Electrochemical Determination of Ascorbic Acid in Pharmaceutical Tablets using Carbon Paste Electrode
Zelalem Bitew and Meareg Amare*
Department of Chemistry, Bahir Dar University, Bahir Dar, Ethiopia
Submission: July 08, 2019;Published: August 30, 2019
*Corresponding author: Meareg Amare, Department of Chemistry, Bahir Dar University, Bahir Dar, Ethiopia
How to cite this article: Zelalem Bitew and Meareg Amare. Electrochemical Determination of Ascorbic Acid in Pharmaceutical Tablets using Carbon Paste
Electrode. Organic & Medicinal Chem IJ. 2019; 8(5): 555749. DOI: 10.19080/OMCIJ.2019.08.555749
Cyclic voltammetry was used to study the electrochemical behavior of ascorbic acid at carbon paste electrode. Ascorbic acid showed an irreversible oxidation peak at about +470 mV. While the observed peak potential shift with pH in the range 0.5 to 6.0 indicated the participation of protons during the oxidation of ascorbic acid, the peak potential shift with scan rate in the range10–300 mV/s confirmed the irreversibility of the oxidation reaction. Under optimized method and solution parameters, an excellent linear dependence of the oxidative peak current on concentration of ascorbic acid was observed in the range 10-100 μM with limits of detection (LOD) and quantification (LOQ) of 1.76 and 5.86 μM, respectively. Detection of 93.33 to 97.58% of the expected ascorbic acid content in two local tablet formulations analyzed using the method confirmed the accuracy of the method. Recovery results in the range 91.65-91.83% for spiked standard ascorbic acid in pharmaceutical tablets further confirmed the potential applicability of the developed method for the determination of ascorbic acid in real samples.
Ascorbic acid, a water-soluble vitamin (Vitamin C), is important in forming collagen, a protein that gives structure to bones, muscles and blood vessels. It is the most common ubiquitous electroactive vitamins ever discovered. Ascorbic acid plays a paramount role as an antioxidant and free radical scavenger [1,2] and hence is a vital component in human diet with the highest concentrations in animal organs like liver, leukocytes, and anterior pituitary . It is widely used in the treatment of certain diseases including scurvy, common cold, anemia, hemorrhagic disorders, wound healing as well as infertility [4,5].
Ascorbic acid (AA) known for its reductive properties is used as an antioxidant agent in foods and drinks, for therapeutic purposes and biological metabolism [6-8]. The human body cannot produce ascorbic acid, and so it must be obtained entirely through one’s diet. Therefore, humans depend on exogenous sources of the vitamin which include fruits and vegetables as well as food supplements and pharmaceutical preparations . The physicochemical and biochemical actions of vitamin C (scheme 1) are accounted for its action as an electron donor [9-11].
The amount of vitamin C required in a healthy diet varies with age and gender. According to Natural Health Product
Monograph, children ages 1-3 require 15 mg/day, adult females 75 mg/day, and adult males need 90 mg/day . Although toxicity of AA is very rare, an intake of AA greater than 2,000 mg/day is not recommended for it may lead to stomach upset and diarrhea. A link between the high dose of AA in human body and increased risk of calcium oxalate kidney stones have also be reported although refutal studies are also reported [12,13].
Titration , chromatography , fluorimetry , and spectrophotometry  are among the analytical methods commonly reported for determination of AA in real samplesincluding pharmaceutical formulations, foods, fruits, and
biological fluids. However, some of these methods are timeconsuming,
while others are costly, require special training for
operators of the equipment, or suffer from insufficient sensitivity
or selectivity [14,17].
On the other hand, recently developed electrochemical
methods are more promising because they possess quick response
times, low cost, simplicity of instrumentation, high sensitivity, and
the possibility of miniaturization [18,19]. Considerable efforts
have been made on the development of electrochemical methods
for determination of AA in pharmaceutical formulations using Pt
electrode , aluminum modified with nickel hexacyanoferrate
film , glassy carbon electrode , and Bi2O3 /Glassy carbon
electrode . Although the reported electrochemical methods
are sensitive with low detection limit, most of them have used
expensive electrodes/modifiers. Thus, the development of
simpler, cost effective, and sensitive method is needed for the
determination of ascorbic acid in pharmaceutical formulations.
