Chemical Evaluation of Vitamin C and Reducing Properties of Selected Fruit Juices

Ascorbic acid is an essential water-soluble vitamin that plays a crucial role in numerous biological functions, including collagen synthesis, immune response enhancement, iron absorption, and protection against oxidative stress due to its strong antioxidant properties [1–3]. Unlike many mammals, humans are unable to synthesize vitamin C endogenously and must therefore obtain it exclusively through dietary sources, primarily fruits and vegetables [4]. Deficiency of vitamin C leads to scurvy and is also associated with impaired wound healing, weakened immunity, and increased susceptibility to infections [5]. Fruits are recognized as one of the richest natural sources of vitamin C, though the concentration varies considerably depending on fruit type, variety, maturity stage, post-harvest handling, and storage conditions [6–8]. Citrus fruits such as orange (Citrus sinensis) and lemon (Citrus limon) are widely regarded as major contributors of dietary vitamin C, while non-citrus fruits like apple (Malus domestica) contribute moderate amounts and are influenced strongly by processing and oxidation [9–11]. Accurate determination of vitamin C content in fruits is therefore important for nutritional labeling, food quality control, dietary assessment, and public health recommendations [12]. A wide range of analytical techniques has been developed for the estimation of ascorbic acid in food matrices. These include titrimetric methods, spectrophotometric assays, high-performance liquid chromatography (HPLC), electrochemical techniques, and enzymatic methods [13–16]. Among these, HPLC is considered highly sensitive and selective; however, it requires expensive instrumentation, skilled personnel, and extensive sample preparation [17]. Electrochemical and spectrophotometric methods offer good sensitivity but may suffer from matrix interferences and reagent instability [18,19]. Classical titrimetric methods, particularly iodometric and dichlorophenolindophenol (DCPIP) titrations, remain widely used due to their simplicity, low cost, rapid execution, and satisfactory accuracy for routine analysis [20–22]. Iodometric titration is based on the quantitative oxidation of ascorbic acid to dehydroascorbic acid by iodine in acidic medium, with starch serving as a sensitive indicator for endpoint detection [23]. The stoichiometric nature of the redox reaction ensures a direct relationship between iodine consumption and ascorbic acid concentration, making the method suitable for educational laboratories and quality assessment studies [24]. Several studies have successfully applied iodine titration to estimate vitamin C in fruit juices and beverages, reporting reproducible and reliable results when experimental conditions are carefully controlled [25–27]. Nevertheless, variations in reported vitamin C values among different studies highlight the influence of sample preparation, dilution, and oxidation during analysis, and interference from other reducing substances naturally present in fruit matrices [28–30]. Therefore, comparative estimation using standardized procedures remains important for generating meaningful nutritional data. In view of these considerations, the present study aims to estimate and compare the vitamin C content of orange, lemon, and apple juices using the iodometric titration method. The study emphasizes methodological simplicity, reproducibility, and comparative nutritional evaluation, thereby contributing to food chemistry research and providing reliable analytical data for academic and applied purposes.

MATERIALS AND METHODS

Fresh fruits, namely orange (Citrus sinensis), lemon (Citrus Limon), and apple (Malus domestica), were procured from the local market. Analytical-grade iodine solution was used as the titrating agent. A freshly prepared 1% (w/v) starch solution was employed as the indicator. Deionized water was used throughout the experiment. Pure ascorbic acid was used for the standardization of iodine solution. All glassware, including burettes, pipettes, and conical flasks, were thoroughly cleaned and rinsed with deionized water before use.To standardize the iodine solution, 11 mg of pure ascorbic acid was dissolved in deionized water and transferred to a conical flask. A few drops of 1% starch solution were added, and the solution was titrated against iodine solution taken in the burette until a permanent blue-black color appeared, indicating the endpoint. The total volume of iodine solution consumed was 58.3 mL. From this standardization, the iodine solution was found to be equivalent to 0.19 mg of ascorbic acid per mL of iodine solution. A 1% starch solution was prepared by dissolving 1 g of soluble starch in a small volume of cold deionized water, followed by gentle heating with continuous stirring until a clear solution was obtained. The solution was cooled and used freshly during titration. The edible portions of each fruit were washed with distilled water and freshly extracted using a clean mechanical juicer. The juice was filtered through muslin cloth to remove suspended solids. Exactly 10 mL of each fruit juice sample was taken for titrimetric analysis followed by the addition of 1% starch indicator. The solution was titrated against standardized iodine solution until a stable blue-black endpoint was observed. Each sample was analyzed in duplicate to ensure reproducibility. The ascorbic acid content was calculated using the equivalence factor obtained during standardization and expressed as mg of vitamin C per 100 mL of fruit juice.

Reaction Mechanism of Iodometric Titration for Vitamin C Estimation

The iodometric titration method for the estimation of vitamin C is based on the redox reaction between ascorbic acid (a strong reducing agent) and iodine (a mild oxidizing agent) in an aqueous medium. Ascorbic acid readily reduces iodine to iodide ions while itself undergoing oxidation.

Step 1: Oxidation–Reduction Reaction

Ascorbic acid (C?H?O?) is oxidized to dehydroascorbic acid (C?H?O?) by iodine. Simultaneously, iodine (I?) is reduced to iodide ions (I?).

