Methods 1. Standard solutions of Ascorbic Acid in 5% MPA solution were prepared at 0.05, 0.10, 015, and 0.20 mg/mL. 2. Both samples of orange juice – the untreated and the microwaved – were mixed in equal parts with 5% MPA solution. This mix was then centrifuged at 3,000 rpm for 10 minutes and 5C. 3. A sample of the centrifugate was then loaded into a disposable C18, Sep-Pak cartridge, which was pretreated with methanol and water. 4. The second portion of the eluate (after the first 2 mL were discarded) was filtered through a 0.45 um Millipore filter. 5. The two filtered solutions, as well as the solutions for the standard curve, were individually injected onto the analytical column of the HPLC. 6. The Vitamin C in the solutions was eluted isocratically with 2% KH2PO4 at a pH of 2.4 and a flow rate of 1 ml/min. This eluate was monitored by UV light at 245 nm and the computer integrator produced peaks with their respective areas and elution times. 7. The Standard Curve was made, the line of best fit was calculated and this was then used to determine the concentrations of Vitamin C in both the control and microwaved OJ.
The conditions of the HPLC were as follows:
Pump, flow rate: 1 mL/min
UV Detector, set @ 245 nm
Mobile phase: 2% KH2PO4 pH = 2.4, adjusted with H3PO4, degassed.
Analytical column: C 18 column (250 x 4.6 mm) coupled with a guard column.