Differential Pulse Anodic Stripping Voltammetric Research Articles

A novel ion-imprinted polymer based on pyrrole as functional monomer for the voltammetric determination of Hg(II) in water samples

A novel selective ion-imprinted polymer (IIP) was synthesized, characterized, and used as part of a carbon paste electrode (CPE) for the electrochemical analysis of Hg(II) in real water samples by differential pulse anodic stripping voltammetric (DPASV). Hg-IIP (mercury-ion imprinted polymer) was synthesized using pyrrole for the first time as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a cross-linker agent, and sodium persulfate as initiator. The CPE was modified with IIP (CPE-IIP) with a composite mixture of Hg-IIP (10%), graphite (90%), and 0.08 g of paraffin oil. A notable difference in the Hg(II) Iap (anodic peak current) between a CPE, CPE-IIP and CPE-NIP (non-imprinted polymer) was observed when optimizing the differential pulse anodic stripping voltammetry (DPASV) parameters by a Box-Behnken design. When analyzing a 50 µmol l−1 Hg(II) solution, the optimal conditions (step potential: 0.11 V, amplitude: 0.15 s, modulation time: 0.005 s, and interval time: 0.15 s) allowed an Iap of 144 µA with a %RSD < 10% for the CPE-IIP response, in comparison to the Iap of 1.47 µA obtained with the CPE-NIP, Iap confirming the existence and performance of the recognition cavities in the IIP. The proposed methodology presents a limit of detection (LOD) of 0.02 mg l−1 within a linear range of 0.09 mg l−1 to 20.05 mg l−1. The inter-day and intra-day precision were measured in two concentration levels (1 mg l−1 and 10 mg l−1) with %RSD < 10%. The developed methodology was successfully applied to determine Hg(II) in three spiked samples (10 mg l−1) of natural water sources without acid digestion, obtaining percentage recoveries close to 99.74%, which demonstrates that digestion treatment is not required for this kind of samples.

Stripping voltammetric analysis of mercury ions at nitrogen-doped reduced graphene oxide modified electrode

Selective and sensitive monitoring of toxic heavy metals is prerequisite to the curb of water contamination. Herein, we present a differential pulse anodic stripping voltammetric (DPASV) method for the determination of mercury in water samples with a glassy carbon electrode (GCE) modified with nitrogen-doped reduced graphene (NRGO). Through thermal treatment of graphene oxide (GO) in NH3 atmosphere, nitrogen atoms can be facilely doped into the hexagonal lattice of graphene to produce NRGO. The NRGO modified GCE (abbreviated as NRGO/GCE) shows an impressively favorable electron transfer activity and a high stripping voltammetric response for mercury compared to that of reduced graphene oxide (RGO) modified electrode. Not only does the NRGO/GCE show an extraordinary low detection limit of 0.58 nM, it also demonstrates a wide linear concentration range from 1 nM to 800 nM and a high sensitivity of 2.69 μA/μM. Due to the excellent selectivity of the NRGO/GCE to mercury, the interference from other heavy metals such as Fe(III), Co(II), Ni(II), Cu(II), Mn(II), Cd(II), Al(III), Ca(II), and Pb(II) were almost negligible. Those outstanding features guarantee the proposed anodic stripping voltammetric method highly applicable to the assay of mercury ions in real water samples, which is validated by the conventional atomic fluorescence spectrometry.

A three-dimensional bimetallic oxide NiCo2O4 derived from ZIF-67 with a cage-like morphology as an electrochemical platform for Hg2+ detection

Selective detection of toxic Hg2+ is of great significance in the control of the environmental and industrial pollution because it poses serious threat to human health. Herein, we present a differential pulse anodic stripping voltammetric (DPASV) method for Hg2+ detection by using hollow and porous three-dimensional nano-structured NiCo2O4 with cage-like morphology as an electrode modifier. The NiCo2O4 nanocages (NiCo2O4 NCs) derived from ZIF-67 (a cobaltic zeolite imidazole framework) possess large specific surface areas that can provide a large number of adsorption sites for Hg2+. The NiCo2O4 NCs modified glassy carbon electrode (NiCo2O4 NCs/GCE) was demonstrated to be an advantageous and high-performance platform for the electrochemical detection of Hg2+ with a wide linear range (0.1 nmol/L–3.5 µmol/L) and low detection limit (0.09 nmol/L). Additionally, the NiCo2O4 NCs/GCE was proved to be highly stable, representing it was benign to environment and could be well applied to detect Hg2+ in real water samples.

