💡 HPLC Troubleshooting: Point-by-Point Guide
• Check injection volume – Was the sample injected correctly?• Verify sample preparation – Ensure correct solvent and concentration.• Inspect flow rate – Ensure mobile phase is flowing at correct rate.• Confirm detector settings – Wavelength, range, and mode should match the compound.
2. 📉 Peak Area Too Low
• Sample might be diluted.• Possible sample degradation before injection.• Incorrect injection volume (too small).
3. 📈 Peak Area Too High
• Sample over-concentrated.• Injection volume too high.• Integration error in software (baseline not properly set).
4. 📊 Baseline Noise
• Caused by air bubbles in the system.• Detector lamp aging (especially UV lamps).• Contaminated mobile phase or solvents.
5. 📉 Baseline Drift
• Temperature fluctuations during run.• Mismatch between sample and mobile phase.• Detector instability or warm-up issues.
6. 👻 Ghost Peaks (Unexpected Peaks)
• Mobile phase contamination (use fresh solvents).• Sample carryover from previous runs.• Dirty injector or loop (perform cleaning).
7. 🔀 Split Peaks
• Incomplete sample dissolution.• Column overload or partial blockage.• Inappropriate solvent compatibility.
8. 🌀 Broad Peaks
• Aged or damaged column.• Poor sample solubility.• Flow rate too slow.
9. 🔻 Tailing Peaks
• Problem with column integrity or packing.• Sample solvent mismatch with mobile phase.• Residual silanol activity (use end-capped columns or buffer).
10. 🔺 Fronting Peaks
• Column overloading.• Too high injection volume.• Highly concentrated sample.
11. ⏱ Retention Time Shift
• Mobile phase composition changed.• Flow rate adjustments during run.• Temperature variations.
12. 🔄 Variable Retention Time
• Mobile phase not mixed consistently.• Pump inconsistencies or mechanical issues.
13. 🚨 High Backpressure
• Blocked column, frit, or inline filter.• Viscous mobile phase or buffer precipitation.• Tubing clogged or narrowed.
14. ⚠️ Low Backpressure
• Leak in system (fittings, pump, or injector).• Column not properly installed or frit missing.
15. 💧 Leaks in System
• Check pump head, injector, and tubing connections.• Tighten any loose fittings.
16. 🌫 Air Bubbles in System
• Degas mobile phase (ultrasonic or vacuum).• Prime the pump before use.
17. 🔊 Noisy UV Detector
• Air in flow cell – degas mobile phase.• Dirty or old UV lamp – check lamp age/hours.• Unstable electrical power supply.
18. 🔁 Non-Reproducible Peaks
• Inconsistent injection volumes.• Errors in sample preparation or handling.• Syringe or injector malfunction.
19. 👯 Double Peaks
• Presence of isomers or closely related compounds.• Column not efficient – check particle size and type.
20. 📉📈 Fluctuating System Pressure
• Air bubbles in pump head.• Leaks in suction line or fittings.• Mobile phase degassing insufficient.
