Analytica Chimica Acta 518 (2004) Received 12 February 2004; received in revised form 7 May 2004; accepted 7 May 2004

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1 Analytica Chimica Acta 518 (2004) Challenges in modelling and optimisation of stability constants in the study of metal complexes with monoprotonated ligands Part III. A glass electrode potentiometric and polarographic study of Cu DIPSO OH system Carina M.M. Machado a, Stefanie Scheerlinck a,b, Ignacy Cukrowski c, Helena M.V.M. Soares a, a REQUIMTE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, Porto, Portugal b KaHo St.-Lieven, Industrial Engineering, Department of Biochemistry Microbiology, Gebroeders Desmetstraat 1, B-9000 Gent, Belgium c Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, P.O. Wits, Johannesburg 2050, South Africa Received 12 February 2004; received in revised form 7 May 2004; accepted 7 May 2004 Abstract The influence of 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO) on solutions containing copper(ii) was studied by two analytical techniques, glass electrode potentiometry (GEP) and direct current polarography (DCP). The readings were taken at fixed total DIPSO to total copper(ii) concentration ratios and various ph values, at 25.0 ± 0.1 C and ionic strength 0.1 M KNO 3. Combined interpretation of data from DCP and GEP indicated the formation of six main species, CuL +, CuL(OH), CuL(OH) 2, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2 2 for which stability constants (as log β) were found to be 4.2 ± 0.2, 11.8 ± 0.2, 17.6 ± 0.1, 8.1 ± 0.1, 14.5 ± 0.2 and 20.6 ± 0.2, respectively. These results warn users of DIPSO that this buffer should not be used for studies involving this metal ion, unless the extent of complexation is accounted for. In addition, the magnitude of stability constants of the species present in the buffer ph range suggests that DIPSO can be used simultaneously as ph and metal ion buffer Elsevier B.V. All rights reserved. Keywords: DIPSO; 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid; Biological buffers; Copper; Stability constants; Electrochemical techniques 1. Introduction Good et al. synthesised more than 20 buffers [1,2], which were originally used in biological/biochemical studies and more recently in environmental speciation studies. In generating these buffers, among several properties, the prevention of metal complexation was one of the desired goals. Nowadays, some of these buffers are presently used routinely in biochemical and speciation studies under the assumption that they undergo little, or if any, interaction with biologically/environmentally important ions. However, several reports have described interferences when some of these buffers, were used in the presence of metal ions; one of these studies described the interference of low concentrations of various Good buffers commonly used in protein determina- Corresponding author. Tel.: ; fax: address: hsoares@fe.up.pt (H.M.V.M. Soares). tion and pointed out that this interference was probably due to chelation of copper [3]. The knowledge of the stability of metal (ph buffer) complexes is of fundamental significance when modelling of natural waters, biological fluids, industrial processes or ligand design strategies are of interest. Therefore, since 1997 a project, whose central aim is to study the interaction of some Good buffers with metal ions and their suitability for metal speciation and biological/biochemical studies involving metal ions, has been carried out in our laboratory. The project aim is to obtain a systematic information on which pairs (Good buffer) (metal ion) do not exhibit complexation and can, therefore, be more adequate for speciation and biological/biochemical studies involving metal ions, as well as to determine and quantify the overall stability constants of the metal buffer systems where complexation occurs. Up till now, several pairs of metal ions buffers have been already studied and important conclusions were drawn. For example, we demonstrated experimentally that HEPPS, /$ see front matter 2004 Elsevier B.V. All rights reserved. doi: /j.aca