Fabrication of a Novel Electrochemical Sensor for Simultaneous Determination of Toxic Lead and Cadmium Ions

Background: Highly sensitive and rapid monitoring of heavy metal ions such as lead and cadmium has attracted considerable interest in analytical chemistry due to serious environmental effects of these ions. Objectives: In this work, clay/carbon nanocomposite modified with an organic ligand was applied as a novel modifier for simultaneous measurement of Pb (II) and Cd (II) in water samples. Methods: The characterization of the modifier was studied by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy. The analytical performance of the method was investigated by the differential pulse anodic stripping voltammetric (DPASV) technique using a carbon paste electrode (CPE) modified with the prepared nanocomposite. Results: Under the selected optimized conditions, a linear voltammetric response in the range of 2.0 - 100.0 ppb was obtained for the both analyte ions. The calculated detection limits (S/N = 3) were found to be 1.1 ppb for Pb (II) and 0.7 ppb for Cd (II), which are below the World Health Organization (WHO) guideline values for lead and cadmium in drinking water. The method was successfully applied for determination of noxious Pb (II) and Cd (II) ions in real waters with acceptable recoveries for the spiked samples. Conclusions: A simple, selective and sensitive electrochemical sensor was prepared and applied for simultaneous detection of Pb (II) and Cd (II). The suggested sensor demonstrated good electrochemical performance and anti-interference capability for electrochemical detection of these ions in real water samples using the DPASV method.

Sensitive voltammetric determination of cadmium at a carbon nanotubes/Fe3O4/eggshell composites modified carbon paste electrode

In the present work, we describe a new method for differential-pulse anodic stripping voltammetric (DPASV) determination of cadmium (II) using a carbon paste electrode modified with magnetic nanocomposite of Fe3O4/eggshell and carbon nanotubes (MWCNT-MES-CPE). In addition to Fourier transform infrared spectroscopy (FT-IR) and vibrating sample magnetometer (VSM) for the characterization of the prepared magnetic nanocomposite, the morphology and structure of Fe3O4/eggshell were studied by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The effective experimental parameters on the preconcentration of Cd (II) ions and voltammetric stripping step such as supporting electrolyte (type and concentration), accumulation and reduction potential, and of time and amount of modifier were investigated by differential pulse anodic stripping voltammetry. The voltammetric peak current of Cd (II) appeared at a potential about −0.7 V, under optimum conditions. The method was then worked out for the determination of cadmium at low levels. Under optimized conditions, the response to Cd (II) is linear in the range from 3.0 to 250 ng mL−1 with a detection limit of 2.4 ng mL−1 for Cd (II) ion. The sensor was applied to the determination of Cd (II) in water, hair samples, and certified reference materials.

A screen-printed carbon electrode modified with a bismuth film and gold nanoparticles for simultaneous stripping voltammetric determination of Zn(II), Pb(II) and Cu(II)

The authors report on a disposable sensor for the differential pulse anodic stripping voltammetric (DPASV) determination of the ions Zn(II), Pb(II) and Cu(II). Simultaneous detection is accomplished by using a screen-printed carbon electrode (SPCE) co-modified with an in-situ plated bismuth (Bi)) film and gold nanoparticles (AuNPs). The synergistic effect of the Bi film, and the large surface and good electrical conductivity of the AuNPs strongly assist in the co-deposition of the three ions. Four well-defined and fully separated anodic stripping peaks, at 540 mV for Zn(II), 50 mV for Pb(II), 140 mV for Bi(III) and 295 mV for Cu(II), all vs. Ag/AgCl, can be seen. The modified SPCE was characterized by scanning electron microscopy, X-ray diffraction, cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimized conditions, the sensor has a good response to these ions. The detection limits (at an S/N ratio of 3) are 50 ng·L−1 for Zn(II), 20 ng·L−1 for Pb(II), and 30 ng·L−1 for Cu(II). The method was applied to the determination of the 3 ions in spiked lake water samples.

