Gel Permeation Chromatography (GPC)

Gel Permeation Chromatography (GPC)

Gel Permeation Chromatography (GPC) is a type of high-performance liquid chromatography. GPC is also referred to as Size Exclusion Chromatography (SEC) and is based on the principle of size exclusion, in which components flowing in solution through a chromatographic column, are separated based on molecular size. The GPC column is packed with semi-permeable, porous polymer gel beads, or with insoluble inorganic porous particles, such as spherical silica. Stagnant liquid present in the pores of beads forms the ‘stationary phase’, while the analyte, dissolved in a suitable solvent, flows through the column as the ‘mobile phase’. A wide range of non-polar organic to aqueous solvents can be used. The mobile phase can flow between the beads and also diffuse into and out of the pores in the beads. Smaller molecules in the mobile phase enter the pores of the gel easily, whereas larger molecules are excluded. The smaller molecules are therefore retained in the column for a longer duration than larger molecules. The molecules exiting the column are detected and characterized using one or more detectors. The elution behavior of the sample is displayed in a chromatogram, showing quantity detected as a function of time. The highest molecular weight components are detected first, followed by successively lower molecular weight components. 

Various types of detectors can be used, such as differential refractive index (DRI), UV, evaporative light scattering (ELS), static light scattering detectors, and viscometers. The other components of the GPC instrument include a pump to push the solvent through the instrument, an injection port to introduce the test sample onto the column and control panel hardware and software. GPC is usually carried out at room temperature, but some instruments have thermostatically controlled heating ovens in which the columns and detectors are placed. Higher temperatures are needed for higher viscosity solvents or to dissolve the components.

The GPC analysis procedure involves several steps, including polymer gel bead selection, sample preparation, column packing, gel swelling, washing with buffer solution, analyte preparation, and then running the column to detect the eluted components.

GPC is primarily used for the characterization of synthetic or natural macro-molecules and the separation of their mixtures into fractions such as polymer, oligomer, monomers, and additives. Since separation is based on molecular size, GPC gives information on the size of polymer molecules in solution, which can be converted into molecular weights using calibration with standard polymers of known molecular weight. GPC can provide the molecular weight distribution of polymers. Applications include analysis of synthetic polymers, oligomers, proteins, enzymes, polysaccharides, lignin, and other biomolecules.

Common Uses of Gel Permeation Chromatography (GPC)

• Proteins fractionation
• Molecular weight determination of synthetic and natural macro-molecules 
• Molecular weight distribution determination
• Separation of polymers or other macromolecules on the basis of their size.

Advantages of Gel Permeation Chromatography (GPC)

• Rapid testing
• Well-defined separation.
• Good sensitivity.
• Wide range of solvents and column packings available
• Less quantity of sample required. 

 Limitations of Gel Permeation Chromatography (GPC)

• Only a limited number of peaks can be resolved within the short time scale of the GPC run.
• Pre-filtration of samples is needed to prevent damage to columns and detectors.

 Industrial Applications of Gel Permeation Chromatography (GPC)

• Research and development
• Polymer Analysis
• Purification of enzymes, polysaccharides, nucleic acids, proteins, and other biological macromolecules

More Details:

5990-6969EN GPC SEC Chrom Guide.pdf (agilent.com) 

https://microbenotes.com/gel-permeation-chromatography/

Gel permeation chromatography – Wikipedia