Effect of storage temperature and time on the vitamin c concnetration in citrus fruit juices

Within a five-day period, the VitC concentration of each sample is determined through iodometry and spectrophotometry. The average values are calculated and used for the analysis (Figure 16).

Preparation of 0.005M iodine (I-) solution

Preparation of a calibration curve

• From the volumetric flask, pour A millilitre of the 0.02 M ferricyanide solution into a cuvette, then cap it. Shaking the cuvettes can introduce air bubbles that will change the absorbance measurements, therefore, avoid doing so. (The cuvettes must be clear of blemishes and scratches to prevent the absorbance reading from being obscured.)
• One millilitre of a 0.02 M ferricyanide solution and one millilitre of an AA solution with the corresponding concentration are added to each of the ten cuvettes to construct the calibration curve. Insert a reference to Figure 14 here. Figures 12-13 depict an example of an absorbance reading.

Calculation of the Vitamin C concentration according to the volume of an iodine solution used for oxidation. (Figure 8)

• 𝑛 (𝐼₂) = 𝑉 × 𝐶 = 0.005𝑙 × 0.005 \({mol \over l}\) = 2.5 × 10⁻⁵𝑚𝑜𝑙
• 𝑛 (𝐶₆𝐻₈𝑂₆) = 𝑛 (𝐼₂) = 2.5 × 10⁻⁵𝑚𝑜𝑙
• 𝑚 (𝐶₆𝐻₈𝑂₆) = 𝑀_𝑟 × 𝑛 = 176.12 \({g \over mol}\) × 2.5 × 10⁻⁵𝑚𝑜𝑙 = 4.4 × 10⁻³ 𝑔 = 4.4 𝑚𝑔
• 𝐶 (𝐶₆𝐻₈𝑂₆) = 4.4𝑚𝑔 × \({100 \over 10}\) ml = 44.0 \({mg \over 100\ ml}\)

Some safety issues must be taken into account. I wear eye protection and hand gloves and keep all the containers containing iodine closed since aqueous and solid iodine release poisonous vapour under room conditions and are harmful to human health in high quantities. The reaction wastes are gathered in a sink, neutralized by the laboratory staff, and then poured into a sewage system to avoid contaminating water and soil. One must adhere to the precise process provided in the technical document to prevent damage caused by excessive voltage when using electrical equipment, such as a UV spectrophotometer.

According to my research into recent statistics regarding vitamin C supplements and contemporary food processing techniques, citrus fruits' declining vitamin C concentration is related to the storage circumstances they are kept in.

VitC is a chemical compound that is water soluble and unstable. With time and a rise in temperature in the presence of water, AA irreversibly breaks down into a number of compounds, such as diketogulonic acid, xylonic acid, chronic acid, and oxalic acid5. Fruits' vitamin C content is highest while they are immature and declines as the fruit ripen. The presence of oxygen and bacteria that use organic molecules as a source of carbon for cell respiration and other metabolic processes are other factors that cause AA to deteriorate. Therefore, ascorbic acid is broken down by bacteria.

In presence of oxidizing agents, AA oxidizes to form L-dehydroascorbic acid 7 (Figure 1)

Many techniques can be utilized to figure out the VitC content. Titration, the enzymatic process, reflectometry, High-Performance Liquid Chromatography (HPLC), and spectrophotometry are a few examples.

A quick, easy, and reliable procedure is needed for the experiment, and the chemicals are readily available at the school.

As a result, I utilized iodine titration to assess the VitC concentration in the juice sample. I also selected the spectrophotometric approach using ferricyanide (Figure 2) as an oxidizing agent to guarantee the data's accuracy. In school lab settings, those are the most practical and reliable techniques, primarily when used jointly to improve the accuracy of outcomes (the reliability of the methods is discussed in the Evaluation part). Iodine functions as a reducing agent during the AA reaction, and the redox reaction proceeds as follows:

