565

Application of a Chelite P Modified Carbon Paste Electrode to Copper Analysis and Speciation Raquel Agruz, Javier de Miguel, Maria-Teresa Sevilla* and Lucas Hernundez

Departamento de Quimica Analitica y Analisis Instrumental, Universidad Autonoma de Madrid, 28049 Madrid, Spain

Received August 10, 1995 Final version: October 5 , 1995

Abstract A carbon paste electrode modified with the commercial resin Chelite P, containing amino-phosphonic groups, was used in the determination and speciation of copper. Copper analysis was performed with cyclic voltammetry and differential pulse voltammetry, allowing copper determination in the pg/L level. A detection limit of 1.9pg/L (30) was found for a 10min preconcentration. Interferences from other ions and organic substances were examined. For speciation purposes, a ligand competition methodology was used and different model ligands in solution were tested. Copper complexes were characterized as a function of their thermodynamic and kinetics properties. Results were compared with those obtained employing a strong chelating resin (containing iminodiacetic acid) as electrode modifier. The method was applied to a water sample from the Jarama river in Madrid, where copper was mainly present as strong low-dissociating complexes.

Keywords: Modified carbon paste electrode, Speciation, Chelite P resin, Copper, River water

1. Introduction

Modified carbon paste electrodes have been widely used in trace analysis. Their easy fabrication and good performance have resulted in many practical applications [ 1-31. Determina- tions of pesticides [4], drugs [5] or trace metals [6] using modified electrodes, represent few examples.. Very selective determina- tions are possible due to the specificity of the chosen modifier. Good sensitivity is also attained due to the analyte preconcen- tration occurring at the electrode surface. The nature and the form on which the modifier is incorporated into the carbon paste may be very diverse. Usually the modifier is added in its solid form [7]. Sometimes, the modifier is incorporated by ionic exchange [8] or it is immobilized in a polymeric matrix via covalent binding [9], or even incorporated as a commercial ion- exchange resin [lo]. In our group, most of the work has been carried out by incorporating a metal complexing agent immobilized in a polymeric matrix. Commercial resins were used for this purposes. In trace metal analysis the use of a commercial resin is advantageous because often they have already been extensively studied in terms of selectivity, kinetics of reaction, preconcentration capabilities, etc. This way of electrode modification presents several advantages: the surface of the electrode has good stability and can easily be reproduced. The electrode has long life and the surface can easily be regenerated [ 1 I].

An important advantage found in using modified electrodes is that the electroanalytical measurement is performed in an independent voltammetric cell and the addition of buffer or electrolyte to correct for pH or ionic strength is not necessary. This can be exploited in trace metal speciation since alteration of the natural conditions of the sample by the addition of chemicals is avoided. Only few examples of metal speciation by means of modified electrodes can be found in the literature. The use of modifiers with different complexing capabilities towards the metal ion has been described [12], but no real sample application or data about the effect of ligands in solution are mentioned. Modification of mercury surfaces with lipid layers [ 131 represents a totally different speciation approach. The study of the influence of different competing ligands in the analytical signal of metal-modifier complexes by varying the hydrodynamic conditions [ 14,151 introduces kinetic terms in the

speciation approach but only a limited number of model ligands are checked.

In a previous article [I61 we presented an approximation of these studies by using an iminodiacetate complexing resin as electrode modifier. The approach for speciation studies is performed using the generally called “ligand competition method”. This method is based on the determination of the extent of the ligand exchange reaction between the natural occurring metal complex in solution and a reacting agent with well-known thermodynamic and kinetics properties [ 171. Such method is now frequently used in metal speciation, where the extent of reaction may be followed by several analytical methodologies such as fluorescence [ 181 cathodic stripping voltammetry [ 191, catalytic cathodic stripping voltammetry [20], liquid-liquid extraction [21], etc. The contribution of modified electrodes to these studies is in the sense of easing the isolation-determination of reaction products.

