Estimation of Ascorbic Acid in Fruit Juices - Research Proposal Example

Estimation Of Ascorbic Acid In Fruit Juices Aim: To determine the amount of ascorbic acid in orange juices Introduction: Ascorbic acid or vitamin C can be determined by oxidation –reduction titration. The dye 2,6-dichlorophenolindophenol (DCP) is used as an oxidising agent to analyse ascorbic acid content in fruit juices. DCP is blue in color. The blue dye DCP is reduced by ascorbic acid. Ascorbic acid is oxidized to dehydroascorbic acid as shown in Figure 1 (Gillete year). The reduced form of DCP is colourless. However, this blue DCP titrant turns pink/red at acidic pH. The fruit juice sample is treated with metaphosphoric acid to precipitate proteins in the juice. This prevents interference of the proteins during analysis. In addition to this, metaphosphoric acid offers stability to ascorbic acid by preventing its decomposition in acidic solution. DCP solution being unstable is stored in dark bottle at 4ºC. DCP titrant has to be standardised using known concentration of vitamin C. This helps in accurate determination of the concentration of vitamin C in an unknown solution. Materials: Apparatus: Volumetric flasks, pipettes (5ml, 10ml), burettes, conical flasks (4), test tubes (5), beakers (3). Reagents and samples: 2,6-dichlorophenolindophenol (DCP), 2- metaphosphoric acid, glacial acetic acid, standard ascorbic acid, freshly squeezed orange juice, Tropicana pure premium and Growers direct organic Method: Preparation of reagents: 1. 2, 6- dichlorophenolindophenol - dye solution Accurately weighed 800 mg of DCP is dissolved in 500ml of previously boiled and cooled distilled water in a 1000 ml volumetric flask. Then the volume is made up to 1000 ml with distilled water. (The solution can last 7 days in fridge, in a dark bottle). 2- Metaphosphoric-acetic acid mixture (MPA) Accurately weighed 15 g of Metaphosphoric acid (HPO) was dissolved in 40 ml of glacial acetic acid and 200 ml water. Then the volume was made to 500ml. The resultant solution was filtered. (MPA lasts 7 days in fridge). 3- Standard ascorbic acid Accurately weighed 200 mg of L-ascorbic acid was dissolved in 10 ml MPA solution, and then volume was made to 100 ml with distilled water (made up in MPA/glacial acetic solution). 10 ml of resultant solution was diluted to 100 ml with distilled water. (20 mg of ascorbic acid per 100 ml i.e. 0.2mg/ml). Procedure: A. Standardisation of DCP solution 1. The clean burette was filled with prepared DCP solution. 2. 5 ml of standard ascorbic acid solution was pipetted out into a conical flask. 3. Then the solution in the flask was titrated with DCP solution from burette slowly with constant stirring until faint pink colour appeared and persisted. Then burette reading was noted. 4. All the titrations were performed in triplicate. 5. The amount of ascorbic acid equivalent to 1 ml dye was calculated. B. Determination of ascorbic acid in fruit juices 1. The clean burette was filled with prepared DCP solution. 2. 10 ml of fruit juice was pipetted out into a conical flask. To this, 40 ml of distilled water and 2 ml of MPA mixture was added. 3. Then the solution in the flask was titrated with DCP solution from burette slowly with constant stirring until faint pink colour appeared and persisted. Then, burette reading was recorded. 4. All the titrations were performed in triplicate. 5. All the steps were repeated for other fruit juices also. 6. The concentration of ascorbic acid in each sample was calculated by comparing the amount of DCP solution required for standard ascorbic acid solution and various fruit juice samples. Results: The titration values of all the samples are presented in Table 1. The amount of ascorbic acid as reported by manufacturer and actual experimental values obtained are tabulated in Table 2. Table 1 Number of titration and its mean. Orange juices Number of Titration ______________________ Samples 1 2 3 Mean Standardisation of dye 3.4ml 3.5ml 3.1ml 3.3ml Freshly squeezed orange juice 10.5ml 8.1ml 9.3ml 9.3ml Tropicana Pure Premium 9.2ml 7.2ml 7.5ml 8.0ml Growers Direct Organic 6.7ml 6.5ml 6.6ml 6.6ml Table 2 Amount of ascorbic acid according to the manufacturers and from titration. Orange juices Ascorbic acid Mg/100 ml Samples Manufacture Found in Differences in Packaging