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Jan 30, 2010 - modification of amalgam electrodes is their covering with a liquid mercury film. The reasons for this kind of modifica- tion (i.e. preparation of ...
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Preparation and Properties of Mercury Film Electrodes on Solid Amalgam Surface Bogdan Yosypchuk,a* Miroslav Fojtab, Jirˇ Barekc a

J. Heyrovsky´ Institute of Physical Chemistry of AS CR, v.v.i., Dolejsˇkova 3, 182 23 Prague 8, Czech Republic Institute of Biophysics, AS CR, v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic c Charles University in Prague, Faculty of Science, Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry, Albertov 6, CZ-128 43 Prague 2, Czech Republic *e-mail: [email protected] b

Received: January 19, 2010 Accepted: January 30, 2010 Abstract A simple apparatus (electrolyzer) and a reliable procedure were developed for the preparation of mercury films of exactly defined thickness on a silver solid amalgam substrate. Constant concentration of Hg(II) in the electrolyzer is ensured by dissolution of an anode from silver paste amalgam. Small volume of electrolyte, which can be used repeatedly many times, and paste amalgam preventing the spillage of liquid mercury substantially decrease the danger of environmental contamination with mercury. Parameters and behavior of mercury film electrodes on silver solid amalgam substrate (MF-AgSAE) were compared with polished silver solid amalgam electrode (p-AgSAE) which does not contain liquid mercury, with mercury meniscus modified silver solid amalgam electrode (m-AgSAE), and with hanging mercury drop electrode (HMDE). The height of anodic stripping voltammetric peaks divided by electrode area was highest for MF-AgSAE and the width of those peaks, which determines the resolution of the method, was minimal at MF-AgSAE. Available potential window of MF-AgSAE in different supporting electrolytes is comparable with that of HMDE. Keywords: Voltammetry, Solid and paste amalgams, Mercury film electrode, Mercury DOI: 10.1002/elan.201000032

Presented at the International Conference on Modern Electroanalytical Methods Prague, December 9 – 14, 2009

1. Introduction Mercury electrodes in polarography [1] and related methods [2] are nearly ideal electrodes, especially for cathodic processes. The application of other electrode materials is complicated by lower overvoltage of hydrogen and worse reproducibility of measurements. It has been shown in our previous papers that solid amalgams are suitable materials for preparation of working electrodes [3 – 6], reference electrodes [7, 8] and a combined voltammetric-potentiometric sensor with a solid amalgam link [9]. Polished surface of a solid amalgam does not contain liquid mercury; it exhibits high hydrogen overvoltage [3, 4] and it is suitable both for direct electrochemical measurements [10 – 14] and for chemical modification with different modifiers [15, 16] which can substantially broaden field of application of amalgam electrodes. One of possible ways of chemical modification of amalgam electrodes is their covering with a liquid mercury film. The reasons for this kind of modification (i.e. preparation of mercury film electrodes (MFE)) are: i) to increase hydrogen overvoltage, i.e. to broaden available potential window; ii) to decrease limit of determination of metal ions by using anodic striping voltammetry (ASV) at Electroanalysis 2010, 22, No. 17-18, 1967 – 1973

MFE, the volume of which is much smaller than the volume of HMDE or the volume of mercury meniscus of m-AgSAE resulting in higher and narrower peaks at MFE compared to HMDE or m-AgSAE; iii) to increase resolution of close ASV peaks because of narrower peaks at MFE; iv) to increase mechanical stability of mercury surface for field measurements or for transfer adsorptive striping voltammetry of nucleic acids, proteins and other biologically important substances, where limited mechanical stability of HMDE can be the source of problems; v) to obtain ideally smooth, stable in time, and easily renewable surface suitable for the preparation of monolayer or multilayer films of biologically important substances; vi) to minimize legal problems limiting the use of liquid mercury. Mercury films can be prepared at different conductive supports. Real, ideally smooth film can be prepared only at a conductive surface which can be wetted by mercury (e.g. at carbonaceous materials an array of mercury micro drops is formed [17]). Ag [18 – 22], Au [23, 24], Pt [17, 25], Ir [26, 27], or Cu [28, 29] are most frequently used supports for mercury films. Solid amalgams of various metals are easily wetted with mercury and thus they are suitable supports for the preparation of MFE. Even though some pilot experiments

