Experiment 4: Chromatography

• What are the main types of chromatography based on different criteria?


  • Based on mechanism of separation
  • Based on the direction of mobile phase movement
  • Based on the nature of mobile and stationary phase
  • Based on the type of support
  • Based on the concentration of the solvent
• What does the term "chromatography" derive from?


  It comes from the Greek words “kroma” meaning color and “graphein” meaning to draw a graph or to write.
• What are the two main phases involved in chromatography?


  The stationary phase and the mobile phase.
• How does the stationary phase function in chromatography?


  It interacts with the analytes and is not moving.
• What is the role of the mobile phase in chromatography?


  It carries the solute across the stationary phase.
• What are the two types of chromatography based on the mechanism of separation?

  • Partition: Separation between liquid mobile and stationary phases (e.g., paper chromatography).
  • Adsorption: Separation between liquid mobile phase and solid stationary phase (e.g., column chromatography).
• In ascending chromatography, how does the solvent move?


  The solvent moves from bottom to top due to capillary action.
• What is the difference between normal phase and reverse phase chromatography?


  Normal phase uses a polar stationary phase and a non-polar mobile phase, while reverse phase uses a non-polar stationary phase and a polar mobile phase.
• What are the two types of chromatography based on the type of support?


  • Planar: Stationary phase on a flat support (e.g., TLC and paper chromatography).
  • Column: Stationary phase in a cylindrical glass support (e.g., HPLC and GC).
• What are the two types of solvent concentration in chromatography?


  • Isocratic: Fixed composition of solvent throughout the process.
  • Gradient: Continuous change in the composition of the mobile phase over time.
• What is the purpose of column chromatography?


  To separate and purify components in a mixture.
• What are the steps involved in column chromatography?


  1. Packing (wet or dry)
  2. Washing
  3. Loading
  4. Development
  5. Collection and Evaluation
• What does retention time (Rt) measure in chromatography?


  The time taken for a solute to pass through a chromatography column.
• How does the polarity of a component affect its retention time (Rt)?


  A lower Rt indicates a more non-polar component, while a higher Rt indicates a more polar component.
• What are the characteristics of paper chromatography?


  • Stationary phase: Water in solid cellulose (polar).
  • Mobile phase: Different solvents (nonpolar).
  • Separation based on affinity between phases.
• In paper chromatography, where do polar components typically travel?

  Polar components travel close to the line of origin due to higher affinity with the polar stationary phase.
• What is the retention factor (Rf) used for in chromatography?


  It aids in the identification of a substance by comparison to standards.
• What are the steps involved in thin layer chromatography?


  1. Sample/Standard Application
  2. Development
  3. Visualization
  4. Evaluation
  5. Documentation
• What is the formula for calculating the retention factor (Rf)?


  $Rf =$$\frac{distance \, of \, component}{distance \, of \, solvent \, front}$
• What are the differences between column chromatography and thin-layer chromatography?


  • Column: Preparative, uses glass cylinder, dropper for sample application, descending solvent front.
  • Thin-Layer: Analytical, uses metal sheet, capillary tube for sample application, ascending solvent front.
• How does the adsorption strength of compounds change with polarity?


  The adsorption strength increases with increasing polarity of functional groups.
• What factors affect the molecular polarity of organic compounds?


  • Number of carbon atoms
  • Presence of electronegative atoms
• What pigments are isolated from Moringa oleifera in decreasing polarity?


  Xanthophylls, Chlorophyll B, Chlorophyll A, Pheophytin, Carotene.
• What pigments are isolated from Capsicum frutescens in decreasing polarity?


  Cabsorbin, Capsanthin, Carotene.