High-Performance Liquid Chromatography (HPLC) Troubleshooting

1. Pressure Fluctuations

• Cause: Often caused by air trapped in the system, partially blocked filters, or poorly mixed solvents.
• Solution: Degas solvents, check for blockages, and ensure proper mixing.

2. Baseline Noise

• Cause: Could be due to a dirty detector, improper mobile phase preparation, or electrical interference.
• Solution: Clean the detector cell, use high-quality solvents, and eliminate any electrical noise sources.

3. Peak Tailing

• Cause: Poor column performance, loose fittings, interaction of analytes with active sites on the stationary phase, or incorrect pH of the mobile phase.
• Solution: Regenerate or replace the column, or adjust the pH of the mobile phase to improve peak shape.

4. Retention Time Drift

• Cause: Fluctuations in temperature, inconsistencies in mobile phase composition, or column degradation.
• Solution: Maintain stable temperature and mobile phase composition; replace the column if necessary.

5. Ghost Peaks

• Cause: Contamination of the system, improper gradient conditions, or issues with sample preparation.
• Solution: Perform thorough system cleaning, optimize gradient methods, and ensure proper sample preparation.

6. Low Sensitivity

• Cause: Detector issues, dirty or degraded column, or incorrect mobile phase pH.
• Solution: Check detector settings, replace the column, and optimize the mobile phase.

7. Clogged Columns

• Cause: Sample particulate matter or precipitated mobile phase. 
• Solution: Filter all samples and solvents, and backflush the column if compatible.

 

8. Split Peaks

• Cause: Can be due to a partially blocked injector, poor sample solubility, or column overload.
• Solution: Clean the injector, dissolve samples in the mobile phase, and ensure proper sample concentration.

9. Broad Peaks

• Cause: Column aging, improper mobile phase composition, or sample overloading.
• Solution: Replace the column, optimize the mobile phase, and reduce the sample volume or concentration.

10. Leaky System

• Cause: Loose fittings, worn seals, or damaged tubing.
• Solution: Tighten fittings, replace seals, and inspect tubing for damage.

Read also: 10 Golden Tips for HPLC Column Regeneration

Reference: 

1. Snyder, L. R., Kirkland, J. J., & Glajch, J. L. (1997). Practical HPLC Method Development. John Wiley & Sons.

2. McMaster, M. C. (2007). HPLC: A Practical User’s Guide. John Wiley & Sons.

3. Kazakevich, Y., & LoBrutto, R. (2007). HPLC for Pharmaceutical Scientists. John Wiley & Sons.

4. Swartz, M. E., & Krull, I. S. (2012). Analytical Method Development and Validation. CRC Press.

5. Meyer, V. R. (2010). Practical High-Performance Liquid Chromatography. John Wiley & Sons.

Resource Person: Bassem Elsayed (MSc)