2 118 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) HEPES, HEPPSO, MES, MOPS, and PIPES do not complex cadmium or zinc [4 7]. Moreover, MES, MOPS and MOPSO do not complex neither copper nor lead [4,5]. On the other hand, slight complexation was verified between lead with PIPES and HEPES [6] and more pronounced with HEPPS [7]. AMPSO and TAPSO revealed to be strong copper complexing agents [8,9]. In this work, we studied the interaction between 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO) and copper, by two electrochemical techniques, Glass electrode potentiometry (GEP) and direct current polarography (DCP), at fixed total ligand to total metal concentration ratios, [L T ]:[M T ], and various ph values, at 25.0 ± 0.1 C and ionic strength 0.1 M KNO 3. Experiments by alternating current polarography (ACP) were also carried out to check if the buffer adsorbs at the mercury electrode surface under the experimental conditions used. 2. Experimental 2.1. Materials and reagents The solutions were prepared with deionised water with resistivity larger than 14 M cm 1. The solid potassium hydroxide was obtained from Merck (Darmstadt, Germany). A stock solution of about 0.1 M KOH, prepared by dissolving solid KOH, was standardised weekly against potassium hydrogen phthalate by potentiometric titration. The equivalence point was determined using the Gran plot method [10]. This method also allowed to verify if the strong base solution was contaminated with carbonate. A stock solution of about 0.1 M nitric acid (prepared from a concentrated acid (Merck)) was standardised potentiometrically against the standardised potassium hydroxide solution. The KNO 3 was supplied by Merck and was used as a background electrolyte to adjust the ionic strength (0.1 M KNO 3 ) of all solutions. The ligand DIPSO (99.1%) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. A standard solution of copper ( M), purchased from Merck, was used. High purity nitrogen was used for deaeration of the sample solutions, i.e. to remove the dissolved oxygen from the solutions. All glassware was cleaned by soaking in 20% (v/v) HNO 3 during 1 day and then rinsed several times with deionised water Apparatus All experiments were carried out in a Metrohm (Herisau, Switzerland) jacketed glass vessel, equipped with a magnetic stirrer, and thermostatted at 25.0±0.1 C using a water bath Polarography Polarographic measurements were performed using a Model 663 VA stand (Metrohm) equipped with a multimode electrode (Metrohm, Model ) as a working electrode, used in the dropping mercury electrode mode. A silver/silver chloride (3 M KCl) and a glassy carbon were used as reference and counter electrodes, respectively (both from Metrohm). The VA stand was attached to a microautolab (Eco Chemie, Utrecht, The Netherlands) system controlled by a personal computer. The ph measurements were conducted with a GLP 22 ph meter Crison (Barcelona, Spain) with a sensitivity of ±0.1 mv (±0.001 ph units), with a silver/silver chloride reference electrode (Metrohm) and a glass electrode (Metrohm) Potentiometry The potentiometric titrations were performed with a PC-controlled system assembled with a Crison MicropH 2002 meter, a Crison MicroBU 2030 micro-burette, a Philips GAH 110 glass electrode and an Orion (double junction) reference electrode with the outer chamber filled with 0.1 M KNO 3. Automatic acquisition of data was done using a home-made program, COPOTISY Procedure Calibration of the glass electrode For all techniques used, the calibration of the glass electrode (ph measured as log[h + ]) was accomplished by addition of the standardised solution of potassium hydroxide to the standardised solution of nitric acid (both solutions adjusted to ionic strength of 0.1 M). From this potentiometric titration, the E and the response slope were established by fitting a straight line in the experimental points collected between ph 2 and Glass electrode potentiometry Glass electrode potentiometry was firstly used to determine the protonation constants of the buffer at 25.0±0.1 C and an ionic strength of 0.1 M KNO 3. Several titrations were performed using different DIPSO solutions containing (3 titrations), (6 titrations using DIPSO from two different lots) or M (3 titrations) DIPSO in 0.1 M KNO 3 adjusted to ph 2.5. These solutions were titrated between ph 2.5 and 10.0 using standardised 0.1 M KOH. Potentiometric titrations of Cu DIPSO OH system were done on several solutions of different ligand to metal concentration ratios, [L T ]:[M T ]. Four titrations, between ph 3.5 and 9.0 (about 50 points were collected for each titration), were carried out with a [L T ]:[M T ] ratio set to 2.9, [Cu 2+ ] = M. With [L T ]:[M T ] ratio set to 4.6 ([Cu 2+ ] = M), four titrations were performed, between ph 3.0 and 11.0, and about 250 points for each titration were collected. For [L T ]:[M T ] ratio 7.0 ([Cu 2+ ] = M), two titrations were carried out, between ph 2.5 and 10.5 and about 70 points were obtained for each titration. Monotonic titrant additions of standardised 0.1 M KOH were used.

