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Size-exclusion Chromatography

  1. Lars Hagel,
  2. Lars Haneskog

Published Online: 17 JUN 2010

DOI: 10.1002/9780470015902.a0002676.pub2

eLS

eLS

How to Cite

Hagel, L. and Haneskog, L. 2010. Size-exclusion Chromatography. eLS. .

Author Information

  1. GE Healthcare Bio-Sciences AB, Uppsala, Sweden

Publication History

  1. Published Online: 17 JUN 2010

Abstract

Size-exclusion chromatography (SEC), also termed gel filtration (GF) and gel permeation chromatography (GPC), separates solutes according to decreasing size. Typical applications are desalting (buffer exchange), fractionation (purification) and size or size-heterogeneity analysis (mass determination). The theory of SEC is well established and simulations of experiments may readily be performed to aid in optimisation of experimental conditions, although optimisation often is not needed. Unlike many other chromatographic techniques separation using SEC allows a limited volume of sample since it is not concentrated on the column. The acceptable volume depends on column size and mode of separation. SEC is possibly the most common final step in protein purification because of its simplicity and its power to remove not only impurities but also aggregates of the target protein. Buffer exchange of protein preparations by SEC is a good alternative to ultrafiltration or dialysis methods because of speed and low risk of protein aggregation.

Key Concepts:

  • Size-exclusion chromatography (SEC) separates substances according to size.

  • SEC is used for desalting (buffer exchange), fractionation (purification) and size or size-heterogeneity analysis.

  • SEC often accepts a broad range of solvent conditions.

  • Only a limited sample volume should be applied in SEC since no concentration of sample components take place on the column. A too large sample volume may compromise separation.

  • Highest separation in SEC requires a long column with small beads and good quality of packing.

  • In method optimisation the choice of SEC material (selectivity and efficiency) is usually more important than running conditions.

  • The peak capacity in SEC is generally much smaller than for other chromatography techniques.

  • SEC is a key technique for the final step (polishing) when purifying proteins. Impurities and aggregated or fragmented target protein can be removed.

  • SEC can be used for analysis of protein size-homogeneity under native conditions, for example for screening purification or storage conditions.

Keywords:

  • gel filtration;
  • size-exclusion chromatography;
  • size separation;
  • molecular mass;
  • protein purification;
  • desalting;
  • buffer exchange;
  • protein analysis;
  • characterisation