Chromatography (LEC)

• Separation of Mixtures:

  A) Particle Size

  B) Molecular Properties

  C) Decantation

  D) Distillation/Evaporation

  E) Chromatography

  F) Liquid-Liquid Extraction

  
• Filtration - based on the size of components as the mixture passes through a filter.
• Pros of Filtration:

  • Simple and effective; low cost
  • Wide range of particle sizes can be filtered depending on the filter used
• Cons of Filtration:

  • Time-consuming when filtering larger volumes
  • Not effective when substances are dissolved (in aqueous phases)
  • Requires multiple filtration step to achieve desired outcomes
• Decantation - used only to remove the liquid layer on top of the sediment/precipitate.
• Separating Mixtures by Molecular Properties:

  A) Physical Properties

  B) Distillation

  C) Evaporation

  D) Density

  E) Flotation

  F) Chromatography

  G) By polarity

  H) Charges

  
• Distillation and Evaporation - utilizes the differences in boiling point of components to separate compounds.
• Flotation - utilizes the differences in the density of particles. Often employed in studies about microplastics.
• Liquid-Liquid Extraction - often used in organic chemistry; separatory funnel is used.
• 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.
• Mikhail Tsvet - Russian botanist who invented chromatography in 1835 while studying chemical compounds in plants to separate plant pigments.
• Guiding Principle of chromatography:

  • Compounds will be separated based on their polarities and affinity towards the mobile phase or stationary phase.
• Mobile phase - liquid or gas that will move the sample through the chromatography medium.
• Stationary phase - solid or liquid material that lines the inside of the chromatography medium and interacts with the sample to separate its components.
• Elution - process of washing a component out of the column using a solvent.
• 1930s - development of paper (partition) chromatography by Martin and Synge; both won the 1952 Nobel Prize in Chemistry.
• 1950s - development of gas chromatography.
• 1960s – development of high-performance liquid chromatography (HPLC).
• Thin Layer Chromatography - separation of liquid components based on their affinity towards the stationary and mobile phases.
• Thin Layer Chromatography

  • Solvent’s polarity can be inferred by the retention factor (RF ) value
• Liquid Chromatography - separates compounds using a liquid as the mobile phase and a stationary phase coated on a column.
• Liquid Chromatography

  • Higher affinity to stationary phase = longer elution time
  • Lower affinity to the stationary phase = shorter elution time
• Types of Liquid Chromatography:

  • Normal Phase Chromatography
  • Reversed Phase Chromatography
• Normal Phase

  • 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 liquid chromatography but the samples are injected into the column with a pump for quicker analysis.
• High-Performance Liquid Chromatography (HPLC):

  • Compounds are detected using UV-Vis (for colored compounds) or refractive index detectors (for sugars, polymers, etc.).
• Gas Chromatography - compounds are vaporized and injected into a column.
• Gas Chromatography Components:

  • Mobile phase - inert gas such as He or N2
  • Stationary phase - depends on the type of analysis
• Detectors in Gas Chromatography Components:

  • Flame ionization detector (FID)
  • Thermal conductivity detector (TCD)
  • Mass spectrometer (MS)
• Practical applications of chromatography:

  A) Drug Discovery

  B) Biotechnology

  C) Chemical Industry

  D) Forensics

  
• Drug Discovery - it is used to identify and isolate new compounds from natural sources, test the purity of drug samples, and analyze the pharmacokinetics of drugs.
• Food and Beverage - chromatography is used to analyze food and beverage samples for quality control and safety purposes
• Forensics - 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.
• Environment - 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.