Gel Filtration Chromatography

By Sushmita Baniya •  Updated: 06/06/22 •  6 min read

Gel filtration chromatography separates the samples based on the molecular size and shape of species present in them. It is also called gel permeation chromatography, molecular sieve chromatography, or elution chromatography.

Principle

Gel filtration chromatography is a separation technique that involves the inclusion and exclusion of solutes through a stationary phase. The stationary phase consists of hetero-porous cross-linked polymeric gel. Separation occurs between the liquid phase within the gel particle and the liquid surrounding the gel particles. By the liquid flow, molecules diffuse in all parts of a gel. A larger molecule appears first, followed by a smaller molecule. This separation is possible due to the size barrier without the gel particle.

Gel chromatography is performed in a column by elution. The small molecules diffuse into the gel and follow a longer path than the larger molecule, which releases from the gel particles. Then compound separation occurs with the large molecules leaving the column first and the small ones last.

Gel Filtration Chromatography
Gel filtration chromatography

First, the large and small molecules mixture is poured into the column. As the molecules pass down, the small molecules diffuse into the gel and follow a longer path than the large molecules. The separation occurs with the large molecules leaving the column first and smaller ones last.

The solid support or gels used in gel chromatography are polymeric organic compounds that possess a three-dimensional network of pores. These gels tend to swell in a suitable solvent, and as a result of the swelling of gel, the space between the polymer gels increases in size. For compounds larger than a gel, there will be a critical molecule size that can just penetrate the interior. Molecules more prominent than gel pores are wholly excluded from the gel. A good gel should be inert and chemically stable. For ordinary laboratory work, powder gel of particle size of 70 µm in diameter is sufficient. Commonly used gels are polymers of dextran, agarose, and polyacrylamide. 

Steps in Gel Filtration Chromatography

Selection of a column

The column used in gel chromatography is usually made of glass material of constant cross-sectional area. It consists of a mobile phase reservoir at the top and the bottom of the column consisting of a straight glass tube with bead support at the bottom of the glass rod. The bead support allows only the liquid or solvent to pass through without disturbing the chromatographic matter. For experimental purposes, use a column of suitable dimensions. Preferable dimensions are 100cm in length and 1-2 cm in diameter.

Gel preparation

Different methods can carry out gel preparation. The two common ways are as follows:

  • Mixing dry power gel with an excess of eluent (solvent). Allow it to swell. And leave until the equilibrium condition is achieved. This process takes a long time, from a few hours to a few days.
  • Mix the powder gel with an excess solvent. And warm the thus obtained slurry to about 100°C for about 30 minutes in a water bath. So the gel swells in a few hours.

Packing at the column

Prepare the slurry of the gel in a beaker. Then clamp the glass column packed with gel in a vertical position and fill with slurry gel slowly. Pour the slurry carefully to avoid air bubbles. Let the suspension settle down. Keep the solvent just above the surface of the chromatographic material.

Application of the sample

Use a glass pipette to pour the sample at the top of the gel surface and allow it to run in the column. Increase the density of the sample by adding sucrose to the concentration to avoid the necessity to train the column. When this solution is layered onto the solvent, above the column bed, it will automatically sink to the surface of the gel and hence can be quickly passed into the column. In all cases, avoid overloading the column with the sample. Otherwise, irregular separation will occur.

The solvent used in gel chromatography has three main functions:

  • The solvent puts the mixture in the column.
  • Solvent brings about the separation of the mixture into different zones.
  • Solvent also elutes the components of each separated zone.

Elution development

After the sample application, add more solvent carefully to the height of 2-5 cm. After that connect the column to a suitable reservoir so that the height of the solvent maintains at the appropriate height.

Then, separate the sample components by the continuous separation of solvent through the column. It is called elution development.

Collection and analysis of eluent

Collect the effluent in a series of test tubes either by automatic infraction or manually on a collector. Keeping the flow rate 1ml/min, collect each fraction.
Use chemical agents to convert non-colored compounds to colored compounds. Analyze the colored compounds by measuring the absorbance by a spectrophotometer or colorimeter.

Carry out Molisch’s or Benedict’s test to detect and analyze monosaccharides. And measure optical density at 510nm wavelength.

Application of Gel Filtration Chromatography

Gel filtration chromatography has many applications in scientific research some of them are:

  • To differentiate sugars, salts, proteins, amino acids, lipids, and different polymers.
  • To purify biological macromolecules, viruses, proteins, enzymes, hormones, antibodies, nucleic acids, and polysaccharides.
  • To concentrate on the solution.
  • To quantify the sample.

Advantages of Gel Filtration Chromatography

Some of the advantages of gel filtration chromatography are as follows:

  1. Short analysis time
  2. Well-defined separation
  3. Narrow bands
  4. Good sensitivity
  5. Small amount of mobile phase
  6. Fixed rate of flow

Limitations of Gel Filtration Chromatography

Although gel filtration chromatography is very useful, it has some limitations. They are:

  • Filter the instrument before using it. It prevents dust and other particulates from ruining the column and interfering with detectors.
  • A reasonable resolution requires at least a 10% difference in molecular weight.

References

  • Prapulla, S. G., & Karanth, N. G. (2014). Fermentation (Industrial): Recovery of Metabolites. In Encyclopedia of Food Microbiology: Second Edition (Second Edition, Vol. 2). Elsevier. https://doi.org/10.1016/B978-0-12-384730-0.00109-9
  • Prapulla, S. G., & Karanth, N. G. (2014). Fermentation (Industrial): Recovery of Metabolites. In Encyclopedia of Food Microbiology: Second Edition (Second Edition, Vol. 2). Elsevier. https://doi.org/10.1016/B978-0-12-384730-0.00109-9

Sushmita Baniya

Hello, I am Sushmita Baniya from Nepal. I am a postgraduate student of M.Sc Medical Microbiology. I am interested in Genetics and Molecular Biology.

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