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Analysis of Vitamin C (ascorbic acid) Contents in Various Fruits and

Vegetables by UV-spectrophotometry

Article in Bangladesh Journal of Scientific and Industrial Research · March 2008

DOI: 10.3329/bjsir.v42i4.749

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Bangladesh J. Sci. Ind. Res. 42(4), 417-424, 2007

Analysis of Vitamin C (ascorbic acid) Contents in Various Fruits and

Vegetables by UV-spectrophotometry

M. Mizanur Rahman,* Mohammad Mizanur Rahman Khan and

Mohammad Mazedul Hosain

Department of Chemistry, Shah Jalal University of Science & Technology, Sylhet - 3114,
Bangladesh. Email : mizan_sust@yahoo.com

Abstract

Total vitamin C (ascorbic acid + dehydroascorbic acid) has been determined by

UV- spectrophotometric method in various fruits and vegetables. In this method
bromine water oxidizes ascorbic acid to dehydroascorbic acid in presence of acetic acid.
After coupling with 2,4-dinitrophenyl hydrazine at 37OC 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 10 mg/100g to 80 mg/100g in fruits and 16 mg/100g to 42 mg/100g
in veg- etables. A loss of 64% at -10OC and 76% at 5OC was observed after two
months stor- age period of one leafy vegetable "Enhydra fluctuans" (Helencha
shak).

Key words: Vitamin C determination, Vegetables, Fruits, UV-spectrophotometer.

Introduction

Vitamin C is one of the most important vita- (ascorbic acid) is an essential micronutrient
min for human nutrition that is supplied by required for normal metabolic function of
fruits and vegetables. L-Ascorbic acid (AA) the body ( Jaffe, 1984). Vitamin C is easily
is the main biologically active form of vita- oxidized, and the majority of its functions in
min C. Ascorbic acid is reversibly oxidized vivo rely on this property. The human body
to form L-dehydroascorbic acid (DHA), cannot produce ascorbic acid, and so it must
which also exhibits biological activity. be obtained entirely through one's diet. A
Since DHA can be easily converted into AA vitamin C deficiency in humans results in
in the human body it is important to the disease called scurvey, whose symptoms
measure both A and DHA in fruits and include hemorrhaging, joint pain and
vegetables to know vitamin C activity (Lee exhaustion (Brody, 1994 and Pauling,
and Kader, 2000). Among the vitamins, 1976). A very small daily intake of vitamin
vitamin C (ascorbic acid) C (10-
*Author to whom correspondence should be made,
418 Analysis of Vitamin C (ascorbic acid) 42(4) 2007

15 mg/day for an adult) is required to avoid ascorbic acid in plants varies greatly,
deficiency and stave off scurvey (Kallner, depending on such factors as the variety,
1986). weather and maturity (Chaney et al., 1979).
Vitamin C content of some fruits and
Vitamin C is the major water-soluble vegeta- bles available in Bangladesh, has
antiox- idant within the body (Sies, et al., been reported by Biswas et al. (Biswas and
1995; Levine, 1986; Levine, 1995). It Mannan 1996). There are many citrus fruits
lowers blood pressure and cholesterol levels and vegetables such as Helencha, Pudina
( Rath, 1993). Numerious analysis have leaf, Punonnoma etc available in different
shown that an ade- quate intake of vitamin region of Bangladesh and vitamin C content
C is effective in low- ering the risk of of these fruits and vegetables are not known
developing cancers of the breast, cervix, as per our knowledge. Recently we have
colon, rectum, lung, mouth, prostate and reported the estimation of vitamin C content
stomach (Levine, 1996; Block, 1992; Block, of locally available some citrous fruits by
1991). Vitamin C is generally non-toxic. For 2,4-DNPH method (Rahman et al. 2005;
maintaining a good and sound health and for Khan et al. 2006). As a part of our ongoing
prevention from common cold, human body study on vitamin C estimation we have col-
should be kept saturated with vitamin C. lected more fruits and vegetables from dif-
Keeping in view its impor- tance, the ferent district of Banglsdesh and estimate
estimation of Vitamin C contain- ing this their vitamin C content. In this paper we
vitamin assumes significance. would like to describe the results of these
samples.
An accurate and specific determination of
the nutrients content of fruits is extremely Materials and Methods
important to understand the relationship of
dietary intake and human health. A wide Several analytical methods have been
variety of food exists that contains vitamin report- ed for the determination of vitamin C
C. Fruits, vegetables, and organ meats are such as titrimetry (Kabasakalis, 2000),
generally the best sources of ascorbic acid; biological, electrochemical, and
muscle, meats and most seeds do not chromatographic method (Arya, et al.,1998;
contain significant amounts of ascorbic acid East et al., 2002; Geigertj et al., 1981;
(Combs, 1992). For better utilization of Veasey et al., 1980). All the method has
fruits and vegetables as a human food, clear great limitation in use for dif- ferent
understanding of their nutrition value as purpose. It is very difficult to choose a
well as the content of vitamin C estimation unique method for determining the content
is essential. A variety of citrus fruits are of total vitamin C in food products, biologi-
avail- able in tropical Bangladesh. The cal samples and pharmaceuticals. Because
amount of
 Rahman, Khan and Hosain 419

