Process for Preparation of Vitamin C and Method for Determination

of Vitamin C

Abstract

Procedure for calcium ascorbate (VITAMIN-C) includes such technological steps as

reaction of ascorbic acid on calcium carbonate in water. In recent years, the
determination of vitamin C has become an important subject in the field of
biochemistry and commercial foods. This is because vitamin C plays an important
role in maintaining human health. Due to the importance of vitamin C in human
beings, the quantitative analysis of vitamin C has gained a significant increase in
several areas of analytical chemistry such as pharmaceutical and food applications.
There are numerous methods for the determination of vitamin C in a variety of
natural samples, biological fluids and pharmaceutical formulations. The prepartaion
method and methods for the determination of vitamin C are spectrophotometric
methods and non-spectrophotometric methods (Arya and Mahajan, 1997). For non-
spectrophotometric methods are such as high-performance liquid chromatography
(HPLC), titration, enzymatic method and fluorometry (Arya, Mahajan and Jain, 2000).
Direct spectrophotometry also has been applied to determine the vitamin C content
in soft drinks, fruit juices, and cordials after correction for background absorption in
the UV region.
Abbreviations: FIA: Flow Injection Analysis, HPLC: High Performance Liquid
Chromatography HMF: Hydroxymethylfurfural, AIDS: Acquired Immuno Deficiency
Syndrome, FDA: Food and Drug Administration
Introduction
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Ascorbic acid (vitamin C) is a water-soluble vitamin. It occurs as a white or slightly

yellow crystal or powder with a slight acidic taste. It is an antiscorbutic product. On
exposure to light, it gradually darkens. In the dry state, it is reasonably stable in air,
but in solution it rapidly oxidizes. Ascorbic acid (vitamin c) is freely soluble in water;
sparingly soluble in alcohol; insoluble in chloroform, in ether, and in benzene. The
chemical name of ascorbic acid (vitamin c) is L-ascorbic acid (vitamin c). The
empirical formula is C6H806, and the molecular weight is 176.13. The synthesis of
ascorbic acid was achieved by Reichstein in 1933, followed by industrial production
of ascorbic acid two years later by Roche. Today, vitamin C identical to that
occurring in nature is produced on a very large industrial scale. The ultimate raw
material for the production of vitamin C (ascorbic acid) is corn or wheat. This is
converted via starch to glucose by specialist companies, and then to sorbitol. We
produce the pure final products from sorbitol in a series of biotechnical, chemical
processing and purification steps.

Vitamin C = Ascorbic Acid

Empirical formula: C6H8O6

Molecular weight: 176.1

Melting point: about 190 °C (with decomposition)

Appearance: white to slightly yellowish crystalline powder, practically odorless, with a

strong acidic taste.

The water-soluble vitamin C is probably the most well-known vitamin. Even before its
discovery in 1932, physicians recognized that there must be a compound in citrus
fruits preventing scurvy, a disease that killed as many as two million sailors between
1500 and 1800. Later researchers. Discovered that man, other primates and the
guinea pig depend on external sources to cover their vitamin C requirements. Most
other animals are able to synthesize vitamin C from glucose and galactose in their
bodies. Nowadays, health has become the most important property of human’s life.
Commonly, diets with high contents of fruits are protective against several human
diseases such as cardiovascular diseases and even cancer. Therefore, people are
putting more and more attention on antioxidant substances such as vitamin C which
is also known as ascorbic acid or more specifically L-ascorbic acid.

Vitamin C is probably one of the most highly well known. Furthermore, people have
become more aware to the importance of vitamin C. Hence, this causes the global
market flooded with vitamin C fortified foods (Arya, Mahajan and Jain, 2000). The
term of vitamin C is used as generic term for all compounds exhibiting qualitatively
the biological activity of ascorbic acid. The molecular structure of vitamin C is
C6H8O6 and the molecular weight is 176.1 (Ball, 2006). Vitamin C is highly polar
and readily soluble in aqueous solution and insoluble in less nonpolar solvents
(Fennema, 1996). It is an acidic compound due to the facile ionization of hydroxyl
group on carbon 3 (pK1 = 4.17) while the hydroxyl group on carbon 2 is much more
resistant to ionization (pK2 = 11.79).

The structure of L-ascorbic acid is shown in Figure 1 (Ball, 2006). Ball (2006) also
stated that ascorbic acid is easily and reversibly oxidized to dehydroascorbic acid,
forming the ascorbyl radical anion which is also known as semidehydoascorbate as
an intermediate as shown in Figure 2. Dehydoascorbic acid possesses full vitamin C
activity because it is readily reduced to ascorbic acid in the animal body. However,
dehydoascorbic acid is not an acid in the chemical sense, as it does not have the
dissociable protons that ascorbic acid has at carbon 2 and carbon 3 positions.
Figure 1: structure of L-ascorbic acid.

Figure 2: Schematic diagram of the rFIA-CL manifold used in this study for the
determination of ascorbic acid.
One of the most important properties of vitamin C is that it is an antioxidant.
Nevertheless, it has a wide range of antioxidant properties outside the body and can
quench most biologically active radicals. It scavenges superoxide, nitroxide,
hydroxide, hydrogen peroxide and will reduce vitamin E (Hickey and Roberts, 2004).
It is also found to be a strong antioxidant as it helps to neutralize harmful free
radicals (Izuagie and Izuagie, 2007). Vitamin C is an almost odorless white or pale
yellow crystalline powder with a pleasant sharp taste and melting point of about
190°C. It is not a carboxylic acid but a lactone and ease of oxidation to the presence
of an enediol grouping (Izuagie and Izuagie, 2007). Vitamin C is highly susceptible to
oxidation, especially when catalyzed by metal ions such as copper (II) ion and
iron(III) ion. The functions and activities of vitamin C are based on its properties as a
reversible biological reductant (Hickey and Roberts, 2004) (Figure 1).

Vitamin C participates for the growth and repair of tissues in all parts of the body
(Kleszczewski and Kleszczewska 2002). Vitamin C is a natural antioxidant that
mostly found in fruits and vegetables. The main sources of vitamin C are citrus fruits,
strawberries, peppers, tomatoes, cabbage, and spinach. Vitamin C plays crucial
roles in electron transport, hydroxylation reactions and oxidative catabolism of
aromatic compounds in animal metabolism (Gazdik, 2008). Vitamin C can help to
prevent and treat common cold, mental illness, infertility, scurvy, cancer and
Acquired Immune Deficiency Syndrome (AIDS) (Yusuf and Gurel, 2005). It is
reported to lower cancer risk and also said to have important interactions with other
vitamins. For example, excessive intake of vitamin A is less toxic to the body when
vitamin C is readily available (Izuagie and Izuagie, 2007). Due to the great
importance of vitamin C in human beings, the quantitative analysis of vitamin C has
gained increased significance in several areas of analytical chemistry such as
pharmaceutical and food applications (Yusuf and Gurel, 2005).

Vitamin C is also used as an index of the nutrient quality for fruit and vegetable
products. This is because it is much more sensitive to various modes of degradation
in food processing and subsequent storage (Ozkan, Kirca and Cemero, 2004). It is
well known that vitamin C is easily oxidized to dehydroascorbic acid in alkaline
solutions, while it is relatively stable in acidic solution. Vitamin C of fruit juices is
readily oxidized and lost during staying of the juices (Kabasakalis, Siopidou and
Moshatou, 2000). In the food industry, vitamin C is used as food additive (Mai and
Mohammed, 2004). It preserves and protects food from any colour changes and act
as an important component of our nutrition as well. Vitamin C helps to prevent the
degradation of soft drinks and juice which helps to retain their flavors. Hence, it
increases the quality of food and nutritional value as well (Burdurlu, Koca and
Karadeniz, 2005)

Degradation of vitamin C undergoes both anaerobic and aerobic pathways.

Qxidation of vitamin C in aerobic pathway occurs mainly during the processing of
food whereas anaerobic degradation of vitamin C mainly during storage.
Hydroxymethylfurfural (HMF) is one of the decomposition products of vitamin C and
acts as precursor of brown pigments (Burdurlu, Koca and Karadeniz, 2005). Vitamin
C degradation in packaged fruit juices depends mainly on storage temperature, pH,
dissolved oxygen level, residual hydrogen peroxide, H2O2 left after the sterilization
of packaging material and trace metal ions (Ozkan, Kirca and Cameroglu, 2004).
Consequently, studies on vitamin C content in foods are important in relation to the
control of nutritional labels, the update of food databases and the establishment of
dietary reference intakes. Orange juice is probably the most globally accepted fruit
juice and it is recognized worldwide as a good source of ascorbic acid (Sharma,
Singh and Saxena, 2006).

In addition, there are many analytical methods used to determine the concentration
of vitamin C in the pharmaceutical samples which are colorimetric method, titration,
enzymatic method, Flow Injection Analysis (FIA) and High Performance Liquid
Chromatography (HPLC) (Arya and Mahajan, 1997). Reflectometer is an instrument
that can used to analyze many different types of test which include ascorbic acid test
that is concerned in this project. It provides a simple and rapid determination of
vitamin C content in many pharmaceuticals product.
Procedure for Calcium Ascorbate
Process

a) In 500.0ml RBF fitted with thermometer pocket and stirrer.

b) Take 200.0ml DM water at 25-30 °C.

c) Charge 30.0gm CaCO3 at 25-30 °C.

d) Charge 0.5ml TGA (Thioglycolic acid)

e) Maintain and stir for 1.0 hr at 25-30 °C.

f) Filter the slurry at 25-30 °C.

g) Suck dry and wash with 100.0ml water.

h) Suck dry and unload wet cake.

Wt. of wet cake 42.0gm

Process

a) In 1000.0ml RBF fitted with thermometer pocket and stirrer.

b) Charge 150.0ml water at 25-30°C.

c) Charge 100.0g Ascorbic acid at 25-30°C.

d) Charge 0.2gm EDTA and 0.2ml TGA at 25-30 °C.

e) Maintain and stir reaction mass for 15.0min at 25-30 °C.

f) Charge above wet cake in 30-45min. at 25-30 °C.

g) Maintain and stir reaction mass for 1.0hr. at 25-30 °C. Solution should be hazy.

h) Clarify the reaction mass at 25-30 °C.

i) Collect filtrate

j) In another 1.0lit. RBF fitted with thermometer pocket and stirrer.

k) Take 500.0ml 95.0% methanol (475.0ml methanol and 25.0ml water).

l) Start addition of above filtrate in 30-45min. at 25-30 °C.

m) Maintain and stir reaction mass for 30.0min. at 25-30 °C.

n) Cool reaction mass to 10°C in 30.0 min.

o) Maintain and stir reaction mass for 1.0hr. at 10-15°C.

p) Filter the reaction mass at 10-15°C.

q) Suck dry and wash with 50.0ml methanol at 25-30°C.

r) Suck dry and unload wet cake.

Wt. of wet cake 100-110.0gm Dry at 40°C under vacuum LOD NMT 0.1% Wt. of dry
material 90.0gm (Tables 1 & 2).
Table 1:

Table 2:
Experimental Procedures
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Ascorbic acid is determined by using an oxidation-reduction reaction. The solubility

of iodine is increased with iodide and triiodide is occurred:

I2 (aq) + I↔ I3 - I3 -

Then oxidizes vitamin C to dehydroascorbic acid: C6H8O6 + I3 + H2O  C6H6O6 +

3I- + 2H+ Vitamin C dehydroascorbic acid The endpoint is production of a blue-black
color which occurs as a result of the reaction of iodine with starch suspension. When
ascorbic acid is present, I3 is converted to iodide and no color change is observed.
However, when all ascorbic acid was utilized, expected blue-black color occurs due
to the reaction between starch and excess tri-iodide. This titration procedure is
widely accepted and is appropriate for testing the amount of vitamin C in the tablets,
liquids and fruits and vegetables [1].
Preparation of Iodine Solution
For preparation of 0.1 M iodine solution, 10g of KI was taken in a 250ml Volumetric
flask and 35 ml of distilled water was added followed by heating the solution; the
mixture was cooled to room temperature and 3.15g of solid Iodine powder was
dissolved. Similarly, to prepare 0.005M of iodine solution 2g of KI was taken in a
500ml beaker and dissolving in 100 ml of distilled water and 1.3 g of iodine powder
was stirred with small quantity of water and qs (quantum satis) to 1 litre [2].
Preparation of Starch Solution
Addition of 0.25g of starch powder in 50ml warm distilled water, As the starch is
insoluble in cold water and needs to be boiled to stay in solution [3].
Preparation of Vitamin C Standard Solution
25mg Ascorbic acid was taken in a 100.00ml beaker and dissolved in 100 ml distilled
water [4].
Preparation of Vitamin C Sample Solution
From the strip of Vitamin C random two tablets were weighed and smashed to form
powder and average value was calculated.

