Effects of Injection volume in HPLC

The relation among injection volume and the peak height is a type of linear relationship. The appropriate injection volume of the sample in HPLC is associated with the volume. The volume of samples in high-performance liquid chromatography is usually fixed or in autosampler, it may be variable. The Rheodyne sample loop is commonly used in HPLC to inject samples without disturbing the flow rate of the mobile phase and the pressure of the HPLC system. The mass overload happens when the chromatographer injects too many analytes and column overload happens when the excess liquid sample is injected into the column. Column and mass overload can reduce column performance due to peak shape and retention time problems.

Here some effects of injection volume in HPLC are mentioned.

  • The column is overloaded if too much sample injected in the column.
  • If injection volume increased then the height of the peak will increases.
  • The peak width increases by an increase in injection volume. 
  • The peak broadening due to volume overloads.
  • Increasing injection volume will decrease the resolution between the separated compounds.
  • When a very large volume of sample is injected in the HPLC column, the peaks begin to tail and the retention time (RT) may increase.

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