CHROMA

• Chromatography


  Components of a mixture are carried through the stationary phase by the flow of the mobile phase, and separations are based on differences in migration rates among the mobile phase components
• Stationary phase


  • Often consists of the water molecules associated with the hydroxyl groups of cellulose
  • Fine porous solid like silica gel (SiO2) which is capable of retaining both solutes and solvents
  • Purified Alumina (Al2O3)
• Mobile phase


  • The solution that will be used as the eluting solvent which may vary in polarity
  • Usually opposite in polar character to that of the stationary phase
  • Examples: Ethanol (CH3CH2OH) - semi polar solvent, Hexane (CH) - non-polar solvent
• Column chromatography


  Stationary phase is held in a narrow tube, and the mobile phase is forced through the tube under pressure or by gravity
• Planar chromatography


  Stationary phase is supported on a flat plate or in the pores of a paper
• Planar chromatography


  • Paper chromatography
• Paper chromatography


  • Solvent soaks upward through paper fibres
  • Sample mix contains A and B but not C
• Partition between stationary and mobile phase

  Solute moves in the direction of the solvent flow at a rate which is affected by its attraction for either the stationary aqueous phase or the moving non-polar phase
• Migration of solute


  Solute which is more attracted to the moving non-polar phase will have a greater velocity of migration than the solute which is more strongly attracted to the stationary phase
• Rate of flow


  • R₁ = Distance traveled by solute (mm) / Distance traveled by solvent (mm)
  • 3:1 (ethanol:water) is better than 1:3 (ethanol:water) because it separated the components of the mixture effectively
• Classification of column chromatographic methods

  • Gas chromatography (GC)
  • Liquid chromatography (LC)
  • Supercritical-fluid chromatography (SFC)
• Gas-liquid chromatography (GLC)


  Gas-liquid chromatography is based on partitioning of the analyte between a gaseous mobile phase and a liquid phase immobilized on the surface of an inert solid packing or on the walls of a capillary tubing
• Carrier gas system in GC

  • Helium is the most common mobile phase, although argon, nitrogen, and hydrogen are also used
  • Pressures at the column inlet usually range from 10 to 50 psi and provide flow rates of 25 to 50 mL/min
• Qualitative analysis of complex samples of unknown composition is limited in GC
• Quantitative GC


  • Based on comparison of either the height or the area of an analyte peak with that of one or more standards
  • Peak area is independent of the broadening effects and is a more satisfactory analytical parameter than peak height
• Applications of GC


  • Amino acid determination
  • Drug test
  • Determination of steroids
  • Analysis of pollutants (carbon monoxide)
  • Food analysis (vitamins)
  • Dairy product analysis- rancidity
• High-performance liquid chromatography (HPLC)


  • The most versatile and widely used type of elution chromatography
  • Includes partition, adsorption, ion-exchange, size-exclusion, and affinity chromatography
• High Pressure Liquid Chromatography is referred to as High Performance Liquid Chromatography due to its high efficiency and performance
• Principle of HPLC


  Separation is based on adsorption, with the difference in the affinity of the compounds for the stationary phase determining the time of elution
• Instruments for HPLC


  • Mobile-phase reservoirs
  • Pumps that force the mobile phase through the column at high pressures up to 6000-9000 psi
  • Columns packed with small particle size (5 to 10μ) stationary phase, usually silica gel
• Normal-phase chromatography


  Highly polar stationary phases and relatively nonpolar mobile phase
• Reversed-phase chromatography


  • Nonpolar stationary phase and relatively polar mobile phase
  • Most polar component elutes first, and increasing mobile phase polarity increases elution time