334mg of the powder tablet was taken in a 100.00ml beaker and dissolved in 100ml
distilled water [5] (Table 3). Standardization of the iodine solution with the vitamin C
standard solution and sample solution. The measured volume of 20ml of both
standard and sample was taken from each solution and equilibrated with 150ml
distilled water separately into distinct two Erlenmeyer flask 250.00ml and titrant
containing iodine solution was run against analyte containing either sample or
standard; 5-6 drops of prepared starch solution were added to the analyte and
titration was started. The burette level for each analyte for distinctive experiment was
noted as mentioned below: For standard solution the volume of iodine solution
required for complete reaction = 45ml Equally, for Sample solution the volume of
iodine solution required = 49ml The endpoint was noted when analyte appears blue
in color [4].
Table 3:

Calculation
For sample solution in the beginning of the experiment 20ml of sample was taken
from 100ml of prepared solution containing 100mg of Ascorbic acid. As 49ml of
iodine is required for the color change containing 20ml ascorbic acid solution, the
dilution was done 5 times to that of the solution. Hence, the final volume of the iodine
solution = 49× 5 = 245 ml

Mole iodine = Mass Ascorbic acid × 1 mole 176.12g of ascorbic acid × 1000ml
volume of iodine M iodine = 0.1g × 1/176.12 × 1000 245ml = 0.00231 mol

For standard solution Mass Ascorbic acid = Mole iodine × Volume of iodine × 176.12
= 0.00231 × 45 × 176.12 = 91.54 mg Initially, the amount of Ascorbic acid was taken
for 100mg and therefore for total amount of ascorbic acid i.e. 250 mg the ratio stands
out to be 2.5 (250/100).
Result
Therefore, a 250mg tablet of ascorbic acid from the ACI LIMITED (Nutrivit® C)
contain = 2.5 × 91.54 = 228.85 mg Amount of Ascorbic acid in ACI LIMITED
(Nutrivit® C) is 8.46% less than the claimed value. %
Flow Injection Analysis (FIA)
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In FIA, there is no air segmentation and it is not necessary for a state of chemical
equilibrium to be reached. The sample is introduced into a carrier stream as a
discrete plug. The presence of a sample-carrier interface allows diffusion-controlled
dispersion of the sample as it is swept through narrow-bore tubing to create a
concentration gradient. The flow-through detector monitors the change in
concentration of the reaction product, which is displayed as a well-defined peak
(Ball, 2006). Flow-Injection analysis permits a simple, rapid and sensitive method for
the determination of vitamin C where its systems allow faster sampling rates and
consumed fewer reagents compared with segmented-flow analysis (Kleszczewki and
Kleszczewska, 2002).

Memon, Dahot and Ansari had proposed a method by using mono 1, 10-
phenanthroline-iron(III) complex as oxidant. This experiment was based on its
reducing reaction on mono(1-10- Phenanthroline)-iron(III) to tris(1,10-
Phenanthroline)-iron(II) (ferroin) and the absorbance of ferroin was monitored at
510nm through spectrophotometer equipped with a flow through cell [6] (Figure 2). In
this analysis single channel manifold is used as shown in Figure 2. The reagent
stream is pumped at the flow rate 1.1mL/min via a peristaltic pump equipped with
PVC pump tubing. The vitamin C sample is introduced into the reagent stream via a
rotary teflon valve. A calibration curve for vitamin C in the range 0-50ppm was
plotted from the results obtained by Memon, Memon, Dahot and Ansari which are
shown in Figure 3. They also studied about the effect of reaction coil and reagent
concentration. From the graph (Figure 3), the maximum intensity was observed at
50cm reaction coil (Figure 4).
Figure 3:

Figure 4:
While the results of the effect of reagent concentration obtained is shown in Figure 5
indicating that the maximum signal could be obtained at 35% reagent (Memon,
Memon, Dahot and Ansari, 2000). This method can be improved within certain limits
by increasing the volume of the injected sample in flow injection analysis. The
sensitivity is increased two fold with the increase of sample volume. As conclusion,
since the time required for sample preparation is short and reagent consumption is
low, hence the method is highly economical and is suitable to use on routine basis
for determination of ascorbic acid in pharmaceutical preparations [7].
Figure 5:

Ultraviolet (UV) Spectrophotometry

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Direct ultraviolet spectrophotometry is a fast, simple and reliable method for the
determination of vitamin C. This method can be done through alkaline treatment and
the maximum absorption of vitamin C falls at 243nm at pH2 (Yanshan, 1997). The
absorption of UV light by the sample matrix was the major problem in this method.
Therefore, alkaline treatment method was found to be used as background
correction in blank. This is because more than 95% of vitamin C will be destroyed in
10 minutes after alkaline treatment which is in the range of pH 12 to 13 (Salkic and
Kubicek, 2008). UV spectrophotometry method was found to be applicable for most
fruits, fruit juices and soft drinks except those that are unstable to alkaline treatment,
and were deeply colored, or contained high concentration of caffeine, saccharin,
caramel and tannic acid (Yanshan, 1997).

To determine the total content of vitamin C in food samples, a well-established

method was investigated by Khan, Rahman, Islam and Begum, 2006 by using the
2,4-dinitrophenyl hydrazine methods (DNPH). This is a simplified method for the
simultaneous determination of total vitamin C employed coupling reaction of 2,4-
dinitrophenyl hydrazine dye with vitamin C and followed by spectrophotometric
determination. The spectrophotometric method involves the oxidation of ascorbic
acid to dehydroascorbic acid by the action of bromine solution in the presence of
acetic acid. Reaction between dehydoascorbic acid and 2,4-dinitrophenyl hydrazine
at 37 °C temperature for three hours will form an osazone. The solution is treated
with 85% H2SO4 to produce a red color complex.

The absorbance of all standards was measured at 521nm by using a UV-

spectrophotometer. The results obtained were taken to contruct a calibration curve
(Khan et al, 2006) (Figure 5). The calibration curve was constructed by plotting the
concentration versus the corresponding absorbance as shown by Figure 6. The
molar absorptivity? Can be obtained using Beer-Lambert plots.

The reliabilty of this method was justified by the calculations of the % of standard
deviation and it was found to be varied within the range from 0.20 to 2.45%. The
reliability of this method was also confirmed from the consideration of the following
expected interferences [8].
Figure 6:
Figure 6.1:

There are a few interferences that might affect the results. First, the interference was
due to the diketogulonic acid. At higher pH, destructive oxidation hydrolysis might
occur (Figure 6). This result in the opening of the lactone ring of the ascorbic acid
and loose the vitamin activity. These processes are naturally occurred in fruits and
some amounts of diketogulonic acid are presence in the fruits. Besides that,
diketogulonic acid has keto group that might form osazone when react with DNPH.
Hence, there is a chance of error in this method which may give false results (Khan
et al, 2006) [9]. Another interference was due to the extracted glucose which
contains similar structure like vitamin C. Therefore, some of the glucose may be
extracted in the meta-phosphoric acid during the extraction of ascorbic acid from
sample. Glucose may also cause the formation of colored complex with DNPH and
gives the false result in the determination of vitamin C. This was proven in Figure 6.1
where there is no absorption peak around the interested peak at 52nm (Khan et al,
2006). As conclusion, the method is simple and excellent for the determination of
total vitamin C in fruits and vegetables (Figure 6.1).
Fluorometric Method
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Fluorometric analysis has been used for ascorbic acid assay in pharmaceutical
preparations, beverages, special dietary foods and even for human serum (Arya,
Mahajan and Jain, 2000). This method had been reported to have successful
application to a wide range of foodstuffs, including liver, milk, fresh and canned fruit,
raw and cooked vegetables, and potato powder (Ball, 2006). Previously, fluorometric
determinations of vitamin C have been developed based on condensation reactions
of vitamin C with o-phenylenediamine and on the oxidation with mercury (II) of
vitamin C to form quinoxaline derivative. The reaction products of these methods
exhibit fluorescensce (Yusuf and Gurel, 2005) [10].

Figure 7 shows the reaction of the dehydroascorbic acid with 1,2-phenylenediamine

dihydrochloride to form the fluorescent quinoxaline derivative 3(1,2dihydroxyethyl)
furol [3,4-b] quinoxaline-1-one. The blank can be prepared by complexing the
oxidized vitamin with boric acid to prevent the formation of the quinoxaline derivative.
It is used to reveal any fluorescence due to interfering substances (Ball, 2006).
(Figures 7 & 8). Yusuf and Gurel have described a method by using Methylene Blue
(MB) for the determination of vitamin C. This experiment was run by using a
spectrofluorimeter to record the spectra and carry out fluorescence measurements
[11].
Figure 7:
Figure 8:

Figure 9:

Figure 10:
This method was used to determine the amount of vitamin C in the purified materials,
specifically vitamin C tablets. MB is a member of thiazine dye group. It is widely used
in many different areas. For example, a photo sensitizer is used to produce singlet
oxygen in photodynamic therapy for the treatment of cancer [12]. The highly colored
oxidized form of MB can be reduced to be colorless leuco form, Leuco-Methylene
Blue (LMB) which is shown in Figure 9. LMB is the reduced and colorless form of
methylene blue (Yusuf and Gurel, 2005) (Figure 9). According to Yusuf and Gurel,
the fluorescence bands of MB were obtained at 664nm for excitation state and
682nm for emission peaks. This was proven by the other researchers who also
examined the emission bands at 682nm for MB and 452nm for LMB. In Figure 10,
the emission peak of MB at 682nm increased due to the increase of its
concentration. A linear relationship between MB concentration and intensity was
obtained over the concentration range of mol L-1 MB (y= 49.082x +
94.46,r2=0.9969). The excitation peak of MB at 664nm also linearly increased
depending on the increase of its concentration (Yusuf and Gurel, 2005) (Figure 10).
The studies of the effect of vitamin C on the fluorescence of MB is made to avoid any
errors that might affect the accuracy of the results. In order to examine the effect of
vitamin C on the fluorescence of MB at 664 nm, mol L-1 MB solutions, each solution
was added with different concentration of vitamin C and were prepared under
nitrogen (N2) atmosphere. This was shown in Figure 11 where the spectra were
recorded at 664nm (Yusuf and Gurel, 2005). Figure 13 above shows the excitation
intensity of mol L-1 without adding vitamin C was about 1000.0 and above. The
intensity was decreased by the increase of vitamin C concentration in MB solutions
(Yusuf and Gurel, 2005) (Figure 11).
Figure 11:
Table 4: Tolerance between Different Substances in Determination of Ascorbic
acida).
Note: Ascorbic acid concentration 1.0 X 10-6 mol -1

Figure 9 shows the emission spectrum of mol L-1 MB as a function of time. Each
spectrum was recorded at 1 minute intervals. The results showed that the
fluorescence was not changed with time, reflecting that the fluorescence spectrum of
MB was highly stable with time (Yusuf and Gurel, 2005) (Figure 12). In the redox
reaction between ascorbic acid and MB, the ascorbic acid is oxidized to
dehydroascorbic acid, while MB was reduced to colorless LMB as shown in the
following: The calibration curve was made based on the concentration of MB (mol L-
1)[13]. The results indicate that the fluorescence intensity of the system is a linear
function of vitamin C concentration in the range of mol L-1 and the regression
coefficient is 0.9941 as shown in Figure 10 (Yusuf and Gurel, 2005) (Figure 13)
(Table 4).
Figure 12:
Figure 13:

Table 4 below shows the tolerance towards different compounds that might cause
interferences in this method. These compounds are usually present in most vitamin
C tablets. The experimental results showed that the presence of hundred-fold excess
of the all contaminant compounds and twenty-fold excess of citric acid did not
significantly influence the determination of vitamin C using this method. Therefore, it
can be concluded that there is no major interference caused by these compounds
(Yusuf and Gurel, 2005). So it is possible to use this method for direct determination
of vitamin C in pharmaceuticals without separating the interfering materials (Table 5).

