Chromatography & Separation Techniques - Week 3

Chromatography & Separation Techniques - Week 3

• Chromatography is a technique for separating mixtures.
• Planar chromatography uses a flat support for the stationary phase.
• The mobile phase moves through the stationary phase by capillary action, potentially aided by gravity or electricity.
• Thin-Layer Chromatography (TLC) uses a thin layer of alumina or silica gel on a glass or plastic plate.
• Paper Chromatography (PC) uses a paper strip (cellulose fibers) with water as the stationary phase.
• Electrophoresis separates molecules based on their migration in an electric field.
• Capillary action is the movement of liquid in the spaces of a porous material, opposing gravity through adhesion, cohesion and surface tension.
• Molecules move up the plate if the adhesive force is stronger than the cohesive force.
• Solubility is the degree a substance dissolves in a solvent. "Like dissolves like" principles are used and different solvents allow separation according to solubility differences.
• Adsorbent materials are finely divided homogeneous solids, with a uniform particle size and a large surface area.
• Resolution refers to the ability of a chromatographic process to isolate pure compounds.
• Rate of Flow is the distance the solute travels divided by the distance the mobile phase travels.
• TLC is typically used for qualitative analysis and often involves a liquid mobile phase.

Thin Layer Chromatography (TLC)

• TLC separates components on a thin layer of a solid phase (like silica gel).
• The solid phase is coated on a flat surface (like glass or plastic).
• A liquid mobile phase is used to carry the components.
• Components separate due to differential affinities with the mobile and stationary phases.
• TLC plates are pre-coated for convenience.
• Plates are dried after development.
• Sample application is critical to quantitative analysis and usually done as a spot a few centimeters from an edge.
• A solvent tank or chamber is used, lined with filter paper to saturate the air with solvent.
• The mobile phase travels via capillary action.
• The plate is removed when the solvent nears the top, then dried.
• Visualization/Detection Methods are used to identify the separated components.

Sample Application (Spotting)

• Applying the sample is critical step, especially in quantitative analysis.
• Spotting is done as a small circle, 1-2cm from an edge.
• Sample solutions are dissolved and applied with capillaries or hypodermic syringes.
• For greater accuracy, automated applicators are used.

Plate Development

• The mobile phase is drawn up the plate through capillary action.
• The plate is immersed in the developing solvent to separate components according to affinities with the stationary and mobile phases.
• The tank or chamber should be saturated with solvent vapors, for example, lined with filter paper.
• The solvent is allowed to travel up the plate until almost to the top.

Locating (Visualizing) Sample Components

• Colored components can be seen directly on the stationary phase.
• Colorless components require visualization via reagents that create spots in the area of the component.
• Various methods exist including using UV light or spraying with specific reagents (e.g., ninhydrin for amino acids, permanganate for oxidizable compounds, 2,4-dinitrophenylhydrazine for aldehydes and ketones).