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Tuesday, January 21, 2020

How to reduce peak broadening in HPLC?

Peak broadening is an occurrence in high-performance liquid chromatography separation that decreases separation efficiency causes leading to poor resolution of analytes. The ideal peaks in chromatography are symmetric and Gaussian. Peak fronting or tailing appears in the asymmetric peaks. Peak fronting or tailing may occur due to poor quality of the HPLC column or dead volume of the system. 

Peak broadening is caused by a number of factors that are mentioned here, and avoiding them may help reduce peak broadening in HPLC. 

  • The peak broadening is influenced by the flow rate of the mobile phase and particle size of the packing material in the HPLC columns. The smaller the particle with the latter, the better is the peak resolution.
  • Small columns and high amounts of sample lead to peak broadening.
  • Ensure the injected volume and concentration of the target component in the sample.
  • The mobile phase composition in gradient mode is steep towers that change from polar to non-polar in very little time.
  • Precipitation in the sample causes slow dissolution and release of the component; you have to centrifuge and filter before loading into the injector.
  • Check the number of theoretical plates for chromatographic columns and regulate the load accordingly.
  • Use the appropriate wavelength of the analyte.
  • If the current flow rate does not work, then you can work with a higher flow rate to get symmetrical peak shape.
  • Ensure that there is no leakage between the column and the detector.
  • Too long tubing between the column and HPLC detector.
  • Sometimes the tubing ID is too large, which causes the peak to broadening.
  • Make it feasible to inject the sample into the same solvent that is used in the mobile phase.
  • Periodically check the performance of columns; older columns reflect peak tailing and other performance issues.
  • While using just water in the mobile phase, it may be essential to manage the pH to control the charge of molecules of interest.
  • Buffers are considered capable of improving the shape of the peak when selected at the proper pH level to control ionization.
  • You can reduce peak broadening by using different types of buffers such as acetate or phosphate buffer solutions, these adjusted to the suitable pH as per the pKa value of the molecule.

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