Table 5 lists the results obtained by the proposed method with triiodide method. It
can be clearly seen that the results are in good agreement with the triiodide method
(Yusuf and Gurel, 2005). Thus, the proposed method provides a simple and
sensitive fluorimetric procedure by using MB for the determination of vitamin C
[14,15]. This experiment also shows that MB could be used for fluorimetric
determination of vitamin C in vitamin C tablets although it has only slightly
fluorescence property compared to LMB. Therefore, as conclusion, it can be
explained that the fluorescence intensity of MB was more sensitive to determine
vitamin C concentration [16].
Table 5: Determination of Ascorbic acid in Pharmaceutical preparations.

Note: Mean for five determinations

Stability of Vitamin C in Orange Juic
Vitamin C is very susceptible to chemical and enzymatic oxidation during the
processing, storage, and cooking of food. The catalyzed oxidation pathway of
vitamin C degradation is the most important reaction pathway for the loss of vitamin
C in foods. Therefore, vitamin C of orange juice is readily oxidized and lost during
staying of the juice (Ball, 2006). On the other hand, there are several factors that will
also affect the stability of vitamin C in orange juice. The factors are such as the effect
of vitamin E, pH, and parameters which include air, heat, water as well as prolonged
storage and overcooking (Kabasakalis, Siopidou, and Moshatou, 2000).
According to Ball, a meta-oxygen-ascorbate complex is formed in the presence of
molecular oxygen and trace amounts of transition metal which particularly are copper
(II) and iron (III). This complex contains a resonance form of a diradical that rapidly
decompose to give the ascorbate radical anion, the original metal ion, and hydrogen
peroxide. This radical anion will in turn reacts with the oxygen to give
dehydroascorbic acid (DHAA). For anaerobic pathway of vitamin C which occurs in
the absence of free oxygen, the degradation is caused by the formation of
diketogulconic acid. As the rate of degradation is maximum at pH 3 to pH 4,
therefore this pathway is mostly responsible for anaerobic loss of vitamin C in
canned grapefruit and orange juices (Ball, 2006) [17].
Effect of Vitamin E on the Stability of Vitamin C in
Orange Juice
Vitamin E is a fat soluble antioxidant that has four tocopherols and four tocotrienols.
In nature, these four tocopherols and four corresponding tocotrienols are designated
as alpha-(?), beta-(?), gamma-(?) and delta-(?) according to the number and position
of methyl substituent in chromonal ring (Ball, 2006). The vitamin E functions as a
biological antioxidant by protecting the vital phospholipids in cellular and subcellular
membranes from peroxidative degeneration. Vitamin E mostly accumulates in body
which are liver and pancreas. But unlike vitamins A and D, vitamin E is essentially
nontoxic (Ball, 2006) [18].

Nagymate and Fodor (2008) have designed a method to study the effect of vitamin E
on the stability of vitamin C. In this experiment, vitamin E stock solution was
prepared by dissolving ?-tocopherol in absolute ethanol. The orange juice which
contained vitamin E and vitamin C was used as sample. The storage temperature of
the vials was 4° C and they were covered with aluminium foil to prevent the effect of
sunlight. Besides, two different temperatures were used to examine the effect of
vitamin E at that temperature which half of the samples were stored at 20° C. On the
other hand, the additive effect of these vitamins was also examined but only cool
samples (4° C) were used for this experiment. Two samples were prepared which
one contained vitamin E stock solution and vitamin C stock solution while another
contained only vitamin C stock solution. The samples were analysed once a week for
five weeks (Nagymate and Fodor, 2008) [19].
The results of the stability of vitamin C show that the presence of vitamin E
influenced the decay of vitamin C. Figure 15 shows that there were differences
between samples with or without vitamin E. From Figure 15, it can be clearly seen
that the concentration of vitamin C without vitamin E fell down to 1.2mg/L on the
second day. However, in the presence of both vitamins, the decay was also
observed, but it was lesser. The concentration of vitamin C in the orange juice with
vitamin E was 13mg/L in the fifth week. As a result, it seems that vitamin E stabilized
vitamin C in orange juice at a determined concentration. This is because vitamin E
delay the oxidation of vitamin C thus, enhances the stability of vitamin C in orange
juice. The combination of vitamin C with vitamin E makes the orange juice more
stable and slower the degradation of orange juice. This concluded that orange juice
with vitamin E addition is a good way to preserve the vitamin C content during
storage (Nagymate and Fodor, 2008).
Effect of Temperature on the Stability of Vitamin C in
Orange Juice
Vitamin C of fruit juice is readily oxidized and lost depends on the conditions of
storage. There are studies about the determination of the amounts of vitamin C
content in fruit juices under different storage conditions. Kabasakalis, Sipadou and
Moshatou had done an experiment to determine the rate loss of vitamin C with
respect to time and temperature of storage [20]. A long-life and short-life commercial
orange juice 100% without preservatives and fresh orange juice were used for
analysis. In this experiment, the days before the expiration date were recorded in
Table 6 to observe the loss of vitamin C in short-life and long-life orange juice 100%
as the expiration date was approached (Kabasakalis, Siopidou and Moshatou, 2000)
(Table 6).

Table 6 shows the loss of vitamin C from fresh and longlife commercial orange juice
100% during a 31 days period, with measurements made every 1 to 3 days. The
samples were refrigerated into containers which after the initial measurement
remained either open or with closed cap until the next measurement. Based on the
results shown in Table 5, the magnitude of vitamin C did not differ significantly
between open and closed cap for both juices. The commercial orange juice lost
higher amounts of vitamin C compared with fresh orange juice. As reported,
decreases of vitamin C upon storage did not correspond to increases of
dehydroascorbic acid levels. In fact, there was an increase of dehydoascorbic acid
levels in aseptically packaged orange juices. This means that the overall nutritional
quality of orange juices is affected upon storage (Kabasakalis, Siopidou and
Moshatou, 2000).
Table 6: Program of the temperature conditions for sample storage.

The loss of the vitamin C in a commercial long-life orange juice 100% stored in
refrigerator and non-refrigerated for a period of 10 days in open containers were
shown in Figure 14 (Kabasakalis, Siopidou and Moshatou, 2000). According to
Figure 14, non-refrigerated samples show higher percentage loss of vitamin C as
compared to refrigerated samples. This is because the dehydoascorbic acid, the
oxidized form of ascorbic acid was more stable at lower temperatures. Thus, the
vitamin C, in the form of dehydroascorbic acid for refrigerated orange juice was well
retained than non-refrigerated orange juice (Kabasakalis, Siopidou and Moshatou,
2000) (Table 7).
Table 7: Assessment of unpasteurized refrigerated orange juice stored under
isothermal and non isothermal conditions for72 hours.
TSS=total titratable acidity; TTA=total soluble solids; TSS,TTA and pH: mean of the
values at time 0,24,48 and72h; condition n01- 4h/80C and 60h/40C; condition n02-
4h/40C, 4h/120C and 64h/80C.
Effect of Hydrogen Peroxide on the Stability of Orange
Juice
Hydrogen peroxide, H2O2 is the primary chemical for sterilization of plastic
packaging material used in aseptic system. Aseptic packaging technology is widely
used by fruit juice industry for the production of shelf-life stable fruit juices. A Food
and Drug Administration (FDA) regulation currently limits the residual of H2O2 to
0.5ppm, leached into distilled water, in finished food packages which stated in Code
of Federal Regulations, 2000 [21]. However, during the sterilization of aseptic
chambers or packaging material with H2O2, some residues will still be left on the
packaging material or vapors generated during drying may get trapped inside the
package upon sealing. These residues will then cause the degradation of vitamin C.

An experiment was proposed by Ozkan, Kirca and Cemeroglu to determine the rates
of vitamin C degradation in orange juice with or without addition of H2O2 at various
storage temperatures. In this experiment, the orange juice sample was thawed at
room temperature and sodium benzoate was added to prevent spoilage. The
degradation studies were done at H2O2 with 0.5ppm concentration at 20°C, 30°C
and 40°C respectively. At regular time intervals, samples were removed from the
water bath or incubator (Ozkan, Kirca and Cemeroglu, 2004).

Then, the predetermined amounts of diluted sodium hydroxide solution were added
rapidly to the samples to halt the reaction between H2O2 and vitamin C. The
samples were then rapidly cooled by plugging into an ice water bath and held at
-30°C until analyzed for vitamin C content. Vitamin C concentration was measured
by using HPLC method. Qzkan, Kirca and Cemeroglu had modified the method by
blending the orange juice sample with metaphosphoric acid. The sample was filtered
through a membrane filter and was analyzed using HPLC (Shimadzu brand) (Ozkan,
Kirca and Cemeroglu, 2004).Vitamin C contents of orange juice were plotted for
various temperatures at 0.5ppm H2O2 concentration.

From Figure 14, the results show that at higher temperature, the rate of vitamin C
degradation also increased. The addition of 0.5ppm H2O2 did not greatly increase
the degradation of vitamin C. However, raising H2O2 concentration from 0.5ppm to
5ppm resulted in a tremendous increase in degradation rates which was recorded in
Table 6. At 0.5ppm H2O2, the antioxidant substances in orange juice which was
flavonols reacted with H2O2, thereby preventing the autoxidation of vitamin C. The
protective mechanism of flavanols was mainly due to chelation of metal ions and
action of antioxidant. Flavanols function as antioxidants by donating the hydrogen
ions to reactive free radicals which may otherwise cause the autoxidation of vitamin
C (Ozkan, Kirca and Cemeroglu, 2004).

Ozkan, Kirca and Cemeroglu also studied the degradation of vitamin C in the
absence of H2O2. In this case, the activation energy, Ea was taken into account to
determine the stability of vitamin C in orange juice. The temperature dependence of
the degradation of vitamin C in orange juice was compared by calculating Ea and
temperature quotients (Q10) at 20° to 40°C from the following equation: These
results clearly indicate that the rate of vitamin C degradation in the presence of
H2O2 was slower at 30°C to 40°C than 20°C to 30°C. This indicates that at 30°C to
40°C, the least effect of temperature rise on vitamin C degradation.

The results obtained for Ea shows that higher Ea in the presence of H2O2. This
means that higher energy needed for the degradation of vitamin C. Therefore, the
reaction time is slower and the degradation of vitamin C also slower. As conclusion,
the effect of temperature on the degradation rates of vitamin C in orange juice was
more pronounced at higher H2O2 concentrations. Therefore, greater vitamin C
losses should be expected as residual H2O2 concentration and storage temperature
increase in aseptically packaged fruit juices (Ozkan, Kirca and Cemeroglu, 2004)
(Figures 14 & 15).
Figure 14:

Figure 15:

Effect of pH on the Stability of Vitamin C

pH is a measure of acidity or basicity of a solution. pH is one of the primary factor
that would affects the stability of vitamin C in orange juice. Hence, the pH value of
the matrix has an influence on the stability of vitamin C. According to FAO/WHO
Expert Consultation on Human Vitamin and Mineral Requirements, Bangkok,
Thailand, 1998, the vitamin C will decay if the pH higher than 4 (Nagymate and
Fodor, 2008). Vitamin C is unstable in neutral and alkaline environments, therefore
the higher the pH value and the longer the exposure, the greater the loss of vitamin
C. This is because the higher the pH value, the faster the oxidation reaction of
vitamin C and causes the degradation of vitamin C. Besides that, the increase in pH
also related to deterioration of fruit characteristic which in this literature review,
orange juice is more concerned. Table 7 below shows the pH value of the fruit juice
with storage time (Ajibola, Babatunde and Suleiman, 2009) (Tables 8 & 9).
Table 8: Reaction rate constants (K) for ascorbic acid degradation in the presence of
hydrogen peroxide in various fruit juices during storage.

