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Monday, August 16, 2021

How to avoid peak tailing in HPLC chromatography

Column contamination is the most common cause of peak tailing in non-active analytes. In HPLC and Gas chromatography, the most usual chromatographic peak shape distortion is peak tailing. Peak tailing shows when the peak asymmetry factor (As) is larger than 1.2, while many assays accept peaks with an asymmetry factor of more than 1.5.

A good sharp symmetrical shape, a Gaussian peak, with sufficient resolution on a flat baseline, is an ideal chromatographic peak. A peak can stray from this ideal for different reasons. It can become asymmetrical, flatten and broader, or have its baseline rise.

The broad peaks, split peaks and variable peak heights in HPLC may be imperfectly filled samples in the sample loop or injector, incompatibility of sample solvents with the mobile phase. It is good to dissolve and inject the sample components in the mobile phase whenever possible. Or else, ensure that the injection solvent is of lower strength as compared with the mobile phase.

Keep in mind that several autosamplers use distinct syringe wash solutions. Ensure that this solution is weaker than the mobile phase and is compatible with it. This is particularly significant while exchanging between reversed and normal phase analyses.

The guard columns and filters are preventing the strongly retain compounds and particles to accumulate on the HPLC column. The lifespan of such devices depends on the composition of the mobile phase, the pH of the mobile phase and purity of the sample, etc. As these products become plugged or contaminated with particles, hence the system pressure increases, and peaks getting broad.

Causes of peak tailing in HPLC chromatography:

  • Don’t overload the column.
  • If the retention time is too long, it shows peak tailing. Modify the method thus that peaks elute earlier.
  • Large amounts of extra-column cause peak tailing hence where possible, reducing tubing diameter and length of the column to protect, especially in post-columns.
  • The precipitation of the sample causes a slow dissolution of the analytes and it causes peak tailing so make sure that the molecule is properly soluble with your solvent.
  • If the detector time is too slow, increase the detector Hz rate.
  • If the column volume is too large it causes tailing hence reduce the lengths and tubing diameters where possible, mainly post-column.
  • Physical damage to the column, injector parts, and loss of inertness in the tubing are also causes of peak tailing and fronting.
  • Coating or obstructions on column frit or particles of column packing.
  • Due to the use of a contaminated column.
  • Obstructed or dirty guard columns and filters cause tail.
  • When you have tailing only for one or two beaks, there may be an interfering contaminant that coexists.
To avoid peak tailing in chromatogram rectify the above-mentioned point, there are a few ways that can be used while performing the experiment or research work.

How to avoid peak tailing:

  • Whenever possible prepare the sample in the same mobile phase that you use for separation.
  • Inject minimum sample in the injector to avoid column overloading.
  • Use a lower pH of the mobile phase to operate the instrument.
  • Use a column that is highly deactivated.
  • The possibility of mass overload should be considered.
  • The possibility of column bed deformation should be considered.
  • Check the pKa value of the analyte and maintain the pH of the mobile phase as required (+/- 2 pH of pKa).
  • Always filter the sample and mobile phase to avoid interfering with contaminants.
  • Make proper use of the sample cleaning process that is required.

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