1. Size exclusion Chromatography

2. Size exclusion Chromatography
Also called:
Gel filtration chromatography (MP water): desalting
Gel permeation chromatography (MP organic solvent)
Molecules are separated according to their size
Small molecules penetrate the small pores in the stationary phase, while large molecules do not.
Large molecules are eluted first.

3. History Applications Species can be: single molecules polymer coils
Dates to the 1960’s.
Porath and Flodin describe the separation of water soluble macromolecules on cross-linked polydextran gels.
Applications
It separates according to the size of species in solution (hydrodynamic volume)
Species can be:
single molecules
polymer coils
aggregates
micelles
etc…

4. Applications Typical SEC applications:
analysis of synthetic polymers and oligomers
lipids
proteins
cellulose derivatives
crude oil alkanes
SEC methods can also be used to:
hydrolysis
refolding of proteins
polymerisation
aggregation
etc

5. Size exclusion Chromatography
Big molecules cannot penetrate the small pores in the stationary phase. They are eluted by a volume of solvent equal to the void volume between beads of the stationary phase 1 2 3 4
Small molecules penetrate the small pores. They are eluted by a volume of solvent equal to the total volume of mobile phase
SEC separation of two macromolecular sizes:
Sample mixture before entering the column packing
Size separation begins
Collecting of the large molecules fraction
Collecting of the small molecules fraction

6. Size exclusion Chromatography
Vt: total void volume
(volume that is penetrable to small molecules is penetrable to small molecules)
V0: external void volume
(volume that is accessible to large molecules. This is the total volume without the pore volume)
Electron microscope picture of a SEC adsorbent

7. Size exclusion Chromatography
Retention of molecules in SEC is characterized by partition coefficient
Kav =
Vr – V0
Vt – V0
Vr: retention volume for a solute
V0: external void volume
Vt : total void volume (total volume of the mobile phase in a column

8. Typical SEC calibration curve: logarithm of molecular weight versus
Big molecules:
Vr = V0; Kav = 0
Kav =
Vr – V0
Vt – V0
Small molecules:
Vr  Vt; Kav  1
FLOW
Total void volume
Exclusion volume
Typical SEC calibration curve:
logarithm of molecular weight versus
retention volume

9. Stationary Phase Polymers of glucose: Cellulose
Dextran (If cross-link is glycerin: Sephadex)
Polymers of agarose
Polyacrylamides
 Crossed-linked gels
Small pore size
Exclude molecules with MM  700
 Crossed-linked gels
Big pore size
Exclude molecules with MM  108
The finer the particle, the greater the resolution, the slower the flow rate of the column
Particles with different pore sizes can be mixed to give a wider molecular size separation range
In general, two types of molecules can be separated provided that there is a 10% difference in their molecular sizes

10. Columns
Unlike the other modes of liquid chromatography, the separation comes from the stationary phase only. The mobile phase should have no effect as long as the sample is well dissolvable.
Separation is carried out on the pores which typically equals 40% of the total column volume.
As a result long columns, or several columns are required.
Usual size of SEC columns:
7-8 mm diameter (analytical)
20-25 mm diameter (preparative)
Length cm
Packing:
porous silica
cross-linked organic gels

11. Columns Selecting SEC columns: Handling SEC columns:
Separation range selected carefully
Small particle size (5µm) provides more theoretical plates
Small particle size more sensitive to contamination
Small particle size can result on shear degradation of large polymers
Combination of packings with different separation range can be achieved by: columns of different porosity or mixed bed columns
Chemical nature of column packing crucial
Handling SEC columns:
A column set should be always run in the same mobile phase
Should not be operated in backward position
No air bubbles in columns during injection and assembly
Always use a pre-column when in doubt about the quality of the sample
Handbook of Size Exclusion Chromatography and Related Techniques: Revised And Expanded
By Chi-san Wu
Edition: 2, illustrated, revised
Published by CRC Press, 2004
694 pages

12. SEC polymers Calibration
Molecular Mass Determination
SEC polymers Calibration

13. Molecular Mass Determination
Molecular Mass calibration graph for polystyrene on Beckman SpherogelTM size exclusion column. Resin pore size ranges from 5 nm to 100 µm.

14. Molecular Mass Determination
Caution:
Branching
Possible adsorption due to occasional charges on the gel
Other adsorption mechanisms (silica must be coated with a hydrophilic phase to minimise solute adsorption)

15. Branching
Molecular size difference between a linear chain and a branched chain polymer with the same molecular weight
At the same molecular weight branched polymers have lower hydrodynamic volume

16. Choosing the pore width
Pore width and separating range of a typical commercial column set (Waters)

17. Separation of proteins
(c) Agilent
Lysozyme 14.3 K
OVA 45 K
Tyrosin 180
Leucin-Enkephalin
Carbonic Anhydrase 29 K
Insulin 5.8 K
Gamma-Globulin 150 K
Cytochrome C 12.4 K
BSA 66 K
Thyroglobulin 660K
Eluent: 0.4 M NaCl, 10mM Na-phosphat, pH 7.4 Factor in molecular weight needed for separation
Eluent: 0.4 M NaCl, 10mM Na-phosphat, pH 7.4 Factor in molecular weight needed for separation

18. Separation of proteins
MATRICES
Conventional
Conventional
Cheap,
Slow flow rate,
Available in bulk
For open column chromatography
High Performance
High Performance
Expensive,
Rapid flow rate,
Available ready-to-use,
For HPLC
Abbreviation: AG, cross-linked agarose; PA, cross-linked polyacrylamide; DX, cross-linked dextran; DX/DA, copolymer of allyl dextran and bisacrylamide; AG/PA, mixture of agarose and polyacrylamide; S, bonded silica.
E. Stellwagen, in: Guide to protein purification Ch. 23

19. Separation of proteins
Mobile Phases
Separation in SEC mostly depends on the stationary phase and does not depend on the properties of an eluent. However precautions must be taken to minimize the binding of proteins to the matrix by electrostatic or by van der Waals interactions. Enzymatic hydrolysis of proteins is another danger.
To face these problems
the ionic strength of the chromatographic solvent should be at least 0.2M
proteolytic inhibitors need be present in the chromatographic solvent
addition of insecticides like 0.02% NaN3 is desirable to prevent the growth of microorganisms

20. Hint
SEC has a relatively low resolution power. Fewer than 10 proteins can be resolved from one another in the effluent from any size-exclusion column. Significant enhancement in purification (-fold) by SEC can be expected if the desired protein has a molecular weight either considerably larger or smaller than that of the majority of proteins in a mixture. Accordingly, it is wise to perform size-exclusion chromatography relatively late in a purification procedure when the numbers of other proteins are small and when the preceding step has fractionated the protein mixture on the basis of a completely different property.
E. Stellwagen, in: Guide to protein purification Ch. 23