plant samples by isotope dilution inductively coupled plasma mass spectrometry

Determination of zinc involved spiking with 68Zn enriched solution, digestion by HNO 3+H 2O 2 in microwave decomposition unit, off-line separation of zinc on Chelex-100 column and measurement of ( 64Zn+ 66Zn)/ 68Zn isotope ratio on ICP-MS spectrometer with a quadrupole mass filter. After optimization of standard operation procedure (details are given) the method was validated. LOD was found to be 0.3 μg g −1 for the procedure without zinc separation and 3.6 μg g −1 for the procedure involving zinc separation, respectively. The accuracy of results was proved by analyses of several CRM and a primary solution of zinc, the concentration of which was verified by gravimetry and complexometric titration. Barium is the only element causing serious interferences and it must be removed from samples. The uncertainty budget is given together with the scheme of combined uncertainty calculation. The main uncertainty components are contamination during zinc separation and uncertainty of isotopic composition of natural zinc.

Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R.

Human serum paraoxonase (PON 1) exists in 2 major polymorphic forms (Q and R), which differ in the amino acid at position 191 (glutamine and arginine, respectively). These PON allozymes hydrolyze organophosphates and aromatic esters, and both also protect LDL from copper ion-induced oxidation. We have compared purified serum PONs of both forms and evaluated their effects on LDL oxidation, in respect to their arylesterase/paraoxonase activities. Copper ion-induced LDL oxidation, measured by the production of peroxides and aldehydes after 4 hours of incubation, were reduced up to 61% and 58%, respectively, by PON Q, but only up to 46% and 38%, respectively, by an equivalent concentration of PON R. These phenomena were PON-concentration dependent. Recombinant PON Q and PON R demonstrated similar patterns to that shown for the purified serum allozymes. PON Q and PON R differences in protection of LDL against oxidation were further evaluated in the presence of glutathione peroxidase (GPx). GPx (0.1 U/mL) alone reduced copper ion-induced LDL oxidation by 20% after 4 hours of incubation. The addition of PON R to the above system resulted in an additive inhibitory effect on LDL oxidation, whereas PON Q had no such additive effect. The 2 PON allozymes also differed by their ability to inhibit initiation, as well as propagation, of LDL oxidation. PON Q was more efficient in blocking LDL oxidation if added when oxidation was initiated, whereas PON R was more potent when added 1 hour after the initiation of LDL oxidation. These data suggest that the 2 allozymes act on different substrates. Both PON allozymes were also able to reduce the oxidation of phospholipids and cholesteryl ester. PON Q arylesterase activity was reduced after 4 hours of LDL oxidation by only 28%, whereas the arylesterase activity of PON R was reduced by up to 55%. Inactivation of the calcium-dependent PON arylesterase activity by using the metal chelator EDTA, or by calcium ion removal on a Chelex column, did not alter PON's ability to inhibit LDL oxidation. However, blockage of the PON free sulfhydryl group at position 283 with p-hydroxymercuribenzoate inhibited both its arylesterase activity and its protection of LDL from oxidation. Recombinant PON mutants in which the PON free sulfhydryl group was replaced by either alanine or serine were no longer able to protect against LDL oxidation, even though they retained paraoxonase and arylesterase activities. Overall, these studies demonstrate that PON's arylesterase/paraoxonase activities and the protection against LDL oxidation do not involve the active site on the enzyme in exactly the same way, and PON's ability to protect LDL from oxidation requires the cysteine residue at position 283.

Additional evidence of the operational character of the lability of complexes of copper(II) with simple organic ligands

A fixed analytical window of observation, 5 μmol l −1 free copper and 100 μmol l −1 total copper, but methods different in nature, operation mode and time scale were applied, in parallel, at pH 6.50 and 16 °C, to solutions with copper complexes of very different thermodynamic stabilities. Ethylenediaminetetraacetic acid (EDTA), l-cysteine, l-lysine, l-proline and succinate were used as metal buffers. Chitin (batch and column modes), chelex-100 (column mode) and normal pulse anodic stripping voltammetry (NPASV, −0.3 V deposition potential) were used as techniques. A strategy, based on using the mass of polymer that provided uptake of free copper similar to that taken up by Pseudomonas syringae bacterial cells in absence of ligands, was used for all but NPASV methods. Flame atomic absorption spectrophotometry was used for metal detection. The lability of copper complexes with simple organic ligands showed a strongly operational character. The time scale of the physicochemical method used for the determination of lability was not the only parameter inherent to the method that influenced the extent of copper complex dissociation. The nature of both initial and competing ligands and the operation mode may also condition the speciation results.

Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction.

This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 l of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 x 10(5)-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 microliters (0.2 PFU/l tap water).

Characterization of the oligopeptides of Parmigiano-Reggiano cheese soluble in 120 g trichloroacetic acid/l.

The non-protein nitrogen (NPN) of samples of Parmigiano-Reggiano cheese ripened for 6 and 15 months was fractionated by ion-exchange chromatography on a Cu(2+)-Chelex column to separate oligopeptides from free amino acids. Peptide components were isolated by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry (FAB-MS). Only the NPN fraction of 6 month old cheese samples contained enough peptides to be further characterized. On the basis of FAB-MNS spectral results, 39 oligopeptides were identified, the main components being phosphopeptides. Two sets of both intact and partly dephosphorylated peptides, accounting for a total of 19 phosphopeptides, were formed by the hydrolysis of beta-casein and belonged to regions 1-20 and 6-28 of beta-casein. The formation and potential role of these peptides in cheese is discussed.

82Sr production from metallic Rb targets and development of an 82Rb generator system

A complete procedure for producing 82Sr/Rb generators is described. 82Sr was produced by bombarding metallic rubidium targets with 60 MeV protons with beam currents as high as 80 μA. The experimentally determined cross section for a thick target (61–48 MeV) agrees with the literature value of 100 mb. Production rates of about 0.2 mCi/μA-h with a 1.8 g/cm 2 target were achieved. The 82Sr was separated from the target by dissolving the rubidium metal in n-butanol then separating the strontium isotopes on a Chelex-100 column. A SnO 2 generator system was built from readily available components. Factors affecting generator design, performance and limitations included SnO 2 particle size, column length and eluant flow-rate.

Studies on metal speciation in the natural environment

A scheme for metal speciation has been developed and applied to samples of river water, rain and snow. The scheme combines physical speciation by size using ultrafiltration of the soluble metal species with chemical characterization of the metal species by their dissociation kinetics. The kinetics of metal complex dissociation was studied by anodic stripping voltammetry and by ion exchange at Chelex-100 cation-exchange resin, using both the Chelex column and the Chelex batch technique. These kinetic studies cover a time scale of measurement of ca. 2 ms for anodic stripping voltammetry and up to 6 days for Chelex batch technique, a range of about 8 orders of magnitude. The kinetic results suggest that the copper in the snow sample (pH 3.9) is probably bound to different sites having different bonding energies in polyfunctional complexing agents. Four different copper species having dissociation rate constants of 3.1 X 10 −2, 1.6 X 10 −3, 6.2 X 10 −5, and 8.8 X 10 −6, s −1 have been observed. All of these species fall in the small size fractions of ultrafiltration. The Al in the Rideau River water sample (pH 8) is probably bound to the different sites in humic materials to yield dissociation rate constants 1.6 X 10 −4, 1.0 X 10 −6 and < 10 −6 s −1. In the Rideau River water sample, most of the soluble Al species have a large molecular size and are probably associated with humic materials. On the other hand, most of the soluble Al species in the small size fraction are Chelex-nonlabile and are probably Al(OH) − 4. Use of graphite platform furnace atomic absorption spectrometer allows direct determination of trace metals in freshwaters, to be done routinely, rapidly, and inexpensively.

