HPLC Troubleshooting: Point-by-Point Guide
No Peak in Chromatogram• 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.
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