# Ascorbic Acid Determination in Commercial Fruit Juice Samples by Cyclic Voltammetry

Authors: Aurelia Magdalena Pisoschi; Andrei Florin Danet; Slawomir Kalinowski
Journal: Journal of Automated Methods and Management in Chemistry
 (2008)

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## Abstract

A method was developed for assessing ascorbic acid concentration in commercial fruit juice by cyclic voltammetry. The anodic oxidation peak for ascorbic acid occurs at about 490 mV on a Pt disc working electrode (versus SCE). The influence of the potential sweep speed on the peak height was studied. The obtained calibration graph shows a linear dependence between peak height and ascorbic acid concentration in the domain (0.1–10 mmol⋅L−1). The equation of the calibration graph was y=6.391x+0.1903 (where y represents the value of intensity measured for the anodic peak height, expressed as μA and x the analyte concentration, as mmol⋅L−1, r2=0.9995, r.s.d.=1.14%, n=10, Cascorbicacid=2 mmol⋅L−1). The developed method was applied to ascorbic acid assessment in fruit juice. The ascorbic acid content determined ranged from 0.83 to 1.67 mmol⋅L−1 for orange juice, from 0.58 to 1.93 mmol⋅L−1 for lemon juice, and from 0.46 to 1.84 mmol⋅L−1 for grapefruit juice. Different ascorbic acid concentrations (from standard solutions) were added to the analysed samples, the degree of recovery being comprised between 94.35% and 104%. Ascorbic acid determination results obtained by cyclic voltammetry were compared with those obtained by the volumetric method with dichlorophenol indophenol. The results obtained by the two methods were in good agreement.