In the present work, we describe the capability of a carbon paste electrode modified with the commercial resin Chelite P, for the determination of copper and the ability of such electrodes to perform metal speciation studies. This amino- phosphonic resin was chosen as modifier to test the capability of these groups for trace metal complexation. This type of functions, including phosphoric, phosphonic, phosphinic and aminophosphonic groups have already been used for metal recovery [22-241. For this purpose, liquid-liquid extraction [25,26] and synthesized resins [27] have been employed. Modified electrodes with quaternary phosphonium resins for anion determination are also found [28]. Phosphonic groups are always present in organisms’ membranes and their capability for trace metal complexation should be considered. We were also interested in testing their ability to compete with other natural ligands for metal complexation. It is already well known that the fate of metals may not only be explained in terms of thermodynamic parameters, but also the kinetics of the reaction play a significant role [29]. In addition, phosphonic groups may offer faster reaction kinetics than other chelating groups [30] and their local concentration in organisms may reach important values. In this work, we try to obtain some conclusions for copper speciation from the comparison of the results obtained with two different modifiers: the chelating agent iminodiacetic acid (see reference

Electroanalysis 1996, 8, No. 6 0 VCH Verlagsgesellschuft mbH. 0-69469 Weinheim, 1996 1040-o397196i0606-565 $ 10.00+.25/0

R. Agraz et al. 566

[ 161 for a detailed description) and the aminophosphonic acid, presented in more detail in this article.

2. Experimental

2.1. Reagents

Ultrapure water obtained with a MilliRO-MilliQ system (Millipore) and analytical-grade reagents were used throughout. Copper stock solution was prepared by dissolving the ultrapure metal in the minimum amount of nitric acid. Chelite P resin (Serva, Heidelberg) was first purified by successive washing with 2N HCI and water and stored in its dry form. Humic acids (Fluka, Switzerland, 600-1000 gimol), were dissolved in the minimum amount of 10p3M KOH until no pH variation was observed and then filtered over a 0.45pm membrane filter. The 0.01 M stock standard solutions of glycine (Gly), thiourea (t-urea) and nitrilotriacetic acids (NTA) were prepared in water. All solutions were stored in polyethylene containers at 4°C in the dark.

2.2. Apparatus

A BAS CV-27 voltammograph equipped with a BAS X-Y recorder was used in cyclic voltammetric studies and an EG&G PAR VersaStat Model 2701250 voltammograph was used in differential pulse voltammetric studies. In both cases, an Ag/ AgCl reference electrode and a platinum counter electrode were used.

The total metal concentrations were measured by flame and graphite furnace atomic absorption spectrometry (AAS) using a Hitachi Zeeman 2-8200 Atomic Absorption Spectrometer.

2.3. Procedure for Characterization of Chelite P Resin

For determination of resin capacity, an exact amount of resin (ca. 0.1 g) in acid form was placed in l0mL solution of 0.1 M NaOH and the suspension was allowed to equilibration until no further pH variation was observed. The resin was then isolated by filtration and the excess of base in the solution was titrated with standard HCI solution and the capacity value was obtained.

The retention of copper on the resin was studied as a function of pH. For this purpose, 0.05 g resin was placed in 20 mL copper solutions containing 10 mg/L copper at different pH values between 1 and 12. The suspensions were allowed to equilibration for 24h. The resin was then isolated from solution and remaining copper concentration in solution was determined by means of flame atomic absorption spectrometry.

2.4. Carbon Paste Electrodes Preparation

Carbon paste was prepared by mixing spectrographic graphite powder and paraffin oil in an agate mortar until an uniform paste was obtained. Modified carbon paste was prepared in the same way but first mixing with the carbon powder the powdered resin in the adequate ratio. Unmodified carbon paste was packed in the end section of a Teflon tube (0.4cm id.) provided with an inner copper contact. Modified carbon paste was placed only at the surface of the electrode (ca. 1 mm deep) to obtain better electrical contact. Appropriate packing of the carbon paste was achieved by pressing the surface electrode against a filter paper. This procedure also helps in discharging the excess of paraffin oil.

2.5. Voltammetric Procedure

The new electrode surface was activated by successive potential sweeps in the measurement electrolyte, followed by a few preconcentration-measurement cycles until a reproducible response was obtained. Preconcentration consisted on immer- sing the electrode in the copper solution in 0.01 M KN03 at neutral pH. During this period, the solution was efficiently stirred and a reaction of copper with active groups at the electrode surface at open circuit was allowed to occur. The electrode was then removed, rinsed with water and placed in the measurement cell containing the supporting electrolyte solution to perform the measurement step. For this purpose, a negative potential was first applied to the electrode and the metal previously accumulated was reduced. A potential sweep towards positive values was then performed, causing metal reoxidation with the subsequent voltammetric signal. Oxidation potential sweep may be completed by cyclic or differential pulse mode. Regeneration of the electrode surface was done by application of 1.00V potential during 2min in the stirred supporting electrolyte. Under these experimental conditions, the electrode can be used during several weeks without further surface renewal.

