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Preparative HPLC System — Standard Operating Procedure

Official SOP

This standard operating procedure governs preparative HPLC system operation. For product inquiries, visit the SENO Biotechnology Official Website.

1. Pre-Operation Safety

  • Wear solvent-resistant gloves (Viton or nitrile) and safety goggles
  • Verify adequate ventilation or connect to exhaust
  • Confirm waste container has ≥50% empty capacity
  • Inspect all tubing connections for leaks

2. Pre-Operation Checks

Item Check Acceptable
Mobile phase A H₂O + 0.1% TFA (filtered 0.22 µm) Prepared fresh, labeled
Mobile phase B ACN + 0.1% TFA (filtered 0.22 µm) Prepared fresh, labeled
System pressure test Pump at 10 mL/min, 50% B, monitor 5 min <5% pressure fluctuation
UV lamp Warm up ≥15 min before use Baseline drift <0.5 mAU/min
Column condition Visual inspection (no end-fitting leakage) Dry exterior
Waste container Volume check ≥50% empty
Fraction collector Tube/bottle positions confirmed Set collection mode

3. System Equilibration

  1. Purge pump heads with mobile phase (5 mL/min each head, 2 min)
  2. Set flow rate to 10 mL/min at initial %B (typically 5% B)
  3. Allow column to equilibrate for 3 – 5 column volumes
  4. Monitor UV baseline at 220 nm (peptide bond) and 280 nm (aromatic)
  5. Accept equilibration when baseline drift <0.5 mAU/min over 2 min
  6. Record equilibration parameters in batch record

4. Sample Preparation

  1. Weigh crude peptide (record mass)
  2. Dissolve in mobile phase A or ACN/H₂O mixture (10 – 50 mg/mL typical)
  3. Filter through 0.45 µm PTFE syringe filter into clean vial
  4. Record sample concentration and volume
  5. Load sample into injection loop using Luer-lock syringe

5. Gradient Purification Method

Step Time (min) Flow (mL/min) %B Curve Purpose
Equilibration 0 – 5 50 5 — Ready column
Injection 0 (event) 50 5 — Inject sample
Gradient 1 0 – 5 50 5 → 20 Linear Wash non-retained
Gradient 2 5 – 45 50 20 → 55 Linear Elute target peptide
Gradient 3 45 – 50 50 55 → 95 Linear Column wash
Hold 50 – 55 50 95 Isocratic Elute strongly retained
Re-equilibration 55 – 65 50 95 → 5 Linear Return to start

6. Fraction Collection Criteria

  1. Set collection threshold: ≥50 mAU at 220 nm (adjust for concentrated samples)
  2. Collect peak apex ±0.5 min for primary fraction
  3. Collect leading and trailing shoulders separately (may contain impurities)
  4. Label each fraction with:
  5. Batch number
  6. Fraction number
  7. Collection time window
  8. UV absorbance at apex

7. Post-Run Fraction Analysis

  1. Transfer 5 – 20 µL from each fraction to auto-sampler vial
  2. Analyze by analytical HPLC (see Analytical HPLC SOP)
  3. Pool fractions meeting purity threshold (≥95% for standard grade)
  4. Record pooled fraction volume, concentration (by UV), and total peptide mass

8. Column Cleaning and Storage

Step Solvent Volume Purpose
Post-run wash 100% ACN 3 CV (50 mL/min) Remove hydrophobic impurities
Intermediate 70% ACN / 30% H₂O 3 CV Transition
Storage 80% ACN / 20% H₂O — Prevent microbial growth