In this the pH values of the orange juice were higher at room temperature and keep
increasing from week to week. This study concluded that, though pH was significant
for the stability of vitamin C, it was not the sole factor in controlling the deterioration
of vitamin C in orange juice with storage life (Ajibola, Babatunde and Suleiman,
2009). On the other hand, the loss of vitamin C activity during oxidative degradation
of vitamin C occurs with the hydrolysis of the dehydroascorbic acid lactone to yield
2,3-diketogulonic acid. This hydrolysis is favored by alkaline solution.
Dehydroascorbic acid is most stable at pH 5.5 but decrease in stability as pH
increases which is more than pH 5.5 (Fennmena, 1996). For example, halftime
values of dehydroasorbic acid hydrolysis at 23°C were 100 and 230 minutes at pH
7.2 and pH 6.6 respectively.
At pH 5.0 or below, dehydroascorbic acid was quite stable which decayed by less
than 3% over 4 hours. This experiment evaluated the effect of hydrogen ion
concentration on delactonization of dehyroascorbic acid over the range of pH 3.0 to
pH 8.0. The possible influence of the presence of oxygen was done by equilibrating
the reaction mixture before and during the incubation with 100% oxygen or with
100% nitrogen. The results indicated no change in the decay rate of dehydoascorbic
acid was obvious with these alterations of atmospheric conditions. The rate of
dehydroascorbic acid hydrolysis markedly increases with increasing temperature but
was unaffected by the presence of oxygen (Bode, Cunningham and Rose, 1990).

Other researchers had proposed a method to determine the effect of pH on the

degradation of vitamin C in orange juice. The aim of their experiment was by
comparing the stability of vitamin C at different concentrations at lower pH value. An
acidic sample was prepared from orange juice with medium alcohol content. The
original pH value of sample was later modified by addition of concentrated
phosphoric acid. After that, different concentrations of vitamin C stock solutions were
added and analysed for five weeks (Nagymate and Fodor, 2008). The results
showed that, for the experiment done in original pH value of the orange juice which
was pH 4.0, the reduction of the amount of vitamin C content decreased with the
increasing ascorbic acid concentration (p> 0.05), so the speed of decay was higher
at lower concentrations. In the case of orange juice, the highest standard deviation of
the repeated data was 3.14% and the lowest standard deviation was 1.48%. This
indicates that the results are accurate and reproducible (Nagymate and Fodor,
2008).
Table 9: Natural Orange Juice Ascorbic acid content (mg/100mL) at different storage
times after squeezing.
When lower pH (pH 3.0) was used, the speed of decay for orange juice grew with the
growing vitamin C concentration, and the highest value was at 50mg/L vitamin C
concentration (p> 0.05). These results were tabulated in Table 9. The highest
standard deviation of the repeated data was 3.42% and the lowest standard
deviation was 1.53%. By comparing the statistical data in both Tables 8 & 9, it shows
that the lower pH values increased the vitamin C content measured at the end of fifth
week (Nagymate and Fodor, 2008). According to Nagymate and Fodor, vitamin C
had a significant decay independently from the storage temperature when the pH
value was more than 4.0. Nevertheless, under this pH limit, low storage temperature
will help in stabilizing this vitamin. Hence, lower pH value was preferred to prolong
the shelf life of orange juice.
Materials and Methods
Go to

Analysis of Vitamin C Content in Vitamin C Tablets

Instrument, Materials and Chemicals

The instrument that used in this research is Reflectometer

The chemicals that used in this research are as below
a) 1 gdm-3 of Ascorbic Acid (Sigma)

b) 0.1M Sodium Hydroxide (R & M Chemicals)

c) Phenolphthalein indicator
d) Potassium acid phthalate (Hamburg Chemical GMBH)

8.2. The materials that used in this research are as below

a) Flavettes Vitamin C-Sugar-Free C

b) Redoxon Orange

c) Cebion Vitamin C

d) Bio C Plus (Anway)

Product Sample Preparation
The vitamin C tablets were crushed into powder by using mortar and pestle. 1gdm-3
of each sample was prepared by dissolving 0.1±0.05g of the samples with distilled
water and top up to 100mL. The solutions were thoroughly mixed. The solutions
were kept away from direct sunlight and stopper was used to minimize the oxidation
of ascorbic acid.
Reagent Solution Preparation
Preparation of Vitamin C Standard Solution

A freshly prepared ascorbic acid solution was prepared by dissolving 0.02g of

ascorbic acid and top up to 100mL with distilled water in a volumetric flask. The
solution was thoroughly mixed. The solution was kept away from direct sunlight and
a stopper was used to minimize the oxidation of ascorbic acid.
Preparation of Sodium Hydroxide Solution
0.1M of sodium hydroxide solution was prepared by adding 2.0g of sodium hydroxide
(NaOH) pellets and was topped up to 500mL into a volumetric flask. This solution
was mixed and was standardized using potassium acid phthalate (KHP) solution.
Experimental Procedure
Go to
Determination of Vitamin C by using Reflectometer
(MERCK)
As control method for ascorbic acid test, 0.02g of ascorbic acid was diluted with
100mL of distilled water. The ascorbic acid solution should be freshly prepared.
Beside that, two additional tests were done by using the same instrument but
different test kits which used to test the glucose and Hydroxymethylfurfural (HMF)
test. The respective strip was immersed into the sample for few seconds. The test
strip was inserted into the strip adapter. At the end of reaction time, the result was
read from the display.
Determination of Vitamin C by using Titration Method
Standardization of 0.1M Sodium Hydroxide (NaOH): Standard potassium acid
phthalate (KHP), KC8H4O4H solution was prepared by weighing 0.5g dried KHP into
an Erlenmeyer flasks and was dissolved by adding 75mL of distilled water. The
molecular weight of KHP is 204.23. Three drops of phenolphthalein indicator were
added into each Erlenmeyer flask. For 0.1M NaOH standardization, KHP solution
was used and titrated with 0.1M NaOH until the faintest pink persists for 30 seconds.
The final volume was recorded. The standardization of NaOH was repeated for
another two times.
Vitamin C Analysis: Quantitative Method
0.2g of vitamin C was weighed and then added into an Erlenmeyer flask. 50mL of
distilled water was added to dissolve the vitamin C tablet. Three drops of
phenolphthalein indicator were added into the Erlenmeyer flask. A burette which
contains 0.1M of NaOH solution was set up as which as shown in Figure 15. The
vitamin C solution was titrated with NaOH solution was until a pink color that persists
for 30 seconds which was the end point. The final volume was recorded. These
procedures were repeated for another two sets of sample.
Result and Discussion
Go to
Determination of Vitamin C by using Reflectometer
(MERCK)
In this project, reflectometer and titration method were used to determine the vitamin
C content in four pharmaceutical products which are Flavette, Cebion, Bio C Plus
and Redoxon Orange. In reflectoquant analysis, Reflectoquant Test Strips are
inserted into the instrument (RQflex 10) which is a highly sensitive reflectrometry
instrument. The combination of these two tests which are the test strips and the
reflectometer help to analyze the vitamin C content for a wide variety of samples in
just few seconds. Reflectometer uses a double optic system which works in
conjunction with a dual reaction zone on Reflectoquant Test Strips to allow for
simultaneous double measurements in one step. It is a portable test system that is
small, compact, and battery-operated for rapid, quantitative analysis of various
samples by evaluation of special test strips. Vitamin C contents in each sample
which is determined using reflectometer do not have much different compared to the
label value. Tablets are labeled according to their vitamin C content and not
according to their weight.

The percentage of deviation of each sample was calculated and the percentages of
deviation obtained for all the samples are less than 5.0%. This indicates that
reflectometer can provide accurate and reproducible results. The difference between
label value and analysis results could be caused by the interference substances
such as the presence of binder. Binders are commonly used when making
conventional tablets. Most binders are polymers which can increase the plastic
deformation of the formulation. Binder can be used to prevent a rapid dissolution of
the effervescent tablet such as Redoxon. Examples of binders are such as methyl
cellulose and gelatin which function to hold the ingredients together to form a tablet
(Swarbrick and Boylan, 1992). For Redoxon, the analysis result is lower than the
label value. This is due to the presence of foreign substances such as zinc citrate
which may influence the concentration of vitamin C.
Determination of Glucose Content and
Hydroxymethylfurfural Content in Vitamin C Tablet
Glucose content in each tablet of different brands is less than 1g/mL except for
Flavette Vitamin C. Flavette Vitamin C contains 22mg/L of glucose content although
this produc is labeled sugar free. Many manufactures use glucose, fructose or
dextrose to sweeten a tablet for commercial purpose even though the tablet labeled
‘no sugar’. For hydroxymethylfurfural (HMF) test, 5-(Hydroxymethyl) furfural (5-
hydroxymethyl-2-furancarbaldehyde, HMF) reacts with a barbituric derivative and an
aminophenazone derivative to form a red-violet compound that is determined
reflectrometrically.
HMF test was done to test the amount of undesirable product such as 5-HMF in
vitamin C tablet. From the results, all the product samples contain about 1.3 to 1.5
mg/L of HMF. Based on the Commission Regulation, the average usage of HMF is
2.0mg/L and the maximum usage is 10.0mg/L for non-alcoholic products (Berger,
2007). HMF is a crystalline product with a pleasant odor. HMF is formed in foods by
thermal treatment during storage (Nollet, 2004). High amount of HMF can cause
vitamin C loss, hence affecting the quality of the product (Damasceno et al, 2008)
[22].
Determination of Vitamin C by using Titration Method
Standardization of Sodium Hydroxide (NaOH) solution using potassium acid
phthalate solution (KHP)

In a titration, it is critical to know the exact concentration of NaOH in order to

determine the concentration of the solution being tested. KHP is a weak acid and
reacts with base in the following way:

In the titration method, phenolphthalein was used as an indicator which will be used
to determine when the reaction reaches its endpoint. Endpoint is the point which the
amount of NaOH added equals the amount of vitamin C. By knowing the strength
and the volume of NaOH required to completely react with vitamin C, the actual
amount of vitamin C present can be calculated. pH meter also helps to determine the
end point which is about pH8.50. As vitamin C is a weak acid, the pH of the end
point is detected by using phenolphthalein indicator with the transition range
between pH8.

0-9.8. Phenolphthalein will changes from colorless to pink when all of the acid has
been neutralized. The samples were analyzed by the reflectometer and the titration
methods are summarized in Table 7. The results show that both methods are in
agreement with the quantities specified on the label. This indicates that the proposed
method was applied successfully for the determination of vitamin C in commercial
pharmaceutical products.
Conclusion
Go to
Vitamin C is required for the optimal activity of several important biosynthetic
enzymes and it is therefore essential for various metabolic pathways in the body.
However, according to RDA for vitamin C, 75mg/day and 90mg/day are required for
normal women and men respectively. This level is believed sufficient enough to
prevent deficiency disease but not chronic disease. Owing to this, vitamin C should
be taken each day to prevent chronic disease and the effective doses are still
remained unclear today. On the other hand, the Tolerable Upper Intake Level (UL) is
2000mg/day where too much of vitamin C may be dangerous due to the adverse
effects such as kidney stone formation, increase of uric acid excretion and overload
iron.

There are few factors that will affect the stability of vitamin C which are vitamin E,
pH, amount of hydrogen peroxide and temperature. Additionally, freshly prepared
orange juice should be taken in as soon as possible. This because vitamin C may be
oxidized during storage even we store it in refrigerator. It was found that vitamin C
loss is the most when the orange juice was stored at room temperature. Well-
pasteurized package juice from market can lose its vitamin C as well due to long
storage time even if it is not opened. The highest loss of vitamin C occurred with
conventional boiling. The research also shows that orange juice with vitamin E can
delay the degradation rate of vitamin C. Besides that, high concentration of hydrogen
peroxide will cause greater loss of vitamin C. Finally, lower pH value was preferred
to prolong the shelf life of orange juice.

