INTRODUCTION:

Vitamin C is a water-soluble vitamin, meaning that our body doesn't store it. We get what we need, instead, from food. Vitamin C is needed for the growth and repair of tissues in all parts of our body is necessary for the treatment and prevention of scurvy. Vitamin C is an antioxidant. Antioxidants block some of the damage caused by free radicals, the build-up of free radicals over time may be largely responsible for the aging process and can contribute to the development of health conditions such as cancer, heart disease, and arthritis. Low levels of vitamin C have been associated with high blood pressure, gallbladder disease, stroke, some cancers, and atherosclerosis. Getting enough vitamin C from our diet by eating lots of fruit and vegetables may help reduce the risk of developing some of these conditions. Though in nearly all cases dietary intake is adequate to prevent deficiency and supplementation is not necessary^1-6.

In the present investigation, an attempt has therefore been made to analyze a simple and reproducible few methods for quantitative estimation of ascorbic acid in orange fruit and respective marketed preparations.

MATERIALS AND METHODS:

Reagents and Chemicals:

All the reagents and chemicals were of analytical grade. All the reagents were prepared in Distilled water. Fresh Orange juice and Lemon juice are their marketed preparations are used as samples for estimation.

Ascorbic acid estimation: Ascorbic acid has been estimated by following methods

1. Vitamin C determination by iodine titration (Iodometry)

2. Vitamin C determinations by iodine titration (Iodimetry)

3. Vitamin C determination by Permanganometric titration

4. Vitamin C determination by 2, 6-dichlorophenol indophenol method

5. Vitamin C determination by sodium thiosulphate method

6. Vitamin C determination by UV spectroscopy method

Method 1: Vitamin C determination by iodine titration⁷

Reagents:

1. Iodine solution- Dissolve 5.0gm Potassium iodide and 0.268gm Potassium iodate in 200ml of distilled water. Add 30ml of 3M sulphuric acid. Dilute it to a final volume of 500ml with distilled water.

2. Vitamin C std. solution-Dissolve 0.100 gm Vitamin C in 100ml distilled water.

3. Starch indicator solution (1%)

Procedure:

25 ml of Fresh fruit juice sample or Vitamin C std. solution was added to 125 ml Erlenmeyer flask and titrated with iodine solution; using starch solution is indicator (10 drops) until a persistent blue-violet color has been obtained.

Calculations:

15.16 ml Iodine solution reacts with 25 mg ascorbic acid.

Method 2: Vitamin C determination by iodine titration⁸

Reagents: Iodine solution (0.001N), Vitamin C std. solution (0.5mg/ml), Starch indicator solution (0.5%) , sulphuric acid (2M), Potassium iodide (1%).

Procedure:

2ml of Vitamin C std. solution or juice sample was pipetted out, added 2ml of sulphuric acid (2M) and 0.5ml Potassium iodide solution. The volume was made up to 20ml with water to a 125ml Erlenmeyer flask. Titrated with iodine solution; using starch solution is indicator (10 drops) until a persistent blue-violet color has been obtained.

Calculations:

2.7ml Iodine solution reacts with 1mg ascorbic acid.

Method 3: Vitamin C determination by Permanganometric titration⁸

Reagents: Potassium permanganate solution (0.001N), Vitamin C std. Solution (0.2 mg/ ml),

Starch indicator solution (0.5%), Sulphuric acid (2M), Potassium iodide (1%).

Procedure:

4 ml and 10ml of juice sample and Vitamin C std. Solution repectively was pipetted out, added 4 ml sulphuric acid and1ml Potassium iodide. Volume was made up to 40 ml with water to a 125 ml Erlenmeyer flask. Titrated with Potassium permanganate solution; using starch solution is indicator (10 drops) until a persistent blue-violet color has been obtained.

Calculations:

0.1ml Potassium permanganate solution reacts with 0.8mg ascorbic acid.

Method 4: Vitamin C determination by 2, 6-dichlorophenol indophenol method⁹

Reagents: 2,6-dichlorophenol indophenol Dye Solution (25mg/100 ml), Vitamin C std. Solution (0.50mg/ml), Metaphosphoric acid (4%).

Procedure:

1. Vit. C Standard solution

1ml of Vitamin C std. solution was pipetted out, added 5ml 4% Metaphosphoric acid to a 125ml Erlenmeyer flask.Titrated with 2, 6-dichlorophenol indophenol solution, until a persistent rose pink color has been obtained.

Omitting the drug carried out same procedure for blank titration.

2. Juice samples

10 ml of juice sample to 50ml Metaphosphoric acid was added. From this 10mlwas pipetted out to 125ml Erlenmeyer flask. Titrated with 2, 6-dichlorophenol indophenol solution, until a persistent rose pink color has been obtained.

Calculations:

0.1ml Dye solution reacts with 0.5mg ascorbic acid.