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were carried out at mercury film electrodes based on silver solid amalgam support (MF-AgSAE) [12, 14], so far there was no attempt to investigate systematically the preparation and properties of such films. Therefore, a simple, reliable and environmentally friendly procedure for the preparation of stable and exactly defined mercury films at silver solid amalgam support was developed and is described in this paper.

2. Experimental 2.1. Apparatus and Chemicals Voltammetric measurements were carried out using computer driven Eco-Tribo Polarograph PC-ETP (PolaroSensors, Prague, Czech Republic) with MultiElchem v. 2.1 software (J. Heyrovsky´ Institute of Physical Chemistry of AS CR, v.v.i.) in differential pulse voltammetry (DPV) mode with pulse height 50 mV, pulse width 100 ms and scan rate 20 mV s1. m-AgSAE with disk diameter D ¼ 0.54 mm and meniscus surface S ¼ 0.46 mm2, p-AgSAE (D ¼ 0.55 mm and S ¼ 0.24 mm2), MF-AgSAE on p-AgSAE support (D ¼ 0.52 mm and S ¼ 0.21 mm2), and HMDE (S ¼ 0.73 mm2) were used as working electrodes. The surface of m-AgSAE, p-AgSAE, and MF-AgSAE was calculated from geometrical parameters of the meniscus measured by means of an optical microscope. Surface of HMDE was calculated from the weight of 100 drops dislodged in the supporting electrolyte used. For calculations, it was assumed that mercury drop has spherical shape, meniscus is a part of a sphere, and mercury film is flat without any curvature. Saturated calomel electrode, to which all potentials are referred to, was used as reference electrode and Pt wire (1 mm diameter and 15 mm long) was used as auxiliary electrode. Oxygen was removed from analyzed solutions by 5 min bubbling with nitrogen. Deionized water was produced by Milli-Qplus system (Millipore). All other chemicals used were of p.a. purity. Calf thymus DNA was isolated, purified and denatured as described [30].

2.2. The Preparation of Mercury Film The apparatus depicted in Figure 1 was used. Into the bottom of plastic tube (2) (diameter ca 10 mm), a glassy carbon rod (diameter 2 – 3 mm) is sealed. Inside a plastic tube sufficient amount of silver paste amalgam (12% Ag) is inserted (4) to completely cover the glassy carbon rod to prevent its contact with electrolyte solution (3). Afterwards, 0.2 mL of the plating solution (0.01 M HgCl2 þ 1 M KI) is added and p-AgSAE to be covered with mercury film (1) is immersed into the solution. Glassy carbon rod is connected to reference electrode socket and p-AgSAE to working electrode socket of PC-ETP potentiostat and the method for measuring of i – t curves is selected in MultiElchem v. 2.1 software. The method enables to record the film formation and to calculate the total charge passed and thus the 1968

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Fig. 1. Scheme of the device for preparation of mercury film on a solid electrode. 1) working electrode (cathode); 2) plastic tube; 3) 0.2 mL of 0.01 M HgCl2 þ 1 M KI; 4) silver paste amalgam (12 % Ag); 5) glassy carbon contact rod (anode).

thickness of the mercury film formed. Optimum potential of electrolysis was found to be Eel ¼  200 mV, time of electrolysis depends on required thickness of the film formed (see Table 1). The program enables to stop the electrolysis after passage of predetermined charge (number of microcoulombs) thus assuring the preparation of the mercury film with exactly predetermined thickness.