Titration % Freshly squeezed orange juice ND 27.9 ND Tropicana Pure Premium 40 24 40 Growers Direct Organic 25 19.8 20.8 Discussion: Most analytical methods exploit ascorbic acid’s ease of oxidation for its estimation. It was seen that the ascorbic acid content of freshly squeezed orange juice was 27.9 mg /100 ml, while the amount of vitamin C in Tropicana pure premium and Growers direct organic was found to be 24 mg / 100 ml and 19.8 mg / 100 ml respectively. The concentration of ascorbic acid as reported by manufacturer was 40 mg / 100 ml for Tropicana pure premium and 25 mg / 100 ml for Growers direct organic fruit juice. Thus, the differences in the ascorbic acid content were observed between the expected (manufacturer’s reported values) and actual results obtained. It has been reported that the storage and handling of ascorbic acid containing samples significantly affects vitamin C content in them. Since vitamin C in a solution is highly unstable and sensitive to atmospheric oxygen, heat and light; it decomposes easily. This might cause reduction in the content of vitamin C in the fruit juice. From analytical point of view, DCP solution and standard ascorbic acid solution should be made accurately and carefully stored to avoid further inaccuracies in the results. DCP solution should be added dropwise to the reaction mixture of fruit juice and should be well mixed. Once all the vitamin C is oxidised, the next drop of DCP in solution will give persistent faint pink colour. This end point of titration should be noted carefully to avoid variation in titer values. The altered titre values might have influenced the amount of ascorbic acid content in the samples (Biology investigations, 2008). Conclusion: The ascorbic acid content of freshly squeezed orange juice was more than commercially available packaged fruit juice samples of Tropicana pure premium and Growers direct organic. *Appendix Titration of standard solution of dye: A = 3.4ml B = 3.5ml C = 3.1ml Calculation of mean: Mean = 3.4ml + 3.5ml + 3.1ml /3 = 3.3ml a) Calculation of standardization of dye: Conc. of standard ascorbic acid (aa) solution: 0.2mg/ml 5ml used so total amount of aa is 1mg Approx: 3.3mlof dye is used in titration Therefore: 1ml of dye = 1/3.3 = 0.30mg aa 1 ml of dye: 0.30mg aa Titration of different samples of orange juice Tropicana Pure Premium Freshly squeezed Orange Juice A = 9.2ml A = 10.5ml B = 7.2ml B = 8.1ml C = 7.5ml C = 9.3ml Calculation of mean: Mean=9.2ml+7.2ml+7.5ml/3=8ml Mean=10.5ml+8.1ml+9.3ml/3= 9.3ml Growers Direct Organic A = 6.7ml B = 6.5ml C = 6.6 ml Mean=6.7ml+6.5ml+6.6ml/3= 6.6ml Calculation b) Determination of aa in orange juice: Tropicana Pure Premium. Typical juice: vol of dye = 10ml 8 x 0.30= 2.4 mg aa in 10 ml 2.4mg X 10ml = 24mg/100ml Therefore 24mg of aa in 100ml of juice. c) Determination of aa in orange juice: Growers Direct Organic Typical juice: vol of dye = 10ml 6.6 x 0.30 = 1.98 mg aa in 10 ml 1.98mg X 10ml = 19.8mg/100ml Therefore 19.8mg of aa in 100ml of juice. d) Determination of aa in orange juice: Freshly Squeezed Typical juice: vol of dye = 10ml 9.3 x 0.30= 2.79 mg aa in 10 ml 2.79mg X 10ml = 27.9mg/100ml Therefore 27.9mg of aa in 100ml of juice. Calculation of Percentage Differences Tropicana Pure Premium Amount of aa in manufacture packaging label: 40mg/100ml Amount of aa determined in experiment: 24mg/100ml (100/40) x 24 = 60% 100 – 60 = 40 % Differences 40% Growers Direct Organic Amount of aa in manufacture packaging label: 25mg/100ml Amount of aa determined in experiment: 19.8mg/100ml (100/25) x 19.8 = 79.2% 100 – 79.2 = 20.8% Differences 20.8% Reference: Gillete M. L. et al. Modular laboratory program in chemistry: Analysing vitamin C in fruit juices. In: Gillete M. L. et al. (eds) ANAL 622. Neidig H. A. p. 1-6. Biology investigations: Teaching notes (2008) URL: https://www.ibid.com.au/ibid/web.nsf/reslookup/312/$file/Biology%20Options%20TN%20for%20www.pdf Vitamin C Content Of Organic Vs. Non Organic Foods Vitamin C or ascorbic acid is an essential vitamin which is required by our body for various purposes. This helps in building connective tissue (by helping to form collagen), healing wounds, and maintaining healthy blood vessels. The antioxidant properties of this soluble vitamin C prevent infections and other diseases. Deficiency of this