In this study, square wave voltammetry determination of ascorbic
acid in tablet samples using carbon paste electrode, which is the
simplest and cheapest electrode material, is presented
Ascorbic acid (Blulux chemicals Ltd., India), graphite powder
(BDH-Laboratory supplies Poole, England), Vitamin C tablets of
two Ethiopian brands (EPHARM, and APF), paraffin oil (Abron
Chemicals), disodium hydrogen phosphate, sodium dihydrogen
phosphate, NaOH, and HCl all (Blulux laboratories (p), Ltd) were
used . Distilled water was used throughout the work.
A BAS 100B electrochemical analyzer [bioanalytical systems
(BAS), USA] with carbon paste electrode as working electrode,
platinum coil wire as auxiliary electrode and Ag/AgCl as
reference electrode was used. A Jenway model 3310 pH meter
and electronic balance (Denver instrument) were used for
measuring the pH of the buffer solutions and mass of chemicals,
Phosphate buffer solutions (PBS) in the pH range 0.5-6.0
were prepared from a mixture of 0.1 M NaH2PO4 and 0.1 M
Na2HPO4 in distilled water. 1 M NaOH and 1 M HCl solutions
were used to adjust the pH of the buffer solutions. Stock solution
of 0.01 M ascorbic acid was prepared by dissolving 0.1761 g of
ascorbic acid in 100 mL of the phosphate buffer solution (pH 2)
from which working standard solutions were prepared through
Vitamin C tablets of two Ethiopian brands, Ethiopian
Pharmaceuticals Manufacturing (EPHARM) and Addis
Pharmaceutical Factory (APF) were purchased from a local
pharmacy. Five tablets (500 mg Ascorbic acid/tablet) from
each brand were accurately weighed and finely powdered
in a porcelain mortar. An adequate amount of this powder
corresponding to a stock solution of 1*10-2 mol L-1 ascorbic acid
was weighed, transferred into a 100 mL flask, and diluted to the
mark with pH 2 PBS. After filtering with Watmann No 1 filter
paper, tablet samples with 60, 100, and 300 μM tablet sample.
Carbon paste electrode was prepared following the reported
procedure . Briefly, 1 g of carbon paste was prepared by
mixing graphite powder with paraffin oil in a ratio of 70:30
(w/w), respectively. The mixture was homogenized with mortar
and pestle for 30 minutes and allowed to rest for 24 hrs. The
homogenized paste was packed into the tip of a plastic tube
of a diameter about 3.5 mm. A copper wire was inserted from
the backside of the plastic tube to provide electrical contact.
The surface of the electrode was smoothed manually against
a smooth white paper until a shiny surface is emerged and the
electrode was made ready for use.
The electrochemical behavior of AA at CPE, effect of scan
rate in the range of 10 to 300 mV s-1 on the peak current and
peak potential, and the influence of pH in the range 0.5 – 6.0
on the peak potential and peak current of ascorbic acid were
investigated using cyclic voltammetry in the potential window
-100 to 1000 mV and scan rate of 50 mV s-1. Square wave
voltammetry in the potential range -100 to +1000 mV was also
used for quantitative determination of ascorbic acid. External
standard addition method was employed for quantification of
the ascorbic acid content in two brands of tablet formulation
Electrochemical Behavior of Ascorbic Acid at CPE
Cyclic voltammetry was used to investigate the
electrochemical behavior of AA at carbon paste electrode.
Figure 1 presents the cyclic voltammograms of CPE in pH 2 PBS
containing (a) no AA and (b) 0.5 mM AA. While no peak was
observed in the absence of AA (curve a of Figure 1), appearanceof an intensive peak centered at about +470 mV in the oxidative
scan direction without a peak in the reductive scan direction
implied an irreversible oxidation of AA at carbon paste electrode.
Figure 2 describes the cyclic voltammograms of 0.5 mM AA
in pH 2 at the scan rate range of 10 to 300 mV/s. As can be seen
from the figure, the increase in anodic peak current (Ipa) with
scan rate accompanied by peak potential shift in the positive
direction confirmed the irreversibility of the oxidation reaction
In order to investigate whether the oxidation process of
ascorbic acid at CPE is predominantly diffusion controlled or
surface confined process, the dependence of peak current on the
scan rate and square root of scan rate was compared. A better
correlation coefficient for the dependence of peak current on the
scan rate (R = 0.99637) (Inset of Figure 2) than on the square
root of scan rate (R = 0.99105) (figure not shown) indicated
that the oxidation of AA at CPE is predominantly governed by
The effect of pH on the oxidation peak current and peak
potential of AA at CPE was studied in the pH range 0.5-6.0. The
cyclic voltammograms of 0.5 mM of AA in PBS of various pH are
shown in Figure 3. As can observed from Fig. 4a, the anodic peak
current increased sharply from pH 0.5 to 2 which then gradually
decreased at pH higher than 2. Thus, pH 2 was selected as the
optimum pH of the buffer solution in the subsequent experiments
which agrees with previously reported works .
The influence of pH on the oxidative peak potential of ascorbic
acid was also examined. The oxidation peak potential shift in
the negative potential direction with increase of pH indicates
participation of protons during oxidation of AA at CPE (Figure
4). As can be observed from the figure, the potential shift showed
linear dependence with the pH in the range 0.5 to 4.0 with a
slope of 0.063 V/pH indicating that the number of protons and
electrons taking part in the electrode reaction were in a 1:1 ratio
. Hence, a reaction mechanism (scheme 2) was proposed for
the oxidation of ascorbic acid at carbon paste electrode which
agrees with previous reports .
Square wave voltammetry, which is one of the most
sensitive voltammetric techniques, was used for the quantitative
determination of ascorbic acid. The electrochemical oxidation of
ascorbic acid at carbon paste electrode was studied by square
wave voltammetry in the potential range from -100 to 1000 mV.
Figure 5 represents the square wave voltammograms of CPE in
pH 2 PBS in the absence (a) and presence (b) of 0.5 mM AA. The
oxidative peak centered at about +410 mV in the presence of
AA (curve b) while no peak in the absence of AA (curve a) was
assigned for oxidation of AA.
The effects of the square wave parameters frequency,
amplitude and step potential, and accumulation parameters
(Eacc and tacc) on the oxidative peak current of AA at CPE
were investigated. As can be seen from the Inset of Figure 6, the
magnitude of the peak current increased with increasing the
square wave frequency. However, the peak current increment
was accompanied by peak broadening and peak potential shift in
the positive direction besides the peak current instability which
affected the reproducibility of the measurement. As a compromise
between the increased peak current and accompanied poor
current reproducibility, frequency of 25 Hz was chosen as
the optimum value in the subsequent experiments. Figure 7
demonstrates the effect of the amplitude on the oxidative peak
current of AA at CPE. As expected, the peak current increased
linearly with amplitude. However, the peak width also increased
with square wave amplitude.
The effect of square wave amplitude on peak current is shown
(Figure 7). Upon increasing the square wave amplitude, a linear
increase in the peak current was observed accompanied by peak
broadening when the amplitude was greater than 50 mV. Thus,
50 mV was chosen as the optimum square wave amplitude which
is a compromise between the peak height and the peak shape.
The effect of square wave step potential on the peak current
response of CPE for ascorbic acid was studied by varying the step
potential from 2 mV to 22 mV (Figure 8). Hence, as a compromise
between the peak current enhancement with increasing step
potential (Inset of Fig. 8) accompanied by peak broadening, a
step potential of 14 mV was chosen as the optimum square wave
step potential for further work.
Since the oxidation of ascorbic acid at CPE is governed
predominantly by surface confined kinetics, the effects of
accumulation time and accumulation potential were investigated,
by varying one of them and maintaining the other constant.
