Mass Spectrometry & Purification Techniques
Open Access

Size-Exclusion Chromatography: A Fundamental Concept and Methodology

Zareena Soni^{* *}Correspondence: Zareena Soni, Department of Chemistry, University of Kolkata, Kolkata, India, Email:

Author info »

Description

Size-Exclusion Chromatography (SEC), also known as gel filtration chromatography, and widely used analytical technique in the field of separation science. It provides valuable information about the size and molecular weight distribution of macromolecules and nanoparticles. This short note aims to provide an overview of size-exclusion chromatography, its principles, methodology, and applications.

The fundamental principle behind size-exclusion chromatography is based on the differential interaction of molecules with a porous stationary phase. In size-exclusion chromatography, a column packed with porous beads is used as the stationary phase. These beads have a range of pore sizes, allowing molecules to enter and diffuse through the pores. The larger molecules, such as proteins or polymers, cannot enter the smaller pores and therefore elute faster from the column, while smaller molecules are more readily trapped within the pores, resulting in delayed elution. The separation process in sizeexclusion chromatography is mainly governed by the size and shape of the molecules. As the sample is injected into the column, it undergoes a size-based separation, with larger molecules eluting first and smaller molecules eluting later. This separation is achieved without any chemical interactions between the sample molecules and the stationary phase, making sizeexclusion chromatography particularly useful for preserving the structural integrity and biological activity of the analyzed molecules.

The methodology of size-exclusion chromatography involves several key steps. First, a suitable stationary phase with a defined pore size distribution is chosen based on the target analytes. Commonly used materials include cross-linked agarose, polyacrylamide, or silica-based gels. The sample, dissolved in an appropriate mobile phase, is then injected onto the column, and molecules are allowed to migrate through the porous beads. The eluted molecules are detected using various detection techniques, such as ultraviolet-visible spectrophotometry, refractive index detection, or multi-angle light scattering, which provides information on the molecular weight and size distribution.

Size-exclusion chromatography finds widespread applications in various scientific fields. In biochemistry and biotechnology, sizeexclusion chromatography is frequently employed for the purification and characterization of proteins, nucleic acids, and other biomolecules. It enables the separation of target molecules from impurities and aggregates, facilitating downstream analyses and maintaining sample integrity. Size-exclusion chromatography is also utilized in polymer chemistry to determine the molecular weight distribution and monitor the polymerization process.

Additionally, size-exclusion chromatography is an essential tool in nanoparticle characterization, allowing researchers to evaluate the size distribution and aggregation behavior of nanoparticles for applications in drug delivery, nanomedicine, and materials science. One of the significant advantages of size-exclusion chromatography is its versatility and compatibility with a wide range of solvents and sample matrices. It can be performed in both analytical and preparative modes, making it suitable for both qualitative and quantitative analyses.

SEC can handle complex mixtures and is often used in combination with other chromatographic techniques, such as high-performance liquid chromatography or mass spectrometry, to achieve comprehensive sample characterization.

In conclusion, size-exclusion chromatography is a powerful analytical technique for the determination of molecular size and weight distribution. Its non-destructive nature, compatibility with various sample types, and broad range of applications have made it an indispensable tool in numerous scientific disciplines. Sizeexclusion chromatography keeps making important contributions to our understanding of macromolecules, polymers, and nanoparticles.