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## Body

## 1. Introduction

Ascorbic acid (vitamin C) is a water-soluble vitamin which can be found in many biological systems and foodstuffs (fresh vegetables and fruits, namely, citrus). Ascorbic acid plays
an important role in collagen biosynthesis, iron absorption, and immune response activation and is involved
in wound healing and osteogenesis. It also acts as a powerful antioxidant which
fights against free-radical induced diseases [1–5]. Nevertheless,
an ascorbic acid excess can lead to gastric irritation, and the metabolic
product of vitamin C (oxalic acid) can cause renal problems [6]. In some cases,
excessive quantities of ascorbic acid may result in the inhibition of natural
processes occurring in food and can contribute to taste deterioration; added to
apple pulp (250 mg/kg), vitamin C inhibits oxidation processes responsible for
apple juice aroma [7]. Ascorbic acid is a labile substance, as it is easily
degraded by enzymes and atmospheric oxygen. Its oxidation can be accelerated by
excessive heat, light, and heavy metal cations [1]. That is why ascorbic acid
content of foodstuffs and beverages represents a relevant indicator of quality
which has to be carefully monitored, regarding its variation during
manufacturing and storage.Many analytical methods
can be used for ascorbic acid determination. Classic (conventional) techniques
are represented by volumetric methods—titration with an
oxidant solution such as dichlorophenol indophenol (DCPIP) [8, 9], potassium
iodate [10], or bromate [11]. Volumetric techniques can suffer from lack of
specificity [12] which limits their use to samples not containing other
reducing agents.Güçlü et al. [13] have proposed a spectrophotometric method based on ascorbic acid
oxidation to dehydroascorbic acid, by using the Cu(II)-neocuproine complex,
which is reduced to Cu(I)-bis(neocuproine), the absorbance of the latter being
determined at 450 nm. Other optical methods for vitamin C estimation include
spectrophotometrical determination of iodine reacted with ascorbic acid [14] and
chemiluminescence [15].Liquid
chromatography is a successful method for vitamin C determination when
selectivity and specificity are concerned [16–18]. HPLC with electrochemical detection has turned out to be a selective
and sensitive method for ascorbic acid assessment in foodstuffs and biological
fluids [19–21].A potentiometric biosensor
[22] for ascorbic acid was made by ascorbate oxidase immobilization in a
polymeric matrix, fixed on a graphite-epoxy
composite electrode.Amperometric biosensors were obtained by ascorbate oxidase
immobilization on a nylon net [23] or on a collagen membrane, using a Clark
oxygen electrode as transducer [24]. Vitamin C analysis was also performed by using
a glassy carbon working electrode as transducer incorporated in a flow system
[25]. Ascorbic and uric acids
were determined by coupling an amperometric technique with flow analysis [26].
Voltammetric and amperometric measurements were performed in a flow cell, using
gold microelectrodes on which Pd was electrochemically deposited.O’Connell et al. [12] developed an amperometric sensor for ascorbic
acid determination from foodstuffs and pharmaceutical preparations. This sensor
was constructed by aniline electropolymerization on a glassy carbon or a screen-printed
working electrode.Kumar and Narayanan
[27] investigated a method for vitamin C assessment based on an amperometric
sensor obtained by graphite electrode modification by cobalt ferrocyanide. The
decrease of the working potential in these amperometric methods based on
electrochemical oxidation of ascorbic acid was possible by using mediators like
ferocene [28] or redox couples like ferri/ferrocyanide [29].Vitamin C determination was also performed in an FIA system
with biamperometric detection, based on ascorbic acid reaction with iodine
[30].Voltammetry is an increasingly popular method applied to
the determination of ascorbic acid in real samples [7], because
it offers low detection limits, even when compared to more expensive
techniques. It requires little or no sample preparation. This technique
provides us with the advantage of a fast analysis as well as with the easiness and
rapidity of the standard addition method application. Because of the low cost
of the required equipment as well as simplicity of the employed procedures necessary
to determine vitamin C, voltammetry appears to offer an attractive alternative
to the titrimetric or instrumental methods mentioned earlier, in particular in
food quality control. It does not require complicated, expensive equipment and
well-qualified personnel nor is it laborious or time consuming like the
previously mentioned instrumental techniques [7].Simultanoeus determination of vitamin C and glucose has
also been performed using a voltammetric biosensor integrated in an automated
SIA system [31].Recently, the use of various voltammetric techniques has
been combined with modified ascorbic acid sensors; square-wave voltammetry was
used to determine ascorbic acid based on its oxidation at a zeolite modified
carbon paste electrode [32], and the method was applied to ascorbic acid
determination in citrus juice. The response of the electrode to ascorbic acid
is linear in the range 4×10−7–1.2×10−3 mol⋅L−1,
with a detection limit of 2×10−8 mol⋅L−1; cyclic and
differential pulse voltammetries
were used for ascorbic acid electrocatalytical determination at a carbon paste
electrode modified with 2,7-bis (ferrocenyl ethynyl) fluoren-9-one [33]. The
detection limits (2σ) were determined as 1.8×10−5 and 4.2×10−6 mol⋅L−1 by CV and DPV, respectively.The results reported in literature regarding the
determination of ascorbic acid by cyclic voltammetry are not numerous. Nevertheless,
cyclic voltammetry has been previously used for antioxidant content assessment,
and in particular low-molecular-weight
antioxidants, including ascorbic acid; this technique has turned out to be a
convenient methodology, validated for the quantification of low-molecular-weight
antioxidant capacity of tissue homogenates, blood plasma, or plant extracts [34].
Cyclic voltammetry and spectrophotometry showed good agreement for the
antioxidant capacity estimation in buckwheat products after hydrothermal
treatment [35]. Ruffien-Ciszak et al. [36] have proposed cyclic voltammetry
using a Pt wire as working electrode to assess the total antioxidant capacity
of skin, based on the reduction capacity of low-molecular-weight antioxidants. Rapta et al.
[37] evaluated the antioxidant capacity of flavonoids by cyclic voltammetry in
acetonitrile, by employing a three-electrode cell with Pt working and auxiliary
electrodes and a calomel electrode as reference. Zielinska et al. [38] used
cyclic voltammetry with glassy carbon working electrode to monitor the total
antioxidant capacity and flavonoid content in onions. H. J. Kim and I. K. Kim [39] evaluated
ascorbic acid content (after isolation on an anion exclusion column) by
amperometric detection at a Pt working electrode operating at 0.6 V (versus
Ag/AgCl). The vitamin C content in apple juice has been monitored by cyclic
voltammetry by means of a Pt working electrode [7]. Campanella et al. [40]
determined the antioxidant capacity of dry vegetal extracts (expressed as mg of
ascorbic acid equivalents) by cyclic voltammetry performed at a glassy carbon
working electrode.The aim of this paper was to investigate a method for
ascorbic acid determination by cyclic voltammetry, taking into account that the
reported data in literature regarding the determination of ascorbic acid by
this method are very scarce. The developed method was applied to the determination
of ascorbic acid in different fruit juice, and the obtained results were
compared with those obtained by a conventional titrimetric method.


