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EXPERIMENT 5 CV: DETERMINATION OF ASCORBIC ACID IN DRINKS AND


NUTRITIONAL SUPLEMENTS.
De Jess M. A.; Vera M; (2010); University of Puerto Rico; Mayagez Campus; Department of
Chemistry; P.O. Box 9000; Mayagez P.R. 00681.
PURPOSE:
Familiarize the student with the cyclic voltammetry technique and its use for the study
of REDOX processes.
Determine the amount of ascorbic acid present in drinks and nutritional supplements.
INTRODUCTION:
Recommended readings:
1. Skoog, D.A.; Holler, F. J.; Nieman, T.A.; Principles of Instrumental Analysis, 6th
ed.; Harcourt Brace: Philadelphia, 2007; Sections 25 B, 25C-2, 25D
2. Mabbott, G. A.; An Introduction to Cyclic Voltammetry; Journal of Chemical
Education (1983); 60 (9), 697-702.
3. James J. Van Benschoten, J.J.; Lewis, J. Y.; Heineman, W. R.; Roston, D. A.;
Kissinger, P. T.; Cyclic Voltammetry Experiment; Journal of Chemical
Education (1983); 60 (9), 773-776.
4. Elving, P. J.; Markowitz, J. M., Rosenthal, I.; Preparation of Buffer Systems of
Constant Ionic Strength; Analytical Chemistry (1956), 28(7), 1179
Ascorbic acid is water soluble organic acid, commonly known as Vitamin C. Ascorbic acid is
essential to the human body due to its paramount role as an antioxidant and free radical
scavenger as well as its role in collagen. It is one of the most ubiquitous vitamins in nature and is
widely as an antioxidant and preservative in the pharmaceutical, chemical, cosmetic and food
industry. Ascorbic acid is also important in a variety of metabolic processes such as amino acid
metabolism, and synthesis of anti-inflammatory agents like steroids hormones and
neurotransmitters.
The oxidation of ascorbic acid involves two electrons and two protons to produce
dehydroascorbic acid, which is followed by its irreversible conversion to 2,3-diketo-L-gulonic
acid at pH lower than 4.0. The accepted oxidation mechanism of Ascorbic acid occurs as
follows:

Scheme 1: Accepted oxidation mechanism of Ascorbic acid

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Cyclic Voltammetry (CV) is considered the most informative electrochemical technique. A key
advantage of electro-analytical techniques is that they are less prone to common interferences
such as coloration, and presence of colloidal suspensions. CV is capable of providing a wealth of
information about an electrochemical system including details on the mechanisms and chemical
reversibility of REDOX processes. Redox properties of drugs can give insights into their
metabolic fate or their in vivo redox processes or pharmacological activity. Information related
to analyte concentration, electrode reaction kinetics, and diffusional contributions is all contained
in a cyclic voltammogram. CV experiments are performed by applying a triangular potential
sweep over time. Refer to readings 2 & 3 as well as Figures 25:23-24 in your Instrumental
Analysis text, for details on how to interpret a CV signal profile.
Modern electroanalytical voltammetric measurements are normally performed with software driven
potentiostats, which perform the voltage changes and record current, and an electrochemical cell
consisting of three electrodes (Figures 1&2).

Figure 1: Electrochemical cell set-up used for voltammetric analyses

V0 2
V0 1

dV0

if V01
V0

Figure 2: Potentiostat circuit diagram

1
R iC

t2
t1

v i dt

0 then,
1
R iC

t2
t1

v i dt

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The first of the three electrodes is the indicator or working electrode. This is the electrode at which
the electrochemical phenomena being investigated takes place. The second electrode is the
reference electrode, whose potential is constant enough that it can be taken as the reference
standard against which the potentials of the other electrodes present in the cell can be measured.
The third electrode is the counter or auxiliary electrode that serves as a source or a sink for
electrons so that current can be passed from the external circuit through the cell. The normal
material for cell construction is Pyrex glass for reasons both of visibility and general chemical
inertness. The cell lid is made from resistant PTFE plastic. A gas line for bubbling (purging) the
solution with nitrogen is also observed. Oxygen is electrochemically active and its solubility in
water is sufficiently great that oxygen reduction can be a problem. As a consequence, most
measurements are carried out under an inert atmosphere of either nitrogen or helium. The peak
current (Ip) in a CV experiment is governed by the Randle-Sevcik relationship:
ip