Three different methods are studied in the review part which is flow-injection
analysis, ultraviolet spectrophotometry and fluorometric method. All these methods
are using spectrophotometer as the detector to determine the content of vitamin C in
a sample. Nevertheless, all these methods required highly cost equipment and
reagent in order to perform an analysis than reflectometric method. In my project, the
proposed method using reflectometer can provide a simple analysis of vitamin C.
The simplicity of the procedure permits rapid analysis for vitamin C content in
pharmaceutical products. This method is found to be more sensitive and reliable.
Besides that, the time required for sample preparation is short and reagent
consumption is also low, hence this method is highly economical.
In addition, by using reflectometer, it is a good alternative method compared to some
of the highly cost instrument method. Therefore, it is suitable to use on routine basis
for the determination of vitamin C in pharmaceutical preparations. It is desirable that
nutrition education should be brought into the public understanding of science.
Moreover, more researches should be done on the field of the relation between
diseases and vitamin C intake. Food or crop engineering is another critical field in
the study of vitamin C where this can improve the concentration of ascorbic acid in
natural food.

Vitamin C Determination by Ultraviolet Spectroscopy

and Multiproduct Calibration

ABSTRACT
In this work the vitamin C was determined in industrialized nectar juices through
ultraviolet (UV) spectroscopy and multiproduct multivariate calibration, based on partial
least squares (PLS) regression. Since samples with different flavors, sugar content (light
or not) were together in the model construction, it can be considered as a multiproduct
and, due to the heterogeneity of the samples, it was necessary to optimize the calibration
and validation sets by outliers elimination. The model was developed and validated by the
evaluation of the figures of merit such as: accuracy, sensitivity, analytical sensitivity,
adjust, linearity, relative prediction deviation, limits of detection and quantification,
indicating that the multiproduct model developed from UV spectroscopy and PLS
regression can be used in the industrial routine analysis as an alternative to titration or
other time and reagent consuming methods. Here, it was evidenced that the UV-PLS
multiproduct model provides advantages as being free of sample preparation steps, is
suitable to be updated in order to measure other parameters, does not generates
residues and is feasible to be implemented for on-line monitoring. Furthermore, the
application of multivariate calibration in multiproduct models is extremely attractive from
the industrial point of view.

Keywords: vitamin C; UV spectroscopy; multiproduct calibration; PLS

INTRODUCTION

Vitamin C or L-ascorbic acid is an essential nutrient for human health, 1 widely known
for its potent antioxidant properties. It can be used in high-doses and has being
pointed out as presenting benefits in the treatment of Alzheimer's disease.2 Among
the multiple roles played by this vitamin, its primary functions are to act as a
cofactor for reactions requiring reduced iron or copper metalloenzyme and as a
protective antioxidant which better reacts in aqueous phases, both intra- and
extracellularly. Scientific evidences show that ascorbic acid plays an important rule
on the corneal epithelium, the stroma and the endothelium, acting over the
maintenance of its functions and ultra-structures.3 Furthermore, epidemiologic
studies have associated greater plasma vitamin C levels with reduced risk of chronic
diseases, including cancer and cardiovascular disease, as well as higher physical
performance in the elderly.4

The wide variety of ready-to-drink beverages has driven market to give special
attention to it,5 and so, fruit nectar, which is by definition a non-fermented ready-to-
drink beverage, obtained from the edible part of the fresh fruit diluted in water, in
which may or not be added of sugars and acids. The preference for beverages
classified as nectars has trend to grow due to its convenience and low price and,
besides the wide range of flavors commercially available, has the possibility for being
used in the production of low-calorie beverages (classified as light ones) in a feasible
way to the industries. These low-calorie beverages are referred to the ones in which
the sugars content, normally added in conventional beverages, are entirely replaced
by natural or artificial sweeteners.6 In both cases, conventional or low-calorie fruit
nectars, vitamin C is used as a conserving reagent.

Nowadays, several methods based on high performance liquid chromatography

(HPLC) have already being proposed in order to determine vitamin C content in
beverages,7,8 and foods.9,10 Nonetheless, methods such as capillary zone
electrophoresis,11 and voltammetry have being used to determine vitamin C in
different samples, as pharmaceutical preparations and fresh fruit juice.12 Recently,
ultra performance liquid chromatography (UPLC) and HPLC were proposed to
determine vitamin C in beverages and vitamin tablets. 13

Ultraviolet (UV) spectroscopy can be used together with multivariate calibration to

determine various compounds, since the spectral region used could bring information
about the chemical structures of the compounds due to chromophore
absorptions.14 By ensuring this, the spectral region can be analyzed with the aid of
chemometric methods of multivariate calibration to quantify different constituents in
samples such as beverages.14,15 Nonetheless, the Association of Official Analytical
Chemists,16 proposes that, when vitamin C is found in low concentration, as in fruit
juices, the Tillman's method is recommended. This method is based on the reduction
of the 2,6-dichlorophenol indophenol by the vitamin C and was used in this research
as reference method, which results were used for multiproduct multivariate
calibration (i.e., only one multivariate calibration model to predict the same property
of interest in different products) to UV spectroscopy in order to determine vitamin C
content in industrialized nectar juices.

From these observations, the aim of the present study was to evaluate a
multiproduct multivariate calibration model derived from UV spectroscopy to
determine vitamin C in beverages from different fruit nectar. In this purpose, the
model is a multiproduct because samples with different flavors and differing on sugar
content (conventional and light) were together in the model construction.

The local calibration models of one-product have some disadvantages as a large

number of computations required for each prediction in routine analysis, besides the
fact that each specific calibration equation can only be used for a small population of
samples and each sample must be clearly identified to be able to select the best
prediction equation.14,17 In addition, maintenance of the one-product multivariate
calibration models can be as laborious as the multi-product ones. Thus, to save time
on updates, it is worthwhile to investigate if multi-product models can be
developed.14,18

Multiproduct multivariate calibration can be used to construct a multivariate

calibration model from different products. In our case, juices of different flavors were
used to provide a particular parameter, the vitamin C content. The early studies
came from nineties decade,19,20 and the goals were evaluations of the performance of
new algorithms. Posteriorly, in 2000 and 2006,17,18 the multiproduct calibration was
recovery but using non-linear calibration methods, which are not easily implemented
in practical applications, such as routine analysis in industry. Recently, preliminary
studies combining UV spectroscopy and linear method based on partial least squares
(PLS) to construct multiproduct multivariate calibration model was proposed. 14 In
those study, vitamin C and carbohydrates were determined in industrialized juices,
but only yellow samples were employed to build the model. In another study the
total acidity was evaluated in nectar juice, suggesting the possibility to build
multiproduct calibration models by the PLS regression method.6

Experimental

Samples and reagents

A total of 123 samples of fruit nectar were purchased in different Brazilian

marketplaces. The flavors employed were: grape (9 samples), white grape (3
samples), light grape (6 samples), peach (9 samples), light peach (6 samples), pear
(3 samples), strawberry (3 samples), passion fruit (9 samples), light passion fruit (3
samples), mango (9 samples), light mango (6 samples), apple (12 samples), lemon
(3 samples), orange (12 samples), light orange (3 samples), guava (6 samples),
light guava (6 samples), cashew (3 samples), light cashew (6 samples) and
pineapple (6 samples). Table S1, in Supplementary Information, shows the sample
compositions.

The following reagents were used for the Tillman's method: ascorbic acid (Impex,
Wood Dale, USA), 2,6-dichlorophenol indophenol sodium salt (Sigma-Aldrich, St.
Louis, USA), indigo carmine (Sigma-Aldrich, St. Louis, USA), metaphosphoric acid,
glacial acetic acid and hydrochloric acid (Vetec, Rio de Janeiro, Brazil), sodium
bicarbonate, sodium hydroxide and potassium biphthalate (Alphatec, Macaé, Brazil),
phenolphthalein 1% (Sigma-Aldrich, St. Louis, USA).

Tillman's method

Initially, 3 solutions were prepared as follows: solution 1: acid solution, prepared by

solubilizing 15 g of metaphosphoric acid in 40 mL of glacial acetic acid and then
adding 450 mL of distilled water, being stirred and filtered; solution 2: vitamin C
solution, prepared by solubilizing 100 mg of vitamin C, previously dried, in 100 mL of
the solution 1 in a volumetric flask (100 mL) and then, diluted 10 times in the same
acid solution; solution 3: Tillman's solution, prepared by solubilizing 42 mg of sodium
bicarbonate in 50 mL of distilled water, adding 50 mg of 2,6-dichlorophenol
indophenol sodium salt was under stirring until total dissolution of the dye. Then, this
solution was filtered and diluted in 200 mL of distilled water in a volumetric flask.

In the standardization of Tillman's solution, 4 mL of the solution 2 was used together

with 6 mL of the solution 1 in an Erlenmeyer flask, where 50 mL of distilled water
was added. This solution was titrated with Tillman's solution (solution 3). A blank
test was performed, replacing vitamin C solution (solution 2) by acid solution
(solution 1) in order to calculate the Tillman's factor (F) according to equation 1:

 (1)

The titration of the fruit nectar was done by using 40 mL of filtered sample mixed
with 40 mL of the solution 1. From this mixture it was taken 10 mL, which was then
titrated with the Tillman's solution. The vitamin C content was calculated through
the equation 2:

 (2)

where, V is the Tillman's solution volume (mL) used in the titration, F is the Tillman's
factor and A is the sample volume (mL).
Tests were carried out to detect interferent ions as Fe 2+, Sn2+ and Cu2+. To this, two
drops of methylene blue 0.05% were added in 10 mL of a solution 1:1 (v/v),
containing fruit nectar and solution 1.

Apparatus, software and multiproduct multivariate calibration

The spectra were obtained with an Ocean Optics spectrophotometer model USB-650
UV-VIS (Dunedin, USA) and using a 1 mm quartz cuvette. All spectra were obtained
in the range from 200 to 400 nm (1 nm step) without any sample preparation. The
spectra were obtained once the sample containers were opened, and then there was
no storage. The data were treated in MATLAB version R2007b (The Math-Works,
Natick, USA). The PLS calculations were carried out with the PLS-Toolbox version
5.2. The outliers detection and figures of merit calculations were carried out with a
homemade program developed in the laboratory.

The PLS method has been discussed in detail in relevant references.21-23 In this case,
the data matrix X was constituted by the UV spectra of nectar fruit samples and the
vector y contained the reference values for vitamin C content, obtained from the
Tillman's method. The model was developed with mean center pre-processing.

Sometimes, samples behaving different from the bulk of the data (outliers) can occur
by different reasons, such as laboratory error, samples from another population,
instrument error and others.22 In this research the outliers were detect based on
leverage,24 unmodeled residuals in spectra,22,24 and unmodeled residuals in
dependent variables.22 The leverage and unmodeled residuals in spectra and
dependent variable were evaluated at the calibration set, while the outliers were
evaluated by the leverage and unmodeled residuals in spectra at the validation set,
as suggested by American Society for Testing and Materials (ASTM E1655-05). 24

It is not uncommon, when outliers are eliminated in a first model and the model is
rebuilt, to find new outliers in this second model.25 In this research, it was also
verified and the outliers test was relaxed: (i) the first model was built on an initial
calibration set; (ii) outliers were detected based on leverage, unmodeled residuals in
spectra and dependent variables were removed; (iii) a second model was built, with
the same variables latent number; (iv) outliers detected based on leverage,
unmodeled residuals in spectra and dependent variables were removed from the
second model; (v) a third model was built, with the same variables latent number;
(vi) outliers in the validation set were evaluated.

The validation of the proposed multiproduct multivariate model was certified through
the determination of figures of merit, such as, accuracy, linearity, sensitivity,
analytical sensitivity, adjustment, limits of detection and quantification. The
determination was done based on previous publications,25,26 and the equations were
presented in the Table 1.

Table 1 Equations 3 to 11 for figures of merit 

The residual prediction deviation (RPD) was utilized to estimate the predictive ability
of the model.27 This parameter is more suitable for absolute comparisons and can be
estimated for calibration (RPDcal) and validation (RPDval) as shown in Table 1.

