ANODIC STRIPPING VOLTAMMETRY
September 1997
The object of this experiment is to gain an understanding of anodic stripping voltammetry through the determinations of trace amounts of copper in a macro-sized biological sample and the amount of copper in micro-sized samples of copper based alloys and to gain an understanding of factors involved in performing quantitative analytical determinations.
REFERENCES
Adrian W. Bott, “Stripping Voltammetry”, Current Separations 1992, 12:3, 141-147. (attached)
Scott R. Peters and Jonathon O. Howell, “A Controlled Growth Mercury Electrode”, Current Separations 1994,13:1, 12-16. (attached)
Morris Bader, “A Systematic Approach to Standard Addition Methods in Instrumental Analysis”, J. Chem. Ed. 1980, 57, 703. (attached)
General Chemistry Lab Manual, PomonaCollege, Claremont, California.
S. B. Adeloju, A. M. Bond, and M. H. Briggs, “Multielement Determination in Biological Materials by Differential Pulse Voltammetry”, Anal. Chem. 1985, 57, 1386-1390. (available in the laboratory to check out)
Giles F. Carter and Hossein Rasi, “Chemical Composition of Copper-Based Coins of the RomanRepublic”, R. O. Allen, Editor, Advances in Chemistry (Archaeological Chemistry-IV) 220, American Chemical Society: Washington, D.C., 1989, Chap. 11. (available in the laboratory to check out)
BAS CV-50W Version 2 MF-9093 Instruction Manual for the Voltammetric Analyzer, July 1995. (available for use in the laboratory only)
BAS CGME MF-9058 Instruction Manual for the Controlled Growth Mercury Electrode, May 1993. (available for use in the laboratory only)
INTRODUCTION
In this experiment, two copper-base alloy samples (a standard alloy sample and a bronze Roman coin) and a biological sample are analyzed for the amount of copper in the sample by anodic stripping voltammetry (ASV). The ASV is done with a controlled growth mercury electrode (CGME) and a BAS CV-50W Voltammetric Analyzer using the method of standard addition. As part of the experiment, an Eppendorf pipet will be calibrated.
Stripping Voltammetry
Stripping voltammetry is a sensitive electroanalytical technique for the determination of trace amounts of metals in solution. The technique consists of three steps. First, metal ions are deposited onto an electrode which is held at a suitable potential. The solution is stirred during this step to maximize the amount of metal deposited. Second, stirring is stopped so that the solution will become quiet. Third, the metal deposits are stripped from the electrode by scanning the potential. The observed current during the stripping step can be related to the amount of the metal in the solution.
The stripping step may consist of a positive or a negative potential scan, creating either an anodic or cathodic current respectively. Hence, Anodic Stripping Voltammetry (ASV) and Cathodic Stripping Voltammetry (CSV) are two specific stripping techniques. The attached paper by Bott discusses stripping voltammetry in detail.
In addition to varying the direction of the scan, the manner in which the potential is scanned may also differ. The simplest technique is Linear Sweep Voltammetry (LSV) where the potential is scanned linearly as a function of time. Another commonly used technique is Differential Pulse Voltammetry (DPV), which has a lower detection limit than LSV. This is due to its pulsed waveform which measures the current in pulses by taking two measurements and recording the difference as the potential is increased. This helps to reduce the background current. The waveforms from each pulse superimpose upon one another to form a staircase waveform since the pulse amplitude is constant while the potential increases in small increments. The potential excitation waveforms of these two methods are shown in the BAS CV-50W Instruction Manual.
Electrodes
Mercury is often used as an electrode material because it is chemically inert in most aqueous solutions and hydrogen is evolved only at negative potentials. Thus, reductions of many species may be studied. It is useful because amalgams are formed with the mercury by the metals produced when the cations are reduced. The metals can also diffuse freely into the electrode because it is liquid. Mercury also forms insoluble salts with cations, which is useful for CSV.
Predominantly, two types of mercury electrodes have been used for the "working" electrode, which is the electrode where the reaction occurs and the responses are recorded. These are the Hanging Mercury Drop Electrode (HMDE), which may also be called Static Drop Mercury Electrode (SDME), and various types of carbon electrodes on which a thin layer of mercury is plated onto the carbon surface either before using the electrode for a determination or simultaneously with the plating of metal ions. The higher surface to volume ratio of the mercury film carbon electrode results in more metal to be deposited for a given amount of time than with the HMDE. Reproducible results, however, are more difficult to attain with carbon electrodes since the carbon needs to be polished and pretreated and the mercury film may not be identically formed. A reference electrode is used to monitor the potential of the working electrode. To complete the electrochemical cell an auxiliary electrode is necessary. The potentials of the working and auxiliary electrodes are adjusted with respect to the reference electrode to maintain the desired potential at the working electrode. A salt bridge is placed between the analyte and the reference electrode to avoid contamination of the analyte.
