What are common causes of peak splitting when running an LC column? - WKB194672

ENVIRONMENT

• UltraPerformance Liquid Chromatography (UPLC)
• Ultra High Performance Liquid Chromatography (UHPLC)
• High Performance Liquid Chromatography (HPLC)
• Peak splitting
• Troubleshooting

ANSWER

If all peaks are splitting, potential reasons include the following:

1. Improper connection somewhere in the flow path between the injector and the detector; for example:

• Tubing slippage when using PEEK fingertight fittings
• Improper ferrule depth on stainless steel fittings or other fittings
• Small void inside the connection
• If all peaks are impacted, the dead volume is often after the column

2. Particulates lodged in the inlet of the column may originate as follows:

• Particulates in the samples
• Particulates in the mobile phase
• Microbial contamination

3. Strong sample solvent effect (may sometimes affect all peaks); for example:

• If the sample diluent contains a higher percentage of nonpolar solvent (reverse-phase) compared with the starting mobile phase
• If the sample diluent contains a higher percentage of polar solvent (normal phase; HILIC) compared with the starting mobile phase

4. If running a mass spectrometer, the emitter could be dirty, causing peak shape issues. There might also be a dead volume in the probe (none tool-free versions).

5. If analyzing sugars with HILIC chromatography, the sugars can form anomers (open chain vs. closed chain).

6. The column might be dirty (buildup of sample matrix).

 

If only some peaks are split within a mixture of several other peaks:

1. Strong sample solvent effect (sometimes affects early eluting peaks); for example:

• If the sample diluent contains a higher percentage of non-polar solvent (reverse-phase) compared with the starting mobile phase
• If the sample diluent contains a higher percentage of polar solvent (normal phase; HILIC) compared with the starting mobile phase

2. If analyzing sugars with HILIC chromatography, the sugars can form anomers (open chain vs. closed chain; not always all peaks).

3. Improper connection somewhere in the flow path between the injector and the column; for example:

• Tubing slippage when using PEEK fingertight fittings
• Improper ferrule depth on stainless steel fittings or other fittings
• Small void inside the connection
• If peak shapes get better in function of retention time, the dead volume is often before the column and peak shape is restored as a result of refocussing on the column

ADDITIONAL INFORMATION