Types of Chromatography Columns

Byipharmacist

Chromatography columns are crucial components in separation techniques, and they vary based on the type of chromatography used. Below are the main types of chromatography columns:

Liquid Chromatography (LC) Columns

Used in HPLC (High-Performance Liquid Chromatography) and UHPLC (Ultra-High-Performance Liquid Chromatography). Common types include:

  • Reversed-Phase Columns (RP-HPLC)
  • C18 (Octadecyl Silane, ODS): Most widely used, retains non-polar compounds.
  • C8: Shorter alkyl chain than C18, leading to shorter retention times.
  • Phenyl Columns: Useful for aromatic compounds.
  • Cyano (CN) Columns: Provide different selectivity than C18 and C8.
  • Amino Columns: Used for carbohydrate analysis.
  • Normal-Phase Columns (NP-HPLC)
  • Silica-based columns used for polar compounds with non-polar mobile phases.
  • Ion-Exchange Columns
  • Cation Exchange: Retains positively charged analytes.
  • Anion Exchange: Retains negatively charged analytes.
  • Size-Exclusion Columns (SEC or GFC/GPC)
  • Separate molecules based on size. Used in protein and polymer analysis.

Gas Chromatography (GC) Columns

  • Packed Columns: Contain solid support with liquid stationary phase.
  • Capillary Columns: Provide higher resolution due to the thin film coating inside a fused silica tube.

Supercritical Fluid Chromatography (SFC) Columns

  • Similar to HPLC columns but use supercritical CO₂ as the mobile phase.

Chiral Columns

  • Used for enantiomeric separations in pharmaceuticals.

Affinity Columns

  • Used in biochromatography for purifying proteins and antibodies.


Common Problems with C18 Columns

Using C18 columns comes with several potential issues:

Column Contamination

  • Caused by sample matrix impurities, proteins, lipids, or polymers.
  • Results in peak tailing, loss of resolution, and increased backpressure.

Peak Fronting or Tailing

  • Often due to improper pH conditions or excessive sample loading.

High Backpressure

  • Caused by particle clogging, buffer precipitation, or bacterial growth.

Baseline Drift and Noise

  • Due to mobile phase instability, contamination, or detector problems.

Irreversible Adsorption

  • Certain compounds strongly bind to the column, leading to carryover issues.
  • Other Causes of Contamination (Guard & Main Column)
  • Particulate Contamination: Due to unfiltered samples or buffer precipitation.
  • pH Instability: Using mobile phases outside the recommended pH range (typically 2-8 for silica-based C18).
  • Bacterial Growth: Occurs if aqueous buffers are left stagnant in the system.
  • Insoluble Sample Components: Lipids, proteins, or polymeric residues can irreversibly bind.
  • Mobile Phase Incompatibility: Incomplete buffer solubility or poor solvent miscibility.


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Resource Person: Hamideh Mousavi

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