3
2

k n AD 0 2 v 0 2 C*0

where the constant, k, has a value of 2.686 x 105; n is the mole of electrons transferred per mole of
electroactive species; A is the area of the electrode in cm2; D is the diffusion coefficient in cm2/s; C
is concentration in mole/L; and v is the scan rate of the potential in volt/s. The ip is linearly
proportional to the bulk concentration C, of the electroactive species and the square root of the scan
rate, v1/2.
The linearity of a plot of the Ip vs V1/2 is a useful tool to confirm that the electrode reaction is
controlled by diffusion, which is the mass transport of electroactive species to the surface of the
electrode across a concentration gradient. The thickness, d, of the "Nernst diffusion" layer can be
approximated by: d ~ [Dt]1/2, where D is the diffusion coefficient and t is time in seconds. A "quiet"
(unstirred solution) is required. The presence of supporting electrolyte, such as the KCl or KNO3, is
required to eliminate movement of the charged electroactive species due to migration in the electric
field gradient. Another important feature to characterize the electrode reaction is the value of the
peak potential, Ep (Figure 3). When the rate of electron transfer is fast, the Ep value will be
independent of the scan rate and thus the value of Ip. Such a reaction is said to be reversible and
the difference between the anodic and cathodic Ep values is equal to 57 mV/n. Irreversibility is
when the rate of electron transfer is sufficiently slow so that the potential no longer reflects the
equilibrium activity of the redox couple at the electrode surface. In such a case, the Ep values will
change as a function of the scan rate. A unique feature of an electrochemical reaction is that a
"reversible" electrode reaction at low scan rates can become "irreversible" at higher scan rates.

Figure 3. Typical Cyclic voltammogram (left), applied potential program (right).

62
As shown in figure 4 CVs of ascorbic acid can exhibit peak currents for its oxidation at
approximately 490 mV versus the calomel electrode (450 mV versus Ag/AgCl). This
experiment, will measure the dependence of these waves on concentration, potential sweep rate,
and pH in McIlvaine buffers at pH 2.2 and 6.0. In addition, the concentration of ascorbic acid in
various commercial drinks and nutritional supplements will be determined

Figure 4. Cyclic voltammograms obtained for different ascorbic acid concentrations


Other useful resources:
1. Mosby; Mosby's nursing drug reference.; 2004; Hodges Reference: RM138 .S59.
2. P.T. Kissinger and W.R. Heineman, J. Chem. Ed. 1983, 60, 702.
3. Bard, A.J.; Faulkner, L.R.; Electrochemical Methods, p.p. 213-222; 228-236; 429434; and 445-465 (highly mathematical).
PRE-LAB EXERCISE:
1. Why is a high supporting electrolyte concentration used in most electroanalytical
procedures? Why is necessary to buffer the solutions in organic voltammetry?
2. What is the recommended daily value of vitamin C?
3. Describe what are the functions of a:
a. working electrode
b. reference electrode
c. auxiliary electrode
4. State the advantages and disadvantages of cyclic voltammetry relative to atomic
absorption spectroscopy.
5. From the electrochemical point of view, define the following terms and state when
they are used in an electrochemical analysis:
a. convection
b. diffusion
c. migration
d. supporting electrolyte

