Using the steps of quantitative analysis to explain from the following below:

Preparation of the reagents: 5% Metaphosphoric acid -10% acetic acid solution: 50g of solid metaphosphoric acid (E.Merck) were dissolved in a mixture of 40 mL glacial acetic acid and 450 mL of distilled water in a 500 mL volumetric flask. 85% Sulphuric acid solution: 180 mL of concentrated sulphuric acid was added to 20 ml of water in an ice bath 10% Thiourea solution: 1g of thiourea was dissolved in 9 mL of distilled water. 2,4- Dinitrophenylhydrazine solution: 2g of 2,4-dinitro- phenylhydrazine were dissolved in 80 mL of distilled water and 14.67 mL of concentrated sulphuric acid (specific gravity 1.84). The solution was filtered and stored. Aqueous Bromine solution: 5 mL of liquid bromine was added to 100 mL of distilled water and shaken vigorously. The solution was filtered and stored.

Preparation of Standard ascorbic acid (vitamin C) solution: 50 mg standard crystalline ascorbic acid was dissolved in 100 mL of 5% metaphosphoric acid -10% acetic acid solution.

Sample Collection: The selected fresh fruits and vegetables were collected from local markets. Care was taken to collect fruits/vegetable samples during their peak seasons. After collection the food samples were cleanly washed with tap water and cut so as to collect edible portion only. Preparation of Samples: The food samples were prepared according by reported methods . Amounts of each sample were noted separately before the extraction of Vit. C. Weights of samples were taken after cooling to room temperature. Thus 1.0 to 2.5 mL of each sample homogenized with about 50 mL of 5% metaphosphoric acid and 10% acetic acid solution. This was then quantitatively transferred to a 100 mL volumetric flask and was shaken gently until a homogeneous dispersion was obtained. Then it was diluted upto the mark with 5% metephosphoric acid and 10% acetic acid solution. The solution was then filtered and the clear filtrate was collected for the determination of vitamin C in the sample.

Estimation of vitamin C: Few drops of bromine water were added to 4 mL of the filtered sample solution until the solution became colored (to confirm the completion of the oxidation of ascorbic acid to dehydroascorbic acid). Then 1 to 2 drops of thiourea solution was added to it to remove the excess bromine and to get a clear solution. The standard solutions of ascorbic acid (5 ppm, 10 ppm, 20 ppm and 25 ppm) were prepared from 500 ppm stock solution of ascorbic acid by proper dilution. 1 mL of 2,4- dinitrophenylhydrazine was mixed thoroughly with all standards and also with the oxidized ascorbic acid. For completion of the reaction all the standards and the samples were kept at 37 o C for three hours in a water bath. After the incubation, the solutions were cooled in an ice bath and were treated with 5 mL of 85% H2SO4 with constant stirring and a colored solution was obtained. The absorbance of these solutions were measured at 522 nm against a reference made identically without ascorbic acid solution. For obtaining a calibration curve the absorbance of the standards were plotted against their corresponding concentrations. The concentration of the sample solution as well as the content of vitamin C of the sample were calculated from the calibration curve for the corresponding absorbance of the sample.

Results and Discussion A calibration graph was plotted using average absorbances for standard solutions versus their respective concentrations. For each standard solution, four duplicates were prepared. This was done to increase the accuracy of the calibration curve. The r2 value obtained (0.9984) was close to the ideal value 1, indicating a good linear correlation between the area under peak of interest and ascorbic acid concentration. This allows good estimates of ascorbic acid content to be made given the absorbance for each sample.

Table 1. Ascorbic acid concentration in Guava samples

Sample - Ascorbic acid concentration in diluted sample (ppm) - Original Ascorbic acid concentration (ppm) - Original Ascorbic acid concentration (mg AA/ 100mL)

1 113 564 56.4

2 116 581 58.1

3 119 596 59.6

4 116 578 57.8

5 199 996 99.6

6 114 570 57.0

7 114 570 57.0

8 120 602 60.2 Sample calculation for sample 2: Ascorbic acid concentration in diluted sample = 116ppm Ascorbic acid concentration in original sample = 116ppm 5 = 581ppm = 58.1 mg / 100mL of juice Average ascorbic acid concentration in original sample = 58.0 mg / 100mL of juice The average ascorbic acid concentration in the guava fruit determined experimentally was 58.0mg/100mL of juice. This was approximately 3.9 times higher than the amount indicated on the packaging (15mg/100mL). As mentioned earlier, As the expiration date is approached, ascorbic acid would be lost to different extents depending on the storage conditions. Manufacturers are known to add ascorbic acid to their products to improve their nutritional value and also to account for the ascorbic acid lost during the manufacturing and storage process. Since the experiment was conducted before the expiration date of the product (March 14, 2014), a higher ascorbic acid content would be expected. The original ascorbic acid concentration for sample 5 was excluded from the calculation as it was almost double of other results and thus likely to be an outlier. A possible reason might be an error in dilution during the preparation of the sample. The other results were found to be precise with a low standard deviation (1.4) and a low coefficient of variation (2.5%).

Conclusion The ascorbic acid content of commercial guava juice determined using spectrophotometer was 58.0mg/100mL of juice.