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Gradient of mobile phases (step 25), column temperature 25°C
| Time (min) | Mobile phase A (%) |
Mobile phase B (%) |
Flow rate (mL/min) | |
|---|---|---|---|---|
| 1 | 0 | 99.8% | 0.2% | 0.3 |
| 2 | 3 | Linear increase 99.8%–0% | 0.2%–100% | 0.3 |
| 3 | 3.5 | 0% | 100% | 0.3 |
| 4 | 4 | 0%–99.8% | 100%–0.2% | 0.3 |
| 5 | 4.5–7 | 99.8% | 0.2% | 0.3 |