3 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) Alternating current polarography (ACP) ph buffer adsorption studies were performed by ACP using an amplitude of sinusoidal alternating current of 5 mv, a frequency of 75 Hz, and a scan rate of 2.5 mv/s; the capacitive current (i ac ) was measured at a phase angle of 90. This study was done for buffer concentrations in a range from to M and at three different ph values: 5.5, 7.5 and 9.5. These ph values were chosen because they correspond to experimental conditions where DIPSO occurs as HL (100%), HL and L (50% of each) and L (100%), respectively. The potential range scanned in the experiments was from 0.2 to 1.2 V versus Ag/AgCl (s), 3 M KCl Direct current polarography The study of the electrochemical behaviour of Cu DIPSO OH system was performed by direct current polarography. A drop time of 1 s and a step potential of 4 mv were used. To make sure that the vessel was not contaminated, a first scan was carried out on a deaerated background solution, ml of 0.1 M of KNO 3. Then, a required amount of copper stock solution was added and another polarogram was recorded after the solution has been deaerated. Two [L T ]:[M T ] ratios were used. The total copper concentration was and M for [L T ]:[M T ] of 100 and 157, respectively. For each independent titration, a required amount of solid ligand was added to the vessel containing the metal ion solution. After deaeration, next polarogram was recorded. With additions of standardised base solution, the ph was varied in ph steps of 0.1 units between ph 3.0 and After each KOH addition, a new polarogram was recorded; in this way, about polarograms were obtained. The equilibrium of the metal ligand solutions was tested and it was reached in a few minutes Data treatment During the Cu DIPSO OH system refinement operations, the K w [11], protonation constant for the ligand DIPSO as well as formation constants for the copper hydroxo species [11] were kept at fixed values (Table 1). The simulation and optimisation of model and refinement of complex formation constants from potentiometric titration data were performed using ESTA program [12,13]; this optimisation was carried out with respect to the objective function using several tasks of optimisation modules. The objective function is related with the number of experimental points, the number of optimised parameters and the sum of squares of the difference between the potential observed and the calculated one. The prediction of the species present in solution was done with Z M plot, whose meaning has been described elsewhere [14]. The refinement of polarographic data was done using the Cukrowski method [15]; this method uses mass balance equations written for [M T ] and [L T ] incorporating all known homogeneous equilibria. These equilibria can be studied at any experimental conditions; in this work data were collected at a fixed [L T ]:[M T ] ratio and varied ph. The model and stability constants were refined by comparing experimental and calculated complex formation curves (ECFC and CCFC, respectively) for the labile (on the polarographic time scale) metal ligand systems. 3. Results and discussion 3.1. Determination of DIPSO protonation constant DIPSO is a zwitterionic buffer, which has two acidic sites, the sulphonic (pk a1 ) and the amino (pk a2 ) groups. After searching the DIPSO s pk a2 values in the literature, it was verified that the published values are very random, vary between 7.45 and 7.63 [16] for the same experimental conditions (25 C and 0.1 M KNO 3 ). This fact can influence the final values of Cu DIPSO OH stability constants, depending on each pk a2 value, we use. So, we decided to determine these values. For this purpose, several solutions were prepared with DIPSO from two different lots, as it was described above. It was not possible to determine the pk a1 value because this value was very low. However, it was tested that at ph values higher than 3.0, the deprotonation coming from sul- Table 1 Protonation constants for water, DIPSO and overall stability constants for [Cu x (OH) y ] (2x y) complexes, at 25 C Equilibrium Log β µ Reference Water H + + OH H 2 O [11] DIPSO L + H + HL 7.47 ± This work Copper Cu 2+ + OH Cu(OH) [11] 2Cu 2+ + OH Cu 2 (OH) [11] Cu OH Cu(OH) [11] Cu OH Cu(OH) [11] Cu OH Cu(OH) [11] 2Cu OH Cu 2 (OH) [11] 3Cu OH Cu 3 (OH) [11] Cu(OH) 2 (s) Cu OH [11]

