Info

HPLC Troubleshooting

Display Info Reason Adjustment
No peaks, flat baseline No peaks, flat baseline
  1. Detector lamp is switched off or defective
  2. Connecting cable between detector and control computer is not plugged in
  3. No solvent flow
  1. Switch on or replace detector lamp
  2. Check all cables and connections
  3. Check solvent supply vessle, start pump/s
No peaks, slight baseline noise No peaks, slight baseline noise
  1. Wrong flow direction
  2. No, too little, wrong sample injected
  3. Wrong detector sensitivity/wave length
  1. Correctly set up and start pumps
  2. Check sample or sample injector
  3. Check detector settings
High noise with periodic spikes High noise with periodic spikes Strongly pulsed solvent flow
  1. Check pump, air bubble in the valve at the pump head possible
  2. Use pulsation damper
High noise with long flat drift High noise with long flat drift Presumably temperature fluctuations due to external radiation
  1. Use column thermostat for the HPLC column
  2. Shield HPLC system from rising ambient temperature
High noise with sharp negative spikes High noise with sharp negative spikes Presumably electromagnetic interference
  1. Use shrouded cables
  2. Power supply with galv. isolation (USV)
  3. Connect the complete HPLC system to a different electric circuit.
Extremely strong noise Extremely strong noise Air bubbles in the detector flow cell
  1. Rinse flow cell until bubble free
  2. Use back pressure restrictor at the flow cell inlet
  3. Use solvent filter and/or degas solvent
Uniform noise Uniform noise
  1. The mixer is defective
  2. Detector lamp is defective
  3. Eluent absorption is too high
  1. Check mixer
  2. Replace lamp
  3. Change eluent/wave length
Aperiodic noise Aperiodic noise Detector flow cell contaminated Rinse flow cell with org. solvent or 20% HNO3
No peaks, no back pressure, no flow in the detector No peaks, no back pressure, no flow in the detector
  1. Pump/s is/are switched off
  2. Air bubble in the valve at the pump head
  3. Leakage at connection or suction tube defective
  4. Supply vessel empty or suction tube not in the eluent
  1. Check and turn on pump/s
  2. Rinse pump head and gently tap the valve
  3. Check/seal or renew connection
  4. Refill eluent, fasten suction tube
No peaks, high back pressure or alarm No peaks, high back pressure or alarm
  1. Suction tube of filter clogged
  2. HPLC column or frit clogged
  3. Sample injection valve or sample loop clogged
  4. Capillary jammed/closed at connection
  1. Remove the blockage, clean filter in ultrasonic bath or replace
  2. Rinse column and frit in ultrasonic bath or replace
  3. Open and clean valve, rinse loop or replace
  4. Cut off defective site and reconnect capillary
Slow pressure increase Slow pressure increase
  1. Frit at column inlet or column clogged
  2. Frit in the mixer clogged
  1. Use/replace pre-column, rinse column as appropriate
  2. Clean frit in ultrasonic bath or replace
Sudden pressure increase Sudden pressure increase
  1. Defective or badly switched valve
  2. Sample precipitation
  1. Check and clean valve
  2. Rinse column and/or system
Pressure fluctuations Pressure fluctuations
  1. Air in pump or outlet tube to the pump
  2. Leaky tube from pump to column
  1. Degas solvent or rinse with helium
  2. Fasten the screws
Pressure loss Pressure loss
  1. No mobile phase flow
  2. Leaky tube from pump to column
  1. Check system for loose screws. Also check eluent supply vessel
  2. Systematically search for leakage and remove
High pressure increase High pressure increase
  1. Clogged column inlet, precipitation of buffer salts
  2. Viscosity of mobile phase too high
  3. Column material particle size too small
  1. Demount column, rinse with strong solvent, replace clogged filter elements and pre-column
  2. Are you using the right eluent for the given buffer concentration?
  3. Change solvent or increase column temperature
  4. Use HPLC column with larger particles (e.g. 8 µm instead of 5 µm particle size)
Tailing, blurred peaks at baseline, often in combination with retention time shift Tailing, blurred peaks at baseline, often in combination with retention time shift HPLC column overloading Reduce injection volume or use column with higher capacity
Fronting, without retention time shift Fronting, without retention time shift
  1. Dead volume between injector and column
  2. HPLC column defective
  1. Check injector/tubing for dead volume
  2. Replace HPLC column
Broad, flat peaks with roof Broad, flat peaks with roof Injection volume too high Use smaller injection volume or smaller sample loop at injection valve
Doubling, all peaks have two maxima Doubling, all peaks have two maxima
  1. Channel formation in the column
  2. Injector defective
  1. Replace HPLC column
  2. Check and repair injector
Retention time shifts in one direction Retention time shifts in one direction
  1. Leaking pump sealing
  2. Column overloading
  3. Column not equilibrated
  1. Replace pump seal
  2. Reduce sample concentration
  3. Equilibrate 2 to 3 column volumes
Arbitrary retention time shifts Arbitrary retention time shifts
  1. Column temperature fluctuation
  2. Pump head valve or proportioning valve defective
  1. Use column thermostat
  2. Check/replace valves
Peaks are significantly smaller than expected Peaks are significantly smaller than expected
  1. Injection volume too small
  2. Eluent with higher absorption
  3. Contaminated detector flow cell
  1. Check injection volume
  2. Use suitable eluent (HPLC-grade)
  3. Rinse detector flow cell
Reproducible ghost peaks Reproducible ghost peaks
  1. Contaminated injection valve
  2. Peak from previous run
  3. Injection with air
  1. Rinse injection valve
  2. Prolong previous run
  3. Check injection volume
Arbitrary ghost peaks Arbitrary ghost peaks
  1. Solvent is contaminated
  2. Air in detector flow cell
  3. HPLC column is contaminated
  1. Use fresh sample
  2. Rinse flow cell, use back pressure restrictor
  3. Replace or rinse HPLC column
Medium leakage at fitting Medium leakage at fitting Tubing at the end of the ferrule is too long
  1. PEEK tubing: shorten to appropriate length and reinstall
  2. Stainless steel tubing: demount tubing with ferrule, remove burr and mount with new ferrule
Slight pre-shoulder at the first peak Slight pre-shoulder at the first peak Dead volume at tubing end, space between tubing end and connection, tubing is too short
  1. PEEK Tubing: Extend to max. length in the unfastened screw fitting and refasten
  2. Stainless steel tubing: remove tubing with ferrule, remove burr and mount with new ferrule