Analytical HPLC System — Standard Operating Procedure

1. Pre-Operation Checks

• [ ] Verify mobile phase composition and filter (0.22 µm)
• [ ] Check solvent reservoir levels
• [ ] Confirm column is within use log limits
• [ ] Perform leak check at operating pressure
• [ ] Verify autosampler vial positions
• [ ] Turn on UV lamp 30 min before analysis

2. System Suitability Test

Parameter	Acceptance Criteria
Theoretical Plates (N)	≥5000 per column
Tailing Factor	0.8 – 1.5
Retention Time RSD (n=5)	≤1.0%
Area RSD (n=5)	≤2.0%
Resolution	≥2.0 between adjacent peaks

3. Sample Analysis Sequence

1. Blank injection (mobile phase)
2. Reference standard (n=2)
3. System suitability check
4. Sample injections (n=2 per sample)
5. Reference standard (end of sequence)
6. Column wash run

4. Data Processing

1. Integrate chromatogram with appropriate parameters
2. Identify target peptide peak by retention time
3. Calculate purity as area% at 220 nm and 280 nm
4. Report peptide content against reference standard
5. Document in batch record

5. Shutdown

1. Flush column with 70% ACN/H₂O (20 min, 1 mL/min)
2. Store column in recommended storage solvent
3. Purge pump with H₂O (10 min)
4. Power down system