Troubleshooting in liquid chromatography / HPLC can be made easier, if one learns to recognize the symptoms produced by an instrument malfunction, leaks, or column problems. Some of the symptoms are described below:
Symptom: Column pressure erratic in an HPLC system
Symptom: Column pressure erratic in an HPLC system
Possible Cause: Leak in high pressure system before column
Action: Locate and repair leak
Possible Cause: Foreign material in column pump check valves
Action: Disconnect liquid inlet and outlet lines. Syringe 20 ml of fresh solvent through liquid end. Reconnect lines. If the above fails, disassemble liquid end and clean check valves
Symptom: No column flow or column pressure
Possible Cause: Leak in system before column
Action: Locate and repair leak
Possible Cause: Air in column pump liquid end
Action: Disconnect liquid outlet line and pump at maximum until no bubbles appear
Symptom: Poor peak shape (Fig. 1)
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| Fig. 1: Poor peak shape in a liquid chromatogram |
Possible Cause: Column overloaded
Action: Inject less sample, less concentrated sample or change diluent to match mobile phase. If possible, change to another column with higher sample capacity.
Symptom: Loss of resolution (Fig. 2)
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| Fig. 2: Poor resolution in a liquid chromatogram |
Possible Cause: Column overloaded
Action: Reduce amount of sample
Possible Cause: Bad column
Action: Replace with a new column
Possible Cause: Strongly retained compound on ion-exchange column
Action: Regenerate the column
Symptom: Increased retention time (Fig. 3)
Possible Cause: Low flow rate
Action: Increase solvent flow rate
Possible Cause: Solvent leak before injector (if same peak height)
Action: Repair leak
Possible Cause: Low column temperature
Action: Increase column temperature
Possible Cause: Improper gradient
Action: Check functioning of gradient system / increase concentration more rapidly
Possible Cause: Column deterioration
Action: Change column
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| Fig. 3: Increasing retention times in a liquid chromatogram |
Symptom: Decreased retention time
Possible Cause: Loss of liquid phase from column
Action: Replace column
Possible Cause: Column not regenerated after gradient elution
Action: Regenerate column
Symptom: Baseline “stepping” baseline does not return to zero, peaks are flat on the top or show steps (Fig. 4)
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| Fig. 4: Baseline stepping and flat-topped peaks |
Possible Cause: Recorder gain and / or damping control improperly adjusted
Action: Adjust recorder gain
Possible Cause: Instrument and /or recorder not properly grounded
Action: Connect instrument and recorder to good earth ground
Symptom: Peak shoulders (Fig. 5)
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| Fig. 5: Peak shoulders in a chromatogram |
Possible Cause: Column overloaded or bad column
Action: Reduce amount of sample or replace the column
Possible Cause: Packing voids/channeling in column
Action: Replace the column
Possible Cause: Poor tubing connection at the head of the column
Action: Connect tubing properly
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