## 2. Experimental

### 2.1. Reagents and Instrumentation

A potentiostat-galvanostat KSP, laboratory made by Slawomir Kalinowski (University Warmia and Mazury, Olsztyn, Poland) was used, as well as the respective soft, Cyclic Voltammetry. A Pt disc electrode
(Metrohm, 2 mm diameter) was used as working electrode. The reference electrode
was a saturated calomel electrode (SCE). The counter electrode was a Pt strip (30mm2 surface). Figure 1 provides a schematic representation of the experimental
setup; the potentiostat enables control of the potential of the working
electrode, with respect to the reference electrode as well as measurement of the
current that flows between the working electrode and counter electrode. A stock
solution of ascorbic acid, 100 mmol⋅L−1, was prepared daily by
dissolving vitamin C (Merck, Haar, Germany, ACS ISO, biochemical grade) in a 0.34 mol⋅L−1 KCl
solution (Reactivul, Bucharest, Romania) used as
supporting electrolyte. Standard solutions of ascorbic acid with concentrations
ranging between 0.1 and 10 mmol⋅L−1 were obtained by diluting the
stock solution with the respective volumes of 0.34 mol⋅L−1 KCl (electrolyte)
solution. Standard solutions of glucose (Reactivul Bucuresti), tartaric acid
(Merck), citric acid (Reactivul Bucuresti), and sodium benzoate (Sigma-Aldrich, Taufkirchen, Germany)
with a concentration of 1 mol⋅L−1 were prepared by dissolution of the
respective amount of reagent in 0.34 mol⋅L−1 KCl solution.Figure 1
Schematic representation of the experimental setup.The
dichlorophenol indophenol (DCPIP) solution,5×10−4 mol⋅L−1,
was prepared by dissolving 145 mg DCPIP, sodium salt (Merck), in 100 mL hot
distilled water and a subsequent addition of 300 mL phosphate buffer, 0.066 mol⋅L−1,
pH=6.98, previously prepared by mixing the respective
volumes of potassium dihydrogen phosphate and sodium monohydrogen phosphate
solutions (2/3 ratio). Distilled water was added to the final volume of 1000 mL.
After homogenization, the solution was kept in a dark place (protected from
light) and filtered [8].All
mentioned solutions were prepared using distilled water which was boiled and
chilled until reached room temperature.


### 2.2. Working Procedure

For voltammetric
measurements, a three-electrodes cell was used equipped with working, counter,
and a reference electrodes [7, 41]. The volume of the analyzed sample was 100 mL,
and all measurements were performed at 295.5 K using 0.34 mol⋅L−1 KCl
solution as supporting electrolyte. All voltammograms were recorded for stirred
solutions. Before each determination, the Pt working electrode was cleaned
mechanically, by polishing it on alumina (Merck, 63–200 μm
granularity) and electrochemically, by applying a −1.5 V potential pulse for 3 seconds. For each
measurement, the potential was scanned within the range −100 and −1000 mV, with
a 50 mV/s scan rate, and the value of the backround current obtained for the KCl 0.34 mol⋅L−1 solution was substracted from the current corresponding
to the analyzed solution/sample. For investigating the potential scan rate
influence, this parameter varied from 50 mV/s to 250 mV/s.


## 2.1. Reagents and Instrumentation

A potentiostat-galvanostat KSP, laboratory made by Slawomir Kalinowski (University Warmia and Mazury, Olsztyn, Poland) was used, as well as the respective soft, Cyclic Voltammetry. A Pt disc electrode
(Metrohm, 2 mm diameter) was used as working electrode. The reference electrode
was a saturated calomel electrode (SCE). The counter electrode was a Pt strip (30mm2 surface). Figure 1 provides a schematic representation of the experimental
setup; the potentiostat enables control of the potential of the working
electrode, with respect to the reference electrode as well as measurement of the
current that flows between the working electrode and counter electrode. A stock
solution of ascorbic acid, 100 mmol⋅L−1, was prepared daily by
dissolving vitamin C (Merck, Haar, Germany, ACS ISO, biochemical grade) in a 0.34 mol⋅L−1 KCl
solution (Reactivul, Bucharest, Romania) used as
supporting electrolyte. Standard solutions of ascorbic acid with concentrations
ranging between 0.1 and 10 mmol⋅L−1 were obtained by diluting the
stock solution with the respective volumes of 0.34 mol⋅L−1 KCl (electrolyte)
solution. Standard solutions of glucose (Reactivul Bucuresti), tartaric acid
(Merck), citric acid (Reactivul Bucuresti), and sodium benzoate (Sigma-Aldrich, Taufkirchen, Germany)
with a concentration of 1 mol⋅L−1 were prepared by dissolution of the
respective amount of reagent in 0.34 mol⋅L−1 KCl solution.Figure 1
Schematic representation of the experimental setup.The
dichlorophenol indophenol (DCPIP) solution,5×10−4 mol⋅L−1,
was prepared by dissolving 145 mg DCPIP, sodium salt (Merck), in 100 mL hot
distilled water and a subsequent addition of 300 mL phosphate buffer, 0.066 mol⋅L−1,
pH=6.98, previously prepared by mixing the respective
volumes of potassium dihydrogen phosphate and sodium monohydrogen phosphate
solutions (2/3 ratio). Distilled water was added to the final volume of 1000 mL.
After homogenization, the solution was kept in a dark place (protected from
light) and filtered [8].All
mentioned solutions were prepared using distilled water which was boiled and
chilled until reached room temperature.