Novel imprinted polymeric nanoparticles prepared by sol–gel technique for electrochemical detection of toxic cadmium(II) ions

In this study, a novel nanostructured cadmium(II) ion imprinted polymer (Cd–IIP) was prepared through sol–gel process. The prepared material was characterized by Fourier transform infrared spectroscopy (FT–IR), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and energy dispersive X-ray (EDX) analysis. The resulting Cd–IIP material was employed for modification of simple and low-cost carbon paste electrode (CPE). The differential pulse anodic stripping voltammetric (DPASV) technique was applied for determination of toxic cadmium(II) ions after closed circuit sorption on the MCPE. The best electrochemical response for Cd(II) ions was obtained with a paste composition of 10% (w/w) of cadmium(II)–IIP, 65% (w/w) of graphite powder and 25% (w/w) of paraffin oil using a solution of 0.1molL−1 phosphate buffer solution (PBS, pH = 5) with accumulation potential of −1.1V and accumulation time of 300s. The proposed electrochemical sensor showed excellent selectivity and stability for Cd(II) analysis. The electrode displayed a linear behavior in the concentration range of 0.5–40µgL−1 Cd(II) with a low detection limit of 0.15μgL−1 (at S/N=3). The designed sensor was further utilized for the determination of cadmium in different water samples with satisfactory results.

Amino-calix[4]arene modified graphite as an electrochemical interface for mercury(II) quantification

A new simple amino-calix[4]arene modified graphitic composite electrode have been prepared and employed in the form of thin film on glassy carbon electrode (GCE) surface as a sensing probe for the measurement of mercury(II) ions. In order to decipher the complexing ability of the modifier towards the metal ions analytical techniques such as cyclic voltammetry and differential pulse anodic stripping voltammetric are used.

Validation of the Polyacrylic acid/Glassy Carbon Differential Pulse Anodic Stripping Voltammetric Sensor for Simultaneous Analysis of Lead(II), Cadmium(II) and Cobalt(II) Ions

Validation of the Polyacrylic acid/Glassy Carbon Differential Pulse Anodic Stripping Voltammetric Sensor for Simultaneous Analysis of Lead(II), Cadmium(II) and Cobalt(II) Ions

Electrochemically induced regioregularity of the binding sites of a polyaniline membrane as a powerful approach to produce selective recognition sites for silver ion

Polyaniline (PANI) modified electrodes based on the electrochemical polymerization of aniline in the presence of a chelating agent, 5-sulfosalicylic acid (SSA), were prepared. The resulting PANI/SSA membrane was templated with respect to silver ion by applying consecutive reduction/oxidation steps. The electrochemically mediated templating causes doping/undoping of silver ions into the polymeric membrane. It seems that repeated addition/removal of silver ions into/from the membrane, induces conformational changes in the binding sites of the PANI and SSA, and increases the number of proper binding sites. This electrochemically induced regioregularity of the binding sites of the membrane components was found to be a powerful approach to produce selective recognition sites for silver ions in the PANI/SSA membrane. The system was able to fulfill four main criteria of the recognition process, including selectivity, sensitivity, rapidity and chemical reversibility in the voltammetric and potentiometric detection of silver ion. The prepared sensor was employed for preconcentration and differential pulse anodic stripping voltammetric (DPASV) measurement of silver ion. The stripping peak current was found to be dependent on the silver ion concentration over the range 1.0×10−10 to 1.0×10−4M. Several metal ions did not show appreciable interference in the voltammetric determination of Ag+, however, mercury ion led to a significant interference. During potentiometric measurements, the sensor demonstrated reproducible response with a near-Nernstian slope of 57.1±0.4mV per decade of silver ion activity over a linear range extending five orders of magnitude (1.0×10−8 to 1.0×10−3M Ag+), with a detection limit of 1.0×10−9M Ag+. The templated electrode provided improved potentiometric selectivity for silver ion with respect to various metal ions and could be used over a pH range of 3.0–5.5.