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APPARATUS AND MATERIALS:
1. Bioanalytical CV-50
2. Three-electrode electrochemical cell, equipped with the following electrodes:
working electrode (glassy carbon)
auxiliary electrode (Pt wire)
reference electrode (saturated calomel electrode (SCE))
3. Citric acid
4. Sodium phosphate dibasic
5. Potassium chloride
6. Juice and vitamin C tablet (PROVIDED BY THE STUDENTS)
EXPERIMENTAL:
I. Dissolution media
1. Prepare 1 liter of pH 2.2, buffer pH 6.0 McIlvaine buffers. The ionic strength of both
buffer solutions must be 0.5 M (see table 1 below).
II. Analytical Standards
1. Prepare a 100 ml stock solution of Ascorbic Acid (AAc) 0.01 M in deionized water.
2. Prepare three AAc standards (2.0x10-3 M) in DI-Water, pH 2.2 buffer and pH 6.0 buffer,
respectively. You will need only approximately 50 mL of each solution.
3. Prepare four additional AAc standards within the 5x10-4 M to 6x10-3 M range in buffer
pH 2.2 (100 mL/sample).
III. Quality Control Standards
1. Prepare one Quality Control standard of AAc (5x10-4 M to 6x10-3 M), diluted in buffer
pH 2.2.
IV. Preparation of the Commercial APAP Sample (Unknown)
1. Determine the mass of a commercial AAc tablet. Dissolve the tablet in pH 2.2 buffer
and quantitatively transfer to a 100 mL volumetric flask and complete to mark with pH
2.2 buffer. Label this sample as AAc Unknown Tablet stock.
2. Transfer enough of the AAc stock to prepare a 3x10-3 M solution of the unknown in a
100 mL volumetric flask and complete to the mark with pH 2.2 buffer. Label this sample
as Unknown Tablet aliquot.
3. Using the reported vitamin C content of the juice, transfer enough of juice to prepare as
close as possible to 3x10-3 M in a 100 mL volumetric flask and complete to the mark
with pH 2.2 buffer. Label this sample as Unknown Juice aliquot.
V. Analysis
1. Begin the measurements with the 1x10-3 M solution in pH 2.2 buffer. When filling the
cell with this (or any other) solution, add enough solution to dip the electrodes between
0.5-1 inch into the solutions. AVOID TILTING OR DRYING THE REFERENCE
ELECTRODE IN THE PROCESS. Purge the solution with N2 for about 5 min. Then,

64
allow N2 to flow over the liquid surface not through the solution for the remainder of the
measurement.
2. Set the potential scan parameters as follows:
Initial potential:
-0.1 V
Sweep segments:
2
High potential:
+1.0 V
Sample interval:
0.001 V
Low Potential:
-0.1 V
Quiet time:
10 s
direction of scan: positive
Sensitivity*:
1E-04 A/V
scan rate:
50 mV/s
* You might need to adjust the sensitivity for experiments at lower concentrations.
3. Connect the three electrodes and purge the solution with nitrogen gas for 5 seconds.
4. Using the second AAc calibration standard, initiate the scan at a sensitivity of 1x10-4
A/V. Then repeat the scan, using 1x10-5, and 1x10-6 A/V. Set the conditions to that
which gives the best voltammetric wave.
5. Obtain CVs for the same solution at scan rates of 100 mV/s and 250 mV/s, respectively.
Set the conditions to that which gives the best voltammetric wave.
6. Obtain CVs for the 2.5x10-3 M AAc standards using the optimized acquisition
parameters.
NOTE: Before removing a particular solution from the cell, disconnect the working
electrode. After removing a solution from the cell, rinse it with distilled water before
adding the next solution. Whenever a new solution is introduced into the cell, follow
the procedure outlined under #'s 1-2 above.
7. Obtain CVs for the three AAc standards (2x10-3 M) in DI-Water, pH 2.2 buffer and pH
6.0 buffer.
8. Perform a replicate analysis (triplicate), on each AAc standard, QC standards, and
Unknown aliquots at pH 2.2. Then, quantify the APAP content of the unknown tablet.
9. Construct an analytical calibration plot for with the pH 2.2 standards and determine the
amount of AAc in each of the original samples.
DATA ANALYSIS:
1. Determine if the electro-active species observed in the cyclic voltammograms are
consistent with those of figure 4.
2. Discuss how changes in sensitivity, scan rate and pH affected your analysis.
3. Use the calibration data to calculate:
correlation coefficient (R2)
equation of the line with its corresponding uncertainty
limit of detection (LOD)
Determine the amount of AAc in each sample with its propagated uncertainty,
confidence level and percent relative error.
4. Calculate the reliability of your experiment based on the results from the QC sample.
QUESTIONS:
1. Discuss the reliability of your findings and what you will do to improve this method.
2. Based on the voltammetric results does the oxidation of AAc was reversible in your study?

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3. Based on the average oxidation potential AAc what would be the observed potential when
a SHE and calomel electrodes are used?
4. Does there was a difference in the precision and accuracy of the QC results vs. those of the
unknowns? Explain
5. Why it is important to clean the working electrode between runs?
Table I. Preparation of Constant Ionic Strength McIlvaine Buffered Solutions:
The desired buffers are the so-called McIlvaine buffers@ (T.C. McIlvaine, J. Biol. Chem. 1921, 49, 183). Directions for preparing the buffers
needed for this experiment are given in the table below (from P.J. Elving et al., Anal. Chem., 1956, 28, 1179).

