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Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
C H A P T E R
10 Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes David Hernández-Santos, Pablo Fanjul-Bolado, María Begoña González-García Metrohm Dropsens, Parque Tecnológico de Asturias, Edificio CEEI, Asturias, Spain
10.1 Background The development of automated systems with high speed and good precision has become essential in Analytical Chemistry owing to the increasing demand for fast analyses of a large number of samples. For this purpose, methods based on flow injection analysis (FIA) or continuous flow systems have received particular attention. FIA methods are being extensively used for several environmental, industrial, and chemical applications, mainly because it provides improvements in versatility, reliability, and sample throughput, coupled with high reproducibility in comparison with wet (batch) chemical assays [1]. However, in 1991, Wang and Taha [2] introduced a technique named batch injection analysis (BIA). In BIA systems with electrochemical detection, a sample plug is injected from a micropipette tip directly on the electrode surface that is immersed in a large-volume blank electrolyte solution. The detector records a transient, peak-shaped response that reflects the passage of the sample zone over its surface. The magnitude of the peak thus reflects the concentration of the injected analyte. Such dynamic measurements performed under batch operation yield an analytical performance similar to that obtained under well-established FIA conditions, but without using additional components such as pumping systems, injection valves, and tubes. In this way, problems with air bubbles and leaks are overcome [3]. So, the BIA system is considered by some researchers as a tubeless FIA system.
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00010-3
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Copyright © 2020 Elsevier Inc. All rights reserved.
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
The first BIA cells reported in the bibliography used three independent conventional electrodes. The combination of BIA cells with screen-printed electrodes (SPEs) has been carried out recently (BIASPE) [4,5]. Similar to BIA systems, SPEs also presented some desirable features useful in portable analytical systems, such as low cost (large-scale production), disposability, rapid responses, simplicity, and robustness (working, counter, and pseudoreference electrodes are printed on a chemically inert substrate). The proposed system shows several requirements of a portable system that include user-friendly, high-speed quantitative and qualitative analysis, excellent cost-effectiveness, and low-power requirements. The coupling of the BIASPE cell with battery-powered accessories (electronic micropipette, potentiostat, and tablet or laptop computer) can be considered as a robust portable electroanalytical system. In Fig. 10.1 a BIASPE system setup is shown. The SPE is placed on the bottom of the cell (Fig. 10.1A). The cell has a tank (maximum volume of 100 mL) with a hole in the base that exactly matches the electrochemical cell of the SPE (see Fig. 10.1B). An O-ring placed around the hole and between the base of the BIA cell and the electrode prevents leaks of the electrolyte. After the tank is filled with the electrolyte, the BIA cell is closed with a cover that had two holes, one of them to put the electronic micropipette and the other one to put a rod stirrer controlled by a stirrer interface connected to the computer. The adapter for micropipette assures that the position of the tip of the pipette is centered on the electrode surface and the distance between the tip of the micropipette and the electrode surface remains constant. These two parameters contribute significantly to the reproducibility of the procedure (Fig. 10.1C). When compared with a standard FIA system, the electronic micropipette (20e200 mL) works as pump and injection valve. Therefore reproducible flow rate and injection volume (electronically controlled) are ensured and problems with air bubbles in the system are
(A) Overview of the opened BIASPE cell with detail of the positioning of the screen-printed electrode (SPE) (B) top view of the batch injection analysis (BIA) cell, and (C) full setup of BIASPE portable system including potentiostat, BIA cell, electronic micropipette, stirrer, and computer.
FIGURE 10.1
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avoided. The micropipette can be programmed with user-friendly and intuitive computer software allowing automatic aliquot dispensing after being programmed. In this experiment a BIASPE system as that shown in Fig. 10.1C is used with SPEs of ruthenium oxide. These electrodes have been already used as transducers for detection of ascorbic acid [6]. This molecule presents an irreversible oxidation process on these electrodes (at around þ0.09 V) at lower potential than this obtained with graphite electrodes. Although other electrochemical techniques can be used, in this experiment amperometry is the technique employed to carry out the measurement, one of the most widely used for the development of (bio)sensors and bioassays. The experiment is directed to undergraduate or Master students of courses related to the development of analytical methodologies (Chemistry, Biotechnology). It will give them, during a 3-h laboratory session, an appreciation of the usefulness of a BIA cell combined with SPEs as well as the main advantages and disadvantages when compared with FIA systems or the main possibilities of using this simple: hydrodynamic system in analytical chemistry.
