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December 11, 2017

How to fix asymmetrical peaks?

By Séverine Lebarq, Global Product Marketing Manager, Protein Purification at GE Healthcare

All good chromatography books say that the peaks in the chromatogram should be symmetrical, but what about real life in the lab? There are many different causes to “fronting” or “tailing” peaks, but most can be easily remedied.


For example, fronting peaks are often caused by column overload or overpacking. Similarly, tailing peaks can be caused by underpacking, or by having a sample that is too viscous.

Undetected peaks can also be a problem, which could be caused by excessive band broadening.

Other common reasons for asymmetrical peaks, and how to fix them, are summarized by GE R&D protein purification experts in the following tables.

Possible cause

Remedy

Column overloaded

  • Decrease sample load and repeat

Column is ”overpacked”

  • Do a column performance test. Repack using lower flow rate
  • Use prepacked columns

Channeling in column

  • Repack column using a thinner slurry of resin. Check column packing

Column contaminated

  • Clean using recommended procedures
Possible cause

Remedy

Column is ”underpacked”

  • Do a column performance test. Repack using higher flow rate
  • Use prepacked columns

Sample is not binding to column due to incorrect start buffer conditions

  • Adjust pH. Check salt concentration in start buffer

Sample too viscous

  • Dilute sample in start buffer

Column contaminated

  • Clean using recommended procedures

Band broadening due to large volume in system

  • Check modules, tubing, and connections for unnecessarily large volumes
Possible cause

Remedy

Sample absorbs poorly at chosen wavelength

  • Use a different wavelength (e.g., 214 nm instead of 280 nm)

Excessive band broadening

  • Check column packing. Repack if necessary or use prepacked columns

UV baseline rises with gradient because of buffer impurities

  • Use high-quality reagents

Download our Protein purification troubleshooting guide.

This guide helps you quickly identify root causes for many of the common issues encountered during protein purification. It gives practical advice on small changes that you can make to fix these problems.