How Does Size Exclusion Chromatography Work: A Comprehensive Guide

how does size exclusion chromatography work


Size exclusion chromatography (SEC), also known as gel filtration chromatography, is a powerful technique used in biochemistry and analytical chemistry to separate and purify molecules based on their size. This comprehensive guide will explain how size exclusion chromatography works and its applications in various fields.

Principle of Size Exclusion Chromatography

Size exclusion chromatography operates on the principle of molecular size exclusion. It utilizes a porous stationary phase, typically a gel matrix, which contains pores of varying sizes. When a sample mixture is injected into the column, smaller molecules can enter the pores and are therefore delayed in their elution, while larger molecules pass through the column more quickly.

Column Setup and Components

A typical size exclusion chromatography setup consists of a column packed with a gel matrix, a sample injection port, a mobile phase (buffer), and a detector to monitor the eluted fractions. The gel matrix is chosen based on the desired separation range, and the mobile phase is selected to ensure optimal separation and stability of the sample.

Sample Preparation and Injection

Before injecting the sample, it is crucial to prepare it appropriately. The sample should be dissolved in a compatible buffer and filtered to remove any particulate matter. A small volume of the prepared sample is then injected into the column using an autosampler or a manual injection valve.

Separation and Elution

As the sample enters the column, molecules of different sizes interact with the gel matrix. Larger molecules cannot enter the pores and, therefore, take a shorter path through the column, eluting first. Smaller molecules, on the other hand, enter the pores and experience a longer path, resulting in delayed elution. This differential elution based on size allows for the separation of molecules within the sample mixture.

Detector and Data Analysis

The eluted fractions are detected by a suitable detector, such as a UV-Vis spectrophotometer or a refractive index detector. The detector generates a signal that is proportional to the concentration of the eluted molecules. The data obtained from the detector is then analyzed to determine the molecular weight and size distribution of the sample components.

Applications of Size Exclusion Chromatography

Size exclusion chromatography finds applications in various fields, including protein purification, polymer characterization, and pharmaceutical analysis. It is commonly used to determine the molecular weight of proteins, separate protein aggregates, analyze polymer size distribution, and assess the purity of pharmaceutical formulations.


Size exclusion chromatography is a versatile technique that allows for the separation and purification of molecules based on their size. By utilizing a porous gel matrix, this technique provides valuable information about the molecular weight and size distribution of the sample components. Understanding the principles and applications of size exclusion chromatography is essential for researchers and scientists working in the fields of biochemistry, analytical chemistry, and pharmaceutical sciences.



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