Gel Filtration

• Gel filtration (GF) - simple and reliable liquid chromatography method for separating molecules according to size.
• Versatile - generally applicable to the purification of all classes of biological substances, including macromolecules not readily fractionated by other techniques.
• Gel Filtration could be used at the early stages of protein

  purification or as a polishing step
• Gel Filtration: Principle

  • Separates molecules according to size
  • Sample passes through a matrix composed of beads
  • smaller molecules take longer time to travel through a porous matrix
  • Larger molecules will
• Gel Filtration: Components

  1. Elution buffer
  2. Gel column
  3. column volume, CV = pi r^2 h
  4. void volume, Vo
  5. elution volume, Ve
  6. Fraction collector
• Gel Filtration: Steps

  1. Equilibration
  2. Sample application,0.3% of bedvolume
  3. Elution - flow rate
• Sephadex G25 - Dextran, 1-5 Fractionation Range
• Sephadex G100- Dextran, 4-150 Fractionation Range
• Sephadex G200 - Dextran, 5-600 Fractionation Range
• Bio-Gel P30 - Polyacrylamide, 2.4-40 Fractionation Range
• Bio-Gel P30- - Polyacrylamide, 60-400 Fractionation Range
• Sepharose 6B - Agarose, 10-4000 Fractionation Range
• Sepharose 4B - Agarose, 60-20000 Fractionation Range
• Principle of Gel Filtration

  • Large particles cannot enter gel and are excluded. They have less volume to traverse and elute sooner.
  • Small particles can enter gel and have more volume to traverse. They elute later.
• Vt or total colum volume - refers to total volume occupied by the gel in the column
• Vo or void volume - space outside the granules
• How to increase resolution

  • Keep within the optimum fractionation range
  • Reduce the sample volume
  • Change to a gel with smaller beads
  • Connect two columns in a series
  • Reduce the flow rate
• Gel Filtration: Applications

  • Determination of molecular weight
  • desalting
  • Removal of contaminating substances
• Gel Filtration: MW determination

  • Somatostatin - 1640
  • Aprotinin, Bovine Lung - 6,500
  • Cytochrome C, Horse Heart - 12,400
  • Carbonic Anhydrase, Bovine Erythrocytes - 29,000
  • Albumin, Bovine Serum - 66,000
  • Alchol Dehydrogenase, Yeast - 150,000
  • β-Amylase, Sweet Potato - 200,000
  • Apoferritin, Horse Spleen - 443,000
  • Thyroglobulin, Bovine - 669,000
  • Blue Dextran - 2,000,000
• Gel Filtration: additional information

  • Area under the curve – amount of the protein
  • Elution time – molecular weight