10.2 Chemicals and supplies Reagents e Phosphate buffer saline (PBS) (0.04 M phosphate, 4.5% NaCl, pH 7.4). e Ascorbic acid: A 0.1 M solution should be prepared in 0.1 M sulfuric acid. Dilutions should be prepared in 0.04 M PBS pH 7.4. e Glucose: Solutions of this compound should be prepared in 0.04 M PBS pH 7.4. e Fructose: Solutions of this compound should be prepared in 0.04 M PBS pH 7.4. e Milli-Q purified water is employed to prepare all solutions. Instrumentation and materials e A potentiostat equipped to perform cyclic voltammetry and chronoamperometry. e SPEs of ruthenium oxide from Metrohm DropSens (DRP-810), with auxiliary electrode of carbon and pseudo-reference electrode of silver. e A specific connector acting as interface between the SPE and the potentiostat. e A BIA cell including an electronic micropipette of 20e200 mL (Metrohm DropSens (DRP-BIASPE02) as that shown in Fig. 10.1. e A Teflon rod stirrer moved by DC motor connected to a stirrer controller and the computer (DRP-STIRBIA from Metrohm DropSens). The controller box has a switch with three positions, 1, 2, and 3 corresponding to 500, 1500, and 3000 rpm, respectively. It is possible to use a conventional magnetic bar and stirrer but the magnetic bar should be placed in the center of the BIA cell, avoiding the contact with the electrode surface. e Microcentrifuge tubes of 1.5 mL, 500 mL (for PBS), and 50 mL (for ascorbic acid standard) volumetric flasks, 100 mL graduate cylinder, and 100 and 1000 mL micropipettes and tips for these micropipettes and for the electronic one (DRP-DTIPD200).
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10.3 Hazards Students are required to wear a lab coat, appropriate gloves, and safety glasses. Special care should be taken when handling concentrated acids or bases.
10.4 Experimental procedure 10.4.1 Preparation of the BIA cell 1. Open the BIA cell and put a ruthenium oxide electrode as it is shown in Fig. 10.1A. 2. Close the tank of the BIA cell and make sure that the electrode is centered on the hole of the bottom of the tank (see Fig. 10.1B). 3. Fill the tank with 80 mL of 0.04 M PBS pH 7.4 buffer (see Fig. 10.1B) using the graduate cylinder. It is not necessary to add an exact volume because this parameter does not affect the reproducibility of the peak currents. If during the filling of the tank, an air bubble is found around the electrode surface, it can be removed aspirating with a micropipette. It is possible to work with a smaller volume of PBS into the tank, for example, 50 mL. This value depends on the concentration of analyte and the volume and number of injections, taking into account that PBS contained in the tank will not be replaced during the experiment. 4. Put the cover of the tank. Then place the micropipette adapter in the corresponding hole (see Fig. 10.1C) and the rod stirrer connected to the stirrer controller box (and this one to the computer). 5. Switch on the stirrer and place the rpm selector in position 2. If it is necessary to work in an unstirred solution, switch off the stirrer. 6. Connect the electrode card to the potentiostat using the adequate cable (this depends on the potentiostat used, for Metrohm DropSens potentiostats, a DRP-CAST cable) 7. Select in the potentiostat the amperometric technique and the desired parameters (applied potential, interval time, run time, etc.) to record the BIA amperometric response. 8. Put the micropipette into the adapter (see next section) 9. Start recording the amperogram. Caution: Put a run time large enough to finish each assay before the end of the amperometric record; it is better to have more time and stop record when the assay finishes. 10. Wait until a stable background is achieved. Then, perform the first injection using the electronic micropipette.
10.4.2 Preparation of the electronic micropipette This is an important part of the procedure because the electronic micropipette works as pump and injection valve, so the management of this device should be carefully done. Caution: Before its use, the battery of the micropipette should be charged. The micropipette has six dispensing modes: reverse, mixed, forward, pipet, repetitive, and programmed, the latter three being the most useful for BIA assays. In this experiment, the
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mode repetitive is used. It consists on the injection of a fixed volume several times. The injection will only be done when the yellow bottom is pushed. Then, students should control the injection time. Taking into account the maximum volume of the micropipette (200 mL) and the volume to be dispensed (e.g., 40 mL), then the number of possible injections is obtained (200/40 injections in this case). The micropipette calculates automatically the maximum number of injections for a fixed volume of injection, but it is possible to select a smaller number of injections (in the last example it is possible to select also 1, 2, 3, or 4 injections). The micropipette has 6 speeds of injection, numbered from 1 to 6. This is a very important parameter because the height of the peaks can be affected by this value. The higher the number in the micropipette, the higher is the speed obtained. Follow the next steps to do injections of analyte (ascorbic acid standards or orange juice samples): 1. Select the repetitive mode in the micropipette and later the desired volume and number of injections. Finally, select the speed of injection (for more detail, check the manual of the micropipette). To start the experiment, the next values are recommended: e Injection volume: 40 mL e Number of injections: 5 (maximum value for this injection volume) e Injection speed: 4 2. Put an adequate tip in the micropipette. 3. Push the yellow bottom. In the display of the micropipette appears the word “Aspirate.” Introduce the tip into the ascorbic acid solution, of desired concentration, and press the yellow bottom. If the repetitive mode has been selected, the micropipette aspirates a little more than 200 mL. Now, in the display it appears the word “Discard.” Push again the yellow bottom without retiring the tip from the solution. Now the total volume into the tip is exactly 200 mL and in the display of the micropipette the volume and the number of injections that the micropipette will do will appear. 4. Place carefully the micropipette with the filled tip into the adapter of the BIA cell until the top. 5. When the baseline is stable, press the yellow bottom to dispense the analyte. For each injection, the yellow bottom should be pressed. It is recommended that the next injection is done when the baseline is recovered. Fig. 10.2 shows an example of 15 consecutive additions for a concentration of 0.2 mM of ascorbic acid.