2.3. Dissolution of Mercury Film Potentiostatic dissolution of the formed mercury film can be used either for the determination of the formed film thickness based on the charge needed for complete film dissolution or for the removal of old mercury film. It is carried out in three-electrode arrangement without stirring and without oxygen removing in the supporting electrolyte 0.1 M acetate buffer and 0.05 M Na2EDTA (pH 4.8) at a constant potential Ediss ¼ þ 250 mV. The time of dissolution depends on the mass of mercury in the film. (For an electrode with disk diameter 0.52 mm and film thickness 1 mm the dissolution time is around 10 min.). In the used MultiElchem v. 2.1 software, a method for measurement of I – t curves is selected which enables to record the process of dissolution with subsequent calculation of the total charge passed from which the film thickness can be calculated.

2.4. Modification of DNA with Os, bipy DNA samples (50 mg mL1) were incubated with 2 mM OsO4 and 2 mM 2,2’-bipyridine in 0.1 M Tris-HCl (pH 7.4) at 37 8C for 3 h. To remove unreacted Os, bipy, the samples

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Mercury Film Electrodes on Solid Amalgam Surface Table 1. Mercury films at polished silver solid amalgam electrode. Electrolyte for film deposition 0.01 M HgCl2 þ 1 M KI. Electrolyte for film dissolution 0.1 M acetate buffer þ 0.05 M Na2EDTA, pH 4.8. Potential for film deposition Edep ¼  200 mV; time of deposition tdep ¼ 60 s; potential for film dissolution Edis ¼ þ 250 mV. N ¼ 7. Parameter

Anode: liquid mercury Film deposition

Average value Confidence interval Standard deviation Relative standard deviation (%)

Anode: silver paste amalgam (12% Ag) Film dissolution

Film deposition

Film dissolution

mm [a]

mC [b]

mm [c]

mC [d]

mm [a]

mC [b]

mm [c]

mC [d]

1.19 0.017 0.019 1.61

3.307 0.046 0.051 1.54

1.19 0.016 0.018 1.49

3.306 0.043 0.047 1.43

1.19 0.013 0.014 1.16

3.313 0.033 0.036 1.10

1.09 0.013 0.014 1.26

3.034 0.032 0.035 1.15

[a] Film thickness calculated from charge passed during film deposition [b] Charge passed during film deposition [c] Film thickness calculated from charge passed during film dissolution [d] Charge passed during film dissolution

were dialyzed against 0.1 M Tris-HCl (pH 7.4) using SlideA-Lyzer MINI Dialysis Units at 5 8C overnight.

3. Results and Discussion Mercury film at a metal substrate can be formed by several ways [28] derived from different ways of amalgam preparation [31, 32]. The simplest way is to amalgamate a metal by its immersion into metallic mercury or sodium amalgam and to suck off the surplus of mercury. This procedure does not ensure good reproducibility of the film thickness and, moreover, some metals (e.g. Pt or Ir) are covered with oxides and without surface modification they are difficult to amalgamate by mercury. On metals electrochemically more active than mercury, the mercury film can be prepared by their immersion into a solution containing mercuric ions. The universal and most precise method is electrolytic deposition of mercury from mercuric ions solution. Under properly selected conditions, it is possible to carry out coulometric measurements and to calculate amount of mercury on the surface of the working electrode. It is necessary to stress out that the last two methods require working with highly toxic mercuric ions. The amount of mercuric ions consumed for the film deposition is not negligible and thus for each new precisely defined film a new fresh solution of mercuric ions should be used. The solutions used and the produced rinse waters must be properly storaged and liquidated because of their high toxicity. The simple apparatus described in experimental part and depicted in Figure 1 markedly decreases the risk of environmental contamination with mercury and its compounds, because the paste amalgam used as anode is not liquid and thus it cannot be spilled [33]. The main advantage of the apparatus is constant concentration of mercuric ions in the electrolyte because of the autoregulation process: the amount of mercury deposited at working electrode (cathode) is exactly equal to the amount of mercury transferred into the solution by anodic dissolution of silver paste amalgam. This statement was verified by two hour electrolElectroanalysis 2010, 22, No. 17-18, 1967 – 1973

