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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 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) Created with MindGenius Business 2005® 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 Created with MindGenius Business 2005® 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®