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• Chromatography


  Laboratory technique for the Separation of mixtures
• Chromatography first employed by Russian scientist Mikhail Tswett


  1900
• Chromatography


  • Separation of plant pigments such as chlorophyll, carotenes, and xanthophylls
  • Colour bands - separation of individual compounds
  • Measured or analysed
• Chromatography


  Method of separation in which the components to be separated are distributed between two phases, one of these is called a stationary phase and the other is a mobile phase which moves on stationary phase in a definite direction
• Chromatographic Terms


  • Eluent - Fluid entering column/ solvent that carries the analyte
  • Stationary phase - Immobilized phase Immobilized on the support particles or on the inner wall of the column tubing
  • Eluate - Mobile phase leaving the column
  • Mobile phase - Moves in a definite direction
  • Retention time - Time takes for a particular analyte to pass through the system
  • Sample (Analyte) - Substance analysed in chromatography
• Gas Chromatography Columns


  • Packed columns
  • Wall coated open tubular columns (WCOT)
  • Support coated open tubular columns (SCOT)
• GC Detectors


  • Thermal conductivity detector
  • Flame ionization detector
  • Electron capture detector
  • Flame photometric detector
  • Photo-ionization detector
• Applications of Chromatography


  • Drug Discovery
  • Clinical Analysis
  • Proteomics
  • Forensic Chemistry
  • Drug Metabolism study
  • Environmental chemistry
  • Diagnostic studies
  • Cosmetic analysis
  • Determination of Green Florescent Protein
  • Structural Determination
  • Pharmaceutical Applications
  • Identification of Bile Acid Metabolite
  • Clinical Applications
  • Biochemical Genetics
  • Qualitative and quantitative analysis
  • Therapeutic Drug Monitoring
• Classification of Ion Exchange Resins

  • Source of the resin
  • Chemical nature
  • Functional group
  • Structural type
  • Physical properties
  • Chemical structure
• Requirements of Ion Exchange Resin

  • Chemically stable
  • Insoluble in common solvents
  • Sufficient degree of cross linking
  • Swollen resin must be denser than water
  • Sufficient no. of ion exchange groups
• Applications of Ion Exchange Chromatography

  • Conversion from one salt to other
  • Household (laundry detergents and water filters) to produce soft water
  • Prepare de-ionized water
  • Separate and purify metals
  • Dealkalization
  • Analysis and purification of immunoglobulin's
  • Separation of inorganic ions
• Gel Filtration Chromatography


  • Group separations - components of a sample are separated into two major groups according to size range
  • High resolution fractionation of biomolecules - Components of a sample are separated according to differences in their molecular size
• Gel Filtration Chromatography Process

  1. A mixture of molecules dissolved in liquid (the mobile phase) is applied to a chromatography column which contains a solid support in the form of microscopic spheres, or "beads" (the stationary phase)

  2. Larger molecules pass around or are "excluded" from the beads, while smaller molecules can access most or all pores

  3. Particles of different sizes will elute (filter) through a stationary phase at different rates
• Applications of Gel Filtration Chromatography

  • Proteins fractionation
  • Purification (viruses, enzymes, hormones, nucleic acids)
  • Molecular weight determination(globular proteins)
  • Separation of sugar, proteins, peptides, rubbers and others on the basis of their size
  • Determining the quaternary structure of purified proteins
• Affinity Chromatography


  • A sample purification technique, used primarily for biological molecules such as proteins
  • Separates a mixture of proteins or nucleic acids by specific interactions of those molecules with a component known as a ligand, which is immobilized on a support
• Chromatographic Media in Affinity Chromatography

  • Insoluble in solvents and buffers employed
  • Chemically and mechanically stable
  • Easily coupled to a ligand or spacer arm
  • Exhibit good flow properties and have a relatively large surface area for attachment
• Ligand in Affinity Chromatography


  • Selected based on the nature of the macromolecule to be isolated
  • Examples: hormone for hormone receptor protein, antigen or hapten for antibody, substrate analog/inhibitor/cofactor/effector for enzyme
• Applications of Affinity Chromatography

  • Isolation and purification of biological macromolecules (nucleic acid, antibodies, enzymes etc.)
  • Noticing which biological compounds bind to a particular substance
  • Reducing an amount of substance in a mixture
• Electrophoresis


  Migration of charged molecules under the influence of an external electric field
• Types of Electrophoresis


  • Zone Electrophoresis
  • Paper Electrophoresis
  • Gel Electrophoresis
  • Thin Layer Electrophoresis
  • Cellulose acetate Electrophoresis
  • Moving Boundary Electrophoresis
  • Capillary Electrophoresis
  • Isotachophoresis
  • Isoelectric Focussing
  • Immuno Electrophoresis