ysis during which more mercury was deposited on the working electrode than it was originally present in the electrolyte solution. The electrolytic current was constant and stable during the whole time period. The procedure of film formation and trouble-free amalgam dissolution was optimized by testing different electrolytes resulting in recommendation of a solution containing sufficiently high concentration of iodides forming in water highly stable and sufficiently soluble complexes with both mercury and silver ions. The obtained dependence of the mercury film thickness on the potential of electrolysis at a constant time of 60 s is depicted in Figure 2. An optimal potential of electrolysis  200 mV was selected which was used in all subsequent experiments. Afterwards, the reproducibility of the mercury film preparation was tested (see Table 1). For the sake of comparison, reproducibility of film preparation using liquid mercury as anode was tested as well. I – t curves were always recorded both during deposition and during dissolution of the mercury film enabling the film thickness calculation on

Fig. 2. The dependence of the thickness of the mercury film l (in mm) formed at p-AgSAE on the potential of electrolysis Eel (in mV) in 0.01 M HgCl2 and 1 M KI at constant time of electrolysis tel ¼ 60 s. The film thickness was calculated from the charge passed.

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Fig. 3. Differential pulse anodic stripping voltammograms of Tl (I) (0.1 mg L1) in 0.1 M acetate buffer, pH 4.8 at different working electrodes. Eac ¼  900 mV, tac ¼ 60 s. Electrochemical regeneration of amalgam working electrodes: Ereg ¼  50 mV, treg ¼ 10 s; 40 cycles Ereg1 ¼  1200 mV, treg1 ¼ 0.2 s and Ereg2 ¼  100 mV, treg2 ¼ 0.3 s. Thickness of mercury films at MF-AgSAE is given in Table 2. Film thickness increases from right to left.

the basis of both cathodic and anodic processes. It can be seen from Table 1 that both film thickness and its reproducibility for both anodes (i.e. amalgam and liquid mercury) and calculated from both processes is nearly identical. Good reproducibility of the film thickness confirms that silver amalgam is a suitable source of mercuric ions and that the chosen electrolyte and optimized parameters of electrolysis ensures 100% current efficiency of mercury film deposition. The influence of mercury film thickness of MF-AgSAE on anodic stripping voltammograms was tested using Tl(I) ions as a model analyte. For the sake of comparison, the same voltammograms were recorded using other working electrodes (see Fig. 3 and Table 2). The used working electrodes differ in the content of liquid mercury from HMDE based completely on liquid mercury to p-AgSAE not containing any liquid mercury at all. As expected, the content of mercury is reflected in peak potentials: The lower is content of liquid mercury (eventually the thinner mercury film), the more negative is peak potential. This shift of peak potential at MFE is general for films at different supports and it is theoretically accounted for [34]. The width of the peak at half of its height (W1/2) is commonly used parameter to characterize how broad/narrow the peak is thus giving an idea of possible resolution. In agreement with theory [28, 34, 35], ASV peaks at MFEs are narrower than at HMDE. It can be seen from Table 2 that the peaks are narrower at MFEs than at m-AgSAE or p-AgSAE. Thus it can be expected that MF-AgSAE gives the best resolution of ASV peaks. Table 2 further shows very good reproducibility of parallel measurements and confirms that MFEs give the highest peaks (recalculated to 1 mm2 of electrode surface). 1970

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Repeatability of ASV peaks of Tl(I) (see Table 3) was tested as follows: At a p-AgSAE a new mercury film was prepared with simultaneous recording of i – t curve from which the film thickens was calculated. Freshly prepared MF-AgSAE was electrochemically activated in 0.2 M KCl (Eact ¼  2200 mV, tact ¼ 300 s) and the first set of 15 anodic stripping voltammograms was recorded during 38 min. The last 11 measurements from this set were statistically evaluated because of slightly anomalous first voltammograms at newly prepared film. Each next set of measurements then started one hour after the beginning of the previous set of measurements. For 8 hours the surface of MF-AgSAE was mirror shiny, however, next day the surface was matted and it structure was easily visible which explains the observed shift of peak potential at this surface. It can be seen from Table 3 that after 3 hrs peak potential starts to shift toward negative values which indicates the decreasing thickness of the film. The comparison of peak heights in Table 2 and 3 can be used for a rough estimation of film thickness in different times after preparation. Peak width expressed as W1/2 and peak heights repeatability does not deteriorate with film ageing. However, peak heights somewhat decrease with mercury film ageing. On freshly prepared films (see Table 1), the peak heights do not depend of mercury film thickness in agreement with theory [28] (The height of ASV peak is directly proportional to the product of film thickness l and analyte concentration in the film c). This product does not depend on film thickness because the thinner is the film the higher is the concentration of amalgam forming metal in this film (presuming constant deposition time). Because of gradual decrease of

