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HPLC Troubleshooting Guide

SCION Instruments have released their LC6000 Troubleshooting guide. This guide will provide end users with common troubleshooting techniques to identify and resolve any HPLC issues, ensuring minimum downtime and maximum productivity.

Introduction

The following guide will demonstrate troubleshooting to identify and solve your HPLC problems. At any one time, only change one component of the instrument configuration.

It is important to document all changes and results observed when troubleshooting and should be referred to if future issues occur. Always compare changed parameters with previous results to ensure best system performance is obtained. Working with a logical and methodical approach will help isolate and rectify possible causes.

Diagnostic and Problem Solving

Retention Time Drifts

Cause Solution
  •  Poor temperature control
  • Use thermostat column oven
  • Change temperature of column oven
  • Incorrect mobile phase composition
  • Prepare fresh mobile phase
  • Poor column equilibration
  • Increase column equilibration time
  • Flow rate change
  • Reset flow rate and test using liquid flow meter
  • Air bubble in system
  • Purge mobile phase

Baseline Drifts

Cause Solution 
  • Column temperature fluctuation
  • Use thermostat column oven
  • Use a thermometer to check set column temperature is accurate
  • 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
  • Replace flow cell
  • Damaged flow cell
  • Check flow cell is not damaged, replace if necessary
  • Pump outlet blocked
  • Replace outlet or remove blockage
  • Flow rate change
  • Reset flow rate and test using flow meter
  • Poor column equilibration
  • Increase column equilibration time
  • f recently changed mobile phase, purge the
    system of old solvent and pump new mobile
    through with 20 column volumes
  • Retained peaks look like baseline drift
  • Use a guard column
  • Thoroughly flush the column with strong
    solvent before next injection

Diagnostic and Problem Solving

Baseline Drifts

Cause  Solution
  • UV Detector not set to maximum absorbance
  • Use maximum absorbance wavelength of target compound(s)
  • Reference wavelength of Diode Array Detector is set to the same absorbance wavelength of target compound
  • Ensure reference wavelength of the detector is different to target compounds

Baseline Pulsing

Cause Solution
  • Proportioning valves are sticking
  • Clean thoroughly
  • Replace values
  • Debris in the detector flow cell
  • Look through the flow cell, if contaminated contact SCION engineer for maintenance

Extra Peaks

Cause  Solution
  • Contamination
  • Flush system with strong solvent
  • Use guard column
  • Change guard column
  • Filter sample
  • Carry Over
  • Increase run time
  • Increase gradient
  • Flush system with strong solvent
  • Ghost peaks
  • Prepare fresh mobile phase
  • Reduce injection volume

Peak Tailing

Cause  Solution
  • Flow path too long
  • Use narrower and shorter PEEK tubing
  • Prolonged retention of target compound(s)
  • Modify mobile phase composition
  • Use appropriate mobile phase buffer
  • Use a different stationary phase column
  • Reduce injection volume
  • Reverse phase flush column with strong solvent
  • Replace column

Diagnostic and Problem Solving

Peak Tailing

Cause  Solution
  • Blocked column
  • Reserve phase flush column with strong 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

Peak Fronting

Cause  Solution
  • Column temperature too low
  • Increase column temperature
  • Use thermostat column oven
  • Sample overload
  • Reduce injection volume
  • Dilute sample concentration
  • Wrong mobile phase composition
  • Prepare fresh mobile phase
  • Column stationary phase depleted
  • Replace the column
  • Use new column with different stationary phase