Size Exclusion Chromatography

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt

Technische Universität München
Size Exclusion Chromatography
PD Dr. J. Graßmann; PD Dr. T. Letzel


• Exclusion chromatography in general encompasses techniques in which
separation is based on exclusion effects like differences in molecular size
and/or shape or charge.
• The term Size Exclusion Chromatography is used when separation is
based on molecular size
• Gel Filtration and Gel Permeation were used earlier to describe techniques
in which the stationary phase is a swollen gel (IUPAC)
• Other definitions use gel filtration as a narrower term for separation of
biomolecules in aqueous or aqueous/organic mobile phase and gel
permeation as a narrower term for separation of organic polymers in non-
aqueous mobile phases


The Media
• Size Exclusion Chromatography (SEC) separates molecules

according to differences in size as they pass through a SEC medium
• The media used for SEC consist of a porous matrix of spherical

particles
• The media are chemical and physical stable an inert (lack of reactivity
and adsorptive properties)
• The matrices are usually composed of naturally occuring polymers

(dextran, agarose) but also synthetic polymers (e.g. polyacrylamide)
are available
• The selectivity of a SEC media depends on its pore size distribution


The polymers are cross-linked and degree of cross-linking determines pore size
Polymer Pores
Cross-linking agent
Different degree
of cross-linking 
Pores Different pore sizes


The Elution
• The packed bed is equilibrated with buffer, which fills the pores of the
matrix and the space between the particles
• The elution then is conducted usually isocratic with buffer
• Instead of retention time often the volume of buffer required to elute

the analyte is used
• There is little to no influence of pH, ionic strength and composition of

buffer on separation since these parameters do not affect size of
molecules


Process of SEC. (A) Schematic
picture of a bead with an electron
microscopic enlargement.
(B) Schematic drawing of sample
molecules diffusing into bead
pores.
(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.
Figure from: Size Exclusion Chromatography Handbook: Principles and Methods; GE Healthcare;
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/de/GELifeSciences-DE/service-and-support/handbooks/

SEC may be used in two different modes:
• Group separation:
To remove small molecules from a group of larger molecules and as a

method of buffer exchange
SEC in group separation mode is often used in protein purification for

desalting and buffer exchange.
• High-resolution fractionation
To separate compounds in a sample based on differences in their size
SEC in fractionation mode may be used to isolate one or more of the

compounds or to analyze the molecular-weight distribution in the sample


Group separation:
The picture illustrates the chromatogram of a group separation used to
desalt a protein solution
Protein = large molecule  elutes first
Salt = small molecule  elutes later
volume


Fractionation:
The picture illustrates a typical chromatogram of a fractionation used to
separate compounds of different size
Column: Superdex Peptide 10/300 GL

Sample:
1. Cytochrome C (Mr 12 384), 1 mg/ml
2. Aprotinin (Mr 6512), 2 mg/ml
3. Vitamin B 12 (Mr 1355) 0.1 mg/ml
4. (Gly)3 (Mr 189), 0.1 mg/ml
5. Gly (Mr 75), 7.8 mg/ml
Sample load: 50 µl
Buffer: 50 mM phosphate, 150 mM NaCl, pH 7.0
Flow rate: 0.5 ml/min, room temperature


Summary
SEC …
• separates molecules on the basis of their size
• columns are packed with media consisting of porous particles
• separation depends on pore size distribution
• can use a wide range of solvents
• can be used to
o remove small molecules from a group of larger molecules (e.g. desalting)
o to separate compounds in a sample based on differences in their size

Size Exclusion Chromatography

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Size Exclusion Chromatography

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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt