QUANTITATIVE ANALYSIS OF ASCORBIC ACID
USING HYDRODYNAMIC MODULATION
VOLTAMMETRY
The goal of this experiment is to gain an understanding of the use of
voltammetry for the quantitative analysis of electroactive species in
solution. In this experiment you'll be introduced to stationary (cyclic)
voltammetry and hydrodynamic (linear sweep) voltammetry.
Hydrodynamic modulation voltammetry will then be used to quantify the
ascorbic acid content of a commercial fruit juice. The calibration method
used in this experiment is the method of standard additions.
REFERENCES/ASSIGNED READING: The following should be read prior to
your pre-lab meeting with Prof. Truttmann
1. Principles of Instrumental Analysis, 5th Edition, Douglas A. Skoog, F. James Holler,
Timothy A. Nieman, Saunders College Publishing, Philadelphia, 1998, Chapter 25
sections A-D and pages 15-18.
2. "Sensitive Electroanalysis Using Solid Electrodes," Joseph Wang, J. Chem. Ed., vol.
59(8), 1982, p. 691-692.
INTRODUCTION:
Electrochemistry comprises a group of important analytical techniques that can
provide quantitative information on the composition of an analyte as well as information
about standard reduction potentials and rates of redox and following chemical reactions.
Due to the variety of information that can be obtained as well as the relatively low cost of
the equipment and the convenience of the experiments, electrochemical techniques are
widely used. Electrochemical detectors are also becoming popular for chromatographic
separations.
There are three main types of electrochemical experiments: potentiometry,
coulometry, and voltammetry. Potentiometry is the measurement of the potential of a
solution under conditions of low current flow. pH and other ion selective electrodes are
common examples of the use of potentiometry to quantify species in solution.
Coulometry involves the quantitative conversion of an analyte via the transfer of
electrons. The total charge or current as a function of time are monitored in a coulometry
experiment in order to quantify the amount of analyte converted. Voltammetry involves
the measurement of the current at an electrode as a function of the applied potential.
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Although more commonly used for studies of redox potentials and kinetics, special forms
of voltammetry can be effectively used for the quantitative analysis of electroactive
species. Polaragraphy and stripping voltammetry using a mercury drop electrode are
common methods.
In this experiment, you'll use hydrodynamic modulation voltammetry (HDMV) at
a platinum disk electrode to quantify the ascorbic acid content of a commercial fruit juice
sample. In HDMV, the electrode potential is held constant while the rate of mass
transport of solution to the electrode surface is modulated via control of the stirring rate.
Because the electrode is held at a constant potential, charging effects due to changing
electrode potentials are diminished. In a stirred solution, a steady-state current is
established that depends on the applied potential, the concentration of the electroactive
species, and the rate of stirring, among other parameters. At high enough potentials, the
surface concentration of the analyte is essentially zero and the current becomes
independent of the applied potential. Quantitative analysis is generally performed in this
limiting current region. The advantages of HDMV for this experiment are the extended
potential range of the platinum electrode (mercury is oxidized at relatively low potentials)
and the lack of mercury waste generation. Solid electrodes are also more useful as
detectors in chromatography units for the above reasons, and thus it is important to
understand how they work.
OH
O
O
HO
HO
OH
Figure 1. Ascorbic Acid. The first pKa is at 4.17 and the second is at 11.571
Ascorbic acid, or vitamin C, is an important nutrient. Various methods exist for
quantifying ascorbic acid in foodstuffs. Although ascorbic acid absorbs light in the UV
region, spectrophotometric methods for the determination of ascorbic acid in foodstuffs
often suffer from interference due to absorbance by other components. Titration methods
are also not very selective and have rather poor detection limits. Chromatography is
commonly used with a variety of detectors including UV and electrochemical. Ascorbic
acid is easily oxidized to dehydroascorbic acid, which undergoes further chemical
reaction to form the gem-diol. The oxidation potential is pH dependent. The
electrochemical reaction can be induced and monitored by voltammetry to quantify the
concentration of ascorbic acid in solution.
1
The Merck Index, 11th Ed., Merck & Co, Inc., 1989, p. 131.
