Chromatography

Separating Mixtures

• Filtration: Separates mixtures based on particle size using a filter.
  • Pros: Simple, effective, low cost, wide range of particle sizes.
  • Cons: Time-consuming for large volumes, ineffective for dissolved substances, may require multiple steps.
• Decantation: Removes the liquid layer from a sediment or precipitate.
• Distillation/Evaporation: Separates mixtures based on boiling points of components.
• Flotation: Separates mixtures based on the density of particles, commonly used in microplastics studies.
• Liquid-Liquid Extraction: Uses a separatory funnel, often used in organic chemistry.
• Chromatography:
  • Separates and identifies components of a mixture.
  • Components are separated based on physical or chemical properties.
  • Invented in 1835 by Mikhail Tsvet to separate plant pigments.

Chromatography

• A 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.

Thin Layer Chromatography (TLC)

• Separates liquid components based on their affinity towards stationary and mobile phases.
• Solvent polarity can be inferred by the retention factor (Rf) value.
  • Stationary phase: paper or silica (polar).
  • Mobile phase: alcohol-water mixture or acetone (less polar).

$R_f$ Value

• $R_f = \frac{\text{solute distance from origin}}{\text{solvent front}}$
• $R_f$ value has no unit.
• Indicates a sample’s affinity to the mobile and stationary phase.
• ↑$R_f$ value, ↑ affinity for the mobile phase.
• ↑$R_f$ value, ↓ affinity for the stationary phase.
• Higher $R<em>f$ means the molecule is less polar, and lower $R</em>f$ means the molecule is highly polar.

Liquid Chromatography (LC)

• Separates compounds using a liquid as the mobile phase and a stationary phase coated on a column.

Types of Liquid Chromatography

• 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.

The Chromatogram

• Compounds with less affinity towards the column have a lower retention time ($R_t$) than compounds with higher affinity.
• Polarity of compounds eluted depends on the type of chromatography (normal or reversed).

Gas Chromatography

• Compounds are vaporized and injected into a column.

Practical Applications of Chromatography

• Drug Discovery: Identifies and isolates new compounds, tests drug purity, and analyzes pharmacokinetics.
• Food and Beverages: Analyzes samples for quality control and safety, detects contaminants and additives.
• Forensics: Analyzes biological samples to identify drugs, poisons, and other chemicals in body fluids.
• Environment: Analyzes pollutants in air, water, and soil to detect and quantify trace amounts of chemicals and identify sources of contamination.
• Chemical Industry
• Biotechnology