𝐶₆𝐻₈𝑂₆ → 𝐶₆𝐻₆𝑂₆ + 2𝐻⁺ + 2𝑒

𝐼₂ + 2𝑒 → 2𝐼⁻

𝐶₆𝐻₈𝑂₆ + 𝐼₂ → 𝐶₆𝐻₆𝑂₆ + 2𝐼⁻ + 2𝐻⁺

The use of a starch indicator in titration with aqueous iodine monitors the oxidation of AA. AA reacts withferricyanide following the scheme:

I employ the serial dilution procedure to create AA solutions with 10-100 mg/100 ml concentrations. First, a 100 mg VitC tablet (NaturaLife, Vitamin C+) is dissolved in 100 ml of distilled water to create a solution with an AA concentration of 100 mg/100 ml. The solution is then increased to 100 ml by transferring 90 ml from the first beaker to the second one (the resulting concentration is 90 mg/100 ml). With reference to Figure 9 and Figure 11, distinct solutions with concentrations ranging from 10 mg/100 ml to 100 mg/100 ml are created.

Preparation of 0.02 M Potassium Ferricyanide ([Fe(CN)6]3-) solution

• It is projected that the vitamin C content of citrus fruit juices will decline with time. Since vitamin C is a water-soluble molecule, an acidic environment and the pring agents during stefficientlyeasily catalyze its destruction, which is why its content depletes over time.
• With a temperature rise, the vitamin C content of citrus fruit juices is anticipated to decline. AA easily breaks down into other acids since it is unstable at an even room temperature of 25°C. The rate of ascorbic acid breakdown increases with temperature. Therefore, the sample has a lower concentration of vitamin C.

To prevent bacterial contamination, the equipment is sanitized, and the procedure is carried out wearing gloves. The extraction of the juice follows the technique.

• Fruit is put on the surface and rolled around with pressure applied.
• The fruit is cut in half, and the juice is squeezed into the glass beaker.
• The extract is filtered with the funnel and filter paper to separate the solid residues from the liquid concentrates.

After juice extraction, iodine titration and spectrophotometry determine the first VitC concentration results. Five juice samples are taken from each fruit (Orange, Tangerine, Lemon, and Grapefruit) and held for five days at varying temperatures (4°C, 15°C, 25°C, 35°C, and 45°C degrees Celsius). The juice samples are kept in amber-coloured bottles with caps (Figure) to reduce exposure to light because VitC readily breaks down when exposed to oxygen or UV (Figure). Oxalic acid is used after extraction to lessen oxidation with oxygen.

To describe VitC breakdown, several kinetic models, including zero-order, first-order, and second-order kinetic reactions, have proved effective. The generic equation: defines the rate of reaction as

𝑅𝑎𝑡𝑒 (𝑉) = − \({\triangle C \over \triangle T}\) = k × C

I want to see if the trend of decomposition coincides with the zero-, first-, or second order kinetic reaction in my experiment to determine the order of AA breakdown with time and temperature, having only five dots on a graph.

First, I hypothesize that the reaction follows a zero-order kinetic model with the equation.

𝐶(𝑡) = 𝐶₀ − 𝑘₀𝑡 (1), where k is an equation constant, t is the time of reaction, and C₀ – is the initial concentration.

The initial concentration of vitamin C in freshly extracted juice varies depending on the variety of fruit, as shown in Figure 15 and Figure 16. Lemon juice has an immense amount of AA (41.21 mg/100 ml), followed by Orange juice (39.94 mg/100 ml), Grapefruit juice (36.84 mg/100 ml), and Tangerine juice (26.34 mg/100 ml). Every day of the trial shows the same trend,*

Ascorbic acid, also known as vitamin C, is a necessary micronutrient for humans. It is also a powerful antioxidant and a cofactor for numerous enzymes. Due to its antioxidant properties, AA prevents multiple malignancies and heart diseases by scavenging free radicals. Particularly during a pathogen invasion, the immune system can produce reactive oxygen species, which harm host body cells. VitC strongly impacts lymphocytes, especially neutrophils, reducing free radical-induced oxidative damage. Although vitamin C does not affect the likelihood of acquiring a cold, it does, on average, 8% for adults and 14% for children, lessen the severity of symptoms and shorten the length of the illness. Vitamin C is a nutritional necessity because the human body cannot produce it. Vitamin C deficiency may result in scurvy, irregular bone growth, and a decreased capacity for wound healing. Teenagers' diets should contain ascorbic acid in particular to ensure optimal physiological function.