each sample has its own specific Chemical and Reagent required
characteris- tics and properties in terms of
extraction, purification, interference of other 5% Metaphosphoric acid-10% acetic acid
com- pounds (such as color, presence of
15g of solid metaphosphoric acid (E.
oxidizing and reducing components etc).
Merck) were dissolved in a mixture of 40 ml
Although some methods are available for of gla- cial acetic acid (BDH) and 450 ml of
determination of ascorbic acid but very few dis- tilled water in a 500 ml volumetric
methods are employed for the determination flask. The solution was filtered and
of both forms (ascorbic acid and dehy- collected.
droascorbic acid) of ascorbic acid. This is
10% Thiourea solution, 2,4-Dinitrophenyl-
because two forms of the vitamin C,
hydrazine solution, 85% Sulphuric acid
ascorbic acid and its oxidized form
dehydroascorbic acid possess the different Standard vitamin C ( ascorbic acid) solution
chemical, optical and electrochemical
properties. For example, the AOAC's Stock standard solution containing 0.5
official method (Kabasakalis, 2000) based mg/ml of ascorbic was prepared in water by
on the titration of AA with 2,6- dissolving 0.05 g of AA in 100 ml of water
dichloroindophenol in acidic solution is not and stored in a glass stoppered bottle.
applicable in all the matrices. Substances Solutions of variable concentrations were
naturally present in fruits such as tannins, prepared by diluting the stock solution in
betannins, Cu(II), Fe(II) and Co(II) are oxi- water.
dized by dye. Moreover, the method is
Sample preparation
appli- cable only when the concentration of
DHA is low. 10 g blended sample was homogenized
with about 50 ml of 5% metaphosphoric
To determine the content of total vitamin C
in food samples, here a well-established acid- 10% acetic acid solution. Then it was
chemical method was used (Riemschneider quan- titatively transferred into a 100 ml
et al., 1976) as a simplified method for the volumet- ric flask and was shaken gently
simultaneous determination of the total vita- until a homo- geneous dispersion was
min C. obtained. Then it was diluted up to the mark
by the 5% metaphosphoric aceid-10% acetic
Instrument acid solu- tion. Then the solution was
A Shimadzu spectrophotometer (model UV- filtered and the clear filtrate was collected
1601) with a pair of 1 cm quartz cells was for the determina- tion of vitamin C in that
used. sample.
420 Analysis of Vitamin C (ascorbic acid) 42(4) 2007

Estimation of vitamin C known amount of vitamin C from a sample.

