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HPLC and Ion Chromatography Retention Time Primer

HPLC and Ion Chromatography Retention Time

What is it?

Retention time (RT) is the time on a chromatogram, at which the apex of a chromatographic peak of a particular analyte is detected.

The actual RT of any analyte will depend on many run factors, such as column type and condition, mobile phase/s composition, gradient used, pump/s flow rate, column temperature, volume and concentration of the injected liquid and sample matrix type.

Under a given set of factors of a validated method, the RT of each analyte within an injected liquid, should be constant in value, for each injection made within a run.  This consistency is needed, so that each analyte is identified by its own RT.

Even when exactly the same run factors are used, the RTs of each chromatographic peak throughout a single run of injected liquids, will show a small degree of variation.  This variation is insignificant.  The analytical column itself is not completely homogenous.  The adsorbance of matter onto column particles and the interstitial column particle spacing will contribute to the variability of retention time.

Consequences  of significant changes of retention time

If RTs change significantly, then the chromatographic peak may be outside of the window of recognition noted for that peak within the software programme.  Hence  the chromatography software will not recognise a visually discernible peak.  In addition, there may be a debate as to the identity of the peak.

Tips for ensuring consistency of retention times

  • Use a column heater/chiller to maintain the analytical column at a constant temperature, particularly when air conditioning causes draughts.
  • Use an autosampler to ensure consistency of injection technique timings.
  • Ensure the mobile phase/s is adequately degassed, as air bubbles can cause sporadic shifts of RT.
  • Try to ensure there are no changes in the mobile phase/s composition through evaporation.
  • Use a guard column to help prevent deterioration of the analytical column.
  • Ensure the pump/s is of a sufficiently high specification, so that flow rate variations are as low as possible.
  • Ensure each chromatogram run time is sufficiently long, so late running peaks of a previous injection are not confused with a subsequent injection.
  • Ensure the detector does not cause significant baseline drift or noise.  For example, with conductivity      detectors ensure the detector temperature is constant, with UV/Visible and fluorescence detectors, ensure the lamps are working well.
  • If mobile phase gradients are used, ensure that each chromatogram run time is sufficiently long, in which to take the system’s dwell volume into account.  The dwell volume is the volume between the start of mixing of mobile phases to the start of the column.  

   For low pressure gradient mixing systems dwell volume equals all of the volume from the proportioning    valve, to the start of the column.  For high pressure gradient mixing systems, dwell volume equals all of    the  volume from the mixing valve to the start of the column.

   If a gradient method is transferred from one instrument to another, the dwell volumes should be as similar    as possible.

Cecil Instruments Limited  

Milton Technical Centre, Cambridge  United Kingdom      telephone  +44 (0)1223 420 821