SEPERATION TECHNIQUES

• Chromatography


  Separation of a mixture on the basis of specific differences of the physical-chemical characteristic of the different components on a supporting medium called adsorbent or sorbent
• Chromatography

  Continuous redistribution between two phases: Stationary phase and Mobile phase (also called eluent or carrier fluid)
• Kinds of Chromatography

  • Paper Chromatography
  • Gel Chromatography
  • Ion-Exchange Chromatography
  • Thin Layer Chromatography
  • Liquid-Liquid Chromatography
  • Column Chromatography
  • Gas Chromatography
  • High Performance Liquid Chromatography (HPLC)
• Paper Chromatography

  1. Spot of substance placed on paper above solvent level

  2. Organic solvent (mobile phase) moves up paper by capillary action

  3. Fractions in sample move at different rates
• Paper Chromatography

  • Special grade of filter paper used as sorbent
  • Example: Whatman Phase Separating Paper
• Paper Chromatography

  • Basis of separation: Rate of diffusion, Solubility of solute, Nature of solvent
• Paper Chromatography

  Clinical use: Fractionation of sugars, amino acids and barbiturates
• Gel Chromatography


  Also called Gel Permeation, Size Exclusion, Molecular Sieve, Gel Filtration, Molecular Exclusion
• Gel Chromatography

  1. Mixture of small and large molecules passed over small particles in a column

  2. Smaller molecules diffuse into gel, larger molecules tend to pass rapidly
• Gel Chromatography

  • Gels with pores of accurately controlled size used as sorbent
  • Hydrophilic gels (water-loving) used for aqueous solutes
  • Hydrophobic gels (water-fearing) used for organic solvent solutes
• Gel Chromatography

  • Basis of separation: Molecular weight and size, Charge of ions, Hydrophobicity of molecules
• Gel Chromatography

  Clinical use: Fractionation of polysaccharides, nucleic acids, proteins including enzymes and isoenzymes
• Ion-Exchange Chromatography (IEC)

  1. Substances passed through ion exchange column

  2. Absorbed from solution based on net charge and pH

  3. Ions with greatest charge densities held most strongly
• Ion-Exchange Chromatography (IEC)


  • Anion or cation resin with functional group used as sorbent
  • Basis of separation: Differences in sign and ionic charge densities
• Ion-Exchange Chromatography (IEC)


  Clinical use: Separation of unwanted substances, Concentration of dilute solutes
• Ion-Exchange Chromatography (IEC)

  • Natural purification of water as it percolates through soil
• Thin Layer Chromatography (TLC)

  1. Sample components identified by comparison with standards on same plate

  2. Each drug has a retention factor (Rf) value that must match standard

  3. Incorrect aqueous to nonaqueous solvent mixture causes all spots to migrate to solvent front

  4. Extraction of drug is pH dependent, pH must be adjusted
• Thin Layer Chromatography (TLC)

  • Thin plastic plates impregnated with silica gel, alumina, polyacrylamide gel or starch gel used as sorbent
  • Basis of separation: Rate of diffusion, Solubility of solute, Nature of solvent
• Thin Layer Chromatography (TLC)


  Used for drug screening, biological samples like blood, urine and gastric fluid can be used
• Liquid-Liquid Chromatography


  Highly polar substances more soluble in polar solvents, less polar substances more soluble in less polar solvents
• Liquid-Liquid Chromatography

  • Basis of separation: Differences in solubility between two liquid phases, Aqueous phase and Organic solvent phase
• Liquid-Liquid Chromatography


  Clinical use: Fractionation of barbiturates, Lipid studies
• Column Chromatography

  • Basis of separation: Difference in pH, Polarity of solvent
  • Clinical use: Fractionation of sugars
• Gas Chromatography


  Capable of separating and measuring nanogram and picogram amounts of volatile substances
• Kinds of Gas Chromatography

  • Gas Solid Chromatography (sorbent is solid of large surface)
  • Gas Liquid Chromatography (sorbent is non-volatile liquid)
• Gas Chromatography

  • Basis of separation: Sample volatility, Rate of diffusion into liquid layer, Solubility of sample gas in liquid layer
• Gas Chromatography


  Clinical use: Drug screening and analysis, Fractionation of steroids, lipids, barbiturates, blood, alcohol and other toxicologic substances
• High Performance Liquid Chromatography (HPLC)


  Follows selective adsorption, applies 4,000 - 10,000 lbs/square inch pressure for rapid identification and separation of high molecular weight and labile biologic compounds
• Electrophoresis


  Migration or movement of charged particles in an electric field
• Electrophoresis


  Charged particle or ion migrates towards anode or cathode depending on isoelectric pH (pI) of solution under influence of applied electric field
• Definition of Terms

  • Iontophoresis - migration of small charged ions
  • Zone electrophoresis - migration of charged macromolecules
• Definition of Terms

  • Amphoteric - molecule whose net charge can be positive or negative depending on pH
  • Electroendosmosis/Endosmosis - movement of buffer ions and solvent relative to fixed support
• Factors Affecting Rate of Migration in Electrophoresis

  • Net Electric Charge
  • Size and Shape of Molecule
  • Electric Fluid Strength
  • Nature of Supporting Media
  • Temperature of Operation
• Net Electric Charge

  Higher charge, faster separation; lower charge, slower separation
• Size and Shape of the Molecule

  Bigger molecules, slower separation; molecular shape also plays a role
• Electric Fluid Strength

  Higher ionic strength, slower movement; mobility depends on buffer type and concentration
• Buffers

  • Barbital (Veronal) buffer pH 8.6
  • Tris-boric EDTA buffer pH 8.7
• Zone Electrophoresis Supporting Media

  • Paper Electrophoresis
  • Starch Gel Electrophoresis
  • Cellulose Acetate Electrophoresis
  • Agar (Agarose) Electrophoresis
  • Polyacrylamide Gel Electrophoresis (PAGE)
• Paper Electrophoresis

  • Earliest supporting media, disadvantages: fragile, easily damaged, variable staining
• Starch Gel Electrophoresis

  • Larger samples could be used, disadvantages: fragile, unable to store results permanently