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.)
Credit@HPLC AND UHPLC FOR PRACTICING SCIENTISTS

Reference:

  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

Read More:

More Topic:

You cannot copy content of this page