What are the common causes of longer than expected retention on a reversed-phase LC column? - WKB236458

ENVIRONMENT

• C18 columns
• Reverse phase
• Retention issues
• longer retention

ANSWER

Various factors are possible. See the "Additional Information" section.

ADDITIONAL INFORMATION

• New C18 columns have full end cap coverage, leading to longer retention in some cases.
  • See: Why would a new C18 column show longer retention than an older, used column of the same part number when running an isocratic method or a gradient method?
• Column heater too cold:
  • If a column heater is not working properly, it is possible that it is colder than expected.
  • Lower temperatures can cause analytes to elute later than expected.
  • Use an external thermometer to measure the actual temperature of the column heater.
• Flow rate too slow:
  • If the system check valves or pumps are not working properly, or if there is a blockage in the flow path, the flow rate could be slower than expected.
  • Slower flow rate causes analytes to elute later than expected.
  • Collect waste into a graduated cylinder during a run, for a timed period. Verify that the volume collected is the expected volume.
• Gradient proportioning valve not working properly:
  • The gradient proportioning valve (GPV) (low-pressure mixing) might be causing too little organic or too much aqueous to be delivered.
  • Either scenario can cause increased retention.
• Gradient mixing (high-pressure mixing) is not working properly:
  • The system may not be mixing the mobile phases properly, which may lead to too little organic or too much aqueous delivered.
  • Either scenario can cause increased retention.
• Incorrect premixed mobile phase composition:
  • Having too little organic (or too much aqueous) in a premixed mobile phase will result in more retention than expected.
• Incorrect mobile phase pH.
  • Weakly basic analytes retain longer when neutralized in a pH that is 2 pH units or higher above their pKa.
  • Weakly acidic analytes retain longer when neutralized in a pH that is 2 pH units or lower below their pKa.
    • Inadvertently using a mobile phase with the wrong pH for the method can cause unexpected retention problems.
• Larger dwell volume (gradient delay):
  • A system with a larger dwell volume (gradient delay) will show longer retention compared with a system with a lower dwell volume (gradient delay):
    • See: Dwell Volume (Gradient Delay) and Extra-Column Volume (Bandspread, System Dispersion): What Are They and How Do They Impact Method Transfer 
• Check valve problem:
  • Sticky check valves can result in mobile phase being pumped back into the mobile phase bottle instead of into the mobile phase flow.
    • This can result in an improper ratio of aqueous to organic in the mobile phase:
      • Longer retention times are seen when less organic enters the mobile phase due to a check valve issue.
• System leak:
  • Check your system for a leak by running a dynamic leak test (or similar test) to ensure that there is no leak causing an incorrect mobile phase aqueous-to-organic ratio.
• There may be additional reasons for longer than expected retention times including mobile phase may have gotten old or a mobile phase modifier is degrading..
• Some pre-mixed mobile phases can partition or separate over time (such as H2O and Isopropanol), water settles on the bottom and Retention Times increase proportionately. (Note: Higher Concentrations of Salt increase the likelihood of Separation. ACN & H2O, can separate at Higher Concentrations of Salt.)
  • The Fix: Allow system to mix, or use a Stir Plate, with a stir bar in the mobile phase.