Determination of Copper in the Samples
In this experiment, a HMDE is used with an Ag/AgCl reference electrode and a platinum auxiliary electrode. These three electrodes are part of the Controlled Growth Mercury Electrode (CGME) unit which is used in conjunction with the BAS CV-50W Voltammetric Analyzer. Differential Pulse Stripping Voltammetry will be performed anodically to detect trace amounts of copper. The attached paper by Peters and Howell further describes the CGME.
Since this is a trace analysis technique, all glassware must be rinsed thoroughly before use with nitric acid and ultra-pure water to minimize contamination. The metal alloy samples are dissolved with acid, the biological sample is digested to destroy organic material, and a standard copper solution is made. After solutions are prepared, the sample in the cell is purged with nitrogen gas to remove oxygen. Then, the sample solution is stirred in a reproducible manner while the working electrode is held at the desired deposition potential for a given length of time. The stirring is stopped and, after a short period of time for the solution to become quiet, the potential of the working electrode is scanned in the positive direction to remove the copper from the electrode. The monitor will display the potential versus the current graph. A typical graph is shown in Figure 1.
To determine the concentration of the copper in the solution, the method of standard addition is used. In this method, as described in the article by Bader, a known amount of a standard solution is added to the sample after the first run and the experiment is rerun. The observed current for the second determination is the sum of the current due to the copper in the sample plus the current due to the added standard. If more than one standard addition is made, then a plot of current versus concentration of added standard solution can be prepared. The unknown concentration can then be found by extrapolating to the x-axis, as seen in Figure 2. With an unknown sample, once the concentration of the copper in the solution has been determined, the percent copper in the sample can be calculated.
Figure 1
Figure 2
EXPERIMENTAL PROCEDURE
BE SURE TO READ ALL OF THESE INSTRUCTIONS THOROUGHLY BEFORE BEGINNING ANY EXPERIMENTAL WORK. PARTNERSHIP INSTRUCTIONS ARE GIVEN AT THE END OF THIS SECTION.
Ultra-pure water is obtained from the Millipore Water Purification System in Room 13. Follow the instructions listed next to the system. Initially, collect two liters of water and discard this water. Then collect four liters of water for use. Record the amount of water collected in the log book.
Because of the sensitivity of the methods employed, much care is needed to ensure that contamination is avoided. All glassware that will be used must be rinsed with a solution of 1:4 concentrated nitric acid and water and then rinsed in the ultra-pure water. Volumetric pipets and flasks will be used extensively throughout this experiment. Please consult your General Chemistry Lab Manual for their proper use. One of the reasons for poor precision and accuracy in this experiment is the improper use of volumetric pipets and flasks.
Proper Techniques for Using the Eppendorf Pipet and the Finnpipette
To obtain the most accurate dispensing values, adjust the volume of the pipet by turning the setting ring so the numbers read a value past the desired volume and then return back to the proper position. Firmly attach a plastic tip to the pipet taking care to keep the instrument and tip as contamination free as possible. For best pipeting results, change the tip with each sample in the experiment. To fill the pipet, press the control button down to the first stop, immerse the pipet into the solution in a vertical fashion, and slowly let the control button return to its initial position. Remove the tip from the solution by sliding it along the inside of the container. If it is necessary, use a Kimwipe to clean off any droplets of solution on the outside of the pipet tip being careful not to touch the end of the tip with the tissue. When emptying the pipet, hold the tip at an angle against the inside of the container (DO NOT immerse in the solution) and slowly press the control button down to the first stop and pause. Then press the button down to the second stop to completely empty the tip. Remove the tip making sure the control button remains completely compressed. During the calibration procedure, it is possible to remove the pipet in the proper manner, by sliding it up the side of the container. However, when pipeting into the cell vial, it is important not to touch the tip to the inside of the white cover since droplets of solution might be deposited and therefore will foul up the concentrations in the vial. Once the tip is moved away from the experimental solutions, the control button can be relaxed (slowly) and the tip removed (using the lateral ejector button) and replaced if desired. If there are any remaining drops of solution inside the pipet tip after it was been emptied, a new tip must be used. In addition, if this occurs, it is necessary for the experiment in process to be restarted because you are dealing with such small volumes that any little fraction of error can significantly affect the experimental results.
***Never lay the pipet down when the tip contains liquid since this has the potential to contaminate and damage the pipet mechanism.
Calibration of the Eppendorf Pipet
Make three separate weighings of 50 microliters of ultra-pure water in a weighing vial to simulate the standard addition technique. Carefully weigh and record each of the additions making sure to cover the weighing vial for each measurement to minimize evaporation. Determine the average mass, standard deviation, and percent relative standard deviation. Be sure to record the temperature of the water in the stock beaker since the density of water varies with temperature. Determine the calculated volume of water using density (The density of water at different temperatures can be found in your General Chemistry Laboratory Manual) and compare the value with the Eppendorf volume. The expected tolerances for Eppendorf pipets 0.5% for accuracy and 0.2% for precision. If your values do not agree reasonably well with these values, repeat your calibration. The Finnpipette does not need to be calibrated. The calibration certificate is taped to the lid of the pipet box.
