A Simple UV-spectrophotometric Method for the Determination of Vitamin C Content in Various Fruits and Vegetables at Sylhet Area in Bangladesh

MATERIALS AND METHODS

An experiment was conducted at Organic Chemistry Laboratory of Shahjalal University of Science and Technology University, Sylhet, Bangladesh during March to October, 2005.

There are many methods that are employed for the quantitative determination of vitamin C such as biological, electrochemical, and chromatographic method (East and Nascimeet, 2002; Geigertj et al., 1981; Veasey and Nieman, 1980). All the method has great limitation in use for different purpose, such as in biological sample, food products, pharmaceuticals etc. It is very difficult to choose a unique method for determining the content of total vitamin C in food products, biological samples and pharmaceuticals. Because each samples type have its own specific characteristics and properties in terms of extraction, purification, interference of other compounds (such as color, presence of oxidizing, reducing components etc).

Although some methods are available for determination of ascorbic acid but very few methods are employed for the determination of both forms (ascorbic acid and oxidized form, dehydroascorbic acid) of ascorbic acid. This is because two forms of the vitamin C, ascorbic acid and its oxidized form dehydroascorbic acid possess the different chemical, optical and electrochemical properties.

On the basis of reducing property of ascorbic acid, it can be determined chemically by titrating against an oxidizing agent such as 2,6-dichlorophenolindole dye Indian Pharmacopoeia, It is not applicable to many pharmaceutical preparations containing Fe (II), Sn (II), Cu (I), SO₂, SO₃^2- and S₂O₃^2- ions which are usually associated with mineral or liver preparations. The method is applicable only when the concentration of dehydroascorbic acid is negligible. The applicability of the method is restricted to only those samples of citrous fruits and multivitamin tables, which do not contain minerals.

To determine content of total vitamin C in food samples, a well-established method is the 2,4-dinitrophenyl hydrazine methods (DNPH) (Riemschneider et al., 1976). This is a simplified method for the simultaneous determination of the total vitamin C employed coupling reaction of 2,4-dinitrophenylhydrazine dye with vitamin C and followed by spectrophotometric determination.

Instrument: A Shimadzu spectrophotometer (model UV-1601) with a pair of 1 cm quartz cells was used.

Reagent required

5% Metaphosphoric acid-10% acetic acid: Fifteen grams of solid metaphosphoric acid (E. Merck) were dissolved in mixture of 40 mL of glacial acetic acid (BDH) and 450 mL of distilled water in a 500 mL volumetric flask. The solution was filtered and collected.

10% Thiourea solution; 2,4-Dinitrophenyl-hydrazine solution; 85% Sulphuric acid:

Standard vitamin C (ascorbic acid) solution: 0.05 g standard crystalline ascorbic acid was dissolved in 100 mL of distilled water to prepare 500 ppm standard stock solution.

Sample preparation: Ten grams of sample was blended or 10 g blended sample was homogenized with about 50 mL of 5% metaphosphoric acid-10% acetic acid solution. Then it was quantitatively transferred into a 100 mL volumetric flask and was shaken gently until a homogeneous dispersion was obtained. Then it was diluted up to the mark by the 5% metaphosphoric acid-10% acetic acid solution. Then the solution was filtered and the clear filtrate was collected for the determination of vitamin C in that sample.

Estimation of vitamin C

Procedure: To the filtered sample solution few drops of bromine water were added until the solution became colored (to confirm the completion of the oxidation of ascorbic acid to dehydroascorbic acid). Then few drops of thiourea was added to it to remove the excess

Table 1:	The total vitamin-C content in fruits and vegetables

bromine and thus the clear solution was obtained. Then 2,4- dinitrophenyl hydrazine solution was added thoroughly with all standards and also with the oxidized ascorbic acid. Total vitamin C employing coupling reaction of 2,4-dinitrophenyl hydrazine dye with vitamin C and followed by spectrophotometric determination.