Method 5: Vitamin C determination by sodium thiosulphate method¹⁰

Reagents: Sodium thiosulphate solution(0.1M), Potassium iodate (0.02M), Sulphuric acid (0.5M), Starch indicator solution (1%).

Procedure:

100 mg of Vitamin C powder or 25ml of fresh fruit juice and 40ml Sulphuric acid was taken in 125ml Erlenmeyer flask, in that 20ml water, 2gm potassium iodide and 25ml potassium iodate was added. Titrated with Sodium thiosulphate solution, using starch solution is indicator (10 drops) until a persistent blue-violet color has been obtained.

same procedure was carried out for blank titration by omitting the standard sample.

Calculations:

15ml Sodium thiosulphate solution reacts with 100mg ascorbic acid.

Method 6: Vitamin C determination by UV spectroscopy method¹¹

Procedure:

Stock solution of 100mg of Standard Ascorbic acid in 100ml of distilled water was prepared. The working standard solutions were obtained by dilution of the stock solution in distilled water. Series of dilutions with conc. 2.5-25μg/ml were prepared. The 10μg/ml dilution was scanned for the wavelength range 200-400nm and λmax of ascorbic acid was determined. It is found to 264nm. Appropriate dilutions of test samples were done. Absorbance was measured for each dilution of standard and test samples at 264nm (Fig.-1).

Calculations:

Equation of graph: - y=0.4796x

Where y= absorbance of juice sample; x=concentration of ascorbic acid

Fig. 1: Calibration curve of Standard Ascorbic Acid

RESULT AND DISCUSSION

The ascorbic acid i.e. vitamin C was estimated by various methods. All methods are easy, simple and accurate. Ammount of Ascorbic acid found by different methods in different samples are reported in (table 1). Orange fresh fruit juice contains higher ammount of Vit. C than Lemon fresh fruit juice.

Table 1: Ammount of Ascorbic acid found by various methods

METHODS	1	2	3	4	5	6
SAMPLE	AMMOUNT OF ASCORBIC ACID (mg/dl)*
Orange ( Fresh Fruit juice)	70.19	70	76	75	74.4	71.11
Brand 1	40.39	31.48	32	32.5	32	32.5
Brand 2	11.92	17.5	12	15	12	12.7
Brand 3	21.85	22.22	20	20	20	20.2
Lemon ( Fresh Fruit juice)	56.06	57.40	60	58	56.25	58
Brand 1	21.4	24.07	24	21.73	21	20
Brand 2	13	14.5	12.0	12.5	12.0	11.5

*Mean of six readings

CONCLUSION:

From above results we came to the conclusion that, the Ascorbic acid is available ample in fruits than marketed juice samples. Therefore, deficiency of vitamin C should be covered from fresh fruits on priority, in other way same will be covered through marketed juice sample during off season.

REFERENCES:

1. U. Satynarayana and U. Chakrapani. Biochemistry, Uppala author-publisher, India, 2006; 3^rd ed: pp. 132-134.

2. Dr. A. C. Deb. Fundamentals of Biochemistry, New central book agency, 2006; 8^th ed: pp.191-192.

3. Government of India ministry of health and family welfare, Indian Pharmacopoeia, published by Indian pharmacopoeia commission Ghaziabad, 2007; vol 2: pp. 742.

4. P.C.Dandia and P.K.Sharma “Biochemistry and clinical pathology (Theory and practicles)”, 2^nd edition-2004, Vallabh prakashan, page no.-68, 69.

5. Afkhami-Ardekani M, Shojaoddiny-Ardekani A. Effect of vitamin C on blood glucose, serum lipids and serum insulin in type 2 diabetes patients. Indian Jounal of Medical Research.. 2007;126(5):471-4

6. Padayatty S. et al. Vitamin C as an Antioxidant: evaluation of its role in disease prevention. Journal of American College of Nutrition 2003; 22(1): 18–35.

7. Anne Marie Helmenstine, Vitamin C determination by iodine titration. available from URL:http://chemistry.about.com/od/demonstrationsexperiments/ss/vitctitration-4htm, visited on 2 September 2010 .

8. K.S.Murty and C.R.Viswanadham Permanganometric estimation of ascorbic acid. available from URL: http://www.new.dli.ernet.in visited on 1 September 2010 .

9. Rodney Boyer. Modern Experimental Biochemistry, 3^rd edn., Pearson Education, South Asia, 2000, pp.393-403.

10. Harris and Skoog. Determination of ascorbic acid in vitamin C tablets by redox titration, 7^th ed., Chap. 16. And Chap. 18 available from URL: http://www.chem.fsu.edu/chemlab/chm3120l/redox/ascorbic_acid_by_redox_titration.pdf visited on 2 September 2010 .

11. Hewitt EJ and Dickes GJ Spectrophotometric measurements on ascorbic acid . Biochemistry Journal 78;1961: 384-391.

Received on 15.09.2011 Modified on 20.09.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1690-1692