Experiment

Equipment

• See the laboratory instructor about what potentiostat is available and the method of data acquisition (i.e., x-y recorder with analog potentiostat or printed output with a computerized potentiostat).
• Electrochemical cell and electrodes
  • 1.0 or 3.0 mm diameter flat tipped glassy carbon electrode
  • Pt auxiliary electrode
  • Ag/AgCl reference electrode
  • Small volume electrochemical cell
  • Polishing kit
• Timer

Chemicals

1. Dopamine [recommend the HCl salt of DA, F. W. 189.64]
2. Norepinephrine [recommend the HCl salt of NE, F. W. 205.64]
3. Na₂HPO₄ and citric acid for making McIlvaine buffer
4. Sulfuric acid (1 M)

Procedure

1. Prepare a solution of dopamine (DA) at a pH near 1.0 by adding approximately 10 mg of solid DA to a 50.00 ml volumetric flask and dissolving it in 1 M sulfuric acid. Record the actual mass of DA. Mix well by shaking the flask. Handle catecholamines with extreme care, as they can have severe physiological effects!
2. Place about 10 ml of this solution into an electrochemical cell and deoxygenate for ~ 10 minutes with nitrogen or argon.
3. Prepare a glassy carbon electrode (1 mM or 3 mM diameter) by polishing it for ~ 1 minute on 0.05 μm alumina. Use a gentle circular motion or trace a figure 8 on a polishing pad that has the alumina on the surface. Clean the electrode carefully with distilled water, sonicate for 10-15 seconds, and then touch the edge with a Kimwipe™ before introducing the electrode into the cell.
4. Please ask laboratory instructor for the directions to set-up and operate the potentiostat.
5. Record cyclic voltammograms for this solution at scan rates of 50 and 100 mV/s between an initial potential of 0.00 V and a positive potential limit of +1.00 V. Make duplicate runs at each scan rate. Measure the pH of the solution before discarding.
6. Prepare a 1 mM solution of dopamine at ~ pH 7.0 by dissolving ~10 mg in a 50.00 ml volumetric flask. Record the actual mass of DA. Pipette 5.0 ml of 0.1 M citric acid, then dilute to mark with 0.2 M disodium phosphate. Together, these ingredients comprise the McIlvaine buffer.
7. Degas the solution and repolish the electrode as previously instructed (see #3 above).
8. Record cyclic voltammograms for dopamine in this pH 7.0 solution at scan rates of 50, 100, 150, 200, 250 and 300 mV/s, adjusting the x-axis (current) sensitivity scale as needed to record the entire I-t curves. Set the scan limits so that you start at -100 mV and scan the potential anodically to the limit of +700 mV, reverse back to -800 mV and end up at -100 mV [sequence of limits: -100, +700, -800 and stop at -100 mV]. Record the pH of the solution before discarding.
9. Prepare a 1 mM solution of norepinephrine (NE) near pH 7.0 by dissolving ~11 mg in 50.00 ml of McIlvaine buffer. Record the actual mass of NE.
10. Degas the solution and repolish the electrode. Record CV scans of NE at 50 mV/s and 400 mV/s with the same potential limits as in step #7. Run duplicates of the CV scans. Record the pH of this solution before discarding.

Calculations:

Table 1

Theoretical Values for the Ratio of Reverse to Forward Peak Currents for Charge Transfer Followed by an Irreversible Chemical Reaction

kft	irev/ ifwd
0.004	1.00
0.023	0.986
0.035	0.967
0.066	0.937
0.105	0.900
0.195	0.828
0.350	0.727
0.525	0.641
0.550	0.628
0.778	0.551
1.050	0.486
1.168	0.466
1.557	0.415

1. Use the Nicholson equation to calculate the value of i_rev/i_fwd from the CV scans for dopamine at pH 1.0 and at pH 7.0, recorded at the scan rates of 50, 100, 200 and 300 mV/s. Next, do the same calculations for the two CV scan rates with norepinephrine.
2. You will need to determine the time, t, in seconds that it takes to scan the potential from the E_½ value to the switching potential, E_λ, of each cyclic voltammogram. This value of t will be different for each of the scans (remember that the time it takes is dependent on the distance along the potential axis and the scan rate). The E_½ value is the potential at ½ the peak current.
3. Table 1 shows the i_rev/i_fwd values as a function of the theoretically calculated k_ft values, as determined by Nicholson. Plot the (i_rev/i_fwd) vs. log(k_ft) to make a working curve. Interpolate the points to obtain a smooth curve.
4. Expand the appropriate region of the working curve corresponding to each experimental value of (i_rev/i_fwd) and measure the value of log(k_ft) for each of your current ratios from the working curve. Next, use the experimentally determined value of t at each of your scan rates to calculate a value of k_f. Calculate the average value of k_f for DA and NE.
5. The literature values for k_f are 0.038 s^-1 and 0.36 s^-1 for DA and NE, respectively [ref. 8].
6. How close are your values to those in the literature?

Postscript: The c step (cyclization) is first-order and irreversible for the oxidized product of both dopamine and norephenephrine. Other examples of this ec mechanism are the compounds of p-aminophenol and catechol – they undergo a 2-electron electrooxidation. The triol, produced by the c step in the case of catechol, is readily oxidized to the quinone form at potentials less positive than the parent.