1. Size Exclusion Chromatography (SEC)
Molecular exclusion Chromatography
Gel filtration chromatography
Gel permeation chromatography.
Principle:
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.

2. History: ’s Porath and Flodin describe the separation of water soluble macro molecules on cross-linked polydextran gels.
Molecules are separated according to their size. Big molecules cannot penetrate the small pores in the stationary phase. They are eluted by a volume of solvent equal to the volume of mobile phase
Kav =
Vr – V0
Vt – V0 Vt
V0: volume of the mobile phase (void volume)
Vr: retention volume for a solute V0
Vt : total volume of the column, r2 x length
V0 found by e.g. Blue Dextran 2000

3. Molecular Exclusion Chromatography Molecules are separated according to their size
SEC separation of two macromolecular sizes:
1.Sample mixture before entering the column
packing
2.Sample mixture upon the head of the column
3.Size separation begins
4.Complete resolution
Typical SEC calibration curve:
logarithm of M.Wt. versus
retention volume

4. CHROMATOGRAPHY
Mobile phase is delivered to a column with the appropriate stationary phase, using a pump at a reproducible and constant flow rate.
mg of polymer sample is injected.
Sample is detected by at least one detector.
Due to the logarithmic relation of mass and elution time, a change in flow-rate of 0.1% can cause an error in molar mass of up to 10%.

5. Stationary Phase Polymers of glucose: Polymers of agarose
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

6. Columns Packing: Usual size of SEC columns:
7-8 mm diameter (analytical)
20-25 mm diameter (preparative)
Length cm
Packing:
porous silica
cross-linked organic gels
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.

7. Selection of SEC columns:
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 (calibration, life of the column)
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

8. Applications Typical SEC applications:
analysis of synthetic polymers and oligomers
coal derived substances
lipids
proteins
cellulose derivatives
crude oil alkanes

9. Separation of proteins
Adenylate kinase
Cytochrome C Enolase kinase Glutamate Dehydrogenase Lactate Dehydrogenase
32000
12400
67000
290000
140000

10. Choosing the pore width
Organic soluble polymers and styrene/divinylbenzene packings
The table lists the molecular weight range of separation for individual pore size columns of styrene/divinylbenzene packings, based on polystyrene chain length exclusion limits (in Angstroms)

11. Instrument parameters
flow rate ( ml/min, default 1 ml/min)
injection volume ( l, default 10 l)
temperature ( ºC, default 40 ºC)
column length ( mm, default 300 mm)
column ID (2-20 mm, default 7.5 mm)
connection (dead) volume ( µl)
detector type (Refractive Index, Viscosity, Density, Lightscatter
detector volume ( µl)
detector path length (1-25 mm)
detector time constant ( s)

12. THANK YOU