## 2.2. Working Procedure

For voltammetric
measurements, a three-electrodes cell was used equipped with working, counter,
and a reference electrodes [7, 41]. The volume of the analyzed sample was 100 mL,
and all measurements were performed at 295.5 K using 0.34 mol⋅L−1 KCl
solution as supporting electrolyte. All voltammograms were recorded for stirred
solutions. Before each determination, the Pt working electrode was cleaned
mechanically, by polishing it on alumina (Merck, 63–200 μm
granularity) and electrochemically, by applying a −1.5 V potential pulse for 3 seconds. For each
measurement, the potential was scanned within the range −100 and −1000 mV, with
a 50 mV/s scan rate, and the value of the backround current obtained for the KCl 0.34 mol⋅L−1 solution was substracted from the current corresponding
to the analyzed solution/sample. For investigating the potential scan rate
influence, this parameter varied from 50 mV/s to 250 mV/s.


## 3. Results and Discussions

In Figure2, several voltammograms, obtained for different
ascorbic acid concentrations, are presented. The peak that appeared at 490 mV
(versus SCE) was attributed to ascorbic acid oxidation. As can be seen from Figure 2, no reduction
peak appears for ascorbic acid. This confirms the data reported in literature
[42, 43] that electrochemical oxidation of ascorbic acid
is an irreversible process.Figure 2
Cyclic
voltammograms obtained for different ascorbic acid concentrations expressed as mmol⋅L−1:
0.1 (10), 0.5 (9), 0.75 (8), 1 (7), 1.5 (6), 2 (5), 4 (4), 6 (3), 8 (2), and 10 mmol⋅L−1 (1).The calibration graph (Figure3)
shows a linear range obtained between 0.1 and 10 mmol⋅L−1 ascorbic
acid (r2=0.9995, y=6.391x+0.1903). The value calculated for r.s.d. was 1.14%, (c=2 mmol⋅L−1 ascorbic acid; n=10). The influence of the potential scan rate on the
anodic peak height was also investigated (Figure 4). The measurements were
performed at 2 mmol⋅L−1 ascorbic acid concentration, and the potential
scan rate varied between 50 and 250 mV/s. The anodic peak height corresponding
to the analyte oxidation increases with the square root of the potential scan
rate and conforms to Randles-Sevcik equation:
(1)Ip=2.69⋅105n3/2AD1/2v1/2c,
where
c represents concentration of the electroactive species, v potential scan rate,
A electrode surface, D diffusion coefficient of the analyte, and n number of
electrons transferred in the redox process.Figure 3
Calibration graph for the
determination of ascorbic acid by cyclic voltammetry within (a) the domain 0.1–10 mmol⋅L−1 and (b) the domain 0.1–2 mmol⋅L−1.Figure 4
The influence of the square
root of the potential scan rate on the anodic peak current;cascorbicacid=2 mmol⋅L−1.


