|
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 dp 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
|
|
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
|
|
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
|