10.4.3 Optimization of parameters that affect the analytical signal 10.4.3.1 Optimization of the potential of detection (hydrodynamic curve) Ascorbic acid presents an irreversible process on ruthenium electrodes around þ0.09 V (vs. silver pseudo-reference). When working in a BIA system, as well as in an FIA system, it is necessary to optimize the potential applied to detect a redox species. With this aim, using a concentration of 0.02 mM of ascorbic acid in 0.04 M PBS pH 7.4, vary the detection potential between 0.0 and þ0.4 V (for example, 0 V, þ0.1 V, þ0.2 V, þ0.3 V, and þ0.4 V). For this study fix an injection volume and a speed of 40 mL and 4, respectively. Put the stirrer speed selector in position 2 (1500 rpm). Make 3 injections for each potential. I. Dynamic electroanalytical techniques
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FIGURE 10.2 Fifteen consecutive injections of a 0.2 mM ascorbic acid solution in 80 mL of 0.04 M phosphate buffer saline pH 7.4. Injection volume: 40 mL; number of injections: 5 (3 aspirations have been done); injection speed: 4; detection potential: þ0.2 V; stirrer speed: 2 (1500 rpm).
Caution: For each potential applied, it is necessary to obtain a stable background before the first injection. After the last injection, wait until the background achieves a stable level and then stop recording. Save the amperogram. Select the next potential and start recording again, applying a new potential. Plot the mean of peak height versus the potential applied. Include also the deviation for each measurement using error bars. From this graph (hydrodynamic curve), select the optimum detection potential. 10.4.3.2 Optimization of speed and volume of injection Both parameters affect the analytical signal and, depending on the concentration assayed, the effects can be more remarkable. For example, if a very low concentration is expected, a higher speed of injection has to be employed if working in a stirred solution. Lower speed can be used for high concentrations. On the other hand, the higher the volume dispensed, the higher the peak height obtained, but the number of injections is limited to a lower number (the maximum volume of the micropipette is 200 mL). Baseline recovering will take more time for higher dispensing volumes, so the number of injections per hour will be lower. However, this effect is minimized if working in a stirred solution. For evaluating the influence of these two parameters, test two concentrations of ascorbic acid (0.02 and 0.5 mM in 0.04 M PBS pH 7.4): 1. Stir the solution fixing the position 2 in the stirrer speed selector (1500 rpm). 2. Fix the speed of dispensing of the micropipette in position 4, the volume of dispensing in 20 (maximum number of injections: 10), 40 (maximum number of injections: 5), or 100 mL (maximum number of injections: 2) for the two concentrations of ascorbic acid. Do three injections for each volume, except volume 100 mL (only two injections are possible). 3. Repeat the last step fixing a speed of dispensing in position 2. 4. Plot the mean of peak height versus the dispensing volume and versus the dispensing speed for each concentration. Include also the deviation for each measurement using error bars. From these graphs, select the best dispensing volume and speed.
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10.4 Experimental procedure
10.4.3.3 Effect of stirrer speed The stirrer used in this experiment has a selector that allows selecting three different speeds: 500 rpm (position 1), 1500 rpm (position 2), and 3000 rpm (position 3). It is very interesting to see the effect on the shape, width, and height of the peaks when working in an unstirred solution or when the electrolyte (PBS) is stirred during the dispensing. To study this effect, follow the next steps: 1. Turn off the stirrer. 2. Put a tip in the micropipette. Select repetitive mode and the next parameters: e Dispensing volume: 40 mL e Dispensing number: 3 e Dispensing speed: 3 3. Aspirate a 2 mM ascorbic acid solution. Discard the excess of volume. 4. Place carefully the micropipette into the cell through the adapter. 5. Start recording the amperogram applying the detection potential selected in Section 10.4.3.1 (fix a long time of measurement and an interval time of 0.1 s). 6. When the baseline achieves a stable current level, start with the dispensing protocol. 7. Make three injections waiting a period of time large enough to recover the background level. 8. After the third addition, remove carefully the micropipette without stopping the recording of the amperogram. 9. Turn on the stirrer. 10. Select the position 1 (you can see that the baseline is recovered faster). 11. Repeat the steps from 3 to 8 (except step 5). 12. Finally, repeat again the procedure selecting position 3 in the stirrer. The study could be repeated for a smaller concentration of ascorbic acid (0.02 mM). An example of the effect of stirrer speed is shown in Fig. 10.3: From the results obtained, select a stir speed that allows recovering the background line faster with a lower noise.
2mM Position 1
Position 2 Position 3
Unstirred
Position 1
Position 2
Position 3
Current (µA)
Current (µA)
Unstirred
0.02 mM Time (s)
Time (s)
FIGURE 10.3 Batch injection analysis amperogram obtained for injections of 0.02 and 2 mM ascorbic acid solutions under stirring conditions.
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
10.4.4 Calibration plot of ascorbic acid Using the experimental conditions optimized in Section 10.4.3, make consecutive injections of increasing concentrations of ascorbic acid comprised between 0.01 and 1 mM. Make five injections for each concentration of ascorbic acid. Plot the mean of peak height versus the concentration of ascorbic acid. Include also the deviation for each measurement using error bars. From this graph, estimate the linear range, the sensitivity (as the slope of the calibration curve), the limit of detection (as the concentration corresponding to a signal that is three times the standard deviation of the intercept [or of the estimate] in the lower range of concentrations), and the limit of quantification (as the concentration corresponding to a signal that is 10 times the standard deviation of the intercept or estimate). An example of the analytical signals obtained and a calibration plot are shown in Fig. 10.4.