Preparation of a 1.0 M Potassium Nitrate Solution and a Blank Solution
To prepare a solution of 1.0 M potassium nitrate weigh out approximately 10.1 g of potassium nitrate and place it in a 100 ml flask. Dilute to volume with ultra-pure water and mix well until all of the potassium nitrate dissolves. This solution can be prepared conveniently while the bovine liver sample is digesting as described below. The blank solution is prepared by pipetting 10 ml of the 1.0 M potassium nitrate solution into a 100 ml volumetric flask and diluting to volume with ultra-pure water. Be sure to mix this solution well.
Digestion and Preparation of the Biological Sample
This procedure is based upon the procedure in the article by Adeloju, Bond, and Briggs.
Weigh to the nearest 0.1 mg approximately 300 mg of oven dried bovine liver, which is stored in a desiccator, and put the bovine liver into a 125 ml Erlenmeyer flask. Add 10 ml of concentrated nitric acid and 1 ml of concentrated sulfuric acid. Insert a glass funnel and heat on a hot plate under the hood. When the brown-orange nitrogen oxide fumes are just given off, cool the flask for about 2 minutes. Add 10 ml of concentrated nitric acid and heat again until brown fumes are just given off. Repeat this process twice more so that a total of 30 ml has been added.
With the final addition of nitric acid, heat until all of the nitrogen oxide fumes are given off. Rinse the funnel with ultra-pure water and remove. Cool for 2 minutes and then add 3 ml of 1:1 concentrated hydrochloric acid and water. Replace the funnel. Heat this mixture for approximately one hour or until the water of the solution has evaporated so that it is less than 25 ml. During this time, the metal samples may be prepared. Cool to room temperature and transfer the solution quantitatively to a 50 ml volumetric flask and carefully dilute to volume.
Pipet 10 ml of this bovine liver digestion into a 100 ml volumetric flask. Be sure to rinse the pipet with the original solution several times before adding the solution to the flask. Add 10 ml of the 1M potassium nitrate solution and then dilute to volume. This is the dilute bovine liver solution which will be used for analysis.
Preparation of the Copper Base Alloy Sample and Standard Copper Solutions.
Weigh to the nearest 0.1 mg, approximately 25 mg of the known copper base alloy sample and place the weighed sample in a 250 ml beaker.
Using a file, obtain approximately 25 mg of the Roman coin sample and weigh to the nearest 0.1 mg. Place the weighed sample in another 250 ml beaker.
Cut a piece of the copper foil of approximately 25 mg and weigh to the nearest 0.1mg. Place this weighed sample in a third 250 ml beaker.
Add 4 ml of ultra-pure water, 6 ml of concentrated hydrochloric acid, and 1 ml of concentrated nitric acid to each of the metal samples. Heat gently on a hot plate under the hood until the samples are dissolved. Do not boil the solution until all of the metal has dissolved.
After the alloy samples and the foil have fully dissolved, dilute the solutions with ultra-pure water to approximately 50 ml and boil briefly to remove oxides of nitrogen and chlorine.
Quantitatively transfer each solution in the beakers to separate 250 ml volumetric flasks. Rinse the beakers with several small portions of ultra-pure water being careful that you do not exceed the capacity of the flask. Dilute the volumetric flasks to volume with ultra-pure water and mix well. The solution made from the copper foil will serve as your standard and will not be diluted further.
Pipet 1 ml of each of your alloy sample solutions with the Finnpipette into 100 ml volumetric flasks and add 10 ml of the 1M potassium nitrate solution. Dilute to volume with the ultra-pure water. These are the dilute solutions that you will be working with and will be referred to as your sample solutions.
Be sure to label clearly all of your prepared solutions using the white tape in the lab.
***This is a good place to stop for the first day. Before returning to lab for the second day, calculate the copper concentration in g/ml in the standard copper foil solution and the concentrations of added copper in 15 ml of sample solution after the addition of 50, 100, and 150 microliters of the standard copper foil solution to the 15 ml of sample solution (25, 50, and 75 microliters for the bovine liver sample). These will be the additions to the cell vial in the standard addition procedure and this data must be entered into the computer before a run can be made. See the first portion of the report form referring to the Standard Solution for assistance with these calculations.
Figure 3
Cleaning and Setting up the CGME
The CGME must be cleaned before it is used. The diagram in Figure 3 of the CGME will be referred to with parentheses indicating the number of the part to be adjusted. The CGME can be turned on by the switch on the back of the instrument.
Begin by making sure that the mercury capillary is in the highest position possible. If not, the height may be adjusted loosening knob 22, which is on the stand to which the electrode assembly is mounted, and carefully moving the electrode assembly. Remove the cell vial (which looks like a shot glass) by sliding the stir motor (17) to the right while holding onto the vial. The two nitrogen purge lines (25) may be carefully pulled out of the white cell cover. The cell cover can then be slid out and rinsed with ultra-pure water. Remove the Teflon tape from the mercury capillary. This electrode as well as the reference electrode, which is stored in a 3 M NaCl solution, and the platinum wire auxiliary electrode should also be rinsed with ultra-pure water. It is also important that all of the electrodes, the tubes, and the white cell cover be rinsed with ultra-pure water between samples.