3. Results and Discussion

3.1. Properties of the Chelite P Resin

From batch studies described above, a resin capacity of 4.9 2~ 0.1 meq/g was obtained. Results for resin/water copper distribution show 95% retention for 3 < pH < 8. These results show the applicability of this resin for trace metal preconcen- tration in natural water samples, where neutral or slightly alkaline pHs are found.

3.2. Voltammetric Measurements

Once checked the ability of Chelite P resin for trace copper recovery, modified carbon paste electrodes were prepared and optimization of analytical and instrumental parameters was done to attain the best analytical signal.

The parameters influencing preconcentration and measure- ment processes were studied. Since the main objective in this work is to perform speciation studies, modification of the water sample is not desirable. Therefore, preconcentration was always done under natural pH and ionic strength of the water sample. Importance of sample composition will be discussed in more detail within the interferences section. Voltammetric measure- ment was performed following medium exchange to a separated voltammetric cell containing the supporting electrolyte. Diverse electrolytes were tested in terms of analytical and background

Table 1 Influence of nature and concentration of measureinent electrolyte on the voltamnietnc signdl Preconcentration in 0 01 M KNO? containing 10Opg/L copper, 20% modifier Measurement by CV with a reduction potential of -0 7 V for 60 5 and 600 mVis scan rate

~~~- ~~~

Peak intensity [ P A ]

Conc-M HCI HC 10, HNO, _ _ _ p ~ _ _ _

0 1 1s 5 1s 0 14 4 0 5 20 5 21 3 I9 2 1 0 22 8 23 0 23 2

~ - _ _

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567 Chelite P Modified CPE for Copper Analysis

14 -r

0 4 I

0 5 10 15 20 25 30 35

Modifier I X

Fig. 1. Influence of the percentage of modifier in the signal-to-noise ratio by means of CV. 2 min preconcentration in a 20 pg/L copper solution. Other parameters as in Table 1.

signals, reproducibility and electrode stability. According to our previous experience, only mineral acids were tested as measure- ment electrolytes because they present lower background currents. Solutions of HCI, HC104 and HN03 acids in concentration ranging from 0.1 to 1.OM were studied. No significant differences (Table 1) were found between the assayed mineral acid solutions. A 1M HN03 solution was chosen for measurements because of its appropriate analytical signal, easy electrode regeneration and low residual current. Modifier percentage was studied by using different electrodes, containing carbon pastes with modifier percentages between 4 and 30%. Different preconcentration-measurement cycles were per- formed for each electrode until a reproducible signal was obtained and optimum response was evaluated in terms of signal-to-noise ratio. Results are shown in Figure 1. As was expected, when the amount of modifier loaded in the electrode increases, the signal increases as well. Nevertheless, when the amount of modifier reaches a certain level, the ratio carbon/ modifier in the electrode is not so favorable for current conduction and the signal-to-noise ratio decreases. In addition, modifier percentages higher than 30% produce physically unstable surfaces. An optimal percentage of 20% was chosen for further measurements.

Instrumental parameters in the measurement step were also studied. Potential and reduction times were first optimized. Reduction potentials between -0.4 and -0.9 V were tested. The signal increases when the reduction potential becomes more negative until a plateau is reached and a -0.7V reduction potential was chosen. Reduction time was varied from 0 to 120 s; again a plateau level is obtained after 60s reduction time, choosing this value for further measurements. The subsequent oxidative potential scan was performed by means of two different voltammetric techniques: cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Results are discussed in terms of analytical response and performance of each technique. In CV, different scan rates were studied, from 50 to 700 mV/s. A linear increment of peak intensity versus scan rate was observed for all the values studied, as it corresponds to the oxidation of a specie deposited at the electrode surface. Background currents increased linearly as well. Nevertheless, signal to noise ratios kept constant in all the scan rates studied and 600mVis scan rate was chosen. Scan rate in DPV was

0.5 V - 0.7 V

Fig. 2. Copper voltammograms obtained with the modified carbon paste electrode, for a 60pg/L copper concentration and 3 min preconcentra- tion time. Other conditions as in Table 1. A) Cyclic voltammetry. B) Differential pulse voltammetry.

