Chromatography - Protein chromatography Ion exchange...

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Protein chromatography 9 Ion exchange chromatography 9 Affinity chromatography Jaana Vesterinen Institute of Biomedicine, University of Helsinki, 2008
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Chromatography ± Separation of biomolecules is based on their physicochemical characteristics ± polarity (solubility, volatility, adsorption) HIC, RP ± size/mass (diffusion, sedimentation) GF ± ionic characteristics (charge) IEX ± shape (ligand binding, affinity) Affinity chr ± based on these properties the molecules differentially separate between the stationary phase and mobile phase
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Liquid chromatography (LC) ± IEX anion exchange cation exchange chromatofocussing ± Affinity chromatography group separations vs. specific interactions ± HIC (hydrophobic interaction chr.) /RP ± Gel filtration/size exclusion; adsorption chromatogr. high resolution
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Adsorptive chromatography ± biomolecule adsorps to the matrix (stationary phase) reversibly ± adsorption is controlled by the mobile phase - elution eg. IEC: proteins in a low-salt mobile phase may be bound to the matrix, but when the composition of the mobile phase is changed to high-salt, the interaction is reversed and the proteins elute
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Liquid chromatography, basics Eluent Resin, stationary phase Protein mix Proteins Buffer, mobile phase Chromatography steps: 1. Eqilibration 2. Injection 3. Elution 4. Washing
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Chromotogram, basics V o V tot Separation area
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Matrix Porous Nonporous cellulose styrene sugar polymers (agarose, dextran) acrylates polymers (acrylamide, styrene) zirkon silica coated with polymers monolith (ProSwift, Dionex) particle size, ~5 um ~2 um pore size eg. 100-200 Å -> chemical and physical stability temperature <80 o Cvs>200 o C) pressure porous<nonporous pH porous<nonporous capacity porous>nonporous
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Diffusion and porous matrixes ± Size exclusion chromatography is based on diffusion and separation based on size, effective size -Small molecules diffuse into smaller pores and travel a longer way, therefore elute last, close to V tot -Large molecules do not fit into pores and elute first, close to V 0 ± In affinity and IE chromatography the functional groups are attached to the particle surfaces inside the pores
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± Composition ± Type of elution ± isocratic vs gradient elution (gradient shape) ± Flow rate ± depends on the matrix ± Affects resolution; in porous matrixes should be
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This note was uploaded on 02/16/2010 for the course CHEM 589 taught by Professor -1 during the Fall '02 term at Pennsylvania State University, University Park.

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Chromatography - Protein chromatography Ion exchange...

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