What are common causes of peak tailing when running a reverse-phase LC column? - WKB237593

ENVIRONMENT

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

ANSWER

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

1. "Basic Tailing" can occur on all peaks if all analytes are basic:

• Basic analytes will tail on a reverse-phase column via weak cation exchange if:
  1. The pH of the mobile phase will allow the basic analyte to carry a positive charge
  2. The pH of the mobile phase will allow residual silanol groups on the particle surface to become negatively charged (usually at pH ~2.5 and higher)
  3. If the above criteria are met, and no ion pairing agent is used

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

• Tubing slippage when using PEEK finger-tight fittings
• Improper ferrule depth on stainless steel or other fittings
• Small void inside the connection

3. Strong sample solvent effect (may sometimes affect all peaks and possibly show tailing, although usually this is seen as peak shape asymetry or splitting); 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 dead volume in the probe (not on tool-free versions).

 

If only some peaks are tailing within a mixture of several other peaks, potential reasons include the following:

1. "Basic Tailing" can occur on only some peaks if those specific analytes are basic and the other analytes are not.

• Basic analytes will tail on a reverse-phase column via weak cation exchange if:
  1. The pH of the mobile phase will allow the basic analyte to carry a positive charge
  2. The pH of the mobile phase will allow residual silanol groups on the particle surface to become negatively charged (usually at pH ~2.5 and higher)
  3. If the above criteria are met and no ion pairing agent is used

2. Strong sample solvent effect (may sometimes affect only earlier eluting peaks and possibly show tailing, although usually this is seen as peak shape asymetry or splitting); 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

 

ADDITIONAL INFORMATION