Figure 9a shows the effect of accumulation potential (Eacc) on
the square wave peak current for 0.5 mM AA over the range of
300-600 mV. As can be shown from the figure, the peak current
increased with increasing the accumulation potential from up to
A peak current decrease was observed at accumulation
potentials higher negative than 350 mV and hence, a
preconcentration potential of 350 mV was taken as the optimum
accumulation potential throughout the present work. Figure 9b
presents the effect of accumulation time on the oxidative peak
current of 0.5 mM AA at constant accumulation potential of 350
mV. As can be seen from the figure, the peak current increased
with the increase in accumulation time up to 20 s and then
almost leveled off. The increase of peak current with increase in
accumulation time indicated that AA can be accumulated at the
surface of the CPE. The leveling off peak current after 20 s could
be ascribed to the saturation of the surface of the electrode.
So, the accumulation time of 20 s was selected as an optimum
accumulation time for this work (Figure 9) (Table 1).
Under the optimum experimental conditions, the
dependence of square wave voltammetric oxidative peak
current on the concentration of AA and inherited sensitivity of
the method was investigated in the range 1 * 10-5 – 6 * 10-4.
While Figure 10 presents the square wave voltammograms of
various concentrations of AA in pH 2 PBS at CPE, Inset of Fig.
10 shows the linear dependence of the oxidative peak current
on concentration of AA in the studied range with regression
equation, and correlation coefficient (R2)) of Ipa (μA) = 0.1343
- 0.0442 C (μM), and 0.99978, respectively. The corresponding
method limit of detection (LOD = 3s/m) and limit of quantification
(LOQ = 10s/m) were calculated to be 1.76 *10-6 and 5.86 *10-6,
respectively where s is the mean blank standard deviation for n
= 8 and m is the slope of the regression equation.
The performance of the developed method for the detection
of AA was compared with other reported methods. The developed
method using CPE, which is the cheapest carbon-based electrode
material, showed a comparable LOD and hence sensitivity with
other reported works which have used expensive electrode
materials (Table 2).
The applicability of carbon paste electrode for determination
of AA was demonstrated by applying it to determine the AA
content in some pharmaceutical preparations. In this work,
two brands of vitamin C tablets (APF, and EPHARM) prepared
following the procedure described under the experimental
section were selected for the analysis of AA. Finally, 70, 100,
and 400 μM tablet sample solutions were prepared from the
corresponding stock solution. Square wave voltammograms
were recorded (Figure 11) following the outlined voltammetric
procedure and optimized conditions as described earlier. Mean
of triplicate measurements was taken for the determination of
AA in these samples.
The amount of AA found in the different concentrations of
the two brands of tablets is summarized in Table 3. For EPHARM
and APF tablets the prescribed levels of AA were 500 mg per
tablet whereas the amount of AA detected relative to the level
were about 91.105% and 91.173%, respectively. Detected values
lower than prescribed value may be due to the oxidation of AA
during preparation or sort of degradation during storage or may
contain lower levels of AA in tablets.
To further evaluate the performance of the method, the
recovery 100 μM standard AA from spiked tablet samples of the
two brands each of 100 μM AA as per to the label. As can be seen
from Table 4, excellent recovery results in the range of 91.65%
to 91.83% with low RSD in the range 0.04-1.14 confirmed the
potential applicability of the developed method for AA analysis
in real samples.
Cyclic voltammetric investigation of Ascorbic acid at CPE
showed that the oxidation of Ascorbic acid over the studied
range of scan rates is irreversible. While peak potential shift
with the scan rate confirmed the irreversibility of the reaction,
peak potential shift with pH also indicated the involvement of
protons in the oxidation process. The ease of preparation of the
electrode in combination with the relatively low detection limit,
better selectivity and sensitivity, very good recoveries relative to
previously reported works which have used expensive electrodes
illustrated the potential applicability of the developed method as
an alternative method for the determination of Ascorbic acid in
real samples like pharmaceutical formulations.
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