3 CHROMOTOGRAPHY

• Chromatography


  A technique used to separate and identify the components of a mixture
• How chromatography works

  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
• 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)
• Uses of chromatography

  • Separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells
  • Separate the colored pigments in plants
• Retardation Factor (RF)


  Measure of how far a component travels up a chromatography strip compared to the solvent

  
• For substances very soluble in the liquid, RF will be close to 1
• For substances rather insoluble in the liquid, RF will be close to 0
• Paper Chromatography

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


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


  The degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties (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 the dye composition of fibers
  • Most samples are not colored and need to be visualized with a UV lamp
• Gas Chromatography

  • Used in airports to detect bombs and in forensics in many different ways
  • Used to analyze fibers on a person's body and also analyze blood found at a crime scene
  • Helium is used to move a gaseous mixture through a column of absorbent material
• Solubility of gases in liquid phases

  • Depends on the volatility of the liquid from which the component vapor drives
  • Gases from low boiling liquids will spend less time dissolved in the liquid phase, whereas gases from less volatile liquids will spend more time in solution with the liquid phase
  • The less time a gas spends dissolved in the liquid phases, the more time it spends flying along with the carrier gas, thus it moves through the stationary phase more quickly
• Liquid Chromatography

  • A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid
  • With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation
  • Used to test water samples to look for pollution in lakes and rivers
  • Used to analyze metal ions and organic compounds in solutions
  • Uses liquids which may incorporate insoluble molecules
• Types of Liquid Chromatography

  • 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 frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds
  • Utilizes a column that holds 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
  • Retention time varies depending on the interactions between the stationary phase, molecules being analyzed, and the solvent(s) used
• 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