### 3.1. Specificity and Interferences

Previously
published studies have proved that compounds commonly found in foodstuffs and juice
(citric acid, tartaric acid, phenylalanine, glutamic acid, aminoacetic acid,
and glucose) do not interfere in the ascorbic acid determination by cyclic
voltammetry performed on glassy carbon working electrodes [44]. A study of
interference for ascorbic acid determination was performed, also at a glassy
carbon working electrode modified with nickel(II) macrocycle containing
dianionic tetraazaannulene ligand [45]. When the permitted relative deviation is
less than ±5%, no interference is observed from citric acid, malic acid, tartaric
acid, and glucose, at a ratio substance/L-ascorbic acid (w/w) of 250 [45].Table1 presents the results obtained at the determination of ascorbic acid by cyclic
voltammetry, in the presence of some common substances usually accompanying ascorbic
acid in citrus juice, namely, glucose, tartaric acid, citric acid, and benzoate
anion. All the determinations were performed by using the reported working
procedure. The studied interferent was added to the analyzed sample as a
concentrated solution, and the final volume of the analyzed sample was of 100 mL.
The ascorbic acid concentration was 2 mmol⋅L−1. The values
presented in Table 1 represent the average of three determinations.Table 1
Study of interference performed on some common chemical species found in citrus juice.
InterferentInterferent/analyte molar ratioInfluence on the analytical peak currentGlucose200less than 1%Citric acid150less than 1%2002.26% decreaseTartaric acid200less than 1%Benzoate anion150less than 1%2004.84% decreaseAs can be seen from Table1, glucose and tartaric acid do not
influence the ascorbic acid analytical signal in concentrations up to 200 times
greater than that of vitamin C. Benzoate anion does not influence the ascorbic
acid analytical signal in concentrations up to 150 times greater than that of
vitamin C. Concentrations of benzoate anion 200 times greater than that of the
analyte produce a decrease of the analytical signal of 4.84%. Interference
tests have proved that citric acid, in concentrations up to 150 times greater
than that of the analyte, has no influence on the analytical peak current. A
citric acid concentration 200 times greater than that of vitamin C produces a
decrease of the ascorbic acid peak current of 2.26%.Therefore, citric acid, tartaric acid, and benzoate anion
do not interfere at ascorbic acid determination (error of determination<5%),
in concentrations commonly found in fresh or commercial fruit juice, for these
organic interferents.


### 3.2. Analysis of Real Samples

Natural orange juice and lemon juice
were obtained by fruit pressing. To this purpose, five average-sized fruits
were peeled and the juice was obtained by using a centrifugal device. Then, the
obtained juice was centrifugated until a clear sample was obtained, which was
subsequently analyzed.Commercial juice containing fruit
pulp (Santal, Tymbark) were centrifugated before analysis, and the obtained clear
sample was analyzed. Solid KCl was added as supporting electrolyte into the
clear fruit juice (without a previous dilution) in order to obtain a
concentration of 0.34 mol⋅L−1 KCl. The working procedure for the standard
ascorbic acid solutions was applied to fruit juice analysis. The ascorbic acid
content was calculated by measuring the peak current and by using the calibration
graph presented in Figure 2. The obtained results are presented in Table 2 together with those obtained by a volumetric technique which uses a
dichlorophenol indophenol (DCPIP) 5×10−4 mol⋅L−1 solution as
titrating agent [8, 9].Table 2
Results
obtained for vitamin C determination in fruit juice and for
the calculation of the degree of recovery of ascorbic acid added in analyzed
samples by using the titrimetric method with DCPIP and the voltammetric method.
The values obtained by the titrimetric method represent the average of three
determinations, whereas those obtained by cyclic voltammetry represent the
average of five determinations.
Juice and producersAscorbic acid concentration—DCPIP methodAscorbic acid concentration— cyclic voltammetryAscorbic acid concentration after 35.2 mg (2 mL standard solution, 0.1 mol⋅L−1, added to 100 mL juice) vitamin C additionRecovery%Ascorbic acid concentration after 70.4 mg (4 mL standard solution, 0.1 mol⋅L−1, added to 100 mL juice) vitamin C additionRecovery%mg/100 mL juicemmol⋅L−1mg/100 mL juicemg/100 mL juicemg/100 mL juiceOrange juice (Greece) obtained by fruit pressing30.481.6729.3964.35103.0195.6199.50Lemon juice (Greece) obtained by fruit pressing35.201.9333.9768.31101.4898.3297Cappy grapefruit (Coca Cola HBC Romania)8.210.468.0940.6794.9075.3099.75Fanta lemon (Coca Cola)10.790.5810.2142.9195.3679.37102.75Tymbark orange (mw La Festa Int’l Romania)22.291.2321.6556.4110288.53100.03Frutti fresh Tutti frutti (European Drinks Romania)14.370.8314.6146.8794.3579.8897.25Prigat orange (Sigat Beverage Company Romania)14.960.8314.6147.5896.4081.0799.01Prigat Peach (Sigat Beverage Company Romania)12.320.7312.8546.8299.1982.75104Fruttia orange (European Drinks Romania)21.121.2421.8255.4498.6989.61101.39Santal grapefruit (Parmalat Romania)31.681.8432.3864.4294.72100.35102.26


