Waters acquity uplc instrument system

The pH dependence of saporin L3 Ala14Cys (pH 4–8.2) was studied by adenine production from A10 RNA and analyzed by ultraperformance liquid chromatography (UPLC). Citric acid buffer (final concentration of 10 mM) was used in the pH range of 4.0–6.0, and sodium phosphate buffer (final concentration of 10 mM) was used in the pH range of 6.5–8.2. A Waters Acquity UPLC instrument system (Waters Co.) equipped with a photo diode array detector (190–400 nm) was used for analysis, and the system was controlled with MassLync version 4.1. Separations were performed using a Waters Acquity UPLC HSS T3 C₁₈ column (2.1 mm × 100 mm, 1.7 μm) with a flow rate of 0.6 mL/min, and a gradient elution of H₂O (A) and acetonitrile (B) with 98% A maintained from 0 to 0.8 min, followed by the level of A being decreased to 40% from 0.8 to 2.2 min, the level of A being increased to 98% from 2.2 to 2.4 min, and this concentration being maintained from 2.4 to 3.0 min. The injection volume of the sample was 10 μL. Adenine release was calculated by interpolation of the observed adenine peak area with the corresponding standard adenine calibration curve. Adenine release was kept below 20% of the substrate RNA.