studied and again a linear relationship between scan rate and peak intensity was observed. A 50mV/s scan rate was chosen. Pulse amplitude was varied between 20 and l00mV and continuous increase on peak intensity when increasing pulse amplitude was observed, but half-width of the peak also increased significantly for values higher than 80mV. An optimum value of 75mV was chosen. Finally, pulse duration was varied from 20 to 100ms. Signal increased with pulse duration up to 50ms after which no further increase was observed. This value was used for further studies. Always, sharp and well-defined reoxidation peaks at 0.02 V for CV and -0.04 V for DPV were obtained (Fig. 2).

3.3. Calibration and Interferences

Copper calibration was performed in the range 10 to 100 pg/L for different preconcentration times by using both electro- analytical techniques. Results with detection and determination limits are described in Table 2. CV offers better results than

Table 2. Calibration and statistical study for copper determination by means of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Preconcentration in lO-'M KN03 containing copper at neutral pH. 20% modifier; measurement in 1 M HNOl solution with a reduction potential of -0.7 V during 60 s, 600 mV/s scan rate in CV and at 50 mV/s with 75mV pulse amplitude in DPV. DL: Detection Limit; QL: Determination Limit; S: Standard Deviation.

0996 2 CV 3 3 2 11.5 10 1 9 5 9 0998 5

DPV 3 11.0 13 8 0.994 8 10 4 7 1 1 6 0992 8

_ _ _ _ _ _ _ _________ ___- ~-

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DPV in terms of detection limits and reproducibility. This fact may be surprising but it can be explained by considering that detection limits are mainly determined by the preconcentration factor attained at the electrode surface during the accumulation step. In addition, it was observed that the response of the electrode in CV is more stable than in DPV, leading also to better reproducibility. Cyclic voltammetry was subsequently used in speciation studies. Detection limits may be improved by using longer preconcentration times.

Inorganic salts commonly present in natural systems were examined for possible interferences in the following way: Different electrolytes were added to the preconcentration cell and the electrode response towards the same copper concentra- tion was evaluated in terms of intensity, reproducibility and background signal. Solutions of KNO?, KCI, NaN03 and NaCl in 10-3M concentration were first checked. As expected, no significant differences in electrode response were obtained. Nitrate does not interact with copper. However, chloride forms inorganic complexes with copper, but metal ions are easily sequestered from these complexes and preconcentrated at the electrode. These results are in agreement with those reported for the use of a chelating resin as modifier [ 161. For the latter, the thermodynamic formation constant of the modifier for metal ion complexation was higher and a strong competition between the modifier (iminodiacetic acid) and the weaker complexing inorganic anions in solution were expected. Here, phosphonic groups are also capable for strong competition with inorganic anions. Salt concentration studies were done in nitrate, phosphate, bicarbonate and chloride solutions in concen- trations from lop4 to 1 M. Results show that salt concentration higher than 1O-’M cause diminution of the electrode response. This effect is due to the increase of ionic strength in the preconcentration cell and diminution of metal recovery because of the Donnan effect. This fact is generally known when working with ion-exchange resins [31]. Nevertheless, this effect will not cause serious interferences in river water samples analysis because ionic strength is usually low. Cation interference was studied for calcium, magnesium, sodium, potassium, iron and zinc. Calcium and magnesium may also be complexed at the electrode, causing then interference when present in concentra- tion higher than 100mg/L. No interference was observed from sodium or potassium at 100 mg/L concentration level or iron at 4mg/L concentration. Other electroactive cations, such as zinc, lead or cadmium can also be simultaneously incorporated at the electrode surface. Nevertheless, their concentration in fresh water is low and hence not competing with copper for the active groups. Simultaneous reduction-reoxidation at the electrode surface occurs, but peak resolution during the reoxidation sweep is always very good and allows simultaneous determina- tion. Organic interferences will be discussed in detail within the speciation section.

Glycine

3.4. Copper Speciation Studies

Theoretical development and experimental procedure were described in detail previously [ 161 and an operationally defined scheme was developed in order to characterize the lability of natural copper complexes under given experimental conditions. In essence, the methodology consists in the comparison of copper (Mso!) reaction with the modifier (Relec) loaded at the electrode surface in absence (Ms,,l + Relec e+ MRelec) and in the presence (ML,,l+ Relrc * MR,,,, + Ls,l) of competing natural ligand in solution (L5,,J. Subscript “sol” and “elec” mean the specie in solution and at the electrode surface, respectively. During the competing ligand reaction, Re,,, concentration at the