Procedure This can be done by addition of different
known amount of vitamin C in this sample.
Bromine water were added to the filtered
For instance, the concentration of the
sample solution to oxidize the ascorbic acid
sample is X ppm and then 5 ppm standard is
to dehydroascorbic acid. Then a few drops
added and the observed concentration of the
of thiourea was added to it to remove the
mix- ture is X' then the % recovery is given
excess bromine and thus the clear solution
by
was obtained.
% recovery = (observed conc. /calculated
Standard solutions of ascorbic acid (5 ppm,
conc.) ´100%
10 ppm, 15 ppm, 20 ppm and 25 ppm) were
= X' /(X+5) ´100%
prepared from 500-ppm stock solution of
ascorbic acid by proper dilution. Then 1 ml Results and Discussion
of 2,4- DNPH solution was added thorough-
ly with all standards and also with the oxi- Calibration curve
dized ascorbic acid. For the completion of
After determination of the max of the col-
the reaction, all the standards, samples and
ored complex (521 nm) using a Shimadzu
blank solution were kept at 37OC tempera-
UV- spectrophotometer the absorbance of
ture for 3 hours in a water bath (thermostat-
the all standards (converted to coloured
ic). After this incubation all of those were
com- plex) were taken to construct a
cooled in an ice bath and treated with 5 ml
calibration curve. The calibration curve was
of 85% H2SO4 with constant stirring. As a
constructed by plotting the concentration
result, a colored solution was obtained
versus the cor- responding absorbance.
whose absorbance was taken at 521 nm.
Molar absorptivity was found 0.0328 L mol-
Reactions: a. Ascorbic acid is oxidized to
1
cm-1 using Beer - Lambert plots (Fig. 1).
dehydroascorbic acid by the action of
bromine solution.

b. 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
Fig. 1. Calibration curve of standard vitamin C at 521 nm
 Rahman, Khan and Hosain 421

Determination of vitamin C in samples

Generally all the parts of a fruit and all Shaluk, Chinichampa and vegetables such
fruits have not equal amounts of edible part. as Punonnoma shak, Helencha shak, sweet
In the comparative study of the vitamin C potato leaf, Pudina leaf etc has been deter-
content in various fruits and vegetables, the mined. Results are summarized in Table I.
percent of edible parts of those must also be
consid- ered. Fruits such as orange, lemon The ascorbic acid concentration vary with
etc con- tain high amount of vitamin C and conditions such as temperature and the stor-
vegeta- bles such as Cabbage, Green pepper, age period on peservation. Therefore,
Red pepper also have relatively high amount changes of ascorbic acid content at one
(7 mg/100g to163 mg/100g) vitamin C.23 In selected vegetables such as Helencha shak
mg/100gm and Sofeda contain 21.72 during frozen storage at different conditions
mg/100gm of vitamin C. By the similar (two months storage at different tempera-
methods vitamin C content of the fruits ture) were also studied. It is observed that
vitamin C content slowly decreases with
temperature and storage period of vegeta-

Table I. Total vitamin C content in fruits and vegetables

Percent of
Condition Temper- Total
Serial Samples Botanical % of edi- standard
ature vitamin-C
No. name ble part deviation
( OC) (mg/100g) (%S)
1 Punonnoma Boerhavia 80 Fresh Room 16.512±0.434 2.629
shak diffusa temp.
(Hogweed)
Shapla(Water Nymphaea 85 Fresh r.t 97.477±0.759 0.779
2
Lilly) noucifera
Sofeda Manilkara 80 r.t 21.719±0.063 0.291
3 Fresh
zapota
Chinichampa Musa spp 90 Fresh r.t. 69.567±0.936 1.346
4
(Banana)
5 Jackfruit Artocarpus 98 Fresh r.t. 80.067±0.251 0.313
Muchi heterophyllus
6 Talakuchi 95 Fresh r.t. 29.615±0.071 0.238
-
7 Sweet potato Ipomoea 95 Fresh r.t. 42.671±0.117 0.274
leaf batatus
8 Shaluk Nymphaeano 95 Fresh r.t. 10.189±0.09 0.885
uchali
9 Laboi - Fresh r.t. 31.274±0.165 0.530
-
10 Palong shak 95 Fresh r.t. 21.439±0.153 0.716
Spinacea
(Spinach) oleracea
11 Leaf of Mentha 95 Fresh r.t. 14.524±0.424 2.921
Pudina(Mint) longifolia
422 Analysis of Vitamin C (ascorbic acid) 42(4) 2007

