2.12. Determination of ascorbic acid
Ascorbic acid (AA) content was measured by using a UV–VIS spectrophotometer according to the literature procedure of Durust et al. (1997).The spectrophotometer was adjusted to zero by using distilled water. The absorbance of mixing of 1 ml of oxalic acid solution with 8 ml of DCPI solution (12 mg of DCPI in 1000 ml of distilled water) and 1 ml of acetate buffer solution (300 g of anhydrous sodium acetate in 700 ml of distilled water with 1000 ml of glacial acetic acid) was recorded at the end of 15 s. This value was recorded as X1. Then, the absorbance of the standard ascorbic acid solution (1 ml) with the acetate buffer solution (1 ml) and DCPI (8 ml) was recorded as X2. X1 was the absorbance of all DCPI, and X2 was the absorbance value of the remaining DCPI, after its reaction with AA. X2 values were similarly recorded for the other standard AA solutions (20, 30, 40, and 50 ppm). X1 − X2 values were the absorbance of each working standard. The calibration graph was constructed by plotting the absorbance values versus the concentration (ppm) of standard AA solutions (r2 = 0.9951).
For the absorbance measurements of the sample extracts, the same procedure was applied. First, an autozero operation for the instrument was performed. Then, the absorbance of 1 ml of the sample extract with 8 ml of distilled water and 1 ml of acetate buffer solution was recorded at the end of 15 s. This was the X2 value of the sample solution. X1 − X2 values represented absorbance of the sample. From the calibration graph, the AA concentration of the sample was determined. All of the measurements were recorded at 520 nm. All determinations were performed in triplicate (n = 3).