‘Strongly bound’ uranium in marine waters: occurrence and analytical implications

In the determination of dissolved 238U in aqueous solutions, a pre-concentration by chelating ion exchange chromatography with a Chelex-100 column at pH 4 (or Chelex- 238U) and co-precipitation with iron hydroxide (or Fe 238U) yielded indistinguishable results for pure solutions of inorganic uranium and samples of open-ocean waters. However, in coastal marine waters, the concentration of Fe 238U could be significantly higher than that of Chelex- 238U. The concentration ratio of Fe 238U. The concentration ratio of Fe 238U to Chelex- 238U seemed to be higher in waters with higher concentration of dissolved organic carbon (DOC). A concentration ratio of Fe 238U to Chelex- 238U as high as 1.27 was observed. The concentration of 238U (UV- 238U) in these coastal waters might be further elevated if dissolved organic matter in the sample was first mineralized, by subjecting it to UV irradiation in the presence of hydrogen peroxide under acidic conditions, before analysis. The ratio of the concentration of UV- 238U to Chelex- 238U increased even more dramatically with increasing concentration of DOC. The highest value observed was 1.83. The concentration of dissolved uranium thus increased in the order: Chelex- 238U ⩽ Fe 238U ⩽ UV 238U. These results are consistent with the notion that: (a) only inorganic and weakly bound species are removed by the Chelex 100 resin so that Chelex-U represents inorganic and weakly bound uranium: (b) iron hydroxide co-precipitation can remove a fraction of dissolved organic matter so that FeU includes inorganic as well as a variable fraction of ‘strongly bound’ uranium; (c) the UV irradiation of a sample of water converts ‘strongly bound’ uranium to the readily detectable inorganic uranium: (d) ‘strongly bound’ uranium is more likely to be found in coastal marine waters where the concentration of dissolved organic matter is higher. Previously published data are almost exclusively Chelex-U and FeU. In the open oceans, if the amount of ‘trongly bound’ uranium is small, they should be essentially identical. In coastal waters, however, Chelex-U and FeU may represent different fractions of dissolved uranium. Thus, some of the conclusions on the behavior of uranium in estuarine and coastal waters drawn from these data may have to be re-examined to take into consideration the speciation of uranium in these waters and the analytical methods used. By determining Chelex- 238U and UV- 238U simultaneously in a sample, ‘strongly bound’ uranium may be estimated as the difference in concentration in these two measurements.

Determination of Lability of Soluble Copper(II) Complex in Natural Water Samples by Chelex Column Method

Determination of Lability of Soluble Copper(II) Complex in Natural Water Samples by Chelex Column Method

Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin

The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots. Evidence for additional copper binding sites in Cp and bSA was obtained suggesting a role for copper ion in the homeostatic regulation of Cu(II) and other metal ions in the serum. In the binding studies the Cp was freed of exogenous Cu(II) by passing it over a Chelex-100 column. Two flow rates were used, 4 ml/hr and 40 ml/hr, which removed Cu(II) of different affinities. Cp passed at the slower flow rate (Cp 4) only contained the prosthetic copper atoms. Cp passed at the faster flow rate (Cp 40) contained one additional copper atom with a Kg a ∼ 10 7M −1. Another 2–6 Cu(II) ion could be added to the Cp 40 with an average affinity of about K a ∼ 10 5M −1. The Cu(II) ions found in Cp provide two distinguishable classes: (1) the prosthetic copper atoms and (2) the exogenous copper atoms that can be removed by Chelex-100. For bSA one copper atom was bound strongly with a K a value approaching 10 12–10 13 M −1 and was not removed by Chelex-100 at any flow rate. A second copper atom was found with a K a = 5.2 × 10 6M −1 and was removed by Chelex-100 at 4 ml/hr. Three additional copper atoms were bound with a K a = 1.6 × 10 5 M −1; they were readily removed by Chelex-100 at 40 ml/hr but were nondialysable.

A low contamination chelex-100 technique for shipboard pre-concentration of heavy metals in seawater

An easy to handle, on-board procedure is proposed which uses a Chelex-100 ion-exchange column to pre-concentrate Cd, Co, Cu, Fe, Mn, Ni, Pb and Zn from seawater with minimal risk of contamination. Immediately after collection, the seawater sample is poured into a polyethylene terephthalate (PET) bottle containing ammonium maleate buffer at pH 6.5. The bottle is then connected to a pre-packed Chelex-100 column set, and hung upside-down to allow the sample to flow through the column in dust-free conditions. Finally, the whole batch of columns is brought back to the land-based laboratory for further elution and analysis. A concentration factor of 250 is achieved.