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.

History: 1960’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

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

CHROMATOGRAPHY Mobile phase is delivered to a column with the appropriate stationary phase, using a pump at a reproducible and constant flow rate. 0.01-1.0 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%.

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

Columns Packing: Usual size of SEC columns: 7-8 mm diameter (analytical) 20-25 mm diameter (preparative) Length 20-60 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.

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

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

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

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)

Instrument parameters flow rate (0.1-10 ml/min, default 1 ml/min) injection volume (1-1000 l, default 10 l) temperature (25-220 ºC, default 40 ºC) column length (50-1200 mm, default 300 mm) column ID (2-20 mm, default 7.5 mm) connection (dead) volume (0-1000 µl) detector type (Refractive Index, Viscosity, Density, Lightscatter detector volume (1-1000 µl) detector path length (1-25 mm) detector time constant (0.01-5 s)

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