A Spectrophotometric Determination of Ascorbic Acid

Kamlesh Shrivas,

a

Kavita Agrawal

a

and Devendra Kumar Patel

b

*

a

School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur, CG, India-492010

b

Department of Botany, Govt., Science College, Raipur, CG, India-492010

A new, simple and sensitive method for the spectrophotometric indirect determination of ascorbic acid in

fruits, beverages, and pharmaceuticals is described. In this method, the ascorbic acid reduces Cu

2+

to Cu

+

and

reacts with 2,9-dimethyl-1,10-phenanothroline (neucoproine) to form Cu (neucoproine)

+

complex, and it was

extracted with N-phenylbenzimidoylthiourea (PBITU) in chloroform. The apparent value of molar absorp-
tivity of the complex in terms of ascorbic acid is (3.52)

´ 10

4

L mole

-1

cm

-1

at

l

max

, 460. The detection limit of

ascorbic acid is 40

mg L

-1

and the method obeys Beer’s law over the concentration range of 0.1-4.0

mg mL

-1

.

The proposed method was successfully applied for the determination of ascorbic acid in various samples. The
validity of the present method was checked by the flow injection analysis (FIA) method.

Keywords:

Ascorbic acid; Determination; Fruits; Beverages; Pharmaceuticals; Spectrophotometry.

INTRODUCTION

Ascorbic acid (vitamin C), is the dienol form of

g-

lactone of 2-desoxy-2-keto-L-gulonic acid, which is present

in citrus fruits, vegetables, milk, beverages and pharmaceuti-

cal products. Ascorbic acid is widely required in the metabo-

lism of living beings. The importance of ascorbic acid for the

organism as well as the problems caused by excess of vitamin

C have been investigated in detail.

1-3

There is some evidence

that large doses of vitamin C increase lymphocyte blast gene-

sis, which is associated with prognosis of cancer.

4

Ascorbic

acid, which increases intestinal absorption of iron, may also

increase absorption of heavy metals such as lead and mer-

cury, accelerating the development of toxicity from these

metals.

5

Many analytical techniques are available for the deter-

mination of ascorbic acid in different matrices, i.e. HPLC,

6

AAS,

7

Flow Injection Analysis,

8-9

Ion exchange,

10

Turbidi-

metric method,

11

etc. A number of organic and inorganic re-

agents 1,2,4-dinitrophenyl hydrazine,

12

2-mercaptoetanol,

13

fast red AL salt

14

have been reported for the spectrophoto-

metric determination of ascorbic acid.

A simple and highly sensitive method is proposed for

the determination of ascorbic acid. The method is free from

the interferences of a number of substances commonly found

in fruits, beverages and pharmaceuticals and has been applied

to the determination of ascorbic acid in fruits, beverages and

pharmaceuticals samples.

EXPERIMENTAL

Apparatus

A Systronic VIS-spectrophotometer type-106 matched

with a 1-cm quartz cell was used for absorbance measure-

ment. A Systronic pH meter model 331 was used for pH mea-

surements.

Reagents

All chemicals used were of analytical grade reagents

(E. Merck). A fresh standard solution containing 1000

mg

mL

-1

of ascorbic acid was prepared by dissolving a known

amount of ascorbic acid in double distilled water in a 1-litre

volumetric flask. The working solution was prepared by the

appropriate dilution of the stock standard solution. A 100

mg

mL

-1

of stock solution of copper was prepared by dissolving a

known amount of CuSO

4

in 0.01 M H

2

SO

4

and then stored in

a PVC bottle. A solution of 30

mg mL

-1

of copper was then

prepared by dilution of the stock. A 0.1%, w/v 2,9-dimethyl-

1,10-phenathroline (neucoproine) solution was prepared in

90%, v/v ethyl alcohol. A pH of 8.0 buffer solutions were

used for pH maintenance. A 0.2%, w/v N-phenylbenzimido-

ylthiourea (PBITU) in chloroform was used for the extraction

of the Cu (neucoproine)

+

complex. N-phenylbenzimidoyl-

thiourea synthesized as described in the literature.

