A Simple UV-spectrophotometric Method for the Determination of Vitamin C Content in Various Fruits and Vegetables at Sylhet Area in Bangladesh
M.M. Rahman Khan,
A simple UV-spectrophotometric method for the determination
of the total vitamin C (ascorbic acid + dehydroascorbic acid) in various
fruits and vegetables at Sylhet area is described. The spectrophotometric
method involves the oxidation of ascorbic acid to dehydroascorbic acid
by bromine water in presence of acetic acid. After coupling with 2,4-dinitrophenyl
hydrazine at 37 °C temperature for three hours, the solution is treated
with 85% H2SO4 to produce a red color complex and
the absorbance was spectrophotometrically measured at 521 nm. The content
of vitamin C were 12 to 118 mg/100 g in fruits and 22 to 135 mg/100 g
in vegetables. The percent recovery, statistical calculation and the possible
interfering factors are also discussed.
Human health is very important to our survival.
Vitamins help the human to maintain a healthy diet. They are serve as
essential components of the specific coenzymes and enzymes participating
in metabolism and other specialized activities. Among the vitamins, vitamin
C (ascorbic acid) is an essential micronutrient required for normal metabolic
function of the body (Jaffe, 1984). Humans and other primates
have lost the ability to synthesize vitamin C as a result of
a mutation in the gene coding for L-gulonolactone oxidase,
an enzyme required for the biosynthesis of vitamin C via the
glucuronic acid pathway (Woodall and Ames, 1997).
Thus, vitamin C must be obtained through the
diet. Vitamin C plays an important role as a component of enzymes involved
in the synthesis of collagens and carnitine. Vitamin C is the major water-soluble
antioxidant within the body (Sies and Wilhlm, 1995; Levine et al.,
1986; Levine et al., 1995). It lowers blood pressure and cholesterol
levels (Rath, 1993). Not only does a vitamin C intake markedly reduce
the severity of a cold, it also effectively prevents secondary viral or
bacterial complications. Numerious analysis have shown that an adequate
intake of vitamin C is effective in lowering the risk of developing cancers
of the breast, cervix, colon, rectum, lung, mouth, prostate and stomach
(Levine et al., 1996; Block, 1992; Frei et al., 1994; Block
G., 1991; Jacobs, 1993). The vitamin is especially
plentiful in fresh fruit, in particular citrus fruit, and vegetables
(Bendich, 1997). A lack of vitamin C in the diet causes the
deficiency disease scurvy (Levine, 1986). This potentially
fatal disease can be prevented with as little as 10 mg vitamin
C/d (Weber et al., 1996), an amount easily obtained through
consumption of fresh fruit and vegetables. Other symptoms of its deficiency
have been reported, but they are not well defined. It participates in
numerous biochemical reactions, suggesting that vitamin C is important
for every body process from bone formation to scar tissue repair (Grrof
et al., 1995). Vitamin C is generally non-toxic. For maintaining
a good and sound health and for prevention from common cold, human body
should be kept saturated with vitamin C (Lehinger, 1993). Keeping
in view its importance, the estimation of vitamin C containing this vitamin
A wide variety of food exists that contains vitamin C.
It is widely known by the general people today that the best sources of
vitamin C are citrus fruits and their juices. Bangladesh is rich in fruits
and vegetables. For better utilization of fruits and vegetables as a human
food, clear understanding of their nutrition value as well as the content
of vitamin C estimation is essential. A variety of citrus fruits are available
in Bangladesh and various types of special fruits are also available in
greater sylhet area. Vitamin C levels in fruits vary considerably due
to the factors, which include species, maturity, portion, soil, climate,
season, handling, method of preparation and consumption. The content of
vitamin C in fruits and vegetables found in Sylhet area may be different
from others because the properties of soil and climate of Sylhet are totally
different from that of the rest part of Bangladesh. These fruits may contain
high content of vitamin C. Among the fruits and vegetables are available
in Sylhet area, vitamin C content of some fruits and vegetables such as
Shatkara, Adalemon are known (Alam, 1996). There are many citrus fruits
and vegetables available at sylhet area such as Dumur, Katajamir, Katadanga
etc which commonly used locally to make pickle and vitamin C content of
these fruits and vegetables are not known. Recently we have reported the
estimation of vitamin C content of locally available some citrous fruits
(Rahman et al., 2005) by 2,4-DNPH method. As a part of our ongoing
study on vitamin C estimation we have collected more citrous fruits and
vegetables from Sylhet area and estimate their vitamin C content. This
study we would like to describe the results of these samples and thus
enable us to evaluate them regarding its role in human health and nutrition.
