Size Exclusion Chromatography (SEC) Overview

Size Exclusion Chromatography (SEC): A Comprehensive Guide

Introduction

Size Exclusion Chromatography (SEC), also known as Gel Filtration or Gel Permeation Chromatography, is a powerful analytical technique used to separate molecules based on their size and molecular weight. This method, which was invented in the early 1960s, is widely used for the purification and analysis of a variety of molecules, including proteins, polymers, and nucleic acids.

Basics of SEC

SEC separates molecules by exploiting the differences in the pores of a porous stationary phase, typically a gel or resin. The stationary phase consists of porous beads, with pores of a specific size distribution. As a molecule passes through the column, it can either enter or be excluded from the pores, depending on its size. Molecules with smaller sizes are able to diffuse into the pores and their flow through the column is retarded, while larger molecules do not enter the pores and are eluted in the column's void volume. This results in the separation of molecules based on their size and molecular weight.

SEC is commonly used for two main applications: fractionation and desalting (or buffer exchange). In fractionation, the goal is to separate mixtures of molecules into individual components. The molecules of interest should fall within the fractionation range of the gel, and the resolution of separation depends on factors such as particle size, pore size, flow rate, column length and diameter, and sample volume. In desalting, the aim is to remove salts and other small molecules from a sample, leaving behind the molecule of interest in the void volume. The gel should have an exclusion limit significantly smaller than the molecule of interest to obtain the desired separation.

SEC Media and Column Selection

Bio-Rad offers a range of media for SEC, including Bio-Gel P polyacrylamide media for MW separations under 100 KD and for desalting, and Bio-Beads S-X media for the separation of lipophilic polymers. Other factors to consider when choosing a column include ensuring the sample doesn't stick to the stationary phase, ensuring the column cannot ionize the sample, and ensuring the column has a sample capacity suited to the experiment's needs.

Applications of SEC

SEC is widely used in various fields, including protein purification, polymer separation, and nucleic acid analysis. It provides a rapid means for separating larger molecules, including polymers and biomolecules. For example, a protein sample can be separated into its individual components based on their size, with smaller proteins eluting first, followed by larger proteins.

Conclusion

In summary, Size Exclusion Chromatography is a versatile and powerful analytical technique used for the separation of molecules based on their size and molecular weight. It has a wide range of applications in various fields, including protein purification, polymer separation, and nucleic acid analysis. The success of SEC depends on factors such as the type of stationary phase, the operating conditions, and the sample's characteristics. By understanding these factors and choosing the appropriate media and conditions, researchers can achieve successful separations and gain valuable insights into their samples.