Lec 8.....Chromatography - 2 د.ذكاء.pdf

Classification of Chromatography Dr. Thukaa Z. Abdul-jalil Lec 8 Classification of chromatography Closed column chromatography ex: column chromatography GLC Open column chromatography ex: paper chromatography (PC) Thin layer chromatography (TLC) physical arrangement of the system Chromatography Adso...

Classification of Chromatography Dr. Thukaa Z. Abdul-jalil Lec 8 Classification of chromatography Closed column chromatography ex: column chromatography GLC Open column chromatography ex: paper chromatography (PC) Thin layer chromatography (TLC) physical arrangement of the system Chromatography Adsorption chromatography Partition chromatography according to the mechanism of separation Gel filtration chromatography Ion-exchange chromatography Affinity chromatography the stationary phase is packed in closed container & the mobile phase flow through it stationary phase is found spreader on a flat surface as an open sheet of paper or a glass plate Adsorption chromatography Partition chromatography 1. the distribution of the solute is between a solid phase which is called Stationary phase & a liquid or gas phase which is called the mobile phase. 1. the distribution of the solute is between a liquid stationary phase & a mobile phase which could be a liquid or a gas. 2. Depends on active sites in stationary and mobile phases. (e.g. solutes can form weak chemical bonds :hydrogen bonds , Van der Waal forces , & dipole-dipole interactions). 2. Depends on relative solubility of the compound between the sorbent (stationary phase) & the solvent (mobile phase). 3. There is a competition of each component for the active site of the adsorbent. ( i.e. in adsorption chromatography the adsorption of sample A will depend on that of sample B & C). 3. The distribution of each solute is independent on the other.( ex: in a mixture of A B & C the distribution of A will be independent on that of B&C as for the others). 4. Ex: Thin layer chromatography (TLC). 4. Ex: Paper chromatography (PC). Wetting Water steam Adsorption (Solid St.Phase) How we can turn the partition in to adsorption and vice versa? Drying Oven (heat) Partition (liquid St.phase) Gel filtration chromatography Size-inclusion/ exclusion chromatography Stationary phase Gel with pores. Mobile phase Any suitable solvent. Sample/ Separation Compound has relative size of its molecules. Ex: Proteins/a.a. Example of this type HPLC. CC. Gel filtration chromatography Ion-exchange chromatography Stationary phase Polymeric resin. Cationexchanger.(-) Anionexchanger.(+) Mobile phase Any suitable solvent. Sample/ Separation Separation is achieved by differences in affinity between ionic components & the stationary phase. Ex.: Salts (NaCl). Ion-exchange chromatography Affinity chromatography This type of chromatography depends on interactions of biological molecules (Sample), such as: 1. antibody-antigen interaction. 2. enzyme-inhibitor interaction. 3. DNA-DNA binding. 4. DNA-protein interaction. 5. Receptor-agonist/antagonist interaction. specific The ligand (receptor) is covalently bound to the packing material