Gel Filtration Chromatography (GFC)

Gel filtration chromatography separates molecules based on size by using a porous gel matrix that allows smaller molecules to enter and be delayed, while larger molecules are excluded and elute faster.

• Key Components and Mechanism
  • Porous Matrix: The stationary phase consists of spherical particles with defined pore sizes. These pores allow smaller molecules to enter while excluding larger ones.
  • Mobile Phase: The buffer solution flows through the gel filtration chromatography column, carrying the sample molecules.

• Separation Process
  • Large Molecules: Those too large to enter the pores of the stationary phase remain in the mobile phase and pass through the column quickly, eluting first in the void volume.
  • Small Molecules: These penetrate the pores, moving through the stationary phase and taking a longer path. As a result, they elute later than larger molecules.
  • Intermediate Molecules: Partially penetrate the pores, eluting between the large and small molecules based on their size.

• Factors Influencing Separation
  • Pore Size: Determines the fractionation range of the column.
  • Molecular Shape: Hydrodynamic radius affects elution order; asymmetrical molecules may behave differently.
  • Buffer Conditions: pH, ionic strength, and composition can influence resolution and elution times.
  • Bead Size: Smaller particles improve resolution by reducing diffusion distances.

HPLC Gel Filtration Chromatography Applications

• Protein Purification and Analysis
  • Separation of Proteins and Peptides: Gel filtration chromatography is widely used to isolate proteins from complex mixtures like cell extracts and to separate monomers from aggregates. By passing the sample through a gel filtration chromatography column, proteins are separated based on their size, ensuring high purity.
  • Molecular Weight Determination: The gel filtration chromatography column helps estimate the molecular weight of proteins by analyzing their elution volume, making it a valuable tool for protein characterization.

• Buffer Exchange and Desalting
  • Buffer Exchange: It allows for efficient buffer exchange, enabling changes in buffer composition without altering the integrity of the sample.
  • Desalting: It is commonly used to remove salts and small molecules from protein samples, a critical step before applications such as crystallization or enzymatic assays.

• Analysis of Other Biomolecules
  • Separation of Nucleic Acids: It is effective for separating DNA and RNA fragments by size, making it useful for nucleic acid analysis.
  • Polysaccharide Analysis: The gel filtration chromatography column can also be used to separate and analyze polysaccharides based on their molecular size.

• Quality Control and Characterization
  • Assessment of Sample Purity: It helps evaluate sample purity by separating contaminants based on size, ensuring accurate analysis.

• Biotechnology and Pharmaceutical Applications
  • Antibody and Immunoglobulin Separation: Gel filtration chromatography is commonly used in biotechnology and pharmaceutical industries to separate and analyze antibodies and immunoglobulins.
  • Protein Complexes and Aggregates: Researchers can study protein-protein interactions and identify protein aggregates, which is crucial for understanding protein stability and function.