Types of HPLC Columns Used in Analysis

Different type of HPLC column used in HPLC:

The column is the heart of the HPLC. The column is classified based on below parameter,

1. Types Based on Chromatographic Mode :

Four primary modes:

  1. Normal Phase Columns
  2. Reverse Phase Columns
  3. Ion Exchange Columns
  4. Size Exclusion Columns
  5. Other specialized modes: Hydrophilic interaction chromatography (HILIC), Hydrophobic interaction chromatography (HIC), affinity, chiral, supercritical fluid extraction (SFE)

2. Column Types Based on Dimension

3. Column Packing Characteristics

Details on HPLC Column:

1. Normal Phase Columns

  • Normal Phase Chromatography is known as liquid–solid chromatography or adsorption chromatography, NPC is the traditional separation mode based on adsorption/desorption of the analyte onto a polar stationary phase (typically silica or alumina).
  • Normal phase HPLC columns are used when the stationary phase is more polar than the mobile phase.
  • Normal phase chromatography (NPC), the polar stationary phase could be silica gel (porous silica particle with silanol groups (Si-OH) residing at the surface and inside its pores) and the less polar mobile phase could be Chloroform, Methylene Chloride & Hexane or another organic solvent.
  • This type of column is commonly used for samples with small molecules, like organic acids or pharmaceuticals.
  • This type of Column used for biomolecules, chiral separations and preparative applications.
  • Some common bonded phases for Normal-phase (NPC)
    1. Si (Silica) –  Si-OH
    2. NH2 (Amino)-  Si(CH3)2-CH2-CH2-CH2-NH2 (with propyl linker)
  • Disadvantages Normal Phase Column : This type of column easily contamination of the polar surfaces area by highly retained sample components execution it a less reproducible technique. This problem is solved by bonding polar functional groups such as amino- or cyano-moiety to the silanol groups of the column.

Related Topics: Theoretical plate numbers (N) and Determination of “N” in Chromatography, Difference Between Isocratic and Gradient Elution

2. Reverse phase columns

  • Reverse phase columns are used in reverse phase chromatography, where the separation is based on analytes’ partition coefficients between a polar mobile phase and a hydrophobic (nonpolar) stationary phase.
  • In comparison to the mobile phase, the stationary phase of this type of HPLC Column is less polar. To put it another way, reverse phase chromatography.
  • Bonded Hydrocarbon C8 & C18 (octadecyl) columns and Water, acetonitrile (ACN), methanol, and tetrahydrofuran (THF) as Mobile Phases are widely used in the analysis.
  • Reverse Phase Chromatography depends on the mechanism of separation and is mainly attributed to hydrophobic or “solvophobic” interaction.
  • The phrase “solvophobic interaction” describes the relatively strong cohesive forces that exist between polar solvent molecules and hydrated analytes, as well as their interaction with the nonpolar stationary phase.
  • This method is more widely used for the analysis of polar (water-soluble), medium-polarity, and some nonpolar analytes.
  • Some common bonded phases for Reversed-phase (RPC)
    1. C18 (Octadecyl)-  Si(CH3)2-(CH2)17-CH3
    2. C8 (Octyl)-   Si(CH3)2-(CH2)7-CH3
    3. CN (Cyano) – Si(CH3)2-CH2-CH2-CH2-CN (with propyl linker)
    4. ϕ (Phenyl)- Si(CH3)2-(CH2)6-C6H5(with hexyl linker)
    5. Polar-embedded – Si(CH3)2-(CH2)3NHCO(CH2)14CH3 (amide group)

3. Ion Exchange Columns

  • In ion-exchange chromatography (IEC), the column separation mode is different; it is based on the exchange of ionic analytes with the counter ions of the ionic groups linked to the solid support.
  • Typical stationary phases are a cationic exchange (sulfonate) or anionic exchange (quaternary ammonium) groups bonded to polymeric or silica materials and the Mobile phases contain of buffers, often with increasing ionic strength (e.g. a higher concentration of NaCl) to force the migration (relocation in stationary phase) of the analytes.
  • The attractive ionic interactions between the molecules in the sample and the charged stationary phase cause separation in this sort of column.
  • Each sample component will be attracted to the charged stationary phase at a different rate, causing the components to separate at various speeds as they pass through the column.
  • This method (IEC) is usually used to distinct carbohydrates, proteins/peptides, polynucleotides & amino acids.

Related: Calibration of HPLC, Gas Chromatography Columns

4. Size Exclusion Columns

  • Size-exclusion chromatography (SEC) is a separation mode based uniquely on the analyte’s molecular size (particle size).
  • It is known as Gel Permeation Chromatography (GPC) when used for the determination of molecular weights of organic polymers & Gel Filtration Chromatography (GFC) termed used when used in the separation of water-soluble biological compounds.
  • It uses a porous stationary phase (HPLC columns are packed with cross-linked polystyrene beads of controlled pore sizes) that only permits small particles into the pores, leaving the bigger molecules to pass through the column sooner (Fast).
  • Because each molecule diffuses into the pores to a different amount, the pore size in the stationary phase controls the retention period and elution profile of each sample component.
  • For size exclusion chromatography (SEC), porous particles are required in the stationary phase, hence molecular sieves (such as zeolites), polysaccharides, and polymers (such as a standard silica column) are typically utilized.
  • This size exclusion chromatography (SEC) technique is used to distinct proteins and carbohydrates.
  • Some common bonded phases for Ion-exchange (IEC)
    1. SP (Sulfopropyl) -Si(CH3)2-CH2-CH2-CH2-SO3
    2. CM (Carboxymethyl)- Si(CH3)2-CO2
    3. DEAE (Diethylaminoethyl)- Si(CH3)2-CH2-CH2-CH2-NH+ (C2H5)2
    4. SAX (Triethylaminopropyl)- Si(CH3)2-CH2-CH2-CH2-N+(C2H5)3

Type of HPLC Columns Based on Inner Diameters (ID.):

HPLC Column Types Based on Inner Diameters (ID.)


  1. HPLC AND UHPLC FOR PRACTICING SCIENTISTS (Second Edition) by Michael W. Dong
  2. Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan(auth.) – Introduction to Modern Liquid Chromatography, Third Edition

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