**4. Experimental conditions**

#### **4.1 Reagents, equipment and analysis**

All solutions were prepared by dissolving the respective analytical grade reagent in ultrapure water (Milli Q, Millipore) with a specific conductivity lower than 0.1 S cm-1, and used without further purification.

A stock solution of 1000 mg l-1 As(V) was prepared by dissolution of Na2HAsO4·7H2O (Panreac, Spain) in water and acidified with concentrated HCl (0.1% v/v). A stock solution of 1000 mg l-1 As(III) was prepared by dissolving As2O3 (Sigma, St Louis, MO, USA) with NaOH 1x10-2 M (Panreac) and then acidified to pH 1 with concentrated HCl (Panreac). Stock solutions were renewed weekly. Standard solutions of As(V) and As(III) of concentrations ranging from 0 to 50 g l-1 were prepared daily by dilution of the respective stock solution.

of time. The current response thus generated is proportional to the concentration of the analyte. During on-line stripping analysis the analyte is pre-concentrated on the surface electrode in flowing conditions, whilst the stripping step can be done in flowing conditions

Different cell designs have been used for electrochemical detection. The cell design must fulfil the requirements of high signal-to-noise ratio, low dead volume, well defined hydrodynamics, small ohmnic drop, high contact area and easy of construction and maintenance. In addition, the reference and counter electrodes should be located downstream next to the working electrode, so that reaction products at counter electrode or leakage from the reference electrode do not interfere with the working electrode detection. The most widely used detectors are based on the wall-jet, thin-layer, and tubular

(a) (b) (c)

In the wall-jet design, the stream flows perpendicularly to the working electrode surface, and then spreads radially over it improving the contact between the analyte and the electrode. The thin-layer cell consists on a thin layer of solution that flows parallel to the planar electrode surface, the main disadvantage is the small contact area. Tubular configuration provides minimal flow disturbance and a higher contact area, compared with thin-layer configuration. This feature has enabled the application of tubular configuration in flow injection systems with sequential determination in which the detector is relocated

All solutions were prepared by dissolving the respective analytical grade reagent in ultrapure water (Milli Q, Millipore) with a specific conductivity lower than 0.1 S cm-1, and

A stock solution of 1000 mg l-1 As(V) was prepared by dissolution of Na2HAsO4·7H2O (Panreac, Spain) in water and acidified with concentrated HCl (0.1% v/v). A stock solution of 1000 mg l-1 As(III) was prepared by dissolving As2O3 (Sigma, St Louis, MO, USA) with NaOH 1x10-2 M (Panreac) and then acidified to pH 1 with concentrated HCl (Panreac). Stock solutions were renewed weekly. Standard solutions of As(V) and As(III) of concentrations ranging from 0 to 50 g l-1 were prepared daily by dilution of the respective stock solution.

Fig. 5. Schematic representation of front and side view of electrochemical flow cells configurations. a) thin-layer, b) wall-jet and c) tubular. WE, working electrode.

**inlet**

**WE WE WE WE**

**inlet outlet**

or stopped flow (Economou, 2010).

configurations (Fig. 5.) (Trojanowicz, 2009):

inside the flow manifold (Catarino et al., 2002).

**4.1 Reagents, equipment and analysis** 

**4. Experimental conditions** 

used without further purification.

**outlet inlet**

**outlet**

**inlet-**

**inlet outlet**

**WE WE**

L-cysteine 1x10-3 M (HOOC-CH(NH2)-CH2-SH, Sigma) was used as reducing agent. A supporting electrolyte solution of 2.0 M HCl was used for all the experiments.

Figure 6 shows a scheme of the sequential injection anodic stripping voltammetry system (SI-ASV) used for inorganic arsenic speciation in water samples. The system consisted of a MicroBu 2030 multisyringe burette with programmable speed (CS, Crison, Spain) used to aspire and dispense the reagent solutions, an eight-way selection valve (SV, Crison), a home-made tubular electrochemical cell (D), and a mixer chamber (MC).

The tubular electrochemical detection cell was made up of a Perspex body in which working and auxiliary electrodes were placed. These electrodes were built from gold and carbon discs (7.0 mm diameter) with length of 1.0 and 2.0 mm, respectively. Both have a tubular channel (0.8 mm diameter) in the centre of the electrode. These electrodes were used in connection with a saturated Ag/AgCl reference electrode (Metrohm, Switzerland). The instrumental devices were controlled by means of the Autoanalysis 5.0 software (Sciware, Spain). All tubing connecting the different components of the flow system was made of Omnifit PTFE with 0.8 mm (i.d.). Electrochemical experiments were performed with an Autolab PGSTAT10 potentiostat/galvanostat (EcoChemie) equipped with GPES 4.6 software. Unless otherwise stated, a frequency (*f*) of 25 Hz, pulse amplitude (Esw) of 50 mV, step height (ΔEs) of 8 mV, and deposition potential (Ed) and time (td) of −0.4 V for 40 s were chosen as the square wave anodic stripping voltammetry (SWASV) parameters. A conditioning potential and time (2 s at 2.0 V) was added to increase the reproducibility (Kopanica & Novotny, 1998).

Fig. 6. Schematic set up of the SI-ASV flow system: CS, carrier solution; R1, holding coil; R2, reaction coil; SV, selection valve; R, reductant ; S, sample; MC, mixer chamber; D, detector; W, waste. Components of the electrochemical cell: a, reference electrode; b, tubular gold electrode; c, glassy carbon counter electrode; d, connector, e, O-ring.

