Ultra micro determination of ascorbic acid in biological samples by coupling reaction

Vitamin C (ascorbic acid, ascorbate, AA) is a water-soluble organic compound which can play a particular role in body function. The important goal of this article was to found of an analytical approach to assessment of Ascorbic acid compound by using organic reaction, depend on coupling reaction with diazotized solution of 4-amino antipyrine with buffer solution Na2HPO4 (pH= 10.8) to produce a color compound yield with orange soluble azo dye with high absorptivity at a wavelength of 450 nm. A group of reactions has been done in order to reach a typical conditions for this method. For a range of concentration (9.14x10−8 – 2.28x10−3 μg/mL) a calibration curve was taken and the value of molar absortivity was 1.6x103 L.mol−1.cm−1, with a relative standard deviation more than 1.15% and a recovery 99.66%. By using the mole ratio method we conclude that the mole ratio for this reaction is 1:1 (Ascorbic acid: 4-amino antipyrine) and the stability factor reach to 1.8x106 L.mol−1. The proposed method showed a group of merits such as very simple, not consume time, ability to verify a variety of examined samples, also screening and confirmatory analyses could be used for routine as well. The result that we obtained revealed that the method was successfully validated to the analysis of the Ascorbic acid in biological samples.