Common Issues and Solutions in Liquid Chromatography (Part 1)

Zero Pressure in HPLC Analysis System
What’s the Reason? How to Solve It?

1. The mobile phase has a high viscosity and is contaminated. It is recommended to choose a suitable mobile phase and proportion, reconfigure, and filter with a 0.45-micron filter membrane.
2. The buffer salt in the mobile phase is causing blockage in the one-way valve. Set the flow rate to 5 mL/min, loosen the exhaust valve, and purge.
3. There is gas in the pump. Set the flow rate to 5 mL/min, loosen the exhaust valve, and purge.
4. The high-pressure sealing gasket is deformed. It is recommended to replace it with a new high-pressure sealing gasket.

When conducting HPLC analysis, unstable column pressure

Why? How to resolve it?

1. If there is gas trapped in the pump, the solution is to remove the gas from the pump and degas the solvent.

2. If the proportioning valve is malfunctioning, replacing the proportioning valve is recommended.

3. If the pump seal gasket is damaged, replacing the seal gasket should resolve the issue.

4. If there are bubbles in the solvent, degassing the solvent and, if necessary, changing the degassing method can help.

5. Perform a system leak check to identify any leakage points and seal them.

6. During gradient elution, pressure fluctuations are normal.

Why is the column pressure too high during HPLC analysis testing?

Possible reasons for high column pressure during HPLC analysis testing include

Excessive column pressure is the most common problem encountered by HPLC column users. There are multiple reasons for this, and often it is not the issue with the column itself. You can follow the steps below to investigate the cause of the problem:

1. Remove the guard column and check if the column pressure is still high. If not, it is an issue with the guard column. If the column pressure remains high, proceed to the next step.
2. Remove the chromatographic column from the instrument and observe if the pressure decreases. If not, there might be a blockage in the tubing, which requires cleaning. If the pressure decreases, proceed to the next step.
3. Reverse the inlet and outlet of the column and flush the column with a mobile phase that is 10 times the column volume. (Do not connect the detector at this point to prevent solid particles from entering the flow cell.) If the column pressure still does not decrease, proceed to the next step. This step is only applicable to used columns.
4. Replace the entrance frit of the column. If the column pressure decreases, it indicates that your solvent or sample contains particle impurities, which block the frit and cause pressure increase. If the column pressure is still high, please contact the manufacturer. In general, placing an online filter between the autosampler and the guard column can prevent excessive column pressure.
5. The autosampler might be blocked. Use a syringe to extract a suitable amount of purified water (more than the quantification loop volume) and inject it into the sample injection port until it flows out from the waste bottle. Repeat this process five times.

Why do irregular peak shapes occur, such as flat-top peaks, split peaks, tailing peaks, and fronting peaks?
Flat-top peak:

1. Contamination in the detector cell, clean the detector cell.
2. UV lamp performance is limited or faulty, replace with a higher-performing UV lamp.
3. Excessive sample injection volume, reduce the injection volume.

Split peak:

1. Contamination in the guard column, replace with a new guard column.
2. Contamination in the chromatographic column, flush the chromatographic column overnight.
3. Contamination in the injection valve, use a syringe to extract a suitable amount of purified water (more than the quantification loop volume) and inject it into the sample injection port until it flows out from the waste bottle. Repeat this process five times.
4. Contamination in the detector cell, clean the detector cell.
5. Inappropriate solvent selection, choose suitable solvents.
6. Excessive sample injection volume, reduce the injection volume.

Tailing peak:

1. Contamination in the chromatographic column, replace with a new chromatographic column.
2. Excessive sample injection volume, reduce the injection volume.
3. Inappropriate mobile phase composition, create a mobile phase with a suitable composition.
4. Inappropriate mobile phase flow rate, adjust the mobile phase flow rate accordingly.
5. Inappropriate selection of the chromatographic column, choose an appropriate chromatographic column.
6. Impure sample, purify the sample.

Fronting peak:

1. Column temperature is too low, adjust the column temperature accordingly.
2. Solvent is not the mobile phase, dissolve the sample in the mobile phase.
3. Inappropriate mobile phase composition, create a mobile phase with a suitable composition.
4. Inappropriate mobile phase flow rate, adjust the mobile phase flow rate accordingly.