CHROMATOGRAPHY (PPT)

• Chromatography


  A technique used to separate and identify the components of a mixture
• Chromatography

  1. Allows the molecules present in the mixture to distribute themselves between a stationary and a mobile medium

  2. Molecules that spend most of their time in the mobile phase are carried along faster
• Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass
• Differential


  Showing a difference, distinctive
• Affinity


  Natural attraction or force between things
• Mobile Medium


  Gas or liquid that carries the components (mobile phase)
• Stationary Medium


  The part of the apparatus that does not move with the sample (stationary phase)
• Chromatography used to separate

  • Inks and dyes
  • Colored pigments in plants
• RF (Retardation Factor)


  Measure of how far a substance travels up a chromatography strip compared to the solvent front
• Substances very soluble in the liquid

  RF close to 1
• Substances rather insoluble in the liquid

  RF close to 0
• Paper Chromatography


  Separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
• Capillary Action


  Movement of liquid within the spaces of a porous material due to adhesion, cohesion, and surface tension
• Solubility


  Degree to which a material (solute) dissolves into a solvent. Like dissolves like.
• Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase
• Thin Layer Chromatography

  • Uses an absorbent material on flat glass or plastic plates
  • Used to detect pesticide or insecticide residues in food
  • Used in forensics to analyze dye composition of fibers
  • Most samples are not colored and need to be visualized with a UV lamp
• Gas Chromatography

  1. A gas mixture (mobile phase) composed of an inert carrier gas and other chemical components in the vapor phase is introduced at the injection port

  2. Passes through a non-moving material (stationary phase) usually tightly packed into a column

  3. Separates the gas stream into its particular components based on how quickly they move through the stationary phase

  4. Components reach a detector at different times which generates an electrical signal recorded
• The stationary phase-mobile phase must be heated to higher temperature in order to vaporize the components of the mobile phase
• Solubility of gases in liquid phases

  • Depends on volatility of the liquid from which the component vapor drives
  • Gases from low boiling liquids will spend less time dissolved in the liquid phase
  • Gases from less volatile liquids will spend more time in solution with the liquid phase
• Uses of Gas Chromatography

  • Detect bombs in airports
  • Analyze fibers on a person's body
  • Analyze blood found at a crime scene
• Liquid Chromatography

  1. A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid

  2. With the proper solvents and packing conditions, some components in the sample will travel the column more slowly than others resulting in separation
• Uses of Liquid Chromatography

  • Test water samples for pollution
  • Analyze metal ions and organic compounds in solutions
• Liquid Chromatography Modes

  • Liquid/Solid Chromatography (adsorption chromatography)
  • Liquid/Liquid Chromatography (partition chromatography)
  • Ion Exchange Chromatography
  • Gel Permeation Chromatography (exclusion chromatography)
• Liquid/Solid Chromatography


  Separation mechanism is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel
• Liquid/Liquid Chromatography


  • The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase
  • Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation
• Ion-Exchange Chromatography


  Separation is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing)
• Gel-Permeation Chromatography


  • Mechanical sorting of molecules based on the size of the molecules in solution
  • Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules
• High Performance Liquid Chromatography (HPLC)


  • A form of column chromatography used to separate, identify, and quantify compounds
  • Utilizes a column with chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules
• Normal Phase Chromatography


  Uses a polar stationary phase and a non-polar mobile phase, and works effectively for relatively polar analytes
• Reversed Phase Chromatography


  Has a non-polar stationary phase and an aqueous, moderately polar mobile phase
• Chromatography is a method of separation and purification of organic compounds