Enzymatic Reaction Analysis Using High-Performance Liquid Chromatography

The reaction was conducted at a moderate temperature of 30°C for a duration of 20 minutes, after which the enzyme was rendered inactive by boiling. Subsequently, the samples were subjected to monolayer analysis using a state-of-the-art Waters high-performance liquid chromatography (HPLC) system, equipped with a Chiralcel IG-3 column (4.6 mm × 250 mm, 3µm, Diacel, Tokyo, Japan). The eluent utilized was a well-balanced mixture of formic acid and methanol (0.1%, 70:30, v/v) at a flow rate of 0.3 mL/min and a temperature of 35°C. The detection wavelength employed was 210 nm.