Sequential Injection Anodic Stripping Voltammetry

**5. Results and discussion** 

is given by the linear model:

can be used to represent the data.

response to be optimized.

As(V) contained in the sample to As(III).

al. 2009).

at Tubular Gold Electrodes for Inorganic Arsenic Speciation 211

The optimization of the variables is a critical step in the design of new analytical methods. Optimization involves the selection of the chemical and instrumental factors which may affect the analytical signal, and the choice of the values of the variables to obtain the best response from the chemical system. For this purpose, two different strategies can be used. In the traditional univariate optimization, all values of the different factors except one are constant, and this one is the object of the examination. The alternative to this strategy is the use of chemometric techniques based mainly on the use of experimental designs (Tarley, et

Factorial designs are used to identify the significant variables (factors) affecting the selected response and as a tool to explore and model the responses as a function of these significant experimental factors. Two-level full factorial designs are a powerful alternative to find the adequate experimental conditions to produce the best response of the chemical system. This type of design fits the response to a linear model. For a two-factor case, the response surface

If the interaction term b12x1x2 is negligible, then the response surface is planar. The more important the interaction term, the greater is the degree of twisting that the planar response surface experiences. Chemometrical optimization commonly uses the following procedure: a) choose a statistical design to investigate the experimental region of interest, b) perform the experiments in random chronological order, c) perform analysis of variance (ANOVA) on the regression results so that the most appropriate model with no evidence of lack of fit

The factors investigated that could affect the response for As(III) and total As determination are listed in Table 1. Two levels for each factor were selected for a complete factorial design (replicates, i.e., 23+1) allowing to identifying the critical factors. Sixteen experiments were performed in random order for each of As(III) and total As optimization. The optimal values obtained for factors B and C from the optimization of SI-ASV for As(III) were fixed for total As determination, and new factors related to the prior reduction of As(V) were included in the second design of experiments. The current of the stripping peak of arsenic (in A) using a 10.0 μg l-1 standard of arsenic was selected as the

The flow rate is critical in on-line stripping methods, as it controls the dispersion between the sample and the carrier solution. This factor and the deposition time contribute to the amount of analyte deposited on the electrode surface. With respect to the reaction coil length, it has to be sufficiently long for loading and pre-treatment of sample previous to the deposition step. In addition, for total As determination the chemical parameters such as reduction time and reducer concentration must guarantee the complete reduction of the

0 1 1 2 2 12 1 2 *y*ˆ *b bx bx b xx* (4)

**5.1 Optimization of the SI-ASV system for inorganic arsenic speciation** 

#### **4.1.1 Sampling**

500 ml polyethylene bottles were conditioned by filling them with 2% v/v HNO3 for at least three days. Once in the sampling site, bottles were rinsed several times with the water to be collected. Groundwater samples were obtained from deep and shallow wells in the area of Tierra de Pinares (Segovia, Spain), affected by arsenic contamination of aquifers. The well was pumped for at least 10 min before a water sample was collected. Bottles were completely filled with the sample to minimize the oxidation of As(III) by air. Immediately after sampling, the bottled water samples were acidified with HCl (pH<2), wrapped in hermetic plastic bags, and transported to the laboratory in iceboxes. Water samples were stored not longer than one week at 4ºC prior analysis.

#### **4.1.2 Analytical cycle**

Initially, a 0.4 ml sample aliquot and a 0.2 ml of the reductant (L-cysteine, R-SH) are aspirated sequentially to the holding coil (R1). The resulting solution is directed towards the mixer chamber MC at 1.2 ml min-1 (50 s), and is allowed to stand for 2 minutes to reduce chemically As(V) to As(III) according to the following reaction:

$$\text{R-SH-SH} + \text{As(V)} \rightarrow \text{R-S-S-R} + \text{As(III)} + \text{2H}^\* \tag{1}$$

Simultaneous to the reduction of As(V) to determine in a second step total As, the measurement of As(III) was carried out. The sample was introduced in the system using a binary sampling strategy consisting on intercalation of multiple small sample segments of 50 l (aspirated to S channel) with small segments (50 l) of carrier solution (dispensed by CS to R2). The total aspirated volume was therefore 400 l of sample and 250 l of carrier solution. The binary sampling strategy creates multiple reaction interfaces which contribute to a faster homogenization of the sample media. The sample mixture is then propelled towards the electrochemical cell by the CS at a flow rate of 0.6 ml min-1 to be electrochemically deposited (40 s at -0.4 V).

$$\text{As(III)} + 3\text{ e} \to \text{As0} \tag{2}$$

After deposition of elemental arsenic, 2.0 ml of the carrier solution are pumped through the detection cell at a flow rate of 30.0 ml min-1. The elemental arsenic is then stripped off in stop flow mode using the CS as clean medium under the SWASV parameters mentioned above. The exchange of the sample solution by the CS electrolyte solution minimizes the interference produced by the chlorine generated at the auxiliary electrode (Billing et al., 2002).

$$\text{As}\\
\bullet \rightarrow \text{As(III)} \; + \text{Эe} \tag{3}$$

For total As determination, the solution contained in the mixing chamber (port 4) is now aspirated to R1 and then propelled through the detection cell at a flow rate of 1.2 ml min-1 (40 s). The resulting As(III) (sum of As(III) plus reduced As(V)) is then electrochemically reduced and stripped in a clean medium as described for As(III). The As(V) is then determined as the difference between total As and As(III).