bles ( Table II). The initial concentration of tions and it was found to be varied within
ascorbic acid was 31mg/100g and decreased the range from 0.08 % to 3.24 % (Table I).
by 76% at 5OC and 64% at -10OC during The reliability of this method is also
two months storage time. Boil at higher confirmed from the consideration of the
temper- ature and frying drastically reduce following expected interferences.
the ascor- bic acid. The results are shown in
Table II. i) Interferences due to diketogulonic acid
Due to the destructive oxidation hydrolysis
The reliability of this method is justified by at higher pH results the opening of the
the calculation of the % of standard devia-

Tabel II. Effect of preservation and cooking of Helencha shak (Enhydra fluctuans)

Sample Botanic Edible Condition Temperature Days Total vitamin- C Percent of

part (mg/100g) standard
al name ( OC) deviation (%S)
(%)
Helencha Enhydra 95 Fresh Room 34.554±0.107 0.310
shak fluctuans temp.
Preserva- 5OC 7 30.067±0.09 0.300
tion 15 25.914±0.048 0.188
21 22.335±0.141 0.633
30 18.146±0.059 0.329
37 14.518±0.019 0.134
45 11.365±0.031 0.278
52 8.426±0.039 0.463
60 4.835±0.039 0.807
-10OC
7 32.658±0.080 0.246
15 30.347±0.026 0.088
21 27.237±0.058 0.214
30 24.829±0.026 0.108
37 22.567±0.053 0.237
45 19.615±0.026 0.136
52 17.945±0.051 0.285
60 15.847±0.041 0.261
Boil 85OC 6.890±0.073 1.061
(in 80 ml
water for
20 min)
Fry 10 min 23.231±0.048 0.209
20 min 13.487±0.051 0.379
30 min 11.347±0.068 0.601
 Rahman, Khan and Hosain 423

lactone ring of the ascorbic acid and loose From the spectrum it is evident that there is
the vitamin activity. These processes are no absorption peak around the interested
nat- urally occurred in fruits and some peak at 521 nm.
amounts of diketogulonic acid is present in
Conclusion
the fruits. (Kabasakalis et al., 2000). As the
diketogu- lonic acid has keto group, it The locally available some fruits and
should give the osazone with DNPH as that vegeta- bles are a valuable and natural
of ascorbic acid and should give the colored sources of vitamin C. The method is simple
complex on treatment with 85% H2SO4. and offers an excellent method for the
Thus there is chance of error in this method. determination of total vitamin C in fruits
But actually this cannot interfere with the and vegetables.
ascorbic acid.
Acknowledgements
Here diketogulonic acid was prepared by the
We are grateful to the Department of
acid hydrolysis (dilute HNO 3) of ascorbic
Chemistry, Shah Jalal University of Science
acid. The spectrum shows that there is no
& Technology, Sylhet-3114, Bangladesh for
considerable absorption peak near the 521
giving necessary facilities.
nm (the absorption maxima of DNPH com-
plex of ascorbic acid). References
ii) Interference due to extracted glucose: Arya, S.P., Mahajan, M., and Jain, P. (1998)
As ascorbic acid is largely similar to the Photometric methods for the determination
glu- cose by structure, some of glucose may of Vitamin C. Analytical Sciences, 14, 889-
be extracted in the meta-phosphoric acid 895.
during the extraction of ascorbic acid from
sample. Because of their structural Brody, T. (1994) Nutritional Biochemistry;
similarity, glucose may also form the Academic Press: san Diogo, CA, pp. x and
colored complex with DNPH as ascorbic 450-9.
acid. But actually no such interference is Block, G.( 1991) Epidemiologic evidence
occurred which is evident from the following regard- ing vitamin C and cancer. American
spectrum are given in fig-2. Journal of Clinical Nutrition, 54: 1310S-
14S.

