Downloaded 46 times
Uploaded byDr.M.Prasad Naidu
Chromatography,
Chromatography separates components of a mixture through interactions between a stationary and mobile phase. Common chromatography techniques include ion exchange, hydrophobic interaction, gel filtration, reverse phase, and affinity chromatography. These techniques separate biomolecules like proteins based on properties like charge, hydrophobicity, size, and specific binding interactions. The document provides details on chromatography components, protocols, equipment, and applications of specific techniques like ion exchange and gel filtration chromatography.
More Related Content
Similar to Chromatography,
Chromatography,
- 1. Chromatography M.Prasad Naidu MSc MedicalBiochemistry, Ph.D,.
- 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.
- 4. Common Media Usedin 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. PrepareColumn () 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
- 7. Ion Exchange Chromatography(IEC) • based on charge-charge interactions between solid matrix and solute
- 8.
- 9. increasing formate ionconcentration
- 10. 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
- 11. Elution from IECColumn • 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
- 12. • collect fractionsas column elutes • analyze fractions for components of interest
- 13. increasing saltingout 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
- 14. HIC vs RPC MobilePhase 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
- 15. Gel Filtration • separationbased 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
- 17. Sephadex Code Range (kDa) G-251-5 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
- 18. 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
- 19. Affinity Chromatography • basedon 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