There is a lot of vitamin C in many organic goods. Berries, kiwis, broccoli, and citrus fruits are among the top food sources of ascorbic acid. Citrus fruits like lemons and oranges are the most accessible food sources of vitamin C for me because I attend a boarding school and reside there. I've learned from various sources that freshly made fruit juices are best for the body because they have the maximum vitamin C concentration. The packed fruit juices, however, are widely available at school. They do not have the best storage or transit conditions because VitC is rapidly degraded when exposed to light or air for an extended period. As a result, I developed a great curiosity and interest in investigating the impact of storage conditions on the vitamin C content in fresh citrus juices. After learning how crucial ascorbic acid is to human health, I decided to look into how temperature and storage duration affect the vitamin C content of citrus fruit juices.

To test the 1st hypothesis, I plot the graph of AA concentration (in mg/ 100 ml) against time (in days). Change in AA concentration in Tangerine juice sample stored at 4°C is shown in Figure 10. If we assume that AA decomposes following zero-order kinetic model, the reaction (for Tangerine at 4°C) follows the equation

𝐶(𝑡) = 29.914 − 3.328𝑡

Where k0 (reaction constant) equalled -3.328, t – day when the concentration was measured, the data strongly correlates with the equation, with Pearson’s correlation coefficient equaling 0.9799.

To make an iodine solution, combine 6.35 x 10^-2 g of iodine with 100 ml of distilled water. I₂ is almost insoluble in water but becomes more soluble when iodide ions are present; therefore, 1 g of KI is added (Figure 7). Tri-iodide ions (I₃) are created when I₂ and KI react. The solution is prepared in a volumetric flask to a maximum of 100 ml and then transferred into a buret (Figure 6).

Preparation of 1% starch indicator

1% starch indicator is prepared by dissolving 1 g of soluble starch in 100 ml of near-boiling distilled water. The solution is filtered and cooled before use.

Titration of citrus fruit juice sample against 0.005M iodine solution

• Pipette a 10 ml aliquot of a juice sample into a 250 ml conical flask
• Add 10 ml of distilled water and drops of 1% starch indicator
• Rinse the burette with a small amount of iodine solution and then fill it. Register the initial reading.
• Start titration, constantly stirring the solution and finish when the end is reached. The endpoint is indicated when the blue colour persists for more than seconds after swirling (Figure 5)
• Repeat titration trice. The results must coincide within 0.1 ml.

What effects do storage time and temperature [4°C, 15°C, 25°C, 35°C, 45°C] have on the amount of vitamin C [mg/100 ml/day] evaluated by iodine titration and spectrophotometry in citrus fruit juices [Tangerine, Orange, Lemon, and Grapefruit]?

𝐶₆𝐻₈𝑂₆ → 𝐶₆𝐻₆𝑂₆ + 2𝐻⁺ + 2𝑒

2[Fe(CN)6]³⁻ + 2𝑒 → 2[Fe(CN)6]⁴⁻

𝐶₆𝐻₈𝑂₆ + 2[Fe(CN)6]³⁻ → 𝐶₆𝐻₆𝑂₆ + 2[Fe(CN)6]⁴⁻ + 2𝐻⁺

Following titration with AA, the reduction of ferricyanide ion, [Fe(CN)₆]^3-, to ferrocyanide, [Fe(CN)₆]^4-, is observed spectro-photochemically. Yellow becomes colourless when the colour shifts. The experiment's UV spectrophotometer sends light through the liquid sample in a cuvette while measuring its absorbance. Therefore, the concentration of vitamin C is lower the higher the absorption. To lower the error margin and compare the results to those obtained from iodine titration, spectrophotometric analysis was employed.

In a volumetric flask, dissolve grammes of potassium ferricyanide (K₃[Fe(CN)6]) in 200 ml of distilled water to create a ferricyanide solution. Once in a cuvette, it is transferred there and combined with a standardized ascorbic acid solution.

Preparation of standard AA solutions