Enzymatic Reaction Analysis using Chiral HPLC: Method and Conditions

The reaction was permitted to proceed at a temperature of 30 °C for a duration of 20 minutes, after which the enzyme was deactivated through boiling. Subsequent to the reaction, the samples were analyzed in a single layer using a Waters HPLC system, equipped with a Chiralcel IG-3 column (4.6 mm × 250 mm, 3 µm, Diacel, Tokyo, Japan). The mobile phase utilized for separation was a mixture of formic acid and methanol (0.1%, 70:30, v/v), delivered at a flow rate of 0.3 mL/min at a constant temperature of 35 °C. Detection was achieved at a wavelength of 210 nm, allowing for the quantification of the reaction products.