HPLC Troubleshooting Guide

Dive into our comprehensive liquid chromatography troubleshooting guide and resolve any issues with your analysis today.

Retention time drift 

• Poor temperature control – Use thermostat column oven. Change column oven temperature. 
• Incorrect mobile phase composition – Prepare fresh mobile phase. Check mixer is working for gradient methods. 
• Poor column equilibration – Increase column equilibration time. Condition the column. 
• Change in flow rate – Reset flow rate. Test using a liquid flow meter. 
• Air bubbles in the system – Degas the mobile phase. Purge the system. 

Baseline noise

•  Leak – Check for loose fittings. Tighten gently. Check pump seals. Replace if worn out. 
• Incorrect mobile phase – Check for the use of only miscible mobile phases and correct preparation. 
• Air bubbles in system – Flush system with a strong organic solvent. Purge the system. Degas mobile phase. 
• Detector cell contaminated – Clean cell flow. 
• Detector lamp low energy – Replace lamp. 
• Stationary phase of column exposed – Replace column. 

 Baseline drifting 

• Column temperature fluctuation – Use a thermostat column oven. Use a thermometer to check the accuracy of the set temperature. 
• Incorrect mobile phase composition – Prepare fresh mobile phase. Check mixer is working for gradient methods. 
• Contamination of detector flow cell – Flush flow cell with a strong organic solvent. If no improvement is seen, change the flow cell. 
• Damaged flow cell – Replace flow cell. 
• Pump outlet blocked – Remove blockage. If no improvement is seen, replace outlet. 
• Flow rate change – Reset flow rate. Test using liquid flow meter. 
• Poor column equilibration – Increase column equilibration time. If recently changed mobile phase, purge the system and pump of old solvent with new mobile phase using 20 column volumes. 
• Retained peaks look like baseline drifts –Use a guard column. Thoroughly flush the column with a strong organic solvent before the next injection.  
• UV detector not set at maximum absorbance – Use maximum absorbance wavelength of target compound(s) 
• Reference wavelength of detector is incorrect – Ensure reference wavelength of the detector is different to target compounds. 
• UV absorbing mobile phase – Use non-UV absorbing HPLC grade solvent. 

 Baseline pulsing 

• Proportioning valves are sticking – Clean thoroughly. If necessary, replace valves (I am not quite sure what is meant by proportioning valves are sticking) 
• Debris in the flow cell – Check flow cell and flush. If contaminated, contact SCION engineer for maintenance.

Broad peaks 

• Mobile phase composition changed – Make new mobile phase. Add buffer to mobile phase. 
• Leaks between column and detector – Check for loose fittings. 
• Flow rate too low – Increase flow rate. 
• Detector not set correctly – Check detector settings and adjust. 
• Column overloading – Decrease injection volume.  
• Tubing between column and detector is too long and/or incorrect internal diameter – Reduce flow path. Use narrower internal diameter tubing. 
• Guard column/ column contaminated – Replace guard column/ column. 
• No resolution of two peaks – Change column to improve separation. 
• Column temperature too low – Increase column temperature.

Peak tailing 

• Flow path too long – Use narrower and shorter PEEK tubing. 
• Prolonged analyte retention – Modify mobile phase composition. Use appropriate mobile phase buffer. Use a different stationary phase column. 
• Blocked column – Reverse phase flush column with a strong organic solvent. Replace column. 
• Interfering peak – Change mobile phase composition. Increase gradient program. Use longer column. 
• Wrong mobile phase pH – Adjust mobile phase pH. Prepare new mobile phase with correct pH. 
• Active sites on column – Change column. 

Extra peaks

•  Contamination – Flush system with a strong organic solvent. Use/replace guard column, filter sample and replace filters in solvent bottle. 
• Carry over – Flush system with a strong organic solvent. Increase run time or gradient. 
• Ghost peaks – Prepare fresh mobile phase. Reduce injection volume. 