### 3.3. Determination of the Degree of Recovery of Vitamin C Added to Analyzed Juice Samples

All
measurements were performed following the working procedure detailed for the
standard ascorbic acid solutions. To 100 mL clear fruit juice, solid KCl was
added to obtain a
concentration of 0.34 mol⋅L−1 electrolyte. Then,
2 mL (35.2 mg) and 4 mL (70.4 mg), respectively, from a concentrated (100 mmol⋅L−1)
ascorbic acid solution containing 0.34 mol⋅L−1 KCl, were added to the
sample. The obtained analytical signal was corrected by taking into account the
sample dilution originating from the addition of standard ascorbic acid
solution. For each addition, the degree of recovery was calculated as follows:
(2)Recovery%=(QDET−QP)×100QADD,
where QDET represents mg determined ascorbic acid in
100 mL juice, QP represents mg ascorbic acid previously
present in 100 mL juice, and QADD represents mg added
ascorbic acid in 100 mL juice.The
obtained results are presented in Table2. As can be seen from Table 2, the
degree of recovery of ascorbic acid varies between 94.35% and 104%, which
indicates a good recovery of the added ascorbic acid amounts.In
order to verify the accuracy of the developed method for ascorbic acid
determination in fruit juice, the standard addition method was applied for an
analyzed sample, namely, Fruttia orange. The following procedure was employed:
to four 100 mL volumetric flasks, 50 mL sample (Fruttia orange) was added. Then, known
amounts from the standard 0.1 mol⋅L−1 ascorbic acid solution were
added in each flask as follows: (1) 0 mL, (2) 2 mL, (3) 4 mL, and (4) 6 mL. Solid
KCl was added in each volumetric flask, as to reach a 0.34 mol⋅L−1 final
electrolyte concentration. Double-distilled water was added to the final
100 mL volume, followed by homogenization. The ascorbic acid content for each
flask was determined, and the obtained results are presented in Figure 5. The determined
concentration in Fruttia was 0.625 mmol⋅L−1 (11 mg/100 mL) ascorbic
acid. Taking into account the dilution degree (1/1), this corresponds to a
concentration of 1.25 mmol⋅L−1 ascorbic acid in the undiluted juice
(Fruttia). The obtained result is in accordance with the one presented in Table 2, which indicates the absence of matrix effects at ascorbic acid determination
by the proposed method.Figure 5
Application of the standard addition method for determination of ascorbic acid in Fruttia orange. The working procedure described in Section2 was used.


### 3.4. Working Procedure for Vitamin C Titrimetric Determination with DCPIP

A sample of 2.5 mL clear juice was
diluted with distilled water to a final volume of 10 mL. Then, it was titrated
with the DCPIP5×10−4 mol⋅L−1 solution until a pink tint appears that persists for about 30 seconds. The
obtained results are presented in Table 2.


## 3.1. Specificity and Interferences

Previously
published studies have proved that compounds commonly found in foodstuffs and juice
(citric acid, tartaric acid, phenylalanine, glutamic acid, aminoacetic acid,
and glucose) do not interfere in the ascorbic acid determination by cyclic
voltammetry performed on glassy carbon working electrodes [44]. A study of
interference for ascorbic acid determination was performed, also at a glassy
carbon working electrode modified with nickel(II) macrocycle containing
dianionic tetraazaannulene ligand [45]. When the permitted relative deviation is
less than ±5%, no interference is observed from citric acid, malic acid, tartaric
acid, and glucose, at a ratio substance/L-ascorbic acid (w/w) of 250 [45].Table1 presents the results obtained at the determination of ascorbic acid by cyclic
voltammetry, in the presence of some common substances usually accompanying ascorbic
acid in citrus juice, namely, glucose, tartaric acid, citric acid, and benzoate
anion. All the determinations were performed by using the reported working
procedure. The studied interferent was added to the analyzed sample as a
concentrated solution, and the final volume of the analyzed sample was of 100 mL.
The ascorbic acid concentration was 2 mmol⋅L−1. The values
presented in Table 1 represent the average of three determinations.Table 1
Study of interference performed on some common chemical species found in citrus juice.
InterferentInterferent/analyte molar ratioInfluence on the analytical peak currentGlucose200less than 1%Citric acid150less than 1%2002.26% decreaseTartaric acid200less than 1%Benzoate anion150less than 1%2004.84% decreaseAs can be seen from Table1, glucose and tartaric acid do not
influence the ascorbic acid analytical signal in concentrations up to 200 times
greater than that of vitamin C. Benzoate anion does not influence the ascorbic
acid analytical signal in concentrations up to 150 times greater than that of
vitamin C. Concentrations of benzoate anion 200 times greater than that of the
analyte produce a decrease of the analytical signal of 4.84%. Interference
tests have proved that citric acid, in concentrations up to 150 times greater
than that of the analyte, has no influence on the analytical peak current. A
citric acid concentration 200 times greater than that of vitamin C produces a
decrease of the ascorbic acid peak current of 2.26%.Therefore, citric acid, tartaric acid, and benzoate anion
do not interfere at ascorbic acid determination (error of determination<5%),
in concentrations commonly found in fresh or commercial fruit juice, for these
organic interferents.