MATERIALS AND METHODS
An experiment was conducted at Organic Chemistry
Laboratory of Shahjalal University of Science and Technology University,
Sylhet, Bangladesh during March to October, 2005.
There are many methods that are employed for the quantitative
determination of vitamin C such as biological, electrochemical, and chromatographic
method (East and Nascimeet, 2002; Geigertj et al., 1981;
Veasey and Nieman, 1980). All the method has great limitation in use for
different purpose, such as in biological sample, food products, pharmaceuticals
etc. It is very difficult to choose a unique method for determining the
content of total vitamin C in food products, biological samples and pharmaceuticals.
Because each samples type have its own specific characteristics and properties
in terms of extraction, purification, interference of other compounds
(such as color, presence of oxidizing, reducing components etc).
Although some methods are available for determination
of ascorbic acid but very few methods are employed for the determination
of both forms (ascorbic acid and oxidized form, dehydroascorbic acid)
of ascorbic acid. This is because two forms of the vitamin C, ascorbic
acid and its oxidized form dehydroascorbic acid possess the different
chemical, optical and electrochemical properties.
On the basis of reducing property of ascorbic acid, it
can be determined chemically by titrating against an oxidizing agent such
as 2,6-dichlorophenolindole dye Indian Pharmacopoeia, It is not applicable
to many pharmaceutical preparations containing Fe (II), Sn (II), Cu (I),
SO2, SO32- and S2O32-
ions which are usually associated with mineral or liver preparations.
The method is applicable only when the concentration of dehydroascorbic
acid is negligible. The applicability of the method is restricted to only
those samples of citrous fruits and multivitamin tables, which do not
To determine content of total vitamin C in food samples,
a well-established method is the 2,4-dinitrophenyl hydrazine methods (DNPH)
(Riemschneider et al., 1976). This is a simplified method for the
simultaneous determination of the total vitamin C employed coupling reaction
of 2,4-dinitrophenylhydrazine dye with vitamin C and followed by spectrophotometric
Instrument: A Shimadzu spectrophotometer (model UV-1601) with
a pair of 1 cm quartz cells was used.
5% Metaphosphoric acid-10% acetic acid: Fifteen grams of solid
metaphosphoric acid (E. Merck) were dissolved in mixture of 40 mL of glacial
acetic acid (BDH) and 450 mL of distilled water in a 500 mL volumetric
flask. The solution was filtered and collected.
10% Thiourea solution; 2,4-Dinitrophenyl-hydrazine solution;
85% Sulphuric acid:
Standard vitamin C (ascorbic acid) solution: 0.05 g standard crystalline
ascorbic acid was dissolved in 100 mL of distilled water to prepare 500
ppm standard stock solution.
Sample preparation: Ten grams of sample was blended or 10 g blended
sample was homogenized with about 50 mL of 5% metaphosphoric acid-10%
acetic acid solution. Then it was quantitatively transferred into a 100
mL volumetric flask and was shaken gently until a homogeneous dispersion
was obtained. Then it was diluted up to the mark by the 5% metaphosphoric
acid-10% acetic acid solution. Then the solution was filtered and the
clear filtrate was collected for the determination of vitamin C in that
Estimation of vitamin C
Procedure: To the filtered sample solution few drops of bromine
water were added until the solution became colored (to confirm the completion
of the oxidation of ascorbic acid to dehydroascorbic acid). Then few drops
of thiourea was added to it to remove the excess
|| The total vitamin-C content in fruits and vegetables
bromine and thus the clear solution was obtained. Then
2,4- dinitrophenyl hydrazine solution was added thoroughly with all standards
and also with the oxidized ascorbic acid. Total vitamin C employing coupling
reaction of 2,4-dinitrophenyl hydrazine dye with vitamin C and followed
by spectrophotometric determination.
||Ascorbic acid is oxidized to dehydroascorbic
acid by the action of bromine solution.
|L-dehydroascorbic acid reacts with 2,4-dinitrophenylhydrazine
and produces an osazone which on treatment with 85% H2SO4
forms red colored solution.
