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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Size Exclusion Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


• 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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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/
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Size Exclusion Chromatography


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

PD Dr. J. Graßmann; PD Dr. T. Letzel