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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

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

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

Peak Distortion

Cause Solution
  • Injection
  • Reduce injection volume
  • Use a weaker injection solvent
  • Dilute sample (too concentrated, sample overload)
  • Carry Over
  • Increase run time
  • Increase gradient
  • Flush system with strong solvent
  • Ghost peaks
  • Prepare fresh mobile phase
  • Reduce injection volume

Split Peaks

Cause Solution
  • Contamination
  • Flush system with strong solvent
  • Use/replace guard column
  • Wrong mobile phase composition
  • Prepare fresh mobile phase
  • Change mobile phase to suit target compound(s)

General Leaks

Cause Solution
  • Fittings are loose, overtightened or damaged
  • Check all fittings
  • Loosen or tighten
  • Replaced damage fittings
  • Incompatible parts
  • Ensure all tubing and fittings are compatible
  • Replace with appropriate fittings

Leaks at Pump

Cause Solution
  • Seal failure
  • Check all seals and replace with new
  • Sample loop blocked
  • Flush with strong solvent
  • Replace sample loop
  • Waste tubing blocked
  • Flush with strong solvent
  • Replace waste tubing
  • Syringe failure
  • Tighten syringe gently
  • Replace syringe

Leaks at Autosampler

Cause  Solution
  • Seal failure
  • Check all seals and replace with new
  • Sample loop blocked
  • Flush with strong solvent
  • Replace sample loop
  • Waste tubing blocked
  • Flush with strong solvent
  • Replace waste tubing
  • Syringe failure
  • Tighten syringe gently
  • Replace syringe

Leaks at Column

Cause Solution
  • End-fittings are loose
  • Tighten gently
  • Wrong sized frit thickness
  • Use correct frit
  • Tubing installed correctly
  • Tighten tubing
  • Replace all tubing with correct size

Leaks at Detector

Cause Solution
  • Flow cell damaged
  • Replace flow cell
  • Flow cell blocked
  • Flush flow cell with solvent
  • Loose fittings
  • Tighten all fittings gently

No Pressure

Cause Solution
  • Power supply OFF
  • Turn on power supply
  • Check fuse/replace fuse
  • Piston damaged
  • Replace piston
  • Air in system
  • Purge system
  • Prime pump with mobile phase
  • No mobile phase
  • Make up new mobile phase
  • Check valves not working
  • Replace check valves
  • Leak
  • Find source of the leak and tighten or replace fittings

High Pressure

Cause Solution
  • Flow rate is set too high
  • Lower flow rate
  • Column is blocked
  • Backflush column
  • Replace column
  • Injector is blocked
  • Flush injector with strong solvent
  • Replace injector
  • Column temperature is too low
  • Increase column temperature
  • Mobile phase precipitation
  • Prepare fresh mobile phase
  • In-line filter is blocked
  • Replace filter

Low Pressure

Cause Solution
  • Flow rate is set too low
  • Increase flow rate
  • Leak
  • Identify leak and tighten/replace all fittings
  • Column temperature too high
  • Decrease column temperature
  • Proportioning valves are sticking

Varying Pressure

Cause  Solution
  • Air in system
  • Degas all solvents
  • Purge pump
  • Check valves are faulty
  • Replace check valves
  • Pump seal failure
  • Replace seal
  • Leak
  • Identify leak and tighten/replace fittings
  • Mobile phase changes
  • Fully purge system with new mobile phase

Baseline Noise

Cause Solution
  • Leak
  • Check for loose fittings, tighten gently
  • Check pump seals, replace if worn
  • Incorrect mobile phase
  • Check that only miscible mobile phases were used
  • Check that the mobile phase was made correctly
  • Air in system
  • Purge the system
  • Flush with strong solvent
  • Degas mobile phase
  • Detector cell contaminated
  • Clean flow cell
  • Detector lamp low energy
  • Replace lamp
  • Stationary phase of column exposed
  • Replace column

Broad Peaks

Cause  Solution
  • Mobile phase composition changed
  • Make new mobile phase
  • Add buffer to mobile phase
  • Leaks – particularly 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
  • Inject smaller volume
  • Tubing between column and detector too long and/or incorrect ID
  • Reduce flow path between column and detector
  • Use a narrower ID tubing
  • Guard column/column contaminated
  • Replace guard column/column
  • No resolution of two peaks
  • Change column to improve seperation
  • Column temperature too low
  • Increase column temperature

Loss of Resolution

Cause Solution
  • Contaminated mobile phase
  • Prepare new mobile phase
  • Contaminated guard column/column
  • Replace guard column/column

Loss of Sensitivity

Cause Solution
  • 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 in system
  • Purge system
  • Degas mobile phase

For more information contact Ashleigh Mellor, LC Product Manager.

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