Chromatography Techniques

• Chromatography is an analytical technique used to separate a mixture of chemical substances into individual components for analysis.

Basic Principles

• All types of chromatography share the same basic principles.

Types of Chromatography

• Liquid chromatography.
• Gas chromatography.
• Ion-exchange chromatography.
• Affinity chromatography.

Reaction Mixture Separation

• Reactants A and B react to form product C, resulting in a mixture of unreacted A, unreacted B, and the desired product C.
• Chromatography separates A, B, and C to isolate pure product C.

Thin Layer Chromatography (TLC)

• A TLC plate is a glass plate coated with a thin layer of silica.
• Apply a spot of the reaction mixture above the base of the plate.
• Place the plate in a jar with an organic solvent to dip the lower edge of the plate.
• The solvent rises by capillary action, separating the mixture into spots with distinct colors.

Glass Column Chromatography

• A glass column with a stopcock is packed with a slurry of silica gel.
• The reaction mixture is poured over the silica bed.
• The solvent runs through the column, separating the mixture into distinct bands.
• Individual bands are collected in separate flasks to obtain pure product C.

Chromatography Terms

• Mobile phase (or carrier): Solvent moving through the column.
• Stationary phase (or adsorbent): Substance fixed inside the column.
• Eluent: Fluid entering the column.
• Eluate: Fluid exiting the column, collected in flasks.
• Elution: Washing out a compound through a column with a solvent.
• Analyte: Mixture to be separated and analyzed.

Principle of Chromatography

• The analyte is loaded onto the silica bed and adheres to the silica (stationary phase).
• Solvent (mobile phase) flows through the silica bed.
• Components of the analyte adhere differently to the silica, causing them to travel at different speeds.
• Components with stronger adhesion travel slower.
• Analytical chromatography can purify compounds from milligram to gram scale.

Experiment: Leaf Pigment Separation

• Crush leaves and spot the extract on chromatographic paper.
• Place the paper in propanone (acetone).
• The solvent rises, separating the leaf pigment into various components.

Separation Principle: Differential Affinities

• Differential affinities of components toward the stationary and mobile phases result in separation.
• Affinity depends on adsorption (sticking to the stationary phase) and solubility (dissolving in the mobile phase).
• Higher adsorption leads to slower movement.
• Higher solubility leads to faster movement.
• Adsorption and solubility are manipulated by choosing appropriate phases.

Polarity and Affinity

• "Polarity” of the compounds dictates their affinities towards the stationary and mobile phases.
• Polar compounds adsorb onto polar stationary phases.
• Non-polar compounds dissolve in non-polar mobile phases.
• The mobile phase is changed to elute different components.

Normal-Phase Chromatography

• Stationary phase: Polar (hydrophilic).
• Mobile phase: Non-polar (hydrophobic).

Reverse-Phase Chromatography

• Stationary phase: Non-polar.
• Mobile phase: Polar.

Chromatography Techniques

• The underlying principle remains the same: differential affinities of the various components of the analyte towards the stationary and mobile phases results in the differential separation of the components.

Paper Chromatography

• Stationary phase: solid (cellulose).
• Mobile phase: liquid.
• Basis of separation: polarity of molecules.
• Compound spotted directly on cellulose paper.

Thin Layer Chromatography (TLC)

• Stationary phase: solid (silica or alumina).
• Mobile phase: liquid.
• Basis of separation: polarity of molecules.
• Glass is coated with thin layer of silica on which is spotted the compound.

Liquid Column Chromatography

• Stationary phase: solid (silica or alumina).
• Mobile phase: liquid.
• Basis of separation: polarity of molecules.
• Glass column is packed with slurry of silica.

Size Exclusion Chromatography

• Stationary phase: solid (microporous beads of silica).
• Mobile phase: liquid.
• Basis of separation: size of molecules.
• Small molecules get trapped in pores, large molecules flow through gaps, eluting first.
• No physical or chemical interaction between analyte and stationary phase.

Ion-Exchange Chromatography

• Stationary phase: solid (cationic or anionic resin).
• Mobile phase: liquid.
• Basis of separation: ionic charge of molecules.
• Molecules with opposite charge bind to the resin, same charge elutes first.

Affinity Chromatography

• Stationary phase: solid (agarose or porous glass beads with immobilized molecules).
• Mobile phase: liquid.
• Basis of separation: binding affinity of analyte to the immobilized molecule (enzymes and antibodies).
• Substrates bind tightly to enzymes, unbound analytes elute, then the substrate is detached and eluted.

Gas Chromatography

• Stationary phase: liquid or solid support.
• Mobile phase: gas (inert gas).
• Basis of separation: boiling point of molecules.
• Samples are volatilized, molecules with lower boiling points elute first.

TLC: Retention Factors (R[_{f}])

• Normal phase TLC uses a silica-coated glass plate.
• Silica is the polar stationary phase, and the solvent is the less polar mobile phase.
• Polar components adhere tightly to silica and travel slowly.
• Non-polar components travel faster with the solvent.

Rule of Thumb

• The component that travels the least distance on the TLC plate is the most polar.
• The component that travels the maximum distance is the least polar.

Retention Factor (R[_{f}])

• Quantitative parameter for each component.
• R[_{f}] = (distance travelled by component) / (total distance travelled by solvent).
• Lower R[_{f}] value indicates a more polar component.