Ec 125 2 nucleodur 100 3 c8 ec column

High-performance liquid chromatography (HPLC) separation of the extracted MELs (50 μL) was performed with an 1100-HPLC system (Agilent) equipped with an EC 125/2 Nucleodur 100-3 C₈ ec column (Macherey-Nagel, Germany). The gradient, applied at a flow rate of 0.2 mL/min with a column temperature of 45°C, was as follows (buffer A is water with 0.05% formic acid, and buffer B is methanol with 0.045% formic acid): a linear gradient from 60% buffer B to 95% buffer B within 30 min and then holding at 95% buffer B for 10 min. MEL-A* variants were separated using a 125/2 Nucleodur C₈ ec column (Macherey-Nagel, Germany) at a flow rate of 0.2 mL/min with a column temperature of 60°C, applying an optimized gradient of solvent A (water) and solvent B (MeOH), starting with 30% solvent B, with a linear increase to 50% solvent B within 25 min, followed by a linear increase to 95% solvent B within 5 min and holding at 95% solvent B for an additional 5 min.