10.4.5 Interference evaluation To study the selectivity of the method, two potential interferences such as fructose and glucose are evaluated. They are chosen as interferences because of its usual presence in orange juice samples. To carry out this study, make 3 injections of a 0.5 mM solution of glucose, followed by 3 injections of a 0.5 mM solution of fructose and other 3 injections of binary mixtures of glucose and ascorbic acid (both in 0.5 mM concentration) and fructose and ascorbic acid (0.5 mM in each compound). Finally make 3 injections of a mixture of the 3 compounds in a concentration of 0.5 mM. Discuss the effect of the presence of these interferences based on the results obtained.
(A)
(B)
Current (µA)
Current (µA)
(f)
(e)
(d) (c)
(b) (a) 0
100
200
300
400
500
600
700
800
900 1,000 1,100
[Ascorbic acid] (mM)
Time (s)
FIGURE 10.4 (A) Batch injection analysis amperogram of five injections of different concentrations of ascorbic acid increasing from (a) 0.01 to (f) 1 mM; (B) the corresponding calibration plot.
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10.4.6 Determination of ascorbic acid in orange juices Taking into account the ascorbic acid content labeled in the orange juices, dilute them with 0.04 M PBS pH 7.4 to a final concentration that is included in the linear range of the calibration plot. Calculate the ascorbic acid concentration in orange juice by using the external calibration curve. Several replicates should be measured to obtain the average and standard deviation of the results.
10.5 Lab report Write a lab report following the typical style of a scientific article. It should include an abstract, a brief introduction explaining the purpose of the experiment, a detailed experimental section, results and discussion, and conclusions. Include tables, graphics, or figures wherever necessary. The following points should be taken into account: 1. In the introduction, include a revision of the different applications of BIA systems with electrochemical techniques. Discuss the main advantages and disadvantages of the BIA systems compared with FIA systems. 2. In the experimental section, explain the protocols to prepare solutions in detail, the equipment used, and the real samples that are going to be analyzed. 3. In the results and discussion section, include figures with representative raw data (chronoamperograms) and results presented in tables and graphs (e.g., hydrodynamic curve, calibration curve) paying special attention to the significant figures in each case. 4. Discuss the values obtained considering the expected results and the incidences during the course of the experiment.
10.6 Additional notes 1. PBS solution can be stored at 4 C for at least 1 week. However, the ascorbic acid stock solution should be prepared daily. 2. The tips used for the electronic micropipette should be those recommended by the supplier. Do not use other tips from other suppliers. The reason is that the distance from the tip to the electrode should be kept constant. 3. If the rod stirrer is not available, it is possible to use a conventional magnetic stirrer placing a magnetic bar stir in the BIA cell. In this case, especial care should be taken because the magnetic bar has to be in the center of the BIA cell without touching the electrode surface. Then, the effect of the stirrer speed on the analytical signal can be done, but taking into account the rpm values employed. 4. Another kind of SPEs could be used. 5. The electronic micropipette has software that allows programming the protocol of dispensing. It should be interesting that the students program the micropipette with the final optimized protocol. For this, check the manual.
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6. It is not necessary to change the electrolyte into the BIA cell except if the liquid overflows. It is possible to use less volume of electrolyte (60 instead of 80 mL as it is recommended). 7. The sample throughput (number of samples per hour) can be also estimated.
10.7 Assessment and discussion questions 1. Explain why the typical signal of the BIA cell is a transient response and the reason of the shape of the peaks. 2. Is it possible to use a BIA cell with other electrochemical techniques? Explain with an example. 3. Indicate the influence of the different operational parameters. 4. What are the parameters that affect sample throughput?
References [1] J. Ruzicka, E.H. Hansen, Flow-injection Analysis, Wiley, New York, 1981. [2] J. Wang, Z. Taha, Batch injection analysis, Anal. Chem. 63 (1991) 1053e1056. [3] M.S.M. Quintino, L. Angnes, Batch injection analysis: an almost unexplored powerful tool, Electroanalysis 16 (2004) 513e523. [4] E.M. Richter, T.F. Tormin, R.R. Cunha, W.P. Silva, A. Pérez-Junquera, P. Fanjul-Bolado, D. Hernández-Santos, R.A.A. Muñoz, A compact batch injection analysis cell for screen-printed electrodes: a portable electrochemical system for on-site analysis, Electroanalysis 28 (2016) 1856e1859. [5] T.F. Tormin, R.R. Cunha, W.P. Silva, R.A. Bezerra da Silva, R.A.A. Muñoz, E.M. Richter, Combination of screenprinted electrodes and batch injection analysis: a simple, robust, high-throughput, and portable electrochemical system, Sens. Actuators, B 202 (2014) 93e98. [6] J. Wu, J. Suls, W. Sansen, Amperometric determination of ascorbic acid on screen-printing ruthenium dioxide electrode, Electrochem. Commun. 2 (2000) 90e93.