4 120 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) phonic group did not influence any more the results. The refinement of DIPSO s pk a2 was performed using points collected between ph 7.0 and 8.0 (about 60 points for the titrations with lower DIPSO concentration and about 130 points for the other titrations). The pk a2 value obtained from 10 titrations was 7.47 ± 0.05 (Table 1). Note that the standard deviation represents the variation of pk a2 in all the titrations. The obtained value is within the values described in the literature, but with a much lower standard deviation DIPSO adsorption studies Adsorption processes can be defined as the accumulation at the interface of a substance present in the bulk of the solution, in such a way that the surface concentration is in excess of the bulk concentration. Uncharged surfactants have a maximum adsorption in the region of zero charge potential (about 0.4 V), i.e. the region where the surface of the mercury electrode has no charge, and the amount of adsorption decreases at potentials both more positive and negative [17]. In this work, the adsorption studies were carried out at three ph values (5.5, 7.5 and 9.5) for evaluating the extent of adsorption of the different ligand forms. At ph 5.5 and 9.5, DIPSO exists totally in the forms HL, and L, respectively, and at ph 7.5, 50% of HL and L forms coexist. To evaluate if DIPSO adsorbs at the mercury electrode, curves of alternating current versus applied potential [i ac = f(e)] were recorded between 0.2 and 1.0 V with a phase angle of 90. Under these experimental conditions, because non-faradaic process is expected, the i ac is mainly capacitive in nature and proportional to the double layer capacity. Fig. 1 shows typical example obtained with increasing concentration of DIPSO at ph 5.5 (Fig. 1A) and 9.5 (Fig. 1B). This figure evidences an insignificant decrease of i ac current at ph 5.5 and a slightly diminution at ph 9.5, in the potential range between 0.2 and 0.6 V. This was due to a decrease of the double layer capacity as a consequence of DIPSO adsorption. These results show that DIPSO adsorbs more strongly in the form of L than HL. The comparison of results obtained with DIPSO (Fig. 1) with those obtained with TAPSO [8] shows that DIPSO adsorbs less than TAPSO, under the same experimental conditions. Fig. 1. Alternating current polarograms at different DIPSO concentrations in 0.1 M KNO 3. (A) ph = 5.5, (B) ph = 9.5; a, b, c, d, e, and f stand for 0.1 M KNO 3, [DIPSO] = , , , , M, respectively.

5 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) Glass electrode potentiometry Three different [L T ]:[M T ] ratios were performed to analyse the Cu DIPSO OH system and to establish the correct model. The modelling of the system and the refinement of the stability constants were done using ESTA program [12]. Final results are summarised in Table 2. For [L T ]:[M T ] ratio 2.9, we only used points collected from ph 3.5 to 6.0 due to the occurrence of precipitation. The fitting of the two simple models (I and II) generated equivalent statistical parameters of the overall fitting, indicating that both models can probably represent the system at this ratio. However, the inclusion of CuL 2 in the model as a major metal containing species is not supported by the graphical analysis of Z-bar function (Fig. 2A), which suggests the formation of mainly CuL and CuL(OH) x species. The analysis of experimental points for [L T ]:[M T ] ratio 4.6 (Fig. 2A) shows that the Z M function grows until 2 without back fanning [14]; this clearly indicates the formation of CuL and CuL 2. Hence for this ratio and higher ones we decided to include CuL 2 in all the models. For [L T ]:[M T ] ratio 4.6, when we try to refine models I and II, ESTA prefers the model containing CuL, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2. However, neither model I nor model II fits the results satisfactory (Fig. 2A), particularly for larger values of Z M. The results obtained with [L T ]:[M T ] ratio 7.0 corroborate those obtained for [L T ]:[M T ] ratio 4.6 (models I and II). However, the values for stability constants of CuL 2 (OH) and CuL 2 (OH) 2 were not reliable since they were correlated. This was probably the reason why the log β of CuL 2 (OH) 2 was very different