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Mercury Film Electrodes on Solid Amalgam Surface Table 2. DPASV determination of Tl( I ) at different working electrodes. The film thickness l was calculated from the charge passed during film deposition. N ¼ 11. W1/2 ( peak width in half of its height) and Ep ( peak potential) were calculated as an average from 11 measurements. A – Average peak current recalculated on 1 mm2 of the working electrode surface. Conditions of measurements are given in the caption for Figure 3. Working electrode

Ep (mV )

W1/2 (mV )

A (nA mm2)

Average peak current value (nA )

Confidence interval (nA )

Standard deviation (nA )

Relative standard deviation (%)

– 30

 698  616

138 91

115.7 262.4

24.3 55.1

0.52 0.69

0.78 1.04

3.19 1.88

60 90 120 180 240 300 – –

 599  585  578  566  555  550  508  494

92 93 90 93 93 93 109 102

278.1 287.6 259.5 269.5 280.0 284.8 85.2 110.1

58.4 60.4 54.5 56.6 58.8 59.8 39.2 80.4

0.27 0.57 0.45 0.94 0.82 0.73 0.55 1.91

0.40 0.87 0.68 1.42 1.23 1.10 0.83 2.88

0.69 1.43 1.25 2.51 2.09 1.84 2.11 3.58

Time of film deposition (s)

(disk diameter, mm; electrode surface, mm2) p-AgSAE (0.55; 0.24) MF – AgSAE (0.52; 0.21) l ¼ 0. 66 mm l ¼ 1.25 mm l ¼ 1.99 mm l ¼ 2.58 mm l ¼ 3.95 mm l ¼ 5.15 mm l ¼ 6.49 mm m-AgSAE (0.54; 0.46) HMDE (0.73 mm2)

Table 3. Stability of current response of DPASV determination of Tl( I ) (0.1 mg L1) at MF-AgSAE. Film thickness 2.67 mm (calculated from charge passed during film deposition); supporting electrolyte 0.1 M acetate buffer, pH 4.8; Eac ¼  900 mV, tac ¼ 60 s. Electrochemical regeneration of MF-AgSAE before each measurement: Ereg ¼  50 mV, treg ¼ 10 s; 40 cycles Ereg1 ¼  1200 mV, treg1 ¼ 0.2 s and Ereg2 ¼  100 mV, treg2 ¼ 0.3 s. W1/2 (peak width in half of its height) and Ep ( peak potential) were calculated as an average from 11 measurements. Time from film deposition (h)

Ep (mV )

W1/2 (mV )

Average peak current value (nA )

Confidence interval (nA )

Standard deviation (nA )

Relative standard deviation (%)

0.1 – 0.5 1 2 3 4 5 6 7 8 24.5 [a]

 578  578  578  580  583  584  585  589  590  613

93 93 93 90 93 90 92 91 90 92

64.19 62.08 60.08 60.00 58.48 57.00 56.97 55.15 52.66 54.06

0.88 0.88 1.22 0.53 1.03 0.92 0.44 0.47 1.10 0.35

1.32 1.33 1.85 0.79 1.55 1.38 0.67 0.71 1.66 0.53

2.06 2.14 3.07 1.32 2.64 2.42 1.17 1.29 3.15 0.98

[a] The surface of MF-AgSAE is matted and clearly structured

current response with time, standard addition method should be preferred similarly as with most stationary electrodes. The range of available working potentials (potential window) is one of the most important characteristic of any working electrode and it determines the range of substances that can be either reduced or oxidized at this electrode. Table 4 contains this characteristic for various electrodes compared in this paper with different content of liquid mercury. It can be seen that hydrogen overvoltage at MFAgSAE is practically the same as that at HMDE. Some differences can be caused by the fact that MF-AgSAE contains at its surface saturated silver amalgam and that its surface is 5 times smaller than that of HMDE. Data in

