# Bioactive Vitamin C Content from Natural Selected Fruit Juices
## 2 Materials and Methods
### Analytical Procedure
#### 2.2.2. Iodometric Determination of Vitamin C Content  
The content of vitamin C was determined directly from the samples of fruit juices in full maturity.  
Iodine titration is based on oxidation-reduction reactions (oxidation-reduction method) according to equation (1) [33; 68; 72]. The titrated sample consisted of 25 cm\({}^{3}\) of fruit juice and 1 cm\({}^{3}\) of starch solution (w = 1%). The samples were titrated with a solution of KIO\({}_{3}\) (c = 0.05 mol\(\cdot\)dm\({}^{-3}\)) until the appearance of blue colouring lasted over one minute. As the iodine is added during titration, the ascorbic acid (AA) is oxidated and becomes dehydroxyic acid (DHA). In contrast, the iodine is reduced to iodide ions (Figure 3) [41; 68]. The greater the activity of the ascorbate oxidase enzyme during the test procedure, the more rapidly vitamin C values decrease owing to rapid oxidation, which affects the evaluation of vitamin C concentration [2; 67; 73; 74; 75; 76].  
\[I_{2}+2e^{-}\leftrightarrow 2I^{-} \tag{1}\]  
The mass of vitamin C (C\({}_{6}\)H\({}_{8}\)O\({}_{6}\)) was calculated according to the amount of iodine solution consumed with Equation (2):  
\[\text{m}=\text{c}\times\text{V}\times\text{M} \tag{2}\]  
Figure 3: The reaction between vitamin C and iodine.  
where c is the iodine concentration (c(KIO\({}_{3}\)) = 0.05 mol\(\cdot\)dm\({}^{-3}\)) used for the titration process, V represents the amount of KIO\({}_{3}\) utilised in the titration process, and M refers to the molecular weight of ascorbic acid (g\(\cdot\)mol\({}^{-1}\)).  
The titration was repeated three times for each fruit juice sample. The obtained vitamin C results in fruit juice were expressed as mean values of three determinations. The values obtained from the analyses were subsequently compared to determine the optimum conditions for retaining the highest possible amount of vitamin C in the stored juice.  
For weighing of chemicals, RADWAG AS 110/C/2 (Max. 110 g, Min. 10 mg, d = 0.1 mg, Libra s.r.o., Bratislava, Slovakia) analytical balance was used.  
Reagents  
Chemicals used: soluble starch (Merck KGaA, Darmstadt, Germany), potassium iodide (KI, LABO, Bratislava, Slovakia), potassium iodate (KIO\({}_{3}\), LABO, Bratislava, Slovakia), sulfuric acid (H\({}_{2}\)SO\({}_{4}\), concentrated, purity p.a., LABO, Bratislava, Slovakia), and ascorbic acid (Merck KGaA, Germany).  
Preparation of Solutions  
One percent starch indicator solution was prepared by adding 0.5 g of soluble starch to 50 cm\({}^{3}\) of near-boiling water.  
Iodine solutions were prepared by dissolving 5.0 g of potassium iodide (KI) and 0.268 g of potassium iodate (KIO\({}_{3}\)) in 200 cm\({}^{3}\) of distilled water, followed by adding 3M sulphuric acid (H\({}_{2}\)SO\({}_{4}\)). The solution was made up to 500 cm\({}^{3}\) in a graduated cylinder and then transferred to a beaker.  
Vitamin C standard solution was prepared by dissolving 0.250 g of vitamin C in 100 cm\({}^{3}\) of water and then diluted to 250 cm\({}^{3}\) with water in a volumetric flask.  
Standardising Solution  
The vitamin C solution (25 cm\({}^{3}\)) was transferred into a 100 cm\({}^{3}\) titration flask, and 10 drops of 1% starch solution were added. The solution was titrated with the iodine solution until the first blue colour, which persisted for about 20 s, was observed. Juice samples (25 cm\({}^{3}\)) were titrated exactly the same way as the standard. The initial and final volume of iodine solution required to produce the colour change at the endpoint was recorded. The titration was performed in triplicate in all cases.  
In all experiments, the chemical reagents (ACS grade) were dissolved using distilled and deionised (DDI) water produced using a MilliporeSigma(tm) Synergy(tm) Ultrapure Water Purification System (Merck Millipore, Bedford, MA, USA).