LC Troubleshooting Guide

Your guide to solving common problems and staying productive

Places to Start Retention Time Drift Possible Cause

Inconsistent online mobile
Solution
Ensure gradient system delivers
Noisy Baseline Possible Cause
Contamination
Solution
Use degassed HPLC-grade solvents;
phase mixing constant composition; compare with flush system; clean up sample
Solvents Pump shutdown manual preparation of mobile phase
–– Use brown borosilicate bottles to avoid algae growth –– Flush all channels to remove salt deposits and particulate matter Detector problems Check number of hours of UV lamp;
replace UV lamp or flow cell
–– Prepare solvent volume to be used up within 1 to 2 days –– Flush the system with appropriate storage solvent and power Variation in column temperature Thermostat or insulate column;
–– Use only HPLC-grade solvents filtered through 0.2 μm filters down the system ensure constant lab temperature

Preparing and powering up the pump Handling of acetonitrile Ghost Peaks Possible Cause Solution
Insufficient equilibration time Make sure at least 10 column
–– Inspect solvent bottles and inlet filters for damage or coloring –– If possible, use 5 to 10% of water in your mobile phase with gradient run or change in volumes pass through column after Peaks from previous injection Flush column to remove
isocratic mobile phase sample run contaminants; check with
–– Always use seal wash when installed and purge the pump –– Be sure to avoid ACN evaporation blank injection
–– Use the appropriate system conditioning method –– Don’t leave ACN on the system for more than 2 to 3 days
Selective evaporation of Less vigorous helium sparging; keep Contamination; unknown Proper sample cleanup
Daily tasks –– Perform a periodic warm water wash (60 to 70 °C) if you interferences in samples
mobile phase component solvent reservoirs covered; prepare
–– Replace aqueous and organic mobile phases every second day face problems fresh mobile phase Ion pair: disequilibrium Prepare sample in actual mobile
phase to minimize disturbance
–– Check seal wash solvent
Contamination buildup Occasionally flush column with
–– Flush the system with the composition of your application strong solvent Contaminated mobile phase Check your mobile phase

Weekly tasks Bubbles in solvent Check and degas your solvents

Column overloaded
–– Change seal wash solvent and bottle and inspect solvent filters with sample
–– Check system backpressure and change
filters if necessary
Pressure Fluctuation Possible Cause
Leak in the system
Buildup of particulates
Bubble in pump
Decrease injection volume
or concentration
Peak Tailing Possible Cause Solution
Solution Unswept dead volumes Minimize number of connections;
Identify the channel and clean ensure injector seal is tight; ensure
or replace check valve; replace fittings are properly seated
pump seals
Column performance Change mobile phase;
Filter sample and mobile phase replace column
Silica-based: Use specialty, polymeric, or sterically
Perform solvent degassing; sparge column degradation protected column
solvent with helium
Silica-based: basic interactions Use stronger mobile phase or add
with stationary phase appropriate base (e.g., TEA)

Pressure Increase Possible Cause Solution

System blockage
Water/organic systems:
buffer precipitation
Check flowpath (needle seat,
capillaries, filter and frits) Peak Broadening Possible Cause Solution
Injection volume too large Decrease injection volume or solvent
Test buffer-organic mixtures strength of injection solvent; use
to ensure compatibility gradient methods

Low sampling rate of Increase data rate

data system
High Column Possible Cause Solution
Detector cell volume too large Use smallest possible cell volume
Column blockage Better sample cleanup;
Backpressure
use guard column
Injection volume too large Decrease injection volume
Mobile phase viscosity Use lower viscosity solvents or
too high higher temperature

Particle size too small Use larger dp packing Sensitivity Problems Possible Cause Solution
Peaks are outside of Dilute/concentrate sample to bring
Plugged inlet frit Replace column sensitivity range of detector into linear region

Sample-related losses Use internal standard during sample

during preparation preparation; optimize sample
Drifting Baseline Possible Cause Solution
preparation method
Positive/negative direction: Flush column; clean up sample;

Maintenance
contaminant buildup/elution use pure solvents

Positive/negative: Use mobile phase for sample solvent Leaks Possible Cause Solution
Agilent Lab Advisor software helps you manage your Agilent LC –– Diagnostic tests to evaluate performance difference in refractive index White powder at fitting/ Tighten fittings; replace capillaries
of injection solvent loose fitting
instruments to achieve high-quality chromatographic results in –– Easier maintenance of all Agilent LC modules
System leak Identify location checking leak
the most efficient way by ensuring high instrument performance, –– Comprehensive reports generated to ease communication Temperature changes Insulate and thermostat column sensors/errors; check flow cell
productivity, and reliability. It is available free-of-charge. with Agilent service and tubing

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This information is subject to change without notice. © Agilent Technologies, Inc. 2019
Printed in the USA, March 1, 2019
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