Block, G. (1992) The data support a role for

antioxidants in reducing cancer risk.
Nutrition Reviews, 50(7): 207-13.

Biswas S.K. and Mannan M.A. (1996)

Determination of vitamin C (ascorbic acid)
in some fruits and vegetables; B. J. Sci. &
Ind. Res. 1: 31.

Fig. 2. Spectrum of DNPH complex of glucose

 424 Analysis of Vitamin C (ascorbic acid) 42(4) 2007

Combs, Jr., G.F. (1992) The Vitamins: Levine, Mark (1995) Determination of optimal
Fundamental Aspects in Nutrition and vitamin C requirements in humans.
Health; Academic Press, San Diego, CA,; American Journal of Clinical Nutrition, 62:
pp. 4-6 and 24-5 and 223-249. 1347.

Chaney, M.S., Ross M.L. and Witschi, J.C. Levine, Mark (1996) Vitamin C pharmacokinet-
(1979) Nutrition, 9th Ed.; Houghton ics in healthy volunteers: Evidence for a
Mifflin: Boston, MA, pp. 283-295. recommended dietary allowance.
Proceedings of the National Academy of
East G. A., Nascimeento E.C. (2002) Microscale
Sciences USA, 9: 3704-09.
determination of vitamin C by weight
tritrimetry. J. of Chemical Education 79(1): Pauling, L. (1976) Vitamin C, the Common
100-102. Cold and the Flu: W. H. Freeman: San
Francisco, pp X, 4-5, 21-2, 33, 60-1, 145.
Jaffe G.M.( 1984) Vitamin C, In: machalinal ed.
Handbook of vitamins. New York: Mercell Rath, Matthias (1993) Eradicating Heart Disease.
Dekker Inc. 199-244. Health Now, San Francisco, CA.

Geigertj., Hirano D.S. and Neidlcman S.L. Rahman M.M., Khan M.M. Rahman, Murad A.
(1981) J. Chromatogra, 206: 396-39. T.M. and Begum S.A. (2005)
Bangladesh J. Environ. Sci. 11(1): 190-
Kallner, A. (1986) Annals of the New York
193.
Academy of Sciences, 498: 418-423.
Riemschneider R., M.Z. Abedin and R.P. (1976)
Khan M.M. Rahman, Rahman M. M., Islam
Mocellin. Qualities and stabilisierungpru-
M.S. and Begum S.A. (2006,) J. Biol.
fung hitzekonservierter Nahrungsmittel
Sciences 6(2): 388-392.
unter verwendung von Vit C als kri kriteri-
Kabasakalis, V. Siopidou, D. and Moshatou, E. um-Mittel. Alimenta 15: 171.
(2000) Ascorbic acid content of commer-
Sies, Helmut and Stahl, Wilhelm (1995)
cial fruit juices and its rate of loss upon Vitamins E and C, beta-carotene and other
storage. Food Chemistry, 70: 325-328). carotenoids as antioxidants. American
Journal of Clinical Nutrition, 62: 1 315S-
Lee S.K., and Kader, A.A. (2000) Preharvest and
21S.
postharvest factors influcing vitamin C
con- tent of horticultural corps; Postharvest Veasey R.L. and Nieman T.A. (1980) J.
Biology and Technology, 20: 207-220. Chromatogra., 200: 153-162.

Levine, Mark (1986) New concepts in the biolo-

gy and biochemisty of ascorbic acid. New Received : May, 07, 2006;
England. Journal of Medicine, 314: 892- Accepted : October, 09, 2007
902 .

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