Graphene/CeO 2 hybrid materials for the simultaneous electrochemical detection of cadmium(II), lead(II), copper(II), and mercury(II)

Abstract The CeO2 nanoparticle-decorated graphene hybrid was prepared by hydro-thermal method and their application in electrochemical detection of heavy metal ions was investigated. It was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscopy (TEM). The electrochemical behavior of four heavy metal ions (cadmium(II), lead(II), copper(II), and mercury(II)) on the graphene/CeO2 hybrid nanocomposite modified glassy carbon electrode (GCE) was investigated in acetate buffer solution by differential pulse anodic stripping voltammetric (DPASV). Compared with the bare GCE, the proposed electrode showed improved analytical performance characteristics in detecting heavy metal ions. By using DPASV method, it shows that the graphene/CeO2 hybrid nanocomposite modified GCE could be used for tracing Cd2 +, Pb2 +, Cu2 +, and Hg2 + simultaneously and selectively. Under optimum conditions, we can detect Cd2 +, Pb2 +, Cu2 +, and Hg2 + simultaneously with high sensitivity in low concentration in the experiments.

Electrochemical sensor based on polystyrene sulfonate–carbon nanopowders composite for Cu (II) determination

A differential pulse anodic stripping voltammetric (DPASV) method, with an open circuit (OC) approach in the pre-concentration step has been developed for copper ion determination at very low concentration level using a sensor based on a polystyrene sulfonate–carbon nanopowders (PSS–CnP) composite. This composite material is easily prepared from ultrasonic assisted dispersions of CnP in aqueous solution of PSS. For preparation of sensor devices, a reproducible and inexpensive drop coating procedure of the surface of home-made pencil graphite electrodes (PGEs) using a CnP dispersion in PSS was performed. At the optimal conditions for accumulation (0.01molL−1 KNO3 at pH 3) and measurement steps (a reduction potential of −0.5V for 60s and then, an anodic DPV scan) and using a pre-concentration time of 300s, the limit of detection was 0.11µgL−1 (1.73nM). This OC-DPASV method using the PSS–CnP–PGE sensor was successfully employed for Cu(II) determination in mineral, river and sea water samples.

Electrochemical studies on lawsone and its determination in henna (Lawsonia inermis) extract using glassy carbon electrode

The electrochemical behaviour of lawsone at glassy carbon electrode (GCE) was investigated by using cyclic and differential pulse anodic stripping voltammetric (DPASV) techniques. Cyclic voltammetry was used to study the influence of pH on the peak current and peak potential. The Mcllavnie’s buffer of pH 3.0 was selected as a suitable analytical medium in which lawsone exhibited sensitive diffusion controlled redox peaks (vs. Ag/AgCl). The peak current varied linearly with lawsone concentration in the range between 0.60 and 1.40 μM with a detection limit of 6 nM. The applicability of the proposed method was illustrated by the determination of lawsone present in real samples. A mean recovery of lawsone in the leaf of Lawsonia inermis was 99.5% with a relative standard deviation of 1.15%.

Electrochemical oxidation behavior of ezetimibe and its adsorptive stripping determination in pharmaceutical dosage forms and biological fluids

Ezetimibe (EZE) is a drug that reduces plasma cholesterol levels and performs by decreasing cholesterol absorption in intestines. In this study, we examined the electrochemical behavior of EZE on a glassy carbon electrode and optimum conditions for its quantitative determination by using voltammetric methods. In addition, some electrochemical parameters such as diffusion coefficient, surface coverage of adsorbed molecules, electron transfer coefficient, standard rate constant and number of electrons were calculated by using the results of cyclic voltammetry. A tentative mechanism for the oxidation for EZE is suggested. As a result of bulk electrolysis and cyclic voltammetric experiments, an intermediate product occurred. The oxidation signal of the EZE molecule was used to develop fully validated, new, rapid, selective and simple square-wave anodic adsorptive stripping voltammetric (AdsSWV) and differential pulse anodic stripping voltammetric (AdsDPV) methods to direct determination of EZE in pharmaceutical dosage forms and biological samples. As a result of these studies, for the AdsDPV and AdsSWV techniques, linear working ranges were found to be 4.0 × 10−7–7.6 × 10−6 and 4.0 × 10−7–6.0 × 10−6 mol L−1, respectively. The detection limits obtained from AdsDPV and AdsSWV were calculated to be 1.529 × 10−7 and 1.185 × 10−7mol L−1, respectively. Moreover, the limit of quantification was evaluated for AdsDPV and AdsSWV methods and found to be 5.098 × 10−7 and 3.951 × 10−7 mol L−1, respectively. The developed methods for EZE were successfully applied to assay the drug in tablets, human blood serum and human urine.