Peak fronting 

• Column temperature too low – Increase temperature. Use thermostat column over. 
• Sample overload – Reduce injection volume. Dilute sample  
• Wrong mobile phase composition – Prepare fresh mobile phase. 
• Solvent incompatibility – Prepare/dilute sample in the mobile phase. 
• Column stationary phase depleted – Replace column. Use new column with different stationary phase. 

Peak Distortion

•  Injection – Reduce injection volume. Dilute sample. Use weaker injection solvent. 
• Ghost peaks – Decrease injection volume. Prepare fresh mobile phase. 
• Carry over – Flush system with a strong organic solvent. Increase run time and gradient. 

Split peaks 

• Contamination – Flush system with a strong organic solvent. Use/replace guard column, filter sample and replace filters in solvent bottle. 
• Wrong mobile phase composition – Prepare fresh mobile phase. Change mobile phase to suit target compound(s). 

Loss of sensitivity 

• Injection volume too low – Check injection volume is correct. 
• Needle blocked – Flush needle. Replace needle. 
• Detector time constant too large – Decrease time constant. 
• Contaminated guard column/ column – Replace guard column/ column. 
• Incorrect mobile phase composition – Prepare new mobile phase. 
• Air bubbles in system – Degas the mobile phase. Purge the system. 

Low resolution

• Contaminated mobile phase – Prepare new mobile phase. 
• Contaminated guard column/ column – Replace guard column/ column. 

Pressure fluctuations 

• Air in system – Degas all solvents. Purge pump. 
• Check valve fault – Replace check valves.  
• Leak – Identify leak. Tighten/replace fittings. 
• Pump seal failure – Replace seal. 
• Blocked flow cell – Clean flow cell. Replace flow cell. 
• Blocked column – Reverse flush column, if possible. Replace guard column and/or column. 
• Blocked injector – Flush needle and tubing. Replace the needle and/or tubing. 
• Blocked pump – Flush with strong solvent. Change in-line filter. 

No pressure 

• Power supply off – Turn on power supply. Check/replace fuse. 
• Piston damage – Replace piston. 
• Air bubbles in the system – Purge system. Prime system with mobile phase. 
• No mobile phase – Prepare new mobile phase. 
• Check valves fault – Check valves. Replace valves. 
• Leak – Identify source of the leak. Tighten fittings. Replace fittings. 

High pressure 

• Flow rate too high – Lower flow rate. 
• Column blockage – Backflush column. Replace column. 
• Injector blockage – Flush injector with a strong organic solvent. Replace injector. 
• Column temperature too low – Increase column temperature. 
• Mobile phase precipitation – Flush the system with a strong organic solvent. Prepare fresh mobile phase. 
• In-liner filter blockage – Replace filter. 

Low pressure 

• Flow rate too low – Increase flow rate. 
• Leak – Identify leak. Tighten fittings. Replace fittings. 
• Column temperature too high – Decrease column temperature. 

General leaks 

• Fittings are loose, overtightened or damaged – Check all fittings. Loosen or tighten. Replace damaged fittings. 
• Incompatible parts – Ensure all tubing and fittings are compatible. Replace with appropriate fittings. 

Leaks at pump and autosampler 

• Seal failure – Check all seals. Replace with new. 
• Sample loop blocked – Flush with a strong organic solvent. Replace sample loop. 
• Waste tubing blocked – Flush with a strong organic solvent. Replace waste tubing. 
• Syringe failure – Tighten syringe gently. Replace syringe. 

Leaks at column 

• End-fittings are loose – Tighten gently. 
• Wrong sized frit thickness. Use correct frit. 
• Tubing installed incorrectly – Replace all tubing with correct size. 

Leaks at detector 

• Flow cell damaged – Replace flow cell. 
• Flow cell blocked – Flush flow cell with solvent. 
• Loose fittings – Tighten all fittings gently. 

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