To resolve the deficiencies, the following modifications to the HPLC program are recommended:
Create clear SOPs covering:
This is the heart of your method. You define: hplc program
The HPLC program must match the physical hardware. You cannot program a 0.5 mL/min flow rate for a standard 4.6 mm column if your pump only handles 1-10 mL/min. Key decisions:
Instrument: [e.g., Agilent 1260 Infinity II, Waters Alliance e2695] Column: [e.g., C18, 150 x 4.6 mm, 5 µm] Detection: [e.g., UV-Vis at 254 nm / PDA / Fluorescence] To resolve the deficiencies, the following modifications to
This is the first decision you make when programming a method.
Gradient Elution:
Start with a generic gradient: 5–95% organic over 20 minutes at 1.0 mL/min. This "scouting run" reveals the retention window.
Create a sequence table that automates: