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Transcript of Chromatography

  • 1.M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.

2. Chromatography Components stationary phase (eg., solid matrix) mobile phase (eg., solvent) solute Solutes which interact differently with the stationary phase can be separated. 3. Apply Sample Continue Developing with Solvent 4. Common Media Used in Liquid Protein Chromatography Media Type Discrimination Ion Exchange Charge Gel Filtration Size and Shape Hydrophobic Surface Hydrophobicity Reverse Phase Total Hydrophobicity Affinity Specific Amino Acids Adsorption Amino Groups? 5. Chromatography Generic Protocol 1. Prepare Column () 2. Apply Sample 3. Wash 4. Elute 5. Analyze Fractions Equipment batch-wise home made work stations FPLC/HPLC HPLC = High Performance (Pressure) Liquid Chromatography FPLC = Fast Protein Liquid Chromatography 6. Ion Exchange Chromatography (IEC) based on charge-charge interactions between solid matrix and solute 7. Basic Principal of IEC 8. increasing formate ion concentration 9. Amino a. pKa Asp, Glu 4.3-4.7 His 7 Lys, Arg > 10 Prepare or purchase column Adjust pH and initial counter ion Apply sample 10. Elution from IEC Column change pH increase counter-ion (ie, salt) concentration in steps (eg, 0.1, 0.2, 0.3, 0.4 M NaCl) gradually (eg, 00.4 M NaCl) with gradient maker 11. collect fractions as column elutes analyze fractions for components of interest 12. increasing salting out effect anions: PO4, SO4, Cl, Br, NO3, Cl04, I, SCN cations: NH4, Rb, K, Na, Li, Mg, Ca, Ba increasingchaotropic effect Hydrophobic Interaction Chromatography (HIC) separates proteins based on differences in hydrophobicity absorb proteins to hydrophobic matrix high salt promotes hydrophobic interactions eg, 1 M (NH4)2SO4 13. HIC vs RPC Mobile Phase Polar Solvent Nonpolar Solvent Conditions Native Denatured Solute Discrimination Surface Residues Total Residues Reverse Phase Chromatography separation based on total hydrophobicity generally used to separate small peptides 14. Gel Filtration separation based on size, aka molecular sieve chromatography size exclusion chromatography media composed of cross- linked polymers pore size of matrix determines degree of interaction larger molecules are excluded and migrate faster smaller molecules are included and are retained longer Dextran (=Sephadex) Agarose (=Sepharose) Polyacrylamide 15. Sephadex Code Range (kDa) G-25 1-5 G-50 2-30 G-100 4-150 G-150 5-300 G-200 5-600 choose matrix with desired characteristics size range does not interact with solute include 0.15-1 M NaCl in buffer load sample in smallest possible volume elute in one column volume Practical Considerations Applications: purification desalting size determination 16. Calculating Size Vo = void volume Vt = total volume Ve = elution volume Ve - Vo Vt - Vo Kav = use size standards (relative MW) migration also affected by shape 17. Affinity Chromatography based on specific binding of protein to ligand ligands can include: substrate analogs, inhibitors natural ligands co-factors, metals binding proteins antibodies Elution: destabilize binding compete with free ligand change pH, ionic strength chaotropic or denaturing agents 18. THANK YOU