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

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A Spectrophotometric Determination of Ascorbic Acid

Kamlesh Shrivas,a Kavita Agrawala and Devendra Kumar Patelb* 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 Cu2+ 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 absorptivity of the complex in terms of ascorbic acid is (3.52) 104 L mole-1 cm-1 at lmax, 460. The detection limit of ascorbic acid is 40 mg L-1 and the method obeys Beers 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 pharmaceutical products. Ascorbic acid is widely required in the metabolism 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 genesis, 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 mercury, accelerating the development of toxicity from these metals.5 Many analytical techniques are available for the determination of ascorbic acid in different matrices, i.e. HPLC,6 AAS,7 Flow Injection Analysis,8-9 Ion exchange,10 Turbidimetric method,11 etc. A number of organic and inorganic reagents 1,2,4-dinitrophenyl hydrazine,12 2-mercaptoetanol,13 fast red AL salt 14 have been reported for the spectrophotometric 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.
* Corresponding author. E-mail: Shrikam@rediffmail.com

EXPERIMENTAL Apparatus A Systronic VIS-spectrophotometer type-106 matched with a 1-cm quartz cell was used for absorbance measurement. A Systronic pH meter model 331 was used for pH measurements. Reagents All chemicals used were of analytical grade reagents (E. Merck). A fresh standard solution containing 1000 m g 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 CuSO4 in 0.01 M H2SO4 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-dimethyl1,10-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-phenylbenzimidoylthiourea (PBITU) in chloroform was used for the extraction of the Cu (neucoproine) + complex. N-phenylbenzimidoylthiourea synthesized as described in the literature.15 Procedure An aliquot of working standard solution containing

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1.0-4.0 mg mL-1 of ascorbic acid was taken in a 125-mL separatory funnel and to this 2 mL copper solution, 2 mL buffer solution and 1 mL neucoproine solution were added. Afterwards 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 aqueous phase was rejected and the color of the extract was measured at lmax 460 nm against blank solution as reference after drying over anhydrous sodium sulfate ( 2 gm). The concentration of the ascorbic acid was evaluated by using the standard 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 ascorbic acid. The juice was separated from the fruits with a mechanical 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 according 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 determination 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 filter 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.

tion maximum at 460 nm, (Fig. 1). Effect of varying reaction conditions Among the studied chemical variables affecting complex 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 copper solution had to be kept above the concentration of ascor-

Fig. 1. Absorption spectra of the Cu (neucoproine) + PBITU complex against the reagent blank.

RESULTS AND DISCUSSION Development of color and absorption spectra Ascorbic acid reduces Cu2+ 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-

Fig. 2. Effect of pH of the aqueous solution on the absorptivity of the complex in chloroform solution.

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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 extraction 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 effect. The absorptivity of the organic extract was stable for at least 2 hrs at room temperature. Addition of strong electrolyte, i.e. KCl up to 0.5 M, did not affect the extraction. Beers law and molar absorptivity The color reaction was found to obey Beers 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) 104 L mole-1 cm-1 at lmax, 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 constituents present in fruits, beverages, and pharmaceuticals. The tolerance limit of some foreign species are Ca2+ (3000), Fe3+, Al3+, Zn2+ (450), Mg2+ (1000), Co2+, PO43-, SO42- (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 present 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 determination of ascorbic acid in fruit and beverage samples. The results 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 determined 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.
Table 1. Determination of ascorbic acid in the fruits Sample* Proposed method mg Apple Orange Lemon Mango 1.30 2.80 2.55 2.25 RSD % 1.4 1.5 1.4 1.7 Reference method8-9 mg 1.35 2.72 2.52 2.32 RSD % 1.7 1.8 2.0 1.9

Fig. 3. Effect of concentration of Cu(II) on the absorptivity of the complex in chloroform solution.

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

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Table 2. Determination of ascorbic acid in the beverages and results of analysis of real samples and recovery from spiked samples Sample */Batch No. Ascorbic acid, mg Proposed Reference method method a Pepsi/500 Coca Cola/17:43 Limica/318 Mirinda/084A Fanta/124:38 0.66 0.48 0.85 0.50 0.78 0.70 0.51 0.83 0.53 0.75
8-9

Ascorbic acid Total ascorbic Difference Recovery, % added acid found (c-a) (c-a) 100 b mg Proposed method, b mg c 1.0 1.0 0.5 1.5 1.0 1.65 1.46 1.33 1.97 1.74 0.99 0.98 0.48 1.47 0.96 99.0 98.0 96.0 97.0 96.0

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

Table 3. Determination of ascorbic acid in the pharmaceuticals Tablet Proposed method mg per tablet 1 2 3 4 099.25 199.80 498.98 499.10 RSD %, (n = 5) 1.4 1.6 1.4 1.6 Claimed value mg per tablet 100 200 500 500

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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