## 3.2. Analysis of Real Samples

Natural orange juice and lemon juice
were obtained by fruit pressing. To this purpose, five average-sized fruits
were peeled and the juice was obtained by using a centrifugal device. Then, the
obtained juice was centrifugated until a clear sample was obtained, which was
subsequently analyzed.Commercial juice containing fruit
pulp (Santal, Tymbark) were centrifugated before analysis, and the obtained clear
sample was analyzed. Solid KCl was added as supporting electrolyte into the
clear fruit juice (without a previous dilution) in order to obtain a
concentration of 0.34 mol⋅L−1 KCl. The working procedure for the standard
ascorbic acid solutions was applied to fruit juice analysis. The ascorbic acid
content was calculated by measuring the peak current and by using the calibration
graph presented in Figure 2. The obtained results are presented in Table 2 together with those obtained by a volumetric technique which uses a
dichlorophenol indophenol (DCPIP) 5×10−4 mol⋅L−1 solution as
titrating agent [8, 9].Table 2
Results
obtained for vitamin C determination in fruit juice and for
the calculation of the degree of recovery of ascorbic acid added in analyzed
samples by using the titrimetric method with DCPIP and the voltammetric method.
The values obtained by the titrimetric method represent the average of three
determinations, whereas those obtained by cyclic voltammetry represent the
average of five determinations.
Juice and producersAscorbic acid concentration—DCPIP methodAscorbic acid concentration— cyclic voltammetryAscorbic acid concentration after 35.2 mg (2 mL standard solution, 0.1 mol⋅L−1, added to 100 mL juice) vitamin C additionRecovery%Ascorbic acid concentration after 70.4 mg (4 mL standard solution, 0.1 mol⋅L−1, added to 100 mL juice) vitamin C additionRecovery%mg/100 mL juicemmol⋅L−1mg/100 mL juicemg/100 mL juicemg/100 mL juiceOrange juice (Greece) obtained by fruit pressing30.481.6729.3964.35103.0195.6199.50Lemon juice (Greece) obtained by fruit pressing35.201.9333.9768.31101.4898.3297Cappy grapefruit (Coca Cola HBC Romania)8.210.468.0940.6794.9075.3099.75Fanta lemon (Coca Cola)10.790.5810.2142.9195.3679.37102.75Tymbark orange (mw La Festa Int’l Romania)22.291.2321.6556.4110288.53100.03Frutti fresh Tutti frutti (European Drinks Romania)14.370.8314.6146.8794.3579.8897.25Prigat orange (Sigat Beverage Company Romania)14.960.8314.6147.5896.4081.0799.01Prigat Peach (Sigat Beverage Company Romania)12.320.7312.8546.8299.1982.75104Fruttia orange (European Drinks Romania)21.121.2421.8255.4498.6989.61101.39Santal grapefruit (Parmalat Romania)31.681.8432.3864.4294.72100.35102.26