15

Procedure

An aliquot of working standard solution containing

Journal of the Chinese Chemical Society, 2005, 52, 503-506

503

* Corresponding author. E-mail: [e-mail]

1.0-4.0

mg mL

-1

of ascorbic acid was taken in a 125-mL sepa-

ratory funnel and to this 2 mL copper solution, 2 mL buffer

solution and 1 mL neucoproine solution were added. After-

wards the aqueous phase was diluted to 10 mL with distilled

water. Subsequently, the complex formed was extracted with

10 mL of chloroform solution of PBITU for 2 min. The aque-

ous phase was rejected and the color of the extract was mea-

sured at

l

max

460 nm against blank solution as reference after

drying over anhydrous sodium sulfate (

» 2 gm). The concen-

tration of the ascorbic acid was evaluated by using the stan-

dard calibration curve.

Determination of ascorbic acid in juice of fruits

Various samples of fruit like oranges, lemons, mangoes

and apples of 5 g each were chosen for the analysis of ascor-

bic acid. The juice was separated from the fruits with a me-

chanical press and centrifuged in order to clarify it. A small

amount of fruit juice was sufficient for the determination of

ascorbic acid in these samples. Separated juice was filtered

with Whatmann No. 41 filter paper and dilution was made ac-

cording to the content of ascorbic acid present in the sample.

Now, a 1 mL aliquot of sample solution was added and then

analyzed as described in the procedure.

Determination of ascorbic acid in beverages

Beverages, i.e. Pepsi, Coca Cola, Limpca and Mirinda

containing carbonate, were degassed before determination of

ascorbic acid. 1 mL of sample was sufficient for determina-

tion of ascorbic acid.

Determination of ascorbic acid in pharmaceuticals

A 0.5 gm tablet or capsule containing ascorbic acid

were weighed, grind into fine powder and stirred for 2-3 min

with 50 mL of distilled water until the clear solution of the

sample. Then it was filtered through a Whatmann No. 41 fil-

ter paper. A known volume was further diluted depending on

the ascorbic acid content and the color of the sample. A 1 mL

of sample solution was added and then analysed as described

above.

RESULTS AND DISCUSSION

Development of color and absorption spectra

Ascorbic acid reduces Cu

2+

to Cu

+

and this reacts with

2,9-dimethyl-1,10-phenanothroline (neucopoine) to form a

yellow color complex in a pH range of 3.5-8.5. The spectrum

of the Cu (Nucoprine)

+

-PBITU complex exhibits an absorp-

tion maximum at 460 nm, (Fig. 1).

Effect of varying reaction conditions

Among the studied chemical variables affecting com-

plex formation, the pH was the most important. The optimum

pH range for full color development of the complex in the

aqueous phase lies in the pH range from 3.5-8.5 (Fig. 2). A

0.01%, 2,9-dimethyl-1,10-phenathroline solution was needed

for the complete reaction. However, the concentration of cop-

per solution had to be kept above the concentration of ascor-

504

J. Chin. Chem. Soc., Vol. 52, No. 3, 2005

Shrivas et al.

Fig. 1. Absorption spectra of the Cu (neucoproine)

+

-

PBITU complex against the reagent blank.

Fig. 2. Effect of pH of the aqueous solution on the ab-

sorptivity of the complex in chloroform solu-
tion.

bic acid in order to assure the total reaction of ascorbic acid,

thus the optimum concentration of copper used was 6.0

mg

mL

-1

(Fig. 3). A 0.02% of N-phenylbenzimidoylthiourea

(PBITU) in chloroform was needed for the complete extrac-

tion complex.

The determination was carried out at room temperature

and the time taken for completion of the reaction was 2 min

and a prolonged extraction up to 5 min caused no adverse ef-

fect. The absorptivity of the organic extract was stable for at

least 2 hrs at room temperature. Addition of strong electro-

lyte, i.e. KCl up to 0.5 M, did not affect the extraction.

Beer’s law and molar absorptivity

The color reaction was found to obey Beer’s law over a

concentration range of 0.1-4.0

mg mL

-1

of ascorbic acid with

slope, intercept, and correlation coefficient of 0.19, 2.0

´ 10

-3

and +0.999, respectively. The molar absorptivity was found

to be (3.52)

´ 10

4

L mole

-1

cm

-1

at

l

max

, 460.