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10 Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes David Hernández-Santos, Pablo Fanjul-Bolado, María Begoña González-García Metrohm Dropsens, Parque Tecnológico de Asturias, Edificio CEEI, Asturias, Spain
10.1 Background The development of automated systems with high speed and good precision has become essential in Analytical Chemistry owing to the increasing demand for fast analyses of a large number of samples. For this purpose, methods based on flow injection analysis (FIA) or continuous flow systems have received particular attention. FIA methods are being extensively used for several environmental, industrial, and chemical applications, mainly because it provides improvements in versatility, reliability, and sample throughput, coupled with high reproducibility in comparison with wet (batch) chemical assays [1]. However, in 1991, Wang and Taha [2] introduced a technique named batch injection analysis (BIA). In BIA systems with electrochemical detection, a sample plug is injected from a micropipette tip directly on the electrode surface that is immersed in a large-volume blank electrolyte solution. The detector records a transient, peak-shaped response that reflects the passage of the sample zone over its surface. The magnitude of the peak thus reflects the concentration of the injected analyte. Such dynamic measurements performed under batch operation yield an analytical performance similar to that obtained under well-established FIA conditions, but without using additional components such as pumping systems, injection valves, and tubes. In this way, problems with air bubbles and leaks are overcome [3]. So, the BIA system is considered by some researchers as a tubeless FIA system.
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00010-3
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Copyright © 2020 Elsevier Inc. All rights reserved.
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
The first BIA cells reported in the bibliography used three independent conventional electrodes. The combination of BIA cells with screen-printed electrodes (SPEs) has been carried out recently (BIASPE) [4,5]. Similar to BIA systems, SPEs also presented some desirable features useful in portable analytical systems, such as low cost (large-scale production), disposability, rapid responses, simplicity, and robustness (working, counter, and pseudoreference electrodes are printed on a chemically inert substrate). The proposed system shows several requirements of a portable system that include user-friendly, high-speed quantitative and qualitative analysis, excellent cost-effectiveness, and low-power requirements. The coupling of the BIASPE cell with battery-powered accessories (electronic micropipette, potentiostat, and tablet or laptop computer) can be considered as a robust portable electroanalytical system. In Fig. 10.1 a BIASPE system setup is shown. The SPE is placed on the bottom of the cell (Fig. 10.1A). The cell has a tank (maximum volume of 100 mL) with a hole in the base that exactly matches the electrochemical cell of the SPE (see Fig. 10.1B). An O-ring placed around the hole and between the base of the BIA cell and the electrode prevents leaks of the electrolyte. After the tank is filled with the electrolyte, the BIA cell is closed with a cover that had two holes, one of them to put the electronic micropipette and the other one to put a rod stirrer controlled by a stirrer interface connected to the computer. The adapter for micropipette assures that the position of the tip of the pipette is centered on the electrode surface and the distance between the tip of the micropipette and the electrode surface remains constant. These two parameters contribute significantly to the reproducibility of the procedure (Fig. 10.1C). When compared with a standard FIA system, the electronic micropipette (20e200 mL) works as pump and injection valve. Therefore reproducible flow rate and injection volume (electronically controlled) are ensured and problems with air bubbles in the system are
(A) Overview of the opened BIASPE cell with detail of the positioning of the screen-printed electrode (SPE) (B) top view of the batch injection analysis (BIA) cell, and (C) full setup of BIASPE portable system including potentiostat, BIA cell, electronic micropipette, stirrer, and computer.
FIGURE 10.1
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avoided. The micropipette can be programmed with user-friendly and intuitive computer software allowing automatic aliquot dispensing after being programmed. In this experiment a BIASPE system as that shown in Fig. 10.1C is used with SPEs of ruthenium oxide. These electrodes have been already used as transducers for detection of ascorbic acid [6]. This molecule presents an irreversible oxidation process on these electrodes (at around þ0.09 V) at lower potential than this obtained with graphite electrodes. Although other electrochemical techniques can be used, in this experiment amperometry is the technique employed to carry out the measurement, one of the most widely used for the development of (bio)sensors and bioassays. The experiment is directed to undergraduate or Master students of courses related to the development of analytical methodologies (Chemistry, Biotechnology). It will give them, during a 3-h laboratory session, an appreciation of the usefulness of a BIA cell combined with SPEs as well as the main advantages and disadvantages when compared with FIA systems or the main possibilities of using this simple: hydrodynamic system in analytical chemistry.
