Transcript Size Exclusion Chromatography (1)

Size Exclusion Chromatography (1)
Separates molecules with a high molecular weight. on the basis of size

Packing consists of small (~10mm) porous particles made of silica or a
polymer
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Separation is dependent on selective penetration of analytes into pores
(requires at least 10% difference in molecular weight)
Theory
Total column volume Vt = Vg + Vi + V0 , where
Vg is the volume occupied by the packing
Vi is the volume of solvent in the pores, and
V0 is the free solvent volume (similar to injection volume)
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Size Exclusion Chromatography (2)
Analytes may:
1) be too large to enter the pores at all, and elute at V0
2) enter the pores completely, and elute at V0 + Vi
3) partially (extent determined by K) interact with the pores and elute at V0 + Kvi
Exclusion limit: the molecular weight beyond which no interaction with the
pores is possible. All analytes beyond exclusion limit elute together at V0*
Permeation limit: the molecular weight below which complete penetration of
the pores occurs. All analytes below permeation limit elute together at (V0 + Vi)*
Selective permeation region: size between permeation limit and exclusion
limit, where 0<K<1 Elution volume dependent on K*
*Assumption: no other interactions taking place
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Size Exclusion Chromatography (3)
Applications
1) Simple separations: e.g. a large protein from low Mw contaminants such as
amino acids and salts
2) Separation of oligomers: e.g. Series of fatty acids of increasing size
3) Separation of homologs: e.g. sugars in fruit juice
4) Determination of molecular weight: e.g. of a polymer with behaviour
calibrated for the conditions used
Advantages
 Short analysis time
 Well defined separation times
 Narrow bands and good sensitivity
 Few problems with column contamination or sample loss
Disadvantages
 Limited number of peaks
 Requires ~10% difference in molecular weight
Created with MindGenius Business 2005®