RESULTS AND DISCUSSION

The calibration and validation data sets were composed by 94 and 29 samples,
respectively, selected by the Kennard-Stone algorithm. 28 According to this algorithm,
the first sample selected is the one with largest distance from the center of the data,
and the next sample is the most far from the first sample, and so on, until
completing the selected number of samples for the calibration set. In this way,
Kennard-Stone algorithm was used within each group of samples to ensure that all
flavors, light and conventional juices were well represented in calibration and
validation sets. The Figure 1 shows the spectra of all samples in the spectral range
used in the model development.
Figure 1 UV spectra of the industrialized nectar juices; (a) calibration data set; and (b) validation
data set. 

The calibration and validation sets were optimized by outliers elimination. In the
calibration set, the outliers were eliminated based on data with extreme leverage in
calibration, unmodelled residuals in spectral data and unmodelled residuals in
property of interest, which in this case was the vitamin C. The outliers in validation
set were eliminated based on extreme leverage and unmodelled residuals in spectral
data. This procedure resulted in 79 and 25 samples for calibration and validation,
respectively.

Root mean square error of cross validation (RMSECV) was utilized to select the
optimum model dimension. In this case, the minimum RMSECV for the calibration
samples, obtained by contiguous block cross validation of nine samples, result the
choice of four latent variables for mean-centered model development. The figures of
merit for the model are shown in Table 2.

Table 2 Analytical figures of merit for the multiproduct PLS model 

Figure of merit UV model

RMSECc 4.2504
a
Accuracy RMSEPd 3.0945
RMSECVe 4.4049
RPDcalf 2.8589
Relative prediction deviationb
RPDvalg 2.4220
Sensitivitya 0.1110
Analytical sensitivity-1 a 0.3315
Fit correlation coefficient 0.8890
Limit of detectiona 1.0941
Limit of quantificationa 3.3153

a
Results in mg per 100 mL-1;

b
dimensionless units;

c
RMSEC = root mean square error of calibration;

d
RMSEP: root mean square error of prediction;

e
RMSECV = root mean square error of cross validation;

f
RDPcal = residual prediction deviation for calibration;

g
RDPval = residual prediction deviation for validation.

Accuracy represented by root mean square error of calibration (RMSEC), root mean
square error of prediction (RMSEP) and RMSECV showed that model dimension was
properly chosen and the model was not over fitted. These parameters incorporate
random and bias errors. Then, accuracy can also be represented by the fit of the
reference values against the predicted ones and the slope, the intercept, correlation
coefficient.26 Figure 2 shows the fit of the multiproduct model, presented by plotting
the reference values against the estimated values for vitamin C. The slope and
intercept for this linear fit are also presented in the Figure 2, while the correlation
coefficient is presented in Table 2. The correlation coefficient value, 0.8890, was
considered satisfactory since previous research reported coefficient value around 0.7
when the reference method is the titration method.6,25,29
Figure 2 Reference values against the values estimated by the PLS multiproduct model.
Calibration samples (•); validation samples (∗). 

Residual prediction deviation (RPD) is the ratio of natural variation in the samples to
the size of probable errors occurring during the prediction, and it is more useful for
comparing models on different data sets or in absolute terms. It was calculated for
the calibration and validation sets and presented values of 2.86 and 2.42 for
calibration and prediction, respectively, which was above 2.4, the lower limit desired
for calibration equations.30

The sensitivity, presented in Table 1, showed appropriate results taking into account
the analytical range of the model (0.74-39.25 mg per 100 mL). However, because of
the preprocessing used in PLS model development, the analytical sensitivity is more
suitable for evaluate the sensitivity of an multivariate calibration method.
Considering a perfect fit of the model and that the spectral noise represent the large
source of error, the inverse of the analytical sensitivity (or analytical sensitivity -1)
allows for the establishment of a minimum concentration difference which is
discernible by the analytical method in the range of concentrations where it was
applied.27 Based on this, it is possible to distinguish samples with concentration
difference of vitamin C of 0.33 mg per 100 mL.

Residuals plot from calibration and validation samples are shown in Figure 3, and
were used to evaluate linearity of the multiproduct model. The absolute and relative
errors (most of samples around 20%) are randomly distributed. These results
suggest that this data set fit on a linear model.22,27 To confirm the model
linearity, Figure 4 show the histogram for the student residuals whose distribution
resemble a Gaussian behavior. The Jarque-Bera test confirm that the student
residuals are normally distributed at 95% of confidence level.24,31
Figure 3 Residuals plot for the multiproduct multivariate PLS model; (a) in terms of the absolute
errors; (b) in terms of relative errors. Calibration samples (•); validation samples (∗). 
Figure 4 Histogram of the student residuals. 

Limits of detection and quantification for the multiproduct model show results
coherent with the measured quantities and the RMSEP obtained. The results obtained
for the figures of merit showed that the proposed method based on UV spectroscopy
and multiproduct multivariate calibration is promising. Furthermore, the UV-PLS is a
low cost, fast and sample preparation free methodology.

CONCLUSIONS

A method to determine vitamin C by UV spectroscopy and multiproduct multivariate

calibration based on PLS is suitable to be constructed. The multiproduct model was
optimized by outlier detection and it was validated by calculation of the figures of
merit, showing promising results. The model showed an appropriate sensitivity
capacity and values for accuracy, limits of detection and quantification, besides other
figures of merit presented results which indicates that the multiproduct model
developed by UV spectroscopy can be used in the industrial routine analysis as an
alternative to titration or even others methods. By comparing with the traditional
ones, the UV-PLS multiproduct model present advantages as no sample preparation,
capacity for being updated to others parameters, is waste free and present
possibilities for on-line monitoring.
Ascorbic Acid Determination in Commercial
Fruit Juice Samples by Cyclic Voltammetry

Abstract
Go to:

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.
Go to:

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 (30 mm2 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.
Go to:
3. RESULTS AND DISCUSSIONS
In Figure 2, 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 (Figure 3) 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:
Ip = 2.69 ⋅ 105n3/2AD1/2v1/2c, 
(1)
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; cascorbic acid = 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].
Table 1 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.

Interferent Interferent/analyte molar Influence on the analytical peak current

ratio

Glucose 200 less than 1%

Citric acid 150 less than 1%

200 2.26% decrease

Tartaric acid 200 less than 1%

Benzoate 150 less than 1%

 Interferent Interferent/analyte molar Influence on the analytical peak current
ratio

anion

200 4.84% decrease

As can be seen from Table 1, 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 Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery

and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

mg/100 mL mmol·xL mg/10 mg/100 mL mg/100 mL

−1
juice 0 mL juice juice
juice

Orange 30.48 1.67 29.39 64.35 103.01 95.61 99.50

juice
(Greece)
obtained
Juice Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery
and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

by fruit
pressing

Lemon 35.20 1.93 33.97 68.31 101.48 98.32 97

juice
(Greece)
obtained
by fruit
pressing

Cappy 8.21 0.46 8.09 40.67 94.90 75.30 99.75

grapefrui
t (Coca
Cola
Juice Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery
and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

HBC
Romania
)

Fanta 10.79 0.58 10.21 42.91 95.36 79.37 102.75

lemon
(Coca
Cola)

Tymbark 22.29 1.23 21.65 56.41 102 88.53 100.03

orange
(mw La
Festa
Int’l
Juice Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery
and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

Romania
)

Frutti 14.37 0.83 14.61 46.87 94.35 79.88 97.25

fresh
Tutti
frutti
(Europea
n Drinks
Romania
)

Prigat 14.96 0.83 14.61 47.58 96.40 81.07 99.01

orange
Juice Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery
and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

(Sigat
Beverage
Compan
y
Romania
)

Prigat 12.32 0.73 12.85 46.82 99.19 82.75 104

Peach
(Sigat
Beverage
Compan
y
Romania
Juice Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery
and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

Fruttia 21.12 1.24 21.82 55.44 98.69 89.61 101.39

orange
(Europea
n Drinks
Romania
)

Santal 31.68 1.84 32.38 64.42 94.72 100.35 102.26

grapefrui
t
(Parmala
t
Juice Ascorbic acid Ascorbic acid Ascorbic Recovery Ascorbic Recovery
and concentration concentration— acid % acid %
produce —DCPIP cyclic concentrati concentrati
rs method voltammetry on after on after
35.2 mg (2 70.4 mg (4
mL mL
standard standard
solution, 0.1 solution, 0.1
mol·L−1, mol·L−1,
added to added to
100 mL 100 mL
juice) juice)
vitamin C vitamin C
addition addition

Romania
)

Open in a separate window

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:

Recovery%=(QDET−QP)×100QADD,
(2)
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 Table 2. 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 Section 2 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 DCPIP 5 × 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.
Go to:

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 of 9 × 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 (Table 2). 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.
Determination of Vitamin C in Foods Using the Iodine-
Turbidimetric Method Combined with an Infrared Camera

Abstract
A novel method was proposed for the determination of vitamin C (VC) using an infrared camera
combined with the iodine-turbidimetric method. Based on the redox between VC and iodine, the
residual iodine was measured using the turbidimetric method with an infrared camera to obtain
VC content. The light emitted by the infrared light-emitting diode (LED) was absorbed and
scattered when it penetrated the residual iodine suspension. The transmitted light was captured
by the infrared camera to form a digital image and the responding color components and
grayscale values were obtained. The obtained color components and log-grayscale were fitted to
the VC concentration, and the fitted relation expressions were used to measure the unknown VC
solution. A VC measuring device equipped with an infrared camera and processing software was
designed to obtain the color components corresponding to the images of the iodine suspensions.
Compared with the spectrophotometry, the method based on the color component of brightness
had a higher accuracy for measuring the VC standard solution. For VC measurements in
tomatoes, nectarines, and VC tablets, our proposed method was highly consistent with
spectrophotometry. Therefore, this method could potentially be implemented in the determination
of VC in fruits and tablets, or other foods.

Keywords:  food analysis; analytical method; vitamin C; iodine suspension; infrared

camera; image processing
1. Introduction
Vitamin C (VC) is an essential micronutrient and appears as a colorless crystal that is
soluble in water. VC is necessary for preventing scurvy and is often called ascorbic acid in
medicine. VC plays an important role in the synthesis of neurotransmitters, steroid hormones,
carnitine, the conversion of cholesterol to bile acids, tyrosine degradation, and metal ion
metabolisms. The role of ascorbic acid as a biological reducing agent may be linked to its
prevention of degenerative diseases, such as cancer and cardiovascular diseases. Thus, VC is of
great importance to one’s overall health. Most plants and animals synthesize ascorbic acid for
their own requirements. However, humans cannot synthesize ascorbic acid due to a lack of the
enzyme gulonolactone oxidase. Hence, ascorbic acid must be supplemented, mainly through
fruits, vegetables, and tablets [1]. Therefore, an accurate measurement of VC in foods is
important to determine VC intake and has a significant influence on dietary health.

There are many methods for the determination of VC, such as high-performance liquid
chromatography (HPLC) [2,3,4,5], fluorescence spectroscopy [6], spectrophotometry [7], and
electrochemical analysis [8,9,10,11]. In recent years, new methods have been proposed,
including resonance Rayleigh scattering (RRS) [12] and flow injection photosensitized
chemiluminescence [13]. However, these methods require expensive instruments, such as
electron microscopes and photomultiplier tubes, or have complicated chemical reaction
processes.

Spectrophotometry is used to determine VC based on the color reaction. By measuring the

absorbance or luminescence intensity of light at a specific wavelength, the concentration of the
product is obtained. It is often used for VC determination with a high sensitivity for 0–8 μg/mL,
and was applied here to compare with our proposed method [7].

The development of computer image processing and imaging technology has made
cameras widely used in various fields. In soil analysis, the RGB (red–green–blue) values of digital
images and the derived soil indices were used to determine iron oxides and fines in soil [14]. A
digital camera was also used for determining the iron and residual chlorine in water using N,N-
diethylphenylenediamine [15]. In addition, image acquisition was carried out using a digital
camera and image processing technology was used to acquire image information to determine
the water turbidity, nitrite, ammonia nitrogen, sulfide, and phosphate contents, etc. [16,17]. The
detection method that combines a digital camera and image processing technology has the
advantages of simple design, convenient operation, and visualization, which verifies the feasibility
of using a digital camera in trace analysis.