electrode surface is constant and we can define the pseudo-first order reaction rate at the initial step of the reaction as Equations I and 2 respectively for both cases.

d(MReiec) = kO(Msol)O(Relec)O vo = dt

Where vo is the pseudo-first order reaction rate, k, is the pseudo-first order reaction rate constant, superscript “L” means in presence of ligand in solution and subscript “0” means at the initial step of the reaction. Substituting (Me& in Equation 2 for the value obtained from its thermodynamic complexation constant, K = (MLso,)’/(Msol)‘ (Lsol)’, that is, free concentration of species in the equilibrium, and provided (%lec)o constant, we come to the equation:

This equation shows how the modifier-metal reaction rate (Lvo) will proportionally decrease in presence of a natural ligand due to metal complexation. The importance of this change will be determined by thermodynamic and kinetic parameters by means of the thermodynamic complexation constant of the ligdnd in solution ( K ) and the reaction rate constant (Lko) respectively. Ligand concentration in solution [(L,ol)o] will influence the resulting reaction rate as well. Experimentally the extension of the competing reaction (Eq. 2) will be followed by controlling the amount of MRelec formed during the reaction. This can be easily done by voltammetric measurement of the amount of trace metal preconcentrated at the electrode surface for a given time. The values for vo and Lvo will be evaluated by means of the variation of the signal versus time of reaction (or preconcentration time), if uO= d(MR)O/dt = d(signal)/dt. Because the interaction between Msol and Relec occurs in the solid phase, the kinetics are retarded in respect to those occurring in solution and the time-signal relationship will

4.5 Humic acids 4 1

1 6 11

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0- 1 6 11

timelrnin

0- 0 2 4 6

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timelrnin

Fig. 3. Aminophosphonic-modified electrode: influence of natural ligands in solution in the initial reaction rate [d(signal)/dt] of the copper-modifier reaction. Meta1:ligand molar ratios 1:O; 0 1 : I ; + 1 : lO; A 1: lOO.

Electroanalysis 1996, 8, No. 6

Chelite P Modified CPE for Comer Analvsis 569

Table 3. Values of the copper-Egand conditional formation constants in solution in the experimental conditions.

Hurnic acids Glycine Thiourea N T A

log K' 14.7 8.7 15.4 10.4 _______ _ _ _ _ _ _ ~

result in a straight line in the initial step of the reaction in the range between 3 and 15 min. Preconcentration (or reaction) times in this range will be suitable. The relative variation of Lvo vs. vo will allow us to evaluate the magnitude of influence of a ligand in solution in thermodynamic and kinetics terms.

For the experimental performance of the speciation studies, humic acids (HA), thiourea (t-urea), glycine (Gly) and nitrilotriacetic acid (NTA) were used as model compounds of the natural aquatic ligands. Experimentally, different solutions containing a given level of copper and increasing ligand concentrations were prepared in 0.01M KN03 at neutral pH and allowed to equilibrate for 24 h a t room temperature. Copper signal was then recorded for each sample employing different preconcentration times and signal vs. time plots were performed at each ligand (L) concentration in solution. The results are shown in Figure 3. From the extent of decrease on the reaction rate in presence of a certain ligand concentration in solution, that is, from the decrease of the slope (d(signal)/dt) in the straight line I, vs. time (Eqs. 2 and 3), we can estimate the level of interaction between copper and the ligand in solution. As it can be seen in the figure, Gly causes almost no interferences, even when present in excess. T-urea causes small interferences also HA. Nevertheless, different behavior is noticed in these two last cases. While t-urea causes an erratic response keeping always the same degree of interaction, humic substances produce a well-characterized increasing effect when the con- centration increases. Finally, NTA causes high interference even when present in low concentration. If we compare these results

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Glycine

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Fig. 4. Iminodiacetate-modified electrode: influence of natural ligands in solution in the initial reaction rate [d(signal)/dt] of the copper-modifier reaction. Meta1:ligand molar ratios 1:O; 0 1:lO; + 1:100; A 1:lOOO and x 1:10000.

with those we could expect from the values of the thermo- dynamic formation constants of copper complexes in solution in the experimental conditions, shown in Table 3, we don't observe a good correlation between them. That is, from the thermo- dynamic point of view we would expect the following interference series: Gly < NTA < HA = t-urea. However, experi- mentally the series Gly < t-urea < HA < NTA is found. This may be explained by considering that Gly and t-urea are small ligands that, although carrying donors atoms capable for copper complexation, dissociate fast in presence of the modifier at the electrode surface, where its concentration is locally higher (molar level). In HA, more complex structures are attained in solution and slower dissociation kinetics have been described. Finally, NTA forms very stable three-dimensional chelates from copper and is not easily liberated. Competition of these last two compounds with the modifier for copper complexation is stronger.