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Table 4 clearly confirm that most determinations developed for HMDE can be carried out at amalgam electrodes as well. Electrochemical processes connected with adsorption and/or catalytic processes are especially sensitive toward material of working electrode and the state of its surface. For electrochemical studies of nucleic acids (NA) and oligonucleotides (ODN) their adsorption on working electrode is frequently employed. These substances are so strongly adsorbed that working electrode with adsorbed substances can be taken out of the solution, rinsed, and transferred into another solution with a suitable supporting electrolyte in which the voltammogram of adsorbed substance is recorded [36, 37]. Previously we studied [12] voltammetric behavior of unmodified nucleic acids at m-AgSAE, p-AgSAE, and MF-AgSAE and observed that presence of the mercury

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Table 4. Available potential window (in V ) of tested electrodes in different supporting electrolytes estimated from DCV currents reaching 1 mA; SCE reference electrode; polarization rate 0.02 V s1; oxygen removed by bubbling with nitrogen. D: disk diameter; A: electrode surface; l: mercury film thickness. Supporting electrolyte

HMDE A ¼ 1.01 mm2

m-AgSAE D ¼ 0.54 mm A ¼ 0.46 mm2

MF-AgSAE D ¼ 0.52 mm A ¼ 0.21 mm2 l ¼ 1.92 mm

p-AgSAE D ¼ 0.55 mm A ¼ 0.24 mm2

0.1 M HClO4 0.1 M HCl 0.2 M acetate buffer pH 4.8 0.05 M Na2EDTA 0.2 M acetate buffer pH 4.8 0.1 M NaClO4 0.05 M Na2B4O7 pH 9.3 0.1 M NaOH

 1.19 to þ 0.44  1.27 to þ 0.11  1.70 to þ 0.31

 1.11 to þ 0.44  1.12 to þ 0.12  1.39 to þ 0.30

 1.28 to þ 0.44  1.19 to þ 0.11  1.47 to þ 0.31

 0.98 to þ 0.44  1.07 to þ 0.13  1.46 to þ 0.47

 1.55 to þ 0.09

 1.39 to þ 0.10

 1.47 to þ 0.10

 1.45 to þ 0.41

 1.97 to þ 0.44  1.98 to þ 0.15

 1.98 to þ 0.44  1.92 to þ 0.15

 2.12 to þ 0.45  1.82 to þ 0.15

 1.86 to þ 0.46  1.70 to þ 0.36

 1.97 to  0.07

 1.99 to  0.06

 2.07 to  0.06

 1.96 to þ 0.19

layer on the solid amalgam surface influences considerably the DNA adsorption-desorption processes. An ongoing study (R. Sˇelesˇovska´ and M. Fojta, unpublished) revealed effects of the mercury film thickness (given by the time of mercury plating) on the DNA tensammetric signals. Here a chemically modified ODN, bearing covalently bound complex of osmium tetroxide with 2,2’-bipyridine (Os, bipy), was used to study the effects of the MF thickness on a signal of catalytic hydrogen evolution accompanying reduction of the osmium moiety [37 – 40]. The DNA-Os, bipy catalytic responses have been measured at the HMDE [37 – 39], MFE at a glassy carbon support [40], m-AgSAE or m-CuSAE [37], or a silver paste amalgam electrode [33]. This catalytic reaction is not observable at non-mercury electrode materials, such as pyrolytic graphite and/or glassy carbon. So far we did not succeed to obtain reliable catalytic signal of NA or ODN at p-AgSAE not containing liquid mercury, either. Because MF-AgSAE belongs according to the volume of present liquid mercury between p-AgSAE and m-AgSAE, we have carried out set of measurements of DNA-Os, bipy modified with osmium tetroxide at MF-AgSAE with different mercury film thickness (see Figure 4). It can be seen that