Buffer
Composition g/Liter
pH
Solution
Desired
Na2HPO4 2
H2O
at 25 C
2.2
1.43
2.4
4.44
2.6
7.80
2.8
11.35
3.0
14.7
3.2
17.7
3.4
20.4
3.6
21.5
3.8
25.4
4.0
27.6
4.2
29.7
4.4
31.6
4.6
33.4
4.8
35.3
5.0
36.9
5.2
38.4
5.4
40.0
5.6
41.5
5.8
43.3
6.0
45.2
6.2
47.5
6.4
49.6
6.6
52.1
6.8
55.4
7.0
58.9
7.2
62.3
7.4
65.0
7.6
67.2
7.8
68.6
8.0
69.6

H3C6H5O7 = citric acid

H3C6H5O7 H2O

20.6
19.7
18.7
17.7
16.7
15.8
15.0
14.2
13.6
12.9
12.3
11.7
11.2
10.7
10.2
9.75
9.29
8.72
8.32
7.74
7.12
6.47
5.72
4.79
3.70
2.74
1.91
1.35
0.893
0.589

System
Ionic
Strength,
M
0.0108
0.0245
0.0410
0.0592
0.0771
0.0934
0.112
0.128
0.142
0.157
0.173
0.190
0.210
0.232
0.256
0.278
0.302
0.321
0.336
0.344
0.358
0.371
0.385
0.392
0.427
0.457
0.488
0.516
0.540
0.559

g KCl Added per


Liter of Solution to
Produce Ionic
Strength of
1.0 M
0.5 M
74.5
37.2
72.7
35.4
71.5
34.2
70.2
32.9
68.7
31.4
67.6
30.3
66.2
28.9
64.9
27.6
64.0
26.7
62.8
25.5
61.7
24.4
60.4
23.1
58.9
21.6
57.2
19.9
55.5
18.2
53.8
16.5
52.1
14.8
50.6
13.3
49.5
12.2
48.9
11.6
47.9
10.6
46.9
9.62
45.8
8.50
44.5
7.23
42.7
5.44
40.4
3.10
38.2
0.488
36.0
34.3
32.9

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OPERATING INSTRUCTIONS FOR THE
BAS CV-50W VOLTAMMETRIC SYSTEM
1.
2.
3.
4.

Turn on the computer.


Choose CV-50W.
Turn ON the potentiostat (switches on the back).
The self-test is done automatically by the instrument, only if the method is chosen with the
potentiostat ON. If not, do the self-test by going to control, choose CV-500W, test, close.
5. Place solution in the sample container, add a magnetic bar (if needed).
6. Connect electrodes according to the legend placed at the cell compartment (Order suggested:
reference (Ag/AgCl), auxiliary (Pt), working electrode (glassy carbon).
7. Open nitrogen cylinder.
8. Go to Set up, select Purge, then OK, to purge (degasify the solution for 8 to 10 minutes).
Samples must be purged for improved performance and reliability. If you already have
the next sample ready, you can take it to the second cell and purge it simultaneously.
9. Go to Mode- Choose type of voltammetry: e.g. CV, DPV, or LSSV.
10. Set parameters, both general and specifics (as
indicated in the experiment). Set initial
voltage, scan rate, etc., according to
experimental parameters required for the
method chosen (see Figure 5). Once all
values have been set, click on OK to
continue.
11. Go to Set Up, and stop the purge by clicking
off the purge button.
12. Go to Run, to start acquiring the
voltammogram.
13. Select Display, single plot, to display the
voltammogram with better resolution.
14. Select Print, to obtain a hard copy of the
voltammogram.
NOTES:
1. Make sure that no air bubbles stay on the Figure 5: CV Parameters
electrode surface after purging, this affects
the analysis.
2. Check cleanliness of the glassy carbon electrode, its surface should be clean and polished.
3. Rinse well all the sample tubes and electrodes prior to analysis. When connecting electrodes,
the working electrode is the last one connected. However, when disconnecting the electrodes,
it is the first one to be disconnected.
4. Please, remember to close the nitrogen cylinder at the end of the experiment, turn off the
computer, potentiostat and plotter.