## 3.3. Determination of the Degree of Recovery of Vitamin C Added to Analyzed Juice Samples

All
measurements were performed following the working procedure detailed for the
standard ascorbic acid solutions. To 100 mL clear fruit juice, solid KCl was
added to obtain a
concentration of 0.34 mol⋅L−1 electrolyte. Then,
2 mL (35.2 mg) and 4 mL (70.4 mg), respectively, from a concentrated (100 mmol⋅L−1)
ascorbic acid solution containing 0.34 mol⋅L−1 KCl, were added to the
sample. The obtained analytical signal was corrected by taking into account the
sample dilution originating from the addition of standard ascorbic acid
solution. For each addition, the degree of recovery was calculated as follows:
(2)Recovery%=(QDET−QP)×100QADD,
where QDET represents mg determined ascorbic acid in
100 mL juice, QP represents mg ascorbic acid previously
present in 100 mL juice, and QADD represents mg added
ascorbic acid in 100 mL juice.The
obtained results are presented in Table2. As can be seen from Table 2, the
degree of recovery of ascorbic acid varies between 94.35% and 104%, which
indicates a good recovery of the added ascorbic acid amounts.In
order to verify the accuracy of the developed method for ascorbic acid
determination in fruit juice, the standard addition method was applied for an
analyzed sample, namely, Fruttia orange. The following procedure was employed:
to four 100 mL volumetric flasks, 50 mL sample (Fruttia orange) was added. Then, known
amounts from the standard 0.1 mol⋅L−1 ascorbic acid solution were
added in each flask as follows: (1) 0 mL, (2) 2 mL, (3) 4 mL, and (4) 6 mL. Solid
KCl was added in each volumetric flask, as to reach a 0.34 mol⋅L−1 final
electrolyte concentration. Double-distilled water was added to the final
100 mL volume, followed by homogenization. The ascorbic acid content for each
flask was determined, and the obtained results are presented in Figure 5. The determined
concentration in Fruttia was 0.625 mmol⋅L−1 (11 mg/100 mL) ascorbic
acid. Taking into account the dilution degree (1/1), this corresponds to a
concentration of 1.25 mmol⋅L−1 ascorbic acid in the undiluted juice
(Fruttia). The obtained result is in accordance with the one presented in Table 2, which indicates the absence of matrix effects at ascorbic acid determination
by the proposed method.Figure 5
Application of the standard addition method for determination of ascorbic acid in Fruttia orange. The working procedure described in Section2 was used.


## 3.4. Working Procedure for Vitamin C Titrimetric Determination with DCPIP

A sample of 2.5 mL clear juice was
diluted with distilled water to a final volume of 10 mL. Then, it was titrated
with the DCPIP5×10−4 mol⋅L−1 solution until a pink tint appears that persists for about 30 seconds. The
obtained results are presented in Table 2.


## 4. Conclusions

The developed method has proved its accuracy in vitamin C
determination in fruit juice, having the value of the recovery of known quantities of ascorbic
acid ranging between 94.35% and 104%. The highest values for ascorbic acid were obtained for natural juice
made by fruit squeezing.The detection limit of the method
was of9×10−5 mol⋅L−1 (calculated as 3x, the standard
deviation of the blank signal), and the limit of quantification was 3×10−4 mol⋅L−1 (calculated
as 10x, the standard deviation of the blank signal).Although other voltammetric methods
(e.g., differential pulse voltammetry or linear sweep voltammetry) are more
sensitive than cyclic voltammetry for determining ascorbic acid, cyclic
voltammetry can be used with very good results to analyze ascorbic acid in
fruit juice.The concentrations of ascorbic acid in fruit juice
determined by cyclic voltammetry are in good agreement with the data obtained by
a classical volumetric method (Table2). The obtained results are also in good
agreement with the data reported in literature regarding the content of
ascorbic acid in citrus fruits. Thus, the reported values for lemon are 44.5 mg/100 mL juice [24] or 48 mg/100 g fruit [46]. Other results indicate a vitamin C
content of 33–50 mg/100 mL for
orange juice (Valencia) obtained by squeezing the fruits [46]. For the
grapefruit juice (Florida), also obtained by fruit pressing, the ascorbic acid
content varies between 38 and 56 mg/100 mL [47]. These values are in accordance
with those we obtained for orange and lemon juice (fruit squeezing), 30.48 mg/100 mL
and 35.2 mg/100 mL, respectively, as well as those obtained for grapefruit juice
(Santal), 31.68 mg/100 mL.The results obtained in this study show that cyclic
voltammetry can be successfully used as part of quality management in food
industry, for assessing the vitamin C content in natural fruit juice and soft
drinks. The results prove why, recently, this technique has been more and more
preferred to the previously applied methods, as it is characterized by accuracy,
rapidity, good specificity, and sensitivity, and also by the simplicity of the required
equipment and procedure.



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*Source: 937651-2009-03-29.xml*