under the given conditions, no catalytic peak is observed at p-AgSAE whilst the highest one was obtained at m-AgSAE. At MF-AgSAE the current response gradually increases with increasing film thickness. At films with thickness of 2 mm and below, the catalytic efficiency of adsorbed osmium complex is much lower compared to thicker films. It can be explained that at the peak potential osmium is reduced to metal which dissolves in mercury film and it is partially fixed at solid support of the working electrode used, i.e. at solid silver amalgam, resulting in the decrease of osmium catalytic activity. The fixation of metals at a solid support in the case of mercury films thinner than 2 mm was previously described for indium and cadmium [28]. Current response calculated to 1 mm2 of the electrode surface is for electrodes with 3.9 mm and thicker films apparently higher than that at m-AgSAE (see Figure 4, stripped columns). For catalytic processes the freshness of electrode surface can play an important role. We have observed earlier [37] that at freshly formed HMDE the catalytic signal of DNA modified with osmium was much higher than that at m-AgSAE or m-CuSAE used for several days. Similar situation can be observed in Figure 4 where

Fig. 4. Dependence of peak-height ip of DNA-Os, bipy on the thickness of mercury film. Black columns: real electrode response; striped columns: electrode response calculated to 1 mm2 of the electrode surface. DPV; supporting electrolyte: 0.1 M acetate buffer, pH 4.8; concentration of DNA-Os, bipy ¼ 13.6 mg L1; Eacc ¼ Ein ¼  900 mV; accumulation time under stirring ¼ 60 s; Efin ¼  1500 mV; electrochemical regeneration of amalgam working electrodes before each measurement in 0.2 M KCl: Ereg ¼  2200 mV, treg ¼ 300 s.

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Mercury Film Electrodes on Solid Amalgam Surface

completely or nearly new MF-AgSAE was used compared to m-AgSAE the meniscus of which was several days old. It was confirmed that MF-AgSAE is suitable for classical adsorptive stripping voltammetric determination of DNA modified with osmium. Preliminary experiments have shown that MF-AgSAE can be successfully used for adsorptive transfer striping voltammetry (AdTSV) in which 1 – 2 mL of the sample solution is applied, the analyte is left to adsorb, the electrode is rinsed with deionized water and transferred into a suitable supporting electrolyte in which the voltammogram of adsorbed DNA modified with osmium is recorded [37, 41]. The biggest advantage of this approach is extremely small volume of the test solution required for this technique.

4. Conclusions A simple, fast and reliable procedure for the production of a uniform mercury film at a conductive support was developed. The used electrolyte and a silver paste amalgam anode (which is electrochemically dissolved during film deposition) ensure very good repeatability of produced films. Small amount of electrolyte solutions which can be used repeatedly many times and a silver paste amalgam which cannot be spilled substantially decrease the risk of environmental contamination. Mercury film at silver solid amalgam support has a mirror shine surface for the whole working day if its thickness is not lower than 2 mm. The height of ASV peaks of Tl(I) ions recalculated on 1 mm2 of electrode surface is higher at MF-AgSAE that at m-AgSAE, pAgSAE or HMDE. Peak width which effects method resolution is lowest at MF-AgSAE as compared to other above mentioned electrode. Available potential window at MF-AgSAE is practically identical with HMDE. Moreover, it was confirmed that MF-AgSAE is suitable for monitoring of adsorption and catalytic processes connected with the study of DNA.

Acknowledgements This work was supported by the Grant Agency of the Czech Republic (Project 203/07/1195), theGrant Agency of AS CR (Project No. IAA400400806), and by the Ministry of Education, Youth and Sports of the Czech Republic (Projects LC 06035, MSM 0021620857 and RP 14/63).

References [1] J. Heyrovsky´, J. Ku˚ta, Principles of Polarography, Publishing House of the Czech Academy of Sciences, Prague 1965.

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