Agilent | LC and LC/MS Troubleshooting

HPLC Troubleshooting

Symptom Type	Possible Cause	Solution
Baseline disturbance at void time	Positive/negative – Difference in refractive index of injection solvent	Use mobile phase for sample solvent
Detector leaks	Plugged inlet frit	Replace seals/gaskets
Drifting baseline	Positive direction – Contaminant buildup/elution	Flush column, cleanup sample, use pure solvents
	Positive/negative – Difference in refractive index of injection solvent	Use mobile phase for sample solvent
	Negative direction (gradient) – Absorbance of “A” mobile phase solvent	Use non-absorbing or HPLC-grade or better solvent
	Negative direction (gradient) – Absorbance of “B” mobile phase solvent	Use non-absorbing or HPLC-grade or better solvent
	Random – Temperature changes	Insulate column and tubing
	Random – Temperature changes	Thermostat column and tubing
	Wavy or undulating – Temperature changes in room	Monitor room temperature and control
Ghost peaks	Peaks from previous injection	Flush column to remove contaminants
	Contamination	Sample cleanup or pre-fractionation
	Unknown interferences in samples	Sample cleanup or pre-fractionation
	Ion pair – Upset equilibrium	Prepare sample in actual mobile phase to minimize disturbance
	Peptide mapping – Oxidation of TFA	Prepare fresh daily; use anti-oxidant
	Reversed phase – Contaminated water	Check suitability of water by running different amount through reversed phase column and measure peak height with elution; use HPLC grade solvents
	Spikes – Bubbles in solvent	De-gas solvents
High column backpressure	Column blockage with irrev, adsorbed sample	Better sample cleanup; use guard column
	Mobile phase viscosity too high	Use lower viscosity solvents or higher temperature
	Particle size too small	Use larger d_p packing
	Plugged inlet frit	Replace column
	Plugged inlet frit	Reverse solvent flow
Leak	Subtle – White powder at fitting/loose fitting	Tighten fitting, cut tubing, or replace ferrule
Leak, injection valve	Catastrophic – Worn valve rotor	Replace rotor in valve
Leak, column or other fittings	Catastrophic – Loose fittings	Tighten or replace fitting
Leak, pump	Catastrophic – Pump seal failure	Replace pump seal
Negative peaks	RI detector – solute refractive index less than solute	No problem; reverse polarity to make positive
Negative peaks	UV detector – solute absorbance less than mobile phase	Use mobile phase with lower UV absorbance; do not recycle solvent too long
Noisy baseline	Random – Contaminant buildup	Flush column; cleanup sample; use HPLC-grade solvent
	Continuous – Detector lamp problem	Replace UV lamp (lasts 1000 hrs)
	Occasional – External electrical interference	Use voltage stabilizer for LC system
	Sample volume too large	Injection volume should be 1/6 when mobile phase used for injection
Peak doubling	Injection solvent too strong	Use weaker injection solvent or mobile phase
	Blocked frit	Replace and use 0.5 µm porosity in-line filter
	Column void or channeling	Replace column; for some columns, fill in void with packing
	Unswept injector flowpath	Replace injector rotor
	Void at head of column	Replace column, top off column with packing
	Column overloaded with sample	Use higher capacity stationary phase Increase column diameter Decrease sample size
	Single peak – interfering components	Sample cleanup; prefractionation
Peak tailing	Beginning of peak doubling	See “peak doubling”
	Unswept dead volumes	Minimize number of connections Ensure injector seal is tight Ensure fittings are properly seated
	Basic compounds – Silanol interactions	Choose endcapped bonded phase Switch to polymeric phase
	Basic substances – Silanol interactions	Use stronger mobile phase or add competing base (e.g. TMA)
	Silica-based – Column degradation	Use speciality column; polymeric column or sterically protected
Peaks are broad	Injection volume too large	Decrease solvent strength of injection solvent to focus solute
	Peak dispersion in injector valve	Introduce air bubble in front/back of sample to decrease dispersion
	Sampling rate of data system too slow	Increase frequency of sampling
	Slow detector time constant	Adjust time constant to match peak width
	Mobile phase viscosity too high	Increase column temperature
	Detector cell volume too large	Use smallest possible cell volume with no heat exchanger in system
	Injector volume too large	Decrease injection volume
	Long retention times	Use gradient elution or stronger mobile phase
Pressure fluctuation	Leaky check valve	Replace check valve
	Pump seal leaks	Replace pump seals
	Buildup of particulates	Filter sample; in-line filter; filter mobile phase
Pressure increasing	Buildup of particulates	Filter sample; in-line filter; filter mobile phase
Pressure increasing	Water/organic systems – buffer precipitation	Test buffer-organic mixtures; ensure compatibility
Retention beyond total permeation volume	Size exclusion – Specific interactions	Add mobile phase modifiers or change solvent
Retention times changing	Column temperature varying	Thermostat column; insulate column; ensure lab temperature constant
	Equilibration time insufficient with gradient run or changes in isocratic mobile phase	Make sure at least 10 column volumes pass through column after solvent change or gradient conclusion
	Selective evaporation of mobile phase component	Less vigorous helium sparging; keep solvent reservoirs covered; prepare fresh mobile phase
	Buffer capacity insufficient	Use >20 mM concentration of buffer
	Inconsistent on-line mobile phase mixing	Ensure gradient system delivering constant composition; check vs. manual prep of mobile phase
	Contamination buildup	Occasionally flush column with strong solvent to remove contaminants
	First few injections – Adsorption on active sites	Condition column by initial injection of concentrated sample
Retention times decreasing	Flow rate increasing	Check pump to make sure correct; if not, reset
	Column overloaded with sample	Decrease sample size
	Loss of bonded stationary phase	Keep mobile phase pH between 2 and 8.5
Retention times increasing	Flow rate is slowing	Fix leaks in liquid lines, replace pump seals, check for pump cavitation or air bubbles
	Active sites on silica packing	Use mobile phase modifier
	Loss of bonded stationary phase	Keep mobile phase pH between 2 and 8.5
	Mobile phase composition changing	Make sure mobile phase container is covered
	Active sites on silica packing	Add competing base to mobile phase
	Active sites on silica packing	Use higher coverage packing for stationary phase
Sensitivity problem	Peaks are outside of linear range of detector	Dilute/concentrate to bring into linear region
	First few sample injections – Absorption of sample in loop or column	Condition loop/column with concentrated sample
	Autosampler flow lines blocked	Check flow and make sure no blockages
	Injector sample loop underfilled	Make sure that loop is overfilled with sample
	Sample-related losses during preparation	Use internal standard during sample prep; optimize sample prep method
Slow column equilibration times (ion pairing)	Equilibration time slow for long-chain ion pairing reagents	Use shorter alkyl chain ion-pair reagent

