Chroma

• Chromatography


  The power of chromatography comes from its ability to separate a mixture of compounds, or "analytes", and determine their respective identity (chemical structure) and concentration
• Three Basic Types of Chromatography

  • Gas
  • Liquid
  • Supercritical fluid
• Branches of Gas Chromatography

  • Gas-solid
  • Gas Liquid
• Branches of Liquid Chromatography

  • Ion exchange
  • Exclusion
  • Partition chromatography
  • Liquid solid absorption
  • Paper chromatography
  • Thin layer
• How chromatography works

  1. Separates the components by differential adsorption between the stationary phase and the mobile phase

  2. Pass a sample-free mobile phase over a stationary phase

  3. Inject the sample into the mobile phase

  4. The solute that spends the most time in the stationary phase takes the longest time to move through the system
• Chromatographic Separation


  • The solute partitions between the mobile phase and the stationary phase
  • A component whose distribution ratio favors the stationary phase requires more time to pass through the system
  • Given sufficient time and sufficient stationary and mobile phase, we can separate solutes even if they have similar distribution ratios
• Ways to identify chromatographic separations

  • Describing the physical state of the mobile phase and the stationary phase
  • Describing how we bring the stationary phase and the mobile phase into contact with each other
  • Describing the chemical or physical interactions between the solute and the stationary phase
• Mobile phase


  Liquid or gas
• Stationary phase


  Solid or liquid film coated on a solid substrate
• We often name chromatographic techniques by listing the type of mobile phase followed by the type of stationary phase
• Example of chromatographic technique naming

  • Gas-liquid chromatography, gas is mobile and liquid is stationary
• Ways the mobile phase and stationary phase can interact

  • Column chromatography- gravity or by applying pressure
  • Planar chromatography- with a reservoir containing the mobile phase which moves by capillary action
• Types of interactions between solute and stationary phase

  • Adsorption chromatography- solutes separate based on their ability to adsorb to a solid stationary phase
  • Partition chromatography- the stationary phase is a thin liquid film on a solid support, separation occurs due to difference in equilibrium partitioning
  • Ion-exchange chromatography- stationary phase has covalently attached anionic or cationic functional groups, ionic solutes attracted by electrostatic forces
  • Size-exclusion chromatography- stationary phase is porous, separation based on size of solutes
• Electrophoretic Separations


  Charged solutes migrate under the influence of an applied potential, larger solutes move more slowly than smaller solutes of equal charge, solutes with larger charge move more quickly than those with smaller charge
• Adsorbents used in column chromatography

  • Silicic acid
  • Charcoal
  • Aluminum oxide
  • Magnesium carbonate
  • Calcium phosphate
  • Cellulose
• Characteristics of Liquid Chromatography

  • Column chromatography
  • Thin-layer
  • HPLC
  • Stationary phase: silica, alumina, etc
  • Mobile phase: organic solvents
  • Important property: polarity
• Characteristics of Gas Chromatography

  • Stationary phase: film of a polymer or a wax
  • Mobile phase: gas, helium as usual carrier gas
  • Important property: boiling point
• Column chromatography


  Stationary phase is packed into a column
• Thin-layer chromatography


  Stationary phase is coated onto a glass, metallic, or plastic plate
• Retention time


  Time between the sample's injection and the maximum response for the solute's peak
• Theoretical plates


  In the original theoretical model, the chromatographic column is divided into discrete sections
• Detection methods

  • UV- Ultraviolet
  • RI- Refractive Index
  • FD- Fluorescence
  • MS- Mass spectrometry
• Types of Partition Chromatography

  • Normal/reverse phase chromatography
  • Ion-exchange chromatography
  • Gel filtration chromatography
  • Affinity chromatography
• Normal-phase HPLC


  Adsorption of analytes on the polar, weakly acidic surface of silica gel, polar solutes elute later than non-polar lypophilic ones
• Reversed-phase HPLC


  Partition of analytes between mobile phase and stagnant phase inside the pore space + adsorption on the surface of bonded phase, separation based on degree of hydrophobicity
• Ion Exchange Liquid chromatography

  Elution order determined by charge density of hydrated ions, for organic acids and bases determined by pKa or pKb
• Gel Permeation Chromatography


  Separation based on molecule size and shape by the molecular sieve properties, also known as size exclusion chromatography
• Affinity chromatography


  Based on interaction between a protein of interest and a ligand
• In column chromatography, the stationary phase is made of solid and the mobile phase is made of liquid
• Liquid chromatography can be performed either in columns or on plane surfaces
• Gas chromatography can be performed only in columns
• Operation after injection of feed in chromatography

  1. Separation in the column

  2. Elution from the column

  3. Detection of components
• Components with least affinity elute first
• Chromatogram


  A detector that responds to concentration solute and is placed at the end of the column, the position of peaks on the time axis can be used to determine components, the area under the peak can be used to determine an amount of component
• In chromatography, molar concentration of solute in stationary phase is directly proportional to molar concentration of solute in mobile phase
• Retention time


  Time taken by the analyte after sample injection to reach the detector
• Dead time


  Time required for a molecule of the mobile phase to pass through the column
• Adjusted retention time


  Remaining retention time after subtracting dead time from retention time, affects the effectiveness of the system
• Retention factor


  Ratio of moles of solute in stationary phase to the moles of solute in mobile phase, used for determining the migration rates of solutes in the column
• Gas chromatography - Mass Spectrometry (GC-MS)


  Hyphenated analytical technique, GC separates the components while MS characterizes each of the components