from the ones obtained from the others ratios. For all [L T ]:[M T ] ratios, the simultaneous refinement of stability constants with selected analytical parameters, like [L], [H + ], [OH ] and the response slope of the glass electrode, did not improve the results (data not shown). Fig. 2. Results from refinement and optimisation operations obtained from GEP (A) and DCP (B) experiments. (A) Observed (points) and calculated (lines) Z M functions obtained from the refinement operations performed on the GEP data. For [L T ]:[M T ] = 2.9, [Cu 2+ ] = M; for [L T ]:[M T ] = 4.6, [Cu 2+ ] = M. The models indicated in the figure correspond to the models described in Table 2. For the model containing CuL, CuL(OH), CuL(OH) 2, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2, the stability constants were 4.2, 11.8, 17.6, 8.1, 14.5 and 20.6, respectively. (B) Experimental (points) and calculated (lines) complex formation curves obtained for the final model: CuL, CuL(OH), CuL(OH) 2, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2.[L T ]:[M T ] = 157, [Cu 2+ ] = M. Dashed line represents the calculated complex formation curve for the system in the absence of complexation with the ligand. For sake of simplicity, charges were omitted Complexation studies of the Cu DIPSO OH system Direct current polarography As it was already described above, two titrations were performed by DCP, corresponding to [L T ]:[M T ] of 100 and 157. Similar results were obtained from both titrations. Next, we will discuss the results obtained from [L T ]:[M T ] ratio of 157. After the addition of the ligand, the DC wave shifted about 1 mv towards anodic potential values, which is within the experimental error of the technique. As with the increase of ph (up to 4.5) no cathodic (or anodic) shift was verified, which shows that no complexation occurred; we decided to use the potential of the metal in the presence of the ligand as the reference potential for the refinement operations. Analysis of DC polarograms shows a decrease in the recorded current (Fig. 3) for ph higher than 5.0 due to the complexation. In addition, the shape of the waves varied significantly (the gamma coefficient varied between 0.9 initially and 0.7 at higher ph). These values indicated that the behaviour of the system, besides the influence of adsorption, could not be regarded as fully reversible. The refinement of polarographic data [15] employed in this work requires that the recorded curves must represent fully reversible electrochemical process if accuracy in obtained stability constants is of interest. Therefore, it was decided to use a simple procedure for correcting the departure from the reversibility of the DCP data reported previously [9]. This procedure corrects the effect of the quasi-reversibility by fixing the gamma coefficient (this parameter indicates the steepness of the waves) at value of 1 and computes the corrected limiting diffusion current and potential of half height, E 1/2 [8,9]. The analysis of polarograms, recorded in the ph range between 3.0 and 10.5, showed that Cu DIPSO OH system behaved as labile on the polarographic time scale as only one DCP wave was observed, which shifted towards more cathodic potential with the increase of ph (Fig. 3). Next, we predicted the species present in solution by the graphic analysis of the DCP data (Fig. 4). According to the analysis of E 1/2 versus ph (Fig. 4A), four regions could be

6 122 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) Table 2 Refinement of the stability constants (as log β) by ESTA for [L T ]:[M T ] ratios: 2.9, about 46 points between ph 3.5 and 6.0; 4.6, about 678 points between ph 3.0 and 11.0; 7.0, about 153 points from ph 2.5 to 10.5 [L T ]:[M T ] Model CuL (Cu + L CuL) CuL(OH) (Cu + L + OH CuL(OH)) CuL(OH) 2 (Cu + L + 2(OH) CuL(OH) 2 ) CuL 2 (Cu + 2L CuL 2 ) CuL 2 (OH) (Cu + 2L + OH CuL 2 (OH)) CuL 2 (OH) 2 (Cu + 2L + 2(OH) CuL 2 (OH) 2 ) 2.9 I 4.04 ± 0.02 NI NI 8.56 ± ± 0.03 R II 4.06 ± ± 0.01 R NI NI NI I 3.56 ± 0.07 NI NI 8.11 ± ± ± II 3.46 ± ± ± 0.03 R NI NI III 3.55 ± 0.07 R ± ± ± 0.03 R IV 3.70 ± (F) ± ± ± 0.03 NI V 3.69 ± (F) 16.4 ± ± ± (F) I a 4.12 ± 0.04 NI NI 8.02 ± ± ± II 4.09 ± ± ± 0.05 R NI NI III a 4.13 ± (F) NI 7.8 ± ± ± IV a 4.13 ± (F) 16.3 (F) 7.8 ± ± ± For all titrations, copper concentration was about M. NI, not included; R, rejected; F, fixed