| Column | Zorbax SB-Aq column, 4.6 × 250 mm(pore size 80 Å, particle size 5 μm) |
| Solvents | A: water, 0.1% (vol/vol) TFA; B: acetonitrile |
| Flow rate | 1 mL/min |
| Gradient | 20–70% (vol/vol) B over 25 min; 70%-98% over 5 min; 98% over 5min; |
| Injection volume | 30–500 μL |
| Wavelength (nm) | 280 (for Fmoc-, Trp- or Tyr-containing peptides), or 494 (FITC-labeled peptides) or 220 (for others) |
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