Size‐exclusion Chromatography

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

Figure 1.

Principle of SEC. (a) SEC beads are composed of porous material, lower picture; electron micrograph of a SEC matrix. (b) Section of a bead shown schematically. During separation, sample components are moving together with the eluent (mobile phase) through the column. Large sample molecules (red) are excluded (if above matrix size‐exclusion limit) from the matrix, and are eluted at the void volume, Vo. Intermediate‐sized molecules (e.g. orange) diffuse in and out of the pores of the beads and become delayed. These molecules thus occupy a larger part of the bead volume than a larger molecule, and therefore have a more delayed elution, VR. Very small molecules occupy the entire pore volume of the bead, and will be eluted at the total volume of the column, Vt. The geometric volume of the column, Vc, is slightly larger than the ‘total volume’, owing to the volume of the matrix material. (c) Graphical description of separation: I. Sample is applied on the column. II. The smallest molecule (yellow) is more delayed than the largest molecule (red). III. The largest molecule is eluted first from the column. Band broadening causes significant dilution of the protein zones during chromatography. (d) Schematic chromatogram. Copyright © 2010 General Electric Company – all rights reserved. Reproduced with kind permission.

Figure 2.

SEC separation examples. (a) Desalting for the removal of excess salt and imidazole from a protein preparation obtained by immobilised metal ion affinity chromatography that involves elution using imidazole. (b) Fractionation of a mouse monoclonal cell supernatant. (c) Analytical SEC of a purified recombinant human growth hormone allows size‐determination and assessment of size‐heterogeneity. Dimers and oligomers can be separated from monomers. Copyright © 2010 General Electric Company – all rights reserved. Reproduced with kind permission.

Figure 3.

Typical columns and devices for desalting ((a)–(e)) and for preparative or analytical fractionation of proteins ((f)–(g)). (a) Column (5 mL) driven by syringe or pump, (b) column (53 mL) driven by pump, (c) column driven by gravity or centrifugation for single or parallel use, (d) column for centrifugation for single or parallel use, (e) devices for parallel desalting driven by centrifugation or vacuum, (f) column for preparative or analytical fractionation and (g) short and narrow column for fast analysis in screening work. Copyright © 2010 General Electric Company – all rights reserved. Reproduced with kind permission.

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References

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Further Reading

Burgess RR and Deutscher MP (2009) Methods in Enzymology 463, Guide to Protein Purification, 2nd edn. London: Academic Press.

Hagel L (1998) Gel filtration. In: Janson J‐C and Rydén L (eds) Protein Purification Principles, High Resolution Methods and Applications, pp. 79–143. New York: Wiley‐VCH.

Striegel AM, Yau WW, Kirkland JJ and Bly DD (2009) Modern Size‐Exclusion Liquid Chromatography: Practice of Gel Permeation and Gel Filtration Chromatography, 2nd edn. New York: Wiley‐Blackwell.

Williams A and Hagel L (1999) Size exclusion for analysis and purification of aqueous macromolecules. In: Wu C‐S (ed.) Column Handbook for Size Exclusion Chromatography, pp. 27–74. London: Academic Press.

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Hagel, Lars, and Haneskog, Lars(Jun 2010) Size‐exclusion Chromatography. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002676.pub2]