value. a CuL 2 (OH) and CuL 2 (OH) 2 correlated. R-factor distinguished; three in the ph-range where ligand exists in its protonated form and one region at higher ph values where ligand is mainly in its deprotonated form. In the first region, which corresponds to ph value up to =4.5, no cathodic shift in E 1/2 was observed, which evidences that no complexation occurred in this range of ph. For the other three regions, different slopes were recorded for which characteristic electrochemical reactions at the DME electrode are illustrated in Fig. 4. For the second and third region, both recorded in a ph range where the ligand is in HL form, slopes of about 28 and 51.5 mv per ph unit were determined. The first slope indicates the involvement of one proton and suggests that CuL and/or Cu(OH) species is (are) present in solution. The latter species, however, is not expected to be formed at any significant concentration level up to ph 6.2. In the third region, if two protons were involved in the electrochemical reaction at the DME, the expected slope should be about 60 mv per ph unit, indicating that CuL 2, CuL(OH) and/or Cu(OH) 2 complexes would predominate. However, the ex- perimental slope was smaller, 51.5 mv/ph, which means that a mixture of complexes, including CuL and one or several of the complexes described above, must be present. In principle, reduction of Cu(OH) 2 should be excluded as a major species since the polarographic signal shifted up to ph 10 and no precipitation was observed, which suggests that the ligand must be involved in the complex formation reaction with copper. Above ph 7.5, the ligand is mainly in the deprotonated form, L. In this region, the shift in E 1/2 continuous to increase and a slope of 59 mv/ph was observed, indicating the formation of CuL x (OH) 2 species that exist in a wide ph-range between 7.5 and 10. In addition, the analysis of E 1/2 versus log[l] (Fig. 4B) clearly supports the presence of CuL (slope of 28 mv/ph) and CuL 2 (slope of 59 mv/ph), as major species in the ph range where they exist. Optimisation of the Cu DIPSO OH model and refinement of the stability constants was performed using the experimental (points) and calculated (lines) complex formation Fig. 3. Experimental DC polarograms obtained from the acid base titration. [Cu 2+ ] = M, [L T ]:[M T ] = 157. The observed decrease in the limiting current is due to the formation of complexes species in solution throughout the experiment.

7 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) Fig. 4. Interpretation of the DCP experimental data involving the corresponding slopes and electrochemical reactions at the DME electrode surface for [L T ]:[M T ] = 157, [Cu 2+ ] = M. Variation in the half-wave potential, E 1/2, with the ph (A) and log [L] (B). curves (Fig. 2B) [18]. During the refinement process, it was possible to fit several M L OH models (Table 3), following a similar pattern as those recorded for [L T ]:[M T ] ratios 4.6 and Combination of the results from GEP and DCP techniques The analysis of all results obtained by GEP and DCP showed that both techniques, when used independently, were not able to provide a conclusive answer related with the most likely model. However, a more detailed analysis of the re- sults evidences that for lower ratios, e.g. [L T ]:[M T ] ratio of 2.9, model CuL, CuL(OH) and CuL(OH) 2 seems to describe better the M L OH system whereas for higher ratios, the model CuL, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2 does it. This suggests that the final model should include all the complexes, CuL, CuL(OH), CuL(OH) 2, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2. Including all the seven species in the model, ESTA program rejected CuL(OH) and CuL 2 (OH) 2 complexes ([L T ]:[M T ] ratio 4.6, model III in Table 2). However, it is important to note that CuL 2 and CuL(OH) exist in the Table 3 Compilation of the DCP results Equilibrium Model I Model II CuL Cu + L CuL 4.50 ± ± 0.02 CuL(OH) Cu + L + OH CuL(OH) NI R CuL(OH) 2 Cu + L + 2(OH) CuL(OH) 2 NI ± 0.01 CuL 2 Cu + 2L CuL ± ± 0.02 CuL 2 (OH) Cu + 2L + OH CuL 2 (OH) ± 0.06 NI CuL 2 (OH) 2 Cu + 2L + 2(OH) CuL 2 (OH) ± 0.01 NI S.D [L T ]:[M T ] = 157, [Cu 2+ ] = M. About 60 points were collected between ph 3.1 and 9.9. Stability constants as log β values. NI, not included; R, rejected.

