The Secrets of Good Peak Shape in HPLC

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Achieving good peak shape in HPLC is essential for accurate quantitation, improved resolution, and extended column lifetime. A well-shaped peak is symmetrical, with a tailing factor of ~1.0, high efficiency, and narrow peak width.

Key Factors Influencing Peak Shape

Column Selection & Chemistry

Silica Purity & Acidity:

  • High-purity, fully hydroxylated silica reduces peak tailing for basic compounds.
  • Bonded Phase & End capping: Double end capped columns (e.g., Eclipse XDB) minimize silanol interactions, improving peak symmetry.

Specialized Phases:

  • Bonus-RP (polar-embedded phase) reduces peak tailing for basic analytes.
  • Extend-C18 is stable at high pH, preventing ion exchange issues.
  • SB-Aq ensures good retention in high aqueous mobile phases.

Mobile Phase Optimization

  • pH Control: Using buffered mobile phases prevents peak tailing by stabilizing analyte ionization.
  • Organic Modifiers: Acetonitrile vs. methanol can influence peak shape by altering secondary interactions.
  • Additives (TEA, TFA, Acetic Acid): Suppress unwanted ion-exchange interactions for basic and acidic compounds.

Injection & Sample Considerations

  • Sample Overload: High concentration can cause peak fronting or tailing.
  • Injection Solvent Strength: Using a stronger solvent than the mobile phase leads to peak broadening or splitting.
  • Metal Contamination: Chelating compounds may interact with metal ions in the system, distorting peaks. Acid washing can mitigate this issue.

Pore Size for Large Molecules

  • 300Å wide-pore columns are ideal for peptides & proteins, ensuring sharper peaks by allowing better analyte diffusion.

Best Practices for Optimal Peak Shape

  • Choose high-purity, fully hydroxylated silica columns (e.g., Stable Bond, Eclipse XDB, Bonus-RP, Extend-C18).
  • Use buffered mobile phases (25–50 mM) to minimize secondary interactions.
  • Adjust pH and mobile phase composition to match analyte properties.
  • Optimize injection volume, solvent strength, and sample concentration to prevent overload issues.
  • Perform acid washes periodically to remove metal contamination.

By following these guidelines, HPLC users can achieve sharper, more reproducible peaks, leading to higher analytical accuracy and longer column lifespan.


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