Troubleshooting and Maintenance in Liquid Chromatography / HPLC

 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

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)

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)

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

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)

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) 

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