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EXPERIMENTAL:
A. Solution Preparations (note: ascorbic acid solutions are oxidized in air, therefore
the entire experiment should be carried out in one day):
In order to complete this experiment, you'll need to prepare the following
solutions. Before coming to lab, determine how you will make each of these solutions,
including amounts to weigh out and what glassware you'll use etc. An important skill of
the analytical chemist is to know what level of accuracy is required for a given task.
Check with your instructor if you're unsure about which equipment to use. Record your
experimental procedure in your notebook and turn in the yellow pages with your lab
report.
1. 250 mL of a 0.10 M phosphate buffer at pH 2 containing 0.5 g/L Na2EDTA to
prevent premature oxidation of the ascorbic acid. Phosphoric acid and sodium
dihydrogen phosphate are available. Deaerate this solution by bubbling a stream
of nitrogen gas through the solution for about 15 minutes.
2. 100 mL of 5 mM ascorbic acid solution using the deaerated buffer prepared in
step one. This solution will be used to explore the electrochemical behavior of
ascorbic acid as well as for making standard additions. Keep tightly closed to
minimize air oxidation of the ascorbic acid.
3. 100 mL of a 3:100 (3%) dilution of a commercial juice drink in the deaerated
buffer prepared in step one. Keep tightly closed to minimize air oxidation of the
ascorbic acid.
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B. Initial Electrochemical Characterization Using Cyclic Voltammetry and Linear
Sweep Voltammetry:
Your instructor will demonstrate proper handling of the electrodes and how to
use the BAS CV50 potentiostat for performing the electrochemical experiments.
The manual for the CV50 is also available in the lab for you to use. The
instrument is connected to a printer from which you can print plots generated by
the CV50. The data for each run should be saved for future reference and/or
plotting (each run overwrites the previous data unless saved). The default format
is .BIN, which is recognized only by the CV50 software. If you'd like to work up
your data in excel, you can save the data as .TXT files on a floppy for later
plotting.
Cyclic voltammetry is often the first electrochemical method chosen to study a
system because it can quickly tell you about the redox potential of the analyte and
whether the reaction is reversible or not. Use your 5mM ascorbic acid solution and
record a cyclic voltammogram using the following parameters
potential region: 200 to +1000 mV
scan rate: 50 mV/sec
sensitivity: 10 A/V
Blanket the solution with nitrogen gas during the experiment. Positioning of the
electrodes is important. The platinum working electrode should be as close as possible to
the stir bar without touching it. The o-ring on the electrode allows you to reproducibly
position the electrode from run to run. The reference and counter electrodes should be
positioned somewhat above the working electrode to minimize turbulence in the
following stirred experiments. Repeat the above experiment while stirring the solution.
This is an example of hydrodynamic voltammetry, also called linear sweep voltammetry.
What do these two scans tell us about the oxidation of ascorbic acid?
Why are the shapes of the two voltammograms so different?
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Ascorbic acid is not the only component in our solution that can undergo redox reactions
at the platinum electrode. Other possible reactions include the oxidation of water, the
reduction of oxygen, and/or redox reactions of the phosphate or EDTA molecules. To
determine the relative importance of these reactions over this potential range, perform
cyclic (unstirred) and linear sweep (stirred) voltammetry for the buffer solution alone.
C. Potential Dependence of Ascorbic Acid Oxidation and Introduction to
Hydrodynamic Modulation Voltammetry:
Because the difference in current for stirred and unstirred solutions is measured for
HDMV, the influence of background charging currents is minimized. Following the
example in reference 2, obtain a hydrodynamic voltammogram for your 5mM ascorbic
acid solution. Use the time-base mode of the BAS CV50 and manually start and stop the
stirring using the switch on the instrument (stir rate = 600 rpm). Precondition the
electrode for one minute at the applied potential with stirring before modulating the
stirring, and complete 3 cycles of on-off stirring at each potential (total run time = 240
sec). Use the results from your cyclic and linear sweep voltammograms to choose
appropriate potentials to apply to the platinum electrode. You should make
measurements at 5 different potentials including 2 in the limiting current region. Perform
similar runs on your blank buffer solution to verify the lack of interfering species. Three
potentials should be sufficient for the blank, but be sure to include the potential at which
you propose to run the concentration dependence experiment (i.e. one of the potentials in
the limiting current region for ascorbic acid). Between each run, remove the platinum
working electrode (being careful not to move the positioning o-ring) and wipe the
electrode clean with a tissue soaked in methanol.