Reproducibility: The reproducibility of this method was checked
by determining the % recovery of known amount of vitamin C from a sample.
This can be done by addition of different known amount of vitamin C in
this sample. For instance, the concentration of the sample is X ppm and
than 5 ppm standard is added and the observed concentration of the mixture
is X' then the % recovery is given by:
% recovery = (observed conc. /calculated conc.)
x100% = X' /(X+5) x100%)
RESULTS AND DISCUSSION
Calibration curve: After determination of the λmax of
the colored complex (521 nm) using a Shimadzu UV- spectrophotometer the
absorbance of the all standards (converted to coloured complex) were taken
to construct a calibration curve. The calibration curve was constructed
by plotting the concentration versus the corresponding absorbance. Molar
absorptivity was found 0.0323 L mol-1 cm-1 using
Beer-Lambert plots (Fig. 1).
Determination of vitamin C in samples: Generally all the parts
of a fruit and all fruits have not equal amounts of edible part, in the
comparative study of the vitamin C content in various fruits and vegetables,
the percent of edible parts of those must also be considered. Fruits such
as orange, lemon etc contain high amount of vitamin C and vegetables such
as Cabbage, Green pepper, Red pepper also have relatively high amount
(7 to 163 mg/100 g) vitamin C (Lidija et al., 2003; Alam 1996).
In study the locally available fruits such as Dumur, Katabadam, Katadenga
etc. collected from Sylhet and the vitamin C content of these fruits were
determined. Dumur is not popular fruit but contain 15 mg/100 g and Katabadam
contain 77 mg/100 g of vitamin C. By the similar methods vitamin C content
of the fruits Arboroi, Peyera (Guava), Katajamir and
||Calibration curve of standard vitamin C at 521 nm
|| Spectrum of DNPH complex of glucose
vegetables such as Rojottepata, Thankoni, Kalmishak etc
is also determined. Since the vitamin C content vary with the storage
period on preservation, therefore, Vitamin C content of some fruits and
vegetables were also determined at frozen conditions. It is observed that
vitamin C content of fruits and vegetables slowly decreases with time
as the storage period increase even at low temperature (Table
The reliability of this method is justified by the calculation
of the % of standard deviations and it was found to be varied within the
range from 0.20 to 2.45 % (Table 1). The reliability
of this method is also confirmed from the consideration of the following
Interferences due to diketogulonic acid: Due to the destructive
oxidation hydrolysis at higher pH results 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 is
present in the fruits (Geigertj et al., 1981). As the diketogulonic
acid has keto group, it should give the osazone with DNPH as that of ascorbic
acid and should give the colored complex on treatment with 85% H2SO4.
Thus there is chance of error in this method. But actually this cannot
interfere with the ascorbic acid.
Here diketogulonic acid was prepared by the acid hydrolysis
(dilute HNO3) of ascorbic acid. The spectrum shows that there
is no considerable absorption peak near the 521 nm (the absorption maxima
of DNPH complex of ascorbic acid).
Interference due to extracted glucose: As ascorbic acid is largely
similar to the glucose by structure, some of glucose may be extracted
in the meta-phosphoric acid during the extraction of ascorbic acid from
sample. because of their structural similarity, glucose may also form
the colored complex with DNPH as ascorbic acid. But actually no such interference
is occurred which is evident from the following spectrum are given in
Fig. 2. From the spectrum it is evident that there is
no absorption peak around the interested peak at 521 nm.
Vitamin C is important to human health, and many species
need a dietary source to stay healthy. The locally available citrous fruits
such Katagamir, Katabadam and Peyar are the excellent sources of vitamin
C. The locally available vegetables which was analyzed contain relatively
good amount of vitamic C and a good source of vitamin C. The method is
simple and offers an excellent method for the determination of total vitamin
C in fruits and vegetables.
We are grateful to the Department of Chemistry,
Shah Jalal University of Science and Technology, Sylhet-3114, Bangladesh
for giving necessary facilities during research.
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