10.2 Chemicals and supplies Reagents e Phosphate buffer saline (PBS) (0.04 M phosphate, 4.5% NaCl, pH 7.4). e Ascorbic acid: A 0.1 M solution should be prepared in 0.1 M sulfuric acid. Dilutions should be prepared in 0.04 M PBS pH 7.4. e Glucose: Solutions of this compound should be prepared in 0.04 M PBS pH 7.4. e Fructose: Solutions of this compound should be prepared in 0.04 M PBS pH 7.4. e Milli-Q purified water is employed to prepare all solutions. Instrumentation and materials e A potentiostat equipped to perform cyclic voltammetry and chronoamperometry. e SPEs of ruthenium oxide from Metrohm DropSens (DRP-810), with auxiliary electrode of carbon and pseudo-reference electrode of silver. e A specific connector acting as interface between the SPE and the potentiostat. e A BIA cell including an electronic micropipette of 20e200 mL (Metrohm DropSens (DRP-BIASPE02) as that shown in Fig. 10.1. e A Teflon rod stirrer moved by DC motor connected to a stirrer controller and the computer (DRP-STIRBIA from Metrohm DropSens). The controller box has a switch with three positions, 1, 2, and 3 corresponding to 500, 1500, and 3000 rpm, respectively. It is possible to use a conventional magnetic bar and stirrer but the magnetic bar should be placed in the center of the BIA cell, avoiding the contact with the electrode surface. e Microcentrifuge tubes of 1.5 mL, 500 mL (for PBS), and 50 mL (for ascorbic acid standard) volumetric flasks, 100 mL graduate cylinder, and 100 and 1000 mL micropipettes and tips for these micropipettes and for the electronic one (DRP-DTIPD200).
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
10.3 Hazards Students are required to wear a lab coat, appropriate gloves, and safety glasses. Special care should be taken when handling concentrated acids or bases.
10.4 Experimental procedure 10.4.1 Preparation of the BIA cell 1. Open the BIA cell and put a ruthenium oxide electrode as it is shown in Fig. 10.1A. 2. Close the tank of the BIA cell and make sure that the electrode is centered on the hole of the bottom of the tank (see Fig. 10.1B). 3. Fill the tank with 80 mL of 0.04 M PBS pH 7.4 buffer (see Fig. 10.1B) using the graduate cylinder. It is not necessary to add an exact volume because this parameter does not affect the reproducibility of the peak currents. If during the filling of the tank, an air bubble is found around the electrode surface, it can be removed aspirating with a micropipette. It is possible to work with a smaller volume of PBS into the tank, for example, 50 mL. This value depends on the concentration of analyte and the volume and number of injections, taking into account that PBS contained in the tank will not be replaced during the experiment. 4. Put the cover of the tank. Then place the micropipette adapter in the corresponding hole (see Fig. 10.1C) and the rod stirrer connected to the stirrer controller box (and this one to the computer). 5. Switch on the stirrer and place the rpm selector in position 2. If it is necessary to work in an unstirred solution, switch off the stirrer. 6. Connect the electrode card to the potentiostat using the adequate cable (this depends on the potentiostat used, for Metrohm DropSens potentiostats, a DRP-CAST cable) 7. Select in the potentiostat the amperometric technique and the desired parameters (applied potential, interval time, run time, etc.) to record the BIA amperometric response. 8. Put the micropipette into the adapter (see next section) 9. Start recording the amperogram. Caution: Put a run time large enough to finish each assay before the end of the amperometric record; it is better to have more time and stop record when the assay finishes. 10. Wait until a stable background is achieved. Then, perform the first injection using the electronic micropipette.
10.4.2 Preparation of the electronic micropipette This is an important part of the procedure because the electronic micropipette works as pump and injection valve, so the management of this device should be carefully done. Caution: Before its use, the battery of the micropipette should be charged. The micropipette has six dispensing modes: reverse, mixed, forward, pipet, repetitive, and programmed, the latter three being the most useful for BIA assays. In this experiment, the
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mode repetitive is used. It consists on the injection of a fixed volume several times. The injection will only be done when the yellow bottom is pushed. Then, students should control the injection time. Taking into account the maximum volume of the micropipette (200 mL) and the volume to be dispensed (e.g., 40 mL), then the number of possible injections is obtained (200/40 injections in this case). The micropipette calculates automatically the maximum number of injections for a fixed volume of injection, but it is possible to select a smaller number of injections (in the last example it is possible to select also 1, 2, 3, or 4 injections). The micropipette has 6 speeds of injection, numbered from 1 to 6. This is a very important parameter because the height of the peaks can be affected by this value. The higher the number in the micropipette, the higher is the speed obtained. Follow the next steps to do injections of analyte (ascorbic acid standards or orange juice samples): 1. Select the repetitive mode in the micropipette and later the desired volume and number of injections. Finally, select the speed of injection (for more detail, check the manual of the micropipette). To start the experiment, the next values are recommended: e Injection volume: 40 mL e Number of injections: 5 (maximum value for this injection volume) e Injection speed: 4 2. Put an adequate tip in the micropipette. 3. Push the yellow bottom. In the display of the micropipette appears the word “Aspirate.” Introduce the tip into the ascorbic acid solution, of desired concentration, and press the yellow bottom. If the repetitive mode has been selected, the micropipette aspirates a little more than 200 mL. Now, in the display it appears the word “Discard.” Push again the yellow bottom without retiring the tip from the solution. Now the total volume into the tip is exactly 200 mL and in the display of the micropipette the volume and the number of injections that the micropipette will do will appear. 4. Place carefully the micropipette with the filled tip into the adapter of the BIA cell until the top. 5. When the baseline is stable, press the yellow bottom to dispense the analyte. For each injection, the yellow bottom should be pressed. It is recommended that the next injection is done when the baseline is recovered. Fig. 10.2 shows an example of 15 consecutive additions for a concentration of 0.2 mM of ascorbic acid.