8 124 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) same ph range, where HL is the major form of the ligand. These complexes involve the same number of protons when reduced at the working electrode (for sake of simplicity charges were omitted): Cu + 2HL CuL 2 + 2H Cu + HL + H 2 O CuL(OH) + 2H This fact makes difficult for GEP to distinguish between these two complexes; then, ESTA converges for either in a random way. The complexes CuL(OH) 2 and CuL 2 (OH) 2 exist at higher ph where the ligand is fully deprotonated: Cu + L + 2H 2 O CuL(OH) 2 + 2H Cu + 2L + 2H 2 O CuL 2 (OH) 2 + 2H Fig. 5. Species distribution diagrams computed for the final model that included all known Cu x (OH) y species (see Table 1) and CuL (1), CuL(OH) (2), CuL(OH) 2 (3), CuL 2 (4), CuL 2 (OH) (5), and CuL 2 (OH) 2 (6) for which stability constants, as log β, were 4.2, 11.8, 17.6, 8.1, 14.5, and 20.6, respectively. (A) [L T ]:[M T ] = 2.9, [Cu 2+ ] = M; (B) [L T ]:[M T ] = 4.6, [Cu 2+ ] = M; (C) [L T ]:[M T ] = 157, [Cu 2+ ] = M.

9 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) Table 4 Comparison between basicity and complex stability for some biological buffers Ligand NH3 AMPSO TAPS TRIS Triethanolamine DIPSO TAPSO BISTRIS Structure NH3 pka Log βml pkm/hl Reference [11] [9] [20] [11] [11] This work [8] [11] For comparison purpose, the values for NH3 and triethanolamine are included. For calculation of pkm/hl see more details in the text. In this case, neither GEP nor DCP are able to distinguish between CuL(OH) 2 and CuL 2 (OH) 2. The inclusion of some complexes with fixed stability constants values did not improve the overall statistical parameter ([L T ]:[M T ] ratio 4.6, models IV and V and [L T ]:[M T ] ratio 7.0, models III and IV, Table 2). It became obvious that a special strategy had to be developed to propose the final values of stability constants. For this purpose, a graphic iterative process, using the Z M function for GEP data (Fig. 2A) and the CCFC/ECFC for DCP data (Fig. 2B), as well as the simultaneous analysis of the species distribution diagrams, was used for all [L T ]:[M T ] ratios. The final values for the stability constants (as log β) are: CuL = 4.2 ± 0.2, CuL(OH) = 11.8 ± 0.2, CuL(OH) 2 = 17.6 ± 0.1, CuL 2 = 8.1 ± 0.1, CuL 2 (OH) = 14.5 ± 0.2 and CuL 2 (OH) 2 = 20.6 ± 0.2. To test the proposed model, species distribution diagrams (SDD) were generated for some [L T ]:[M T ] ratios used at GEP and DCP experimental conditions (Fig. 5). Looking at Fig. 5A, this figure predicts that, at [L T ]:[M T ] ratio 2.9, CuL and CuL(OH) are the major metal containing species present in solution prior the precipitation to occur at about ph 7.0. The GEP experiment, at this [L T ]:[M T ] ratio, confirmed the occurrence of precipitation around this ph value. A significant increase in the concentration of CuL 2 and CuL 2 (OH) 2 is predicted in Fig. 5B for [L T ]:[M T ] ratio of 4.6 and these species together with CuL and CuL 2 (OH) were refined without problem ([L T ]:[M T ] ratio 4.6, model I in Table 2). In addition, CuL, CuL 2, CuL 2 (OH) and CuL 2 (OH) 2 are predicted as major species at [L T ]:[M T ] ratio of 157 (Fig. 5C) and this was exactly established from the DCP experiments. The comparison between log β for CuL(OH) (11.8 ± 0.2) and CuL(OH) 2 (17.6 ± 0.1) with the theoretical ones [CuL(OH) = CuL + Cu(OH) = = 10.3; CuL(OH) 2 = CuL + Cu(OH) 2 = = 16.0] shows that the first ones are significantly larger than the computed value of 10.3 and On the other hand, log β values for CuL 2 (OH) (14.5 ± 0.2) and CuL 2 (OH) 2 (20.6 ± 0.2) are closer to the expected ones [CuL 2 (OH) = CuL 2 + Cu(OH) = = 14.2; CuL 2 (OH) 2 = CuL 2 + Cu(OH) 2 = = 19.9]. A similar occurrence was also detected with other two ligands, TAPSO [8] and AMPSO [9], which suggests that the structure of the complexes formed with the three buffers should be similar. This fact can be related with different mechanisms of CuL n hydrolyses; the lost of the proton can be from the coordinated water molecules or the bond is formed between the central metal ion and one of the OH groups of the ligand; in the later case the stability constant would increase above the theoretical predicted value [19]. It is not possible from the potentiometric or polarographic data to deduce if this is a hydroxy complex, CuL(OH), or a complex where DIPSO acts as a chelate, CuLH 1. If the deprotonation of the coordinated water is the mechanism present, these results suggest that deprotonation does change to some extent the geometry of CuL complex but not of the CuL 2 complex.