Step 2: Role of Starch Indicator

Starch does not react with iodine in the presence of ascorbic acid because iodine is immediately reduced to iodide ions. As long as ascorbic acid is present in the solution, no free iodine remains. Once all the ascorbic acid is completely oxidized, any further addition of iodine remains unreacted and forms a blue–black complex with starch.

I2+Starch→Blue-Black-Starch-Iodine Complex

This permanent blue–black coloration indicates the end point of the titration.

1. Standardization of Iodine Solution

Mass of ascorbic acid taken = 11 mg
Volume of iodine solution used = 58.3 mL

1mL iodine solution =0.19 mg of ascorbic acid

2. Estimation of Vitamin C in Fruit Juices

Ascorbic acid in sample (mg) =V×0.19

Where, V = Volume of iodine solution used (mL)

Vitamin C (mg/100mL) = Ascorbic acid in 10 mL×10

RESULTS AND DISCUSSION

The quantitative estimation of vitamin C in orange, lemon, and apple juices was performed using the iodometric titration method after prior standardization of the iodine solution. The analytical principle of the method involves the oxidation of ascorbic acid to dehydroascorbic acid by iodine in acidic medium, with starch used as an indicator to detect the endpoint through the formation of a blue-colored starch–iodine complex. For the standardization procedure, 11 mg of pure ascorbic acid was accurately weighed, dissolved in deionized water, and titrated against the iodine solution in the presence of 1% starch indicator. The average volume of iodine solution required to reach the endpoint was 58.3 mL. From this titration, it was determined that one milliliter of the iodine solution was equivalent to 0.19 mg of ascorbic acid is presented in Table 1. This equivalence factor was applied throughout the analysis of fruit juice samples. Freshly prepared fruit juices of orange, lemon, and apple were analyzed, and 10 mL of each juice sample was used for titration. Two independent titrations were conducted for each fruit juice to ensure reproducibility of results. In the case of orange juice, the volumes of iodine solution consumed were 14.5 mL and 12.0 mL in the two trials are presented in Table 2. These volumes corresponded to 2.755 mg and 2.08 mg of ascorbic acid in the titrated 10 mL sample. When extrapolated to a 100 mL basis, the vitamin C content was calculated to be 275.5 mg and 208 mg, respectively, yielding an average value of 241.75 mg per 100 mL of orange juice. The experimentally determined vitamin C content of orange juice confirms its role as a rich dietary source of ascorbic acid. The minor variation observed between trials may be attributed to slight differences in endpoint detection, exposure of the sample to air during handling, and partial oxidation of vitamin C prior to titration. For lemon juice, iodine volumes of 1.5 mL and 1.5 mL were recorded in the two trials. These values corresponded to 0.285 mg of ascorbic acid in the titrated 10 mL sample. When expressed on a 100 mL basis, the vitamin C content was found to be 285 mg, for both the trial, with an average value of 28.25 mg per 100 mL is presented in Table 3. The comparatively lower measured value may be influenced by factors such as dilution of juice, rapid oxidative degradation of vitamin C, and the sensitivity of the iodometric endpoint in acidic media. Apple juice required substantially higher volumes of iodine solution, namely 64 mL and 68 mL in the two trials are presented in Table 4. These volumes corresponded to 12.16 mg and 12.92 mg of ascorbic acid in the 10 mL titrated sample. On conversion to a 100 mL basis, the calculated values were 1216 mg and 1292 mg, respectively, resulting in an average apparent vitamin C content of 1254 mg per 100 mL. This exceptionally high value does not necessarily represent true ascorbic acid concentration but instead reflects the strong reducing nature of apple juice. Apple juice contains significant amounts of polyphenols and other antioxidant compounds that can react with iodine, thereby increasing iodine consumption and leading to an overestimation of vitamin C when using iodometric titration. A comparative evaluation of the three fruit juices reveals marked differences in iodine consumption and calculated vitamin C content. Orange juice exhibited consistent and nutritionally meaningful levels of vitamin C, lemon juice showed relatively lower experimental values, and apple juice demonstrated a very high apparent vitamin C content due to matrix interference. The pie chart illustrates the relative contribution of vitamin C content in different fruit juices presented in Table 5. Apple juice accounts for the largest proportion of total vitamin C, contributing approximately 82.3%, followed by orange juice at about 15.9%, while lemon juice contributes only 1.9% as shown in figure 1. This graphical representation highlights the significantly higher apparent reducing capacity of apple juice compared to orange and lemon juices under iodometric titration conditions. These observations highlight an important limitation of iodometric titration, as the method measures total reducing capacity rather than exclusively ascorbic acid. Further more, the results demonstrate that iodometric titration is a simple, cost-effective, and reproducible method for comparative estimation of vitamin C in fruit juices. However, the presence of other reducing substances must be carefully considered while interpreting the results, particularly in complex fruit matrices.

Table 1: Standardization of Iodine Solution

Table 2: Estimation of Vitamin C in Orange Juice

Table 3: Estimation of Vitamin C in Lemon Juice

Table 4: Estimation of Vitamin C in Apple Juice

Table 5. Average Vitamin C Content in Fruit Juices

Figure1. Relative Vitamin Ccontent in different fruits juice (mg/100 mL)