10.4.3 Optimization of parameters that affect the analytical signal 10.4.3.1 Optimization of the potential of detection (hydrodynamic curve) Ascorbic acid presents an irreversible process on ruthenium electrodes around þ0.09 V (vs. silver pseudo-reference). When working in a BIA system, as well as in an FIA system, it is necessary to optimize the potential applied to detect a redox species. With this aim, using a concentration of 0.02 mM of ascorbic acid in 0.04 M PBS pH 7.4, vary the detection potential between 0.0 and þ0.4 V (for example, 0 V, þ0.1 V, þ0.2 V, þ0.3 V, and þ0.4 V). For this study fix an injection volume and a speed of 40 mL and 4, respectively. Put the stirrer speed selector in position 2 (1500 rpm). Make 3 injections for each potential. I. Dynamic electroanalytical techniques
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
FIGURE 10.2 Fifteen consecutive injections of a 0.2 mM ascorbic acid solution in 80 mL of 0.04 M phosphate buffer saline pH 7.4. Injection volume: 40 mL; number of injections: 5 (3 aspirations have been done); injection speed: 4; detection potential: þ0.2 V; stirrer speed: 2 (1500 rpm).
Caution: For each potential applied, it is necessary to obtain a stable background before the first injection. After the last injection, wait until the background achieves a stable level and then stop recording. Save the amperogram. Select the next potential and start recording again, applying a new potential. Plot the mean of peak height versus the potential applied. Include also the deviation for each measurement using error bars. From this graph (hydrodynamic curve), select the optimum detection potential. 10.4.3.2 Optimization of speed and volume of injection Both parameters affect the analytical signal and, depending on the concentration assayed, the effects can be more remarkable. For example, if a very low concentration is expected, a higher speed of injection has to be employed if working in a stirred solution. Lower speed can be used for high concentrations. On the other hand, the higher the volume dispensed, the higher the peak height obtained, but the number of injections is limited to a lower number (the maximum volume of the micropipette is 200 mL). Baseline recovering will take more time for higher dispensing volumes, so the number of injections per hour will be lower. However, this effect is minimized if working in a stirred solution. For evaluating the influence of these two parameters, test two concentrations of ascorbic acid (0.02 and 0.5 mM in 0.04 M PBS pH 7.4): 1. Stir the solution fixing the position 2 in the stirrer speed selector (1500 rpm). 2. Fix the speed of dispensing of the micropipette in position 4, the volume of dispensing in 20 (maximum number of injections: 10), 40 (maximum number of injections: 5), or 100 mL (maximum number of injections: 2) for the two concentrations of ascorbic acid. Do three injections for each volume, except volume 100 mL (only two injections are possible). 3. Repeat the last step fixing a speed of dispensing in position 2. 4. Plot the mean of peak height versus the dispensing volume and versus the dispensing speed for each concentration. Include also the deviation for each measurement using error bars. From these graphs, select the best dispensing volume and speed.
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10.4 Experimental procedure
10.4.3.3 Effect of stirrer speed The stirrer used in this experiment has a selector that allows selecting three different speeds: 500 rpm (position 1), 1500 rpm (position 2), and 3000 rpm (position 3). It is very interesting to see the effect on the shape, width, and height of the peaks when working in an unstirred solution or when the electrolyte (PBS) is stirred during the dispensing. To study this effect, follow the next steps: 1. Turn off the stirrer. 2. Put a tip in the micropipette. Select repetitive mode and the next parameters: e Dispensing volume: 40 mL e Dispensing number: 3 e Dispensing speed: 3 3. Aspirate a 2 mM ascorbic acid solution. Discard the excess of volume. 4. Place carefully the micropipette into the cell through the adapter. 5. Start recording the amperogram applying the detection potential selected in Section 10.4.3.1 (fix a long time of measurement and an interval time of 0.1 s). 6. When the baseline achieves a stable current level, start with the dispensing protocol. 7. Make three injections waiting a period of time large enough to recover the background level. 8. After the third addition, remove carefully the micropipette without stopping the recording of the amperogram. 9. Turn on the stirrer. 10. Select the position 1 (you can see that the baseline is recovered faster). 11. Repeat the steps from 3 to 8 (except step 5). 12. Finally, repeat again the procedure selecting position 3 in the stirrer. The study could be repeated for a smaller concentration of ascorbic acid (0.02 mM). An example of the effect of stirrer speed is shown in Fig. 10.3: From the results obtained, select a stir speed that allows recovering the background line faster with a lower noise.
2mM Position 1
Position 2 Position 3
Unstirred
Position 1
Position 2
Position 3
Current (µA)
Current (µA)
Unstirred
0.02 mM Time (s)
Time (s)
FIGURE 10.3 Batch injection analysis amperogram obtained for injections of 0.02 and 2 mM ascorbic acid solutions under stirring conditions.
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
10.4.4 Calibration plot of ascorbic acid Using the experimental conditions optimized in Section 10.4.3, make consecutive injections of increasing concentrations of ascorbic acid comprised between 0.01 and 1 mM. Make five injections for each concentration of ascorbic acid. Plot the mean of peak height versus the concentration of ascorbic acid. Include also the deviation for each measurement using error bars. From this graph, estimate the linear range, the sensitivity (as the slope of the calibration curve), the limit of detection (as the concentration corresponding to a signal that is three times the standard deviation of the intercept [or of the estimate] in the lower range of concentrations), and the limit of quantification (as the concentration corresponding to a signal that is 10 times the standard deviation of the intercept or estimate). An example of the analytical signals obtained and a calibration plot are shown in Fig. 10.4.