Precision of the method

Analyzing 1.0

mg mL

-1

of ascorbic acid for seven days

checked the precision of the method. The standard deviation

and relative standard deviation were found to be

± 0.0029 and

± 1.5%, respectively. The detection limit (amount of ascorbic
acid causing an absorbance more than thrice of std. dev.) was

found to be 40

mg mL

-1

.

Effect of foreign species

The effect of diverse ions in the determination of 1.0

mg

mL

-1

of ascorbic was examined as described in the procedure.

The method was also free from the interference of major con-

stituents present in fruits, beverages, and pharmaceuticals.

The tolerance limit of some foreign species are Ca

2+

(3000),

Fe

3+

, Al

3+

, Zn

2+

(450), Mg

2+

(1000), Co

2+

, PO

4

3-

, SO

4

2-

(1500),

and species such as folic acid (1500), ferrous fumarate (2500),

vitamins B

1

, B

2

(600) B

6

(350), B

12

(300), nicotinamide

(1000) and calcium pentothenate (1500) are commonly pres-

ent in the pharmaceutical preparation and a number of foreign

species such as citric acid (250), tartaric acid (500) malic acid

(450), glucose (600), sucrose (800) and that are known to be

present in the fresh juices do not interfere with the proposed

method.

Application

The present method has been applied for the determina-

tion of ascorbic acid in fruit and beverage samples. The re-

sults obtained by the proposed method agreed well with the

reference method (Table 1), i.e. Flow Injection Analysis

(FIA) method.

8-9

To check the validity of the method, known amounts of

ascorbic acid were added to samples of beverages and deter-

mined by the proposed method as well as reference method.

8-9

The recoveries of ascorbic acid added to beverages samples

were found to be 96-99% which were close to the established

method (Table 2), and also the results obtained in the tablets

were in good agreement with the claimed value on the labels

(Table 3).

CONCLUSION

The proposed method is simple, selective, and rapid

and can be further automated for routine measurements. It is

found applicable for the analysis of ascorbic acid in fruits,

beverage, and pharmaceutical products and also is a method

free from interferences present in the sample. The detection

limit of the method is 40

mg L

-1

of ascorbic acid.

A Spectrophotometric Determination of Ascorbic Acid

J. Chin. Chem. Soc., Vol. 52, No. 3, 2005

505

Fig. 3. Effect of concentration of Cu(II) on the absorp-

tivity of the complex in chloroform solution.

Table 1. Determination of ascorbic acid in the fruits

Proposed method

Reference method

8-9

Sample*

mg

RSD

± %

mg

RSD

± %

Apple

1.30

1.4

1.35

1.7

Orange

2.80

1.5

2.72

1.8

Lemon

2.55

1.4

2.52

2.0

Mango

2.25

1.7

2.32

1.9

* Amount of sample taken for analysis purpose-1 mL.

ACKNOWLEDGEMENT

The authors are thankful to Pt. Ravishankar Shukla

University, Raipur, and Govt. Science College, Raipur for

providing laboratory facilities.

Received April 23, 2004.

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J. Chin. Chem. Soc., Vol. 52, No. 3, 2005

Shrivas et al.

Table 2. Determination of ascorbic acid in the beverages and results of analysis of real samples and recovery

from spiked samples

Ascorbic acid,

mg

Sample *

/

Batch No

.

Proposed

method

a

Reference

8-9

method

Ascorbic acid

added

mg

b

Total ascorbic

acid found

Proposed method,

mg

c

Difference

(c-a)

Recovery, %

(c-a)

b

Pepsi/500

0.66

0.70

1.0

1.65

0.99

99.0

Coca Cola/17:43

0.48

0.51

1.0

1.46

0.98

98.0

Limica/318

0.85

0.83

0.5

1.33

0.48

96.0

Mirinda/084A

0.50

0.53

1.5

1.97

1.47

97.0

Fanta/124:38

0.78

0.75

1.0

1.74

0.96

96.0

* Amount of sample taken for analysis purpose-1 mL.

´ 100

Table 3. Determination of ascorbic acid in the pharmaceuticals

Proposed method

Tablet

mg per tablet

RSD

± %, (n = 5)

Claimed value

mg per tablet

1

0

99.25

1.4

100

2

199.80

1.6

200

3

498.98

1.4

500

4

499.10

1.6

500