The turbidimetric method is often used for medical and biological detection [18,19]. The
principle is that suspended particles absorb and scatter the incident light, and the intensity of
scattering and absorption is positively proportional to the concentration of suspended particles.
The concentration of the detected substances in the solution can be deduced by measuring the
intensity of transmitted light. However, there is no turbidimetric method on the basis of iodine
suspensions using a camera.

Most of the existing VC measuring methods require specialized instruments, or complicated

operations, and cannot easily measure VC. There is a need for a VC measuring method that is
easy to operate and does not require expensive instruments. The goal of the present work was to
measure VC content using a typical infrared camera. In this work, an infrared camera was used to
capture images of light penetrating through iodine suspensions that were prepared by the
reaction between the aqueous VC solution and iodine ethanol solution. The VC concentration
was obtained from the attenuation of incident light, which was indicated by the color component
and gray information of the image.
2. Experimental Section
2.1. Measuring Device
The designed experimental device is as shown in Figure 1, and consists of a constant
current driving circuit board for a light-emitting diode (LED), an infrared light source, a sample
cell, and an infrared camera. The sampling area was sealed by a box that was made of light-
excluding black acrylic, the thickness of which was 3 mm to isolate external light and, thus, avoid
the effect of external light on the sample. The light source consisted of six 850 nm infrared LEDs,
which were driven by a constant current circuit. Although the light from the LEDs was non-
uniform, the measurement error was eliminated by calculating the average value of the whole
imaging area. Compared with a single LED, the image sampling area was expanded, which
reduced the impact of random errors, including the uneven distribution of suspended iodine and
sampling area movements due to slight shakes of the device. When the infrared light passed
through the suspension, it was absorbed and scattered by the suspended substance. Due to the
different turbidity in the suspensions, the absorption and scattering varied and the image captured
by the camera responded to the variation.

Figure 1. Structure diagram of the vitamin C (VC) measuring device based on an infrared
camera.

2.1.1. Constant Current Circuit

The infrared LED was driven by a constant current circuit, as shown in the schematic
diagram in Figure 2. U2 generated the reference voltage buffered by U1A, which was compared
with the voltage on the sampling resistance R6. The comparison results controlled Q1 to realize
the constant current driving of the LED. Adjusting W1 could change the working current of the
LED. The circuit was powered by a universal serial bus (USB) port. After the circuit was powered,
it required preheating for 20 min before the experiment could be carried out.
Figure 2. Light-emitting diode (LED) constant current driving circuit diagram.

2.1.2. Infrared Camera

The digital camera used for measurements is shown in Figure 3. It was a commercial
ordinary infrared camera consisting of an optical lense, a CMOS (complementary metal oxide
semiconductor) image sensor, and a signal processing circuit board. The camera model was
KS1.3A142, produced by Shenzhen Kingsen Technology Co., Ltd. of China, and the maximum
resolution of the camera was 1280 × 960 pixels. The lense was equipped with an 850 nm narrow-
band filter, which could manually adjust the focal length to increase the clarity of the suspension
image. The image acquired by the infrared camera was a single-channel image. These images
are similar to grayscale images and avoid color interference from the test solution.

Figure 3. Camera printed circuit board (PCB) details.

With the designed PC software, we could change the properties of the image by adjusting
the camera parameters, such as the hue, saturation, and white balance, and ensure that the
whole measurement range had a good discrimination by adjusting the camera parameters of
exposure, brightness, and contrast. These parameters of the camera were automatically saved to
the registry of the system after setting. The same parameters should be used in the calibration
and measurement process to ensure the accuracy of the measurement. Regarding camera
selection, as long as the camera can manually change the focal length, disable the function of
automatic exposure and brightness, and can adjust the exposure to ensure the consistency of the
parameters during the measurement process, it can be used in the measurement system.

The CMOS sensor used in the digital camera was a semiconductor component that was
used to record light changes. Each pixel of the camera is equivalent to a photo detection element.
When the light from the light source passed through the suspension, it was filtered by the 850 nm
filter, and then projected onto the CMOS sensor. The signal processing component integrated in
the camera processed the voltage data from the CMOS sensor to obtain the digital signal, and
the digital signal was transmitted to the upper computer software through the USB port for further
processing. The use of the digital camera replaces the photoelectric sensor, optical system,
signal amplification, signal acquisition, analog-to-digital conversion circuit, and signal processing
circuit that are required by other optical instruments. The design of the instrument is simplified,
and measurement process can be visualized.

2.1.3. Software Design

The image acquisition software interface is shown in Figure S1 which was developed with
C# and Camera_NET library in VS2012 platform. “Camera selection” was used to select the
infrared camera. “Camera settings” were used to set the brightness, white balance, hue,
saturation, and exposure of the camera. The software can automatically save the set parameters.
“Snapshot the frame” obtained a frame image and took the average RGB value of the region of
interest (ROI). In this way, the RGB value corresponding to the VC solution was obtained. Then,
the corresponding grayscale and log-grayscale values were calculated and the corresponding
Commission Internationale de L’Eclairage (CIE) Lab (CIE L*a*b*, L* for the lightness from black
(0) to white (100), a* from green (-) to red (+), and b* from blue (-) to yellow (+)) values were
obtained by converting the RGB color space to the CIE Lab color space. “Determine” opened the
measurement part, which included zero calibration, span calibration, and measurement functions.

Grayscale used black tones to represent objects, and quantified the gray values from 0 to
255. “L” represents the brightness or the luminance of the light through suspension, “a”
represents the range from red to green, and “b” represents the range from yellow to blue. L
ranges from 0 to 100, and a and b range from +127 to −128.

Equation (1) was used to compute the grayscale and was calculated by a logarithmic
grayscale:

grayscale=R×0.299+G×0.587+B×0.114.

(1)

An approximate conversion algorithm was used to transform the RGB color space to the
CIE Lab color space as follows [20].

First, the RGB color space was converted to CIE XYZ color space (X, Y, and Z are
extrapolations of RGB created mathematically to avoid negative numbers. Y means luminance, Z
is somewhat equal to blue, and X is a mix of cone response curves chosen to be orthogonal to
luminance and non-negative):

⎡⎣⎢XYZ⎤⎦⎥=⎡⎣⎢0.4124530.2136710.0193340.3575800.7151600.191930.804230.0721690.950227⎤⎦⎥⎡⎣⎢
RGB⎤⎦⎥
 (2)

⎧⎩⎨⎪⎪⎪⎪⎪⎪XYZ===X255×0.950456Y255Z255×1.088754.

(3)

Then, the XYZ color space was converted to the CIE Lab color space,

⎧⎩⎨⎪⎪Lab===116f(Y)−16500[f(X)−f(Y)]200[f(Y)−f(Z)]

(4)

f(t)=⎧⎩⎨⎪⎪⎪⎪t1313(296)2t+429ift>(629)3otherwise.

(5)

2.2. Chemical Reagents and Materials

In this design, sucrose, fructose, glucose, alumina, ammonium chloride, ferrous sulfate,
copper sulfate, anhydrous calcium chloride, sodium chloride, ferric sulfate, magnesium chloride,
vitamin C (ascorbic acid), vitamin B1, vitamin B2, L-methionine, L-tryptophan, L-lysine, L-
cysteine, acetic acid, and kaolin were purchased from Sinopharm Chemical Reagent Co., Ltd.
(https://www.reagent.com.cn, Shanghai, China). Vitamin E was purchased from Shanghai
Ruichu Biotech Co., Ltd. All chemicals used were of analytical grade and used directly without
further purification. The deionized water obtained from Shanghai Jingchun Water Technologies
Co., Ltd. was used throughout the experiment.

Iodine ethanol solution: 0.7 g of iodine was added into 30 mL of 95% ethanol, with a FSH-
2A adjustable high-speed homogenizer (Changzhou Guowang Instrument Manufacturing Co.,
Ltd., China; http://www.gwyq.net/) to accelerate dissolving. Then, the solution was transferred to
a 50 mL volumetric flask and diluted to 50 mL with 95% ethanol.

VC standard solution: VC is stable in acidic solution but easily oxidized in alkaline and
neutral solutions. Thus, 200 mg of VC was added to 200 mL of 0.25 M acetic acid to prepare 1
g/L of VC. Various VC standard solutions were prepared by diluting the 1 g/L of VC.

2.3. Sample Preparation

At 20 °C, 10 g of pant tissue of tomato or cucumber, or a crushed VC tablet, were put into a
FSH-2A adjustable high-speed homogenizer. Then, 30 mL of 0.25 M acetic acid was added, and
the mixture was quickly minced and homogenized with internal cutting for 4 min at 10,000 r/min
into a homogenate. The homogenate was transferred to a 50 mL volumetric flask, and 20 mL of
0.25 M acetic acid added to dilute to the scale of 50 mL, before we shook well to obtain a sample
solution. For a comparison of spectrophotometry, 0.5 g of kaolin was added to the sample
solution, and then was filtered to eliminate color interference. The filtrate was centrifuged for 6
min at 4000 r/min. The VC sample solutions were obtained.

2.4. The Principle of Measuring Vc Using the Iodine-Turbidimetric Method

The schematic presentation of the detection of VC is shown in Figure 4.
Figure 4. Schematic presentation of the detection of vitamin C (VC) with our proposed method.

When a VC solution is added to iodine ethanol solution, the solubility of iodine will decrease
significantly due to the decrease of alcohol resulting from the VC solution, and an iodine
suspension is formed [21]. Iodine is reduced to iodide ions and then iodide ions react with iodine
to form triiodide, which is soluble. This reaction process is shown in Equations (6) and (7). The
turbidity of the iodine suspension is relative to the VC content:

C6H8O6+I2=C6H6O6+2H++2I−

(6)

I2+I−⇌I−3.

(7)

The 850 nm light source used in this study belongs to near-infrared (NIR, 780–1000 nm).
NIR is widely used in turbidity measurements, and commercial turbidity instruments are based on
850 nm/860 nm light [22]. NIR light is less influenced by the color of the sample and is not
sensitive to soluble particles, which essentially do not absorb and scatter infrared light, and the
light band of the 850 nm light source is narrow ( Δλ=50 nm when I =20 mA); thus, it is not
absorbed by most organic substances [23]. Additionally, Maki et al. proved that I−3 has no
sensitive light absorption in the NIR band [24], and J. G. Bayly et al. reported that the absorption
of 850 nm light by water was small, and did not affect turbidity measurements [ 25]. Therefore, in
the suspension, only suspended particles reduce the intensity of the transmitted light at 850 nm.
With the exception of substances used to precipitate I− or with strong oxidation or reducibility, the
soluble chemicals (such as pigments, inorganic salts, and sugars) do not interfere with the
measurement [23], which is in keeping with our Interference Experiments. These results
demonstrated the selectivity and feasibility of the method.

When light passes through a suspension, the suspended particles block the propagation of
light in the suspension. To what degree the propagation of light is affected by the suspended
substances depends on the size, shape, and composition of particles, and the wavelength of the
incident light. In addition to the scattering effect, the transmitted light is absorbed by the particles
and the light intensity reduces [23], and the change in the light intensity follows the Lambert–Beer
law Equation (8) or Equation (9).

I=I0e−[αa+αb]xc

(8)

lnI=lnI0−(αa+αb)xc

(9)

where I is the intensity of the transmitted light, I0 is the intensity of the incident light, X is the
length of the solution the light passes through, c is the particle concentration, αa is the absorption
coefficient, and αb is the scattering coefficient.

The suspensions with different turbidity were obtained by adding the VC solution to a certain
amount of iodine ethanol solution. When light passes through the suspension, light is absorbed
and scattered. The absorption and scattering affects the image, and this effect is mainly related to
the turbidity of the suspension. After using the infrared camera to obtain the images of the
incident light, the RGB values of the suspension were obtained by image processing. The
grayscale and log-grayscale were calculated and the Lab values of the images were obtained by
the color space conversion from RGB to Lab. By analyzing the relationship between the VC and
RGB, and Lab and grayscale values, the relation expressions between the VC concentration and
L, and grayscale and log-grayscale values were obtained, which were used to determine the VC
in real samples.