vitamin leads to disease called scurvey. Other clinical symptoms like include colds, depression, high blood pressure, arthritis, allergies and cholesterol gallstones can also appear when the requirement of vitamin C is not met in our diet. The daily amount of vitamin C which is must in our diet is referred as Recommended Dietary Amounts, RDA. The daily dosage is 75-90 mg of ascorbic acid for the average person. These RDA vary depending on health, age, weight, activity, energy level and metabolism. This can not be prepared by human being, so we have to include it in our healthy diet. It is found in high concentrations in citrus fruits like oranges, kiwi, peach etc and green plants such as green peppers and spinach (Boyer, 1993). Organic and nonorganic foods are grown, handled and processed differently. The researchers have claimed that organically produced food provide greater health benefits since they contain fewer harmful chemicals like pesticides. Pesticides are sprayed on nonorganic food crops to protct them from pests. The nutritional value of organically grown food is always better than the conventionally grown (nonorganic) food since they contain high amounts of vitamins and minerals. For nonorganic food crops synthetic fertilizer (like nitrogen fertilizers) is used, which absorb more water from the soil which increases water content and make the fruits and vegetables look bigger. This in turn has a dilution effect of the nutrients which are present in fruits and leafy vegetables. Vitamin C being water soluble is present in aqueous part of the fruits and vegetables. Therefore, naturally the concentration of vitamin C will be less in these nonorganic foods. In contrast to this, organic food which is grown from environmental perspective, absorbs less water. Therefore, though organic food products may look smaller due to less water content they are rich in water soluble nutrients due to less water content in fruits and vegetables. The organic food market in 2007 is reported to increase six times in the span of 10 years. (Organic foods and children 2007). When the mineral and vitamin C contents of organic and non-organic crops are compared for 27 different food crops, it has been found that 14 showed significantly higher levels in organic food crops. The researchers have reported that ascorbic acid content was higher (0.33 g/kg) in the organically produced kiwi fruit when stored at 0oC than conventionally grown kiwi fruit (0.29 g/kg) (Amodio et al. 2007). When ascorbic acid content of freeze dried and Air-Dried Marionberry, Strawberry, and Corn Grown Using Conventional, Organic, and Sustainable Agricultural Practices are compared, it was found that the levels of vitamin C in organically grown and sustainably grown crops were higher than the levels for the non organic crops (Asami et al. 2003). It has been reported that organically grown peaches has higher concentration of vitamin C than their nonorganic counterparts (Carbonaro et al. 2002). Same trend was observed in organic tomatoes (Caris-Veyrat et al. 2004). Therefore, beneficial effects of organic foods overweigh their exorbitant cost. Amodio et al. (2007) A comparative study of composition and postharvest performance of organically and conventionally grown kiwifruits, Chemistry & Industry, 26 March 2007, pp. 8. Asami et al. (2003) Comparison of the Total Phenolic and Ascorbic Acid Content of Freeze-Dried and Air-Dried Marionberry, Strawberry, and Corn Grown Using Conventional, Organic, and Sustainable Agricultural Practices, Journal of Agricultural and Food Chemistry, 51, January 2003, pp. 1237-1241. Boyer, Rodney F. (1993) Modern Experimental Biochemistry, 2 nd ed, Benjamin Cummings. Carbonaro et al. (2002) Modulation of antioxidant compounds in organic vs. conventional fruit (peach, Prunus persica L., and pear, Pyrus communis L.), Journal of Agricultural and Food Chemistry, 50, January 2002, pp. 5458-5462. Caris-Veyrat et al. (2004) Influence of organic versus conventional agricultural practice on the antioxidant microconstituent content of tomatoes and derived purees; consequences on antioxidant plasma status in humans, Journal of Agricultural and Food Chemistry, 52, January 2004, pp. 6503-6509. Organic Foods and Children URL: http://www.thedietchannel.com/organic-food-and-kids [19 October 2007]. Read More