Classification of Chromatographic Methods Classification of Chromatographic Methods (Geometry) •Planar –Paper – Thin layer • Column –GC –HPLC –IC – SFC Classification of Chromatographic Methods (Mobile Phase) • Gas Chromatography • Liquid Chromatography • Supercritical Fluid Chromatography Classification of Chromatographic Methods (Stationary Phase) • Adsorption • Partition • Ion-Exchange • Molecular Exclusion • Affinity Example of Affinity Chromatography Purification of monoclonal antibody on a 5 cm x 4.6 mm column with protein A covalently attached to a polymer support A Hot Idea: Molecular Imprinting An “artificial antibody” can be constructed by synthesizing a polymer in the presence of a template molecule. When the template is removed, the polymer is “imprinted” with the shape of the template and with complementary functional groups that can bind to the template. The imprinted polymer can be used as a stationary phase in affinity chromatography. Molecular Imprinting Hydrophobic Interaction Chromatography (HIC) • Based on the interaction of a hydrophobic stationary phase with a hydrophobic region of a solute such as a protein • A protein can have hydrophilic regions that make it soluble in water and hydrophobic regions interacting with a hydrophobic stationary phase • Proteins are adsorbed on the hydrophobic surface when the aqueous mobile phase contains high salt concentration • Eluent strength is increased by decreasing the salt concentration Hydrophilic Interaction Chromatography (HILIC) • Most useful for small molecules too polar to be retained by reversed-phase HPLC • Stationary phases are strongly polar • The mobile phase contains ≥ 80% polar organic solvent mixed with water Hydrophilic Interaction Chromatography (cont.) • Solute equilibrates between the mobile phase and a layer of aqueous phase on the surface of the stationary phase • Eluent strength is increased by increasing the fraction of water in the mobile phase. (In NP HPLC, the solvent is nonaqueous. In RP HPLC, eluent strength is increased by decreasing the fraction of water in the mobile phase). Modes of Chromatographic Separation: • Frontal analysis • Displacement development • Elution development Frontal Analysis • The liquid or gas mixture is fed into a column containing a solid packing • The mixture acts as its own mobile phase • The separation depends on the ability of each component to be sorbed • Once the packing is saturated, the mixture flows through with its original composition • The least-sorbed component breaks through first and is the only component obtained in a pure form Displacement Development • The developer is contained in the mobile phase • The mobile phase must be more sorbed than any sample component • A single pure band of the first component is obtained • There is always an overlap zone for each succeeding component Elution Development • Sample components travel through the column at rates determined by their retention on the solid packing • A complete separation is possible C K = S CG Isotherms An isotherm is a graphical representation of the interaction of an adsorbent and a solute in solution (gas or liquid) at a specified temperature. The isotherm is a graphical representation of the partition coefficient or distribution constant K: C K = S CG Process Types in Chromatography • The process of chromatographic separation is defined by two conditions: – The distribution isotherms may be linear or nonlinear – The chromatographic system is either ideal or nonideal. Ideal Chromatography • The exchange between the two phases is thermodynamically reversible • The equilibrium between the two phases is immediate (longitudinal diffusion can be ignored and the mass transfer is very high) • In nonideal chromatography, these assumptions cannot be made Partition chromatography Liquid-solid chromatography Gas-solid chromatography.