10 126 C.M.M. Machado et al. / Analytica Chimica Acta 518 (2004) The comparison of the stability constants for CuL complex with various biological buffers (DIPSO, TAPSO [8], AMPSO [9], TAPS [20], triethanolamine [11], TRIS [11], BISTRIS [11] and ammonia [11] (Table 4)) shows the reinforcement of the complexing ability of the ethanolamines with respect to ammonia due to most likely a chelate effect. The fact that the corresponding stability constants of CuL (where L can be each one of the biological ligands defined above) and (CuNH 3 ) 2+ are about the same, even though the basicity of all biological ligands is smaller than NH 3 (Table 4), is a clear indication that at least one hydroxo group of each ligand should be involved in the formation of the bond with the central metal ion. If the stability of CuL complexes were only determined by the basicity of the amino group, their stability should be considered below the stability of the ammonia complex. It is evident that the hydroxo groups of the biological buffers participate in complex formation, probably involving different number of hydroxo groups depending on the ligand. To account for the different basicity of these ligands, pk M/HL, defined as the difference between the pk a of the ligand and the log β ML, was calculated for all the systems (Table 4) according to the following equilibrium: Cu + HL CuL + H, where K Cu/HL = [CuL][H] [Cu][HL] The analysis of the pk M/HL data shows the following pattern: NH 3 > AMPSO > TAPS > TRIS > triethanolamine > DIPSO > TAPSO > BISTRIS. These results suggest an increasing coordination tendency with the number of hydroxo groups. Table 4 also evidences that the coordination tendency does not correlate with the basicity of the ligand and the type of amine. Once again, the importance of using [L T ]:[M T ] ratios of various orders of magnitude in order to characterise properly the model was demonstrated. The combination of both techniques (DCP and GEP) allowed us to vary the concentration of the total metal ion concentration between about 10 5 and 10 3 M, depending on the technique used (DCP or GEP, respectively). The highest concentration of the ligand, and thus the larger [L T ]:[M T ] ratio, is conditioned, in the case of DCP, by adsorption of the ligand at the surface of the mercury electrode and/or the solubility of the ligand. On the other hand, for GEP, in principle, one works at very low [L T ]:[M T ] ratios, in order to make sure that the liberated protons are coming mainly from complexation reactions and not from the deprotonation of the ligand. This should be taken into consideration, especially when the protonation constants are close to each other, e.g. about three log units or less. An important lesson to be learned from this study is that copper forms strong complexes with the DIPSO in this buffer ph range (Fig. 5). When these types of compounds, such as is the case of DIPSO, are employed as a buffer then their concentrations are very large (about 10 3 and 0.1 M). Consequently, the addition of buffer may strongly disturb the metal mass balance of the system under study and thus modify the answer of biochemical/environmental reactions containing these metal ions, unless the extent of complexation is taken into account for. The knowledge of the metal stability constants allows us to define correctly the metal speciation for a specific system. Because DIPSO forms strong complexes with copper in the buffer ph range, as well as AMPSO [9] and TAPSO [8], this can be an advantage as these buffers can be used as ph and metal ion buffer simultaneously. Acknowledgements The authors thank the Fundação para a Ciência e a Tecnologia from Portuguese Government for the financial support of this work with FEDER funds, by the project POCTI/39950/QUI/2001. 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