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Size Exclusion Chromatography (SEC)

Introduction

Size-exculsion chromatography (SEC), also called gel-filtration or gel-permeation chromatography (GPC), uses porous particles to separate molecules of different sizes. It is generally used to separate biological molecules, and to determine molecular weights and molecular weight distributions of polymers. Molecules that are smaller than the pore size can enter the particles and therefore have a longer path and longer transist time than larger molecules that cannot enter the particles.

Schematic of a size-exclusion chromatography column

Molecules larger than the pore size can not enter the pores and elute together as the first peak in the chromatogram. This condition is called total exclusion. Molecules that can enter the pores will have an average residence time in the particles that depends on the molecules size and shape. Different molecules therefore have different total transit times through the column. This portion of a chromatogram is called the selective permeation region. Molecules that are smaller than the pore size can enter all pores, and have the longest residence time on the column and elute together as the last peak in the chromatogram. This last peak in the chromatogram determines the total permeation limit.


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