Baseline is fluctuating or sloping

System not equilibrated. Equilibrate with 10 volumes of mobile phase.
Bubbles in flow cell. De-gas the mobile phase and pass degassed solvent through the flow-cell. Do not exceed the cell's pressure limit.
Contaminated guard. Replace the guard cartridge.
Contaminated column. Wash the column using the appropriate steps detailed in the manual or technical guide. If this does not resolve the problem, replace the column.
Detector contamination. Clean the flow cell according to the manufacturer's instructions.
Contaminated solvents. Use freshly prepared solvents of HPLC grade.

Low pressure

Low viscosity mobile phase. Confirm expected pressure using the Lozeny-Carmen or similar equation.
Piston seals leaking. Check for evidence of leaking or wear; replace where necessary.
Leak in the system. Check for and replace any leaking tubing or fittings.
Air in solvent lines or pump. Ensure that the reservoirs and solvent lines are fully primed and the purge valve is fully closed.

High pressure

High viscosity mobile phase. Confirm expected pressure using the Lozeny-Carmen or similar equation.
Pump flow-rate malfunction. Contact instrument manufacturer.
Tubing blocked. Working backwards from detector outlet, check source of blockage and replace tubing as necessary.
Guard blocked. Replace guard cartridge.
Sample precipitation. Consider sample clarification steps such as filtration or SPE.
Detector blockage. Clean the flow cell according to the manufacturer's instructions.

Quick links

Broad peaks

System not equilibrated. Equilibrate the column with 10 volumes of mobile phase.
Injection solvent too strong. Ensure that the injection solvent is the same or weaker strength than the mobile phase.
Injection volume too high. Reduce the injection volumes to avoid overload. Typically, injection volumes of less than 40% of the expected peak width should be used.
Injected mass too high. Reduce the sample concentration to avoid mass overload.
Extra column volume too high. Reduce diameter and length of connecting tubing. Reduce the volume of the flow cell where possible.
Temperature fluctuations. Use a thermostatically controlled column oven. Higher temperatures will produce sharper peaks.
Old guard cartridge. Replace the guard cartridge.
Old column. Do not use columns that have been used with ion-pair reagents for reverse-phase methods. Old columns give much lower efficiencies than new columns. Replace the column if necessary.
Contaminated column. Wash the column using the appropriate steps detailed in the manual or technical guide. If this does not resolve the problem, replace the column.
Voided column. Replace the column. Do not use the column outside the recommended pH range.

Double peaks

Old guard cartridge. Replace the guard cartridge.
Contaminated column. Wash the column using the appropriate steps detailed in the manual or technical guide. If this does not resolve the problem, replace the column.
Voided column. Replace the column. Do not use outside the recommended pH range.

Negative peaks

Contaminated solvents. Use freshly prepared solvents of HPLC grade.
Wrongly wired detector. Check the signal polarity from the detector to the recording device.
Unbalanced RI detector optics. Refer to manufacturer's instructions.
Ion pair method. Inject the sample in the mobile phase.

Flat-topped peaks

Detector overload. Reduce the sample concentration.
Detector set-up. Check the detector attenuation and re-zero.

Tailing peaks

Old guard cartridge. Replace the guard cartridge.
Injection solvent too strong. Ensure that the injection solvent is the same or weaker strength than the mobile phase.
Injection volume too high. Reduce the injection to avoid overload. Typically injection volumes of less than 40% of the expected peak width should be used.
Injected mass too high. Reduce the sample concentration to avoid mass overload.
Old column. Do not use columns that have been used with ion-pair reagents for reversed phase methods. Old columns give much lower efficiencies than new columns. Replace the column if necessary.
Contaminated column. Wash the column using the appropriate steps detailed in the manual or technical guide. If this does not resolve the problem, replace the column.
Voided column. Replace the column. Do not use outside the recommended pH range.

Fronting peaks

Old or damaged column. Replace the column.

No peaks

Sample vial empty. Inject a fresh sample.
Leak in system. Check for and replace any leaking tubing or fittings.
Pump not mixing solvents properly. Where being used, ensure that the proportioning valve is mixing the solvents correctly. If the method is isocratic, blend the solvents manually.
Damaged or blocked syringe. Replace the syringe.
Different dwell volume. For gradient methods, check that the dwell volume of any new system is not very different from any previous system. Apply a final hold time if necessary.
Old detector lamp. Replace the lamp, particularly when this has been in use for greater than 2000 hours.

Quick links

Small peaks

Degraded sample. Inject a fresh sample.
Low analyte concentration. Increase the analyte concentration.
Detector set-up. Check the detector attenuation and re-zero.
No wash solvent. Check that the solvent wash reservoir is filled with a miscible solvent and that the injector is set to wash between injections.
Damaged or blocked syringe. Replace the syringe.
Incorrect amount injected. Check the injector loop size. No more than 50% of this volume is used for partial loop injection.
Viscous injection solvent. Reduce the syringe draw-time.
Old detector lamp. Replace the lamp, particularly when this has been in use for greater than 2000 hours.

Missing peaks

Degraded sample. Inject a fresh sample.
Immiscible mobile phase. Check that any solvent already in the column is miscible with the mobile phase. Flush with propan-2-ol or ethanol where necessary.
Fluctuations in pH. Buffer the mobile phase so that retention of ionizable compounds is controlled.

Extra peaks

Degraded sample. Inject a fresh sample.
Contaminated solvents. Use freshly prepared solvents of HPLC grade. Gradient methods often shoe "ghost peaks".
Immiscible mobile phase. Check that any solvent already in the column is miscible with the mobile phase. Flush with propan-2-ol or ethanol where necessary.
Fluctuations in pH. Buffer the mobile phase so that retention of ionizable compounds is controlled.
Contaminated guard cartridge. Replace the guard cartridge.
Contaminated column. Wash the column using the appropriate steps detailed in the manual or technical guide. If this does not resolve the problem, replace the column.

Varying retention

System not equilibrated. Equilibrate the column with 10 volumes of mobile phase.
Leak in system. Check for and replace any leaking tubing or fittings.
Temperature fluctuations. Use a thermostatically controlled column oven. Higher temperatures will produce sharper peaks.
Contaminated column. Wash the column using an appropriate solvent. If this does not resolve the problem, replace the column.
Blocked solvent reservoir frits. Ultrasonicate the reservoir frits in water and then methanol.
Pump not mixing solvents properly. Where being used, ensure that the proportioning valve is mixing the solvents correctly. If the method is isocratic, blend the solvents manually.
Different dwell volume. For gradient methods, check that the dwell volume of any new system is not very different form any previous system. Apply a final hold time if necessary.
Piston seals leaking. Check for evidence of leaking or wear and replace where necessary.
Air in solvent lines or pump. Ensure that the reservoirs and solvent lines are fully primed and that the purge valve is fully closed.

No peaks

Sample vial empty. Inject a fresh sample.
Leak in system. Check for and replace any leaking tubing or fittings.
Pump not mixing solvents properly. Where being used, ensure that the proportioning valve is mixing the solvents correctly. If the method is isocratic, blend the solvents manually.
Damaged or blocked syringe. Replace the syringe.
Different dwell volume. For gradient methods, check that the dwell volume of any new system is not very different from any previous system. Apply a final hold time if necessary.
Old detector lamp. Replace the lamp, particularly when this has been in use for greater than 2000 hours.