30/12/2025
6 non-negligible critical points in hplc operation
HPLC Operation Pitfalls to Avoid! 🧪
HPLC users, listen up! Small operational mistakes can ruin your instrument, mess up data, or even cause costly damage. Here are 6 KEY pitfalls to skip & quick fixes to keep your lab work smooth:
🔹 1. Unfiltered Mobile Phase
Pitfall: Dust/impurities scratch core parts & clog pipelines, causing pressure issues.
Fix: Use 0.2μm (normal-phase) / 0.45μm (reverse-phase) syringe filters; add a sand filter at the pump inlet & clean/replace built-in filters regularly.
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🔹 2. Skipping Post-Use Pump Cleaning
Pitfall: Salt crystals form from leftover buffer, damaging seals/plungers.
Fix: 2-step clean - flush with ultrapure water first, then switch to storage solvent (methanol for reversed-phase; n-hexane-isopropanol for normal-phase). Never leave corrosive/buffer solutions in the pump long-term.
🔹 3. Mobile Phase Exhaustion
Pitfall: Dry pumping wears parts & causes air bubbles/leaks.
Fix: Check mobile phase level before use; mark min level. If no flow/pressure, purge bubbles (5mL/min flow) or replace worn seals.
🔹 4. Unstable Pressure/Flow
Pitfall: Bubbles or clogged check valves/sand filters.
Fix: Degas mobile phase (15-20min超声/online); purge flow path. Clean check valves with acetone (ultrasonic); unclog sand filters with mobile phase/nitric acid.
🔹 5. Abnormal Pressure (Too High/Low)
Pitfall: High = clogging; Low = leakage.
Fix: Check pipelines, valves, columns & detector flow cell section by section. Flush clogs with suitable solvent; tighten/replace seals at leaks.
🔹 6. Improper Gradient Elution
Pitfall: Solvent stratification, salt crystals, baseline drift.
Fix: Use miscible solvents; test buffer-organic compatibility. Do blank gradient elution first; degas solvents thoroughly.
💡 Pro Tip: HPLC success = detail control! Follow SOPs for mobile phase prep, instrument cleaning & gradient verification to ensure accurate, reproducible data.