2.5. Statistical Methods

The independent-sample T-test and one-way analysis of variance (ANOVA) were used with
MATLAB2014 in this work to verify the reliability of the proposed method. After the independent-
sample T-test between the standard concentrations and the data obtained by the proposed
method, the P-value was obtained. The P-value—the level of significance—is the probability that
hypothesis H0 is true but refused according to the sample information. The test level α is usually
taken as 0.05, and P <α, H0 was rejected, indicating that the measuring error of the proposed
method should be considered. One-way ANOVA was used to analyze two data sets from two
methods. For any given significance level α, if the probability P-value was greater than α, the
original hypothesis should be accepted, that is there was no significant difference between the
concentrations determined by the two measuring methods. In this study, α was 0.05.

2.6. Measuring Procedure

The aqueous VC solution (10 mL) was added to the iodine ethanol solution (1 mL) and
mixed them uniformly to prepare the suspension. After transferring to a cuvette, the solution was
placed into the measuring device and measured at room temperature. An image of the
suspension was taken within 30 s, and the RGB and Lab values of the image were calculated
automatically by the software. Before measurement, the measuring device was preheated for 20
min.

By measuring and fitting the Lab, RGB, grayscale, and log-grayscale values corresponding
to a sequence of VC concentrations, the relation expressions were obtained. The L, grayscale,
and log-grayscale that had higher goodness of fit and consistency were used in the actual
sample’s measurement.

3. Results and Discussion

 The RGB values and Lab values of the suspensions were measured corresponding to
different VC concentrations. A total of 16 standard aqueous VC samples prepared by diluting 1
g/L of VC solution using 0.25 M acetic acid were 0.5–5 μg/mL. All experiments were performed at
a stable room temperature of 20 °C. Three groups of randomly selected experimental data were
used to obtain a set of average data.

3.1. Experiment Data and Analysis

The color component data (R, G, B, L, a, and b), grayscale, and log-grayscale of the
suspension image were obtained by the measuring device and analysis software. Figure S2a–
c are the relationships between the R, G, and B values and the VC concentrations. The infrared
images were similar to the gray images, and in theory, the RGB values are equal. However, the
adjustment of camera parameters, including the white balance, hue, and saturation caused the
observed difference between the R, G, and B values in Figure S2. The instability of the RGB to
camera parameters is detrimental to the accuracy of the measurement. Thus, the RGB values
were not suitable for the VC measurements.

Figure 5 and Figure S3 show the relationship between the L, a, and b values and VC
concentrations. Figure 5 shows an approximately linear relationship between L and the VC
concentration in the range of 0–5 μg/mL, and a linear relationship is suitable for measurement. In
addition, L is not disturbed by the camera parameters of white balance, hue, saturation, etc.,
because L represents lightness and is not affected by color. Therefore, the L value is a good
choice for measuring the VC concentration. Figure S3a,b shows that different VC concentrations
can correspond to the same a and b values in the range of 0–5 μg/mL. The relationship of the VC
concentration to a and b cannot be expressed by simple functions therefore, the a and b values
cannot be used to measure the VC concentration.
Figure 5. The relationship between the L value and VC concentrations.

Figure 6a,b show the relationships between the grayscale, and log-grayscale values with
VC concentrations. Figure 6a shows that the grayscale value and VC concentration have a
logarithmic relationship. In Figure 6b, the logarithmic grayscale and VC concentration are shown
to be linearly related. The changes of grayscale and logarithmic grayscale conform to I and lnI in
the Lambert–Beer law of Equations (8) and (9), therefore, grayscale is considered to linearly
correlate to I. Therefore, grayscale and log-grayscale were used to measure the VC
concentration.
Figure 6. (a) The relationship between grayscale and VC concentrations. (b) The relationship
between the ln(grayscale) and VC concentrations.

In Figure 6b and Figure 5, the log-grayscale and L show a linear and approximately linear
relationship to the VC concentration, and the comparison between L and log-grayscale after
normalization is shown in Figure S4. According to Equations (6) and (7), the VC concentration is
linearly related to the suspended iodine particle concentration. In Equation (8), there is a negative
exponential relationship between the transmitted light intensity and the concentration of the
suspended iodine particles. However, Figure S4 shows that L has a similar trend with the log-
grayscale, linearly relating to the VC concentration. The reason for this is that L obtained by the
imaging method is different from the light intensity obtained by the photocell of the turbidimeter.
When L is converted from the RGB color space to the CIE Lab color space, the operation of
Equation (2)–(5) is close to a logarithmic operation thus, the exponential relationship becomes
approximately linear [23].

3.2. Fitting Results of Vc to L, Grayscale, and Log-Grayscale

The above experimental results show that the L, grayscale, and log-grayscale values have a
high correlation to the VC concentration, and after fitting L, grayscale, and log-grayscale to the
VC concentration, the fitting curves are shown in Figure 7. The adjusted R-Squared (Adj R-
square), relation expressions, limit of detection (LODs), limit of quantification (LOQs), and ranges
are shown in Table 1.
Figure 7. (a) The fitting curves of the VC concentration with the L value. (b) The fitting curves of
the VC concentration with the grayscale value. (c) The fitting curves of the VC concentration with
the log-grayscale value.

Table 1. The relation expressions, adjusted R-Squared (Adj R-square), relation expressions, limit
of detection (LODs), limit of quantification (LOQs), and ranges.

Adj R-square is a statistical indicator to reflect the degree of correlation between the
variables. Adj R-square is calculated as a product-moment method, based on the same
respective means of two variables based on the deviation, multiplied by two dispersions to reflect
the degree of correlation between two variables. R-Square is the square of the correlation
coefficient between the expression value (measured data) and the estimated value (calculated by
the fitting model). Adj R-square is obtained by adjusting R-square according to the degree of
freedom error. The closer to 1, the better the fit results. According to the fitting results, the L,
grayscale, and log-grayscale values all had a high goodness of fit, with the log-grayscale value
having the highest.
 LOD, expressed as a concentration or quantity, is derived from the smallest measure that
can be detected with reasonable certainty for a given analytical procedure. LOQ refers to the
smallest concentration or the mass that can be quantitatively analyzed with reasonable reliability
by a given procedure. According to International Union of Pure and Applied Chemistry (IUPAC)
regulation, the LOD and LOQ were calculated as LOD =3SD/k and LOQ =10SD/k and were
confirmed experimentally, where SD is the standard deviation of the measured signal values of
the blank samples (n = 20) and k is the standard curve slope. In Table 1, the L, grayscale, and
log-grayscale values all had a LOD lower than 0.5 μg/mL, and the L value had the lowest.

3.3. Interference Experiments

The influence of foreign substances, such as sugars, metal ions, amino acids, vitamins, and
reductive acids was tested in the 4 μg/mL VC solution. The tolerance ratios of the maximum
concentrations of the foreign substances that caused an approximately ±5% relative error in the
VC concentration are listed in Table 2.

Table 2. Effects of foreign substances ([VC] = 4 μg / mL, n = 3).

The interference experiment demonstrated that light metal ions, sugars, Fe2+,

and NH+4 could be allowed at very high concentrations, while some substances, such as those
with reducibility and heavy metal ions, could be allowed only at low concentrations. However, the
common plants generally contain little or no heavy metal ions, and thus do not affect the
measurement. In addition, in the study combining the voltammetric technique with iodometry (of
which the main chemical process was also the redox between I2 and VC), Roxana A. Verdini et
al. treated natural samples with ascorbate oxidase and carried out iodometric titration [26].

Compared with the average value of untreated samples (113 mg/100 g), the mean value of
the treated samples was only 1.5 mg/100 g. This result proves that, although there are other
reducing substances, the degree of reaction was very low, accounting for only 1.3% of the total
reaction. Therefore, the interference of the reducing substances could be ignored.

3.4. Comparison with Spectrophotometry

Based on the above analysis results, we proposed a measurement method for measuring
the VC concentration based on L, grayscale, and log-grayscale. In order to verify the accuracy
and reliability of the method, the VC concentrations of standard solutions, fruits, and VC tablets
were determined by the proposed method and compared with the measurement results by
spectrophotometry. The spectrophotometry experiment used for contrast was based on the
reduction of iron (III) by ascorbic acid to iron (II). Then the solution was complexed with 1,10-
phenanthroline and the absorbance was measured at a 510 nm wavelength [7]. The
spectrophotometer used for comparison was the 721G visible spectrophotometer (INESA
Analytical Instrument Co., Ltd., China; https://www.instrument.com.cn).

3.4.1. Comparison Results for Standard Solution

The proposed method and spectrophotometry were used to determine the standard
solutions of VC with 1, 1.5, 2, 2.5, 3, 3.5, and 4 μg/mL, respectively. After independent-sample T-
testing of the VC concentrations, the P-value was obtained. The results show that the data from
the measuring methods of L, grayscale, log-grayscale, and spectrophotometry were not
significantly different from the standard VC concentrations. Table 3 shows the measured
concentrations, P-values, and standard deviation (SD) of the four methods.
Table 3. The measured concentration values of L, grayscale, log-grayscale,
and spectrophotometry on the standard solutions (n = 3), and the results of the independent-
sample T-test and the standard deviation (SD) of L, gray, log-grayscale, and spectrophotometry.

In Table 3, the method L was closer to the standard VC concentration and had a higher
accuracy. The methods of L, grayscale, and log-grayscale can be all used for the VC
concentration measurement, and although gray and log-grayscale were less accurate than other
methods, we suggest using gray or log-grayscale, as the transformation from RGB to Lab space
was very time-consuming.

3.4.2. Comparison Results for Fruit and Tablet Samples

Three tomatoes and three nectarines selected from the market and a bottle of VC tablets,
which can be purchased as an over-the-counter (OTC) drug (100 mg VC per tablet, made from
Northeast Pharmaceutical Group Co., Ltd.), were used as test samples and treated to obtain the
actual samples. The proposed methods and spectrophotometry were use to measure the
samples and compare the VC concentrations measured by spectrophotometry with those of the
proposed methods. The results are shown in Table 4. The VC concentrations in the VC tablets
were measured as 90.325, 86.275, 85.850, and 85.108 mg per tablet (calculated from the
concentrations of the sample solution in Table 4) by L, grayscale, log-grayscale, and
spectrophotometry, respectively. These results can be regarded as normal for over-the-counter
(OTC) drugs.

Table 4. Measurement of VC in fruits and tablets by the proposed methods and

spectrophotometry (n = 3) and contrast results of L, grayscale, and log-grayscale,
and spectrophotometry by one-way analysis of variance (ANOVA).

One-way ANOVA was used to respectively analyze the seven sets of data measured based
on L, grayscale, and log-grayscale with the data of spectrophotometry. The probability P was also
obtained. The P-value of one-way ANOVA for the seven sets of data is shown in Table 4, and all
P-values were greater than 0.05, so there was no significant difference between L, grayscale, and
log-grayscale and spectrophotometry, which verified the feasibility of the proposed method. In
addition, recovery tests were performed with the standard addition method, the results of which
are listed in Table 5.

Table 5. Analytical results of the recovery tests.

4. Conclusions
Our method using an infrared camera combined with the iodine-turbidimetric method was
able to simplify the instrument design and replace the traditional optical instruments used in the
VC analytics of fruits and VC tablets. This demonstrated the feasibility of a new method for VC
measurements. By adding a VC solution to an iodine ethanol solution to form a suspension with
the remaining iodine, the VC concentration was obtained by measuring the turbidity of the
suspension with an infrared camera. The designed measuring device and image processing
software were able to accurately acquire the corresponding color components of the suspension.
The proposed method had high accuracy in standard solution measurements and no significant
difference to the spectrophotometry in the VC measurements of the fruits and VC tablet,
therefore, the method was proven to be effective.

Compared to the established VC measuring methods, the proposed method had the
advantages of simple design and operation, visualization, as well as easy miniaturization and
broadened the application scope of the image detection and turbidimetric methods. However, a
disadvantage is that temperature had a great influence on the experimental results, which was
found experimentally. This is because temperature can affect the solubility of iodine and,
therefore, the reaction between iodine and VC. The method was not implemented on more tissue-
dense foods. Therefore, following works should test the feasibility of this method on a wider range
of foods and complete the compensation algorithm for different temperatures, which will provide
more functionality to the method