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Common HPLC Problems & Troubleshooting

⚠️ Common HPLC Problems & Troubleshooting

1. High Back Pressure 🚧

Possible causes

Blocked column

Dirty mobile phase

Blocked inline filter

Precipitated buffer salts



Troubleshooting

Flush column with strong solvent (e.g., high % organic)

Replace guard column or inline filter

Filter and degas mobile phase (0.45 µm or 0.22 µm)

Check tubing for blockage



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2. Low Pressure 📉

Possible causes

Leak in system

Pump seal worn out

Empty solvent reservoir

Air in pump


Troubleshooting

Check for leaks in fittings

Prime the pump properly

Replace pump seals

Ensure solvent bottle has enough mobile phase



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3. Baseline Noise 🔊

Possible causes

Air bubbles in detector

Dirty flow cell

Poor degassing

Contaminated mobile phase


Troubleshooting

Degas mobile phase (sonication or helium sparging)

Clean detector flow cell

Purge detector

Use fresh solvents



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4. Baseline Drift 📈

Possible causes

Temperature fluctuations

Mobile phase composition change

Column not equilibrated

Detector lamp aging


Troubleshooting

Allow system to equilibrate (20–30 min)

Maintain constant column temperature

Prepare fresh mobile phase

Check detector lamp intensity



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5. Peak Tailing 🧪➡️

Possible causes

Column contamination

Secondary interactions

Incorrect pH

Dead volume in fittings


Troubleshooting

Flush column with strong solvent

Adjust mobile phase pH

Replace column if old

Tighten fittings properly



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6. Peak Fronting ⬅️🧪

Possible causes

Column overload

Injection volume too high

Sample solvent stronger than mobile phase


Troubleshooting

Reduce injection volume

Dilute sample

Match sample solvent with mobile phase



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7. No Peaks Detected ❌

Possible causes

Wrong wavelength

No injection

Detector off

Sample degradation


Troubleshooting

Check detector wavelength

Confirm injector working

Check sample preparation

Verify standard solution



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8. Ghost Peaks 👻

Possible causes

Contaminated mobile phase

Carryover from previous injection

Dirty injector


Troubleshooting

Use fresh solvents

Wash injector thoroughly

Run blank injection

Clean autosampler needle



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9. Retention Time Shift ⏱️

Possible causes

Flow rate variation

Column temperature change

Mobile phase composition change


Troubleshooting

Calibrate pump flow rate

Maintain column oven temperature

Prepare mobile phase accurately



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10. Irregular Peak Shape 📉📈

Possible causes

Air bubbles

Column void

Damaged column packing


Troubleshooting

Degas mobile phase

Reverse flush column (if allowed)

Replace column



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🧰 Quick Daily Preventive Tips

Filter mobile phase 🧴

Degas solvents 💨

Flush system after buffer use 🔄

Use guard column 🛡️

Maintain logbook 📒

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