Ascorbic Acid Determination
Two principles have been employed in the important chemical methods for vitamin C determination. In the first the strong and fairly rapid reducing property of the vitamin is determined by titration against a standard oxidizing solution. At present the dye 2, 6-dichlorobenzenoneindophenol (2, 6-dichlorophenolindophenol) is the oxidant of choice. Rather than titrate ascorbic acid, an excess of the dye solution may be added to the ascorbic acid solution, and the loss of color due to reduction of the dye, determined by use of a photoelectric colorimeter.
Many modifications of each of these approaches have been used. The vitamin is generally extracted from animal or vegetable tissues with metaphosphoric acid (HPOs) or trichloroacetic acid. In the case of vegetable tissue high in ascorbic acid oxidase the enzyme can be destroyed by extracting with hot HPOs' Dehydroascorbic acid does not reduce the 2,6 dye, and, in plant tissue especially, appreciable amounts of the vitamin may exist in this form. Treatment of the acid extract with H2S converts the vitamin C to the reduced form. This is an important consideration, since both forms have vitamin activity. The direct titration methods, the photoelectric method and the xylene extraction method each have certain advantages. The latter is especially adapted to turbid or highly colored ascorbic acid-containing solutions. Another chemical principle employed in ascorbic acid determination involves the conversion of the vitamins into a soluble colored complex by treatment with 2, 4-dinitro-phenylhydrazine.
The color intensity can be determined in any of the various instruments for this purpose.
A critical review of various methods is available. In the various biological methods the guinea pig is the animal of choice. Practical methods have been developed employing growth, tooth structure, and growth of odontoblasts of incisors as the criteria ofthe ascorbic add content of the test material. Details can be found in the sixteenth revision of the United States Pharmacopoeia.
