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 |
|
- Use thermostat column oven
- Change temperature of column oven
|
- Incorrect mobile phase composition
|
- Prepare fresh mobile phase
|
- Poor column equilibration
|
- Increase column equilibration time
|
|
- Reset flow rate and test using liquid flow meter
|
|
|
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
|
|
- Check flow cell is not damaged, replace if necessary
|
|
- Replace outlet or remove blockage
|
|
- 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 |
|
- Flush system with strong solvent
- Use guard column
- Change guard column
- Filter sample
|
|
- Increase run time
- Increase gradient
- Flush system with strong solvent
|
|
- Prepare fresh mobile phase
- Reduce injection volume
|
Peak Tailing
Cause |
Solution |
|
- 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
|
|
- Reverse phase flush column with strong solvent
- Replace column
|
Peak Tailing
Cause |
Solution |
|
- Reserve phase flush column with strong solvent
- Replace column
|
|
- Change mobile phase composition
- Increase gradient program
- Use longer column
|
|
- Adjust mobile phase pH
- Prepare new mobile phase with correct pH
|
|
|
Peak Fronting
Cause |
Solution |
- Column temperature too low
|
- Increase column temperature
- Use thermostat column oven
|
|
- 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 |
|
- Reduce injection volume
- Use a weaker injection solvent
- Dilute sample (too concentrated, sample overload)
|
|
- Increase run time
- Increase gradient
- Flush system with strong solvent
|
|
- Prepare fresh mobile phase
- Reduce injection volume
|
Split Peaks
Cause |
Solution |
|
- 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
|
|
- Ensure all tubing and fittings are compatible
- Replace with appropriate fittings
|
Leaks at Pump
Cause |
Solution |
|
- Check all seals and replace with new
|
|
- Flush with strong solvent
- Replace sample loop
|
|
- Flush with strong solvent
- Replace waste tubing
|
|
- Tighten syringe gently
- Replace syringe
|
Leaks at Autosampler
Cause |
Solution |
|
- Check all seals and replace with new
|
|
- Flush with strong solvent
- Replace sample loop
|
|
- Flush with strong solvent
- Replace waste tubing
|
|
- Tighten syringe gently
- Replace syringe
|
Leaks at Column
Cause |
Solution |
|
|
- Wrong sized frit thickness
|
|
- Tubing installed correctly
|
- Tighten tubing
- Replace all tubing with correct size
|
Leaks at Detector
Cause |
Solution |
|
|
|
- Flush flow cell with solvent
|
|
- Tighten all fittings gently
|
No Pressure
Cause |
Solution |
|
- Turn on power supply
- Check fuse/replace fuse
|
|
|
|
- Purge system
- Prime pump with mobile phase
|
|
|
|
|
|
- Find source of the leak and tighten or replace fittings
|
High Pressure
Cause |
Solution |
- Flow rate is set too high
|
|
|
- Backflush column
- Replace column
|
|
- 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
|
|
Low Pressure
Cause |
Solution |
|
|
|
- Identify leak and tighten/replace all fittings
|
- Column temperature too high
|
- Decrease column temperature
|
- Proportioning valves are sticking
|
|
Varying Pressure
Cause |
Solution |
|
- Degas all solvents
- Purge pump
|
|
|
|
|
|
- Identify leak and tighten/replace fittings
|
|
- Fully purge system with new mobile phase
|
Baseline Noise
Cause |
Solution |
|
- Check for loose fittings, tighten gently
- Check pump seals, replace if worn
|
|
- Check that only miscible mobile phases were used
- Check that the mobile phase was made correctly
|
|
- Purge the system
- Flush with strong solvent
- Degas mobile phase
|
- Detector cell contaminated
|
|
|
|
- Stationary phase of column exposed
|
|
Broad Peaks
Cause |
Solution |
- Mobile phase composition changed
|
- Make new mobile phase
- Add buffer to mobile phase
|
- Leaks – particularly between column and detector
|
|
|
|
- Detector not set correctly
|
- Check detector settings and adjust
|
|
|
- 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
|
|
- Contaminated guard column/column
|
- Replace guard column/column
|
Loss of Sensitivity
Cause |
Solution |
|
- Check injection volume is correct
|
|
- Flush needle
- Replace needle
|
- Detector time constant too large
|
|
- Contaminated guard column/column
|
- Replace guard column/column
|
- Incorrect mobile phase composition
|
|
|
- Purge system
- Degas mobile phase
|
For more information contact Ashleigh Mellor, LC Product Manager.
Download Guide
Download the complete guide: HPLC Troubleshooting Guide
Keep in Touch
If you wish to keep up to date with SCION Instruments latest research and articles, why not join us on social media and sign up to our newsletters today?