LC/MS Troubleshooting

Symptom Type	Solution
No peaks	Spray from the nebulizer
	Make sure capillary voltage is set correctly
	Make sure LC/MSD is tuned correctly
	Make sure LC/MSD pressures are within normal ranges
	Check drying gas flow and temperature
	Make sure fragmentor is set correctly
Poor mass accuracy	Recalibrate the mass axis
Poor mass accuracy	Make sure ions used for tuning span mass range of sample ions and show strong table signals
Low signal	Check the solution chemistry; make sure solvent is appropriate for sample
	Make sure sample is fresh and has been stored correctly
	Make sure LC/MSD is tuned correctly
	Check the nebulizer condition
	Clean the capillary entrance
	Check the capillary for damage and contamination
Unstable signal	Make sure drying gas flow and temperature are correct for the solvent flow
	Make sure solvent is thoroughly degassed
	Make sure LC backpressure is steady; this indicates a steady solvent flow
High spectral noise	Use appropriate mass filter values
	Check spray shape; nebulizer may be damaged or set incorrectly
	Make sure drying gas flow and temperature are correct for the solvent flow
	Make sure solvent is thoroughly degassed
	Make sure LC backpressure is steady; this indicates a steady solvent flow
	If you are using water as part of the mobile phase, make sure it is de-ionized (>18MW)
Droplets, not spray, exiting the nebulizer	Make sure nebulizing gas pressure is set high enough for the LC flow
	Check position of needle in nebulizer
	Stop solvent flow and remove nebulizer assembly
	Examine end of nebulizer for damage
No flow	Make sure LC is on and there is sufficient solvent in correct bottle
	Check for LC error messages
	Check for blockages
	Repair or replace any blocked components
	Check for leaks
	Make sure MS stream selector valve is set to LC to MSD
Undesired fragmentation	(APCI vs. Electrospray)
	APCI temperature is too high
	Fragmentor voltage is set too high