Gel filtration chromatography is one of the chromatography methods that facilitate particles separation based on the molecular size. It also called size exclusion and gel permeation chromatography.
The source of particle separation is achieved by employing a filtration technique via gel beads. The gel beads possess specific porosity that retains or exclude the components with varying sizes when passed through this column.
Content: Gel Filtration Chromatography
Definition of Gel Filtration Chromatography
Gel filtration chromatography can define as the method of chromatography that makes the use of porous gel beads of specific porosity to isolate components depending upon their molecular sizes. This technique principally retains or excludes particles based on size difference, that’s why it is also known as size exclusion or gel exclusion chromatography. It mainly helps in the isolation of biomolecules like proteins, peptides and oligonucleotides.
Phases in Gel Filtration Chromatography
Mobile phase: The solvent running through the column is the mobile phase. The test sample must be diluted in an appropriate organic solvents, then filtered and finally passed onto the column. The separation of a multi-component mixture takes place in the column.
Stationary phase: The column packed with microporous gel beads acts as the stationary phase. Hydroxypropylated Sephadex, cross-linked polyacrylamide, agarose gel and are mainly used to separate different biomolecules.
The principle of size exclusion chromatography mainly based on the isolation of biomolecules based on differences in molecular weight or size. Gel filtration technique employs spherical gel beads with definite porosity as the packing material in the chromatography column.
In gel filtration chromatography, the components in a liquid mixture pass through a column of porous gel beads, where some will elute earlier or later through the column, depending on the elution limit. The elution limit is a factor that decides the retention or exclusion of molecules eluted through the column.
Molecules possess high molecular weight compared to the elution limit will elute early, while those molecules that possess low molecular weight or size than the elution limit will elute later. Therefore, in this way, the particles are separated in the gel filtration chromatography.
The process of gel filtration chromatography involves the following sequential steps:
- Firstly, a column of spherical gel beads is prepared for the gel filtration chromatography.
- The packed bed is equilibrated with a buffer.
- Then the test solution (mobile phase) is eluted through the column.
- After that, the particles in the test sample will enter into or diffuse out of the porous gel matrix (stationary phase).
- The molecules that are having small molecular size will enter into the pores of gel, i.e. small molecules will cover a longer path or stay longer on the column.
- The molecules possess large molecular size cannot get into the pores of gel bead or easily pass through the column.
- Thus, the separation of the particles occurs at different intervals, by following isolation and identification of the components separated.
Fractionation and desalting are the methods that accomplish the separation of components in size exclusion chromatography.
Desalting: The heavy molecules are eluted, by keeping the exclusion limit of the gel smaller, so that the smaller molecules can trap into the pores of gel, while the larger particles get separated.
The molecules separated from the gel filtration chromatography unit depends on the size distribution of microporous gel. It helps to construct a calibration curve that in turn determines the molecular weight of an unknown analyte in the sample by comparing the molecular weight of known analytes.
- Gel permeation chromatography is a robust method for the purification of biomolecules like enzymes, polysaccharides, nucleic acids, proteins etc.
- Gel filtration unit can enable renaturation of denatured proteins.
- It is used in protein fractionation experiments.
- By employing gel filtration chromatography, the molecular weight of the separated particles can be examined.
- It is method that can examine the quaternary structure of purified proteins.
- Gel filtration can separate the biomolecules, sensitive to varying pH, temperature, the concentration of metal ions etc.
- The particles do not stick to the chromatography medium, like in an ion-exchange chromatography.
- The explication of result can be made within a minimal time.
- It gives a well-defined separation.
- Small amount of test sample is enough to conclude the results.
- The flow rate can be set.
- The test sample must be filtered before passing it through the gel filtration column to prevent the clogging of particles within the instrument.
- The instrument must be dust free and other particulate matter that may interfere with the result interpretation.
Therefore, the gel filtration chromatography is a technique that depends upon the molecular weight of the biomolecule, size distribution of gel beads and elution limit. The movement of small molecules is slower than the large molecules, due to rapid diffusion into the pores. Oppositely, the larger particles unable to move into the pores, so move more quickly and are eluted out.