RESULTS
Table 1: Standardization of 0.001M 2,6-dichlorophenolindophenol, C12H7O2NCl2 solution
Table 2: Determination of the ascorbic acid concentration of fruit juice.
Table 3 : CuSO4 and bubbling to air
CALCULATION
The molarity of ascorbic acid solution was calculated.
Number of moles of ascorbic acid = Weight____
Molecular weight
= 0.1_______
(12x6)+(1x8)+(16x6)
= _0.1_
176 moles
= 5.6818x10-4 moles
Molarity of ascorbic acid = Mole x 1000
Volume
= 5.6818x10-4 moles x 1000
500 cm3
= 1.1364x10-3 mol/dm3
= 0.001134 mol/dm3
Thus, the molarity of the ascorbic acid is 0.001134 mol/dm3 which is approximately the same as the 0.001 M as labeled on the ascorbic acid.
The molarity of the dye was calculated.
M1 x V1= M2 x V2 M1 = Molarity of DCPIP
V1 = Volume of DCPIP
M1 x (25.40ml)= (0.001 mol/L) x (25ml) M2 = Molarity of ascorbic acid
V2 = Volume of ascorbic acid
M1= 9.8425 ×10-4 mol/L
Thus, the molarity of the dye solution is 9.8425 ×10-4 mol/L which is approximately the same as the 0.001 M as labeled on the 2,6-dichlorophenolindophenol.
The concentration of ascorbic acid in the fruit juice was calculated.
M1 x V1= M2 x V2
(0.001 mol/ml) x (9.20 ml) = M2 x (50ml)
M2 = 0.000184 mol/L
M2 = 0.000184mol/L x [(12x6)+(1x8)+(16x6)]
= 0.000184 x 176g/L
=0.0324g/L
= 32.7 mg/L
Thus, the concentration of the ascorbic acid in the fruit juice is 32.7 mg/L.
DISCUSSION
The wide use of ascorbic acid (AA) in canned fruits, vegetables, animal foods and drugs, to enable the determination of AA in different matrices and at different levels many analytical techniques are available. In this experiment, the quantitative determination of ascorbic acid is based on its reaction with the mild oxidizing agent, 2,6- dichlorophenolindophenol (DCPIP).
DCPIP is a chemical compound used as a redox dye. This dye is blue in base (DCPIP-) and pink in acid (DCPIPH) and the pink form can be reduced by ascorbic acid to a colorless form (DCPIPH2).
Titration with 0.001M 2,6- dichlorophenolindophenol solution
Reaction 1: DCPIP- (blue) +H+ → DCPIPH (pink)
Reaction 2: DCPIPH (pink) + Ascorbic acid → DCPIPH2 (clear) +Dehydroascorbate
If a drop of blue DCPIP dye is added to a low pH solution (pH<4.0), it will turn pink (Reaction 1). If a suitable electron donor such as ascorbic acid is present in that solution, it will turn colorless (Reaction 2). When all of the ascorbic acid in the solution has been oxidized to dehydroascorbate, no more electrons will be available to reduce a drop of DCPIPH to the colorless form and the solution will remain pink (Reaction 2 will not take place). The end-point was a faint pink colour that persisted for 15 seconds.
(http://www.bbc.co.uk/schools/ks3bitesize/sosteacher/science/45432.shtml)
Acetic acid added in Step 2 (iii) and Step 4 of the method will reduce the oxidation of the ascorbic acid by lower the pH of the orange juice to retard the action of the enzyme polyphenol oxidase. If the pH is reduced below 3.0, the polyphenol oxidase will be inactivated. Acetic acid also reduces interference from any iron present, and thereby facilitates subsequent clarification of the extract. Since the ascorbic acid is not oxidized, it was existed in L-enantiomer form. Therefore, the L-enantiomer form of ascorbic acid was determined in this experiment.
( James , 1999 )
(http://ag.udel.edu/other_websites/foodworkshop/WSFWorkshop/Enzymatic%20Browning%20(Ch1).htm)
From the experiment, the molarity of the ascorbic acid and the dye solution computed are 0.001134 mol/L and 9.8425 ×10-4 mol/L respectively. Hence, the concentration of the ascorbic acid in the fruit juice is 3.27mg/100ml which is much more lower compared to the amount stated in the product label (Sunkist) in which the ascorbic acid content is 150mg/100ml. The high discrepancy between these two values might be due to the oxidization of ascorbic acid, which was exposed to the oxygen for a period of time due to the insufficient of apparatus in the laboratory. This can be improved by not exposing ascorbic acid to oxygen, metals, light and heat, as it can be oxidized easily. Therefore, it must be stored in dark and cold and but not in a metal containment.
The mechanisms of ascorbic acids degradation is commonly due to the effect of metal ions and the presence or absence of oxygen. The rate of oxidative degradation of ascorbic acids is commonly proportional to the concentration of ascorbate monoanion (HA-), molecular oxygen and the metal ion. It is known that uncatalyzed oxidation is essentially negligible but the presence of trace metals in food are responsible for most of the oxidative degradations. The potency of metal ions in catalyzing ascorbate degradation depends on the metal involved, its oxidation state, and the presence of cheletors. For example, Cu(II) is about 80 times more potent than Fe(III) while te chelate of Fe(II) and ethylenediaminetetraacetic acid (ETDA) complex is about 4 times more catalytic than free Fe(III). (Fennema , 1996)
In this experiment, the potency of copper (II) sulfate in catalyzing ascorbate degradation was tested. One of the conical flask with only fruit juice act as control. It is titrated with 44.70ml of DCPIP for oxidation to occur. On the other hand, another conical flask with fruit juice and 1 mg of copper sulfate titrated with only 11.70ml of DCPIP for oxidation to occur. It is proven that the presence of metal ions responsible for accelerates the rate of degradation of ascorbic acid in an air-saturated fruit juice as less DCPIP is needed. During the step 5, bubbling through air is applied to the ascorbic acid to enhance the oxidation of ascorbic acid by the catalyst, copper. If not, it will consume a lot of time before the reaction can take place.
Advantages
- 2,6-Dichlorophenolindophenol served as a good electron acceptor.
- DCPIP is used as the titrant because it only oxidises ascorbic acid and not other substances that might be present and it acts as a self-indicator in the titration
- It is reasonably accurate, rapid, and convenient.
- Can be applied to many different types of samples.
Disadvantages
- The end point of a titration for this reaction is difficult to ascertain due to the lack of complete decolourisation of the DCPIP.
- These methods are not specific or are not very sensitive.
- The reagent itself is not stable and needs standardization before use.
- If the sample solution is intensely coloured (fruit juice or syrup), end point detection will be difficult.
Better choice for vitamin C
According to the hypothesis, content of vitamin C in fresh fruit is suppose to be higher than commercial packet fruit juice. Due to a lot of processing, most of the vitamin C in commercial fruit juice are destroy.
The fewer amounts of millilitres of juice it took to turn DCPIP from blue to clear, the larger the amount of vitamin C there was in the drink. Many of the commercial fruit juice are heavily fortified with vitamin C.
Therefore, it is recommended that vitamin C should take in through the fruits instead of processing fruit juice. The table below showing ascorbic acid content of various fruits:
Conclusion
The molarity of the ascorbic acid and the dye solution computed are 0.001134 mol/L and 9.8425 ×10-4 mol/L respectively. The concentration of the ascorbic acid in the fruit juice is 3.27 mg/100mL which is much more lower than the ascorbic acid content of the label product (15 mg/100mL). this might be due to the oxidation of ascorbic acid.
Ascorbic acid (vitamin C) is essential to humans. It is involved in the synthesis of collagen, which is the main constituent of skin, connective tissue, and the organic substance of bones and teeth. A deficiency of vitamin C results in a disease called scurvy. A quantity of 60 mg vitamin C per day is enough to prevent the disease, and this is the recommended daily dietary allowance (RDA).
(http://a-s.clayton.edu/ptodebus/CHEM1211/lab/experiments/Vitamin%20C/Vit%20C%20titration%20v1.doc)
REFERENCES
- Ceirwyn S.James , 1999, Analytical Chemistry of Foods , An Aspen Publications , page 138,139
- Owen R. Fennema, 1996, Food Chemistry, Third Ed., Marcel Deeker,Inc., pg 561,562
- http://a-s.clayton.edu/ptodebus/CHEM1211/lab/experiments/Vitamin%20C/Vit%20C%20titration%20v1.doc