To determine ascorbic acid content in a sample of fruit juice by using titration method with 0.001M 2,6-dichlorophenolindophenol, C12H7O2NCl2.
Structure of Ascorbic acid
Ascorbic acid, the chemical term for vitamin C, are found naturally in fruits and green vegetables. It is a dietary requirement for normal metabolism, formation of collagen, wound healing, and tissue repair. Ascorbic acid is often used as an antioxidant to help prevent free radical damage in the skin, builds resistance to infection, aids in the prevention treatment of the common cold, and aids in the absorption of iron.
Yet, vitamin C cannot be synthesized by the body, and needs to be ingested. A lack of vitamin C can cause abnormalities of the spine, scurvy, and a reduction in the ability of the body to heal wounds.
The determining factors as to whether organic substances can be determined in an aqueous medium depend primarily on the functional groups that characterise the redox properties. The determination of ascorbic acid content is based on the oxidation of ascorbic acid to dehydroascorbic acid: C6H8O6→ C6H6O6 + 2e- + 2H+
The redox potential depends on the pH and without adequate buffering the pH at the electrode surface can be displaced by the oxidation reaction of the ascorbic acid leading to peak broadening.
Vitamin C is found in fruit drinks such as orange juice and also other sources like vegetables, liver and kidney meat. Vitamin C in food can be destroyed by cooking, leaching out from fruits and vegetable during washing, and being oxidized when expose to the air. Thus, food that rich in vitamin C needs to be stored and prepared well.
1. Standardization of 0.001M 2, 6-dichlorophenolindophenol solution. 25.00mL aliquot of ascorbic acid solution was pipette into a 100mL conical flask. 0.001M 2, 6-dichlorophenolindophenol solution is titrated until a faint colour persisted for at least 15 seconds.
The molarity of the dye solution is calculated by the result obtained.
2. Ascorbic acid concentration of fruit juice is determined.
5mL of fruit juice was pipette into a 100mL conical flask. 10mL of 5M acetic acid, 5mL of acetone (prevent interference of SO2) and 30mL of water was added, then the mixture was allowed to stand for 5 minutes and titrated with 2, 6-dichlorophenolindophenol solution.
3. CuSO4 and bubbling to air.
2 flasks is set up and filled with 25mL of fruit juice in each flask. 1mg of copper sulfate is added to one of the flasks and both are put to bubbled air for 20 minutes. The titration is repeated at the completion of the 20 minutes of air bubbling.
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.
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 )
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.
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.
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.
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 is3.27 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).
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