Separations and Purifications: Chromatography

Overview

Chromatography: Utilizes physical and chemical properties to separate and identify compounds from a complex mixture.
Core Idea: The more similar a compound is to its surroundings (by polarity, charge, etc.), the more it will adhere and move slowly through the stationary phase.

Stationary Phase: Solid medium where the sample is placed.
Mobile Phase: Liquid or gas that runs through the stationary phase.
Partitioning: Represents equilibrium; different compounds elute at different rates based on their partition coefficients.
Result: Separation of substances allowing isolation of individual compounds.

Thin Layer Chromatography: Uses a thin layer of silica gel or alumina on an inert carrier sheet.
Paper Chromatography: Uses paper, typically cellulose.
Spotting: Placing the sample directly on the absorbent.
- Development: Placing the absorbent in a developing chamber with a shallow pool of solvent.
- Capillary Action: Solvent creeps up the plate, carrying compounds at varying rates.
Detection: UV light or staining to view white spots on the paper/TLC plate.
RF Factor: Ratio of the distance the spot moved to the distance the solvent front moved.

Difference from TLC: Uses an entire column filled with silica or alumina beads.
- Movement: Gravity moves the solvent and compounds down the column.
- Enhancement: Flash column chromatography uses gas pressure to speed up the process.
Polarity Adjustment: Changing solvent polarity to help elute the desired compound.
Fraction Collection: Different compounds are collected over time, solvent evaporated, leaving compounds.

Ion Exchange Chromatography: Beads in the column are coated with charged substances attracting oppositely charged compounds.
- Example: Positively charged beads attract negatively charged DNA backbone.
- Elution: Use of a salt gradient.
Size Exclusion Chromatography: Beads contain pores of varying sizes; small compounds enter pores and are slowed down, large compounds move faster.
Affinity Chromatography: Uses beads with receptors for the target protein.
- Elution: Different solvent to remove bound proteins from the receptors.

Main Difference: Mobile phase is a gas instead of a liquid.
Absorbent: Crushed metal or polymer inside a coiled column.
Process: Mixture injected, vaporized, and compounds travel at different rates.
Volatile Compounds: Must be vaporizable liquids or have low melting points.
Detection: Detector records peaks of compounds at the end of the column.
Application: Pure molecules can be injected into a mass spectrometer for molecular weight determination.

Original Name: High pressure liquid chromatography.
Mobile Phase: Liquid runs through a defined composition column.
Process: Small sample injected, compounds separated while traveling through the column.
Detection: Compounds pass through a detector and are collected.
Interface: Computerized, similar to gas chromatography.

Chromatography uses two phases to separate compounds based on physical or chemical properties.
Key Takeaway: Stationary phase (usually polar solid), Mobile phase (liquid or gas).
Techniques Covered: TLC, paper chromatography, column chromatography (with ion exchange, size exclusion, affinity), gas chromatography, and HPLC.

Final Note: Good luck with your studies and happy learning!