Anodic Stripping Voltammetric Determination of Lead (II) and Cadmium (II) by Using a Carbon Nanotubes Paste Electrode Modified with Ion Exchange Synthetic Resin

This work describes an electroanalytical method for simultaneous determination of cadmium (II) and lead (II) ions using a carbon paste modified with carbon nanotubes and an ion exchange synthetic resin (Amberlite IR120) electrode under Differential Pulse Anodic stripping voltammetric (DPASV) conditions. The procedure is based on the effective preconcentration of metal ions on the electrode surface containing ion exchange resin under open circuit condition. Using optimized conditions of 600 s preconcentration time for an electrode composition of 5% w/w Amberlite IR 120 and 30 % w/w carbon nanotubes in the paste, a scan rate of 10 mVs-1, and a potential pulse amplitude of 75 mV, linear calibration graphs can be obtained by were monitoring the peaks at -0.75 V for cadmium (II) and -0.58 V for lead (II) from 0.07 – 2.50 μmol L-1 and 0.01 – 5.80 μmol L-1, respectively. The proposed method was applied for the determination of lead (II) and cadmium (II) in ethanol fuel, gasoline for aviation, and spiked water. For all samples analyzed, recoveries ranged from 95.8 to 106.3%. Interferences were also evaluated.

Optimization of analytical procedures for the simultaneous voltammetric determination of total Hg(II) in presence of Cu(II) in environmental matrices

Abstract The present work reports the critical comparison about the employment of three different supporting electrolytes (0.1 mol L−1 HClO4, 0.01 mol L−1 EDTA-Na2 + 0.06 mol L−1 NaCl + 2.0 mol L−1 HClO4 and 0.1 mol L−1 KSCN + 0.001 mol L−1 HClO4) and their instrumental and chemical optimisation for the simultaneous voltammetric determination of total mercury(II) and copper(II) in sediments and sea water at gold electrode, especially discussing the reciprocal interference problems. The differential pulse anodic stripping voltammetric (DPASV) measurements were carried out using a conventional three-electrode cell: a gold electrode (GE) as working electrode, a platinum wire and an Ag‖AgCl‖KClsat as auxiliary and reference electrodes, respectively. The analytical procedure was verified by the analysis of standard reference materials: Estuarine Sediment BCR-CRM 277, River Sediment BCR-CRM 320 and Mercury in Water NIST-SRM 1641d. Once set up on the standard reference materials, the analytical procedure was transferred and applied to sediments and sea waters sampled in a lagoon ecosystem connected with Adriatic Sea (Ravenna area, Italy).

Electrochemical oxidation of atorvastatin and its adsorptive stripping determination in pharmaceutical dosage forms and biological fluids

Electrochemical behavior of atorvastatin (AT) and optimum conditions to its quantitative determination were investigated using voltammetric methods. Some electrochemical parameters such as diffusion coefficient, surface coverage of adsorbed molecules, electron transfer coefficient, standard rate constant and number of electrons were calculated using the results of cyclic voltammetry. A tentative mechanism for the oxidation for AT has been suggested. The oxidation signal of AT molecule was used to develop fully validated, new, rapid, selective and simple square-wave anodic adsorptive stripping voltammetric (AdsSWV) and differential pulse anodic stripping voltammetric (AdsDPV) methods to direct determination of AT in pharmaceutical dosage forms and biological samples. For the AdsDPV and AdsSWV techniques, linear working ranges were found to be 1.0 × 10–7–5.0 × 10–6and 3.0 × 10–7–5.0 × 10–6mol l–1, respectively. The detection limits obtained from AdsDPV and AdsSWV were calculated to be 6.55 × 10–8and 1.53 × 10–7mol l–1, respectively. The methods were successfully applied to assay the drug in tablets, human blood serum and human urine.

Preparation of a carbon ceramic electrode modified by 4-(2-pyridylazo)-resorcinol for determination of trace amounts of silver

A 4-(2-pyridylazo)-resorcinol (PAR)-modified carbon ceramic electrode (CCE) prepared by the sol-gel technique has been reported for the first time in this paper. By immersing the CCE in aqueous solution of PAR (0.001molL(-1)), after a short period of time, a thin film of PAR was rapidly formed on the surface of the electrode due to its strong adsorption properties. A differential pulse anodic stripping voltammetric (DPASV) method was developed for determination of Ag(I) at the modified carbon ceramic electrode. The analysis procedure consisted of an open circuit accumulation step in a sample solution which was continuously stirred for 12 min. This was followed by replacing the medium with a clean solution where the accumulated Ag(I) was reduced for 15s in -0.6 V. Then, the potential was scanned from -0.2 to +0.2V to obtain the voltammetric peak. The detection limit of silver(I) was 0.123 microg L(-1), and for seven successive determinations of 10, 100 and 200 microg L(-1) Ag(I), the relative standard deviations were 2.1, 1.4 and 1.03%, respectively. The calibration curve was linear for 0.5-300 microg L(-1) silver(I). The procedure was applied to determine silver(I) in X-ray photographic films and super-alloy samples.

Amplified electrochemical aptasensor for thrombin based on bio-barcode method

In the present study, an electrochemical aptasensor for highly sensitive detection of thrombin was developed based on bio-barcode amplification assay. For this proposed aptasensor, capture DNA aptamerI was immobilized on the Au electrode. The functional Au nanoparticles (DNA–AuNPs) are loaded with barcode binding DNA and aptamerII. Through the specific recognition for thrombin, a sandwich format of Au/aptamerI/thrombin/DNA–AuNPs was fabricated. After hybridization with the PbSNPs-labeled barcode DNA, the assembled sensor was obtained. The concentration of thrombin was monitored based on the concentration of lead ions dissolved through differential pulse anodic stripping voltammetric (DPASV). Under optimum conditions, a detection limit of 6.2 × 10 −15 mol L −1 (M) thrombin was achieved. In addition, the sensor exhibited excellent selectivity against other proteins.

Kinetic and equilibrium studies of copper-dissolved organic matter complexation in water column of the stratified Krka River estuary (Croatia)

An interaction of dissolved natural organic matter (DNOM) with copper ions in the water column of the stratified Krka River estuary (Croatia) was studied. The experimental methodology was based on the differential pulse anodic stripping voltammetric (DPASV) determination of labile copper species by titrating the sample using increments of copper additions uniformly distributed on the logarithmic scale. A classical at-equilibrium approach (determination of copper complexing capacity, CuCC) and a kinetic approach (tracing of equilibrium reconstitution) of copper complexation were considered and compared. A model of discrete distribution of organic ligands forming inert copper complexes was applied. For both approaches, a home-written fitting program was used for the determination of apparent stability constants ( K i equ), total ligands concentration ( L iT ) and association/dissociation rate constants ( k i 1, k i - 1 ). A non-conservative behaviour of dissolved organic matter (DOC) and total copper concentration in a water column was registered. An enhanced biological activity at the freshwater–seawater interface (FSI) triggered an increase of total copper concentration and total ligand concentration in this water layer. The copper complexation in fresh water of Krka River was characterised by one type of binding ligands, while in most of the estuarine and marine samples two classes of ligands were identified. The distribution of apparent stability constants (log K 1 equ : 11.2–13.0, log K 2 equ :8.8–10.0) showed increasing trend towards higher salinities, indicating stronger copper complexation by autochthonous seawater organic matter. Copper complexation parameters (ligand concentrations and apparent stability constants) obtained by at-equilibrium model are in very good accordance with those of kinetic model. Calculated association rate constants ( k 1 1:6.1–20 × 10 3 (M s) − 1 , k 2 1: 1.3–6.3 × 10 3 (M s) − 1 ) indicate that copper complexation by DNOM takes place relatively slowly. The time needed to achieve a new pseudo-equilibrium induced by an increase of copper concentration (which is common for Krka River estuary during summer period due to the nautical traffic), is estimated to be from 2 to 4 h. It is found that in such oligotrophic environment (dissolved organic carbon content under 83 µM C, i.e. 1 mg CL − 1 ) an increase of the total copper concentration above 12 nM could enhance a free copper concentration exceeding the level considered as potentially toxic for microorganisms (10 pM).