Standard Operating Procedures and Safety Guidelines for High Performance Liquid Chromatography (HPLC) Systems

HPLC systems are precision analytical instruments widely used in pharmaceuticals, chemicals, and food testing. To ensure data accuracy and instrument longevity, operators must strictly adhere to the following protocols:

I. Pre-Operation Preparations

1. Mobile Phase Preparation

   • Use HPLC-grade reagents
   • Aqueous phase: Prepare fresh and filter (0.45 μm membrane)
   • Organic phase: Degas via sonication (15-20 min)

2. Column Inspection

   • Verify column compatibility with analytical method
   • Check pressure against manufacturer specifications

3. System Purge

   • Condition new columns per manufacturer guidelines
   • Gradual solvent transition when changing buffers

II. Operational Protocols

1. Startup Sequence
   Power → Degasser → Pump → Column Oven → Detector

2. Parameter Setup

   • Ramp flow rate incrementally to target
   • Maintain column temperature within ±2°C

3. Sample Injection

   • Filter all samples (0.22 μm)
   • Injection volume ≤80% of loop capacity

III. Maintenance Procedures

1. Daily Maintenance

   • Flush system with compatible solvent (water first for salt buffers)
   • Document baseline noise/drift

2. Long-Term Storage

   • Store columns in prescribed solvents
   • Preserve system with 10% methanol/water

3. Troubleshooting

   • Investigate if pressure fluctuates >±10%
   • Ghost peaks: Check seals and solvent purity

IV. Safety Precautions

• Never disconnect high-pressure fittings under pressure
• Segregate waste streams (organic vs. aqueous)
• Allow UV lamp to cool 30 min before shutdown

Compliance & Validation

• Aligns with Chinese Pharmacopoeia 2020 guidelines
• Quarterly system suitability tests recommended
• Annual professional calibration required

Key Performance Metrics

Note: Always consult instrument-specific manuals for parameter adjustments.

Best Practices
✓ Use only HPLC-grade solvents
✓ Maintain detailed logbooks (column usage, pressure trends)
✓ Implement preventive maintenance schedules

Adherence to these protocols can extend HPLC service life by 3-5 years while ensuring regulatory compliance (USP <621>, ICH Q2).

Pro Tip: For methods using ion-pair reagents (e.g., TFA), include weekly 0.1% phosphoric acid flushes to prevent buildup.