10.4.5 Interference evaluation To study the selectivity of the method, two potential interferences such as fructose and glucose are evaluated. They are chosen as interferences because of its usual presence in orange juice samples. To carry out this study, make 3 injections of a 0.5 mM solution of glucose, followed by 3 injections of a 0.5 mM solution of fructose and other 3 injections of binary mixtures of glucose and ascorbic acid (both in 0.5 mM concentration) and fructose and ascorbic acid (0.5 mM in each compound). Finally make 3 injections of a mixture of the 3 compounds in a concentration of 0.5 mM. Discuss the effect of the presence of these interferences based on the results obtained.
(A)
(B)
Current (µA)
Current (µA)
(f)
(e)
(d) (c)
(b) (a) 0
100
200
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500
600
700
800
900 1,000 1,100
[Ascorbic acid] (mM)
Time (s)
FIGURE 10.4 (A) Batch injection analysis amperogram of five injections of different concentrations of ascorbic acid increasing from (a) 0.01 to (f) 1 mM; (B) the corresponding calibration plot.
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10.4.6 Determination of ascorbic acid in orange juices Taking into account the ascorbic acid content labeled in the orange juices, dilute them with 0.04 M PBS pH 7.4 to a final concentration that is included in the linear range of the calibration plot. Calculate the ascorbic acid concentration in orange juice by using the external calibration curve. Several replicates should be measured to obtain the average and standard deviation of the results.
10.5 Lab report Write a lab report following the typical style of a scientific article. It should include an abstract, a brief introduction explaining the purpose of the experiment, a detailed experimental section, results and discussion, and conclusions. Include tables, graphics, or figures wherever necessary. The following points should be taken into account: 1. In the introduction, include a revision of the different applications of BIA systems with electrochemical techniques. Discuss the main advantages and disadvantages of the BIA systems compared with FIA systems. 2. In the experimental section, explain the protocols to prepare solutions in detail, the equipment used, and the real samples that are going to be analyzed. 3. In the results and discussion section, include figures with representative raw data (chronoamperograms) and results presented in tables and graphs (e.g., hydrodynamic curve, calibration curve) paying special attention to the significant figures in each case. 4. Discuss the values obtained considering the expected results and the incidences during the course of the experiment.
10.6 Additional notes 1. PBS solution can be stored at 4 C for at least 1 week. However, the ascorbic acid stock solution should be prepared daily. 2. The tips used for the electronic micropipette should be those recommended by the supplier. Do not use other tips from other suppliers. The reason is that the distance from the tip to the electrode should be kept constant. 3. If the rod stirrer is not available, it is possible to use a conventional magnetic stirrer placing a magnetic bar stir in the BIA cell. In this case, especial care should be taken because the magnetic bar has to be in the center of the BIA cell without touching the electrode surface. Then, the effect of the stirrer speed on the analytical signal can be done, but taking into account the rpm values employed. 4. Another kind of SPEs could be used. 5. The electronic micropipette has software that allows programming the protocol of dispensing. It should be interesting that the students program the micropipette with the final optimized protocol. For this, check the manual.
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10. Batch injection analysis for amperometric determination of ascorbic acid at ruthenium dioxide screen-printed electrodes
6. It is not necessary to change the electrolyte into the BIA cell except if the liquid overflows. It is possible to use less volume of electrolyte (60 instead of 80 mL as it is recommended). 7. The sample throughput (number of samples per hour) can be also estimated.
10.7 Assessment and discussion questions 1. Explain why the typical signal of the BIA cell is a transient response and the reason of the shape of the peaks. 2. Is it possible to use a BIA cell with other electrochemical techniques? Explain with an example. 3. Indicate the influence of the different operational parameters. 4. What are the parameters that affect sample throughput?
References [1] J. Ruzicka, E.H. Hansen, Flow-injection Analysis, Wiley, New York, 1981. [2] J. Wang, Z. Taha, Batch injection analysis, Anal. Chem. 63 (1991) 1053e1056. [3] M.S.M. Quintino, L. Angnes, Batch injection analysis: an almost unexplored powerful tool, Electroanalysis 16 (2004) 513e523. [4] E.M. Richter, T.F. Tormin, R.R. Cunha, W.P. Silva, A. Pérez-Junquera, P. Fanjul-Bolado, D. Hernández-Santos, R.A.A. Muñoz, A compact batch injection analysis cell for screen-printed electrodes: a portable electrochemical system for on-site analysis, Electroanalysis 28 (2016) 1856e1859. [5] T.F. Tormin, R.R. Cunha, W.P. Silva, R.A. Bezerra da Silva, R.A.A. Muñoz, E.M. Richter, Combination of screenprinted electrodes and batch injection analysis: a simple, robust, high-throughput, and portable electrochemical system, Sens. Actuators, B 202 (2014) 93e98. [6] J. Wu, J. Suls, W. Sansen, Amperometric determination of ascorbic acid on screen-printing ruthenium dioxide electrode, Electrochem. Commun. 2 (2000) 90e93.
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