Chapter 7

• Chromatography


  Method of separating and identifying components of a mixture by passing it through a medium that separates the components based on their physical or chemical properties
• Chromatography invented by Mikhail Tsvet (Russian botanist) while studying chemical compounds in plants to separate plant pigments


  1835
• Development of paper (partition) chromatography by Martin and Synge; both won the 1952 Nobel Prize in Chemistry


  1930s
• Development of gas chromatography


  1950s
• Development of high-performance liquid chromatography (HPLC)

  1960s
• Mobile phase


  Liquid or gas that will move the sample through the chromatography medium (paper or column)
• Stationary phase


  Solid or liquid material that lines the inside of the chromatography medium (paper or column) and interacts with the sample to separate its components
• Elution


  Process of washing a component out of the column using a solvent
• Thin Layer Chromatography (TLC)


  • Separation of liquid components based on their affinity towards the stationary and mobile phases
  • Solvent's polarity can be inferred by the retention factor (RF) value
• TLC Setup


  1. Stationary phase: paper or silica (polar)

  2. Mobile phase: alcohol-water mixture or acetone (less polar)
• Liquid Chromatography


  • Higher affinity to stationary phase = longer elution time
  • Lower affinity to the stationary phase = shorter elution time
• Normal Phase Chromatography


  • Mobile Phase: nonpolar such as hexane
  • Stationary Phase: polar such as silica
• Reversed Phase Chromatography


  • Mobile Phase: polar such as water
  • Stationary Phase: nonpolar such as C18 column
• High-Performance Liquid Chromatography (HPLC)


  • Follows the principles of LC but the samples are injected into the column with a pump for quicker analysis
  • Compounds are detected using UV-Vis (for colored compounds) or refractive index detectors (for sugars, polymers, etc.)

  
• Compounds with less affinity towards the column


  Have a lower retention time (Rt) than compounds with higher affinity
• Polarity of compounds eluted


  Depends on the type of chromatography (normal or reversed)
• Chromatography is used to identify and isolate new compounds from natural sources, test the purity of drug samples, and analyze the pharmacokinetics of drugs
• Chromatography is used to analyze food and beverage samples for quality control and safety purposes. It is used to detect contaminants, additives, and other substances in food and beverages
• Chromatography is used in the analysis of biological samples in forensic investigations. It is used to identify drugs, poisons, and other chemicals in blood, urine, and other body fluids
• Chromatography is used to analyze pollutants in the air, water, and soil. It is used to detect and quantify trace amounts of chemicals and to identify the sources of contamination