Reactions

Reproducibility: The reproducibility of this method was checked by determining the % recovery of known amount of vitamin C from a sample. This can be done by addition of different known amount of vitamin C in this sample. For instance, the concentration of the sample is X ppm and than 5 ppm standard is added and the observed concentration of the mixture is X^' then the % recovery is given by:

RESULTS AND DISCUSSION

Calibration curve: After determination of the λ_max of the colored complex (521 nm) using a Shimadzu UV- spectrophotometer the absorbance of the all standards (converted to coloured complex) were taken to construct a calibration curve. The calibration curve was constructed by plotting the concentration versus the corresponding absorbance. Molar absorptivity was found 0.0323 L mol^-1 cm^-1 using Beer-Lambert plots (Fig. 1).

Determination of vitamin C in samples: Generally all the parts of a fruit and all fruits have not equal amounts of edible part, in the comparative study of the vitamin C content in various fruits and vegetables, the percent of edible parts of those must also be considered. Fruits such as orange, lemon etc contain high amount of vitamin C and vegetables such as Cabbage, Green pepper, Red pepper also have relatively high amount (7 to 163 mg/100 g) vitamin C (Lidija et al., 2003; Alam 1996). In study the locally available fruits such as Dumur, Katabadam, Katadenga etc. collected from Sylhet and the vitamin C content of these fruits were determined. Dumur is not popular fruit but contain 15 mg/100 g and Katabadam contain 77 mg/100 g of vitamin C. By the similar methods vitamin C content of the fruits Arboroi, Peyera (Guava), Katajamir and

Fig. 1:	Calibration curve of standard vitamin C at 521 nm

Fig. 2:	Spectrum of DNPH complex of glucose

vegetables such as Rojottepata, Thankoni, Kalmishak etc is also determined. Since the vitamin C content vary with the storage period on preservation, therefore, Vitamin C content of some fruits and vegetables were also determined at frozen conditions. It is observed that vitamin C content of fruits and vegetables slowly decreases with time as the storage period increase even at low temperature (Table 1).

The reliability of this method is justified by the calculation of the % of standard deviations and it was found to be varied within the range from 0.20 to 2.45 % (Table 1). The reliability of this method is also confirmed from the consideration of the following expected interferences.

Interferences due to diketogulonic acid: Due to the destructive oxidation hydrolysis at higher pH results the opening of the lactone ring of the ascorbic acid and loose the vitamin activity. These processes are naturally occurred in fruits and some amounts of diketogulonic acid is present in the fruits (Geigertj et al., 1981). As the diketogulonic acid has keto group, it should give the osazone with DNPH as that of ascorbic acid and should give the colored complex on treatment with 85% H₂SO₄. Thus there is chance of error in this method. But actually this cannot interfere with the ascorbic acid.

Here diketogulonic acid was prepared by the acid hydrolysis (dilute HNO₃) of ascorbic acid. The spectrum shows that there is no considerable absorption peak near the 521 nm (the absorption maxima of DNPH complex of ascorbic acid).

Interference due to extracted glucose: As ascorbic acid is largely similar to the glucose by structure, some of glucose may be extracted in the meta-phosphoric acid during the extraction of ascorbic acid from sample. because of their structural similarity, glucose may also form the colored complex with DNPH as ascorbic acid. But actually no such interference is occurred which is evident from the following spectrum are given in Fig. 2. From the spectrum it is evident that there is no absorption peak around the interested peak at 521 nm.

Vitamin C is important to human health, and many species need a dietary source to stay healthy. The locally available citrous fruits such Katagamir, Katabadam and Peyar are the excellent sources of vitamin C. The locally available vegetables which was analyzed contain relatively good amount of vitamic C and a good source of vitamin C. The method is simple and offers an excellent method for the determination of total vitamin C in fruits and vegetables.

ACKNOWLEDGMENTS

We are grateful to the Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet-3114, Bangladesh for giving necessary facilities during research.