A gradient system was developed at a flow rate of 1.0 mL/minute for 60 minutes: 0–5 minutes, isocratic gradient 100% (B); 5–35 minutes, linear gradient 100–60% (B); 35–45 minutes, linear gradient 60–20% (B); 45–50 minutes, isocratic gradient 20% (B); 50–55 minutes, linear gradient 20–100% (B); and 55–60 minutes, isocratic gradient 100% (B).