If we compare these results with those obtained in the same conditions, but in presence of a chelating iminodiacetate group as electrode modifier (Fig. 4), we observe a few different behaviors. First, Gly and HA show stronger influence in the chelating iminodiacetate agent. This fact is surprising if we consider that this complexing agent has higher affinity for copper ions than the phosphonic one and, in principle, stronger competence in equal conditions could be expected. Nevertheless, iminodiacetate resin posses slower kinetics of reaction because chelate formation within the resin matrix is the rate determining step of the reaction [32], and may also explain these results. T- urea, however, doesn't show this behavior, showing that copper is easily displaced. NTA shows in both cases strong copper association, as corresponds to its high copper affinity. Imino- diacetate modified electrode shows then slower reaction rates than the phosphonic modified one. Therefore, the presence of a certain ligand in solution may affect it more easily. Phosphonic modified electrode, although possessing lower affinity for copper, can compete more efficiently with other copper complexes in solution with medium stability. Further considera- tions about the biodisponibility of every specie can be extrapolated as a function of their capabilities for reaction against the test reagent. Complexes having fast reaction rate, even when presenting low thermodynamic equilibrium constant, will show strong interaction with competing groups presently in the membrane of the organisms. The same can be applied from the point of view of the competing ligand ,which even when its affinity for metal ions may not be very high, fast reaction rates and high local concentrations may cause strong competition for metal complexation.

3.5. Applications

The methodology described above was tested for the speciation of copper in a natural water sample from the Jarama river (Algete, Madrid). The sample was taken and immediately filtered over a 0.45 pm filter. An aliquot for ion's analysis was adjusted to pH2 with nitric acid and stored in a polyethylene bottle (at 4" in the dark). Another aliquot was stored in the same conditions in a glass bottle for organic compound analysis. A third aliquot for speciation analysis was stored in a polyethylene container without pH modification and analyzed within 24h. In this sample, the total copper concentration was 3 pg/L, as measured by graphite furnace AAS. The speciation method was applied to the filtered non- treated sample. Not quantifiable response was obtained even for 15 min preconcentration time, showing that the whole copper amount present in the sample was present as strong

Elrctroanalysis 1996, H , No 6

R. Agraz et al. 570

2 4 6 8 1 0

preconcentration time I min

Fig. 5. Variation of the initial reaction rate for the reaction aminopho- sphonic modified electrode for a spiked river water sample.: (m) standard copper solution, spiked (20 pg/l) river water without (A) equilibration time and (0) after 24h equilibration time.

non-dissociating compounds. This fact is not surprising if we consider the high content of dissolved organic matter (3.5 mg/L) and dissolved iron(rn) (10.05 pg/L), probably in colloidal form and with high affinity for trace metals. To test the applicability of the method, untreated samples were spiked to a total copper concentration of 20 pg/L and copper analysis method was applied after a few minutes and 24 hours equilibration time. The results are shown in Figure 5. Strong evidence of the importance of the kinetics of the processes is then obtained. When the method is applied after few minutes of equilibration, copper recovery is higher, while after 24 hours copper is present in form of complexes with slower rate of dissociation and recovery is more inefficient. In the first case, 4.0 pg/L copper were recovered and only 1.4 pg/L (7%) copper was recovered after 24 h equilibration. Copper recovery was very small even when using only a few minutes equilibration time and showing again the primary presence of strong low- dissociating compounds.

These results agree with those found in the literature for speciation of copper, using different analytical techniques, in fresh waters where the percentage of organically bound is usually high 133,341.

4. Acknowledgements

To Comunidad Aut6noma of Madrid (project C-09091) and Ministry of Education and Science of Spain (grant to R. Agraz) and DGlCyT Grant PB94-0178 for financial support.

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