D. Concentration Dependence of Ascorbic Acid Oxidation- Hydrodynamic Modulation
Voltammetry:
In this part of the experiment you will verify the linear dependence of the limiting
current on concentration. Based on your results from part C, choose a potential in the
limiting current region with minimal interference from the blank. Starting with 10.00 mL
of the buffer solution, perform an HDMV at the chosen potential. Add 200-L of your
5mM stock ascorbic acid solution and repeat the run. Make four more 200-L additions,
recording an HDMV after each one. Remember to clean the electrode with methanol as
in part C. Plot the observed change in current versus concentration and perform a linear
regression. Check with your instructor if linear behavior is not observed.
HDMV
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E. Hydrodynamic Modulation Voltammetry--Determination of Ascorbic Acid Content
of Fruit Juice (to be completed on the second day):
In this part of the experiment, you'll determine the concentration of ascorbic acid in a
sample of processed fruit juice. Perform an HDMV run on 10.00 mL of your diluted
juice sample and then make appropriate additions of your 5mM ascorbic acid stock
solution and further HDMV runs to obtain a standard addition calibration plot.
Remember to clean the electrode with methanol as in part C. Report the concentration of
the original fruit juice sample on your report sheet.
REPORT:
Your lab report for this experiment includes your yellow sheets (which describe your
experimental work and show sample calculations), any plots or graphs that you generate,
and the attached report sheets including answers to the discussion questions (these may
be typed and attached as an additional sheet). The following graphs should be included
with your report:
1.
2.
3.
4.
5.
cyclic voltammogram of 5mM ascorbic acid solution
linear sweep voltammogram of 5mM ascorbic acid solution
cyclic voltammogram of buffer solution
linear sweep voltammogram of buffer solution
multigraph plot of HDMV runs of 5 mM ascorbic acid solution at different
potentials
6. multigraph plot of HDMV runs of buffer solution at different potentials
7. multigraph plot of HDMV runs of the concentration dependence of ascorbic acid
8. multigraph plot of HDMV runs standard additions to diluted juice sample
9. plot of current difference versus potential for the 5 mM ascorbic acid solution
10. plot of current difference versus potential for the buffer solution
11. plot of the current difference versus concentration for the ascorbic acid sample
12. plot of the current difference versus added concentration of ascorbic acid for the
diluted juice sample (standard addition plot)
QUANTITATIVE ANALYSIS OF ASCORBIC ACID USING
HYDRODYNAMIC MODULATION VOLTAMMETRY
NAME
DATE
A. Solution Preparations
1. mass NaH2PO4
volume H3PO4
mass EDTA
2. mass NaOH
mass KHP
volume NaOH to titrate KHP
standardized [NaOH]
3. mass ascorbic acid
volume NaOH to titrate ascorbic acid
standardized [ascorbic acid] in stock solution
4. [ascorbic acid] in diluted solution
B. Electrochemical Characterization
Estimated E1/2 for oxidation of ascorbic acid in pH2 phosphate buffer
Discuss the shapes of the cyclic and linear sweep voltammograms (e.g. why are they
different?) and what information can be obtained from them.
HDMV
C. HDMV-Potential Dependence
Limiting current region
Chosen potential for concentration studies
D. HDMV-Concentration Dependence
slope and 95%CI
y-intercept and 95%CI
R2
E. HDMV-Fruit Juice Sample
slope and 95%CI
x-intercept and 95%CI
R2
[ascorbic acid] in diluted sample
[ascorbic acid] in original sample
What other ingredients are present in the fruit juice sample that might undergo redox
reactions in this potential range? Does the method of standard additions reduce the
influence of these potential interferences?
Attach all graphs and plots, and show sample calculations.