Size exclusion chromatography

• Gel permeation is also called

  • Gel filtration
  • Size exclusion chromatography
• Stationary phase


  • Porous matrix, made up of compounds like Cross-linked polystyrene, cross like dextrans, polyacrylamide gels, agarose gel, etc.
• Separation principle


  Based on the analyte molecular size since the gel behaves like a molecular Seive
• Separation of


  • Proteins
  • Polysaccarides
  • Enzymes
  • Synthetic polymers
• Size exclusion chromatography was first developed by Lathe Ruthven


  1955
• Principle of Gel Permeation Chromatography


  Separation of components based on the difference in molecular weight or size
• Stationary phase


  • Porous polymer matrix whose pores are completely filled with the solvent to be used as the mobile phase
• Separation process


  1. Molecules in sample are pumped through specialized columns packing containing microporous material (gel)

  2. Molecules above a certain size are totally excluded from the pores

  3. Smaller molecules will be retarded according to their penetration of gel
• Components/Instrumentation of Gel Permeation Chromatography

  • Stationary phase
  • Mobile phase
  • Column
  • Pump
  • Detectors
• Stationary phase


  • Semi-permeable, porous, polymer gel beads with a well-defined range of pore size
  • Chemically inert
  • Mechanically stable
  • With ideal & homogeneous porous structure
  • A uniform particle pore size
• Examples of gel


  • Dextran (Sephadex) gels
  • Agarose
  • Acrylamide
• Mobile phase


  Liquid to dissolve the bio-molecules used to make the mobile phase permit high detection response and wet the packing surface
• Columns


  • Analytical Column 7.5-8mm diameters
  • Preparative Columns 22-25 mm
  • Usual Column lengths - 25, 30, 50, and 60 cm
  • Narrow bore molecules 2-3mm have been introduced
• Pumps


  Syringe pumps or reciprocating pumps with a high constant flow rate
• Detectors


  • Concentration sensitive detectors
  • Bulk property detectors
  • Refractive index (RI) detectors
• Steps in Gel permeation Chromatography

  1. Preparation of column - Swelling of gel

  2. Packing the column - Semi-permeable, porous polymer gel beads

  3. Washing column - Pass buffer solution to remove air bubbles and test column homogeneity

  4. Loading sample onto the column

  5. Eluting the sample and detection of components
• Applications of Gel permeation Chromatography

  • Proteins Fractionation
  • Purification
  • Molecular Weight determination
  • Separation of sugar, proteins, Peptides, rubbers on the basis of their size
  • Determine quaternary structure of purified proteins
• Gel permeation chromatography


  A technique used for the separation, purification, and molecular weight determination of proteins, sugars, peptides, and other molecules based on their size
• Gel permeation chromatography


  • Short analysis time
  • Well-defined separation
  • Narrow bands and good sensitivity
  • No sample loss
  • Small amount of mobile phase required
  • Flow rate can be controlled