In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.

Silica particles offer advantages such as rigidity, stability, and compatibility with various solvents. In contrast, polymer beads come in different pore sizes and can be hydrophilic (water-loving) or hydrophobic (water-repelling). Hydrophilic gels like agar and polyacrylamide are used for aqueous separations, while hydrophobic gels such as polystyrene-divinylbenzene are preferred for non-polar solvents. Silica particles used for size exclusion are deactivated to prevent unwanted interactions, while polymer resins are synthesized without exchange sites.

Smaller solutes spend more time within the pores of the stationary phase, resulting in a longer elution time from the column. The retention volume for a solute depends on its distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. This reflects how well the solute penetrates the pores.

SEC is a notable application in the analysis of protein mixtures and the determination of formula weights. Calibration curves can be prepared between the exclusion and inclusion limits, but accurate formula weight determinations require carefully chosen standards to minimize the effect of shape.

This technique can be carried out using conventional HPLC instrumentation by replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is typically used to obtain chromatograms.

From Chapter 11:

article

Now Playing

11.22 : Size-Exclusion Chromatography

Principles of Chromatography

302 Views

article

11.1 : Chromatographic Methods: Terminology

Principles of Chromatography

452 Views

article

11.2 : Chromatographic Methods: Classification

Principles of Chromatography

529 Views

article

11.3 : Analyte Adsorption and Distribution

Principles of Chromatography

360 Views

article

11.4 : Diffusion on Chromatography Columns

Principles of Chromatography

230 Views

article

11.5 : Chromatographic Resolution

Principles of Chromatography

196 Views

article

11.6 : Column Efficiency: Plate Theory

Principles of Chromatography

244 Views

article

11.7 : Column Efficiency: Rate Theory

Principles of Chromatography

162 Views

article

11.8 : Optimizing Chromatographic Separations

Principles of Chromatography

235 Views

article

11.9 : Silica Gel Column Chromatography: Overview

Principles of Chromatography

665 Views

article

11.10 : Thin-Layer Chromatography (TLC): Overview

Principles of Chromatography

658 Views

article

11.11 : Gas Chromatography: Introduction

Principles of Chromatography

327 Views

article

11.12 : Gas Chromatography: Types of Columns and Stationary Phases

Principles of Chromatography

219 Views

article

11.13 : Gas Chromatography: Sample Injection Systems

Principles of Chromatography

218 Views

article

11.14 : Gas Chromatography: Overview of Detectors

Principles of Chromatography

203 Views

See More

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved